On Plantain and Banana Seasonal Fluctuations of R

INFOINFOMUSAMUSA The International Magazine on Banana and Plantain

Vol. 11 N° 1 June 2002 IN THIS ISSUE Integrated crop management strategies for plantain production and control of black Sigatoka in DRC The spread of black Sigatoka throughout Venezuela, 1997-2000 Frequency of Paracercospora fijiensis and Pseudocercospora musae in Dominico hartón plantain Effects of the natural fungicide F20 on black Sigatoka disease on plantain and banana Seasonal fluctuations of R. similis and P. coffeae in certain cultivars of banana Host plant response of Pisang Jari Buaya and Mysore bananas to R. similis Effect of three arbuscular mycorrhizal fungi on root-knot nematode infection of Musa Endophytic fungal species associated with root necrosis of banana in Cuba Effects of mycorrhization on micropropagated banana Arachis pintoi: a cover crop for bananas? Dynamics of boron in a soil cultivated with plantain in Colombia Evaluation of the agronomic characteristics of plantain hybrids Options for in vitro propagation of the banana hybrid FHIA-20 Multiplication rate and regeneration potential of somatic embryos from a cell suspension of banana (Musa AAA) Introduction, multiplication and distribution of improved bananas and plantains in Nicaragua Using RAPD technique for identifying and classifying some banana cultivars in Vietnam Consumption and expenditure patterns of banana and plantain consumers in Nigeria Thesis MusaNews MusaForum INIBAP News Books etc. Announcements

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INFOMUSA is published with the support of the Technical Center for Agricultural and Rural Cooperation (CTA) The mission of the International Network for the Improvement of Banana and Plantain is to sustainably increase the productivity of banana and plantain grown on smallholdings for domestic consumption and for local and export markets. The Programme has four specific objectives: •To organize and coordinate a global research effort on banana and plantain, aimed at the development, evaluation and dissemination of improved cultivars and at the con- INFOMUSA servation and use of Musa diversity The International Magazine on Banana and Plantain •To promote and strengthen collaboration and partnerships in banana-related Vol. 11 N° 1 June 2002 IN THIS ISSUE Integrated crop management strategies for plantain production research activities at the national, regional and global levels and control of black Sigatoka in DRC The spread of black Sigatoka throughout Venezuela, 1997-2000 Frequency of Paracercospora fijiensis and Pseudocercospora musae in Dominico hartón plantain Effects of the natural fungicide F20 •To strengthen the ability of NARS to conduct research and development activities on on black Sigatoka disease on plantain and banana Seasonal fluctuations of R. similis and P. coffeae in certain cultivars of banana Host plant response of Pisang Jari bananas and plantains Buaya and Mysore bananas to R. similis Effect of three arbuscular mycorrhizal fungi on root-knot nematode infection of Musa Endophytic fungal species associated with root necrosis of •To coordinate, facilitate and support the production, collection and exchange of banana in Cuba Effects of mycorrhization on micropropagated banana Arachis pintoi: a cover crop for bananas? Dynamics of boron in a soil cultivated with plantain in Colombia information and documentation related to banana and plantain. Evaluation of the agronomic characteristics of plantain hybrids Options for in vitro propagation of the banana hybrid FHIA-20 Multiplication rate and regeneration potential of somatic embryos from INIBAP is a programme of the International Plant Genetic Resources Institute a cell suspension of banana (Musa AAA) Introduction, multiplication and distribution of improved bananas and plantains in Nicaragua Using RAPD technique for identifying and classifying some (IPGRI), a Future Harvest center. banana cultivars in Vietnam Consumption and expenditure patterns of banana and plantain consumers in Nigeria Thesis MusaNews MusaForum INIBAP News Books etc. Announcements PROMUSA News

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INFOMUSA is published with the support of the Technical Center for Agricultural and Rural Cooperation (CTA)

Vol. 11, N° 1 INFOMUSA Vol. 11, N° 1 Cover photo: In Tanzania, banana corms are frequently distributed through schools. CONTENTS (D. Mowbray, Baobab Productions) Integrated crop management strategies for plantain production and control Publisher: of black leaf streak (black Sigatoka) disease in the Democratic Republic International Network for the Impro- of Congo...... 3 vement of Banana and Plantain The spread of black Sigatoka throughout Venezuela, 1997-2000 ...... 6 Managing editor: Frequency of Paracercospora fijiensis and Pseudocercospora musae Claudine Picq in Dominico hartón plantain...... 9 Editorial Committee: Effects of the natural fungicide F20 on black Sigatoka disease (Mycosphaerella Emile Frison, Jean-Vincent Escalant, fijiensis Morelet) on plantain (AAB) and banana (AAA) ...... 14 Suzanne Sharrock, Elinor Lipman, Seasonal fluctuations of Radopholus similis and Pratylenchus coffeae in certain Charlotte Lusty cultivars of banana ...... 16 Printed in France Host plant response of Pisang Jari Buaya and Mysore bananas to Radopholus ISSN 1023-0076 similis ...... 19 Editorial Office: Effect of three arbuscular mycorrhizal fungi on root-knot nematode INFOMUSA, INIBAP, Parc Scientifique (Meloidogyne spp.) infection of Musa ...... 21 Agropolis II, 34397 Montpellier Cedex 5, A study of endophytic fungal species associated with root necrosis of banana in France. Telephone + 33-(0)4 67 61 13 banana and plantain plantations in Cuba...... 23 02; Telefax: + 33-(0)4 67 61 03 34; Effects of mycorrhization on the development of two cultivars of E-mail: [email protected] http//:www.inibap.org micropropagated banana...... 25 Subscriptions are free for developing Arachis pintoi: a cover crop for bananas? Advantages and disadvantages as countries readers. Article contributions regards nematology...... 28 and letters to the editor are welcomed. Dynamics of boron in a soil cultivated with plantain (Musa AAB cv. Dominico Articles accepted for publication may be hartón) in the Quindío, Colombia ...... 30 edited for length and clarity. INFOMUSA Evaluation of the agronomic characteristics of plantain hybrids (Musa spp.).. 34 is not responsible for unsolicited mater- Options for in vitro propagation of the banana hybrid cultivar FHIA-20...... 35 ial, however, every effort will be made to Multiplication rate and regeneration potential of somatic embryos from a cell respond to queries. Please allow three months for replies. Unless accompanied suspension of banana (Musa AAA cv. ‘Grande naine’)...... 38 by a copyright notice, articles appearing Introduction and multiplication of improved bananas and plantains in in INFOMUSA may be quoted or repro- Nicaragua and distribution to farmers ...... 44 duced without charge, provided acknowl- Using RAPD technique for identifying and classifying some banana cultivars edgement is given of the source. in Vietnam ...... 48 French-language and Spanish-language Consumption and expenditure patterns of banana and plantain consumers editions of INFOMUSA are also published. in Nsukka Urban, Nigeria ...... 50 To avoid missing issues of INFOMUSA, Thesis ...... 54 notify the editorial office at least six weeks in advance of a change of address. MusaNews ...... 56 INIBAP New ...... 60 Views expressed in articles are those Books etc...... 65 of the authors and do not necessarily Announcements...... 66 reflect those of INIBAP. Diseases Control of black Sigatoka Integrated crop management strategies for plantain production and control of black leaf streak (black Sigatoka) disease in the Democratic Republic of Congo

P. Mobambo Kitume Ngongo unsound in the framework of the Plants were grown in half-shade condi- resource-poor smallholders growing tions and watered regularly. They were plantain. Chemicals are very expensive transplanted in the field 3 months later, lantain (Musa spp., AAB group) is and their applications may be hazardous when they had 3-4 true leaves an important staple food in many to health in the village homesteads Four different treatments to prevent Pcountries of the humid tropics. It is where the bulk of plantain is grown. infection by microorganisms, based on among the most important carbohydrate Therefore, proper soil management cultural practices, were compared: crop sources in the diet of people in these using several crop residues mulches to residues mulches (wood sawdust or rice regions. Its low labour requirement and improve the organic matter and nutrient husk), cover crop (Vigna unguiculata) relatively high-energy output make plan- content of the soil could reduce the soil- and fertilizers (NPK). Non-treated plants tain a suitable staple for areas where disease-pest complex effects on plantain were used as control. labour shortage is usually the main con- with low inputs. straint to production. The crop is mainly The objectives of the research reported Field layout and cultural practices grown by small-scale farmers and it is an here were to compare field performance The experimental design was a random- integral component of most farming sys- and yield of plantain under different ized complete block with five plot- tems in West and Central Africa, where practices of soil fertility management and treatments and five replications. The plot about 50% of the world’s plantain is pro- disease control. size was 15 m x 10 m with 25 plants spaced duced (Wilson 1987, FAO 1990). by 3 x 2 m, resulting in a plant density of In spite of its importance to local peo- Materials and methods 1667 per ha. Data were recorded only on ple, plantain has long been ignored by the nine central competitive plants. agricultural researchers in the region, Location of the experiment Every 3 months, crop residues mulches since it had no major disease problems Investigations were carried out at were applied to the soil around the stem until the 1970s and was therefore Kinshasa (4°22’S, 15°21’E, western in mulched plots using one head-pan regarded as a disease-free crop in Africa Congo), which is at 390 m above sea level (10 kg). In fertilized plots, 300 kg N, 60 kg (Wilson 1987). Twenty-five years ago, (Anonymous 1985). The soil of the exper- P2O and 550 kg K2O per ha per year were however, the crop was threatened by imental site is a latosol derived from split into six applications during the rainy black leaf streak (black Sigatoka), an deposited sands, well drained, but poor in season: urea at a rate of 65 g per plant per air-borne leaf spot disease caused by the nutrients and highly acidic. Annual rain- application, phosphorus at a rate of 20 g fungus Mycosphaerella fijiensis fall averages 1800 mm and average temper plant per application, and muriate of Morelet. The disease spread rapidly into perature is 24.5°C. potash at a rate of 89 g per plant per all plantain-producing regions of Africa. application. Black Sigatoka is the most destructive Plant materials and treatments For each treatment, soil samples were leaf disease of plantain, as it is spreading Musa AAB cv. ‘Yumba’, locally wide- taken at about 50% flowering stage using inexorably to all major lowland plantain- spread, was used in this experiment. a soil hand auger up to 20 cm depth, growing regions as the dominant leaf spot Planting materials still constitute a con- where plantain has the majority of its (Meredith and Lawrence 1970). Plantain straint for plantain production in rural roots (Swennen 1984, Purseglove 1988). yield loss of 76% due to black Sigatoka has areas. As it is impossible to get many These samples were air-dried in the labo- been reported during the second crop- and uniform plantain suckers at once, ratory, crushed, passed through 0.5 and ping cycle, while the whole complex of the investigation started by vegetative 2 mm sieves and analyzed. disease, pests and soil fertility decline multiplication of planting materials together reduced yield by 93% (Mobambo (technique described by Auboiron 1997) Evaluation of host response et al. 1996a). As a perennial starchy crop, in order to obtain 625 plants for the to black Sigatoka, growth plantain requires a considerable time to experiment: 5 treatments x 5 replications and yield parameters mature, resulting in longer exposure to x 25 plants per treatment. The disease development was evaluated diseases, pests and in depletion of soil Plantain corm stumps were split into every week using the “symptom evolution nutrients. sets of 50 g each and treated with wood time”, which is the number of days The soil-disease-pest complex can be ashes. They were air-dried for 24 hours between the appearance of symptoms of controlled by the combination of inor- before being planted in 15 cm-diameter stage 1 of the disease development ganic fertilizers, fungicides and insecti- plastic bags almost filled with forest top- (Fouré 1982) assimilated to stage b of the cides/nematicides. In Africa however, soil. New sprouts emerged after 4 weeks cigar (Brun 1963) and the appearance of chemical control strategies are socio- from the date of planting and up to spots with dry centres (stage 6 of the dis- economically and environmentally 20 new plants were obtained from a corm. ease, Fouré 1982, 1987). The “youngest

INFOMUSA — Vol 11, N° 1 3 leaf spotted” which is the leaf with 10 or Table 1. Selected soil chemical properties under different plantain management more discrete necrotic lesions with dry practices at Kinshasa, western Congo, 1998. centres (Meredith and Lawrence 1970, Treatment pH Organic Carbon Total Nitrogen Exchangeable cations Fouré 1982, 1987) and the “life time of (%) (%) (meq/100 g) the leaf”, which is the number of days Ca Mg K between the cigar-stage b of the leaf and Control 4.2 a 1.15 a 0.11 a 1.22 a 0.21 a 0.15 a leaf death (100% leaf area necrotic), Wood sawdust 6.2 c 3.51 c 0.25 b 6.51 c 1.87 c 0.87 c either due to senescence or black Rice husk 6.8 d 3.79 d 0.28 b 7.52 d 2.10 c 0.98 d Sigatoka (Mobambo et al. 1994) were also Vigna unguiculata 5.2 b 2.15 b 0.22 b 4.07 b 0.78 b 0.46 b recorded. N-P-K 5.6 b 2.23 b 0.26 b 5.63 c 1.06 b 0.96 c Within columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to the Disease severity was evaluated every Duncan Multiple Range test. two weeks, from two months after planting until flowering. The percentage of leaf area with symptoms was recorded using the modified scale of Stover and the mulched plots than in the non- With respect to the youngest leaf spot- Dickson (1970) as described at the mulched plots. Crop residues mulches ted (YLS), results show the same trend as International Institute of Tropical constitute better sources of nutrients and for the symptom evolution time (Table 2). Agriculture (Mobambo et al. 1993a). act therefore as a fertilizer. As pointed There were significant differences Growth parameters evaluated include out by Lal and Kang (1982), organic mat- between the plantain mulched with crop height of pseudostem, girth of pseudo- ter constitutes a key component of soil residues and the non-mulched plantain. stem, number of leaves emerged and fertility, as a reservoir of nutrients, as a On mulched plantain, YLS was 11 for rice height of the tallest sucker. They were main source of cation exchange capacity husk and 9 for wood sawdust, whereas on recorded on each plant from 2 months and as major promoter of aggregate struc- both the fertilizer-treated plantain and after planting until flowering as tural stability. on the cover-cropped plantain the YLS described by Swennen and De Langhe was 8. The non-treated plantain (control) (1985). Host response to black Sigatoka had the lowest YLS value, 6. Yield parameters evaluated were num- Significant differences were found These results indicate that when using ber of hands per bunch, number of fruits between plantain mulched with crop the rice husk mulch the plant gains three per bunch and bunch weight. residues (wood sawdust and rice husk) healthy leaves comparing with the fertil- Data collected were analyzed using the and the non-mulched plantain (control, izers or cover crop treatments, and five ANOVA procedures of Statistical Analysis cover crop and fertilizer) regarding symp- healthy leaves against the control. System (SAS 1988) for randomized com- tom evolution time, youngest leaf spot- Therefore, with a leaf emergence time of plete block design. The Duncan Multiple ted, percentage of leaf area with symp- about one per week for plantain in gen- Range (DMR) test at the 0.05 significance toms and life time of leaf (Table 2). The eral, the soil fertility (Table 1) due to rice level was used to compare treatment severity of black Sigatoka on plantain was husk mulch slowed the symptom evolu- means for each parameter. much lower in the mulched plantain than tion by 3 and 5 weeks compared respec- in the non-mulched plantain. Among crop tively to that of the fertilized and cover- residues mulches, however, rice husk was cropped plots and the control. Results and discussion statistically the best for slowing disease Pronounced differences were also development. obtained between mulched and non- Soil conditions Black Sigatoka symptom development mulched plants regarding the percentage Soil analysis results presented in Table 1 in the mulched plantain was slower than of leaf area with symptoms (Table 2). showed significant differences in the in the non-mulched plantain. In the plan- While in cover-cropped and fertilized amounts of nutrients between crop tain mulched with crop residues, the dis- plots, plantain presented respectively residues mulches (wood sawdust and rice ease needed almost one month more to 10.3% and 6.9% of leaf area infected by husk) and other management practices, develop the last symptom stage compared black Sigatoka, in plots mulched with such as cover crop (Vigna unguiculata) to the control. For the fertilized and crop residues it lost only 3.8% to 4.2% of and mineral fertilizer (NPK). Meanwhile, cover-cropped plantain, symptom evolu- its leaf area. The slower spread of the dis- statistical differences were found tion time values were respectively 40 and ease in mulched plots is facilitated by an between rice husk and wood sawdust, 36 days, i.e. 2-3 weeks less than for the increased functional leaf area compared with rice husk as the best improving soil mulched plantain. to that of non-mulched plots. fertility level. According to the scales of Black (1965) and Brady (1984), in plots mulched with crop residues the soil was in general moderately acidic with very Table 2. Host-plant response to black Sigatoka of plantain under different high organic carbon, high total nitrogen, management practices at Kinshasa, western Congo, 1998. moderate calcium, moderate magnesium Treatment Symptom Youngest % Leaf area Life time and high potassium. In the non-mulched evolution time leaf spotted with symptom of leaf plantain plots however, the soil was (SET, days) (YLS) (% LAWS) (LTL, days) extremely acidic with low organic carbon, Control 23.0 a 5.5 a 19.6 d 62.5 a moderately low total nitrogen, low cal- Wood sawdust 50.0 c 9.3 c 4.2 a 125.7 d cium, very low magnesium and very low Rice husk 56.8 c 10.9 d 3.8 a 130.3 d potassium. Vigna unguiculata 35.5 b 7.5 b 10.3 c 80.3 b These results indicate that the N-P-K 40.0 b 8.1 b 6.9 b 103.3 c Within columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to amounts of soil nutrients are higher in the Duncan Multiple Range test.

4 INFOMUSA — Vol 11, N° 1 Regarding leaf life time, significant Table 3. Growth parameters of plantain under different management practices differences were also found between at Kinshasa, western Congo, 1998. plants mulched with crop residues and Treatment PH PG NEL DF DFF DH HTS those that were not mulched (Table 2). (cm) (cm) at harvest Slower disease development on mulched (cm) plantain prolonged the lifetime of Control 360.3 b 65.4 a 44 d 370 d 76 a 446 d 78.0 a leaves. In plantain treated with rice Wood sawdust 345.5 a 74.4 b 35 a 255 b 103 c 358 b 105.0 b husk and wood sawdust, black Sigatoka Rice husk 340.0 a 76.6 b 34 a 232 a 110 d 342 a 145.0 c needed almost 9, 7 and 4 weeks longer to Vigna unguiculata 349.5 a 68.8 a 41 c 295 c 86 b 381 c 80.5 a destroy the leaves as compared respec- N-P-K 342.2 a 66.8 a 38 b 268 b 102 c 370 bc 86.8 a Within columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to tively to the control, the cover-cropped the Duncan Multiple Range test. Legend: PH: Plant height; PG: Plant girth; NEL: Number of emerged leaves; DF: Days to flowering; DFF: Days for fruit filling; and the fertilized plantain. The control DH: Days to harvest; HTS: Height of the tallest sucker. plantain was the most affected by the disease. As already reported, all plantain cultivars (Musa spp., AAB group) over the world are susceptible to black Sigatoka (Fouré 1987, Mobambo et al. Table 4. Yield parameters of plantain under different management practices 1996b). at Kinshasa, western Congo, 1998. The difference in the host response to Treatment No. of hands No. of fruits Bunch weight Yield black Sigatoka between the plantain per bunch per bunch (kg) (t/ha) mulched with crop residues and non- Control 6.0 a 75 a 9.5 a 15.8 a mulched plantain is mainly attributed to Wood sawdust 6.5 b 88 c 15.0 c 25.0 c the difference in soil fertility. The higher Rice husk 6.5 b 90 c 17.5 d 29.2 d the soil fertility level, the lower the black Vigna unguiculata 6.2 a 82 b 11.0 a 18.3 a Sigatoka severity. On better soils this is N-P-K 6.2 a 87 bc 13.0 b 21.7 b expressed in a slower symptom develop- Within columns, means followed by the same letter are not significantly different at the 0.05 probability level, according to the Duncan Multiple Range test. ment, older leaves bearing dry spots, less leaf area with black Sigatoka symptoms and longer life time of leaves (Mobambo et al. 1994). mulched with rice husk was harvested rice husk was 14% higher than that 16 days earlier than that mulched with obtained with wood sawdust. Growth and yield performances wood sawdust. It also showed better These results indicate that crop resi- Results presented in Table 3 show sig- suckering, the tallest sucker, i.e. the dues mulches confer important advan- nificant differences for all the parame- sucker that will continue as the next tages to plantain cultivation: higher yield, ters studied: plant height (PH), plant production cycle, being significantly earlier maturity or shorter production girth (PG), number of emerged leaves taller than for other treatments. This cycle and bigger girth allowing reduction (NEL), days to flowering (DF), days should normally result in a shorter of losses from wind-damage, another for fruit filling (DFF), days to harvest ratoon cycle for the plantain treated important constraint to plantain produc- (DH) and height of the tallest sucker with rice husk as compared to other tion (Mobambo et al. 1996a). (HTS). treatments. For all treatments (crop residues The yield components evaluated were mulches, fertilizer or cover crop) the the number of hands per bunch, number Conclusion plants had a similar height, whereas of fruits per bunch and bunch weight The research reported here compares dif- they were shorter than the control. (Table 4). ferent management practices of plantain However, regarding plant girth and num- There were significant differences production. The effects of the crop ber of emerged leaves, the plantain between the plantain mulched with crop residues mulches (wood sawdust and rice mulched with crop residues performed residues (wood sawdust and rice husk) husk) were compared to those of fertil- better than the non-mulched plantain. and the non-mulched plantain (control, izer application and cover crop for soil Bigger plant girth and lower number of cover crop and fertilizer) regarding the fertility, black Sigatoka severity, growth leaves were obtained on plants treated number of hands per bunch and number and yield parameters of plantain. with rice husk and wood sawdust than of fruits per bunch (Table 4). The For all parameters evaluated, the plan- on the non-mulched plants. Plants mulched plantain had a higher number of tain mulched with crop residues per- mulched with rice husk flowered signifi- hands and fruits per bunch than the non- formed better than the non-mulched cantly earlier and had a longer fruit-fill- mulched. plantain. Soil fertility is the critical facing period than those under other treat- Yield per hectare was calculated from tor responsible for the difference ments. They flowered 5 months earlier the average bunch weight multiplied by between crop residues mulches, cover than the control and about 1 to 2 months plant density. Yield was significantly dif- crop and fertilizer. Because of the high earlier than the fertilized and cover- ferent between the plantain mulched level of fertility due to the application of cropped plantains. The combined effect with rice husk and other treatments. The crop residues mulches, plantain was less resulted in a shorter production cycle yield of the best-performing plantain affected by black Sigatoka and conse- for the plantain mulched with rice husk, treated with rice husk was 46%, 37% and quently better growing than when receiv- whose bunches were harvested 104 and 26% higher than those of control, covering no mulch. Among crop residues 28 days earlier than in the control and cropped and fertilized plantain, respec- mulches, rice husk was statistically bet- fertilized plantain respectively. Plantain tively. The yield of plantain mulched with ter than wood sawdust.

INFOMUSA — Vol 11, N° 1 5 Therefore, proper management of Pp. 152-178 in Non-symbiotic nitrogen fixation Purseglove J.W. 1988. Tropical crops: organic matter is essential for the sus- and organic matter in the tropics. Symposia Monocotyledons. Longman Scientific & Technical, tainable productivity of plantain, by mini- papers 1. 12th International Congress of Soil Singapore. 607pp. mizing the black Sigatoka severity with Science, 8-16 Feb. 1982, New Delhi. SAS. 1988. Statistical Analysis Systems Procedures low inputs. Since plantain is grown Meredith D.S. & J.S. Lawrence. 1970. Black leaf Guide, Release 6.03. SAS Institute, Cary, NC. mainly by small-scale farmers in Africa, streak disease of bananas (Mycosphaerella 441pp. chemical fertilizers are not readily and fijiensis): susceptibility of cultivars. Tropical Swennen R. 1984. A physiological study of the suck- economically available. Thus, the poten- Agriculture 27: 275-287. ering behaviour in plantain (Musa cv. AAB). PhD tial of traditional organic fertilizers such Mobambo K.N., F. Gauhl, D. Vuylsteke, R. Ortiz, Thesis. Katholieke Universiteit te Leuven, as compost, farmyard manure and crop C. Pasberg-Gauhl & R. Swennen. 1993a. Yield loss Belgium. 180pp. residues mulches need to be better in plantain from black Sigatoka leaf spot and field Swennen R. & E. De Langhe. 1985. Growth parame- exploited. A study integrating organic performance of resistant hybrids. Field Crops ters of yield of plantain (Musa cv. AAB). Annals of resources and soil fauna may help to Research 35: 35-42. Botany 56: 197-204. understand the mechanisms regulating Mobambo K.N., M. Naku & Z. Nganga. 1993b. Black Wilson G.F. 1987. Status of bananas and plantains in the biological processes for the improve- leaf streak disease in banana and plantain in West Africa. Pp. 29-35 in Banana and plantain ment of soil fertility in relation with the Zaïre. INFOMUSA 2: 14-15. breeding strategies (G.J. Persley and E.A. De sustainability of plantain production, dis- Mobambo K.N., K. Zuofa, F. Gauhl, M.O. Adeniji & Langhe, eds). Proceedings of an International ease and pest severity. C. Pasberg-Gauhl. 1994. Effect of soil fertility on Workshop, 13-17 October 1986, Cairns, Australia. host response to black leaf streak of plantain ACIAR Proceedings No. 21. (Musa spp., AAB group) under traditional farming Acknowledgements systems in southeastern Nigeria. International This research was fully supported by the Journal of Pest Management 40: 75-80. International Foundation for Science Mobambo K.N., F. Gauhl, R. Swennen & C. Pasberg- (IFS), Stockholm, Sweden. I am espe- Gauhl. 1996a. Assessment of the cropping cycle cially grateful to Ms Ingrid Lindhe effects on black leaf streak severity and yield (Assistant, Crop Sciences Area) and Ms decline of plantain and plantain hybrids. Josiane Lindberg (Purchasing services) International Journal of Pest Management for their availability, assistance and effi- 42: 1-7. cient help during my research. ■ Mobambo K.N., F. Gauhl, C. Pasberg-Gauhl & The author is a Professor in the Faculty of Agriculture, K. Zuofa. 1996b. Season and plant age affect eval- University of Kinshasa, BP 785 Kinshasa XI, Democratic uation of plantain for response to black Sigatoka Republic of Congo. Tel. +243 99 18257; References disease. Crop Protection 15: 609-614. E-mail: [email protected] Anonymous. 1985. Caractéristiques climatiques de Kinshasa. Institut national de géographie. 85pp. Auboiron E. 1997. Propagation rapide de matériel de plantation de bananiers et plantains. La multipli- Diseases Distribution of black Sigatoka cation sur souche décortiquée. Fiche technique. CRBP, Douala, Cameroun. Black N.C. 1965. Physical and mineralogical properties, including statistics of measurement and sam- The spread of black Sigatoka pling. Pp. 515-545 in Methods of soil analysis. Part I: Physical methods (C.A. Black, ed.). American throughout Venezuela, Society of Agronomy, Madison, Wisconsin. Brady N.C. 1984. The nature and properties of soils. 1997-2000 MacMillan Publishing Company, New York. 750pp. Brun J. 1963. La cercosporiose du bananier en Guinée. Etude de la phase ascosporée de G. Martínez, J. Hernández, Estévez 1992, Stover 1993, Guzman et al. Mycosphaerella musicola Leach. Thesis. Faculté O. Tremont, R. Pargas 2000, Romero 2000). des Sciences, Université Paris-Orsay. 196pp. and E. Manzanilla This situation makes clear the extent FAO. 1990. Production Yearbook. Food and of the problem posed by this disease and Agriculture Organization of the United Nations, necessitates the introduction of inte- Rome. ince the first report of black grated control measures using resistant Fouré E. 1982. Les cercosporioses du bananier et Sigatoka (Mycosphaerella fijiensis clones with a high yield potential (Rowe leurs traitements. Comportement des variétés. 1: SMorelet) in Venezuela, great uncer- and Rosales 1993). The existence of a Incubation et évolution de la maladie. Fruits 37: tainty has existed as to the future of close relationship between certain cli- 749-766. banana and plantain production there. matic factors (relative humidity, temper- Fouré E. 1987. Varietal reactions of bananas and The complex nature of the pathogen gives ature and rainfall) and the pathogen plantains to black leaf streak. Pp. 110-113 in it considerable potential for adaptation influences the incidence and severity of Banana and plantain breeding strategies (G.J. to new climatic conditions, fungicides the disease (Fouré 1994, Gauhl 1994). It Persley and E.A. De Langhe, eds). Proceedings of and host genotypes (Ploetz 2000). This is is this which has enabled us to map the an International Workshop, 13-17 October 1986, clearly demonstrated by the loss in effi- spread of the disease across the country Cairns, Australia. ACIAR Proceedings No. 21. cacy of certain products used for chemi- and also to take steps against its medium- Lal R. & B.T. Kang. 1982. Management of organic cal control such as the benzimidazoles term incidence in zones where its pres- matter in soils of the tropics and subtropics. and triazoles (Douglas and Ching 1992, ence has yet to be recorded, such as was

6 INFOMUSA — Vol 11, N° 1 CARIBBEAN SEA FALCON

YARACUI DE SUCRE ZULIA LARA CARABOBO ARAGUA MIRANDA TRUJILLO DELTA CO PORTUGUESA MONAGAS MERIDA JE DE GUARICO AMACURO C BARINAS ANZOATEGUI O TACHIRA L APURE O BOLIVAR M B I GUYANA A 1991 - 1994 AMAZONAS 1994 - 1996 BRASIL 1996 - 1998 1998 - 2000

Figure 1. Spread of black Sigatoka in Venezuela from 1991 to 2000.

done in 1997 and 1998 (Martínez 1997, Martínez et al. 1998), these regions were infection. The liberation of ascospores is Martínez et al. 1998). declared to be at high risk of potential rapid during rain because of the presence The work presented here aims to infection in the short term (Figure 1). of a film of residual water on the upper describe the current situation of black They are characterized by rainfall in surface of the leaves, whose lower surface Sigatoka in Venezuela, the course of its excess of 1500 mm/yr, a relative humidity exhibits more lesions. Dead leaves which spread, its relationships with various cli- above 79% and a mean temperature remain attached to the plant therefore matic factors which determine its aggres- between 25 and 28ºC (Figures 2 and 3), represent an excellent source of inocu- siveness and measures taken for its con- which is significant in view of the rela- lum (Gauhl 1994). As to the temperature, trol. For this purpose, an expedition was tionship established between climate and it is estimated that the ascospores of made into different areas in the south- the incidence and development of the Mycosphaerella fijiensis germinate east of Venezuela, collecting samples disease (Fouré 1994, Gauhl 1994, between 10 and 38ºC, with an optimum at showing typical symptoms of the disease Mobambo 1995). They are very different 27ºC, and noting that the relative growth for identification, questioning growers from the Maracay region where the mean rate of the germination tubes (hyphae) and analyzing weather data (relative rainfall is 922 mm/yr with a 6-month dry falls rapidly at temperatures below 20ºC humidity, rainfall and temperature). season. This situation has enabled the (Pérez and Mauri, cited by Pérez 1996). establishment of a model for comparing Concerning the effect of wind, it has been Current situation and spread two totally different agro-ecological shown that the concentration of conidia of the disease states with which are correlated critical in plantations is higher in the lowest air Black Sigatoka was detected for the first levels of the severity reached by the dis- layers than on the leaves whereas the time in Venezuela in 1991 in the state of ease. This model serves as a reference for concentration of ascospores in the air is Zulia in the western region (Haddad et al. introducing control measures on the the same: this confirms the importance 1992, Escobar and Ramirez 1995), and basis of climatic conditions and to pre- of the ascospores in the life cycle of the then it spread into various zones and vent possible spread into zones possess- disease (Stover 1984, Gauhl 1994). states (Martínez 1997, Martínez et al. ing similar characteristics (Martínez It remains to emphasize the presence 1998). Mention is made in this report of et al. 2000). of topographic accidents in well-defined its arrival in the state of Bolivar and, Fouré (1994) mentions relationships geographical areas which is apparently between 1999 and 2000, in the states of existing between climatic parameters correlated with the variation in climatic Delta Amacuro and Amazonas, in the and the spread of the disease which per- factors mentioned above. These topo- extreme east and south of the country, mit a better understanding of the dynam- graphic accidents therefore also affect respectively. On the basis of their rainfall ics of the epidemic in production zones the development and severity of the dis- and relative humidity (Martínez 1997, and of its potential for initiating future ease. The first report of the disease

INFOMUSA — Vol 11, N° 1 7 products obtained. This has been intro- 2500 duced thanks to research work carried 2000 Barinas out by INIA, and also to the growing 1500 Maracay importance of the organization among El Vigía the producers. 1000 San Felipe Bolivar In the course of all the field experi- 500 ments the accent has been placed on effi- Precipitations (mm) Precipitations cient application of basic cultural prac-

87 88 89 90 91 92 93 94 95 96 tices, such as removal of hanging dead leaves and the use of fertilizer, practices Years which are not being applied at present even though it has been demonstrated Figure 2. Rainfall in Barinas, Maracay, El Vigia, San Felipe and Bolivar (Source: FAV.MARNR, DANAC). that they help to reduce the amount of inoculum of the pathogen in the plantation and that they render the plant less

90 vulnerable to fungal attack (Gauhl 1994). Quite clearly one should try to reduce 85 San Felipe Barinas the use of chemicals and aim for the best 80 Maracay possible way of living with the pathogen. 75 El Vigía An alternative solution which may be 70 adopted is the use of resistant clones

Relative humidity (%) 65 which can either be grown in commercial 88 89 90 91 92 93 94 95 96 plantations in rows between the clones

Years traditionally grown in the country (so as to reduce the quantity of inoculum avail- Figure 3. Relative humidity in Barinas, Maracay, El Vigia and San Felipe (Source: FAV.MARNR, DANAC). able), or else as an entirely separate plantation, as is seen in the Ocumare region of Costa, in Aragua state. There they have chosen to grow plantain FHIA-21, whose occurred in the Lake Maracaibo area, ducers’ organizations. With a low yield fruit has a softer texture than that of where the high relative humidity may be which is all consumed by the family, the ‘Hartón gigante’ and can be used for mak- due to the proximity of the lake and to alternatives for the small producer are to ing “tostones”, or chips, of excellent qual- the topography of the landscape of the sell his plantation, change the crop or ity, much appreciated by the consumers, region. These conditions are similar to simply to abandon the farm completely which has facilitated its introduction to those around Lake Valencia (the point of (Martínez et al. 2000). the market. Likewise it should be noted entry of the disease into the state of Medium-sized producers tend to adjust that there are other possibilities for pro- Aragua) and in the sectors close to the the area of their plantation if production duction such as the use of the hybrids Caroni river, Hato Gil (Bolivar state). In costs increase, allowing them to obtain FHIA-01, FHIA-02 and FHIA-03 which the same way, the presence of the Andes yields which are dependent on the yield well and a have a very good response cordillera and the interior mountain amount of investment. Large producers to the disease. chain, which constitute a natural barrier succeed in living with the disease, as can to the passage of the fungal spores to be seen to the south of Lake Maracaibo, Conclusions adjacent regions, ought to have pre- where there are associations of produc- 1. The speed of spread of the pathogen vented the spread of the disease into ers and firms which improve the quality through the country has increased these zones. However this has happened, of the product in the plantations where it rapidly: its passage from the western and its appearance in these regions can is destined for the international market. zone to the central zone took five years only be due to the transport of infected That which is rejected for export is sold while the spread from the central zone material. on the national or local market where to the eastern zone and the south there is no quality control (Martínez et needed only one. It seems clear that this Influence of the disease al. 2000). development has been encouraged by on plantation management man. Causing an increase of 40-45% in Farm surveys and visits to different areas Changes in crop management the costs of production, the disease has of the country have shown us that the observed in the presence particularly affected small producers biggest losses have occurred in fields of black Sigatoka and cast doubt over the survival of their where there was no control of weeds, The presence of black Sigatoka in the holdings. The advance of the disease nematodes or insects. Hanging, dried-up country has resulted in radical changes through the national territory continued leaves were not removed and no fertilizer in the way bananas are grown. The tradi- into the states of Bolivar, Delta was used. There were also problems of tional approach, which is to manage the Amacuro and Amazonas between 1997 irrigation and drainage and an unsatis- plantations as perennial crops, tends to and 2000. factory spacing of plants in the field. The have been gradually replaced by semi- 2. The case of the state of Amazonas, on growers are not in the habit of removing perennial, and in some cases annual man- the frontier with Brazil, is particular. side shoots, nor of using chemicals to agement of the crop, with high planting The banana and the plantain, grown by control diseases. They lack technical sup- densities possibly in association with the indigenous communities, are major port and resources to buy agrochemicals other short-term crops, giving an increase elements of their diet. The ecosystem and equipment. Finally, there are no pro- both in yield and in the diversity of the of the region is fragile and there is com-

8 INFOMUSA — Vol 11, N° 1 plex genetic and biological diversity. XIV Congreso Venezolano de Fitopatología. Leach ex Mulder) en banano y plátano. Proyecto For this reason it is undesirable to use Universidad de los Andes, Mérida, Venezuela. TCP/CUB/4454. 27pp. chemical control products and prefer- Estévez M. 1992. Monitoreo sobre la resistencia de la Ploetz R. 2000. La enfermedad más importante del able to recommend introducing resis- Sigatoka Negra a fungicidas sistémicos, pene- banano y el plátano: Una breve introducción a la tant clones which do not need the trantes, inhibidores de esteroles. Revista P.N.B. historia, importancia y manejo de la Sigatoka application of fungicides, even if the (Ecu): 32-33. negra. P. 117 in Reunión ACORBAT 2000. cultivars are not fully acceptable to the Fouré E. 1994. Leaf spot diseases of banana and plan- Memorias. Mesa redonda Sigatoka negra. native consumers. tain caused by Mycosphaerella fijiensis and Romero R. 2000. Podemos evitar o disminuir el riesgo 3. Today it is evident that the presence of M. musicola. Pp. 37-46 in The improvement and de desarrollo de resistencia a los funguicidas en black Sigatoka in the country has testing of Musa: a global partnership. Proceedings las poblaciones de M. fijiensis? P. 118 in Reunión brought about radical changes in the of the first Global conference of the International ACORBAT 2000. Memorias. Mesa redonda agronomic management of plantations. Musa Testing Programme held at FHIA, Honduras Sigatoka negra. The effective use of basic cultural prac- (D. Jones, ed.).INIBAP, Montpellier, France. Rowe P. & Rosales F. 1993. Diploid breeding at FHIA tices, within the framework of inte- Gauhl F. 1994. Epidemiology and ecology of black and the development of Goldfinger (FHIA-01). grated control, makes it possible to live Sigatoka (Mycosphaerella fijiensis Morelet) on INFOMUSA 2(2): 9-11. with the pathogen, as has been shown plantain and banana in Costa Rica, Central Stover R. 1984. Las manchas producidas por las by numerous research studies carried America. PhD thesis originally presented in Sigatokas en hojas de bananos y plátanos. Curso out by INIA. German. INIBAP, Montpellier, France. 120pp. internacional de reconocimiento, diagnostico y Guzmán M., A. Jiménez, R. Vargas & R. Romero. 2000. control de Sigatoka negra del plátano y banano. Acknowledgements Caracterización de cepas de M. fijiensis, causante 14-18 May, Tulenapa, Colombia. 15pp. We thank particularly Mr Daniel Muñoz de la Sigatoka negra, con menor sensibilidad a Stover R. 1993. Cambios en la sensibilidad de for his valuable collaboration in collect- funguicidas triazoles. P. 64 in Reunión ACORBAT Mycosphaerella fijiensis al tilt. Informe UPEB ing information in the field, Mr Marcos 2000. Memorias. 16(97): 41-44. Sanoja, a specialist at the Corporación Haddad O., M. Bosque, J. Osorio & L. Chávez. 1992. Venezolana de Guayana (CVG), Aspectos fitosanitarios: Sigatoka negra medidas Fundacite-Guayana for logistic support, de prevención y control. FONAIAP Divulga 40: the staff of the climatology section of the 44-45. Venezuelan Air Force (FAV) at Maracay, Martínez G. 1997. The present situation with regard the Ministry of the Environment and to black Sigatoka in Venezuela. INFOMUSA Natural Renewable Resources (MARNR) 6(1): 16-17. and the Polar Foundation, through Martínez G., R. Pargas, E. Manzanilla & D. Muñoz. DANAC. ■ 1998. Report on black Sigatoka status in Venezuela in 1997. INFOMUSA 7 (1): 31-32. References Martínez G., J. Hernández & A. Aponte. 2000. Douglas M. & L. Ching. 1992. Monitoreo de sensibili- Distribución y epidemiología de la Sigatoka negra dad de Mycosphaerella fijiensis al Benomil. en Venezuela. Serie C.48. FONAIAP. Fundacite Pp. 17-19 in Informe anual Corbana. Guayana. 50pp. The authors work in different centres of Instituto Escobar C. & M. Ramírez. 1995. Avance y establec- Mobambo K. 1995. Factors influencing the develop- Nacional de Investigaciones Agrícola, INIA (former imiento de la Sigatoka Negra (Mycosphaerella ment of black Sigatoka disease on plantain and FONAIAP), Venezuela. Gustavo Martínez, Rafael Pargas and Edwuard Manzanilla at CENIAP- fijiensis) en el occidente de Venezuela. plantain hybrids. INFOMUSA 4(1): 16-17. Maracay, apartado postal 4653, Maracay; E-mail: Universidad Nacional Experimental del Táchira Pérez L. 1996. Manual para el control integrado de [email protected] and [email protected], y Ministerio de Agricultura y Cría-SASA. Sigatoka negra (Mycosphaerella fijiensis) y Julitt Hernández at CIAE-Yaracuy and Omar Tremont VIII Congreso Latinoamericano de Fitopatología. Sigatoka amarilla (Mycosphaerella musicola at CIAE-Amazonas.

Diseases Variability in leaf spot diseases

Frequency of Paracercospora fijiensis and Pseudocercospora musae in Dominico hartón plantain

C. Lorena Cardona-Sanchez minimal cultivation and as a result yellow mercial acceptance being due to their and J. Castaño-Zapata Sigatoka and black Sigatoka have flavour and other qualities and size; it is increased in severity and dissemination highly susceptible to black and yellow (Merchán 1998), reducing production Sigatokas in the marginal coffee-growing lantain (Musa sp.) is a subsistence by 50% (Burt et al. 1997). zone (Merchán 1992). crop and in many areas is the sta- Nationally 384 957 ha are cultivated, Today, these diseases are found Pple food for the population espe- 75% with plantain Hartón and Dominico competing at altitudes greater than cially in rural areas, with an estimated hartón, the latter cultivar increasingly 1000 m above seal level. According to national per capita consumption of cultivated in plantain plantations some reports, black Sigatoka, caused 68.5 kg/annum. Production is mostly with throughout the country, the high com- by Mycosphaerella fijiensis Morelet, is

INFOMUSA — Vol 11, N° 1 9 found attacking plantain Dominico on 13 March 1999. Impressions were removed by pressing the agar surface hartón in the district of Victoria (Caldas) taken weekly from leaves attacked by the against the lesion at leaf stage 4-5 of the 100 m above sea level, and is more aggres- Sigatokas with the aim of quantifying youngest leaf spotted. The cylinder of sive than yellow Sigatoka, caused by spore populations of the anamorph crystal violet agar and conidia was placed Mycosphaerella musicola Leach, a dis- state of M. fijiensis (Figure 1) and on a slide and transferred to a tray lined ease which was displaced in less than six M. musicola (Figure 2). with a paper towel moistened with sterile months (Merchán 1992). A similar behav- Leaf impressions were made with water. The trays were covered with plas- iour was observed in the district of syringes of solidified agar in the form of a tic bags and transferred to a hermetic Pueblo Rico (Risaralda) 1560 m above dispenser, which was prepared from a polystyrene container (icopor). sea level (Merchán 1992). According to 5 ml disposable syringe from which the Both fungi were identified and the the latest reports, black Sigatoka can far end was removed forming a cylinder conidia/cm2 counted by means of a com- affect plantain from sea level up to an 1.26 cm diameter. The dispenser was pound microscope (Olympus) with a 40 x altitude of 1940 m (Belacázar et al. 1994). filled with crystal violet agar, prepared by objective. The Sigatokas are difficult to differen- mixing 1 g bacteriological agar with15 ml The variables analyzed were the num- tiate from their external symptoms in the of a 1% solution of crystal violet in dis- bers of conidia/cm2 of P. fijiensis and field, and hence it is not possible to estab- tilled water and 100 ml water. The mix- P. musae, temperature (maxima, means lish clearly which of the two diseases is ture was autoclaved at 121oC for 15 min., and minima), relative humidity and more frequent when they occur together after which 1 mg benomyl and two sensi- rainfall. (Aguirre et al. 1998b). Microscopically, tivity discs of streptomycin (10 µg) were Each variable was subjected to analysis M. fijiensis and M. musicola are distin- added (Aguirre et al. 1998b). of variance, descriptive procedures for guished mainly by the morphological dif- Conidial populations were quantified maximum, minimum and mean values, ferences of the anamorphs, in particular weekly by making impressions of each regression, Pearson’s correlation and the conidiophores and conidia character- material evaluated, the conidia being Chi-square test using the SAS (Statistical istics, especially by the presence of scars present on conidiophores and conidia of Paracercospora fijiensis but absent from Pseudocercospora musae (Aguirre et al. 1998b). Management of these diseases may be with chemical products, a practice which is not really applicable to the traditional system of crop production. In order to solve this problem more economical alternatives have been sought, such as host resistance to both diseases which results in a reduced sporulation of the causal organisms. The study was carried out with the objective of determining the frequency of P. fijiensis and P. musae spores in plantain Dominico hartón which is susceptible to black and yellow Sigatoka diseases.

Materials and methods The investigation was carried out in the Tolima department, 7 km from the Figure 1. Conidia of Paracercospora fijiensis,anamorph of Mycosphaerella fijiensis. District of Fresno, on the road from Manizales (Caldas) to Mariquita (Tolima) in the village of La Ceiba, Campoalegre estate, located at an altitude of 1250 masl and a temperature range of 18-25oC, a relative humidity of 65-100% and a rainfall of 1800 mm/annum. First, 1600 plants of the clone Dominico hartón, produced in vitro and multiplied in the tissue culture laboratory, Departamento de Fitotecnía de la Facultad de Ciencias Agropecuaria de la Universidad de Caldas, were transplanted then acclimatized at the Montelindo farm of the University. Evaluations were carried out weekly on 53 clones selected at random. Records were taken starting 13 September 1998, the time of flower initiation, until harvest Figure 2. Conidia of Pseudocercospora musae, anamorph of Mycosphaerella musicola.

10 INFOMUSA — Vol 11, N° 1 Analysis System) statistical programme Table 1. ANOVA components for the number of conidia of Paracercospora fijiensis (SAS Institute 1980). Conidial numbers and Pseudocercospora musae. were transformed with Lnx+1, which best ANOVA P. fijiensis fits the behaviour of the data, where x is G.L. C.M. F Pr > F R2 C.V. (%) the number of conidia/cm2. Model 985 6.83 3.74 0.0237* 0.99** 35.8 Error 8 183 Results and discussion Clones 53 10.67 5.84 0.0061** Analysis of variance of P. fijiensis and Dates of evaluation 18 56.59 30.97 0.0001** P. musae counts suggested highly signifi- Interaction of clone-date 859 5.53 3.03 0.0456* cant differences for clones and dates of Total 943 evaluation. The interactions between ANOVA P. musae the two factors were significant for G.L. C.M. F Pr > F R2 C.V. (%) P. fijiensis and highly significant for P. musae, indicating that a high or low Model 936 3.79 11.93 0.0004** 0.99** 23.31 inoculum production depends on the Error 8 0.32 planting material and the effects of envi- Clones 53 3.39 10.67 0.0007** ronmental conditions on the develop- Dates of evaluation 18 40.50 127.67 0.0001** ment of each material (Table 1). Interaction of clone-date 860 3.04 9.59 0.0009** The processes of infection and inocu- Total 944 * : Denotes significant differences, p = 5% lum production were favoured by rainy ** : Denotes highly significant differences, p = 1% periods, and as rainfall increased so Note: Data transformed as square root number of conidia. did the numbers of conidia shown by P. fijiensis and P. musae with two periods of maximum conidial production at 30000 350 334 and 424 days after planting (dap). A Precipitation These coincided with the maximum 300 P. fijiensis rainfall recorded during the study, with 24000 P. musae an accumulated rainfall of 211.8 mm 250 and 296.2 mm respectively (Figure 3A), 18000 which was in agreement with the stud- 200 ies of Aguirre et al. (1998a). The authors 150 observed that accumulated rainfall was 12000

inversely related to the incubation Number of conidia Precipitations (mm) Precipitations period and development of black and 100 yellow Sigatokas, and directly related to 6000 sporulation. This suggests that as weekly 50 accumulated volume of rainfall 0 0 increases, the incubation period and 334 361 389 424 452 473 development of both Sigatokas declines and results in an increased disease 23 B severity and hence greater inoculum production of the causal fungi. From 22 C) 424 dap the relationship between coni- ° 22 dial number and rainfall started to 21 decline, being particularly evident at

473 dap when, although there was an ( mperature 21 Te increased rainfall (192.5 mm), conidial 20 production was very low because foliage was severely necrotic, and no healthy 20 tissue remained available for infection. 334 361 389 424 452 473 Temperature and relative humidity remained fairly constant with an aver- 84 C age of 21.5oC (Figure 3B) and 81% 81 (figure 3C), conditions that are opti- 78 mum for conidial production. In agree- 75 ment with Mouliom Pefoura and 72 Mourichon (1990) and Tapia (1993), cited by Porras and Pérez (1997), tem- Relative humidity (%) 69 334 361 389 424 452 473 peratures higher than 20oC favour conidial development in P. fijiensis. Days after planting (dap) According to Stover (1965), temperatures higher than 22oC favour conidial Figure 3. Total conidia of P. fijiensis and P. musae in relation to time for 53 clones and their relationship production in P. fijiensis, with a temper- to climatic conditions. ature of 26°C being optimum (Stover A. Rainfall, B. Temperature and C. Relative humidity.

INFOMUSA — Vol 11, N° 1 11 Table 2. Mean conidia/cm2 with time for P. fijiensis and P. musae and their standard At the time of flowering, which coin- deviations, and their agreement with rainfall (September 1998 – March 1999). cided with the start of the study, the standard deviation remained high up to Days after Correlation Conidia Standard Conidia Standard Weekly planting (r) of deviation of deviation rainfall 452 dap, when buds had emerged from (dap) P. fijiensis P. musae (mm) most plants; the high standard deviation 319 0.7782 ** 45 65.96 37 58.55 81.60 was due mainly to the effects of rainfall 326 0.7466 ** 42 55.10 16 25.65 34.40 on conidial production as mentioned for 334 0.8464 ** 26 46.98 12 24.87 95.80 the results where P. fijiensis had a higher 340 0.8115 ** 32 58.84 13 20.71 9.70 frequency than P. musae (Table 2). 347 0.6622 ** 23 29.70 12 27.17 21.00 From 319 to 347 dap the accumulated 361 0.8557 ** 13 11.35 7 8.61 44.00 rainfall was 242.5 mm, with a high fre- 375 0.6407 ** 31 37.79 9 14.34 51.80 quency of conidia of both fungi. However, 389 0.7373 ** 11 22.61 3 4.80 109.10 between 361and 404 dap, the accumu- 404 0.8552 ** 22 41.92 8 14.96 173.70 lated rainfall of 378.6 mm was excessive, 411 0.6903 ** 15 20.72 5 8.69 42.20 reducing the conidial populations of both 418 0.7697 ** 16 18.44 4 6.55 25.30 pathogens. It is noteworthy that at 438 424 0.6966 ** 14 19.26 5 11.66 55.00 dap the highest correlation (r = 0.8575) 438 0.8575 ** 14 21.65 2 3.78 51.90 occurred between conidial populations 452 0.6361 ** 10 13.14 2 4.78 57.60 and rainfall (Table 2). 466 0.6028 ** 5 6.05 2 3.68 103.20 Between 411 and 452 dap the majority 473 0.8074 ** 4 6.08 2 4.35 89.30 of leaf tissue started to become necrotic. 493 0.5474 ** 3 3.62 1 2.50 220.80 During this period rainfall was 232 mm Mean 19 8 81.32 and conidial populations an average of ** Correlations highly significant between the number of conidia of P. fijiensis and P. musae with time in relation to rainfall. 14 conidia/cm2/clone for P. fijiensis and 4 conidia/cm2/clone for P. musae. From 466 dap onwards, when the cultivation cycle was complete, the standard Table 3. Comparison of the mean number of conidia/cm2 of P. fijiensis deviations were low because the majority and P. musae in clones evaluated in relation to rainfall. of clones were severely infected and no more leaf tissue was available for Days after planting Mean number conidia Mean number conidia Rainfall infection, conidial populations of both (dap) of P. fijiensis of P. musae (mm) fungi were low, resulting in a low stan- 319 45ª 37 81.60 dard deviation. At harvest, there were an 326 42 17 34.40 average of 4 and 2 P. fijiensis and 334 26 12 95.80 P. musae conidia/cm2/clone respectively. 340 32 13 9.70 The conidial populations of P. fijiensis 347 23 13 21.00 were always higher than those of P. musae, 361 13 6 44.00 but populations of both fungi declined due 375 31 9 51.80 to a lack of healthy susceptible tissue 389 11 3 109.10 (Figure 4). 404 22 8 173.70 411 15 5 42.20 Finally, the relationship observed 418 16 4 25.30 between the two pathogens followed 424 14 5 55.00 a regression between the numbers of 2 438 14 2 51.90 conidia/cm of P. fijiensis and P. musae 452 10 2 57.60 in all clones evaluated. The correlation 466 5 2 103.20 coefficient was very low (r = 0.40656) 473 4 1 89.30 (Figure 5), suggesting that the number of 2 493 3 0 220.80 conidia/cm of P. musae did not depend Total 281 139 1266.00 on the behaviour of P. fijiensis and vice versa, and that conidial production in each of the clones depended on the susceptibility of the material and envi- 1965). A relative humidity of about 100% (1998a) who demonstrated that black ronmental conditions, particularly rain- favours production and viability of Sigatoka was more aggressive, occurring fall. In spite of the higher numbers spores, particularly when a water film is at times of the year when yellow of P. musae conidia, the numbers not present on the leaf surface (Jacome and Sigatoka disappeared, tending to be dis- always increasing or decreasing, the coni- Schuh 1992). placed by black Sigatoka. In general dia of P. musae followed the same pat- Conidial populations of P. fijiensis there was a marked direct correlation tern, but there was no direct or strong were always greater than those of between conidial numbers of P. fijiensis relationship between inoculum produc- P. musae in a ratio of 2.3:1 (Table 2) and P. musae and rainfall, which is also tion of the two pathogens. hence confirming that black Sigatoka in agreement with the studies of Aguirre In general, the clones evaluated pro- tends to displace yellow Sigatoka et al. (1998a) who observed that fluc- duced higher total numbers of conidia because of its greater aggressiveness; tuations in the numbers of conidia of P. fijiensis, confirming that black this is in agreement with the studies in trapped each week is highly correlated Sigatoka tends to displace yellow the same region by Aguirre et al. with rainfall. Sigatoka (Table 3). ■

12 INFOMUSA — Vol 11, N° 1 20000 250 Merchán V.V.M. 1992. Informe de actividades convenio ICA-IRFA. Subdirección Investigación

10000 Estratégica. CORPOICA Regional 9. Pp. 5-10. 2 200 Merchán V.V. M. 1998. Manejo de problemas fitosanitarios del cultivo del plátano en la zona 8000 central cafetera. Pp. 177-191 in Memorias del 150 Seminario Internacional sobre Producción 6000 de Plátano. Universidad del Quindío – Comité

100 de Cafeteros del Quindío – SENA – INIBAP –

4000 (mm) Precipitations CORPOICA.

otal number of conidia/cm Porras A. & L. Pérez. 1997. The role of temperature T 50 in the growth of the germ tubes of ascospores 2000 of Mycosphaerella spp., responsible for leaf spot diseases of banana. INFOMUSA 6(2): 27-31. 0 0 319 326 334 340 347 361 375 389 404 411 410 424 490 452 466 479 499 SAS Institute Inc. 1980. The SAS applications guide. (K.A. Council, ed.). SAS Inst., North Carolina. Age of the plant (dap) 195pp. Precipitations P. fijiensis P. musae Stover R.H. 1965. Leaf spot of bananas caused by Mycosphaerella musicola: Effect of tempera- Figure 4. Frequency of P. fijiensis and P. musae with time and the relationship with rainfall. ture on germination, hyphal growth, and conidia production. Tropical Agriculture (Trinidad) 42(4): 351-360. 12000

10000 2 8000

6000 P. fijiensis = 0.79**

4000 otal number of conidia/cm T 2000

0 1 2345678910111213141516171819 Weeks P. fijiensis P. musae

Figure 5. Relationship between the number of conidia of P. musae and P. fijiensis.

References de la producción del cultivo del plátano. Aguirre M.C., J. Castaño-Zapata, J.A. Valencia, Segundo informe técnico 1984-1994. Región 9 L.E. Zuluaga & C. Arce. 1988a. Interacción de ICA-CORPOICA. Creced-Quindio, Armenia, Mycosphaerella fijiensis Morelet y M. musicola Colombia. Leach en siete genotipos de Musa spp. en un área Burt J.A., J. Rutter & H. González. 1997. Short dis- límite de expansión de la Sigatoka negra en la tance wind dispersal of the fungal pathogens caus- zona cafetera colombiana. Pp. 192-220 in ing Sigatoka diseases in banana and plantain. Memorias del Seminario Internacional sobre Plant Pathology 46(4): 451-458. Producción de Plátano. Universidad del Quindío – Hernández G.J.C., J.A.C. Gómez & P.M.I. Arcila. 1994. Comité de Cafeteros del Quindío – SENA – Comportamiento agroeconómico de plántulas de INABAP – CORPOICA. plátano clon Dominico hartón Musa AAB Aguirre M.C., J.Castaño-Zapata & L.E. Zuluaga. Simmonds, manejados bajo condiciones de almá- 1998b. Método rápido de diagnóstico de cigo. Pp. 41-54 in Mejoramiento de la producción Mycosphaerella musicola Leach y M. fijiensis del cultivo del plátano. Segundo informe técnico Morelet, agentes causales de la Sigatoka amarilla 1984-1994. Región 9 ICA-CORPOICA. Creced- Claudia Lorena Cardona-Sanchez is an undergrad- y Sigatoka negra. Agronomía 8(2): 26-30. Quindio, Armenia, Colombia. uate student, Programa de Agronomía, Facultad Belalcázar C.S., V.F. Salazar, M.J.A. Valencia, Jacome L.H. & W. Schuh. 1992. Effects of leaf wet- de Ciencias Agropecuarias, Universidad de Caldas and Jairo Castaño-Zapata is Associate Professor, S.C.H. Silva, P.M.I. Arcila & R. Jaramillo. 1994. ness duration and temperature on development Departamento de Fitotecnia, Faculdad de Ciencias Reacción de variedades mejoradas de plátano of black Sigatoka disease on banana infected Agropecuarias, Universidad de Caldas, Apartado aéreo al ataque de Sigatoka negra (Mycosphaerella by Mycosphaerella fijiensis var. difformis. 275, Manizales (Caldas), Colombia. fijiensis Morelet). Pp. 192-214 in Mejoramiento Phytopathology 82(5): 515-520. Email: [email protected]

INFOMUSA — Vol 11, N° 1 13 Diseases Control of black Sigatoka Effects of the natural fungicide F20 on black Sigatoka disease (Mycosphaerella fijiensis Morelet) on plantain (AAB) and banana (AAA)

R. Sánchez Rodríguez, There appear to be no publications on Each clone separately, plantain 3 J.A. Pino Algora, C. Vallin Plous, the use of streptothricins for the control ‘CEMSA /4’ and banana ‘Parecido al Rey’, M.E. Pérez Rodríguez, Y. Iznaga Sosa of plant pathogens; specifically there were arranged in an experimental design and F. Malpartida Romero have been no reports up to the present on of randomized blocks with six plants per the use of any antibiotic produced by plot, and four replications. microorganisms against diseases of The effect of F20 on black Sigatoka was he presence of black Sigatoka dis- banana and plantain. compared with the effect without phy- ease (Mycosphaerella fijiensis) in In this work we demonstrate the possi- tosanitary treatment and with plots TCuba since 1990 has resulted in ble use of streptothricins for the control treated chemically with propiconazole increased costs of production in planta- of black Sigatoka disease in clones of (Tilt 250 EC) at 400 ml/ha. F20 was 3 tions of plantain and banana because of plantain (Musa AAB) cv. ‘CEMSA /4’ and applied at a similar dose to propiconazole. the increased frequency of aerial and of banana (Musa AAA) cv. ‘Parecido al The effect of the fungicides was deter- ground-based sprays to control the causal Rey’. mined weekly by recording the develop- organism. Thus, there is an urgent need ment stage (DS) of the disease, the to find alternatives with nationally pro- Material and methods youngest leaf with symptoms or streak- duced products in order to reduce the The study was carried out at the Instituto ing (YLSS), and the youngest leaf spot- costs of disease control. de Investigaciones en Viandas Tropicales ted (YLS) (Fouré 1982, Pérez 1996, The indiscriminate use of chemical (INIVIT). Product F20, whose main active Orjeda 1998). products has resulted in side effects ingredients are the streptothricins B and including, inter alia, the appearance of F, was obtained from the Centro de Results and discussion fungicide resistance in the causal organ- Química Farmacéutica (CQF) in collabo- Sprays with F20 and propiconazole (Tilt) ism, the formation of strains more viru- ration with the Centro Nacional de with mineral oil, resulted in a reduction lent than indigenous strains and environ- Biotecnología de la Universidad Nacional in the disease index DS in comparison mental contamination (Rodríguez and Autónoma, Canto Blanco, Madrid, Spain, with untreated plots (UT) in clones 3 Jiménez 1985, Fullerton and Olsen 1991, by fermentation of strains of Streptomyces ‘Parecido al Rey’ and ‘CEMSA /4’. Mouliom Pefoura 1999). lavendofoliae var. 383 (which produces Figure 1 shows the results of phyto- The use of natural products obtained streptothricin B) and Streptomyces rochei sanitary applications in both clones. from microorganisms presents consider- var. f20 (which produces streptothricin F), Graphs A and B show the similar able advantages in comparison with com- isolated from Cuban soils. The fermenta- behaviour of F20 and Tilt with DS values mercial products, since their production tion broth was centrifuged, the super- not significantly different (P>0.05), and is much less damaging to the ecosystem natant submitted to chromatography on a significant difference (P<0.01) of both and their in situ biodegradability results ion exchange resin IRC-50, finally giving a applications in comparison with the in compounds that are not toxic to the solution saturated with sodium acetate control treatment. indigenous microflora. The search for after elutriation with acetic acid. The effects of disease control of the new and different products of natural ori- The product was applied after the satu- products were also evident in the graphs gin that do not contaminate the environ- rated sodium acetate solution containing as a reduction in the numbers of oscilla- ment, for the control of pests and dis- the antibiotics was dissolved in an aque- tions in DS values and by the amplitude eases, is an important alternative for ous solution of 0.2 g/L commercial deter- of their fluctuations in both clones. For sustainable agriculture. gent as emulsifier and 60 ml/L mineral example, when treated in week 5, DS Product F20 comprises two antibiotics: oil, to give a final concentration of 5-13 g values declined steadily from weeks 5 the streptothricins B and F. These anti- streptothricin/L, corresponding to a dose and 10, from about DS = 3000 to about 3 biotics are produced mainly by micro- of 80-200 g streptothricin/ha. Application DS = 50 in clone ‘CEMSA /4’; the rate of organisms of the genus Streptomyces. was with a knapsack sprayer, with the disease development declined, whereas The structure has an aminosugar spray lance modified to simulate an aer- for control plants, DS values varied (glucosamine) joined to a ß-lysine pep- ial spray of 12 L/h. The product was between 1500 and 2500. tide chain. Streptothricins F to A differ applied in weeks 8, 13, 17 and 22 Analysis of the variable YLSS (Figure 2) in the numbers of ß-lysine residues in to banana, and in weeks 5, 11 and 16 to showed no significant differences the peptide chain, from 1-ß-lysine in plantain. Infected leaves were removed (P>0.05) between treatments with F20 streptothricin F to 6-ß-lysine in strep- at 2 week-intervals for all treatments or Tilt. However, with clone ‘Parecido al tothricin A. and mineral oil with 0.2 g/L commercial Rey’ and F20, minor disease symptoms, The physicochemical properties of detergent, as emulsifier, applied to stage 1, were evident on leaf 9 (Pérez streptothricins, their spectrum of antimi- the control treatment in order to ensure 1996). crobial activity and toxicity are well their maintenance for the whole of the Figure 3 showed that YLS could reach a known (Wienstein and Wagmans 1978). biological cycle. value equal or greater than 9 before

14 INFOMUSA — Vol 11, N° 1 DS

5000 DS

3500 4000 A B 3000

3000 2500 2000

2000 1500

1000 1000 500

0 0 0101214161820 22 24 26 28 5 791113 15 17 19 21 23 Weeks Weeks UT TILT F20

Figure 1. Effect of the treatments F20 and Tilt on the development stage (DS) of black Sigatoka disease (M. fijiensis) in clones, A: ‘Parecido al Rey’ (AAA), 3 and B: ‘CEMSA /4’ (AAB). Arrows indicate the times of application. UT: Control.

YLSS B 7 YLSS A 6 10 5 8 4 6 3 4 2

2 1

0 0 26 27 28 29 17 18 19 20 21 Weeks Weeks UT TILT F20

Figure 2. Youngest leaf with streak symptoms, (YLSS), weeks before flowering, after application of product to clones, A: ‘Parecido al Rey’ (AAA), 3 and B: ‘CEMSA /4’ (AAB). UT: Control.

YLS A 12 YLS B 10 10 8 8 6 6

4 4

2 2

0 0 26 27 28 29 17 18 19 20 21 Weeks Weeks UT TILT F20

3 Figure 3. Youngest leaf spotted (YLS), weeks before flowering, after application of product in clones, A: Parecido al Rey’ (AAA), and B: ‘CEMSA /4’ (AAB). UT: Control. flower initiation, thus confirming that YLS has been observed (Pérez et al. 1993, cial detergent as an emulsion, for the there were no effects on weight or prema- Pérez 1996). control of black Sigatoka disease in ture fruit maturity in either clone; in All the data analyzed above suggest the banana and plantain crops. F20 is supe- Cuba a strong negative correlation use of F20, produced from natural origins rior to synthetic chemicals in terms between the leaf area affected and the and mixed with mineral oil and commer- of environmental effects. Attention is

INFOMUSA — Vol 11, N° 1 15 drawn to the difference in behaviour References Pérez L. 1996. Manual para manejo integrado 3 between the two clones, with ‘CEMSA /4’ Fouré E. 1982. Les cercosporioses du bananier et de Sigatoka negra (Mycosphaerella fijiensis having the higher infection. However, in leurs traitements : Etude de la sensibilité varié- Morelet) y Sigatoka amarilla (Mycospharella order to avoid the possibility of fungal tale des bananiers et des plantains à M. fjiensis musicola Leach ex Mulder) en banano y plátano. resistance, it is important that this prod- Morelet au Gabon. Fruits 37(12): 749-771. FAO, Representación de la FAO en Cuba, La uct should form part of an integrated Fullerton R.A. & T. Olsen. 1991. Pathogenic vari- Habana. 54pp. control programme in combination with ability in Mycosphaerella fijiensis Morelet. Romero R.A. 1997. Avances en epidemiología y other antifungal products (Pérez 1996, Pp. 105-114 in Banana diseases in Asia and the manejo de la Sigatoka negra del banano. Romero 1997). Pacific. Proceedings of a Regional technical Agronomía Costarricense 21(1): 77-81. meeting on diseases affecting banana and plan- Rodríguez R. & L. Jiménez. 1985. El problema de Conclusions tain in Asia and Pacific. (R.V. Valmayor, la tolerancia de Mycosphaerella fijiensis al • Product F20 showed no significant dif- B.E. Umali and C.P. Bejosano, eds). INIBAP- fungicida Benomil en plantaciones bananeras ferences in comparison with the com- ASPNET Book Series No. 3. de Costa Rica. ASBANA. 16pp. mercial product Tilt in its effectiveness Mouliom-Pefoura A. 1999. First observation of the Wienstein M.J. & G.H. Wagman. 1978. Antibiotics. to control black Sigatoka. breakdown of high resistance in ‘Yangambí km 5’ Isolation, separation and purification. Journal •Maximum effectiveness of F20 was (Musa sp.) to the black leaf streak disease in of Chromatography Library 15: 625-680. obtained with an emulsion of mineral Cameroon. Plant Disease 83(1): 78. oil and commercial detergent, and the Orjeda G. 1998. Evaluation of Musa germplasm for effect was maintained for 3 or 4 weeks resistance to Sigatoka diseases and Fusarium wilt. INIBAP Technical Guidelines No. 3. INIBAP, from the time of application. R. Sánchez Rodríguez and J.A. Pino Algora work at • Application at doses of 80-200 g strep- Montpellier, France. the Instituto de Investigaciones en Viandas Tropicales tothricin/ha controlled black Sigatoka Pérez L., A. Hernández & F. Mauri. 1993. Efficacy (INIVIT), Apdo. 6, Santo Domingo, Villa Clara, Cuba, in field plantations, irrespective of time of a biological warning system for timing CP 53000; C. Vallin Plous, M.E. Pérez Rodríguez of the year. There are advantages in fungicide treatments for the control of black and Y. Iznaga Sosa work at the Centro de Química comparison with synthetic chemical Sigatoka disease Mycosphaerella fijiensis Farmacéutica (CQF), Calle 2000 y 21, Atabey 16042, Playa, Ciudad Habana, Cuba and F. Malpartida products because its biodegradability Morelet in banana plantations in Cuba. Romero at the Centro Nacional de Biotecnología, in situ results in compounds that are in Proceedings VI International Congress Campus de la Universidad Nacional Autónoma, 28049 not toxic to the indigenous microflora of Plant Pathology, 28/07-06/08/1993, Montreal, Canto Blanco, Madrid, Spain. Author for correspon- and it has a lower toxicity. ■ Canada. dence: Robersy Sánchez, E-mail: [email protected]

Pests Nematode species in southern India Seasonal fluctuations of Radopholus similis and Pratylenchus coffeae in certain cultivars of banana

P. Sundararaju Lakshadweep Islands (Sundararaju trees, months and years. However, there 1990), Manipur (Anandi and Dhanchand were definite periods for occurrence of 1992), Orissa (Mohanty et al. 1992), maximum and minimum populations he lesion-producing nematodes Tripura (Mukherjee et al. 1994) and within a year. An extensive survey carried such as Radopholus similis and Bihar, Uttar Pradesh and Nagaland out by Sundararaju (1996) from different TPratylenchus coffeae are consid- (Khan 1999). banana-growing regions in the country ered to be the economically important The root-lesion nematode, Pratylenchus indicated the presence of 17 genera of nematode pests of banana and are coffeae is reported to have spread to differ- plant parasitic nematodes. Among them, widely distributed in South India (Koshy ent banana-growing regions through the the lesion nematodes, Radopholus et al. 1978, Rajendran et al. 1979). The infested corm. In India, the nematode is similis and Pratylenchus coffeae are the burrowing nematode, Radopholus sim- known to occur on plantain (AAB) in predominant species found to occur in ilis enjoys wide geographical distribu- South India, Gujarat, Orissa, Bihar and different cultivars of banana in various tion in the tropical and subtropical Assam (Sundararaju 1996). P. thornei, the intensities. The burrowing nematode- banana-growing regions of the world. In other important species was found to infested field exhibited severe root rot- India, the first occurrence of the nema- infest banana plants from Assam only ting, resulting in serious economic losses. tode was reported on banana from (Choudhury and Phukan 1990). Yield was reduced up to 25-35% in the Palghat District of Kerala (Nair et al. Crop losses caused by nematodes to burrowing nematode-infested field com- 1966), causing up to 41% yield losses. bananas are very high, with average pared to nematode-free plantations. Crop Subsequently this nematode was annual yield losses estimated at about losses due to the root-lesion nematode, reported from banana in South India 20% worldwide (Sasser and Freckman Pratylenchus coffeae in banana cv. (Koshy et al. 1978), Gujarat (Sethi et al. 1987). Soil temperature at a depth of Nendran were reported to be 25.4% 1981), Maharashtra (Darekar et al. 30 cm did not influence population size (Sundararaju et al. 1999). Therefore, 1981), Madhya Pradesh (Tiwari et al. (Jimenez 1972). The populations were studies were initiated to determine the 2000), Goa (Koshy and Sosamma 1988), found to fluctuate between samples, seasonal fluctuations of these nematode

16 INFOMUSA — Vol 11, N° 1 populations in different cultivars of 100 banana roots by periodic sampling of nematode-infested banana plants at the 90 National Research Centre for Banana 80 (NRCB) Farm. The main objective of the 70 study was to find out the activity peaks, in terms of highest and lowest populations 60 of these nematode parasites, in the rhi- 50 zosphere so that while devising the management schedules, the findings of the 40 work could be considered. 30

Mean no. of nematode population 20 Materials and methods In order to study the population fluctua- 10 tion of lesion nematodes, the three 0 banana cultivars Kalyan bale (AB), Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Alukkal (ABB) and Kalibow (AAB), which Kalibow Kalyan Bale Alukkal are highly susceptible to R. similis, and a Figure 1. Population fluctuation of Radopholus similis in roots of banana. variety Nendran (ABB), which is highly susceptible to P. coffeae, were selected at NRCB Farm, Podhavur, Trichy, Tamil 100 Nadu. The nematode-infested field was 90 selected for this study and the tested banana cultivars were grown in the field 80 and alluvial soil condition. Samples of 70 both soil (250 cc) and root (10 g) were collected from the base of the mother 60 plants at monthly interval from the 5th 50 month up to harvesting stage during 40 1997-98. Tender, main feeder roots of white to creamy-white colour, with 30 reddish-brown cortical lesions, were col- 20 lected from the base of the plants. Care Mean no. of nematode population 10 was also taken to collect only the abovementioned type of roots known to har- 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec bour the maximum number of lesion Month nematodes. Root samples washed thor- Figure 2. Population fluctuation of Pratylenchus coffeae in roots of banana (cv. Nendran). oughly and cut into 2-2.5 cm pieces and later sliced into 8 longitudinal pieces were left in 15 cm-Petri dishes containing 150 ml of tap water for 72 hrs at 10-14°C 250 in a refrigerator for nematode extraction

(Koshy et al. 1975). Soil samples were 200 processed as per Cobb’s sieving method followed by the modified Baermann’s fun- nel method for estimation of nematode 150 populations. Soil temperature at 15 cm depth was recorded from the fields daily 100 at 7 am. The mean temperature, soil moisture and data on cumulative rainfall were correlated with the nematode popu- 50 lation density in the sample.

0 Results and discussion Apr-97 May-97 Jun-97 Jul-97 Aug-97 Sep-97 Oct-97 Nov-97 Dec-97 Jan-98 Feb-98 Mar-98 It is seen from Figure 1 that a drastic increase of R. similis population was Rainfall (mm) Temperature (°C) Soil mosture (%) Relative humidity (%) noticed in all three cultivars during the months of November to April; it later Figure 3. Total monthly rainfall, mean temperature, soil moisture and relative humidity at NRCB Farm, Podhavur, during the experimental period. decreased to a negligible level from May to October which was in agreement with Shafice and Mendez (1975). It is Kalibow (68/g root), and the minimum occuring during the month of November interesting to observe that the maximum in July in cv. Kalyan bale (20/g root). to April with maximum rainfall and soil nematode population was recorded Analysis of the soil samples also revealed moisture during the period. In the case in April from all the cultivars: Kalayan the same trend as in the case of root of P. coffeae on variety Nendran the maxi- bale (86/g root), Alukkal (78/g root) and samples with maximum population mum population was recorded from

INFOMUSA — Vol 11, N° 1 17 October to December and the minimum and gradually decreased from January banana and pepper in wide areas of Orissa, India. population from May to August (Figure to June (Figure 2). Afro Asian Nematol. Network. 1: 25-26. 2). Regarding the average population for This clearly shows that the population Mukherjee B., R.C. Nath & M.K. Dasgupta. 1994. a month, it is seen that a maximum of build-up of R. similis and P. coffeae would New record on the occurrence of burrowing nema- 92 per gram root was recorded in greatly vary depending upon the season tode, Radopholus similis (Cobb, 1893) Thorne, December, whereas it was only 23 per and other ecological conditions such as 1949 on banana in Tripura. Indian J. Ent. 28: gram in June. The root-lesion nematode rainfall, soil temperature, soil moisture 553-554. population from soil samples also and availability of susceptible roots Nair M.R.G.K., M.N. Das & M.R. Menon. 1966. On the showed the same trend as in the case of which play their own roles in the popula- occurrence of the burrowing nematode root samples with maximum population tion build-up. Radopholus similis (Cobb, 1893) Thorne, 1949 during the months October-December on banana in Kerala. Indian J.Ent. 28: 553-554. and minimum population during the Acknowledgement Rajendran G., T.N. Naganathan & S. Vadivelu. 1979. months May-August. Peak rainfall The author is thankful to Dr H.P. Singh, Studies on banana nematodes. Indian J. Nematol. occurred during the Northeast monsoon former Director, NRCB, Trichy for provid- 9: 54. (September to December) with an average ing necessary facilities. Technical assis- Sasser J. N & D.W. Freckman. 1987. A world perspec- rainfall of 140 mm. The soil temperature tance given by Mr T. Sekar is duly tive on nematology: the role of the society. Pp. 7- at 15 cm depth recorded from fields var- acknowledged. This research work was 14 in Vistas on Nematology (J.A. Veech and D.W. ied from 18-37.5°C. Analysis of the mois- carried out within the framework of the Dickson, eds). Society of Nematologist Inc., ture content revealed that it was maxi- NRCB research programmes. ■ Hyattsville, USA. mal in those months where maximum Sethi C.L., Siyandand & N. Srivastava. 1981. nematode populations were recorded References Occurrence of Radopholus similis (Cobb, 1893) (Figure 3). Rainfall also influences the Anandi Y. & D. Dhandachand. 1992. Nematodes of Thorne, 1949 in Gujarat. Indian J. Nematol. growth of roots. Thus, with the increase banana plantation in Imphal district, Manipur. 11: 116. in the availability of root system, there Curr. Nematol. 3: 153-58. Shafice M. F. & J.M. Mendez. 1975. Seasonal fluctua- was an increase in the activity Choudhury B.N. & P.N. Phukan. 1990. Distribution tions in Radopholus similis on three varieties of R. similis during November to April and occurrence of certain plant parasitic nema- of Musa species. Universidad de la Habana, Serie and of P. coffeae during October to todes in different cultivars of banana. Curr. 11, Sanidad vegetal No. 12. 12pp. December. Nematol. 1: 153-156. Sundararaju P. 1990. Occurrence of the burrowing Fluctuations in the populations of Cooke D.A. & Draycott. 1971. The effect of soil fumi- nematode, Radopholus similis in the Union Pratylenchus spp. were correlated with gation and nitrogen fertilization on nematodes Territory of Lakshadweep Island. Indian J. rainfall (Cooke and Draycott 1971). The in sugar-beet in sandy soils. Ann. Appl. Biol. 65: Nematol. 18: 112. behaviour of P. coffeae in relation to soil 253-254. Sundararaju P. 1996. Nematode pests of banana and temperature and rainfall was similar DuCharme E.P. & R.F. Suit. 1967. Population fluctua- their management. Pp. 17-19 in Souvenir, to that of P. crenatus and P. penetrans tion of burrowing nematodes in Florida citrus “Conference on challenges for banana production in corn (Miller et al. 1972). Lack of mois- groves. Proc. Fla. St. Hort. Soc. 80: 63-67. and utilisation in 21st Century” held at Trichy ture coupled with high summer tempera- Jiménez M.F. 1972. Seasonal fluctuations of on 24-25 September 1996. ture during April to August was found to Radopholus similis in the banana growing area of Sundararaju P. & K. Ratnakaran. In press. Factors be unfavourable for the prevalence of Pococo, Costa Rica. Nematropica 2: 6. influencing the prevalence of the root-lesion P. coffeae in oil palm (Sundararaju and Khan R.M. 1999. Distribution of Radopholus similis nematode Pratylenchus coffeae on oil palm Ratnakaran, in press). The present inves- in India, its spread in new regions and an analysis (Elaeis guineensis Jacq.). Indian J. Nematol. tigation is in agreement with Kumar of the nematofauna of banana crop pathosystem. Sundararaju P., B. Padmanaban & S. Sathiamoorthy. (1984) who reported that higher popula- Nematol. Medit. 27: 239-245. 1999. Control of root-lesion nematode tion of P. coffeae was recorded during the Koshy P.K. & V.K Sosamma. 1978. Studies on the Pratylenchus coffeae in certain cultivars of month of October to December which is population fluctuations of Radopholus similis banana. Pp. 46-47 in National Seminar on the period of high rainfall and increased in coconut and arecanut roots. Indian Phytopath. Nematological Research in India held at C.S. Azad root activity in coffee plants. 31: 180-183. University of Agri. & Tech. 17 December 1999 Similar observations were reported in Koshy P.K. & V.K Sosamma. 1988. Occurrence of the (Abstract). the burrowing nematode R. similis on burrowing nematode Radopholus similis in the Tiwari S.P., I. Vadhera & G.S. Dave. 2000. Burrowing citrus (DuCharme and Suit 1967), banana state of Goa. Indian J. Nematol. 18: 130. nematode Radopholus similis associated with (Vilardebo 1976), coconut and arecanut Koshy P.K., V.K Sosamma & C.P. Radhakrishnan Nair. banana crop in Madhya Pradesh. Indian J. (Koshy and Sosamma 1978). 1975. Preliminary studies on Radopholus similis Nematol. 30(1)38: 41. Figure 1 indicates that the R. similis (Cobb, 1893) Thorne, 1949 infesting coconut and Vilardebo A. 1976. Population dynamics of Radopholus population fluctuates between the arecanut palms in South India. Indian J. Nematol. similis in relation to climate factors and the months. A steady increase of R. similis 5: 26-35. physiology of the plant. Nematropica 6: 54-55. population was recorded during the Koshy P.K., P. Sundararaju & V.K Sosamma. 1978. months of November-January and grad- Occurrence and distribution of Radopholus sim- ual decrease was recorded in February ilis (Cobb, 1893) Thorne, 1949 in South India. and March, whereas a drastic increase of Indian J. Nematol. 8: 49-58. nematode population was recorded in Kumar A.C. 1984. Investigations on Cannoncado April in cv. Kalyan bale (Figure 1). A sim- dieback in coffee. J. Coffee Res. 14: 85-103. ilar trend was noticed in cvs. Alukkal and Miller R.E., C.W. Bothroyd &W.F. Mai. 1972. Plant Kalibow (Figure 1). parasitic nematodes associated with corn roots in In the case of P. coffeae a steady New York. Phytopath. 52: 22 (Abstr.). The author works at the Crop Protection National increase of nematode population was Mohanty K., N.K. Sahoo & S. Ray. 1992. Occurrence of Research Centre for Banana, 44 Ramalinga Nagar recorded from September to December Radopholus similis (Cobb, 1893) Tgirbe 1949 on South Vayalur Road,Tiruchirapalli – 620 017, India.

18 INFOMUSA — Vol 11, N° 1 Pests Nematode resistance Host plant response of Pisang Jari Buaya and Mysore bananas to Radopholus similis

Duong Thi Minh Nguyet, in Musa as described by Speijer and interval) and poured through 106 and A. Elsen, Nguyen Thi Tuyet De Waele (1997) were used. 25 µm pore sieves. Carrot tissue collected and D. De Waele on the 106 µm pore sieve was discarded, Materials and methods while the nematodes were collected from the 25 µm pore sieve. lant parasitic nematodes are a Preparation of banana plants A suspension of 1000 living vermiform major constraint of banana produc- Thirteen diploid (AA) banana genotypes nematodes was poured in three holes Ption worldwide (Gowen and belonging to the Pisang Jari Buaya group made in the substrate around the base of Quénéhervé 1990). Nematode infection (Experiments 1 and 2, see Tables 1 and each plant. After inoculation, the holes can interfere with nutrient and water Table 2) and five triploid (AAB) banana were covered. uptake and transportation, resulting genotypes from the Mysore group in slow growth, reduced fruit filling and (Experiment 3, see Table 3) were Host plant response observations sensitivity to wind lodging. Among included in the study. Two triploid (Musa Eight weeks after inoculation, the plants the nematodes attacking banana, AAA) bananas, ‘Grande Naine’ and were harvested to observe the response Radopholus similis (Cobb) Thorne is ‘Yangambi Km5’, were included as refer- of the different banana genotypes to considered the most destructive species ence genotypes because of their high sus- R. similis. The following data were (Sarah et al. 1996). ceptibility and resistance to R. similis, recorded: The possibilities of controlling nema- respectively. The Musa genotypes used in todes in bananas are limited because the experiments were provided by the Root necrosis percentage bananas are usually grown as a perma- INIBAP Transit Centre (ITC) at the The procedure followed was that nent crop by small-scale farmers and Catholic University of Leuven. After pro- described by Speijer and De Waele sources of resistance have proved hard to liferation, regeneration and rooting (1997). Five 10 cm-pieces of functional find. Resistance to R. similis has been (Banerjee and De Langhe 1985), each primary roots were collected and sliced reported in ‘Pisang Jari Buaya’ (Musa in vitro propagated banana plantlet with longitudinally. The percentage of root AA—Pisang Jari Buaya group) and 3-4 leaves and 5-6 roots was transplanted cortex showing necrosis was scored for a ‘Yangambi Km5’ (Musa AAA—Ibota in a 1-litre (12 cm-diameter) plastic pot half of each root. The maximum root group) (Pinochet 1988, Viaene et al. 1998, containing about 1000 cm3 autoclaved necrosis per root half is 20%, giving Fogain and Gowen 1998, Stoffelen 2000). substrate of peat and quarts (2:1). To a maximum root necrosis of 100% for The clone ‘SH-3142’ derived from a geno- keep a high humidity, the pots were the five root-halves together. type belonging to the Pisang Jari Buaya placed under a plastic cover, which was group and ‘SH-1734’ was found to be slightly opened after 2 weeks and Nematode population densities highly resistant to R. similis (Pinochet removed after 4 weeks. The greenhouse The entire root system, including the and Rowe 1979, Pinochet 1988). conditions were maintained at 25-30°C 5 roots segments observed for necrosis, Moreover, some ‘Pisang Jari Buaya’ and 70-80% relative humidity with a was weighed and cut into 2 cm-pieces. expressed favourable agronomic features 12-hour photoperiod. The pots were irri- Fifteen grams of fresh roots were taken similar to those of commercial banana. gated as needed and fertilized with a randomly and macerated three times for The Mysore banana (Musa AAB) is a hydroponics solution (Swennen et al. 10 s with 5 s intervals. The mixture was very popular and delicious dessert. 1986) every 3 weeks after nematode inoc- poured through a series of 250-106-40 µm Information on resistance and/or toler- ulation. The plants were inoculated with pore sieves and the sieves were rinsed ance to R. similis of Mysore bananas is nematodes either 4 weeks after planting with tap water. Nematodes remaining on scarce. When testing 17 AAB Musa geno- for the Pisang Jari Buaya group, or the 40 µm pore sieve were collected in a types, Fogain (1996) reported that none 8 weeks after planting for the Mysore beaker with distilled water. Nematodes of the plants were immune, including group, since the number of nematodes were counted in 6 ml aliquots of each ‘Pisang Ceylan’, the only cultivar belong- was too low in the experiment with sample using a binocular microscope. ing to the Mysore group. The objective of Mysore genotypes. our study was to further investigate the Experimental design and data analysis host plant response of Musa genotypes Preparation of nematode inoculum Three experiments were conducted, from the Pisang Jari Buaya and Mysore The R. similis population used in the based on a completely randomized groups to a R. similis population from experiments was obtained from infected design, with either eight replicates for Costa Rica, to find additional sources of banana roots of ‘Valery’ (Musa AAA) at each genotype (Pisang Jari Buaya group, resistance to the burrowing nematode. Talamanca in Costa Rica. The population Experiment 1, Table 1; Mysore group, Throughout the study, the terminology was reared monoxenically on carrot discs Experiment 3, Table 3) or nine repli- of Bos and Parlevliet (1995) concerning and incubated at 28°C in the dark for sev- cates (Pisang Jari Buaya group, resistance and susceptibility of host eral generations (Moody et al. 1973, Experiment 2, Table 2). Prior to statis- plants to pathogens and the methodology Pinochet et al. 1995). The carrot discs tical analysis, the percentage of root for nematode resistance screening were blended twice for 10 s (with 5 s necrosis was transformed to arcsin

INFOMUSA — Vol 11, N° 1 19 Table 1. Reproduction of Radopholus similis (Costa Rica population) on 8 diploid Table 2, reproduction of R. similis was (Musa AA) banana genotypes belonging to the Pisang Jari Buaya group and on observed in all the genotypes tested. In the reference genotype ‘Grande Naine’ measured 8 weeks after inoculation with general, the nematode populations col- 1000 vermiform nematodes per plant. lected from roots of the Pisang Jari Buaya Musa genotype Genome ITC number Fresh root Root necrosis Nematodes Nematodes genotypes, including Pisang Jari Buaya weight (%) per 1 g per root accession ITC0312, were not significantly (g) fresh roots system different from those recovered from Huwundu AA 0308 35.3 22.4 ab 1050 a 36188 a ‘Grande Naine’ but significantly higher Morong Datu AA 0309 41.0 14.5 ab 851 a 33526 a compared to ‘Yangambi Km5’ and Morong Princessa AA 0310 29.9 30.5 b 972 a 26022 a ‘SH-3142’. Only for ‘Pisang Sipulu’ was the Pisang Rotan AA 0313 41.1 16.6 ab 794 a 29033 a number of nematodes per 1 g fresh roots Pisang Tunjiuk AA 0315 44.2 7.9 a 255 a 10770 a not significantly different from ‘Yangambi Saing Todloh AA 0316 36.0 6.8 a 297 a 10038 a Km5’. The lowest nematode numbers Unnamed AA 0318 43.2 11.3 ab 442 a 17151 a were recorded on ‘SH-3142’ and Umbarim AA 0317 32.6 17.1 ab 495 a 16030 a ‘Yangambi Km5’. The root necrosis per- Grande Naine AAA 1256 52.0 9.3 ab 528 a 23052 a ITC= INIBAP Transit Centre. centages of all Pisang Jari Buaya geno-

Original data are presented, but data of nematode numbers were transformed to log10 (x+1) and data of root necrosis types, ‘Yangambi Km5’ and ‘SH-3142’ percentage were converted to arcsin (x/100) for statistical analysis. Means in the same column followed by the same letter were significantly lower compared with are not significantly different (P ≤ 0.05) according to the Tukey HSD test. ‘Grande Naine’. These results show that all Pisang Jari Buaya genotypes tested are as suscepti- Table 2. Reproduction of Radopholus similis (Costa Rica population) on ble to R. similis as ‘Grande Naine’. They 5 Pisang Jari Buaya genotypes, Yangambi Km5 and on the reference genotype confirm a previous report (Wehunt et al. ‘Grande Naine’ measured 8 weeks after inoculation with 1000 vermiform 1978) that ‘Pisang Jari Buaya’, ‘Gabah nematodes per plant. Gabah’, ‘Pisang Sipulu’ and ‘Pisang Gigi Musa genotype Genome ITC number Fresh root Root necrosis Nematodes Nematodes Buaya’ are significantly less sensitive to weight (%) per 1 g per root root damage (expressed as root necrosis (g) fresh roots system percentage) compared with ‘Grande Gabah Gabah AA 0307 74.4 11 a 588 c 44571 b Naine’. Surprisingly, ‘Pisang Jari Buaya’, Pisang Gigi Buaya AA 0310 64.0 8.4 a 741 c 45914 b which has previously been confirmed Pisang Jari Buaya AA 0312 54.9 11.9 a 1053 c 56541 b resistant to R. similis (Pinochet 1988, SH-3142 AA 0425 52.7 8.9 a 108 a 5941 a Pisang Sipulu AA 1308 60.6 8.9 a 579 bc 34355 b Viaene et al. 1998, Fogain and Gowen Yangambi Km5 AAA 1123 57.2 7.8 a 120 ab 6384 a 1998, Stoffelen 2000), did not appear to Grande Naine AAA 1256 43.9 25 b 2041 c 87763 b be so in our study. Also, ‘Pisang Sipulu’, ITC= INIBAP Transit Centre. considered a promising banana genotype See note in Table 1. because less susceptible to R. similis (Wehunt et al. 1978, Binks and Gowen 1996), did not show resistance either to Table 3. Reproduction of Radopholus similis (Costa Rica population) on 5 triploid R. similis in our study. (Musa AAB) banana genotypes belonging to the Mysore group and on The results obtained from the Mysore the reference genotype ‘Grande Naine’ measured 8 weeks after inoculation group are presented in Table 3. The with 1000 vermiform nematodes per plant. nematode numbers per root system and Musa genotype Genome ITC number Fresh root Root necrosis Nematodes Nematodes per 1 g fresh roots of ‘Gorolo’ and ‘Lady weight (%) per 1 g per root Finger’ (South Johnstone) were signifi- (g) fresh roots system cantly lower compared with ‘Grande Thap Maeo AAB 1301 101.3 17.3 a 852 ab 82849 abc Naine’, while those recovered from the Gorolo AAB 0723 45.6 22.8 ab 579 a 32562 a other Mysore genotypes were not signifi- Pisang Ceylan AAB 0650 58.3 29.6 abc 804 ab 46827 abc cantly different compared with the ref- Lady Finger (South Johnstone) AAB 0583 51.8 36.9 c 679 a 38616 ab erence genotype. The root necrosis per- Lady Finger (Nelson) AAB 0582 87.8 33.1 bc 1128 ab 99009 bc centage observed in ‘Thap Maeo’ and Grande Naine AAA 1256 83.9 42.5 c 1552 b 127439 c ‘Gorolo’ was significantly lower com- ITC= INIBAP Transit Centre. pared with ‘Grande Naine’. In contrast, See note in Table 1. the root necrosis percentages of ‘Pisang Ceylan’, ‘Lady Finger’ (South Johnstone) and ‘Lady Finger’ (Nelson) (x/100) and the nematode numbers and 2. In Table 1, no significant differ- did not differ significantly from those in were converted to log10 (x+1). All data ences were observed in nematode num- ‘Grande Naine’. were subjected to analysis of variance bers per root system or per 1 g fresh roots According to Price (1994) and Price (ANOVA) and means of the parameters and root necrosis percentage between and McLaren (1995), AAB Musa geno- were compared using the Tukey HSD the Pisang Jari Buaya genotypes and types are susceptible to R. similis when test at P ≤ 0.05. ‘Grande Naine’. Among the Pisang examined in field trials. Unfortunately, Jari Buaya genotypes, root necrosis per- genotypes of the Mysore group were not Results and discussions centage was significantly higher in included in their trials. Our study con- The results obtained from the Pisang Jari ‘Morong Princessa’ compared with firms previous reports (Stanton 1994, Buaya group are presented in Tables 1 ‘Pisang Tunjiuk’ and ‘Saing Todloh’. In Fogain et al. 1996) that ‘Lady Finger’

20 INFOMUSA — Vol 11, N° 1 (Nelson), ‘Lady Finger’ (South Johnstone) Fogain R., S.R. Gowen & F. Mekemda. 1996. Stanton J.M. 1994. Status of nematode and weevil and ‘Pisang Ceylan’ are susceptible Screening for susceptibility to Radopholus borer problems affecting banana in Australia. Pp. to R. similis. similis: evaluation of plantain AAB and diploid 48-56 in Banana nematodes and weevil borers in AA, AB and BB. Tropical Agriculture, Trinidad Asia and the Pacific. Proceedings of a conference- Acknowledgements 73: 281-285. workshop on Nematodes and weevil borers affect- The authors would like to thank the Gowen S.R. & P. Quénéhervé. 1990. Nematode para- ing bananas in Asia and the Pacific, 18-22 April INIBAP Transit Centre (ITC) at the sites of bananas, plantain and abaca. Pp. 431-460 1994, Serdang, Selangor, Malaysia (R.V. Valmayor, Catholic University of Leuven for supply- in Plant parasitic nematodes in subtropical and R.G. Davide, J.M. Stanton, N.L. Treverrow & V.N. ing the Musa genotypes and the equip- tropical agriculture (M. Luc, R.A. Sikora & Roa, eds). INIBAP/ASPNET, Los Baños, ment for completing this research. The J. Bridge, eds.). CAB International, Wallingford, Philippines. ASPNET Book Series No. 5. Flemish Interuniversity Council (VL.I.R.) United Kingdom. Speijer P.R. & D. De Waele. 1997. Screening of Musa is gratefully acknowledged for funding Moody E.H., B.F. Lownsberry & J.M. Ahmed. 1973. germplasm for resistance and tolerance to nema- scholarships for Ms Duong Thi Minh Culture of the root-lesion nematode Pratylenchus todes. INIBAP Technical Guidelines (1). INIBAP, Nguyet and Ms Nguyen Thi Tuyet to com- vulnus on carrot disks. Journal of Nematology Montpellier, France. 42pp. plete this study as part of their MSc 5: 225-226. Stoffelen R. 2000. Early screening of Eumusa and thesis in the Postgraduate International Pinochet J. 1988. Nematodes problem in Musa spp.: Australimusa bananas against root-lesion and Nematology Course. ■ pathotypes of R. similis and breeding for resis- root-knot nematodes. Doctoral thesis, Universiteit tance. Pp. 66-70 in Proceeding of a workshop on K.U. Leuven, Belgium. References Nematodes and the borer weevil in bananas: pre- Swennen R., E. De Langhe, J. Janssen & D. Decoene. Banerjee N. & E. De Langhe. 1985. A tissue culture sent status of research and outlook, 7-11 1986. Study of the root development of some Musa technique for rapid clonal propagation and storage December 1987, Bujumbura, Burundi. INIBAP, cultivars in hydroponics. Fruits 41: 515-524. under minimal growth conditions of Musa (banana Montpellier, France. Viaene N., J. Duenas & D. De Waele. 1998. Screening and plantain). Plant Cell Reports 4: 351-354. Pinochet J. & P.R. Rowe. 1979. Progress in breeding for resistance and tolerance to Radopholus Binks R.H. & S.R. Gowen. 1996. Field evaluation for resistance to Radopholus similis on bananas. similis and Pratylenchus coffeae in banana and of nematode infestations in Musa germplasm Nematropica 9: 76-78. plantain [Abstract]. P. 125 in Programme and at FHIA (La Lima, Honduras). INFOMUSA 5(2): Pinochet J., C. Fernandez & J.L. Sarah. 1995. abstracts of the 24th International Symposium of 15-17. Influence of temperature on in vitro reproduction the European Society of Nematologists, 5-8 Aug. Bos L. & J.E. Parlevliet. 1995. Concept and terminol- of Pratylenchus coffeae, P. goodeyi, and 1998, Dundee, Scotland (D. Brown, ed.). ogy on plant/pest relationships: toward consensus Radopholus similis. Fundamental and Applied Wehunt E.J., D.J. Hutchinson & D.I. Edwards. 1978. in plant pathology and crop protection. Annual Nematology 18: 391-392. Reaction of banana cultivars to the burrowing Review of Phytopathology 33: 69-102. Price N.S. 1994. Field trial evaluation of nematode nematode (Radopholus similis). Journal of Fogain R. 1996. Screenhouse evaluation of Musa for susceptibility within Musa. Fundamental and Nematology 10: 368-370. susceptibility to Radopholus similis: evaluation Applied Nematology 17: 391-396. of plantains AAB and diploid AA, AB and BB. Price N.S. & C.G. McLaren. 1995. Technique for field Pp. 79-86 in Proceedings of a workshop on New screening of Musa germplasm. Pp. 87-105 in frontiers in resistance breeding for nematode, Proceedings of a workshop on New frontiers in Duong Thi Minh Nguyet and Nguyen Thi Tuyet Fusarium and Sigatoka, 2-5 Oct. 1995, Kuala resistance breeding for nematode, Fusarium and work at the Vietnam Agricultural Science Institute Lumpur, Malaysia (E.A. Frison, J.P. Horry & Sigatoka, 2-5 Oct. 1995, Kuala Lumpur, Malaysia (VASI), Van Dien, Thanh Tri, Hanoi, Vietnam. Tel: (84) D. De Waele, eds). INIBAP, Montpellier, France. (E.A. Frison, J.P. Horry & D. De Waele, eds). 4 861 43 25, Fax: (84) 4 861 71 67. Annemie Elsen Fogain R. & S.R. Gowen. 1998. ‘Yangambi Km5’ INIBAP, Montpellier, France. and Dirk De Waele work at the Laboratory of Tropical Crop Improvement, Catholic University of Leuven, (Musa AAA, Ibota subgroup): a possible source Sarah J.L., J. Pinochet & J. Stanton. 1996. The bur- Kasteelpark Arenberg 13, 3001 Heverlee, Belgium. of resistance to Radopholus similis and rowing nematode of bananas, Radopholus similis Tel: (32) 16 32 96 03, Fax: (32) 16 32 19 93. Pratylenchus goodeyi. Fundamental and Applied Cobb, 1913. Musa Pest Fact Sheets (1). INIBAP, Corresponding author: e-mail: Nematology 21: 75-80. Montpellier, France. 2pp. [email protected] or [email protected]

Pests Nematode control

Effect of three arbuscular mycorrhizal fungi on root-knot nematode (Meloidogyne spp.) infection of Musa

A. Elsen, S. Declerck and D. De Waele water and mineral nutrients from the on temperate agricultural crops. Many soil. AM fungi also may protect plants mycorrhizal associations are reported to against soil-borne plant pathogens, have a suppressive effect over sedentary rbuscular mycorrhizal (AM) fungi including nematodes. Several studies endoparasitic nematodes. In some crops are obligate symbionts of plants have addressed the associations between this effect is significant enough to con- Athat biotrophically colonize the AM fungi and root-knot nematodes, sider mycorrhizal infection as a more or root cortex and develop an extramatrical which are considered the most important less effective means of biological control mycelium which helps the plant acquire nematodes in the western hemisphere (Pinochet et al. 1996).

INFOMUSA — Vol 11, N° 1 21 In bananas, only a few studies were car- tral effect on plant growth as long as the no effect on the percentage root coloniza- ried out on the effects of AM fungi on mycorrhizal colonization was not well tion in mycorrhizal plants (Pinochet et al. nematode development. Radopholus developed (Jakobsen 1998). Therefore 1997, Jaizme-Vega et al. 1997). similis populations in the roots as well as at the time of harvest, the root coloniza- in the soil were suppressed in mycor- tion by the three Glomus strains tested rhizal plants compared to non-mycor- was relatively low. This may partly Conclusion rhizal plants (Umesh et al. 1988). Under explain why in this experiment no effect The results of this experiment suggest a in vitro conditions, using Ri T-DNA trans- on plant growth was observed. In addi- suppressive effect of the three Glomus formed Daucus carota roots, a R. similis tion, it is important to note the differ- strains studied over the rook-not nema- population was suppressed with 50% in ences in colonization among the Glomus tode M. javanica. Mechanisms involved the presence of AMF (Elsen et al. 2001). species in the plants without nematodes. in nematode suppression are still a Pinochet et al. (1997) reported that Higher colonization was observed with matter of speculation. However some mycorrhizal colonization did not effect G. mosseae as compared to G. caledo- major factors are likely to be involved: nematode build-up in the roots, although nium and G. macrocarpum. Such dif- enhanced nutrient status of the plant, plants infected with both Meloidogyne ferences were also reported in literature biochemical changes in plant tissue javanica and Glomus intraradices were (Declerck et al. 1994, 1995). Glomus (increase in chitinase, amino acids, per- more galled. mosseae was shown the more infective oxidase and phytoalexins), anatomical In this experiment, three Glomus on ‘Williams’ and other cultivars, as com- changes (increased lignification), stress species (G. mosseae, G. macrocarpum pared to G. macrocarpum (Declerck alleviation, microbial changes in the rhi- and G. caledonium) were tested on the et al. 1995). zosphere and induced changes to root Musa cultivar Williams (ITC0570) for morphology (increased branching, their effect on Meloidogyne javanica, a larger proportion of higher order roots) root-knot nematode population isolated Effect of AM fungi on nematode (Hooker et al. 1994). Further study from banana in Morocco. Tissue-culture reproduction is needed to confirm the suppressive derived plantlets were acclimatized in Glomus caledonium and G. macro- effect of the AM fungi over the root-knot 1-litre pots filled with sterilized soil in carpum significantly reduced galling in nematodes and to reveal the mecha- the greenhouse. During transplant the the roots, while for G. mosseae this nisms involved. ■ plantlets of the mycorrhizal treatment reducing effect was not significant were mycorrhized with soil inoculum, (Table 1). In literature, results are con- consisting of ± 1850 spores and 0.25 g tradictory: according to Pinochet et al. mycorrhized roots from Allium porrum. (1997), Glomus intraradices did not References After one month, the plants were inocu- reduce nematode build-up of M. javan- Declerck S., B. Devos, B. Delvaux & C. Plenchette. lated with a mixture of 5000 M. javanica ica and resulted in more galled roots 1994. Growth response of micropropagated plants juveniles and eggs. The experiment was compared to non-mycorrhized roots. In to VAM inoculation. Fruits 49: 103-109. planned as a 4 x 2 randomly factorial contrast, G. mosseae suppressed root Declerck S., C. Plenchette & D.G. Strullu. 1995. design with 8 replicates per treatment: galling and nematode build up of Mycorrhizal dependency of banana (Musa AM fungi (- AM, G. mosseae, G. macro- Meloidogyne incognita (Jaizme-Vega acuminata AAA group) cultivar. Plant and Soil carpum and G. caledonium) x M. javan- et al. 1997). 176: 183-187. ica (+ M. javanica, - M javanica). Three Elsen A., S. Declerck & D. De Waele. 2001. Effects of months after planting, the ‘Williams’ Glomus intraradices on the reproduction of the plants were harvested and assessed for Effect of M. javanica on burrowing nematode (Radopholus similis) in dix- mycorrhizal colonization and nematode mycorrhizal development enic culture. Mycorrhiza 11: 49-51. damage/development. A sub-sample of Meloidogyne javanica significantly Hadisoeganda W.W. & J.N. Sasser. 1982. Resistance the roots was stained with 0.05% trypan decreased the intraradical development of tomato, bean, southern pea and garden pea cul- blue in lactic acid (Koske and Gemma of G. mosseae. For G. macrocarpum and tivars to root-knot nematodes based on host suit- 1989), in order to determine the mycor- G. caledonium no such effect was ability. Plant Disease 66: 145-150. rhizal colonization. The galls on the roots observed: the presence or absence of the Hooker J.E., M.C. Jaizme-Vega & D. Atkinson. 1994. were counted in a 5 g sub-sample after root-knot nematode had no effect on Biocontrol of plant pathogens using arbuscular staining with phloxine B (Hadisoeganda internal root colonization. In similar mycorrhizal fungi. Pp. 191-200 in Impact of and Sasser 1982). experiments, root-knot nematodes had arbuscular mycorrhizas on sustainable agricul-

Effect of the AM fungi on plant Table 1. Mycorrhization and effect of mycorrhization on reaction of “Williams” growth roots to infection with Meloidogyne javanica. The AM fungi had no effect on the plant growth since shoot weight, shoot diame- % mycorrhized root tissue Galls / 5 g roots ter, plant height and root weight did not - AM – M. javanica // differ among the treatments (data not - AM + M. javanica / 41 ± 12 b shown). In general, mycorrhization of G. mosseae – M. javanica 29 ± 10 b / banana plants resulted in a better plant G. mosseae + M. javanica 23 ± 12 a 29 ± 16 ab growth compared to non-mycorrhizal G. macrocarpum – M. javanica 14 ± 3 a / plants (Declerck et al. 1994, 1995). G. macrocarpum + M. javanica 15 ± 5 a 25 ± 17 a Although, in some cases, it has been G. caledonium – M. javanica 22 ± 5 a / observed that the establishment of the G. caledonium + M. javanica 16 ± 6 a 16 ± 8 a Data are means of 8 replications. Means in the same columns followed by the same letter do not differ according Tukey’s symbiosis resulted in a negative or neu- multiple range test (P ≤ 0.05).

22 INFOMUSA — Vol 11, N° 1 ture and natural ecosystems (S. Gianinazzi & Koske R.E. & J.N. Gemma. 1989. A modified proce- Umesh K.C., K. Krishnappa & D.J. Bagyaraj. 1988. H. Schüepp, eds). Birkhäuser Verlag, Basel, dure for staining roots to detect VA mycorrhizas. Interaction of burrowing nematode, Radopholus Switzerland. Mycological Research 92: 486-488. similis (Cobb, 1893) Thorne 199, and VA mycor- Jaizme-Vega M.C., P. Tenoury, J. Pinochet & Pinochet J., C. Calvet, A. Camprubi & C. Fernandez. rhiza, Glomus fasciculatum (THAXT) Gerd. and M. Jaumot. 1997. Interactions between the root- 1996. Interaction between migratory endopara- Trappe in banana (Musa acuminata colla.). knot nematode Meloidogyne incognita and sitic nematodes and arbuscular mycorrhizal Indian Journal of Nematology 18: 6-11. Glomus mosseae in banana. Plant and Soil 196: fungi in perennial crops. Plant and Soil 185: 27-35. 183-190. Annemie Elsen and Dirk De Waele work at the Jakobsen L. 1998. Transport of phosphorus and car- Pinochet J., C. Fernandez, M. Jaizme & P. Tenoury. Laboratory of Tropical Crop Improvement, KULeuven Kasteelpark Arenberg 13, 3001 Leuven, Belgium; bon in arbuscular mycorrhizas. Pp. 305-332 in 1997. Micropropagated banana infected with Stéphane Declerck works at the Mycothèque de Mycorrhiza structure, function, molecular biology Meloidogyne javanica responds to Glomus l’Université catholique de Louvain (MUCL). Unité de and biotechnology (A. Varma & J.E. Hooker, eds). intraradices and phosphorus. Hortscience 32: microbiologie, Place Croix du Sud 3, 1348 Louvain- Springer-Verlag, New York, USA. 101-103. la-Neuve, Belgium.

Agronomy Pests and pathogens of roots A study of endophytic fungal species associated with root necrosis of banana in banana and plantain plantations in Cuba

A. Battle-Viera and L. Pérez-Vicente to the genera Cylindrocladium and The roots were washed and necrotic Acremonium, which contribute to or fragments, typical of R. similis attack, increase damage by the nematode, are disinfected in 1% hypochlorite for two n Cuba a total of 108 700 ha are cul- well documented (Booth and Stover 1981, minutes and cultured on water-agar sup- tivated with Musaceae, 32 800 ha Loridat 1989, Sarah 1990). Such associa- plemented with 50 µg/ml streptomycin. Iwith cultivars of banana subgroup tions have been found in the majority of Blocks of agar and fungal growth were Cavendish (AAA), 13 800 ha with plan- soils infested with nematodes in some of transferred to tubes of PDA, and incu- tain (AAB) and 62 000 ha with varieties the Antilles islands. In Cuba there have bated when the fungal species were ready of type Burro/Bluggoe (ABB). Of the been no studies to investigate and quan- for identification. Fusarium species 32 800 ha of banana plantations, tify such relationships at the level of the were identified according to the key of 13 800 ha are cultivated with localized root. However the relationship between Booth (1981). Cylindrocarpon species microjet irrigation systems, and they nematode populations, root damage and were identified according to the keys of must therefore remain in place for plant development is not strong. the CMI edited by CAB. the next five years. However there is The purpose of the present study was The relative frequency of each species interest in replanting these areas with to identify the species of fungi associ- present at each site was determined in tetraploid hybrids developed by the ated with root necrosis of different relation to the total numbers of isolates Fondación Hondureña de Investigación clones of banana and plantain in planta- obtained from the different sites. Agrícola (FHIA), and which are resis- tions in Cuba. tant to pests and diseases. Results and discussion The nematode species most common- Material and methods A total of 59 isolates of endophytic fungi ly found in our plantations are Samples were taken from banana plan- were obtained from the tissue of roots Radopholus similis, Pratylenchus tations located in the provinces of Pinar apparently necrotic as a result of coffeae, Helycotylenchus multicinctus, del Río, La Habana, Matanzas, Villa R. similis. The species are described in Meloidogyne spp. and Rotylenchulus Clara, Ciego de Avila, Camagüey, Table 1. reniformis, the first three being the most Cienfuegos, Santiago de Cuba and Species of Cylindrocarpon musae and important in Cuba (Pérez et al. 1984). Guantánamo. Fusarium oxysporum Schlect. were iso- The pathogenicity of nematodes has usu- Samples were taken from necrotic lated from almost all samples from all the ally been established as the population roots of plants of Gran enano (AAA), Gros sites. F. oxysporum was the most fre- density found in the roots. However, evi- Michel (AAA), CEMSA 3/4 (AAB) and quently isolated species (45.6% total iso- dence on the relationship of population Burro CEMSA/ Bluggoe (ABB), some of lates), followed by C. musae (19.2% total density of nematodes to damage in the them associated with plants that had isolates). F. equiseti (Corda) Sacc. was crop is contradictory. Over the last years fallen over (lodged) apparently as a also isolated but with less frequency. The plant lodging and root necrosis in Cuba result of nematode attack. From each results were similar to those of have been recorded where low popula- field 10 plants were selected at random, Pocasangre (2000) who found that tions of nematodes were very low. holes 20 x 20 x 20 cm excavated 10 cm Fusarium species were predominant in Interactions at the root level between from the pseudostem and five affected soils from Cuba, Costa Rica, Guatemala R. similis and species of fungi belonging roots removed. and Honduras.

INFOMUSA — Vol 11, N° 1 23 Table 1. Species of endophytic fungi associated with roots of banana and plantain in plantations on different locations, Cuba.

Isolate Species Clone Location

1.1 F. equiseti (Corda) Sacc. Gran enano UBPC 14 La Cuba, Ciego de Avila 1.2 F. oxysporum Schlecht. Gran enano UBPC 14 La Cuba, Ciego de Avila 1.5 C. musae B. & St. Gran enano UBPC 14 La Cuba, Ciego de Avila 2.1 F. oxysporum Schlecht. Gran enano UBPC 1 La Cuba, Ciego de Avila 2.2 F. oxysporum Schlecht. Gran enano UBPC 1 La Cuba, Ciego de Avila 2.3 Unidentified dark colonies Gran enano UBPC 1 La Cuba, Ciego de Avila Figure 1. Roots of banana with necrosis caused 2.4 Unidentified dark colonies Gran enano UBPC 1 La Cuba, Ciego de Avila by nematode attack. 3.1 C. musae B. & St. Gran enano UBPC 7 La Cuba, Ciego de Avila . 3.2 F. equiseti (Corda) Sacc. Gran enano UBPC 7 La Cuba, Ciego de Avila 4.1 Unidentified dark colonies Gran enano UBPC 5 La Cuba, Ciego de Avila 4.2 C. musae B. & St. Gran enano UBPC 5 La Cuba, Ciego de Avila 5.1 Unidentified dark colonies Gran enano Sola, Camagüey 5.2 C. musae B. & St. Gran enano Sola, Camagüey 5.5 Unidentified Monilia Gran enano Sola, Camagüey 6.1 F. equiseti (Corda) Sacc. Gran enano La Esperanza, Quemado de Güines, Villa Clara 6.3 C. musae B. & St. Gran enano La Esperanza, Quemado de Güines, Villa Clara

6.5 F. oxysporum Schlecht. Gran enano La Esperanza, Quemado de Güines, Villa Clara Figure 2. Cylindrocarpon musae. Macroconidia 7.1 F. semitectum Gran enano Margarita, Quemado de Güines, Villa Clara and chlamydospores. 7.2 C. musae B. & St. Gran enano Margarita, Quemado de Güines, Villa Clara 8.1 C. musae B. & St Parecido al Rey Lutgardita, Quemado de Güines, Villa Clara 8.2 Basidiocarp Parecido al Rey Lutgardita, Quemado de Güines, Villa Clara 9.1 Unidentified dark colonies Gran enano Güines, Quemado de Güines, Villa Clara 9.2 F. oxysporum Schlecht. Gran enano Güines, Quemado de Güines, Villa Clara Booth and Stover (1871) reported the 9.3 Unidentified dark colonies Gran enano Güines, Quemado de Güines, Villa Clara presence of C. musae associated with 9.4 Unidentified Monilia Gran enano Güines, Quemado de Güines, Villa Clara root necrosis in banana in Costa Rica, 10.1 F. oxysporum Schlecht. Gran enano Horquita, Cuban 11, Cienfuegos however the fungus did not have the par- 10.2 F. oxysporum Schlecht. Gran enano Horquita, Cuban 11, Cienfuegos asitic capacity to cause lesions on healthy 11.1 F. oxysporum Schlecht. FHIA-03 Lenin, Matanzas roots. Other species of Cylindrocarpon 11.2 C. musae B. & St. FHIA-03 Lenin, Matanzas are important pathogens of plants. For 11.3 F. oxysporum Schlecht. FHIA-03 Lenin, Matanzas example C. destructans causes root 12.2 Unidentified dark colonies Parecido al Rey Lenin, Campo 48, Matanzas necrosis and death in pine (Pinus sp.) 13.1 Unidentified dark colonies Gran enano Lenin, Campo 52, Matanzas (Chakravarty and Unestam 1987). 13.2 F. oxysporum Schlecht. Gran enano Lenin, Campo 52, Matanzas Recently there have been bioassays of 14.1 F. equiseti (Corda) Sacc. Gran enano Pinar del Río, Vitroplantas artificial inoculation of C. musae alone or 15.2 F. oxysporum Schlecht. Robusta La Maya, Santiago de Cuba of C. musae in co-inoculation with R. sim- 15.3 F. oxysporum Schlecht. Robusta La Maya, Santiago de Cuba ilis. The studies provided important 15.4 C. musae B. & St. Robusta La Maya, Santiago de Cuba information on the effects of the patho- 16.1 F. oxysporum Schlecht. Gros Michel La Ciénaga, Baracoa, Guantánamo genicity of these species on root necrosis 16.2 F. oxysporum Schlecht. Gros Michel La Ciénaga, Baracoa, Guantánamo in the crop. 17.1 F. oxysporum Schlecht. Gran enano Imías, Guantánamo None of the sites yielded species of 17.2 Unidentified dark colonies Gran enano Imías, Guantánamo Cylindrocladium or Zythia as reported 17.3 F. oxysporum Schlecht. Gran enano Imías, Guantánamo by Loridat (1989), Mourichon (1993) 18.1 F. oxysporum Schlecht. Gros Michel Vega del Jobo, Imías, Guantánamo and Risède (1994). These species have 18.2 F. oxysporum Schlecht. Gros Michel Vega del Jobo, Imías, Guantánamo been associated with necrosis of banana 18.3 F. oxysporum Schlecht. Gros Michel Vega del Jobo, Imias, Guantánamo in Martinique and Guadeloupe, and lat- 19 F. oxysporum Schlecht. Burro Palma Soriano, Santiago de Cuba terly in Cameroon (Abadie 1998, pers. 20 Fusarium oxysporum Schlecht. Burro Antero Regalado, Artemisa, La Habana comm.) and Côte d’Ivoire (Kobenan 21 Rhizoctonia sp. Burro UBPC Emilio Hernández, Artemisa 1991). 22 F. oxysporum Schlecht. Burro CPA Niceto Pérez, Güira La Habana 24 Rhizoctonia sp. Burro Ojo de Agua, San Antonio, La Habana Conclusions 25 F. oxysporum Schlecht. Pelipita CPA Niceto Pérez, Güira La Habana 1. F. oxysporum and C. musae were the 26.1 Rhizoctonia sp. FHIA La Palma, Alquízar, La Habana species most frequently associated 26.2 F. oxysporum Schlecht. FHIA La Palma, Alquízar, La Habana with necrosis caused by nematodes in 27 C. musae B. & St. Burro Rpto. Hnos. Cruz, Pinar del Río plantations of banana and plantain in 28 Unidentified dark fungus Burro San Juan y Martínez, Pinar del Río different parts of Cuba. F. equiseti and 29 C. musae B. & St. Cavendish enano Coifa, Boyeros, La Habana Rhizoctonia spp. were found less fre- 30 Unidentified fungus Burro CEMSA CCS Pedro Lantigua, Bauta, La Habana quently. 2. F. oxysporum was the most frequent 31 F. oxysporum Schlecht. Consejo de Estado, Plaza, La Habana isolate accounting for 45.6% total iso- 32 F. oxysporum Schlecht. Robusta Fca. Govín, Caimito, La Habana lates, followed by C. musae.

24 INFOMUSA — Vol 11, N° 1 Cylindrocladium and Zythia spp., Loridat P. 1989. Etude de la microflore fongique et Risède J.M. 1994. Eléments de caractérisation de reported in other countries to be associ- des nématodes associés aux nécroses de l’appareil Cylindrocladium sp. agent des nécroses raci- ated with root necrosis, were never souterrain du bananier en Martinique. Mise en naires du bananier en Martinique. Fruits 49(3): found. évidence du pouvoir pathogène du genre 167-178. Cylindrocladium. Fruits 44(11): 587-598. Sarah J.L. 1990. Les nématodes et le parasitisme des References Mourichon X. 1993. Parasites fongiques du bananier. racines de bananiers. Fruits. (Numéro Spécial Booth C. 1981. The genus Fusarium. CAB, Farnham Fruits 48(1): 26-28. Bananes): 60-67. Royal, England, 237pp. Pérez J., O. García & E. Fernández. 1984. Booth C. & R.H. Stover. 1971. Cylindrocarpon musae Distribución de los principales nematodos parási- sp. nov., commonly associated with burrowing tos del plátano en Cuba. Ciencia y Técnica en la nematode (Radopholus similis) lesions on Agricultura, Serie Protección de Plantas. 7(1): banana. Trans. British Mycol. Soc. 63: 503-507. 27-58. Chakravarty P. & T. Unestam. 1987. Mycorrhizal fungi Pocasangre L. 2000. Biological enhancement of prevent disease in stressed pine seedlings. banana tissue culture plantlets with endophytic Journal of Phytopathology 118(4): 335-340. fungi for the control of the burrowing nematode Alicia Battle-Viera and Luis Pérez-Vicente work at Kobenan K. 1991. Parasites du système racinaire Radopholus similis and Panama disease the Instituto de Investigaciones de Sanidad Vegetal des bananiers en Côte d’Ivoire. Fruits 46(6): (Fusarium oxysporum f. sp. cubense). PhD (INISAV), Gaveta 634, Zona Postal 13, Playa, Ciudad 633-641. Thesis, Bonn University. 95pp. de la Habana 11300, Cuba.

Agronomy Effets of micorrhizae

Effects of mycorrhization on the development of two cultivars of micropropagated banana

M.C. Jaizme-Vega, banana plants, which gives rise to plants per tray (capacity of tray 24 kg) with the M. Esquivel Delamo, that are well developed and have an following isolates: P. Tenoury Domínguez increased tolerance to attack by soil- • Glomus intraradices Schenck & and A.S. Rodríguez Romero borne pathogens. However, at present Smith, from stock collection, multi- there is no information on the effects of plied on sorghum, and giving 68% colo- such symbiotic fungi on the banana nization; he likelihood of using arbuscular plant during the later stages of develop- • Glomus manihotis Howeler, Sieverding mycorrhiza (AM) in crop produc- ment and with fertilizer regimes similar and Schenck, from stock collection, Ttion systems is increasingly more to those practised in commercial crops. multiplied on tomato, and giving 70% realistic and studies have increased con- Therefore, the sequential effects of colonization. siderably in the last few years. early mycorrhization on the growth of At inoculation, plants were 10 cm Banana (Musa AAA) in its early stages micropropagated banana plants were ±2cm and had approximately three of development is readily colonized by studied from the earliest stages of devel- developed leaves. Inoculation was in poly- mycorrhiza and is moderately (40-50%) opment until nine months after trans- ethylene (PE) trays (40 x 60 cm, H x L), dependent on them (Jaizme-Vega et al. planting to the field in microplots. each tray containing one cultivar/fungus 1998). Mycorrhization in vivo has combination with an additional two con- resulted in large increases in the growth Materials and methods trol trays with non-inoculated plants, one and nutrition of this species (Lin and tray per cultivar. Thus there was a total of Chang 1987, Rizzardi 1990, Declerk et al. Host plant six trays each with 35 plants. 1995, Jaizme-Vega and Azcón 1995) Micropropagated material of the two The substrate comprised a steam-ster- including in the presence of standard most widespread commercial cultivars of ilized mixture of dark-coloured volcanic fertilization regimes in commercial nurs- banana Musa acuminata Colla AAA, cvs. soil and amended peat (TKS1®, Instant, eries (Tenoury 1996, Sosa Hernández ‘Grande naine’ and ‘Gruesa’ (a local Floragard, GmbH) in a proportion of 1997), with favourable effects on plant selection of ‘Dwarf Cavendish’) was used. 5:2:1. This phase lasted six weeks in a behaviour when confronted with various glasshouse and under a tunnel of black soil-borne pathogens such as Meloidogyne Rooting stage mesh for acclimatization. Irrigation was incognita (Jaizme-Vega et al. 1997), with distilled water according to the Pratylenchus goodeyi (Jaizme-Vega and Inoculation with AM fungi needs of the plants. Pinochet 1997) and Fusarium oxyspo- Mycorrhization was done during harden- rum f.sp. cubense (Jaizme-Vega et al. ing off. Inoculum comprised a homoge- Nursery phase 1998). These results demonstrate the neous mixture of rhizosphere soil, spores At the end of root production and before advantages of applying inoculum of fun- and rootlets of the host plant. transplanting to individual containers, gal AMs during root production and Each cultivar was inoculated with one of 10 plants of each treatment/cultivar com- acclimatization of micropropagated two AM fungi, each with 1500 g inoculum bination were selected and the effects

INFOMUSA — Vol 11, N° 1 25 RECORDING OF DATA RECORDING OF DATA RECORDING OF DATA

Inoculum of AM fungi

“In vitro” Rooting phase Nursery phase Microplots phase (6 weeks) (14 weeks) (9 months)

Commercial fertilization

Figure 1. The different development phases of the banana plant in the trial.

evaluated of inoculation with mycorrhiza (1:1:1) of steam sterilized soil (‘picón’) O.M. Tarragona). Once in position in their on plant development, the mycorrhizal and enriched peat (TKS1®). This phase new pots (10 per cultivar and treatment), dependency under the fixed conditions of took 14 weeks in glasshouse conditions at the plants were placed amongst other fertilizer inputs, and the extent of colo- temperatures of 27-32°C, and a relative similarly sized pots previously buried up nization by the AM fungi. humidity of 70-80%. to the upper edge of pot, in the trial plot. Parameters relevant to the growth of Fertilization was according to the fer- Plants were fertilized weekly (1 L/plant), the plant in general were evaluated at tilizer regime of a commercial banana via the localized irrigation system, with each stage of the investigation as follows: nursery. Plants were fertilized twice the two combinations of fertilizer treat- fresh weight (g) of roots and aerial parts, weekly (100 cc/plant) on alternate days. ment described previously for banana dry weight (g) of aerial parts, length and One of the fertilizer applications was with plants after the first transplanting. Foliar diameter (cm) of pseudostem, leaf num- (NO3)2Ca (3 g/L) and NO3H (0.4 cc/L), fertilizers were applied fortnightly. The 2 bers and area (cm ). Leaf area was calcu- and the other application was with SO4K2 days on which fertilizers were not lated with an area meter Li-COR, inc. (3 g/L) and PO4H3 (0.2 cc/L). The days on applied, plants were irrigated according Lincoln, Nebraska, USA, model Li-3100. which fertilizer was not applied alter- to the needs of the plants. The relative mycorrhizal dependency nated with irrigation with running water Plants remained in position for nine (RMD), defined by Gerdeman (1975) as according to the needs of the crop. Plants months. The trial was then terminated the degree of mycorrhization needed by received a weekly foliar application of and the effects of symbiosis on develop- plants to produce the maximum growth micronutrients consisting of 3% Wuxal® ment of the banana plants evaluated. or yield depending on fertility of the soil, Super AA 8-8-6 (Argos Shering, Agrevo, The following experimental variables was calculated according to the formula S.A., Valencia, Spain). were studied: fresh weight of roots and proposed by Plenchette et al. (1983) as aerial parts, numbers of suckers, num- the numerical expression of this concept: Microplot phase bers of leaves, leaf area, N, P and K con- After growth for 3.5 months, plants were tent, and dependency for mycorrhiza. DW of plant with AM–DW of plant without AM RMD = x 100 transferred to larger containers, and On completion of the foliar analyses, DW of plants without AM buried in a plot within the boundaries of the samples were transferred to a heater (DW : dry weight) the ICIA estate situated 300 masl. The for 24 hours at 70°C after which nitrogen, Infection by the mycorrhiza was con- site was chosen on the basis of aspect, cli- phosphorous and potassium contents firmed by observation with a light micro- matic conditions and as an area marginal were determined. For N determination, scope. Root samples were bleached with for this crop. Prior to this, and as with the the sample was mineralized “via humid 10% KOH and then stained with 0.05% try- first transplanting, 10 plants per cultivar process”, P was determined colourimetri- pan blue in lactic acid as described by and treatment were evaluated for the cally and K by spectrophotometry of Phillips and Hayman (1970) and modified effects of the AM fungi, that is the extent atomic absorption. by Koske and Gemma (1989). Percentage of root infection by mycorrhiza and myc- Data were analyzed by means of root colonization was determined on 20 orrhizal dependency. ANOVA (Systat). Means were compared 1-cm sections of stained root, mounted For this last phase of the trial, PE pots by Fisher’s test of least significant differ- on slides and examined with a light 35 cm diameter and 50 L volume were ences (LSD) using the statistical pack- microscope as described by Brundett selected and filled with non-sterilized age Systat version 5.0 (SPSS Inc., et al. (1985). medium of the same materials and in the Chicago, USA). Once the determinations were com- same proportions as described for the plete 20 plants of each treatment were previous transplanting (1:1:1), and Results and discussion transferred to 2 L PE bags containing a amended with 1.5 g/L of slow release fer- By completion of the rooting stage, both substrate comprising equal volumes tilizer (Osmocote 17:10:10, Scotts, cultivars showed a positive response to

26 INFOMUSA — Vol 11, N° 1 Table 1. Effect of Glomus manihotis and G. intraradices on the development, colonization and mycorrhizal dependency of micropropagated banana cv. ‘Grande naine’ at a) 6 weeks after inoculation, b) 14 weeks after inoculation and c) 9 months after transplanting to microplots.

Fresh weight (g) Dry weight (g) Pseudostem Leaf area Colonization RMD** Root Aerial parts Aerial parts Diameter Length No. leaves (cm2)(%) a) 6 weeks after inoculation (rooting phase) Control 2.6 b* 8.6 b 0.5 b 0.9 b 10.4 b 5.2 b 143 b — — G. manihotis 6.4 a 17.5 a 1.1 a 1.2 a 12.9 a 6.3 a 261 a 26 51 G. intraradices 5.5 a 17.8 a 1.0 a 1.2 a 12.1 a 6.0 a 269 a 37 46 b) 14 weeks after inoculation (nursery phase) Control 13.1 b* 38.1 b 2.6 b 1.8 b 15.4 b 7.3 b 494 b 15 — G. manihotis 29.4 a 66.5 a 4.4 a 2.6 a 23.5 a 8.5 a 777 a 59 40 G. intraradices 26.7 a 63.7 a 4.3 a 2.4 a 22.1 a 8.7 a 805 a 38 38

Table 2. Effect of Glomus manihotis and G. intraradices on the development, colonization and mycorrhizal dependency of micropropagated banana cv. ‘Gruesa’ at: a) 6 weeks after inoculation, b) 14 weeks after inoculation, and c) 9 months after transplanting to microplots.

Fresh weight (g) Dry weight (g) Pseudostem Leaf area Colonization RMD** Root Aerial parts Aerial parts Diameter Length No. leaves (cm2)(%) a) 6 weeks after inoculation (rooting phase) Control 3.1 b* 8.2 b 0.50 b 0.97 b 8.7 b 5.5 a 155 b – – G. manihotis 5.5 a 12.9 a 0.80 a 1.15 a 8.7 b 6.3 a 223 a 27 38 G. intraradices 6.0 a 11.9 a 0.75 a 1.18 a 9.8 a 6.5 a 216 a 24 34 b) 14 weeks after inoculation (nursery phase) Control 22.8 b* 40.5 b 2.8 b 2.0 b 14.7 b 7.7 a 514 b 14 – G. manihotis 33.9 a 57.5 a 3.9 a 2.5 a 16.3 a 8.5 a 722 a 26 29 G. intraradices 36.4 a 50.7 a 3.5 ab 2.4 a 15.9 ab 8.0 a 662 a 30 19

Fresh weight (g) No. leaves No. suckers Leaf area Colonization RMD Macronutrient content Root Aerial parts (cm2)(%)(%)N P K c) 9 months after transplanting to microplots Control 7.1 a* 7.8 a 14.9 b 2.4 a 41845 b 59 – 2.84 a 0.176 a 2.65 a G. manihotis 7.7 a 11.5 ab 19.1 ab 3.6 a 57733 ab 72 31 3.03 a 0.189 a 3.00 a G. intraradices 9.5 a 13.6 a 23.2 a 4.0 a 61660 a 83 42 3.00 a 0.184 a 3.03 a *Means of 10 replicates. Within each column, differences between numbers followed by the same letter are not statistically different with Fisher’s test (P ≤ 0.05). ** RMD: relative mycorrhizal dependency. the two AM fungi used for inoculation phase of the trial with averages of 40% cant effects on plant development. These (Tables 1a and 2a). In this phase, the rel- for both AM fungi on ‘Grande naine’, and endophytes are able to disperse in irriga- ative mycorrhizal dependency (RMD) of 30% and 20% respectively for Glomus tion water or by uncontrolled contamina- both cultivars to Glomus manihotis and manihotis and Glomus intraradices on tion in the nursery containing the plants. Glomus intraradices were the highest Gruesa (Tables 1b and 2b). These data confirm those already pub- throughout the trial and were 35% and Root colonization of banana plants by lished on the benefits of early mycor- 50% respectively. In this first phase the mycorrhizae tended to differ depending rhization of plants in the first phases of percentage colonization by the two inoc- on the cultivar. Thus, roots of ‘Grande development of this crop (Declerck et al. ulated AM fungi was similar for the two naine’ inoculated with G. manihotis had 1995, Tenoury 1996, Sosa-Hernández cultivars. twice the mycorrhiza infection in com- 1997, Jaizme-Vega et al. 1997, 1998). Following transplanting, the positive parison with the beginning of the study, The results of the second phase of the effect of the AM fungi on plant develop- similar results being maintained on roots trial in which the effects of the AM fungi ment was maintained for 3.5 months colonized by G. intraradices. However on mycorrhiza-treated plants in the after mycorrhization. For inoculated with plants of cv. ‘Gruesa’ no changes in in vivo phase for three months and trans- plants of both cultivars, the majority of root colonization were observed in com- planted to non-sterile medium, showed experimental variables were signifi- parison to the first transplanting. During that after nine months in microplot con- cantly different for both cultivars in com- the trial, from 14 weeks onwards 15% root ditions and a standard fertilizer regime, parison with the controls (Tables 1b and infection by contaminant AM fungi was banana plants inoculated with 2b). The development of RMD was simi- noted in control plants of both cultivars G. intraradices usually, particularly with lar for both cultivars completing this (Tables 1b and 2b) but without signifi- cv. ‘Gruesa’, showed a beneficial effect of

INFOMUSA — Vol 11, N° 1 27 symbiosis on plant development, with This study formed part of project Lin Ch. & D.C.N. Chang. 1987. Effect of three Glomus RMDs of approximately 40%. These values INCO-DEV (International Cooperation endomycorrhizal fungi on the growth of micro- are considered to be relatively high for with Developing Countries) (1998-2002), propagated banana plantlets. Trans. Mycol. Soc. the conditions of the trial (Tables 1c and Contract No. ERB IC 18 CT97-0208. Rep. China 2(1): 37-45. 2c); moreover there was an increase in Phillips J.M. & D.S. Hayman. 1970. Improve proce- the other experimental variables. References dures for cleaning roots and stain parasitic and However, data on macronutrients (N, P Brundrett M.S., Y. Piche & R.L. Peterson. 1985. A vesicular-arbuscular mycorrhizal fungi for rapid and K) although noticeably higher, did development study of the early stages in vesicular- assessment of infection. Trans. Br. Mycol. Soc. 55: not differ statistically (Tables 1c and 2c). arbuscular mycorrhizal formation. Canad. J. Bot. 158-161. This lack of response in nutrient content 63: 184-194. Plenchette C., J.A. Fortin & V. Furlan. 1983. Growth of aerial parts can be interpreted as typi- Declerck S., C. Plenchette & D.G. Strullu. 1995. responses of several plant species to mycorrhiza cal for a mycorrhiza-treated plant receiv- Mycorrhizal dependency of banana (Musa acumi- in a soil of moderate P fertility. I. Mycorrhizal ing soluble fertilizer. nata, AAA group) cultivar. Plant and Soil 176(1): dependency under field conditions. Plant and Soil Plants of cultivar ‘Grande naine’ showed 183-187. 70: 191-209. a smaller response to the AM fungi after Gerdemann J.W. 1975. Vesicular-arbuscular mycor- Rizzardi V. 1990. Effect of inoculation with vesicular- the microplot phase, plants inoculated rhizae. Pp. 575-591 in The development and arbuscular mycorrhizal fungi on the growth of with G. manihotis showing a develop- Function of Roots. (J.G. Torrey and D.T. micropropagated Musa acuminata clone “Grand ment and nutritional state equal or Clarkson, eds). Academic Press, New York and Nain”. Revista de Agricultura Subtropicale e slightly less than control plants. London. Tropicale 84(3): 473-484. At the end of this phase, root coloniza- Jaizme-Vega M.C. & R. Azcón. 1995. Responses of Sosa Hernandez B. 1997. Estudio de la interacción de tion by both Glomus species was rela- some tropical and subtropical cultures to endomy- los hongos formadores de micorrizas arbusculares tively important in both cultivars (greater corrhizal fungi. Mycorrhiza 5: 213-217. (MA) y el patógeno vascular Fusarium oxysporum than 79%). Attention is drawn to the high Jaizme-Vega M.C. & J. Pinochet. 1997. Growth f. sp. cubense sobre platanera en fase de vivero. level of colonization of roots of control response of banana to three mycorrhizal fungi in Proyecto Fin de Carrera. Universidad de La Laguna. plants. This part of the trial used non- Pratylenchus goodeyi infested soil. Nematropica Centro Superior de Ciencias Agrarias. 155pp. sterilized substrate which, together with 27(1): 69-76. Tenoury P. 1996. Estudio de la interacción del hongo other conditions in the trial, explained Jaizme-Vega M.C., P. Tenoury, J. Pinochet & M. formador de micorrizas arbusculares (MA) the data. Jaumot. 1997. Interaction between the root-knot Glomus mosseae y el nematodo agallador In conclusion, in general and particu- nematode Meloidogyne incognita and the mycor- Meloidogyne incognita en platanera. Proyecto larly in the last phase of the trial, we can rhizal association of Glomus mossae and Grande Fin de Carrera. Universidad de La Laguna. Centro confirm that, at the later stages of the Naine banana. Plant and Soil 196: 27-35. Superior de Ciencias Agrarias. 159pp. crop, this biotechnological resource Jaizme-Vega M.C., B. Sosa-Hernández & J. Hernández. showed promise for the improvement of 1998. Efecto de Fusarium oxysporum f.sp. production. cubense (Foc) en platanera micorrizada bajo dos niveles de fertilización fosforada. Acta Acknowledgements Horticulturae 490: 285-295. The authors work in the Departamento de Protección Koske R.E. y J.M. Gemma. 1989. A modified proce- The authors thank Ana Rosa Socorro del Instituto Canario de Investigaciones Agrarias, Monzón, head of the Laboratory for soils dure for staining roots to detect VA mycorrhizas. Apartado 60, 38200 La Laguna, Tenerife, Canary and irrigation, ICIA, for the leaf analyses. Mycol. Res. 92: 486 –505. Islands, Spain.

Agronomy Use of cover crops

Arachis pintoi: a cover crop for bananas? Advantages and disadvantages as regards nematology

P. Quénéhervé, Y. Bertin tion in Meloidogyne attacks on tomatoes 1998). Lastly, in 1999, Jonathan et al. and C. Chabrier was observed in an intercropping experi- showed from an artificial inoculation ment (Marban Mendoza et al. 1992). In experiment that the legume Arachis Costa Rica, a field experiment showed pintoi is not a host for certain species of he legume Arachis pintoi L. (the that Arachis pintoi is a good host for Meloidogyne Goeldi 1892 (M. incognita, perennial, wild or pinto peanut) has Radopholus similis (Cobb 1893, Thorne M. arenaria, M. javanica) nor for Tbeen used for many years as a cover 1949) with a concomitant mean infection Rotylenchus reniformis Lindford & crop in many tropical countries, notably rate of about 30 individuals per g of root Oliveira 1940. Central America (Kerridge 1993). Its (Araya 1996). Also in Costa Rica, trials Before experimenting with, and per- response to nematodes is still barely carried out with bananas and plantains haps recommending the use of Arachis documented. Possible resistance of have shown a beneficial effect of wild pintoi as a possible cover crop for cv. Amarillo to Meloidogyne spp. in peanut used as a cover crop in reducing bananas, we wanted to check its behav- Australia has been mentioned (Cook et the density of Radopholus similis on iour towards nematodes of banana in al. 1990). In Mexico a noticeable reduc- neighbouring banana plants (Vargas Martinique. A controlled inoculation trial

28 INFOMUSA — Vol 11, N° 1 using the main species present tion in Arachis pintoi is extremely slow, (Radopholus similis, Pratylenchus with a shoot/root ratio of about 7.5 in our coffeae, Hoplolaimus seinhorsti, Meloi- experiment, and it would be interesting dogyne incognita) together with to quantify the real “reservoir” capacity Meloidogyne mayaguensis, a species of this plant for nematodes in the field as which is very pathogenic in Martinique was done by Araya in 1996. However one although not yet observed on bananas, can already regard it as a “non-host” for was therefore carried out in a controlled Meloidogyne spp., and especially environment chamber at the IRD M. mayaguensis, and reconsider this Nematology laboratory before attempting crop as a “cleaning fallow crop” (for reha- any field experiments. bilitation) which provides protection against nematodes before a susceptible annual or perennial crop. Materials and methods For many years agronomists have Seeds of Arachis pintoi cv. Amarillo sought plants useful as fallows of short, from Costa Rica were inoculated by coat- medium or long term, or as cover crops ing at the moment of sowing with their which can, inter alia, reduce parasitic symbiotic bacterium Rhizobium sp. pressure (for example by nematodes) and These seeds were then grown in 237 cm3 also reduce the effects of weeds, improve PVC culture tubes filled with sterile soil soil fertility and limit erosion (Terisien (steam sterilization for 1 h at 100ºC). and Melin 1989). In the Caribbean zone, The substrate was a volcanic andosol of two species have been used for their pH 6.2, with 7.3% organic matter and a Figure 1. Root system of Arachis pintoi. activity against nematodes, with both cation exchange capacity of 10.3 meq per advantages and disadvantages: the forage 100g soil. The experiment was carried grass Digitaria decumbens and the for- out in a controlled environment cham- age legume Mucuna pruriens cv. utilis, ber with eight replicates, using a 14-h of African origin. photoperiod and a temperature in the Each of these plants has its value light of 27±1ºC, and in the dark of earlier results on the inability of this according to the cropping system being 22±1ºC, daily watering and weekly appli- peanut to act as host to the main species considered. Digitaria decumbens fits into cation of a Hoagland nutrient solution. of root-knot nematodes, with the excep- long-term rotations combining livestock Four weeks after sowing and develop- tion of M. hapla (Jonathan et al. 1999). and field vegetable growing, as practised ment of the peanut, the five nematode Arachis pintoi does however act as on the vertisols in the south of Martinique. species, meanwhile grown in the labora- host to three other species, and accord- Mucuna pruriens, widely grown in the tory (Radopholus similis, Pratylenchus ing to the criteria applied to banana southeastern United States and in Africa, coffeae, Hoplolaimus seinhorsti, weeds (Quénéhervé et al. 2002), one might can also find a place in Martinique as a Meloidogyne incognita and Meloidogyne say that it is a bad host for R. similis short-term intercrop or in certain inten- mayaguensis) were individually inocu- but a very good host for H. seinhorsti and sive vegetable systems to control nema- lated at a rate of 400 individuals per P. coffeae. The hosting capacity of todes, particularly Meloidogyne spp. plant. The infestation of the root system Arachis pintoi to R. similis already (Quénéhervé et al. 1998). was checked 45 days later after extrac- observed (Araya 1996) is thus confirmed, This third plant, Arachis pintoi, tion of the nematodes from the roots by but also (which is new) its great suscep- recently introduced by CIRAD-FLHOR into spraying (Seinhorst 1950). The nema- tibility to P. coffeae and H. seinhorsti, Martinique, seems to offer certain advan- tode densities were then expressed as two species of nematodes whose patho- tages, but also possesses disadvantages: numbers of nematodes per root system genicity to bananas was demonstrated • advantages: commercially available and per g of dry root (after oven-drying for one (P. coffeae) and seems very likely seed, propagation by seed, or vegeta- at 60ºC for 24h). for the other. tive; “non-host” plant to several nema- These results may be compared with tode species including Meloidogyne others from the field under conditions of spp.; suitable as a cover crop; supplies Results and discussion natural infestation. In fact, root produc- nitrogen (about 60 kg/ha/year). The results of this experiment (Table 1) show that at 45 days only three species of nematode are supported: R. similis, H. seinhorsti and P. coffeae. The inoculation of the different species of nematode Table 1. Results of nematode counts and Arachis pintoi weighings 45 days after has had no effect on the growth either of inoculation. shoots or roots of the peanut, which No./g roots Roots (mg) Shoot (mg) Host quality1 therefore appears, over this short period Control - 260 ± 10 1470 ± 140 - of time, to be tolerant of attack by these Hoplolaimus seinhorsti 382 ± 132 240 ± 40 1880 ± 110 *** nematodes. Pratylenchus coffeae 2918 ± 447 240 ± 30 1630 ± 350 *** Arachis pintoi was unable to maintain Radopholus similis 112 ± 95 250 ± 50 1820 ± 50 * or permit the multiplication of the two Meloidogyne mayaguensis 0 240 ± 60 1600 ± 300 NH species of Meloidogyne, M. incognita Meloidogyne incognita 0 270 ± 30 2010 ± 180 NH and M. mayaguensis. This result con- ANOVA NS NS firms and completes for M. mayaguensis 1 Very good host = ***; Good host = **; Poor host = *; Non-host = NH

INFOMUSA — Vol 11, N° 1 29 • disadvantages: plants are host to seri- References Quénéhervé P., C. Chabrier, A. Auwerkerken, ous migratory endoparasites, including Araya M. 1996. Capacidad hospedante de Arachis P. Topart, B. Martiny & S. Marie-Luce. 2002. R. similis and P. coffeae; slow to estab- pintoi a Radopholus similis. CORBANA 21: 19-24. Status of weeds as reservoirs of nematodes in lish; requires inoculation with a spe- Cook B.G., R.G. Williams & G.P.M. Wilson. 1990. banana fields in Martinique. Nematropica (sub- cific associated bacterium. Arachis pintoi Krap et Greg. nom. nud (pinto mitted). The introduction and use of Arachis peanut) cv. Amarillo. Tropical Grasslands 24: Seinhorst J.W. 1950. De betekenis van de toestand pintoi as a cover crop for bananas could 124-125. van de grond voor het optreden van aanstasting therefore take place under certain con- Jonathan E.J., K.R. Barker & T.B. Sutton. 1999. door het stengelaattje (Ditylenchus dipsaci) ditions: Host status of wild peanut Arachis pentoi for (Kühn) Filipjev). Tijdschr. Plziekt. 5: 291-349. • in the absence of the nematodes root-knot and reniform nematodes. INFO MUSA Ternisien E. & Ph. Melin. 1989. Etude des rotations R. similis and P. coffeae, which would 8: 9-10. culturales en bananeraie. Première partie : bilan limit its use immediately after banana Kerridge P.C. 1993. Biology and agronomy of forage des cultures de rotation. Fruits 44: 373-383. or another crop infested by P. coffeae, Arachis. International Center for Tropical Vargas A. 1998. Banana (Musa AAA) and plantain such as yam or dasheen; Agriculture (CIAT), Colombia. (Musa AAB) cultivation in the presence and • after crop rotation but in the presence Marban-Mendoza N., M.B. Dicklow & B. Zuckerman. absence of a green cover crop (Arachis pintoi of Meloidogyne spp. so as to reduce the 1992. Control of Meloidogyne incognita on tomato CIAT-18748). CORBANA 22: 23-39. infestation potential of these root-knot by two leguminous plants. Fundam. Appl. nematodes before replanting with Nematol. 15: 97-100. tissue-cultured banana plants. Quénéhervé P., P. Topart & B. Martiny. 1998. This plant could also find a place in Mucuna pruriens and other rotational crops Martinique and elsewhere in the West for control of Meloidogyne incognita and Indies in various other ecosystems, which Rotylenchulus reniformis in vegetables in remain to be experimented with: polytunnels in Martinique. Nematropica 28: • in orchards such as citrus and espe- 19-30. cially guavas which suffer serious Quénéhervé P., Y. Bertin & A. Kermarrec. 2002. Patrick Quénéhervé works at the Institut pour la recherche et le développement (IRD, former attacks from M. mayaguensis in the Meloidogyne mayaguensis: a root knot nematode ORSTOM), BP 8006, 97259 Fort-de-France Cedex, West Indies (Quénéhervé et al. 2001); causing severe decline of guava trees in the Martinique, FWI; Yves Bertin and Christian • as a fallow crop or intercropped cover Caribbean (Abstr.). African Plant Protection Chabrier work at CIRAD-FLHOR, BP 153, 94202 Fort- crop for vegetables. ■ (in press). de-France Cedex, Martinique, FWI.

Agronomy Micronutrient studies Dynamics of boron in a soil cultivated with plantain (Musa AAB cv. ‘Dominico hartón’) in the Quindío, Colombia

M.M. Bolaños Benavides not taken up by plants. Compared with flowering and fruiting, germination of and A. Garcia Alzate other micronutrients, boron has several pollen grains, cell division, cell wall syn- special features, thus in soil solution it is thesis and the metabolism of nitrogen, always found in combination with oxygen, carbohydrates and pectic substances. oron (B) is the only non-metallic behaving as an anion (borate) in all reac- These substances are reported to element of the six essential tions. The borate anion is highly mobile increase in plants that are deficient in Bmicronutrients; it has a constant and hence is easily lost by leaching. The boron (Rajaratnam and Lowry 1974). valence of +3, and has the smallest ionic available boron in soil can be considered Another function of boron is the radius. It is found mainly in sedimentary as belonging to a cycle where a small absorption of water by protoplasm and rocks. Of the igneous rocks it is most amount originates from tourmaline and a the absorption of mineral salts. The abundant in granites, in the form of large part from soil organic matter. main function of boron is reported to borosilicates, with tourmaline (3-4% Organic material is decomposed by facilitate the transport of highly polar boron) the most common of the minerals. microorganisms which liberate available sugar molecules across the cell wall. It is found in soil in four states: a) form- boron to the soil solution, from whence it Boron is a constituent of cell membranes ing part of the crystalline structure of is taken up by plants; part can be washed and is immobile in the plant; therefore minerals; b) adsorbed or bound by soil out by percolating water, and a small part any boron deficiency is immediately colloids; c) as an anion in the soil solu- is fixed or bound by clays (Berger and reflected in a change in the metabolism tion and d) associated with soil organic Pratt, cited by Bonilla et al. 1994). of carbohydrates (which accumulate in matter (Bonilla et al. 1994). The multiple functions involving boron leaves). This condition could be the The total content of boron in soils in plant metabolism include the follow- cause of almost all the remaining func- varies from 2 to 200 ppm most of which is ing: it affects, inter alia, the processes of tions attributed to boron (Gómez and

30 INFOMUSA — Vol 11, N° 1 Table 1. Methods of chemical analysis Table 2. Changes in chemical properties of soil under study (1990–2000). of soils. Changes in soil fertility Factor Analytical methods PH Potentiometer, Year pH MO K Ca Mg P B ratio 1:2.5 (%) (meq/100 g) (meq/100 g) (meq/100 g) (ppm) (ppm) EA (exchangeable acidity) KCl 1N 1990 6.08 3.79 0.95 5.2 0.93 22 0.40 MO Walkley–Black 1993 5.18 3.66 0.69 4.4 1.03 71.6 0.12 P (ppm) Bray II 1995 5.72 3.72 1.22 2.8 0.64 34.6 0.19 Exchangeable bases Ammonium acetate (1N) 1997 5.78 4.80 1.30 3.8 0.94 61.0 0.06 and neutral (pH 7) 2000 5.10 4.80 1.79 6.0 0.60 29.0 0.01

Leguizamón 1975). In spite of the impor- soil organic matter, and boron-pH. The between boron content and pH, are based tant advances in mineral nutrition, the relationships between Ca/Mg, Mg/K, Ca/K on the following: role of boron in plant metabolism still and (Ca+Mg)/K were also analyzed. • Profoundly influences many biological raises many questions. processes in soil, At present, in the coffee production Results and discussion •Affects the availability of micronu- area of central Colombia, many plantain The data obtained from soil analyses, trients, crops show symptoms associated with averaged over the five replicates, changed • Alters the absorption of an element boron deficiency. According to León et al. over the years as shown in Table 2. and its effect on microbial activity, (1985) ten examples of boron deficiency • Results in changes in the ability of were reported in the country. In the pre- Boron content roots to absorb or transport ions once sent study we tried to obtain a sound As can be seen from the chemical analy- they have been taken up, foundation in order to deal more clearly ses during the 10 years of the study, • Causes variations in the stability of with the problems above. The objective boron content declined considerably soluble and insoluble organic com- was to determine the importance of from levels sufficient for cultivation of plexes, boron in the cultivation of plantain plantain, according to Buriticá (1985) • Changes the solubility of antagonistic (Musa AAB cv. Dominico hartón) in the from 0.4 ppm to 0.01 ppm boron, a value ions and changes conditions in the rhi- Quindío, and to study the dynamics over a which gave rise to deficiencies. However, zosphere. period of ten years, in a soil fertilized one must consider the edaphic cycle of with the major elements. boron which determines its concentra- Relations between nutrients tion in soil solution and hence the avail- The results also show a close inverse cor- Materials and methods ability for uptake by plants (Mengel relation between K and boron (Table 3) The study plot was located at the 1980). explicable because the K content over the Experimental station El Agrado, District years had reached levels greater than of Montenegro, Department of Quindío, Relationship between pH and boron 0.3 meq/100 g soil, which, according to Colombia. The station was 1320 m above content Gómez and Leguizamón (1975) can sea level, with an average rainfall of As can be seen from Table 3, boron induce boron deficiency. 2000 mm/annum, a mean annual tem- showed a direct and close correlation The potassium-boron interaction does perature of 22oC and a relative humidity with pH, hence this is in a range optimum not appear to follow a particular pattern. of 76%. for absorption of boron; fixation of this Revé and Shive (1944) cited by According to the classification of microelement to hydroxides of Fe and Al, Domínguez (1988), demonstrated that in Holdridge, the ecosystem corresponds to as is with clays, increases with pH being a a boron-rich medium, absorption of boron premontane humid woodland. The soils maximum between pH 8 and 9 and a min- increased as the soil became enriched are derived from volcanic ash (andisols) imum at about pH 5 (Lora 1994). with K but, in contrast, when boron levels and have an average natural fertility, a According to Domínguez (1988) the in the medium are low, boron deficiency medium to heavy texture, a low moisture increase in pH reduces the availability of becomes worse as K increases. The direc- retention capacity, and are leached and boron but this does not become evident tion of the interaction between K and susceptible to erosion. until more than pH 6 which did not occur boron appears to depend on the amount For the study the soils were taken for after the start of the experiment. of boron in soil solution. The trend in this analysis from 2 May 1990 until 2 March According to Marschner (1986) the study showed that increasing applica- 2000. Soil samples, replicated five times, availability of boron to plants decreases were taken every two years. Rainfall data with increased soil pH, as happens in cal- was analyzed for the same period. careous soils or in soils with high clay Table 3. Correlations between soil boron, Samples were analyzed for pH, organic content, presumably as a result of the for- pH, K, Ca, P and weight of bunches matter content, exchangeable calcium, mation and absorption of B(OH)4. (WB) in kg. phosphorus (P), magnesium (Mg), potas- In agreement with the soil chemical sium (K) and boron (B). The analytical analyses, the pH value (5.1-6.08) fluctu- Correlations methods are described in Table 1. The ated in the range sufficient for the pH – B 0.82 data were analyzed to determine the cor- micronutrient to be available. This K – B -1 relation between: boron-weight of bunch explains why symptoms of boron defi- Ca – B 0.8 (for each production cycle), boron-potas- ciency only became evident in the last P – B -0.86 sium, boron-calcium, boron-percentage years. The explanation for the correlation WB – B 1

INFOMUSA — Vol 11, N° 1 31 tions of K resulted in a small reduction in Table 4. Relationships between cations in the experimental soil (1990-2000). boron availability. 1990 1993 1995 1999 2000 When boron interacts with other ele- Relationship ments, it is necessary to consider the pos- Mg/K 1.58 1.93 0.87 0.73 1.00 sibility of nutritional imbalances in soil, Ca/Mg 5.72 4.14 5.00 4.88 5.00 given that this implies an antagonism Ca/K 8.97 7.96 5.37 3.58 6.66 that affects the plant directly, as one or (Ca+Mg)/K 10.50 9.88 6.26 4.33 7.66 other elements would not then be available. Such is the situation with potassium that is absorbed in smaller amounts when boron content is very low. which is explained by the large quantities contributes to Mg deficiency, resulting in As regards calcium, the levels increase of potassium containing fertilizers that an imbalance in the relationship Mg/K when boron is deficient. In the soil under were applied over the years, as well as by and as a result an inhibition of Mg study, where we did not find a high avail- the recycling that occurs with this ele- uptake. It should be noted that in the ability of Ca, this could favour boron ment in residues from the harvest of the zone where this study took place, it is uptake. However, the interaction cal- plantain. According to Belalcázar (1991), common to come across crops with symp- cium-boron has been studied with rather cultivation of plantain removes a large toms of magnesium deficiency. In accor- high concentrations, and with the rela- percentage of elements such as potas- dance with Fried and Dean (1952) nutri- tionship of Ca/boron in the plant. Revé sium (76.02%) and calcium (13.62%), fol- tional deficiencies resulting from an and Shive (1944) cited by Domínguez lowed by nitrogen, magnesium and phos- imbalance can be corrected by a pro- (1988), indicated that high concentra- phorus. Those with the higher percentage gramme of balanced fertilisation. tions of calcium exacerbate symptoms of that are removed are nitrogen (25.55%) boron deficiency in tomato. The toxicity followed by magnesium (20.09%) and Soil organic matter and boron in soil of boron, in a medium with too much of phosphorus (19.80%), whilst those that In accordance with the results of this the element, can on the other hand have are reincorporated or recycled in higher study there was no correlation between a reduced increase in the amount of cal- amounts are calcium (94.47%) and potas- organic matter and boron, however, it cium in the medium. It is possible that sium (89.77%). should be noted that various authors with all the preceding, boron deficiencies With reference to magnesium, amongst (Gómez and Leguizamón 1975) claim that in the cultivation of plantain studied, only the factors that gave rise to the deficiency in mineral soils rich in organic matter, became apparent in the last years (1999- in this macronutrient, were unsuitable boron deficiency is rarely seen because 2000) even when the low levels of this ele- relationships with the other bases in the soil organic matter is a major source of ment were present from 1993. soil, mainly potassium (Table 4). boron. Similarly, Berger and Truog (1945) Table 3 shows an inverse correlation of The relationship Mg/K appeared to be cited by Domínguez (1988), obtained a P to boron. According to studies by unbalanced, explaining a deficiency of positive relationship between assimilable Robertson and Loughman (1974), it was Mg. Therefore, the high levels of K func- boron (water soluble boron) and the evident that there was a clear reduction tion in a manner antagonistic to Mg, organic matter content of soil. More in the uptake of phosphorus in boron- implying a low absorption for this ele- recently, Olsen and Berger (1946) cited deficient plants. This idea is based on the ment. The losses of magnesium in soil are by Domínguez (1988), demonstrated that role of boron as a stimulant of the utilisa- greater when added with potassium fertil- mineralization of organic material leads tion of glucose1-phosphate. This suggests izers. Many authors consider that a soil to to a release of assimilable boron. that when boron is low, the P found in soil be low in magnesium when it has less On the other hand, boron absorbed on will be taken up very slowly, leading to a than 1.0 meq/100 g is present, whilst oth- organic and inorganic soil colloids consti- steady accumulation of P in soil. ers classify soils as poor in magnesium tutes a reserve that maintains the con- According to the relationships obtained when there is less than 1.5 or even centration of boron in solution; this helps between the different cations (Table 4) 2.0 meq /100 g (Suárez and Carrillo 1984). to replenish the demand by crops and and later comparisons with the critical Intensive and continual fertilisation reduces losses by washing. Furthermore, levels, K was never seen to be deficient, with K, as employed in this zone, possibly soils with higher organic matter content

25 production 0,5 precipitation 3500 0,5 boron 20 boron 0,4 3000 0,4 2500 15 0,3 2000 0,3 10 0,2 1500 0,2 1000 0,1 Precipitation (mm) Production (t/ha) 5 0,1 500 0 Boron concentration (ppm) 0 0 Boron concentration (ppm) 90 90 95 97 0 90 95 95 97 Years Years

Figure 1. Production of plantain in relation to boron content in soil. Figure 2. Boron content of soil vs. rainfall.

32 INFOMUSA — Vol 11, N° 1 have higher concentrations of boron, that the addition of boron aids root Buriticá C.P. 1985. Situación del plátano en since an important fraction of soil boron growth, and that if this continues in the Colombia y el sistema de producción de tecnología comes from soil organic matter. coat with organic matter and roots do not para su cultivo. Rev. ICA – Informa (Colombia) 13: want to penetrate the soil, there may not 24-31. Production of plantain and soil boron be sufficient boron. Cohen M.S. & Lepper R. Jr. 1977. Effect of boron on There was a close and direct correlation Boron deficiency in plants is not easily cell elongation and division in squash roots. Plant between yield and boron (Table 3). This identified except by leaf or soil analysis. Physiol. 59: 884-887. is explicable as a result of the chemical This is important in the cultivation of Domínguez A. 1988. El boro. Pp. 155-181 in Los degradation of the soil, as shown in plantain because the micronutrient plays microelementos en agricultura (A. Loué, ed.). Table 2. The gradual loss of the micronu- a key role in the transport of sugars, as a Mundi-Prensa, Madrid, Spain. trient boron, drastically affects the filling result of the transformation of boron- Fried M. & L. Dean. 1952. A concept concerning the of the fruit (Figure 1) to the point where sugar complexes (Marschner 1986) and measurement of available soil nutrients. Soil the young fruits become deformed, therefore affects the filling of the fruits; Science 73: 263-271. mature prematurely and size is reduced, in such situations, a deficiency directly Gómez A. & J. Leguizamón. 1975. Importancia del hence yield declines and they are diffi- and adversely affects the quality and boro en las plantas. Cenicafé (Colombia). Avance cult to market. Clearly productive capac- quantity of the plantain harvest. técnico No.43. ity of plantain is harmed. The reduction Leguizamón J. 1975. Deficiencia de boro en cultivos in yield can also be associated with the Conclusions de plátano en el Valle del Cauca. Cenicafé regulation of uptake and translocation of •Boron is crucial for optimal yields of (Colombia). Avance técnico No.39. boron by the plants, which is more lim- plantain, as well as for the quality and León L.A., A.S. López & P.L.G. Vlek. 1985. ited in comparison with other minerals. quantity of fruit. This was confirmed by Micronutrient problems in tropical Latin America. At the same time, the low quantity and the correlation between bunch weight Pp. 95-129 in Micronutrients in tropical foods quality of production could be caused by and boron. (P.L.G. Vleck, ed.). Martinus Nijhoff/Dr. W. Junk an early boron deficiency, which checks • The availability of boron in soil solution Publ., The Netherlands. growth of the apices and restricts cell is closely linked to rainfall and the loose Lora S., R. 1994. Factores que afectan la disponibili- elongation (Lovatt et al. 1981, Robertson sandy loam texture of the soil and the dad de nutrientes para las plantas. Pp. 29-55 in and Loughman, 1974b) and cell division definite mobility of the borate anion. Fertilidad de suelos: diagnóstico y control (Cohen and Lepper 1977). • In the experimental soil, from 1990 to (F. Silva M., ed.). Soc. Col. Ciencia del Suelo According to Leguizamón (1975) in 2000, as soil potassium content (SCCS). many situations the affected developing increased so boron content declined, Lovatt C.J., L.S. Albert & G.C. Tremblay. 1981. bunches do not produce a yield and in and as a result yields of the experimen- Synthesis, salvage and catabolism of uridine this situation, bunches are small and tal crop of plantain declined. This could nucleotides in boron-deficient squash roots. Plant deformed. be associated with the repeated appli- Physiol. 68: 1389-1394. From the above it can be concluded cation of potassium fertilizer to the soil. Marschner H. 1986. Mineral nutrition of higher that boron is a nutrient fundamental to plants. San Diego CA Academic Press. 674pp. good production as well as fruit quality Recommendation Mengel U.S. 1980. Borax plant food. Borate Meeting, and in quantity. Further investigations are needed to Lafayette, Ind. improve accuracy of the recommendation Primavessi A. 2000. Manejo ecológico del suelo. Rainfall and boron for fertilization with boron in systems of Presentación al Simposio “Biología de Suelos Boron content tends to decline as a result plantain cultivation, in relation to boron Tropicales”, 14-18 agosto 2000, Universidad de of the high rainfall which occurs in the content of leaves and based on different Caldas, Manizales, Colombia. area of the study as shown in Figure 2; critical levels of extraction of the borate Rajaratnam J.A. & J.B. Lowry. 1974. The role of boron this, linked to the sandy loam type tex- anion. in the oil-palm (Elaeis guineensis). Ann. Bot. ture, and the definite mobility of the (London) (N.S.) 38: 193-200. boron anion, gave rise to an increased Acknowledgements Robertson G.A. & B.C. Loughman. 1974a. Reversible rate in leaching of boron. Thus, the nutri- The authors would like to thank the effects of boron on the absorption and incorpora- ent should be applied in a more finely Comité de Cafeteros del Quindío, for the tion of phosphate in Vicia faba L. New Phytol. 73: divided form. economic support for the soil analyses, 291-298. This result was in agreement with which were essential to the present Robertson G.A. & B.C. Loughman. 1974b. Response Marschner (1986) who suggested that study; Dr Fabio Aranzazu H., research to boron deficiency: a comparison with responses under conditions of high rainfall boron is scientist at CORPOICA, Regional 9; produced by chemical methods of retarding root washed out as B(OH)3. and Huberto Morales Osorno and Luz elongation. New Phytol. 73: 821-832. Dary Celis García, research assistants, Suárez S & I.F. Carrillo. 1984. Comportamiento de Boron and the physiology CORPOICA, Regional 9. ■ tres fertilizantes potásicos en un dystrandept of the plant típico. Cenicafé (Colombia) 35(2): 31-39. A general aspect of boron deficiency is References the poor development of meristematic Belalcázar C.S., C.A. Salazar M., G. Cayón S., J.E. tissues, as found at the tips of young roots Lozada Z., L.E. Castillo & J.A. Valencia M. 1991. and in the buds. With boron deficiency, Manejo de plantaciones. Pp. 147-214 in El cultivo Martha M. Bolaños Benavides is a research biologist irregularities in development are the first del plátano en el trópico. Manual de Asistencia with CORPOICA, Avenida Bolíva, Sector Regivit 28 symptoms (Domínguez 1988). This check Técnica No. 50. Instituto Colombiano Agro- Norte. A.A. 1807, Armenia, Colombia. Email: corpoarm @armenia.multi.net.co and Alexander García to the growth of the root tips possibly con- pecuario, Colombia. Alzarte is an undergraduate, Facultad de Ciencias tributes to one of the main problems of Bonilla C.R., A. García, L.E. Castillo & Ch.F. Salazar. Agropecuarias, Programa de Agronomía, Universidad the cultivation of plantain, which harms 1994. Boro y zinc: dos elementos limitantes en de Caldas, Manizales, Caldas, Colombia. Email: the plant. Primavessi (2000) confirmed Colombia. ICA Programa de Suelos, Colombia. [email protected]

INFOMUSA — Vol 11, N° 1 33 Evaluation Performance of plantain hybrids Evaluation of the agronomic characteristics of plantain hybrids (Musa spp.)

P. Orellana Pérez, I. Bermúdez flowering, the following characters were the weight in the first hands is a charac- Caraballoso, L. García Rodríguez recorded: teristic of the plantain. It justifies the and N. Veitía Rodríguez • Plant height removal of the terminal hands of hybrids • Number of functional leaves (with more that have developed more than eight than 75% of green leaf area) at the start hands per bunch in order to encourage lantains are an important food of flowering (NFLF) greater development of the fingers in source for the populations of Latin • Number of leaves showing lesions typical terms of length and diameter. This latter PAmerica and certain African coun- of black leaf streak (Stage 5 on the Sto- aspect is very important if the hybrids tries. Plantains of the ‘Horn’ type, tradi- ver scale modified by Gauhl (1984) at are to claim to rival the ‘Horn’ type tionally the most commonly grown, are the start of flowering (NLSF) plantains. No differences were observed seriously affected by black leaf streak • Number of functional leaves (with more between the hybrids as regards other (Mycosphaerella fijiensis Morelet), than 75% of green leaf area) at harvest bunch characteristics. Arcila et al. which has greatly reduced the supply of (NFLH) (2000) recommended leaving five hands this product both in local markets and for • Number of leaves with black leaf streak and removing the rest 20 days after the export. This is the main disease which lesions at harvest (NLSH) start of flowering. threatens the production of this source of • Number of hands per bunch It is important to emphasize that the both food and income (Jacome 1998). • Length and diameter of the central fin- hybrids with the longest interval between The fact that plantains are usually grown ger of the first and penultimate hands harvest and ripening under natural on smallholdings, sometimes in moun- • Number of days between harvest and ri- conditions (11 days) are FHIA-20 and tainous areas and very often interplanted pening (second hand at ripening degree FHIA-22; for FHIA-21 it is only 8 days. with other crops, makes chemical control 1, using the scale in ‘Descriptors for ba- This shows that the first two have advan- of the disease difficult. The consequence, nana’ (IPGRI-INIBAP/CIRAD 1996)) tages for local sale and for export over not only on volume of production but also • Growing period in days from planting to short distances. on the quality of the product, is that cur- flowering and until harvest. As for response to black leaf streak, the rent levels of production fail to meet the Using the results of counts of the num- FLRI shows that FHIA-04, which has only growing demand of certain local and ber of functional leaves showing typical 1.3 functional leaves at harvest (FLRI = export markets. black leaf streak lesions, two formulae 9.31) is the hybrid whose leaf area was In the 1990s, the first plantain hybrids were devised to serve as indicators of the most reduced during the filling process of resistant to black leaf streak and destined reduction in functional leaf area: the the fingers, resulting in insufficient fill- for commercial use, developed by the Functional Leaf Reduction Index (FLRI) ing of these fingers. The other hybrids Fundación Hondureña de Investigación and Relative Infection Index (RII), had lower values of this index and similar Agrícola (FHIA), offered the possibility reflecting the damage caused by the dis- for them all (Table 2). For these hybrids, of introducing new clones into commer- ease. The latter index depends on the the number of functional leaves at har- cial production and of restoring adequate number of functional leaves showing typi- vest was not less than four, allowing fill- production levels at lower cost. cal lesions at the start of flowering and ing of the fingers. However, because of their genetic con- when the bunch is harvested. The results indicate that hybrid stitution, which includes a contribution Formulae: FHIA-04 is also the most affected by black from clones of the tall ‘French’ type, the • FLRI = NFLF/NFLH leaf streak, with an RII of 9.31 due to the new hybrids must be characterized mor- •RII = FLRI X NLSH/NFLH fact that all its functional leaves bore typ- phologically and studied agronomically = NFLF X NLSH/(NFLH)2 ical lesions of the disease, which devel- before being exploited commercially. As there is only one production cycle in oped rapidly after flowering. At the time The work presented here shows the Cuban plantains, the counts all apply to of harvest, FHIA-20 and FHIA-22, with results of evaluation of various agronomic the mother plant. more than two functional leaves unaf- characters of the FHIA hybrids in the fected by the pathogen, had the lowest central region of Cuba. Results and discussion values of RII: 1.38 and 1.40 respectively. The results show that with the exception FHIA-05, FHIA-19 and FHIA-21, although Materials and methods of FHIA-19, which has the lightest having higher values, responded well to The studies were made on tissue-cultured bunches, the hybrids did not differ in the disease itself even though all their plants produced by micropropagation their bunch weight. For all the hybrids, functional leaves at the time of harvest using the method proposed by Orellana the majority of the weight of the bunch is bore typical lesions. The results confirm (1994), planted out on the multi-crop concentrated in the first four hands that the time needed for the development farm “La Cuba”, situated in the province (59.71% of the total weight). FHIA-19 of the disease on FHIA-04 was very much of Ciego de Avila. had the highest proportion in these less than on the other hybrids, as already Fifty plants of each hybrid were hands (71%), which is confirmed by the reported by Jones (1994). planted 3 metres apart, in two furrows observation of the length and breadth of The results suggest the possibility of also situated 3 m apart. On 20 plants of the fingers of the first hand (Table 1). using FLRI as an expression of the reduc- each hybrid, starting at the beginning of The concentration of the majority of tion of leaf area during the process of fill-

34 INFOMUSA — Vol 11, N° 1 Table 1. Bunch characteristics and yields of the hybrids studied.

Hybrid Bunch Weight of % of total Number of Finger length Finger diameter Number of weight first four bunch hands per (mm) (mm) days from (kg) hands (kg) weight bunch harvest to First Penult. First Penult. ripening FHIA-04 20.3 a 12.8 63 8.5 21.4 14.8 39.3 32.4 8 FHIA-05 21.5 a 13.5 63 8.6 20.8 15.5 39.0 33.5 8 FHIA-19 16.8 b 12.0 71 8.0 22.0 13.8 40.2 30.0 9 FHIA-20 20.6 a 12.1 59 9.7 19.0 14.0 39.8 32.0 11 FHIA-21 21.3 a 13.1 62 8.7 21.1 14.8 38.7 31.5 7 FHIA-22 22.2 a 14.0 61 8.6 20.0 14.0 41.0 31.0 11 (a, b): means of values followed by different letters are significantly different according to Duncan’s multiple range test (P ≤ 0.05). First: Central finger of the first hand; Penult.: Central finger of the penultimate hand.

ing of the fingers, and RII as an expres- Table 2. Response of hybrids to black leaf streak attack. sion of the time needed for the development of the disease as a function of the Hybrid At flowering At harvest NFLF NLSF NFLH NLSH FLRI RII leaf area affected, given by the number of FHIA-04 12.1 3.13 1.3 1.3 9.31 9.31 functional leaves and that of leaves which FHIA-05 10.2 3.80 4.0 4.0 2.55 2.55 are necrotic at harvest time, a relation- FHIA-19 9.0 4.0 4.0 4.0 2.25 2.25 ship which has always been difficult to FHIA-20 12.7 4.0 5.6 3.4 2.27 1.38 quantify numerically. FHIA-21 11.5 4.8 4.5 4.5 2.56 2.56 According to Ortiz and Vuylsteke FHIA-22 12.5 4.6 5.5 3.4 2.27 1.40 (1994), cited by Craenen (1998), it NFLF: Number of Functioning Leaves at Flowering; NLSF: Number of Leaves with Streak at Flowering; NFLH: Number of requires at least eight functional leaves Functioning Leaves at Harvest; NLSH: Number of Leaves with Streak at Harvest; FLRI: Functional Leaf Reduction Index; RII: Relative Infection Index. during the whole growth period and a similar number of healthy leaves before flowering to guarantee a good yield. Craenen K. 1998. Black Sigatoka disease of banana Negra (M.M. Robles et al., compil.). 8-10 de julio From this point of view, FHIA-20 has and plantain: a reference manual. IITA, Ibadan, 1998, Manzanillo, México. SAGAR, INIFAP, INI- the shortest growth period from planting Nigeria. 60pp. BAP, Universidad de Colima, IICA. to harvest, at 481 days, whereas for the Gauhl F. 1994. Epidemiology and ecology of black Jones D.R. 1994. International Musa Testing other hybrids this period varies from 493 Sigatoka (Mycosphaerella fijiensis Morelet) on Programme Phase I. Pp. 12-20 in The improve- to 518 days. plantain and banana in Costa Rica. INIBAP, ment and testing of Musa: a global partnership Montpellier, France. 120pp. (D.R. Jones, ed.). Proceedings of the first global Conclusions IPGRI-INIBAP/CIRAD. 1996. Descriptors for banana conference of the International Musa Testing • The hybrids FHIA-20 and FHIA-22 should (Musa spp.). International Plant Genetic Programme held at FHIA, Honduras 27-30 April have good yield potential owing to the Resources Institute, Rome, Italy/International 1994. INIBAP, Montpellier, France. longer period of time between harvest Network for the Improvement of Banana and Orellana P.P. 1994. Tecnología para la micropropa- and fruit ripening. FHIA-20 also has the Plantain, Montpellier, France/Centre de coopéra- gación in vitro de clones de Musa spp. Thesis for shortest growth period. tion internationale en recherche agronomique the obtention of a PhD in Agronomic Siciences, • The hybrids FHIA-05, FHIA-19 and FH-21 pour le développement, Montpellier, France. 55pp. Instituto de Biotecnología de Las Plantas. also have good yield potential. However, Jacome L. 1998. Sigatoka negra, la situación en Universidad Central de Las Villas, Cuba. 120pp. the RII shows us that by the time of har- América Latina y el Caribe. Pp. 18-23 in Memorias The authors work at the Instituto de Biotecnología vest all their functional leaves are affec- Primer Simposio Internacional sobre Sigatoka de Plantas, Santa Clara, Villa Clara, Cuba. ted by black leaf streak, which in extreme infection conditions can have repercussions on yield. Tissue culture Mass propagation • The indicators FLRI (functional leaf reduction index) and RII (relative infection index) proposed in this study, seem to be suitable for comparing the reduc- Options for in vitro propagation tion in active leaf area and the time needed for black leaf streak to develop in of the banana hybrid cultivar different plantain clones during the period between flowering and harvest. ■ FHIA-20

References L. García Águila, B. Pérez Mederos, nique is the basis of mass propagation Arcila M.I., J.A. Valencia & S. Belalcázar. 2000. Efecto Z. Sarría Hernández, of bananas and plantains with the aim, del desmane sobre la calidad y la produccin this and J. Clavero García for many countries at the present time, study, seem to be suitable for compari in of large-scale commercial distribution Postcosecha y agroindustria del plátano en el eje of completely disease-free plants (Afza cafetero de Colombia. (D.G. Cayón & G. Giraldo, urrently, several Musa cultivars et al. 1996). eds). CORPOICA. Universidad del Quindío, ASIPLAT, are propagated by direct organo- Genotype is known to have an influence Comité departamental de Cafeteros del Quindío, Cgenesis from axillary buds by in on the efficacy of in vitro propagation Colciencias. Fudesco, Armenia, Colombia. vitro culture (Vasil 1994). This tech- and so, when new varieties or hybrid

INFOMUSA — Vol 11, N° 1 35 clones are introduced into production ages was the comparison of proportions - Five explants were inoculated in 250 ml programmes, it is necessary to modify the ANOVA. The analysis of the variable flasks containing 30 ml of semi-solid cul- micropropagation techniques used. “number of buds per apex” was a simple ture medium (2 mg.L-1 of Gellan gum Banerjee et al. (1986) (cited by Afza et al. analysis of variance and the comparison (Spectrum). There were 10 replicates. 1996) found considerable differences of means was made using Tukey’s test at The data were analyzed by multifactorial between clones in shoot formation. This P < 0.05%. variance and the means compared with seems to be correlated with the presence 14.5 x 2.0 cm test tubes were used con- Tukey’s test. Results in percentages were of one or two B genomes. taining 10 ml of culture medium. For liq- analyzed as in the previous experiment. In vitro propagation of the hybrid uid culture media, a paper filter support FHIA-20 (AAAB) has proved difficult. was used, forming a bridge on which the Results and discussion Conversion of apices into plants has apices were placed. In the case of semi- been observed during the initiation solid media, 2 mg.L-1 of the gelling agent Influence of the size of the apex phase, as well as buds growing out in the Gellan gum (Spectrum) was added. and the physical state of the culture form of rosettes and presenting white The plants obtained during the initia- medium during the initiation phase bulbous structures during the multipli- tion phase were transferred, after separa- The use during the initiation phase of cation phase, resulting in a reduction of tion and decapitation, onto multiplica- apices of 1 cm2, cut in halves and placed the multiplication coefficient. In view of tion culture media. It was observed that on a semi-solid culture medium, gave 85% these problems and the need to multiply the growth of the buds continued regeneration after 20 days of in vitro cul- the hybrid FHIA-20 efficiently in vitro, throughout this phase as small rosettes ture. On each section of the apex axillary the development of alternative protocols and presented white bulbous structures. buds were noted, guaranteeing a larger for the manipulation of apices during the This behaviour of the buds of FHIA-20 number of future explants at the multipli- initiation phase, and of axillary buds during the multiplication phase resulted cation phase, significantly different from during the multiplication phase, appear in a reduction in the multiplication coef- that obtained with the other treatments necessary. ficients (buds obtained/buds initiated). (Table 1). The technique of decapitating the api- Materials and methods cal dome proved necessary to induce the For the study, young plants with a mean Effects of doses formation of new buds from the axillary height of 25.6 cm, grown in the green- of 6-benzylaminopurine and type buds, normally inhibited by apical domi- house, were selected (Figure 1). The of manipulation on the growth nance (Ma and Shi (1972) and Swami et process of introduction into the labora- of buds during the multiplication phase al. (1983) (cited by Afza et al. (1996)). tory, including manipulation of the With the aim of resolving problems Pérez et al. (1998) emphasized the plants, disinfection of the corms, culture encountered during the growth of buds in importance of increasing the multiplica- media for initiation and multiplication, the multiplication phase, the effect of a tion coefficient during in vitro propaga- together with the culture conditions, dose of 2 mg.L-1 of 6-benzylaminopurine tion of plantains, because each increase were as described by Orellana (1994). (BAP) was studied, the rate of 4 mg.L-1, of this indicator by one unit corresponds The plants were cultivated in chambers proposed by Orellana (1994) serving as to a cost reduction of about 10%. under natural light conditions at a tem- control. Each dose was combined with In this study, mortality occurred only perature of 27 ± 2ºC. In every case, the two manipulation protocols. in apices cut and grown in liquid base of the apex or bud was placed down- Protocol 1. The buds were separated, medium, possibly because the cutting wards on the culture medium. decapitated 0.5 cm from the top and cut procedure produced pieces which were in two. too small to be grown in a liquid medium Influence of the size of the apex Protocol 2. Buds which had not (Table 1). According to Orellana (1998), and the physical state of the culture reached 1 cm length were left in groups there are differences in tissue growth medium during the initiation phase of two or were not separated from the depending on the physical state of the This study was made in order to establish mother plant, and there was no decapita- culture medium: it proceeds differently the conditions for manipulation and tion. Buds of more than 1 cm were sepa- according to whether the medium is solid growth of the apex during the initiation rated, decapitated at this height and cut or liquid. phase. For this purpose, the following into two when the pseudostem was com- The incidence of infection during this treatments were studied (Figure 2): posed of more than three leaves. phase did not differ significantly with the 1. 0.5 cm2 apex cultured in liquid medium Four treatments were thus obtained: treatment. However, various authors (control) 1. Multiplication medium with 4 mg.L-1 mention the influence of the size of the 2. 0.5 cm2 apex cultured in a semi-solid BAP combined with protocol 1 (control). initial explant on the incidence of infec- medium 2. Multiplication medium with 4 mg.L-1 tion and have noticed that the smaller 3. 1.0 cm2 apex cut in halves and cultured BAP combined with protocol 2. the explant, and the closer it is located to in liquid medium 3. Multiplication medium with 2 mg.L-1 the apical meristem, the more the popu- 4. 1.0 cm2 apex cut in halves and cultured BAP combined with protocol 1. lations of micro-organisms fall (Garcia in semi-solid medium 4. Multiplication medium with 4 mg.L-1 and Noa 1998, Leifert et al. 1994). At the end of 20 days’ culture, the fol- BAP combined with protocol 2. lowing variables were recorded: The variables recorded were the num- Effect of doses of 6-BAP •Percentage regeneration of apices ber of buds per initial explant and the and type of manipulation on the growth •Percentage infection of apices percentage of buds growing as rosettes. of buds in the multiplication phase •Percentage mortality of apices The counts were made after three subcul- By reducing the dose of cytokinin in the • Number of buds per apex tures carried out every 21 days (culture multiplication medium, the differentia- There were 20 replicates and the stain growth chambers with natural light tion of buds into plantlets could be stimu- tistical method used to compare percent- and a temperature of 27 ± 2ºC). lated whilst little by little the growth of

36 INFOMUSA — Vol 11, N° 1 Figure 1. Young plants of the clone FHIA-20 Figure 2. Treatments studied during the initiation phase in vitro. used for introduction into the laboratory.

rosettes disappeared (inasmuch as the Table 1. Behaviour of apices during the initiation phase after 20 days’ in vitro three subcultures were made with the culture. 2 mg.L-1 dose of BAP). The manipulations Treatments Regeneration Number Infection Mortality or cuts made during this phase, combined of apices (%) of buds per apex (%) (%) with the reduction in the dose of 1 (control) 40.0 b 0.25 c 15 a 0.0 b cytokinin encouraged the biological 2 40.0 b 1.10 b 20 a 0.0 b response of the plants and caused the 3 35.0 b 0.00 c 15 a 40.0 a appearance of a larger number of buds 4 85.0 a 2.42 a 15 a 0.0 b per initial explant when using protocol 2 *identical letters in the same column indicate that the results do not differ significantly at P < 0.05%. (Table 2). These buds, once transferred onto rooting media, had no difficulty in Treatments continuing their growth and reached the 1. 0.5 cm2 apex cultured in liquid medium (control). height, girth, and number of leaves 2. 0.5 cm2 apex cultured in semi-solid medium. needed to allow their transfer to the 3. 1.0 cm2 apex cut in two and cultured in liquid medium. acclimatization phase. 4. 1.0 cm2 apex cut in two and cultured in semi-solid medium. On the other hand, for the treatments receiving the 4 mg.L-1 dose of BAP, we continued to note the appearance of rosette growth for both protocols, Table 2. Growth behaviour of buds during the multiplication phase. although the highest percentage of this type of growth occurred with the normal Treatments Number of buds per explant Buds with rosetted growth (%) protocol (i.e. separation, decapitation at 1 (control) 1.24 b 44.0 a 0.5 cm and cutting buds into two). It 2 2.10 b 24.0 b seems that this treatment accentuates 3 2.20 b 6.00 c 4 4.70 a 2.00 c the presence of this particular growth in *identical letters in the same column indicate that the results do not differ significantly at P < 0.05%. the clone FHIA-20: in fact, the presence of rosettes tended to diminish when Treatments -1 protocol 2 was used (Table 2). 1. Multiplication medium with 4.0 mg.L of BAP + protocol 1 (control). -1 The development of cultures in vitro 2. Multiplication medium with 4.0 mg.L of BAP + protocol 2. 3. Multiplication medium with 2.0 mg.L-1 of BAP + protocol 1. requires an adequate ratio between -1 auxins and cytokinins in the culture 4. Multiplication medium with 2.0 mg.L of BAP + protocol 2. medium. One must also consider the endogenous concentrations of these hormones in the different types of explants or species (Jiménez 1998). Certain species are cultivated without the addition of any 85% of them regenerate plants by the end References external regulator, probably because there of 20 days’ culture. During the multiplica- Afza R., M. Van Duren, R. Morpurgo & F.J. Novak. is enough endogenous hormone present. tion phase, the cytokinin dose should be 1996. Banana tissue culture and its prospective reduced to 2 mg.L-1 in the culture medium use in the developing countries. Pp. 58-70 in Plant Conclusions and the explants separated into well- Tissue Culture (A.S. Islam, ed.). Oxford & IBH The results obtained from this work make defined buds which are not less than 1 cm Publishing Co. Pvt. Ltd., New Delhi/Calcutta. it possible to propagate the hybrid tall (those which are should be kept as García L. & J.C. Noa. 1998. Obtención de plantas FHIA-20 in vitro with a distinct improve- pairs or should remain on the mother libres de patógenos. Pp. 135-148 in Propagación y ment in the efficiency of the process of plant). Buds of 1.5-3.0 cm having more mejora genética de plantas por biotecnología propagation by organogenesis by virtue of than three leaves can be decapitated at a (J.N. Pérez Ponce, ed.). Instituto de Biotecnología an increase in the number of buds. height of 1.0 cm and cut in half. This way de las Plantas. Universidad Central de las Villas, During the initiation phase it is necessary rosette growth is reduced by 2% and on Santa Clara, Cuba. to culture apices of 1 cm2, cut into two, average 4.7 buds per explant are obtained Jimenéz E. 1998. Generalidades del cultivo in vitro. on a semi-solid medium. In this way, in the multiplication phase. ■ Pp. 13-22 in Propagación y mejora genética de

INFOMUSA — Vol 11, N° 1 37 plantas por biotecnología (J.N. Pérez Ponce, ed.). Instituto de Biotecnología de las Plantas. culture and field-grown plants: reasons for conta- Capitulo 8. Instituto de Biotecnología de las Universidad Central de las Villas, Santa Clara, mination problems in vitro. Critical Reviews in Plantas. Universidad Central de las Villas, Santa Cuba. Plant Sciences 13(2): 1 39-183. Clara, Cuba. Pérez Ponce J.N., E. Jiménez & D. Agramonte. 1998. Vasil I. 1994. Automation in plant propagation. Plant Orellana P. 1994. Tecnología para la propagación Aumento de la eficiencia en la micropropa- Cell, Tissue and Organ Culture 39(2): 105-109. in vitro de clones de Musa spp. Tesis para optar gación. Pp. 179-190 in Propagación y mejora por el grado de Doctor en Ciencias Agrícolas. genética de plantas por biotecnología (J.N. Pérez Instituto de Biotecnología de las Plantas, Ponce, ed.). Instituto de Biotecnología de las The authors work at the Instituto de Biotecnología Universidad Central de las Villas. 104pp. Plantas. Universidad Central de las Villas, Santa de Plantas, Universidad Central “Marta Abreu” de Las Orellana P. 1998. Propagación vía organogénesis. Pp. Clara, Cuba. Villas, Carretera a Camajuaní Km. 51/2, CP 54830, 151-176 in Propagación y mejora genética de plan- Leifert C., C. Morris & W.M. Waites. 1994. Ecology of Santa Clara, Villa Clara, Cuba. tas por biotecnología (J.N. Pérez Ponce, ed.). microbial saprophytes and pathogens in tissue E-mail: [email protected]

Tissue culture Cell suspensions Multiplication rate and regeneration potential of somatic embryos from a cell suspension of banana (Musa AAA cv. “Grande naine”)

S.L. Lerma, P. Acuña, A.S. Riveros important plant material (Escalant et al. transferred into M2 cell suspension and J.A. Sandoval 1994, Gómez-Kosky et al. 2000), or of medium [MS salts, 100 mg/L glutamine their use in genetic improvement pro- and malt extract, 1 mg/L 2,4-D, 45 g/L sac- grammes by the induction of mutations, charose, pH 5.3] until its establishment. he production of bananas and plan- the study of selections in vitro (by means This technique for somatic embryogene- tains is widely distributed in tropi- of fungal toxins or plant extracts) and sis was originally developed by Escalant Tcal and sub-tropical regions. The genetic transformation by particle bom- et al. (1994) and it is currently applied area cultivated, estimated to be around bardment. Despite all the research car- in the biotechnology laboratory of 10 million hectares, gives an annual yield ried out internationally in various labora- CORBANA on this same clone (Acuña and in the order of 88 million tonnes. This tories, it is still difficult to maintain cell Sandoval 2000). crop, whose fruit forms part of the diet of suspensions effectively. The setting up of From this initial suspension, new cul- more than 400 million people, ranks banana cell cultures free from bacterial tures were started during the phase of fourth in the world in the category of sta- contamination, changes due to oxidation maintenance in M2 medium onto a ple food products, after rice, wheat and or possible fungal attacks requires a lot medium made up of 35 ml of fresh M2 milk (FAO 1999). of time, and their maintenance therefore medium and 13 ml of the previous M2 In view of the interest in banana grow- proves to be difficult (Schoofs et al. medium (in which the suspension had ing, considerable research effort has 1999). The aim of the work presented been maintained during the preceding been devoted to the improvement and here was to determine, by using sources cycle), a mixture into which were intro- control of its mass propagation by means of carbon and growth regulators, the opti- duced 2 ml of cells made up to a total of biotechnological techniques such as mum experimental conditions for the set- volume of 50 ml per erlenmeyer flask. somatic embryogenesis, for which three up and multiplication of a cell suspension These suspensions were subjected to protocols have been described using on the one hand, and for the regeneration four treatments: T0 = 45 g saccharose; vegetative tissue such as fragments of the of the somatic embryos on the other. T1 = 45 g saccharose + 100 mg/L corm and leaf bases (Novak et al. 1989, myoinositol; T2 = 30 g saccharose Ganapathi et al. 1999), cultures of prolif- Materials and methods + 100 mg/L myoinositol; T3 = 15 g sac- erating meristems (Dhed’a et al. 1991, charose + 100 mg/L myoinositol. There Dhed’a 1992, Schoofs 1997, Schoofs et al. Maintenance of cell suspensions were 10 replicates (Figure 1). 1998), and immature male or female and homogenization of cultures Four subcultures were made which flowers (Escalant et al. 1994, Grapin The plant material used to initiate the were each incubated for 14 days as pro- et al. 1996). cell suspensions consisted of immature posed by Escalant et al. (1994). The The use of cell suspensions in somatic male flowers of Musa AAA cv “Grande number of cells and the percentage via- embryogenesis and the discovery of the naine” which had been placed on M1 bility of the suspensions were recorded factors involved in the metabolic synchro- induction medium [Murashige & Skoog on the 1st, 7th, and 14th days of the cul- nization of cell suspensions and its tim- (1962) salts - MS, 1 mg/L biotin, ANA and ture using a haemocytometer. There ing constitute two fundamental aspects AIA, 4 mg/L 2,4-D, 6 g/L agarose, 30 g/L were three replicates and 5 counts for either of the procedures for applying saccharose, pH 5.71] proposed by Grapin each, making a total of 15 readings per methods of temporary immersion for et al. (1998) for forming calluses. The fri- treatment. Also, every 15 days, the mass micropropagation of economically able embryogenic tissue obtained was increase in cell volume was measured by

38 INFOMUSA — Vol 11, N° 1 the sedimentation method (SCV) proposed by Schoof (1997) and the packed cell volume (PCV) as used by Reinert M2 Initial suspension and Yeoman (1982). Four extra replicates were also made for monitoring the pH (2 in the inoculated medium and 2 in the non-inoculated medium), the mea- surements being made at the beginning No. 14 T0 T1 T2 T3 and end of each subculture. In order to evaluate the effect of the pH pH pH pH growth regulators on the quality of the a) cell suspension in the M2 medium, the S 45 gr. S 45 gr. + S 30 gr. + S 15 gr. + treatment was selected which showed the 100M mg/L 100M mg/L 100M mg/L 100M mg/L highest multiplication rate and cell viability during the first four subcultures of No. 14 the maintenance phase. For this study, we added to the selected M2 medium: A1 A1 A2 A3 = 0.5 mg/L 2,4-D, A2 = 1 mg/L 2,4-D and A3 = 2 mg/L 2,4-D. The material was han- pH pH pH dled the same way as the treatments b) involving different saccharose concentra- 2,4-D + 2,4-D + 2,4-D + tions. For evaluation, the same parame- 0,5 mg/L 1,0 mg/L 02,0 mg/L ters were used as in the maintenance phase of the cell suspensions (mentioned above). The cell morphology was also c) noted, as clusters or solid masses, and photographs were taken using both opti- Figure 1. General scheme of the protocol followed to study a cell suspension of banana (cultivar cal and electron microscopes. “Grande naine”). a) Experiment 1: M2 cell suspension medium; S = saccharose; M = myoinositol; T = treatment; No. = replications. b) Experiment 2: Different concentrations of 2,4-D. Regeneration of somatic embryos c) Evaluation of embryo formation. The viability of the process was evaluated by observing the embryos obtained on the culture medium of Schenk and Results and discussion on the treatment (P = 0.0040). This dif- Hildebrandt (1972), called modified M3 ference in behaviour of distinct cell lines [10 mg/L biotin, 100 mg/L of glutamine Effect of different saccharose of a single clone may be an intrinsic char- and malt extract, 230 mg/L proline, or saccharose + myoinositol acteristic of the material (Schoofs et al. 1 mg/L ANA, zeatin and 2-IP, 10 g/L lac- concentrations on the maintenance 1999), which would indicate the need to tose, 45 g/L saccharose and a pH of 5.3]. of cell suspensions and redouble efforts towards improving these The M3 medium was put into Petri dishes the homogenization of the cultures methods. and sterile filter papers were placed on The results for the increase in numbers Treatment T3 (15 g saccharose + the surface on which were inoculated of cells, presented in Figure 2, indicate myoinositol) was eliminated because it aliquots of 1 ml of the cells of treatments that the dose of 30 g of saccharose pro- showed progressive diminution of 5.18, corresponding to the different growth vides enough carbon for the suspension, 4.20 and 2.06 ml in subcultures 1,2 and 3 regulator concentrations. The type of as its behaviour differed little from that respectively. This low success rate of cell material regenerated was evaluated by of the suspension maintained with 45 g of proliferation may be attributable to the making three evaluations per Petri dish saccharose. In general, the addition of low availability of sugars in the medium from the zones where the distribution of myoinositol (T1-T2) did not alter the compared with the demand of the cells in the suspension was most homogeneous. behaviour of the cells and a tendency was phase G1 of the cell cycle or to the All the cultures were maintained under noted to stabilization in subculture 4 osmotic shock due to the medium. conditions of controlled temperature (relation of T1 and T2 with T10). Whichever it is, it is well known that sac- (27ºC), relative humidity (80%) and pho- There were no significant differences charose as a source of carbon is a stabi- toperiod (12 hours). between the percentages of viability of lizer of culture media (Takeuchi and the treatments with and without Komamine 1982, Vardi et al. 1982, Smith Statistical analysis myoinositol (T1 and T0 respectively). No et al. 1984). The results obtained for the phase of differences were noted between the The differences in cell volume found maintenance of cell suspensions and observations at different times (7 or between treatments T0, T1 and T2 homogenization of cultures for variation 14 days) nor any interactions between (P = 0.02602) were much reduced in sub- in pH, cell volume, number of cells and the subcultures and time of observation cultures 1 and 2 (Figure 3a and b) but percentage viability, were analyzed by a (P = 0.1574). were accentuated in subcultures 3 and 4 linear modelling scheme and subjected On the other hand, as for the behaviour (Figure 3c and d) for which the differ- to analysis of variance using the SAS pro- of this same percentage viability for ence between treatments T0 and T1 can gramme (1990). Results showing hetero- treatments with myoinositol associated be attributed to the action of myoinositol. geneity of variance were adjusted to with different saccharose concentrations Subcultures 2, 3 and 4 of T1 (with homogeneity by using a square root trans- (T1 and T2), it was found that the differ- myoinositol) produced a mean cell vol- formation. ences for the four subcultures depended ume 0.67 ml greater (P = 0.0188) than

INFOMUSA — Vol 11, N° 1 39 Subculture 1 Subculture 2 36 36 32 32 28 28 /ml) /ml)

4 7 days 4 24 24 7 days 20 14 days 20 14 days 16 16 12 12 8 8

Number of cells (10 6 Number of cells (10 6 0 0 T0 T1 T2 T0 T1 T2 Treatments Treatments

Subculture 3 Subculture 4 36 36 32 32 28 28 /ml)

4 7 days

24 /ml) 4 24 7 days 20 14 days 20 14 days 16 16 12 12 8 8 Number of cells (10

6 Number of cells (10 6 0 0 T0 T1 T2 T0 T1 T2 Treatments Treatments

Figure 2. Number of cells obtained with three treatments of multiplication media for banana cell suspensions (Musa AAA cv. “Grande naine”). Treatment mean for four subcultures n = 3. T0 = 45 saccharose; T1 = 45 g saccharose + myoinositol; T2 = 30g saccharose + myoinositol. Bars represent standard errors.

that reached in the T0 subculture homo- the pH varied with time, treatment and is a useful parameter for carrying out logues. These results agree with those their interaction (P = 0.0001): similar studies of the cell cycle or cell metabo- from other research on banana and other behaviour was observed by Skirvin et al. lism and other phenomena connected crops: for example Cronauer and (1986). These same authors suggested with synchronized cell populations. Krikorian (1983) and Aftab et al. (1999) that acidification of the medium could The final results from subculture 4 confirm the stimulatory action of be due to ionic exchange between the measured by the PCV method show that myoinositol on mitosis and morphogene- cell and the culture medium, leading to all the treatments have progressively sis of plant cells. an optimum pH for the normal function- increased the cell volume without large The difference between the four sub- ing of the cell wall. fluctuations during the 14 days of incuba- cultures was on average 0.23 ml in favour tion and that this volume doubled on the of treatment 1. The treatment which Response of the suspension to different sixth day, when the cells begin a phase of responded best was that containing 45 g concentrations of 2,4-D active division (Figure 5). These results saccharose and 100 mg/L myoinositol. It Analysis of the results obtained for the agree with those obtained by Bieberach was also noted that the cell volume of variables “number of cells” and “percent- (1995) for various Musa clones. subculture 1 was 5.95 ml and that of sub- age viability” on media containing differ- As to the use of 2,4-D and appropriate culture 4 was 7.59 ml, representing a ent concentrations of 2,4-D did not show doses, the results given here supplement mean increase of 0.65 ml for each. There any marked differences in their behav- the information about the action of this is a positive correlation (Figure 3e) iour. Treatments A1, A2 and A3 of the four growth regulator on the embryogenic between the number of subcultures and subcultures had mean numbers of cells of process and the doses required by differ- the cell volume, since this latter 7.9, 6.0 and 7.0 and percentage viabilities ent plant species. Lazzeri et al. (1987) increases with the number of subcul- of 59, 62 and 59 respectively. emphasize the importance of auxins in tures, finally to stabilize at the fourth When one studies the cell volume the regulation of the somatic embryogen- subculture. obtained with varying concentrations of esis of soyabean and show that there is After mixing, the 35 ml of fresh 2,4-D (Figure 4) it is seen that treatment better somatic embryo production when medium and the 13 ml of old medium had A1 (1 mg/L of 2,4-D) is that which main- 2,4-D is used alone than in combination a pH of 4.74. During the 14 days of cul- tains the best cell suspension with a with acetic a-naphthalene. ture, the non-inoculated media remained mean volume of 7.6 ml and a maximum of The morphology of cells in suspension between pH 4.1 and 4.2 and the inocu- 8.8 ml in subculture 3. The 2 mg/L dose of was observed by optical microscopy at lated media between 4.4 and 4.6 (unpub- 2,4-D was the best for standardizing the magnifications of 20x and 40x. The lished results). In the cell suspensions, cell volume of several subcultures, which preparations showed cell clusters and

40 INFOMUSA — Vol 11, N° 1 Subculture 1 a Subculture 2 b 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 Cells volume (ml) 1 Cells volume (ml) 1 0 0 T0 T1 T2 T0 T1 T2 Treatments Treatments

Subculture 3 c Subculture 4 d 10 9 10 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 Cells volume (ml) Cells volume (ml) 1 1 0 0 T0 T1 T2 T0 T1 T2 Treatments Treatments

10 Treatments T0, T1, T2 e 9 8 7 6 5 4 3 Cells volume (ml) 2 1 0 1234 Subculture

Figure 3. Cell volumes in the multiplication media of cell suspensions of banana (Musa AAA cv. “Grande naine”); a: means of treatments in subculture 1 (n = 10); b: means of treatments in subculture 2 (n = 9);c: means of treatments in subculture 3 (n = 9); d: means of treatments in subculture (n = 6); e: means of treatments in subcultures 0, 1, 2 (n = 23); T0 = 45 g saccharose; T1 = 45 g saccharose + myoinositol; T2 = 30 g saccharose + myoinositol; T3 = 15 g saccharose + myoinositol. Bars represent the standard errors. isolated cells (Figures 6a and 6b), which Treatments A1, A2 and A3 conforms with descriptions by Grapin 9,4 9,0 (1996) who reported that in suspensions 8,6 of ‘French Sombre’ clusters were seen 8,2 7,8 which could reach 70 to 80% of the vol- 7,4 ume of the suspension very similar to 7,0 6,6 what we observed in the course of this 6,2 work. The clusters are formed by pre- Cells volume (ml) 5,8 5,4 embryogenic cells (Figure 6c) possess- 5,0 ing partitions or cellular plates typical of 00,511,5 2 2,5 the last stage of mitosis and of cells Dosis of 2,4-D (mg/L) which are empty or in the course of dif- Sub 1 Sub 2 Sub 3 Sub 4 mean ferentiation. The isolated cells are round, with a Figure 4. Cell volumes in the multiplication media of banana (Musa AAA cv, “Grande naine”). dense cytoplasm and a well-defined Mean of treatments A1, A2 and A3 containing 2,4-D in subcultures 1, 2, 3 and 4. nucleus: one may regard them as proto- Bars represent the standard errors.

INFOMUSA — Vol 11, N° 1 41 a 12 b 12 11 11 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3

2 Cells volume (ml) 2 Cells volume (ml) 1 1 0 0 02468101214 02468101214 Time (days) Time (days) A1 = 2,4-D 0.5 mg/L A2 = 2,4-D 1.0 mg/L

12 11 c 10 9 8 7 6 5 4 3

Cells volume (ml) 2 1 0 02468101214 Time (days) A3 = 2,4-D 2.0 mg/L

Figure 5. Increase in cell volume (PCV) of a suspension of banana (Musa AAA cv, “Grande naine”) with different growth regulator concentrations. a, b, c: means of treatments (n = 10) in subculture 4. plasts, initial cells with a primary cell and “Gros Michel” and also by Sannasgala lacunae are non-viable cells in a suspen- wall that is characteristic of undifferenti- (1989) who described pre-embryos made sion because they have already formed ated cells and with an active cell cycle. up of protein bodies and starch. their secondary cell wall. These observations are shared both by The characters described above are a Under the scanning electron micro- Bieberach (1995) who noted the pres- factor indicating the embryogenic condi- scope, round cells were seen of 50-80µm ence of cells with identical morphologi- tion of the cell suspension (Williams and diameter, with rough walls with irregular cal characteristics in cell suspensions of Maheswaren 1986). Certain isolated cells ornamentations surrounded by a polysac- the cultivars “Dominico”, “Grande naine” with elongated cytoplasm containing charide mucus (Figures 7a and 7b).

Regeneration of somatic embryos Mixtures of cell samples treated with growth regulators were inoculated and maintained for 55 days on the semi-solid M3 medium to develop embryos. By the end of 22 days their growth began to be apparent, without traces of oxidation. The presence was detected of small clusters of 1 cm2 containing globular embryos in the shape of a heart or torpedo. The embryos were sorted with a view to later regeneration of the plants (Figure 8a). A total of 200 “torpedo-type” embryos were transferred into eight Petri dishes at the rate of 25 embryos per dish. At the end of 20 days there was 63% germination, and after 41 days the plants possessed normal morphological characteristics. Until then the percentage germination for somatic embryos of the genus Musa oscillated Figure 6. Photomicrograph of cells and clusters, between 45% and 80% according to the a: viable isolated cells, b: pre-embryogenic cells, genotype and culture medium (Bieberach c: clusters. N = nucleus, T = cell plates, C = clusters, CD = differentiating cells, 1995, Escalant et al. 1995, Côte et al. CI = individual cells, CP = pre-embryogenic cell. 1996, Schoof 1997, Grapin et al. 1998).

42 INFOMUSA — Vol 11, N° 1 Thesis submitted for the Magister Scientiae degree. CATIE, Turrialba, Costa Rica. 84pp. Cronauer-Mitra S. & A.D. Krikorian. 1983. Somatic embryos from cultured tissues of triploid plantains (Musa ABB) Plant Cell Reports (2): 289-291. Côte F., R. Domergue, S. Monmarson, J. Schwendiman, C. Teisson & J.V. Escalant. 1996. Embryogenic cell suspensions from the male flowers of Musa AAA cv. Grand Naine. Physiologia Plantarum 97: 285-290. Dhed’a D. 1992. Culture de suspensions cellulaires embryogéniques et régénération en plantules par embryogenèse somatique chez le bananier et le bananier plantain (Musa spp.) Thèse PhD, Figure 7. Scanning electron microscope photographs. a: viable cells, b: external surface of a cell. KuLeuven, Belgique. 171pp. Dhed’a D., F. Dumortier & B. Panis. 1991. Plant regeneration in cell suspension cultures of the cooking bananas cv. Bluggoe (Musa spp. ABB group). Fruits 46: 125-135. Escalant J.V., C. Teisson & F. Côte. 1994. Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.). In Vitro Plant Cell. and Dev. Biol. 30: 181-186. Escalant J.V., C. Bieberach, L.E. Pocasangre, L. del S. Espinoza, R.G. Kosky, J.L. Ortiz & C. Teisson. 1995. Regeneration through somatic embryogenesis from male flowers of banana and plantain: 1. Amplification by temporary immersion. (Abstract). in Simposio CIRAD/CATIE. Mejoramiento genético y desarrollo de los cultivos tropicales. Resúmenes. CATIE, Turrialba, Costa Rica. 54pp. FAO. 1999. FAO Quarterly Bulletin of Statistics 12(3-4). Figure 8. Formation of embryos of banana Musa AAA cv, “Grande naine” on M3 medium; Ganapathi T.R., N. Higgs, J. Van Eck, P. Balint-Kurti a: 55-day old embryo cluster. (*) indicates “torpedo-type” embryos. b: germination and growth of embryos on M3 medium. & G.D.May. 1999. Transformation and regeneration of the banana cultivar “Rasthali” (AAB). P. 34 in Abstracts of the International symposium on Figure 8b shows the potential regene- Acknowledgements the molecular and cellular biology of banana, ration of somatic embryos from cell sus- The authors are pleased to thank the 22-25/03/1999, Cornell University, Ithaca, NY, pensions. Escalant et al. (1994) have Biotechnology Unit of the National USA. Boyce Thompson Institute for Plant obtained the highest germination per- Banana Corporation (CORBANA) of Research, Inc., Ithaca. centages, achieved by using temporary Costa Rica for permitting the experimen- Gómez-Kosky R., T. Gilliard., L.A. Barranco & M. Reyes. immersion systems with other banana tal part of the work, and the University of 2000. Somatic embryogenesis in liquid media: cultivars. Costa Rica, where the photomicrographs maturation and enhancement of germination of shown in this article were taken. the hybrid cultivar FHIA-18 (AAAB). INFOMUSA Conclusions Note: Extract from the Biology thesis 9(l): 12-16. These experiments have enabled us to of Sandra Liliana Lerma defended Grapin A., J. Schwendiman & C. Teisson. 1996. standardize a protocol for obtaining before the Faculty of Sciences, Somatic embryogenesis in plantain banana. In embryos of Musa AAA cv. “Grande naine” University of Tolima, April 2001. Ibagué, Vitro Cell. Dev. Biol. Plant. 32: 66-71. from cell suspensions and by using Tolima, Colombia. ■ Grapin A., J-L. Ortiz, R. Domergue, J. Babeau, S. growth regulators. The initial cell suspen- Monmarson, J.V. Escalant, C. Teisson & F. Côte. sion was maintained with 45 g saccharose References 1998. Establishment of embryogenic callus and + 100 mg/L myoinositol. An initial pH of Acuña P. & J. Sandoval. 2000. Embriogénesis initiation and regeneration of embryogenic cell 4.74 and four subcultures of 14 days each somática en banano (cv. “Gran enano”) a partir de suspensions from female and male immature flow- guaranteed a sufficient cell volume and flores masculinas. Pp. 20-22 in Informe Anual. ers of Musa. INFOMUSA 7(1): 13-15. embryos with a good percentage viability. Dirección de Investigaciones, CORBANA, San Lazzeri P., D Hildebrand & G. Collins. 1987. Soybean The optimal dose of growth regulator for José‚ Costa Rica. somatic embryogenesis. Effects of hormones and the efficacy of the process is 1 mg/L of Aftab F. & J. Iqbal. 1999. Plant regeneration from culture manipulations. Plant Cell, Tissue and 2,4-D applied as an exogenous hormone. protoplasts derived from cell suspension of Organ Culture 10: 197-208. The morphological observations reveal adventive somatic embryos in sugarcane Murashige T. & F. Skoog. 1962. A revised medium for that the protocol has allowed the devel- (Saccharum spp. hybrid cv. CoL-54 and cv. CP- rapid growth and biossays with tobacco tissue cul- opment of viable cells which are easily 43/33). Plant Cell Tissue and Organ Culture tures. Physiol. Plant. 15: 473-497. transformed into embryos. The germina- 56(3): 155-162. Novak F.J., R. Afza, D.M. Van, D.M. Perea, B.V. tion of embryos has validated the entire Bieberach C. 1995. Embriogénesis somática y regen- Conger & X. Tang. 1989. Somatic embryogenesis method and the doses used. eración de plantas en cultivares de Musa spp. and plant regeneration in suspension cultures of

INFOMUSA — Vol 11, N° 1 43 dessert (AA and AAA) and cooking (ABB) Schoofs H., B. Panis, H. Strosse, A. Moyo, J. Lopez, N. in methodological requirements among cultivars bananas (Musa spp.) Bio/Technology 7: 154-159. Roux, J. Colezel & R. Swennen. 1999. Bottlenecks and species. Theor. Appl. Genet. 62: 171-176. Reinert J. & M.M. Yeoman. 1982. Plant cell and tis- in the generation and maintenance of mor- Williams E.G. & G. Maheswaran. 1986. Somatic sue culture. A laboratory manual. Springer Verlag, phogenic banana cell suspensions and plant embryogenesis: factors influencing coordinated Berlin-Heidelberg-New York. regeneration via somatic embryogenesis there- behaviour of cells as an embryonic group. Annals Sannasgala K. 1989. In vitro somatic embryogenesis from. INFOMUSA 8(2): 3-7. of Botany 57: 443-462. in Musa. PhD Thesis, KULeuven, Belgium. 189pp. Skirvin R.M., M.C. Chu, M.L. Mann, H. Young, J.G. SAS Institute. 1990. SAS/STAT User’s Guide. Version Sullivan & T.W. Fermanian. 1986. Stability of tis- 6.4. SAS Institute, Inc., Cary, NC. sue culture medium pH as a function of autoclav- Schenk R.U. & A.C. Hildebrandt. 1972. Medium and ing, time, and cultured plant material. Plant Cell techniques for induction and growth of mono- Reports 5: 292-294. Sandra Liliana Lerma works in the Laboratorio de cotyledonous and dicotyledonous plant cell cul- Smith M.A.L., J.P. Palta & B.H. Mc Cown. 1984. The Protección de Plantas, Departamento de Biología, tures. Can. J. Bot. 50: 199-204. measurement of isotonicity and maintenance of Universidad del Tolima, Ibagué (Tolima), Colombia, Schoofs H. 1997. The origin of embryogenic cells in osmotic balance in plant protoplast manipula- e-mail: [email protected]; Pablo Acuña is Assesor Musa. PhD Thesis, KULeuven, Belgium. 258pp. tions. Plant Sci. Let. 33: 249-258. in Plant Biotechnology, Guápiles, Costa Rica, e-mail: + annexes. Takeuchi Y. & A. Komamine. 1982. Effects of culture [email protected]; Alba Stella Riveros is Schoofs H., B. Panis & R. Swennen. 1998. conditions on cell division and composition of Associate Researcher in the framework of the Convenio Universidad del Tolima-CATIE, Unidad de Competence of scalps for somatic embryogenesis regenerated cell walls in Vinca rosea protoplasts. Fitoprotección, CATIE, Turrialba, Costa Rica, e-mail: in Musa. Acta Horticulturae 490: 475-483. Plant Cell Physiol. 23: 249-255. [email protected] and Jorge Arturo Sandoval is rst Proceedings of the 1 International symposium Vardi A., P. Spiegel-Roy & E. Galum. 1982. Plant Subdirector of Research at CORBANA, Guápiles, Costa on banana in the subtropics. ISHS. regeneration from Citrus protoplasts: variability Rica. e-mail: [email protected].

Improved varieties Partnerships for distribution in Nicaragua

Introduction and multiplication of improved bananas and plantains in Nicaragua and distribution to farmers

K. Dens, M. Vargas, G. Matton, (Musa cv. AAA) bananas are found in plantains caused by poor cultivation prac- S. Coessens, I. Van den Houwe some regions of Nicaragua. tices, drought and pests and diseases. and R. Swennen Plantain (Musa cv. AAB) is most pre- Diseases and pests are the main prob- ferred because it is an attractive cash lems; black Sigatoka (Mourichon et al. crop. The most common landraces are 1997) and plant material contaminated Banana and plantain in Nicaragua False horn plantain with an average of by weevils (Gold and Messiaen 2000) are Unlike most other Central-American only 25 fingers. The price of plantain on the major constraints affecting the countries, the production of banana and the local market is much higher than the small-scale plantain producer. Another plantain in Nicaragua is low (Table 1). price of the other bananas (Gros Michel, important problem, especially in the The major production zones of bananas Bluggoe, Silk) because of its much larger Leon-Chinandega region, is the uneven and plantains are located in the coastal finger size and longer green life. distribution of annual rainfall. Without area near the Pacific Ocean. In the Cavendish only enters the local market as irrigation banana and plantain yields are Chinandega region (North-West) an esti- after being rejected on the export planta- reduced due to the long dry season. mated area of 2000 ha Cavendish tions. Its price is even lower than that of (Musa cv. AAA) is grown for export, while Gros Michel. Over the last five years Creating multipliying effects in the Rivas region (South of Managua) prices of plantain have continued to The objective of the intervention is to 13 000 ha plantain are cultivated for local increase, which reflects the high demand contribute to food security and food consumption. For many small- and and insufficient supply of bananas and quality of resource-poor farmers by giv- medium-scale farmers in Rivas, plantain is the most important crop. Gros Michel (Musa cv. AAA), Bluggoe (Musa cv. ABB) Table 1. Production, export and consumption data of banana and plantain in five and Silk (Musa cv. AAB) bananas are cul- Central American countries. tivated all over the country, mainly by Population in 1999 Production in 2000 Export in 1999 Consumption in 1999 small-scale farmers in backyards. In the (millions) (metric tonnes) (metric tonnes) (kg/cap/year) higher regions of Central Nicaragua, with Guatemala 10.8 802 545 576 900 4.5 altitudes up to 1300 masl, bananas are Honduras 6.1 702 578 155 200 63.9 grown in combination with coffee or Nicaragua 4.8 13 636 37 846 14.5 cacao. Bananas and plantains are also Costa Rica 3.8 2 790 000 2 557 000 29.5 important for the people at the Atlantic Panama 2.8 918 382 596 900 43.7 Coast. Pelipita (Musa cv. ABB) and Red Source: FAO on INIBAP’s Web site (http://www.inibap.org/network/statistics_eng.htm).

44 INFOMUSA — Vol 11, N° 1 were built with about 20 national and international organizations operating in Nicaragua (Table 3), to accelerate the distribution of improved plants and technologies, and to obtain a maximum feedback from the farmers.

Achievements The project started in mid-1996. Rooted plantlets were sent by KULeuven for weaning in the small nursery of the UNAN Figure 1. Operational area of the project farm in Leon, located a few kilometres showing the location of demonstration fields in from Leon City centre. These plants were the Leon-Chinandega region. ● represents 10 farmers’ fields; used for the first test plots at the ● represents 25 backyards. University farm. The tissue culture laboratory of UNAN was built in 1997. Tissue culture techniques were transferred from KULeuven to UNAN that produced plantlets to extend the test fields at the University farm. In cooperation with the Centro de Enseñanza Técnica Agropecuaria (CETA), workshops were organized in six communities in Chinandega. In 1998, five extension brochures in cartoon style were developed for distribution to participating farmers (Figure 2). At the test farm of UNAN, a field collection was established containing both the introduced and the locally grown varieties (40 in total), and 2 plots of 36 plants of each variety were evaluated (Table 2). In 1999, two trained Nicaraguan technicians of the tissue culture laboratory produced 6500 plants. One hundred and forty new test plots were planted in the northwestern region of Nicaragua, mostly in Chinandega because of the cooperation with CETA and the larger agriculture activity of that region. In 2000, 20 000 plants have been distributed to 370 new farmers, comprising farmers of the Leon region as well (Figure 1). Ten thousands plantlets were ing support to banana and plantain cul- Investigación Agrícola (FHIA) and the imported from KULeuven to accelerate tivation. Food insecurity is very high in International Institute of Tropical the distribution of the new varieties. Nicaragua and the number of under- Agriculture (IITA) via the INIBAP Transit Oxfam-Belgium contracted the UNAN to nourished people increased from Centre (Diekmann and Putter 1996). The distribute 25 000 plants of superior vari- 1.2 million in 1991 to 1.4 million in 1998 company Bananic supported this inter- eties to nearly 1000 families that were (FAO 2001). The project focuses on the vention by covering the operational costs relocated after the hurricane Mitch in Leon-Chinandega region (Figure 1), of the laboratory and field activities. October 1998 and urgently needed new where the poorest farmers live and Tissue culture facilities were set up at plant material. Therefore the shade where bananas and plantains could be UNAN to produce high-value banana and house was extended to 700 m2. part of more diversified agriculture sys- plantain varieties. Selected varieties are In 2001, a few test fields were also tems, now that the cotton monoculture evaluated at UNAN’s test farm before planted in the Rivas, central Nicaragua has disappeared. distribution to small-scale farmers and Atlantic coast regions, where a selec- In 1996, the Universidad Nacional (Table 2). They are sorted in four main tion of farmers received in vitro banana Autónoma de Nicaragua (UNAN), based categories by local farmers according to and plantain plants. at Leon, and the Flemish Office for their comparison with Bluggoe, the The number of plants produced and Development Aid and Technical locally known False horn plantain, Gros distributed by UNAN’s tissue culture lab- Assistance (VVOB) started their inter- Michel and Silk. At harvest, palatability oratory increased from 2000 in 1998 to vention with technical assistance from tests are conducted with the farmers. 15 000 in 2001. The number of farmers the Katholieke Universiteit Leuven Extension personnel of the project participating also increased considerably (KULeuven). Improved germplasm came teach relevant cultivation and field mul- – from 40 at the start of the project in from the Fundación Hondureña de tiplication techniques. Partnerships 1998, to a total of 820 having received

INFOMUSA — Vol 11, N° 1 45 Table 2. Harvest characteristics of 23 varieties obtained from test fields during their improved varieties and participated in first and second cycles (C1-2) and third and fourth cycles (C3-4). the project in 2001. During 2001, the Varieties are grouped according to consumers’ preference. planting material was sold to cooperating ITC Plant height Bunch weight No. No. of institutes who distributed the plants in Number Genome (cm) (kg) of hands fingers their own development programmes. Name C1-2 C3-4 C1-2 C3-4 C3-4 C3-4 A total of 2757 farmers were trained in Cooking bananas different workshops and 1500 brochures Cuadrado1 (Bluggoe) ABB 310 356 19.5 20.5 6.5 102 dealing with field selection and prepara- FHIA-03 0506 AABB 305 381 29.3 42.1 13.2 204 tion and plant material were distributed. Pelipita 0396 ABB 420 392 22.9 23.8 10.0 152 Workshops for farmers were organized in Cardaba 0394 ABB 344 - 11.3 - *7.1 *90 collaboration with local NGOs, govern- Saba 1138 ABB 375 - 25.2 - *8.8 *131 mental and international organizations, Plantains which drastically increased the contact Cuerno1 (False horn) AAB 283 400 9.1 11.7 7.4 39 with farmers (Table 3). The project also TMPx 1621 1205 AAAB - 352 - 15.8 6.0 88 participated in the organization of TMPx 4479 (PITA 17) 1293 AAAB 325 361 12.7 14.8 6.3 89 regional and national workshops for TMPx 7002 1272 AAAB - 325 - 14.6 6.0 80 extension workers. Six new brochures TMPx 7152 (PITA 14) 1294 AAAB 299 350 16.8 13.5 6.2 78 were developed about diseases and pest TMBx 5295 (BITA 2) 1297 AABB - 396 - 16.8 10.6 101 control. A catalogue of the new acces- Dessert bananas sions following the format of Musalogue Patriota1 (Gros Michel) AAA 286 355 20.5 22.1 10.0 161 (Daniells et al. 2001) was made available. FHIA-01 0504 AAAB 254 342 26.4 30.2 10.5 162 Close contact is being maintained with FHIA-02 0505 AAAB 238 300 15.9 18.2 10.0 143 the farmers who are growing the new FHIA-17 1264 AAAA 334 - 37.5 - *12.5 *213 varieties (Figure 3) to assess their reac- FHIA-23 1265 AAAA 339 - 20.1 - *10.3 *159 tion and improve the efficiency of the Dessert bananas intervention. Interviews are conducted Rosa1 (Silk) AAB 332 358 17.0 18.6 8.5 151 to determine the acceptance rate of the Pisang ceylan 0650 AAB - 382 - 24.5 14.4 193 new varieties and to identify the under- Yangambi km5 1123 AAA 259 339 15.4 19.5 9.9 171 lying reasons, e.g. appearance, taste, cul- TMBx 1378 (BITA 3) 1296 ABBB 382 418 20.5 22.0 10.7 151 tivation as a cash crop and/or food crop, Pisang mas 0653 AA 329 361 6.6 9.5 9.8 147 etc. (Table 4). The most popular variety AA cv. Rose 0712 AA 265 289 6.9 11.1 12.3 199 so far is FHIA-03, because of its drought Pisang lidi 0395 AA 267 306 5.0 8.6 7.4 125 resistance and large bunches that are 1 local varieties; * data of cycle 1-2; - data not available. comparable to the local Bluggoe cooking banana. Tasting sessions are organized Table 3. National and international partners involved in the project. on a regular basis and the new varieties Name Description are prepared according to prevailing Nicaraguan customs i.e. fried, green and Project coordinating and executing institutes ripe plantains, plantain chips, cooked INIBAP International Network for the improvement of Banana and Plantain KULeuven Katholieke Universiteit Leuven green and ripe plantain, and banana as a UNAN Universidad Nacional Autónoma de Nicaragua dessert. People are asked to compare the new fruits with the local fruits (False Organizations involved in plant distribution horn plantain, Bluggoe or Silk). First Nicaraguan NGOs ALISTAR Foundation for community development results confirm the acceptability of most ATC Asociación de Trabajadores del Campo varieties but also show that palatability BLOQUE Evangelistic Association for Education of Farmers tests are absolutely necessary since CIPRES Centro de Investigación y Promoción para el Desarrollo Rural y Social visual aspects can determine consumers SGJRH Association of Garmendia Jiron with Limited Responsibility (Table 5). UNAG Unión Nacional de Agricultores y Ganaderos UNAPA Unión Nacional Agropecuaria de Productores Asociados Plans for the future Xochilt Acalt Women Association of Malpaisillo Governmental organizations Currently the laboratory has the capacity CETA Centro de Enseñanza Técnica y Agropecuaria to produce 50 000 in vitro plants per INTA Instituto Nicaragüense de Tecnología Agropecuaria annum. Plans are being made to further MAG-FOR Ministerio de Agricultura, Ganaderia y Forestales upscale the production capacity to International organizations ensure the sustainability of the tissue CARE North-American NGO culture laboratory by selling the plant CATIE Centro Agronómico Tropical de Investigación y Enseñanza material. Small-scale farmers will CLUSA (USAID) Cooperative League of USA receive plant material at subsidized EU European Union Project León-Chinandega FAO Food and Agriculture Organization of the United Nations prices while commercial growers will OXFAM-Solidarity Oxford Committee for Famine Relief – Belgium have to pay a higher price. SI Solidaridad Internacional – Spain The best accepted banana varieties will Private companies be produced in large amounts as well as BANANIC Corporación Bananera Nicaragüense other food crops for which a demand SETAGRO Servicios Técnicos Agropecuarios de Occidente exists in Nicaragua.

46 INFOMUSA — Vol 11, N° 1 Figure 2. Extension brochures distributed Distribution and extension work will be to farmers. increasingly coordinated by local organizations and NGOs. To facilitate this, the UNAN/VVOB staff participated in 2001 in the foundation of a national Musa network, MUSANIC. A baseline study about the socioeco- nomic situation of the collaborating farmers has been carried out to be able to measure the project-impact within a few years.

References Daniells J., C. Jenny, D. Karamura & K. Tomekpe. 2001. Musalogue: a catalogue of Musa germplasm. Diversity in the genus Musa. INIBAP, Montpellier, France. Diekmann M. & C.A.J. Putter.1996. FAO/IPGRI Technical guidelines for the safe movement of germplasm. No. 15. Musa (2nd edition). Food and Agriculture Organization of the United Nations, Rome/International Plant Genetic Resources Institute, Rome. FAO. 2001. The state of food insecurity in the world 2001. Food and Agriculture Organization of the United Nations, Rome, Italy. (Available on-line at http://www.fao.org/SOF/sofi/index_en.htm). Gold C. & S. Messiaen. 2000. The banana weevil Cosmopolites sordidus. Musa Pest Fact Sheet No. 4. INIBAP, Montpellier, France. Mourichon X., J. Carlier & E. Fouré. 1997. Sigatoka leaf spot disease: black leaf streak disease (black Sigatoka) and Sigatoka disease (yellow Sigatoka). Musa Disease Fact Sheet No. 8. INIBAP, Montpellier, France.

Figure 3. Farmer growing FHIA-03 in his backyard in the Leon region.

Table 4. Most preferred varieties and reasons for it (N = 80).

Variety Most important reason False horn Plantain* (Cuerno) Market FHIA-03 Drought-resistant, bunch size TMBx 5295 Good taste Bluggoe* (Cuadrado) Drought-resistant, firmness of fruit, taste TMBx 1378 Shape of fruit, taste Pelipita Firmness of fruit, taste * local varieties.

Table 5. Acceptability of the fruit taste and visual aspect (N = 80).

Fruit taste Fruit visual aspect Koen Dens, G. Matton and S. Coessens work as cooperants of VVOB at the UNAN; M. Vargas is Mode of Compared % better Compared % better Variety preparation with or the same with or the same Head of the Musa project of the UNAN, Laboratorio de Cultivo de Tejidos; Iglesia la Merced 1/2 C al N; FHIA-03 Ripe Bluggoe 95 Gros Michel 44 Facultad de Ciencias, UNAN-León, Nicaragua; FHIA-01 Ripe Gros Michel 62 Bluggoe 68 E-mail: [email protected] ; Pisang lidi Ripe Silk 37 Silk 13 http://www.unanleon. edu.ni/~vitro/; TMBx 1378 Ripe Silk 99 Ines Van den Houwe is germplasm conservation offi- TMBx 5295 Ripe fried False horn 86 False horn 57 cer at the INIBAP Transit Centre, Kasteelpark Arenberg TMBx 5295 Green fried False horn 67 13 – 3001 Leuven, Belgium. TMPx 4479 Ripe fried False horn 75 E-mail: [email protected] TMPx 4479 Green fried False horn 85 False horn 49 and Rony Swennen, Head of the Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven, Pelipita Green fried False horn 80 False horn 12 Kasteelpark Arenberg 13 – 3001 Leuven, Belgium. Pelipita Ripe fried False horn 37 E-mail: [email protected]; Pisang ceylan Ripe Silk 72 Silk 91 http:// www.agr.kuleuven.ac.be/dtp/tro/home.htm

INFOMUSA — Vol 11, N° 1 47 Genetic resources Classification of Vietnamese germplasm Using RAPD technique for identifying and classifying some banana cultivars in Vietnam

Nguyen Xuan Thu, Le Thi Lan Oanh [1,5% cetyltrimethylammonium bromide • The dendogram of cultivars studied was and Ho Huu Nhi (CTAB); 100 mM Tris-HCl) (pH 8); produced using the NTsyspc 2.0 compu- 20mM ethylenediaminetetraacetic acid ter programme. (EDTA) (pH 8); 1.4 mM NaCl; 0.2% mer- anana is an important plant in captoethanol] heated to 65°C was Results and discussion tropical countries. Banana origi- added, and the mixture was then incu- Bnates from Musa acuminata bated at 65°C for 30 min. The mixture RAPD-PCR (AA) and Musa balbisiana (BB). Ten was shaken lightly with 1.5 volume of Twelve primers were used to amplify the groups of cultivars with levels of ploidy chloroform: isoamyl (24:1) for 20 min at banana genome DNA. Nine of them ranging from diploid (2n = 2x = 22) to room temperature. The sediment was amplified to give multiple PCR amplifica- tetraploid (2n = 4x = 44) and different removed by centrifugation at 3000 rpm tion products (Figure 1: example with genomes are recognized. The following for 20 min. The DNA was precipitated by primer H08), and three primers (G6, Y14, genome configurations are known to the addition of 0.8 volume of freezing Y15) did not. exist: diploid AA, BB and AB; triploid propanol (or 1.5 volume of 96% ethanol). There were two kinds of bands: AAA, AAB, ABB; and tetraploid AAAA, The pellet was washed 2-3 times in 70% monomorphic bands which were present AAAB, AABB, ABBB (Simmonds and ethanol. At the end, DNA was redis- in all cultivars and polymorphic bands Weatherup 1990). Therefore, a wide vari- solved in a minimum volume of TE which were asynchronously present ety of classification and identification (about 200 ml). or absent in all cultivars. Nine primers systems exists for banana. Up to now, the were amplified into 79 bands, of which traditional classification and identifica- DNA amplification 67 bands (84.81%) were polymorphic and tion have been based only on morphol- Twelve primers from Operon 12 bands (15.19%) monomorphic. The ogy and quantitative traits. Recently, Technologies, each ten bases in length, high proportion of polymorphic bands molecular markers have been used to were used to amplify the DNA (Table 2). was due to the very different origin of the study diversity on plants, animals and PCR was carried out in 25 ml reactions cultivars studied. Two primers (D07, G14) microorganisms. containing 20 ng of template (genome amplified 5 bands only, while H07 ampli- The random amplified polymorphic DNA), 200 mM of each dNTP, 2.5 units of fied 17 bands. The size of the bands DNA (RAPD) technique, which utilizes Taq-polymerase, 15 ng of primers, 10 mM ranged from 360 Kb to 3200 Kb. polymerase chain reaction (PCR) ampli- Tris-HCl (pH 8.3), 50 mM KCl, 1.5mM fication with single primers of arbitrary MgCl2, 0.001% (w/v) gelatine and 20 ml Genetic similarity nucleotide sequence, has been deve- of mineral oil overlay. Forty-five amplifi- The Nei and Li formula allowed to calcu- loped by Williams et al. (1990) and Welsh cation cycles were performed, each con- late coefficients of similarity among cul- and McClelland (1990) to produce mole- sisting of 94°C for 30 s, 36°C for 1 min, tivars based on RAPD data. Similarity cular markers for genetic analysis. 72°C for 2 min. Products were analyzed coefficients reflected the relationship RAPDs have been shown to be useful in by electrophoresis in 1.1% agarose gels between cultivars. Similarity coeffi- genetic fingerprinting (Yang and Quiros at 100 V for 3 h, stained with 0.01% ethid- cients between the original cultivars 1993, Orozco-Castillo et al. 1994, Lanham ium bromide and photographed under from M. acuminata ranged between et al. 1995). In this study, we have used UV light. 0.764-0.826, while those between the RAPDs for identifying and classifying original cultivars from M. balbisiana some banana cultivars. Data analysis ranged between 0.696-0.835 (Table 3). • Coefficients of similarity among cultivars Cultivars belonging to the two groups Materials and methods were calculated by using the formula of had low similarity coefficients, ranging Nei and Li (1979): between 0.317 and 0.461. Materials 2Nij In this study, six indigenous banana culti- Sij = Specific RAPD markers of cultivars vars of Vietnam (Table 1), which were Ni + Nj Specific RAPD markers are bands that obtained from the Institute of where: are present only in one cultivar. In this Agricultural Genetics (Vietnam) were Nij = number of bands in common study, we found 12 specific markers for screened for RAPD markers. between cultivars i and j, and 4 cultivars (Table 4). These results sug- Ni and Nj = number of bands for culti- gest that RAPDs can be used for the Isolation of DNA vars i and j, respectively. selection of banana breeds in agriculture. DNA was isolated from banana leaves using the method of Murray and Thompson (1980) with some modifica- Table 1. Cultivars and genotypes employed in the study. tions. Four grams of fresh leaf material were grounded in liquid nitrogen in Cultivar Genotype Cultivar Genotype presence of glass-sand. The powdered 1 Chuoi Tieu Xanh AAA (2n = 3x = 33) 4 Chuoi Tay ABB (2n = 3x = 33) leaf tissue was stored at -20°C for two 2 Chuoi Tieu Hong AAA (2n = 3x = 33) 5 Chuoi La ABB (2n = 3x = 33) hours. Ten milliliters of extraction buffer 3 Chuoi Ngu AA (2n = 2x = 22) 6 Chuoi Hot BB (2n = 2x = 22)

48 INFOMUSA — Vol 11, N° 1 Table 2. Primers used in the study.

Primer Nucleotide sequence Primer Nucleotide sequence AA10 5’AGACGGCTCC 3’ H07 5’CTGCATCGTG 3’ AA14 5’AACGGGCCAA 3’ H08 5’GAAACACCCC 3’ B17 5’AGGGAACGAG 3’ U01 5’ ACGGACGTCA 3’ D07 5’TTGGCACGGG 3’ Y14 5’GGTCGATCTG 3’ G06 5’GTGCCTAACC 3’ Y15 5’AGTCGCCCTT 3’ G14 5’GGATGAGACC 3’ Y18 5’GTGGAGTCAG 3’

Table 3. Similarity coefficients among banana cultivars calculated using the Nei and Li formula.

Cultivar Tay Hot La Tieu Xanh Tieu Hong Ngu Tay 1.00 Hot 0.826 1.00 La 0.764 0.829 1.00 Tieu Xanh 0.577 0.461 0.586 1.00 Tieu Hong 0.500 0.373 0.489 0.835 1.00 Ngu 0.477 0.317 0.422 0.782 0.696 1.00

Table 4. Specific markers of some banana cultivars studied.

Chuoi La Chuoi Hot Chuoi Ngu Tieu Hong AA10-950 H07-500 H07-900 H07-800 H07-1270 H07-400 U01-1400 U01-700 Y18-800

Figure 1. Results of RAPD-PCR with primer H08. 1: 1Kb scale; 2: Chuoi Tay; 3: Chuoi Tieu Xanh; 4: Chuoi Tieu Xanh; 5. Chuoi Ngu; 6: Chuoi Tieu Hong; 7: Chuoi La; 8: Chuoi Hot. Chuoitay

Chuoihot Phylogenetic tree of banana cultivars Based on RAPD data, a phylogenetic tree Chuoila of banana cultivars was constructed using the NTsyspc 2.0 program. The phylogenetic tree had two branches: the cultivars Tieuxanh originating from M. acuminata were on one branch, and the cultivars originating Chuoingu from M. balbisiana were on the other branch (Figure 2). These results are in agreement with the cytological analysis Tieuhong of these banana cultivars.

Acknowledgements 0.35 0.49 0.63 0.76 0.90 The authors thank the Program of Coefficient Fundamental Researches for supporting this research, and Inge Van den Bergh for Figure 2. Dendogram of banana cultivars produced using the NTsyspc 2.0 computer program. reviewing this article. ■

References Orozco-Catstillo C., K.J. Chalmers, R. Waugh & by arbitrary primers are useful as genetic mark- Lanham P.G., R.M. Brennan, C. Hackett & R.J. McNicol. W. Powell. 1994. Detection of genetic diversity and ers. Nucleic Acids Res. 18: 6531-6535. 1995. RAPD fingerprinting of blackcurrant (Ribes selective gene introgression in coffee using RAPD Yang X. & C. Quiros. 1993. Identification and classifi- nigrum L.) cultivars. Theor. Appl. Genet. 90: markers. Theor. Appl. Genet. 87: 934-940. cation of celery cultivars with RAPD markers. 166-172. Simmonds N.W. & S.T.C. Weatherup. 1990. Numerical Theor. Appl. Genet. 86: 205-212. Murray M.G. & W.F. Thompson. 1980. Rapid isolation taxonomy of the cultivated bananas. Tropical of high molecular weight plant DNA. Nucleic Acids Agriculture 67: 90-92. Nguyen Xuan Thu and Le Thi Lan Oanh work at the Institute of Biotechnology (IBT), National Centre for Res. 8: 4321- 4325. Welsh J. & M. McClelland. 1990. Fingerprinting Natural Science and Technology of Vietnam (NCST), Nei M. &W.H. Li. 1979. Mathematical model for study- genomes using PCR with arbitrary primers. Hoang Quoc Viet Street, Cau Giay, Ha Noi, Vietnam. ing genetical variation in term of restriction Nucleic Acids Res. 18(24): 7213-7218. E-mail: [email protected]; Ho Huu Nhi works at the endonucleases. Proc. Natl. Acad. Sci. USA 74: Williams J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski Vietnam Agricultural Science Institute (VASI), Van 5267-5273. & S.V. Tingey. 1990. DNA polymorphism amplified Dien, Ha Noi, Vietnam. E-mail: [email protected]

INFOMUSA — Vol 11, N° 1 49 Marketing Consumer studies in Nigeria

Consumption and expenditure patterns of banana and plantain consumers in Nsukka Urban, Nigeria

A.R. Ajayi and M.O. Aneke change, the results that can be achieved, and consumption of bananas and plan- the process through which change is tains in the study area. achieved, and the uncertainties inher- n Nigeria, banana and plantain have ent in change (Williams 1978). Methodology always been very important tradi- Expenditure patterns of households in Nsukka Urban lies within the centre of Itional staple foods for both rural and Nigeria vary from place to place. Apart Nsukka Local Government Area, Enugu urban population. They serve as a source from the income of the households, fac- State, Nigeria. The land area of Nsukka of revenue for smallholders who produce tors such as preference of a particular Urban is about 45.38 km2 (Oformata them at the compound farms, mixed- product by a member of the household, 1995). It is made up of the following dif- cropping farms and small-scale sole crop- quality and quantity of the product sold, ferent sections (clusters): the University ping farms (Baiyeri 1996, Ajayi and environment under which the product of Nigeria campus, Onuiyi, Odenigbo, Baiyeri 1999). was processed and sold, and the relative Government Reserved Area (GRA), Nsukka Urban is heavily populated. It price of the products, also influence the Odenigwe, Ugwoye, Umuyo, Ngwuru, has a large market centre, operated on a expenditure patterns of the households Owerre, Makashi and Isiakpu. daily basis. Men, women and the youths (Anyanwu 1985). Out of the above-listed 11 clusters, five within Nsukka Urban and neighbouring Food consumption pattern, in a broad were selected through simple random communities converge at the market cen- sense, means not only what the people sampling. From each of the five clusters, tre to buy and sell. Agricultural products eat or consume, but also the quantities as 12 households were selected, using clus- such as banana, plantain, vegetables, well as the forms in which these foods are tering and simple random sampling tech- pepper, mangoes, palm oil, other fruits, consumed (Dury et al. 1999). According niques. In all, a total of 60 households honey, yam, livestock, etc., are sold. In to Olagoke (1989), food consumption pat- were involved in the study, and the head of the area, banana and plantain are found terns vary from one place to another due each of the households was interviewed. in compound farms and they are mixed to factors such as household size, educa- A structured questionnaire schedule with other crops. Each of the banana tional levels of members of the house- was developed and used in obtaining rel- and/or plantain growers in the area has hold, relative prices of the food items, evant information from the consumers less than 50 stands and a greater propor- environment in which the consumers are of bananas and plantains. The data tion of them grow more banana than living, social values attached to some food collected were analysed through the use plantain (Baiyeri and Ajayi 2000). items, nutritive values of the food items, of percentage distribution and bar However, banana and plantain marketing type or status of job performed by mem- charts. is most prominent among women, espe- bers of the household, household’s tastes cially within Nsukka Urban, University and preferences, season/period of the Results of the survey campus and the neighbouring communi- year, and culture/religion of the house- Consumption and expenditure patterns ties. The sale of banana and plantain pro- hold members. of banana and plantain among house- vides means of livelihood for many house- The study was designed to assess the holds in Nsukka Urban are presented in holds in the area. consumption and expenditure patterns of the following figures and tables: In view of the significant contributions banana and plantain consumers in of banana and plantain to the economic, Nsukka Urban in Enugu State, Nigeria. Consumption rate of banana health and nutritional well-being of both Specifically, the study was designed to: and plantain rural and urban households in Nigeria, it 1. determine the consumption patterns of Figure 1 shows that the consumption rate is very important that efforts should be bananas and plantains among house- of bananas is higher than the consump- made continually to improved their mar- holds in Nsukka Urban; tion of plantains. keting and consumption patterns. In 2. determine the expenditure patterns of planning such a national banana and banana and plantain consumers in Sources of banana and plantain plantain marketing and consumption Nsukka Urban; for consumption improvement programme, data based on 3. determine the decision-making role of Most of the consumers depend on the the consumption and expenditure pat- household members in banana and market for their banana and plantain terns of their consumers in both rural plantain consumption in Nsukka supply. A very small proportion of con- and urban areas would be necessary. The Urban; sumers produce their fruits regularly role of Agricultural Extension (AE) in 4. determine the major problems militat- (Figure 2). the gathering of data, planning, imple- ing against effective consumption of mentation, monitoring and evaluation of bananas and plantains in the study Period of the day that bananas such a programme cannot be overem- area; and and plantains are mostly consumed phasized. AE is a primary process 5. draw implications for extension pro- It clearly appears in Figure 3 that people through which the households can learn gramme on improved and efficient prefer to eat plantain (consumed boiled, the reasons for change, the value of preservation, processing, marketing roasted or fried) in the morning and at

50 INFOMUSA — Vol 11, N° 1 70 Banana Banana (%) 60 40 Plantain Plantain (%) 35 50

30 40 25 30 20 % of respondents 15 20 % of respondents 10 10 5 0 0 Daily Once Twice Thrice Purchased Produced Both a week a week a week

Figure 1. Frequency of consumption of banana and plantain. Figure 2. Percentage distribution of respondents on the basis of their source of banana and plantain fruits.

Banana (%) Banana

100 Plantain (%) 50 Plantain 90 45 80 40 70 35 60 30 50 25 40 20 % of respondents 30 15 20 10 10 5 0 0 Morning Afternoon Evening Night Daily Weekly Monthly Occasionally

Figure 3. Percentage distribution of consumers on the basis of the time of Figure 4. Expenditure rate of banana and plantain consumers. the day that banana and plantain are mostly preferred.

Table 1. Forms of plantain meal among the households. Proportion of monthly income spent on banana and plantain Form of meal Breakfast (%)* Lunch (%)* Dinner (%)* consumption Fried plantain + pap 28.5 4.3 1.7 Most of the respondents (Figure 5) Pottage plantain 19.2 3.7 11.0 spend only 1% of their income on Plantain + beans 3.5 10.6 18.9 bananas and plantains. The main factors Plantain + rice 2.3 9.6 21.1 that determine the percentage of their Boiled plantain + stew 15.1 5.3 13.3 monthly income spent on the purchase Pounded plantain + soup 1.2 25.5 5.6 of bananas and plantains are the avail- Plantain with yam (pounded) 4.7 10.6 17.8 Roasted plantain 3.5 24.0 5.0 ability of money, closely followed by the Boiled plantain 22.0 6.4 5.6 family interest, and then the price of the *Multiple responses fruits (Figure 6 – NB: more than one factor was given).

Forms in which bananas and plantains are purchased in the market night, and banana as a ‘snack’ in the Expenditure rate of banana Bananas are mostly purchased ripe, afternoon. and plantain consumers whereas the respondents prefer to buy It is to be noted that plantains are more unripe plantains (Figure 7). Common forms of plantain meal expensive than bananas (N121 or N15 per among households plantain finger and N5 per banana fin- Response of households to changes Respondents preferred fried plantain for ger). This could explain why the majority in banana and plantain prices breakfast. For lunch, pounded and of consumers surveyed buy banana more Table 2 shows that most of the respon- roasted plantains are the most eaten. regularly than plantain (Figure 4). dents do not change their habits in For dinner, plantain accompanied buying bananas when the price with rice, beans or yams is preferred increases, but they buy more if the price (Table1). decreases. In the case of plantain, more 1 10 N (Nigerian Naira) = 0.085 USD, March 2002. than half of the respondents would buy

INFOMUSA — Vol 11, N° 1 51 10 6,7 Availability of 65 Availability of fruits 3% Quality of fruits 13% 18,3 money (cash) 33%

Family interest 31% about1% about2% Price of fruits 20% about3% 4 to 5%

Figure 5. Percentage distribution of respondents on the basis of the Figure 6. Percentage distribution of the respondents on the basis of the proportion (%) of their monthly income spent on banana and plantain factors determining the proportion of their monthly income spent on consumption. banana and plantain consumption.

90 80 Banana (%) Transportation without any 70 Plantain (%) bruise 3% Processing Storage 60 17% 80% 50 40 30 20 10 0 Ripe Unripe Over-ripe Form

Figure 7. Percentage distribution of respondents on the basis of the forms Figure 8. Major problems militating against effective consumption of in which bananas and plantains are purchased. banana and plantain fruits.

Table 2. Percentage distribution of respondents according to their response fewer plantains in case of increased to change in banana and plantain price. price and 75% would buy more in case of decreased price. Response to change in price 10% increase in price 10% decrease in price

Banana (%) Plantain (%) Banana (%) Plantain (%) Household decision-making role Buy same quantity 75.0 41.7 25.0 20.0 in banana and plantain consumption Buy more quantity 0.0 0.0 75.0 80.0 processes Buy less quantity 25.0 58.3 0.0 0.0 According to Table 3, wives play the most Total 100.0 100.0 100.0 100.0 important decision-making role in banana and plantain purchasing and consumption process. On the other hand, the husband plays the major role in the utilization of the peels, while children play the greatest role in determin- Table 3. Household decision-making role in banana and plantain consumption ing the purchasing interval and storage processes. period. Decision-making role Banana Plantain

H (%) W (%) C (%) H (%) W (%) C (%) Major problems militating against Proportion of monthly income 43.3 56.7 0.0 30.0 70.0 0.0 effective consumption of bananas Quantity purchased 21.0 70.7 8.3 35.0 50.0 15.0 and plantains Form in which fruits are purchased 8.3 75.0 16.7 30.0 60.0 10.0 Three major problems militating against Processing 0.0 85.5 14.5 10.0 64.6 25.4 the effective consumption of banana and Storage 0.0 75.3 24.7 0.0 86.2 13.8 plantain fruits were identified by the Utilization of peels 50.0 11.7 38.3 70.0 4.7 25.3 respondents (Figure 8). These included Storage period 16.7 25.0 58.3 11.7 21.7 66.6 storage problems such as pest (house rats Purchasing interval 33.3 41.7 25.0 13.3 33.3 53.4 and insects) attacks, over-ripening and Quality purchased 18.0 82.0 0.0 27.0 73.0 0.0 mould formation due to sustained H = Husband, W = Wife and C = Children bruises; processing problems such as lack

52 INFOMUSA — Vol 11, N° 1 of technological know-how, unfavourable References weather conditions, unavailability of pro- Ajayi A.R. & K.P. Baiyeri. 1999. Household decision- cessing mills and poor supply/lack of making role in backyard banana and plantain pro- electricity, etc.; and transportation with- duction in the Nsukka agroecological zone in out any bruise. Chukwu (1996) observed southeastern Nigaria. Pp. 719-727 in Bananas and that inadequate storage, insufficient dis- Food Security. Les productions bananières: un tribution and lack of processing tech- enjeu économique majeur pour la sécurité alimen- niques result in large proportions of taire (C. Picq, E. Fouré and E.A. Frison, eds). banana and plantain being transformed Proceedings of an International symposium held into wastage. in Douala, Cameroon, 10-14 November 1998. Anyanwu C.U. 1985. An evaluation of the relationship Conclusions between income level, expenditure and food con- The analysis of these results leads to the sumption pattern in UNN. B. Agric. Research following conclusions: Project, Department of Agricultural Economics, 1. the consumption rate of bananas was University of Nigeria, Nsukka. higher than that of plantain; Baiyeri K.P. 1996. Characterization, correlation, 2. a greater proportion of the respon- path-analysis and selection indices of Musa geno- dents depended on the market for types under different environments. A PhD their supply of bananas and plantains; Research Proposal, Department of Crop Science, 3. bananas were consumed mostly in the University of Nigeria, Nsukka. afternoon, while plantains were Baiyeri K.P. & A.R. Ajayi. 2000. Status and con- mostly consumed in the morning; In Nigeria, False horn plantains are very straints of Musa spp. production in a sub-humid 4. plantain meals were prepared and appreciated because of theit long finger size zone of Nigeria. Pp. 73–77 in Proceedings of the (Photo: R. Swennen, IITA). consumed in various forms; First International Conference on Banana and 5. plantains were more expensive than Plantain for Africa (K. Craenen, R. Ortiz, E. B. bananas; relevant pieces of information if and Karamura and D.R. Vuylsteke, eds). Acta 6. the major factors that determined the when necessary. Horticulturae 540. proportion (%) of the monthly income 2. To ensure effective preservation, pro- Chukwu U.E. 1996. Effect of post-harvest injury on spent on bananas and plantains cessing and utilization of bananas and shelf-life and extrusion processing of Musa spp. included availability of physical cash, plantains, the State Agricultural fruits. A PhD Research Proposal, Department of family interest, price, quality and avail- Development Project (ADP) should Food Science, University of Ibadan, Ibadan, Oyo ability of fruits; organize workshops for the women State, Nigeria. 7. bananas were mostly purchased in a marketers on methods of preservation, Dury S., N. Bricas, J. Tchango Tchango & A. Bikoï. ripe form, while plantains were mostly processing and efficient utilization of 1999. La consommation et les critères de qualité purchased unripe; banana and plantain. du plantain à Douala et à Yaoundé. Pp. 507-523 8. households responded accordingly to 3. The fact that a greater proportion of in Bananas and Food Security. Les productions change in banana and plantain prices; the respondents purchased the bananières: un enjeu économique majeur pour 9. wives played the greatest decision- needed bananas and plantains from la sécurité alimentaire (C. Picq, E. Fouré and making role in banana and plantain the market centres and hawkers E.A. Frison, eds). Proceedings of an International purchasing and consumption shows that there should be efficient symposium held in Douala, Cameroon, 10-14 processes than their husbands and distributing and marketing channels November 1998. children; and to enhance consumers’ access to the Ofomata G.E. 1975. Nigeria in Maps. Eastern State 10.the major problems militating against fruits as of when due. Thus, the Enugu Ethiope Publishing Company, Benin City, Nigeria. effective consumption of bananas State ADP should embark on efficient Olagoke M.A. 1975. Food consumption patterns in and plantains in the area included Musa postharvest handling, distribut- the Obafemi Awolowo University. BA Research storage, processing and transporta- ing and marketing strategy pro- Project, Faculty of Agriculture, Ile-Ife, Osun State, tion problems. gramme for the rural farmers and the Nigeria. sellers. Williams S.K.T. 1978. Rural development in Nigeria. Implications of the findings 4. Even though the wives could be spe- Obafemi Awolowo University Press, Ile-Ife, Osun for extension programme cially targeted (because of their State, Nigeria. on improved and efficient largest decision-making role in preservation, processing, banana and plantain consumption) by marketing and consumption the Enugu State ADP to improve on of bananas and plantains the banana and plantain consumption 1. Since fluctuation in the market price and expenditure patterns among of bananas and plantains affected the households through appropriate edu- consumption patterns of the house- cational activities, the importance of holds, there is a need for the establish- the household as a working unit in ment of a consumer cooperative orga- extension practice should not be over- nization for bulk purchase and retail looked. Therefore, all members of the sale of the fruits. Capable extension household should be intensively agents should be attached to each of involved in any extension programme The authors work at the Department of Agricultural the so formed organizations for the designed to improve consumption and Extension, Faculty of Agriculture, University of Nigeria purpose of monitoring and evaluating expenditure patterns of banana and Nsukka. Tel.: +234 042 770815 or 771019, the activities of members and giving plantain in the study area. ■ Fax: +234 02 770664; E-mail: MISUNN @ aol.com

INFOMUSA — Vol 11, N° 1 53 Thesis

Molecular characterization of dwarf banana plants (Musa spp.) using AFLP

Katholieke Universiteit Leuven, Faculty of Agricultural and Applied Biological Sciences, Laboratory of Tropical Crop Improvement, Leuven, Belgium Dissertationes de Agricultura No. 515, 2002

Iris Engelborghs the amplified fragments. With this several genes are involved in the process. method, sets of restriction fragments are Differential fragments were cloned and visualized by PCR without knowledge of sequenced. Primers were designed on the nucleotide sequence. The power of this obtained sequences and used with DNA fingerprinting technique was genomic DNA of the respective variety to assessed on a few world species, but not confirm the differential and unique on Musa spp. Therefore, it was here first nature of the fragments. However, speci- optimized for banana. In addition, the ficity was lost. technique was adapted for non-radioac- Besides the above-described analyses tive detection of the AFLP patterns using at the DNA level, some physiological a more recent detection method. assays were performed on the dwarf-nor- Fluorescein labelled primers were used mal banana pairs. The relation between in the PCR reactions which allow the dwarfism and gibberellic acid (GA) is computer based separation and detection described for several mutant dwarf on a sequencing gel. species, e.g. rice and wheat, as well as The assessment of the power of the some Musa spp. Two categories of dwarf anana plants are the most impor- AFLP technique and its variants three- types are described, i.e. a GA-(in)sensi- tant world fruit crop and show a endonuclease-(TE)-AFLP, cDNA-AFLP tive group and a GA-deficient group. The Blarge diversity in shapes and sizes, and the methylation sensitive amplified influence of GA was tested in this study of which one is the dwarf type. Unlike the polymorphism (MSAP) technique for the on the in vitro growth of the dwarf-nor- normal type, the dwarf variant has a characterization and the early detection mal banana pairs. The (in)sensitivity shorter pseudostem and wider leafs. of the dwarf type was performed in this appeared to be variety dependent, sug- Because these plants have the same num- study on a range of dwarf-normal banana gesting that the different dwarf varieties ber of leaves as the normal variant, their pairs. The dwarf ‘Curare enano’ and its in originated in a different manner and that photosynthetic ability is not reduced and vitro generated normal-sized off-type other mechanisms may be involved than therefore the bunch size almost identi- were first used for the optimization of the the GA pathway alone. When grown on cal. In addition, its reduced height pre- AFLP technique. Later, the analysis was ancymidol (a GA-synthesis inhibitor) in vents it from toppling during tropical extended to three naturally occurring vitro growth of all the tested dwarf vari- storms. These characteristics make the dwarf varieties ‘Cachaco enano’, ‘Figue eties was inhibited, which indicates that dwarf variant a valuable plant for the rose naine’ and ‘Prata ana’, which have a these plants are not GA-deficient, and the tropical banana farmer. Different natu- normal variant called ‘Cachaco’, ‘Figue signal transduction after the synthesis rally occurring dwarf varieties exist rose’ and ‘Prata’, respectively. In addi- must be impaired. which have a normal variant, but this tion, the extra dwarf ‘Dwarf parfitt’, the From these results we can conclude phenotype is often obtained by in vitro normal ‘Cavendish’ and the giant ‘Giant that the AFLP technique allows a fast and culture too, e.g. in an in vitro germplasm cavendish’ were analyzed. early fingerprinting of these particular collection or during rapid in vitro multi- Differential AFLP, TE-AFLP, cDNA- dwarf banana types, that the mechanism plication. AFLP, cDNA-TE-AFLP and MSAP patterns behind dwarfism is complex and seems to Vos et al. (1995) described the ampli- were obtained and different levels of involve gibberellic acid, (de)methyla- fied fragment length polymorphism polymorphisms were observed between tion… and that the dwarf varieties here (AFLP) technique as ‘a novel and very the dwarf and normal type depending on analyzed probably originated via differ- powerful DNA fingerprinting technique the technique, the primer combination ent mechanisms. ■ for DNAs of any origin or complexity’. and the variety. For each variety, a dis- This DNA fingerprinting technique is tinction could be made between the based on the selective PCR amplification dwarf and the normal type. However, no of restriction fragments from a total dwarf specific fragment was found to be Reference digest of genomic DNA and involves three common for all dwarf varieties, which is Vos P., R. Hogers, M. Bleeker, M. Reijans, T. van de steps: (i) restriction of the DNA and liga- an indication that (i) either the observed Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, tion of oligonucleotide adapters, (ii) polymorphisms are not related to the M. Kuiper & M. Zabeau. 1995. AFLP: a new tech- selective amplification of sets of restric- phenotype, (ii) or the different varieties nique for DNA fingerprinting. Nucl. Acids Res. tion fragments, and (iii) gel analysis of originated in a different way, (iii) or that 23: 4407-4414.

54 INFOMUSA — Vol 11, N° 1 Thesis

Study of the interaction between arbuscular mycorrhizal fungi and plant-parasitic nematodes in Musa spp.

Katholieke Universiteit Leuven, Faculty of Agricultural and Applied Biological Sciences, Laboratory of Tropical Crop Improvement, Leuven, Belgium Dissertationes de Agricultura No. 517, 2002

Annemie Elsen ‘Pisang jari buaya’, no suppression was studies and AMF-nematode interaction observed. However, when reproduction is studies. Secondly, three model systems already very low (due to low reproductive were developed: Ri T-DNA transformed iseases and pests are the major fitness of the nematode population and/or Daucus carota roots, in vitro Musa constraints to the productivity of the resistant host plant response of the plants and in vitro Arabidopsis thaliana Dbananas and plantains. Nema- tested genotype), the presence of the plants. todes cause important yield losses in AMF has no effect on the nematode repro- Finally, the transformed D. carota roots Latin America, West and East Africa and duction. The AMF reduced the root necro- were used to study the AMF-nematode Asia. Usually, banana nematodes are con- sis, caused by P. coffeae. For R. similis, no interaction under sterile conditions. The trolled by nematicides. These are not only reduction was observed. The nematodes results reported in this study confirmed very expensive but also extremely toxic reduced the frequency of mycorrhization, the suppressive effect of AMF on nema- for non-target organisms, including the without reducing the intensity of the tode reproduction. Glomus intraradices user, and they pollute the environment. mycorrhizal association. could suppress the R. similis, P. coffeae Arbuscular mycorrhizal fungi (AMF) are In the second part, the RMD and the and to a lesser extent M. javanica popu- obligate symbionts that biotrophically AMF-nematode interaction were studied lation in the roots. The internal and colonize the root cortex and develop an in Musa genotypes differing in root mor- external developments of the AMF were extramatrical mycelium which helps the phology. The influence of the AMF on the not affected by the presence of these plant to acquire water and mineral root system and the influence of the plant-parasitic nematodes. nutrients from the soil, in exchange for altered root system on the nematode Although this in vitro system has sev- carbon as an energy source. In addition, reproduction were examined. Mycorrhi- eral limitations, there are still many legit- AMF increase the ability of the plant zation with G. mosseae resulted in a sig- imate reasons to use this system to study to control the spread of soil-borne nificant better plant growth, even in the the AMF-nematode interaction. The AMF pathogens. In Musa the association presence of P. coffeae. The effect of AMF develops appressoria, arbuscules and occurs naturally when plants are trans- on the root system was related to the vesicules in the root cortex, produces pro- planted into the field. The association of RMD of the genotype. Musa genotypes fuse extraradical mycelium and spores AMF with plant-parasitic nematodes and with a low RMD will not experience and is completing its life cycle in vitro. the beneficial effect of the mycorrhizal changes in the branching of their root The early colonization occurs in a similar symbiosis on plant growth and nematode system in response to mycorrhization. way as under in vivo conditions. The resistance/tolerance led to investigations But in genotypes with a high RMD, the nematodes, R. similis and P. coffeae, can into the potential of AMF to limit yield root system will be more branched. We infect and reproduce in the roots, and losses due to nematodes. showed that P. coffeae also affects the cause similar damage in the in vitro roots In the first part of our study, the rela- root system, by reducing the branching. as in in vivo roots. In addition, the effects tive mycorrhizal dependency (RMD) and The effect of AMF on the nematode repro- of the interaction reflect those observed the AMF-nematode interaction were duction was not very clear. The nematode in vivo. Although the dixenic system used studied in four Musa genotypes, selected population density tended to be reduced, is artificial, it may represent a valuable for their known host plant response to but was not significant in the experiment tool for studying the AMF-nematode nematodes (i.e. ‘Grande naine’, ‘Gros with ‘Obino l’ewai’. In the root system, it interaction, complementary to classical Michel’, ‘Pisang jari buaya’ and appeared that the decreased branching experimental approaches. ■ ‘Yangambi km5’). Mycorrhization with caused by the nematodes was counterbal- Glomus mosseae (AMF) resulted in a sig- anced by the increased branching caused nificant better plant growth, even in the by the AMF. Therefore application of AMF presence of Radopholus similis and could be used as a strategy to decrease Pratylenchus coffeae. No differences in susceptibility to nematodes. RMD were observed among the four geno- In the third part of our study, AMF- types. Glomus mosseae could protect nematode interactions were studied ‘Grande naine’ and ‘Pisang jari buaya’ under in vitro conditions. Firstly, aseptic against R. similis and P. coffeae, since the nematode cultures were established nematode reproduction was suppressed. using alfalfa callus as a host tissue. Until Only in the case of R. similis (Indonesian now the lack of completely sterile culture population with low pathogenicity) in systems limited in vitro nematode-host

INFOMUSA — Vol 11, N° 1 55 MusaNews from all over the world who presented ‘Africa’ (biovar 1, 2), and phylotype IV more than 100 papers either orally or as ‘Indonesia’ (biovar 1, 2, Pseudomonas posters. Aspects discussed were: epi- syzygii, and blood disease bacterium – World demiology, disease management, breed- BDB). This shows that strains of ing and deployment for disease resis- Indonesia, including P. syzygii which Farmer-participatory evaluation tance, host response and disease causes Sumatra disease of clove, and the and dissemination of improved development, pathogen genetics, diver- BDB on banana, are separated from Musa germplasm sity and diagnosis. other strains. INIBAP is the executive agency of an Bacterial wilt caused by Ralstonia important four-year project of the solanacearum is reported to be one of Detection Common Fund for Commodities (CFC) the major constraints for many important Various diagnostic kits have been devel- which started in November 2001. The aim crops such as potato, tomato, groundnut, oped, especially for quarantine purposes of this project is to contribute to banana, tobacco and ginger. In many and for monitoring the pathogen in symp- improved food and income security for cases, the disease causes very significant tomatic and latently infected plant mate- small-scale farmers in banana-based yield losses. rials, surface water, soil, vegetable wash- farming systems, through the distribution There is still a big gap in the research ings, and processing waste. Methods used and evaluation of improved Musa hybrids progress between developed and devel- are selective isolation and enrichment, suitable for local consumption and mar- oping countries. In developed countries, bioassay, immunofluorescence, serology, keting. The project involves seven coun- scientists are generally more interested and polymerase chain reaction (PCR). tries namely Democratic Republic of in the molecular aspects of the pathogen For use in developing countries, serology Congo, Ecuador, Guinea, Haiti, Honduras, such as pathogen genetics, diversity and methods such as ELISA are more appro- Nicaragua, and Uganda. diagnosis. Except for diagnostics, other priate as they are cheaper. The project will be implemented in two aspects under study are not directly The 4th Symposium will be held in 2007, phases. The first phase includes the mul- related to control of the disease. possibly in the UK. tiplication of planting material of at least Therefore, research being carried out on The American Plant Pathology Society ten improved Musa varieties in each disease control by scientists in develop- will publish the papers presented in the country and the distribution of these ing countries, where the disease is more Symposium. plants to farmers for on-farm evaluation. serious and widespread, needs to be Further information about the symposium At least 150 farmers per country partici- strengthened. Some work on breeding is available from Dr Supriadi, Research Institute pate in the trials. The second phase will and deployment for disease resistance for Spice and Medicinal Crops, Jalan Tentara Pelajar No. 3, Bogor 16111, Indonesia. consist of funding support in the form of has been carried out and good results Fax: (0251) 327010. loans to small-scale farmers to enable have been obtained for groundnut and them to purchase planting material and potato. However, very little has so far essential inputs for the more wide scale been done for many other important Asia and the Pacific production of improved hybrids. The pro- crops such as banana and ginger. ject will also include market studies on Musa acuminata in Northern Borneo the improved hybrids and the training of Pathogen genetics A preliminary report on the status of farmers in improved production tech- Significant progress has been made in Musa acuminata in Northern Borneo niques, focusing on the integrated man- studies of the genome of R. solanacearum. has recently been prepared by Markku agement of pests and diseases. The main The pathogen has a 3,716,413 base pair Häkkinen and Edmond De Langhe. This result from the project will be the (bp) chromosome and a 2,094,509 bp report is based on a survey of Musa in increased production of improved Musa megaplasmid, which taken together Sabah, Sarawak and Brunei carried out hybrids by small-scale farmers. These encode over 5000 proteins. The chromo- by the first author in August 2001. varieties will produce higher yields and some harbours all essential genes, Although the main focus of the survey will not require chemicals for pest and whereas the megaplasmid is involved in was the section Callimusa, a large num- disease control. In addition, farmers and the biosynthesis of various amino acids, ber of photographs of Musa acuminata entrepreneurs will be assisted to set up cofactors, and fitness to environments. were also taken. With the expertise of banana-related businesses (the produc- There are about 200 candidate genes for Edmond de Langhe, a tentative taxo- tion of planting material for sale etc.), pathogenicity distributed both in the nomic identification of the plants has thus contributing to increased income chromosome and megaplasmid. This been made. generation for rural communities. The information is essential in understanding The photographs showed that all the major beneficiaries will be small-scale the biodiversity in R. solanacearum in plants exhibited the basic characteristics banana farmers. relation to host specificity. Traditionally, of M. acuminata, with the typical top-like R. solanacearum strains are grouped male bud, the horizontal-to-oblique inflo- For more information on the project, please contact Suzanne Sharrock, project coordinator, into five races, based on host range, and rescence and bunch, and rather slender at INIBAP Headquarters. five biovars based on the oxidation on fingers. The flowers appeared to be certain carbon sources. white-to-creamy, but the details were not A new classification scheme has been visible in the photos. Africa proposed based on the molecular analy- The specimens in question were found ses of R. solanacearum. Strains of to fall into four main categories: The 3rd International bacterial wilt R. solanacearum are classified into four • M. acuminata ssp. microcarpa or symposium took place phylotypes such as Phylotype I ‘Asia’ truncata in South Africa – 4-8 February 2002 (include biovars 3 and 4, race 1, 4 • M. acuminata of uncertain status The 3rd International bacterial wilt sym- and 5), phylotype II ‘America’ (biovar 1 • Edible AA diploids posium was attended by 110 scientists and 2, race 1, 2,and 3), phylotype III • Unclassified accessions.

56 INFOMUSA — Vol 11, N° 1 A microcarpa-truncata cluster? The project was funded by ACIAR species in natural habitats in North The most important result of the (Australian Centre for International Vietnam Häkkinen visit to Northern Borneo, an Agricultural Research), INIBAP and Three survey trips were undertaken in area previously little explored for wild VVOB (Flemish Association for natural habitats in North Vietnam and bananas, is the domination of a wild Development Cooperation and Technical root samples from three species of wild M. acuminata population with a combi- Assistance) bananas were taken. With the exception nation of characteristics typical of the The project had two main goals: of R. similis, the most important Musa subspecies microcarpa and truncata. 1. capacity building: to improve the local nematode species, i.e. Meloidogyne spp., The occurrence of yellow-green male bud infrastructure for nematological P. coffeae and Helicotylenchus multicinc- types is the first time this character has research and to train local scientists in tus, were found. This indicates that the been recorded for these subspecies. It is the field of nematology; natural soils in Vietnam are infested with possible that the two supposed sub- 2. scientific research: to gain more these nematode species and that the species may actually form one large and insight into the different aspects of the three wild banana species are suscepti- compound population, and further stud- association between nematodes and ble to these species. ies on this material in a field genebank bananas in Vietnam, in order to are required to confirm the status of the improve local banana production. 2. Assessment of the occurrence and dis- accessions in this group. Specifically: tribution of nematodes on Musa culti- • to obtain a more detailed picture of the vars in North and Central Vietnam M. acuminata of uncertain status occurrence of different nematode Five survey trips were undertaken in six A number of accessions were character- species on bananas in various regions provinces in North and Central Vietnam ized by a moderately to strongly imbri- of Vietnam; and root samples of three commonly culti- cated male bud and a trend for the bract • to increase the knowledge about popu- vated banana genotypes were taken. colour to be pink/red/purple. Male buds lation-dynamics and the damage and Again, Meloidogyne spp., P. coffeae and with visibly imbricate bracts are charac- yield loss potential of the most impor- H. multicinctus were found, but not teristic for the subspecies siamea and tant nematode species on bananas; R. similis. Damage parameters showed burmannica, but are not expected for • to screen Vietnamese Musa germplasm a clear relation with the presence of the acuminata’s in Borneo or the for resistance to the most important certain nematode species in the roots. Indonesian islands, with the exception of nematode species. For example, root-knot galling was posi- Java. Further studies are required to con- tively correlated with the number of firm if these are edible diploids or indeed Meloidogyne spp. in the roots, while root truly wild plants. Activities undertaken and results necrosis was positively correlated with the achieved number of P. coffeae found in the roots. Edible AA diploids A number of plants are recorded as edi- Capacity building 3. Influence of a Pratylenchus coffeae ble AA diploids. Amongst these are plants The general infrastructure of the labora- population and Meloidogyne spp. on that were found to be populating large tories was improved and various items of plant growth and yield of banana areas along the sides of roads, as truly equipment were bought. An e-mail/inter- A field was planted with over 150 banana wild populations do. Since there were no net connection was established in order plants, of which one third was inocu- villages in the proximity, these may be the to improve the communication between lated with P. coffeae, one third with remnants of human population from the different project partners and with Meloidogyne spp. and one third was kept remote time. other nematologists in the world, and to nematode-free (control plants). The pre- have access to information from the liminary results showed that infection Conclusions worldwide web. with P. coffeae and Meloidogyne spp. The findings presented in this report are During the time of the project, two staff can reduce the plant height and the clearly of a preliminary and tentative members of the Agrobiotechnology number of standing leaves in compari- nature, as they are based on studies of pho- Department of VASI followed the post- son with the uninfected control plants. tographs only. However, it does provide a graduate international nematology basis for further studies, which the authors course in Belgium. In this respect: 4. Population-dynamics of a Pratylenchus hope will be stimulated by this report. Duong Thi Minh Nguyet defended her coffeae population collected on Musa MSc thesis entitled: «In vivo and in vitro in North Vietnam Copies of the report, including colour photographs of all the accessions, are available studies of Radopholus similis and The reproduction of P. coffeae on carrot in PDF format from the INIBAP web site Pratylenchus coffeae associated with discs under in vitro conditions could be (http://www.inibap.org/publications/borneo.pdf) or in printed form from INIBAP Headquarters, banana» in 1999 (Promotor: Prof. D. De described by the Gompertz equation: log Montpellier. Waele). (nem + 1) = 0.725 + 2.561 exp [-exp Nguyen Thi Tuyet defended her MSc (1.742 (5.044 - time))]. Study of the association between thesis entitled: « In vitro and in vivo From a greenhouse experiment that nematodes and bananas in Vietnam screening for Radopholus similis resis- was repeated monthly over a period of Inge Van den Bergh, a VVOB/INIBAP tance in Musa» in 2000 (Promotor: Prof. one year, it could be seen that tempera- Visiting Associate Scientist, was based D. De Waele). ture has a strong effect on the reproduc- at the Agro-biotechnology Department tion rate of P. coffeae on bananas. During of the Vietnam Agricultural Science Scientific research carried out by the the winter months, the reproduction was Institute (VASI), Hanoi, Vietnam from VVOB/INIBAP Visiting Associate Scientist very low, while during the summer October 1997 to December 2001 to carry months, the population increased signifi- out a study on the association between 1. Assessment of the occurrence and dis- cantly. The extent of root-necrosis fol- nematodes and bananas in Vietnam. tribution of nematodes on wild Musa lowed more or less the same pattern.

INFOMUSA — Vol 11, N° 1 57 A greenhouse experiment was set up to found. The number of nematodes in the assess the influence of irrigation on the roots seems to be related to the physio- reproduction of P. coffeae. A shortage of logical stage of the plants. The highest water had a very strong negative effect on nematode numbers were found during the plant growth, while the nematodes flowering. could still reproduce well. A very high application of water reduced the general Scientific research by Duong Thi Minh plant growth slightly, but the nematodes Nguyet and Nguyen Thi Tuyet could barely reproduce. An intermediate Duong Thi Minh Nguyet started a water volume was best for the growth of research programme on the occurrence the plants, but also favourable for the of Radopholus similis in Vietnam and its nematode reproduction. morphological and biological aspects. The reproduction of a P. coffeae popula- Two surveys were carried out in Tay tion on banana plants in the field was fol- Nguyen (Western Highlands) to assess the lowed for more than one year. From pre- occurrence of R. similis on coffee, durian, liminary results, it can be seen that bananas, etc. One R. similis population temperature and rainfall have an effect on was collected from durian roots and is the reproduction rate of the nematodes. being maintained on carrot discs under in vitro conditions. Since R. similis is 5. Screening of Vietnamese Musa still a quarantine pathogen in Vietnam, germplasm for resistance to a Duong Thi Minh Nguyet went to Belgium Pratylenchus coffeae population in the for a period of three months to study the

greenhouse collected population. She determined Longitudinal splitting of a banana pseudostem Twenty-four Vietnamese banana geno- that the optimal temperature for repro- due to Panama wilt. types were screened for resistance to duction of R. similis from Vietnam on P. coffeae in the greenhouse. The most carrot discs was 25°C. She also compared promising genotypes are ‘Tieu xanh’, the reproduction of the Vietnamese popu- ‘Tieu mien nam’, ‘Com chua’, ‘Com lua’, lation with populations from Indonesia serious damage to the crop. It was also ‘Man’ and ‘Ngu thoc’. and Uganda. noticed that ‘Nendran’, the most impor- Nguyen Thi Tuyet is studying the biodi- tant commercial variety in the State was 6. Screening of Vietnamese Musa versity of Pratylenchus coffeae in susceptible to Panama wilt while germplasm for resistance to Vietnam. She is collecting P. coffeae pop- Cavendish group, ‘Palayankodan’, Meloidogyne spp. in the greenhouse ulations from various crops and places in ‘Karpooravally’ and cooking varieties Twenty-two Vietnamese banana geno- Vietnam to study the morphological, bio- such as ‘Monthan’, ‘Kanchikela’, etc. types were screened for resistance to logical and genetic diversity between the were not seen to be affected by the Meloidogyne spp. in the greenhouse. No different populations. disease. sources of resistance were found. Several studies were conducted at the Integrated management Banana Research Station, Kannara under 7. Screening of Vietnamese Musa of Panama wilt disease of banana Kerala Agricultural University on inte- germplasm for resistance to in Kerala grated management practices for Meloidogyne spp. in the field Banana wilt caused by Fusarium oxys- Panama wilt disease. The pathogen was Eight Vietnamese banana genotypes, porum f. sp. cubense is one of the serious found to be soil borne and its entry to the ‘FHIA-01’, ‘FHIA-02’ and ‘Yangambi km 5’ threats to banana cultivation in Kerala. host plant was through roots. Since the were evaluated for their host plant The acidic soils of the state and the sus- conidia can survive in the soil for as long response to Meloidogyne spp. under field ceptibility of the major commercial vari- as seven years, a package of agronomic conditions. ‘FHIA-01’, ‘Ngu thoc’, ‘Tay’ eties offer an easy spread of the disease practices are recommended based on the and ‘Com lua’ were found to be less sus- throughout the region, causing a yield findings of the studies. ceptible to Meloidogyne spp. ‘FHIA-01’, loss of 10-15%. Field preparation should be carried out ‘Ben tre’ and ‘Bom’ were less sensitive to The symptoms of the disease appear in a systematic way. In disease affected the knot-forming activity of Meloidogyne with the yellowing of older leaves which locations, weathering the pits for a week spp. The number of juveniles recovered extends rapidly from the margin towards or more and burning the soil with dry from the roots was strongly influenced the midrib. These leaves hang withered leaves is recommended. Field sanitation, by the weather. During the cold and dry around the pseudostem and the infection especially removal of grass weeds is nec- season, the numbers dropped very signif- spreads to all leaves except the top, essary as they become critical alternate icantly. The number of egg-laying which hang down. The heartleaf also hosts. females in the roots (ELF) was much withers after 3-4 weeks. The plant Cultivation of tolerant varieties is sug- less influenced by the environmental exhibits longitudinal splitting with gested for disease prone areas. In other conditions: there was a stagnation dur- bulging and elongation of the pseu- cases, suckers should be selected from ing the cold and dry season but no dostem. When the rhizome is cut open, disease free areas. Dipping pared suckers decline. Meloidogyne spp. seem to over- the discolouration of the vascular bun- in 0.2% solution of carbendazim was also winter as eggs in egg-masses. Root-knot dles can be seen and the cut stem smells found to be an effective prophylactic galling and ELF can be used as easy of rotten fish. measure. parameters to estimate the infection of A survey conducted by Estelitta et al. It was also found that the application Musa with Meloidogyne spp. No effects revealed that the disease was prevalent of lime at 1 kg per plant as a soil amend- of the nematodes on plant growth were in all the districts in Kerala causing ment at the onset of the monsoon and

58 INFOMUSA — Vol 11, N° 1 good drainage were helpful in checking Table 2. Mean seed set in successful crosses. the disease. The studies further indicated that use Name of cross No. of flowers No. of seeds Mean seeds / fruit of organic manures in banana cultivation pollinated obtained could give a better stand of the crop 3x x 3x against the disease, probably due to Karpooravalli x Robusta 150 187 1.250 improved soil structure with more aera- 3x x 2x tion. Karpooravalli x Pisang lilin 185 5 0.027 In the case of disease occurrence, Karpooravalli x H-110 120 8 0.067 removal and destruction of the diseased 2x x 2x plants is recommended to check its Matti x Pisang lilin 30 30 1.000 further spread. Application of lime at H-201 x Anaikomban 102 152 1.490 0.5-1 kg in the diseased plant pits and in H-201 x Pisang lilin 79 427 5.405 the basins of surrounding plants also gave H-201 x H-110 53 90 1.700 encouraging results in checking the fur- H-201 x Anaikomban 11 24 2.182 ther spread of the pathogen. H-201 x Pisang lilin 12 4 0.333 From experiments carried out on dif- H-201 x Robusta 11 1 0.091 ferent chemicals to control Panama wilt, it was found that corm injections with 2% solution of carbendazim at 3ml/corm during the 5th, 7th and 9th month after varieties, including commercial triploids, ticularly in the synthesis of triploids of planting could help to control the dis- diploids and TNAU-bred synthetic bispecific origin. Seed production was ease. Drenching the soil with 0.2% car- hybrids were included in the study (see maximum in H-201 x Pisang lilin followed bendazim was also found to be effective. Table 1). Anthers were collected from the by H-201 x Anaikomban and Karpooravalli Since the commercial banana varieties male parents just prior to dehiscence and x Robusta. In other successful combina- in Kerala are often cultivated in exten- the pollen grains were extracted and tions, seed production was very low sive wetlands, studies indicated that crop smeared on the stigma of the female flow- despite the crosses being compatible. rotation, intermittent fallowing or flood- ers of female parents on the day of opening followed by fallowing are also effec- ing in the early morning between 6.00 to References tive ways of reducing the spread of the 9.00 a.m., when the receptivity of stigma Alexander M.P. 1970. Mega and microsporophyte fer- disease. was good and ensured by stickiness by tility of some banana varieties. Pp. 27-28 in touch. After pollination, the flowers were Proceedings of the 3rd International Symposium Further information is available from S. Estelitta, Associate Professor, Kerala Agricultural covered with perforated paper bags. Once on Tropical and Subtropical Horticulture. Today University, Mannuthy, Thrissur, Kerala, S. India. ripe, the fingers were longitudinally cut and Tomorrow Publishers, New Delhi. and the seeds, if present, were extracted Sathiamoorthy S. & T.N. Balamohan. 1993. Cross compatibility of some banana carefully. Improvement of banana. in Advances in clones Among the 74 cross combinations Horticulture Vol. I - Fruit Crops Part I. (K.L. Before initiating a hybridization pro- tested, compatibility was found only in Chadha and O.P. Pareek, eds). Malhotra gramme in banana breeding, cross-com- eight combinations (Table 2), thus indi- Publishing House, New Delhi, India. patibility between desirable parents has cating the existence of compatibility More information about this work is available to be assessed. Such work is presently on- between clones. The successful crosses from: V. Krishnamoorthy, Dept of Fruit Crops, going at Tamil Nadu Agricultural were between diploid x diploid and Horticultural College and Research Institute, University (TNAU) in India. Seventeen triploid x diploid. Out of the 10 male TNAU, Coimbatore-641003, Tamil Nadu, India. parents tested, ‘Pisang lilin’ and ‘Anaikomban’ were compatible with all Collecting banana germplasm in female parents. ‘Nendran’ which was ear- northeastern India Table 1. Details of parents used. lier reported as female sterile (Alexander The northeastern states of India, namely Female parents Male parents 1970) was confirmed as female sterile in Assam, Arunachal Pradesh, Megalaya, Triploids Triploids this investigation. The study indicated Tripura, Mizoram and Manipur are a rich Karpooravalli (ABB) Robusta (AAA) that with the exception of ‘Karpooravalli’, source of natural diversity in Musa. Since Red banana (AAA) Red Banana (AAA) the clones of commercial importance 1998, INIBAP has been supporting a Rasthali (AAB) have a very low percentage of female fer- series of Musa collecting missions in this Nendran (AAB) tility, and diploids provide the best region. These have been conducted by the Diploids Diploids female fertile clones. Seed set was how- National Research Centre for Banana Nivediyakadalli (AA) Nivediyakadalli (AA) ever good in the triploid x triploid cross (NRCB), Trichy. Specimens of the wild Matti (AA) Pisang lilin (AA) of Karpooravalli x Robusta, which indi- species Ensete glaucum, Musa bal- Sannachengadalli (AA) Sannachengadalli (AA) cates the possibility of new line in banana bisiana, M. acuminata, M. ornata and Anaikomban (AA) Anaikomban (AA) breeding, bringing the ‘Cavendish’ other Rhodochlamys species have been Ambalakadalli (AA) Ambalakadalli (AA) genome into new hybrids. H-201 collected, together with a range of culti- Ney poovan (AB) Erachivazhi (AA) (Pedigree: Bareli chinia x Pisang lilin x vated varieties. All the collected material Synthetic hybrids Synthetic hybrids Robusta) is a good female parent and has has been established in the NRCB H-59 (AA) H-59 (AA) hybridized with diploid as well as triploid genebank for formal identification and H-97 (AA) H-97 (AA) parents (viz. Robusta). Sathiamoorthy characterization. H-66 (AAA) H-66 (AAA) and Balamohan (1993) reported that In Arunchal Pradesh, it was noted that H-201(AB) H-201(AB) H-201 was a potential female parent par- where Eumusa and Rhodochlamys

INFOMUSA — Vol 11, N° 1 59 Table 3. Details of wild Musa species collected during the exploration in Musaid.win, a software developed by northeastern India. CIRAD to assist in the identification of unknown varieties, was also explained by Genus Section Species Site of collection No. of accessions Use Xavier Perrier from the Biometry Unit of Ensete E. glaucum Assam, Tripur, Mizoram 1 Fibre, CIRAD. This software is provided to all vegetable, ornamental participants in MGIS. Musa Eumusa M. balbisiana Assam, Tripur, Mizoram 1 Fruit, vegetable Participants agreed that the training M. acuminata Assam, Tripur, Mizoram 9 Fruit, vegetable provided a useful tool for the manage- Rhodochlamys M. ornata Assam, Mizoram 1 Ornamental ment of genetic resource information and Unidentified Assam, Tripur, Mizoram 5 - highlighted the importance of collecting and managing this information using a standard format. The workshop also provided a valuable opportunity for curators bananas grew together, the Rhodochlamys Training course on MGIS in Africa to make contact with their colleagues had a closer eco-compatibility with from the region as a whole, and also to M. acuminata than M. balbisiana, 22–27 April 2002, CARBAP, Nyombé, identify resource people who can help although M. acuminata was clearly domi- Cameroon them in their future work. nant over the Rhodochlamys species. In A training course on the use of the Musa Following the MGIS training course, a Assam, Bhimkol, a seeded balbisiana Germplasm Information System (MGIS) workshop on the “Names and synonyms clone is widely grown in backyards for its for the management of information of plantains” was held with the partici- medicinal qualities. Although seeded, the related to genetic resources of bananas pants from West and Central Africa. seeds are soft enough that the fruit can be and plantains (Musa spp.) was held Dr Kodjo Tomekpe conducted the work- consumed along with the seeds. recently for Musa germplasm curators shop using data and photos from MGIS. A Throughout the region, the unusual prac- from Africa. The objective of this training first draft list of variety names has been tice of selling male buds was noticed. The course was to provide these curators with established that should be confirmed by immature male buds of wild bananas are the expertise and tools to better manage further studies of the varieties in the field. harvested even before shooting and are information related to the accession in INIBAP gratefully acknowledges the used for the preparation of special dishes. their collections. The use of MGIS will valuable support of CTA and CARBAP in A summary of the wild species col- also allow them to exchange genetic the organization of the training course. lected during the missions is provided in resource information with other Table 3. researchers and curators throughout the List of participants world. This training course was held thanks to funding support provided by the From national organizations INIBAP News Technical Centre for Agricultural and Sylvestre M. Rogers, Rice Research Rural Cooperation (CTA). Station, Rokupr, Sierra Leone; New staff The training course gathered 23 partic- Guilavogui Zeze, Institut de recherche Hélène Laurence, an intern funded by ipants from West, Central, Eastern and agricole de Guinée (IRAG), Guinea; the Ministère des Relations interna- Southern Africa (see list below). The Simplice Koffi Kouassi, Centre national tionales du Québec joined INIBAP as a course was held in French and English, de recherche agronomique (CNRA), Côte Research Assistant in January 2002. She with translation ensured by the trainers. d’Ivoire; is based at the Regional Office of INIBAP All documents and training materials Lawrence Aboagye, Plant Genetic in Kampala and will begin work on a were provided in both languages. Resources Centre, Niaouli, Ghana; 3-year study of the impact of improved The course included both field and Flore Sindemion, Institut national de banana varieties (Musa) on the liveli- computer-based training. Exercises on recherches agricoles du Bénin (INRAB), hoods of households in Eastern Africa. the taxonomical and botanical identifica- Benin; She will work closely with INIBAP scien- tion of varieties were held in the field Antoine Mputu Kena Kudia, Institut tists and regional NARS partners. Hélène using the list of descriptors published by national pour l’étude et la recherche has a BSc in geography and a MSc in IPGRI, INIBAP and CIRAD. The large agronomique (INERA), DRC; Agrometeorology. germplasm collection maintained by Clotilde Ngnigone Ella, Institut de Olivier Guinard, also funded by the CARBAP provided an excellent resource recherches agronomiques et forestières Ministère des Relations internationales for the field exercises. This collection (IRAF), Gabon; du Québec, joined the INIBAP pro- consists of over 400 accessions, repre- Fernand Mouketo, Centre de recherche gramme in Montpellier as an intern in senting a very large range of African vari- agronomique de Loudima (CRAL), April 2002. During his 6-month internship eties, especially plantains, but also Congo; he will be working on a project using the including some East African highland Selome Y. Dogbe, Institut togolais de la MGIS database (Musa Germplasm varieties. recherche agronomique (ITRA), Togo; Information System) to carry out a series With regard to the computer-based Olagorite Adetula, National Horticultural of tracer studies on important germplasm training, participants learnt how to Research Institute (NIHORT), Nigeria; accessions as well as visualizing geo- install the MGIS software on their com- Robert Muhwezi, National Agricultural graphical assignments for the different puters, how to create users accounts, Research Organization (NARO), Uganda; accessions. Olivier comes from Québec enter new records, and carry out informa- Mkulila Shaban, Agricultural Research where he studied for his BSc in Biology, tion searches in the global database. They and Development Institute (ARDI), specializing in molecular biology/biotech- were also trained in the procedures for Tanzania; nology at the University of Quebec in data exchange through the global data- Margaret Onyango, Kenya Agricultural Montréal and has just completed his MSc. base. Research Institute (KARI), Kenya;

60 INFOMUSA — Vol 11, N° 1 Antoine Nsabimana, Institut des sciences • Identification of where gaps exist in plantains and Musa production in Benin agronomiques du Rwanda (ISAR), the coverage of collecting missions has increased from 22 000 tonnes in 1998 Rwanda; (comparison with production areas); to 45 000 in 2001. He hoped that the Ferdinand Ngezahayo, Institut de • Documentation of the potential effects research carried out in the network will recherches agronomique et zootechnique of climate change and other potential help find solutions to constraints facing (IRAZ), Burundi; genetic erosion factors on wild species farmers in Benin such as lack of clean Dickson L.N. Banda, Department of distribution; planting materials, pests and diseases Agricultural Research and Technical • Comparison of species distribution in and post-harvest transformation of the Services, Malawi; relation to pests and diseases; fruit. Dejene Abera, Ethiopian Agricultural • Predict and assess impact, e.g. of new Research Organization (EARO), varieties. Adoption of the report of the Accra Ethiopia; More information about the DIVA soft- meeting Connie Fraser, Institute for Tropical and ware is available from the CIP website The first order of business was the adop- Sub-Tropical Crops (ITSC), South Africa. (http://www.cipotato.org/diva/). The soft- tion of the minutes of the last regional ware is freely available and can be down- steering committee meeting held in From regional/international loaded, along with the user’s manual, Accra in April 2001. Before members organizations from the website. would do that, they wanted to know if the William Nguefack, Centre africain de recommendations of that meeting had recherches sur bananiers et plantains Fifth meeting of the regional been implemented. The President and (CARBAP), Cameroon; steering committee of MUSACO secretary provided the following informa- Chyka Okarter and Perpetua Udu, From 11 to 12 February 2002, the regional tion on the various recommendations International Institute of Tropical steering committee of the banana and that were made in Accra: Agriculture (IITA), Onne research sta- plantain research network for west and • The training course on Mycosphaerella tion, Nigeria; central Africa, MUSACO, met in Cotonou, leaf spot diseases was held in Malaysia Emmanuel Njukwe, International Benin for its annual meeting. In atten- in June 2001. Drs Kobenan Kouman of Institute of Tropical Agriculture (IITA), dance were representatives from Benin, CNRA, Côte d’Ivoire and Ekow Mbalmayo, Cameroon; Cameroon, Congo Republic, Côte d’Ivoire, Akyeampong were the two partici- Deborah Karamura, INIBAP-ESA, Gabon, the Democratic Republic of pants from West and Central Africa. A Uganda. Congo, Ghana, Guinea, and Sierra Leone second training course on the post- as were those of CARBAP (formerly laboratory handling of tissue-cultured Resources persons: CRBP) IITA and INIBAP. Togo was admit- plantlets and on rapid multiplication Kodjo Tomekpe CARBAP, Cameroon; ted as the 13th member of the network at of Musa planting materials was orga- Ekow Akyeampong, INIBAP – MUSACO, this meeting. nized at CARBAP, Nyombé from 2 to Cameroon; In a speech to officially open the meet- 7 December 2001 for francophone Elizabeth Arnaud and Suzanne Sharrock, ing, Dr David Arodokoun, Scientific member countries of the network. INIBAP Headquarters, France; Director of the Institut national des • The nine countries which received recherches agricoles du Bénin acting on funds from INIBAP have collected and Xavier Perrier, CIRAD, France. behalf of his Director General stressed sent the secondary baseline informa- that banana and plantain are crops that tion to the network secretariat. A Geographical Information could contribute to food and nutritional report is being prepared by a young Systems (GIS) and Musa diversity security, alleviation of poverty, and the professional officer seconded to the INIBAP is investigating the possibility of creation of employment in Benin. Banana office by the FAO. using a newly developed GIS software and plantain are two of the crops that • Concerning participation in PROMUSA (DIVA) for the spatial analysis of Musa Benin is now promoting to diversify the working groups by scientists from the genetic resource information. DIVA has food crop base. He reported that 33% of sub-region, only Benin, Côte d’Ivoire, been developed by the International households in Benin regularly consume Gabon and Ghana sent names of Potato Centre (CIP) with the support of FAO, the CGIAR’s System-wide genetic resources programme (SGRP) and IPGRI. A training course was held recently at INIBAP’s headquarters in Montpellier to train staff in the use of the software and to assess the potential to use this software in association with data available in the Musa Germplasm Information System (MGIS), in particular with data recorded during collecting missions. INIBAP hopes to use the DIVA- GIS software for the following tasks: •Prediction of where particular species/varieties can be found; • Prediction of where germplasm with specific traits is likely to be found; • Identification of high-diversity areas at genetic and species level; Participants to the 5th MUSACO meeting.

INFOMUSA — Vol 11, N° 1 61 researchers to the PROMUSA secre- annual crop at densities from 2500 to hybrid, ‘CRBP-39’, which maintained six tariat through Dr Adiko, the West and 5000 plants per hectare. green leaves to harvest. Tests to deter- Central Africa representative on the Ghana’s new representative on the mine consumer acceptance of the fruits steering committee of PROMUSA. steering committee, Dr Anno-Nyako are being conducted but the results are • As at the time of the meeting, nothing informed the meeting that Musa breed- not yet available. had been heard from CORAF regarding ing has started in Ghana. A plant biology The periurban Musa production project the eight initiatives written in collabo- laboratory with a tissue culture unit has is making good progress in Benin with the ration with CARBAP that were sent in been established in Gabon. vitroplantlets provided by CARBAP. response to its call for projects. Being the first time to participate in Among the hybrids established in Benin •On the question of IITA scholarships, the meeting, the Togolese representative, are ‘FHIA-25’, ‘FHIA-18’, ‘FHIA-23’ and members were informed that Mr Ben Dr S. Dogbe, gave an overview of the ‘CRBP-39’. Survival of plants in the field Banful, former representative of Ghana research on banana and plantain in his was very high so is the enthusiasm of the on the MUSACO steering committee country. Bananas and plantains are culti- 40 farmer participants. The visit to some has received an IITA scholarship for vated in the cocoa/coffee zone as a shade of the farms generated a lot of interest as PhD studies. for the cash crops. The programme has a the plants were doing very well. The field genebank consisting of 32 acces- researchers and farmers exchanged Country Musa news sions many of which originate from views on farmer participatory research Each country representative gave a brief Ghana. A major problem confronting the approaches. These discussions continued report on new activities that are taking production of the crops in Togo is the lack when the scientists returned to the meet- place in their countries. To popularize of good quality planting materials. ing place the next day. A survey on periur- banana and plantain production in Sierra ban Musa production conducted in Benin Leone, nurseries and demonstration plots Updates on regional projects revealed that 56% of Musa farmers culti- have been established in four districts. Members reported on the two regional vate plantain on an average plot size of These will be extended to four more dis- projects, Musa germplasm evaluation 0.8 ha. In Benin also, inadequate supply tricts. Also, local varieties have been col- and periurban Musa production. of planting materials is cited a bottleneck lected and planted for characterization Germplasm evaluation trials have been to expanded production purposes. established in almost all the network The periurban project has not really As in Sierra Leone, to re-launch plan- countries but it is only in Côte d’Ivoire taken off in Ghana as the national insti- tain and banana production in Littoral that the first crop has been harvested. It tution that was supposed to supply the and in the Forest regions of Guinea was reported that ‘FHIA-23’ is the most planting materials failed to do so. (Conakry), nurseries have been estab- productive among the bananas (‘FHIA-01’, Materials imported from South Africa lished to produce clean planting materials ‘FHIA-18’ and ‘SH-3460’) that are being have been weaned and will be supplied that will be distributed to farmers. evaluated in Côte d’Ivoire, but it also has to farmers at the beginning of the rainy Among the varieties to be given to farm- the longest crop cycle. The inoculum season. ers are hybrids of IITA and CARBAP pressure of black leaf streak disease Studies are being planned in Côte (BLSD) was so high that at harvest, International and regional institutions d’Ivoire to attempt to explain the obser- ‘Orishele’, the susceptible plantain vari- CARBAP and IITA each sent a delegation vation that Pratylenchus spp. seems to ety had virtually no functional leaves. of several scientists to present the differ- be replacing Radopholus similis as the Consequently, bunch weights of ‘Orishele’ ent areas of Musa research at their cen- main nematode on Musa. IITA is inter- were low compared to the resistant tres. Dr Lutaladio representing FAO ested in the issue of changes in species talked about collaboration with the net- composition and will consider offering a work. doctoral fellowship for work on this in CARBAP presented the advances of collaboration with KULeuven. It was rec- their programmes regarding plantain ommended that a nematological survey breeding, agronomy and integrated sys- be conducted in all member countries to tems, plant pathology and pest control determine if the nematode diversity and with an integrated pest management the relative abundance of the species approach (leaf spot diseases, nematodes remain the same as before. and weevil), post-harvest technologies The other new activity in Côte d’Ivoire and socio-economics. Achievements of was the high planting density technology the centre include the development and that Ivorian scientists are testing on-sta- transfer of in vivo multiplication tech- tion. The high planting density trials niques to produce large numbers of clean being conducted in Côte d’Ivoire and also planting materials, and ‘CRBP-39’, a plan- in Cameroon are the follow-up of a visit tain-like hybrid that has been released made by ten scientists, farmers and and distributed within the MUSACO net- extension agents from West and Central work and to more than 40 countries Africa to the Dominican Republic and around the world in the framework of Costa Rica to study the high density plan- the 3rd International Musa Testing tain plantation production technologies Programme coordinated by INIBAP. being used there. The delegation was Secondary triploids with resistance to impressed by the up to 60% yield diseases have been created by different increases that have been obtained in the breeding schemes and short and early

Latin America and Caribbean region from ‘CRBP-39’, one of the hybrids selected for the yielding plantain hybrids are also under managing ‘False horn’ plantain as an periurban project. (Photo: CRBP) evaluation. Banana and plantain-based

62 INFOMUSA — Vol 11, N° 1 snack foods, infant formulas and flour the different tasks he was given at the profitable plantain production systems have been developed. beginning of his assignment. and (c) farmer participatory research Presentations from IITA were on par- In the immediate future the FAO will should be created immediately within ticipatory research, integrated pest man- assist at least two MUSACO member the network; agement, Musa breeding and agronomy. countries in designing projects for 5. A grant proposal should be developed In farmer participatory evaluations con- improvement of banana and plantain pro- to look for funds to equip all national ducted in eastern Nigeria, farmers pre- duction for small-scale growers through programmes with basic information ferred ‘PITA-14’ one of IITA’s plantain- the setting up of efficient and cost-effec- technology equipment and Internet type hybrids over ‘Agbagba’, the local tive multiplication systems for produc- access. variety. ‘PITA-14’ gave higher financial tion of disease-free planting materials. returns as well. The meeting was The FAO will collaborate with MUSACO, MUSACO 2003 informed of a pilot project supported by CARBAP and IITA to collect and charac- The next steering committee meeting will USAID in which hybrids developed at terize Musa germplasm in the Congo be hosted by IRAG in Guinea (Conakry) CARBAP, IITA and FHIA are being evalu- basin, to upgrade tissue culture and nurs- during the first week of March 2003, ated in farmers’ fields in Nigeria. It is ery facilities in certain countries, and to under the presidency of Mr Bernadin hoped that after the pilot phase, the pro- train researchers in handling tissue cul- Lokossou, the representative of Benin ject will be expanded to other countries ture plantlets, virus indexing, and rapid who was elected to replace Madam Adèle in the West and Central Africa. IITA con- production of planting materials. Finally, Sambo of Gabon. tinues to develop plantains hybrids with the FAO will assist in the development of superior resistance or tolerance to dis- a protocol for mass propagation and dis- 2nd International workshop eases, including a practical physiological- tribution of quality planting materials. on Mycosphaerella leaf spot genetic and biotechnological approach to diseases of bananas control banana streak virus, and good Functioning of the network agronomic characteristics such as earli- The delegates discussed ways to improve San José, Costa Rica, 20–23 May 2002 ness, short stature, and good rooting. the operation of the network. For a start, This workshop was organized by INIBAP Integrated pest management research it was agreed that working groups based in collaboration with the Corporación has included developing and testing on identified research priorities should Bananera Nacional (CORBANA), the methods such as using hot or boiling be set up. These will meet as often as nec- Escuela de Agricultura de la Región water to clean suckers contaminated essary depending on available resources. Tropical Húmeda (EARTH) and the with pests. There are also studies to Working groups are to be formed immedi- Centro Agronómico Tropical de determine the efficacy of nematicidal ately on (1) rapid multiplication of clean Investigación y Enseñanza (CATIE). plants such as Flemingia interplanted in planting materials; (2) profitable plan- Coming 13 years after the last interna- plantain fields. tain production systems and (3) farmer tional meeting on this topic, it provided a The four projects of INIBAP, namely participatory research. The secretary of timely opportunity to analyse the current germplasm management, germplasm the network was asked to identify leaders situation regarding Mycosphaerella leaf improvement, information and communi- for each of the three working groups. spot diseases at the global level. The cations and regional networks were Activities proposed by the working groups meeting also allowed new lines of investi- briefly described and activities in Africa will be approved and their implementa- gation to be suggested and facilitated the under each were mentioned. The objec- tion monitored by the regional steering re-orientation of breeding programmes tives and modus operandi of PROMUSA, committee. The current unsatisfactory and biotechnology strategies for the the global programme for Musa improve- communication links among members genetic improvement of bananas and ment, coordinated from INIBAP head- and between members and the secre- plantains. quarters were also described. tariat was attributed to the lack of infor- More than 60 scientists attended the The FAO representative at the meeting, mation technology equipment in many of workshop from both the private and pub- Dr Lutaladio, traced the history of the the research stations where the Musa lic sectors, representing more than 16 dif- collaboration between his department programmes are based. ferent countries from Latin America and and INIBAP. In 1999 AGPC/FAO and INI- the Caribbean, Europe, Africa, Asia and BAP discussed collaboration on the gath- Recommendations the Pacific, including Australia. ering and exchange of information and The assembly passed the following rec- In order to maximize the outputs of the the transfer of technology. A young pro- ommendations: meeting and to guarantee the development fessional officer (YPO) was recruited and 1. A survey should be conducted in all of new strategies in the control of the dif- posted to the INIBAP/MUSACO secre- member countries to determine the ferent Mycosphaerella leaf spot diseases, tariat to develop instruments for the col- diversity and prevalence of Musa participation in this workshop was by invi- lection and compilation of baseline infor- nematodes in West and Central Africa; tation only. The results of the meeting will mation and to incorporate collected 2. INIBAP, IITA and CARBAP should however be widely disseminated through information into HORTIVAR, a database assist MUSACO to organize training the publication of the proceedings. on performances of horticulture cultivars courses on germplasm evaluation and The meeting was inaugurated by in relation to environmental conditions on participatory research methods; Dr Jorge Sauma, Director of CORBANA. and cultivation practices. In addition, the 3. All should wait for results on the high- Dr Emile Frison, Director of INIBAP, wel- YPO was to provide inputs in the urban density planting technology trials being comed all the participants and paid trib- and periurban horticulture programme in conducted by CNRA in Côte d’Ivoire ute to Ramiro Jaramillo Celis, former relation to food security and was to assist and CARBAP in Cameroon before dis- INIBAP regional coordinator for Latin in project proposal development. Dr seminating the technology to farmers; America and the Caribbean, in recogni- Lutaladio informed the meeting that the 4. Working groups on (a) rapid multipli- tion of his invaluable contribution and YPO has prepared preliminary reports on cation of clean planting materials; (b) tireless efforts to the development of the

INFOMUSA — Vol 11, N° 1 63 regional Musa research network. The official opening was made by Dr Salvador Monge, Executive Director of the Secretary of sector-based planning of the Ministry of Agriculture of Costa Rica. The workshop was organized around five main topics: 1) Impact of Mycosphaerella leaf spot diseases of bananas; 2) Population biology and epidemiology; 3) Host-pathogen interactions; 4) Genetic improvement for a management of durable resistance and 5) Integrated disease management. During the workshop, participants had the opportunity to learn about the distribution and impact of the different Mycosphaerella leaf spot diseases in several countries around the world. Discussions were held at the end of the Sharing information on Mycosphaerella leafspot diseases during the visit at the CORBANA research each session to allow research priorities station in Guápiles. (Photo: C. Picq, INIBAP) and corresponding activities required at the global level to be identified or refined, gated. Further surveys in South and Session 3. Host-pathogen interactions in order to significantly reduce the impact Southeast Asia to determine where Several cases of an unexpected level of of these diseases and thus make Musa a M. musicola, M. fijiensis and M. eumusae susceptibility to black leaf streak disease more sustainably productive crop. occur are necessary. More information on (BLSD) have been reported. Although the effect of M. eumusae on the growth different reasons have been offered to Session 1. Impact of Mycosphaerella and yield of banana clones is needed. explain the phenomenon (poor nutrition, leaf spot diseases of bananas Information suggests that Cavendish and environmental stress), the problem of the Introductory papers presented informa- Plantain cultivars are very susceptible. erosion of resistance cannot be ignored tion on the global spread, current dis- and requires a precise characterization tribution and impact of the three Session 2. Population biology of the pathogen population. A greater Mycosphaerella leaf spot pathogens - and epidemiology understanding of the mechanisms M. musicola, M. fijiensis and M. eumusae. Pathogenicity and distribution variabil- involved in plant-pathogen interactions Other papers described techniques devel- ity, sources of resistance, epidemiology continues to be needed to ensure the long oped in Australia for the rapid diagnosis and population structure of the main term success of breeding programmes. of M. musicola and M. fijiensis, the species (M. fijiensis, M. musicola and Further studies are also needed to com- effects of M. musicola and M. eumusae M. eumusae) at the national, regional pare the effect of infection by each of the on banana cultivation in South Africa and and international levels were defined as three pathogens (M. fijiensis, M. musi- the impact of M. fijiensis in Cuba, Brazil fundamental information for the contin- cola and M. eumusae) on the host plants. and tropical Asia. The latest taxonomic ued success of banana production. Such Other pathosystems (such as work undertaken on the anamorph of studies are particularly necessary in Magnaporthe grisea) have shown the M. eumusae and on other Mycosphaerella Asia, which is the centre of diversity of powerful nature of the genetic approach species was also described. M. fijiensis the three pathogens and where little to identify without any a priori the path- continues to spread to new areas. From research has so far been conducted. A ogenicity factors. These approaches 2000 to 2002, the pathogen was identified study of the evolution of host–pathogen include the study of gene expression dur- for the first time in Madagascar, the relationships for the three pathogens, ing production of pathogenicity mutants, Bahamas, and the Galapagos Islands of particularly involving resistant cultivars, comparative genomic and gene function Ecuador and in the north Queensland is of a special concern, in order to iden- validation techniques. Consequently, the banana growing area where eradication tify pathogen populations that could development of genetic and molecular is being attempted. M. eumusae leaf spot break down plant resistance and to eval- biology tools for M. fijiensis in collabora- has also been observed on ‘Mysore’ (AAB) uate selection pressure. Molecular tools tion with M. graminicola groups and the in Sri Lanka. As this clone has strong such as microsatellites have been rec- launching of a genomic initiative to resistance to M. musicola and M. fijiensis, ommended to monitor the genetic access genomic tools and set up a this is the cause of some concern. variability of the pathogen populations genomic-wide comparison of M. fijiensis It was agreed that more taxonomic and pathogenicity should be evaluated with M. graminicola have been recom- information about Mycosphaerella spp. is concurrently. Epidemiological studies, mended. needed, as well as information on other including disease dispersal, are needed It is also recommended that the differ- related genera that either form or occur to better understand the distribution ent mechanisms of resistance (partial or in banana leaf lesions. A greater knowl- and the spread of the pathogen and will vertical) should be studied. edge of Mycosphaerella pathogens complement, together with the studies /saprophytes and those in related genera on pathogenicity and genetic variability, Session 4. Genetic improvement for a is a prerequisite to the development of all the information required to antici- management of durable resistance rapid diagnostic tests to distinguish leaf pate the evolution of pathogen popula- During this session, progress that has spot pathogens. The exact distribution of tions and to define resistance manage- been made towards the creation of new M. eumusae also needs to be investi- ment strategies. varieties resistant to BLSD, either

64 INFOMUSA — Vol 11, N° 1 through conventional and/or modern expected that the proceedings will remove the subjective nature of colour technologies, was presented. New become a reference document of recording, and leads to a common under- tetraploids hybrids resistant to BLSD are Mycosphaerella diseases for the next ten standing of such characters. already available and some of these are years. Copies of the descriptors and their widely grown around the world. However, addendum are available from INIBAP because of the lack of new sources of Headquarters. resistance and due to the presence of the Books etc. activable form of the banana streak virus Resúmenes analíticos de (BSV) in interspecific hybrids (A x B), la investigación sobre plátano the production of a new generation of en Colombia triploid hybrids is seriously jeopardized. Strategy for the Global Musa Edited by D.G. Cayón and F. Salazar Good progress has been reported in the Genomics Consortium Alonso development of a molecular toolbox for Report of a meeting held in Arlington, ISBN: 958-96885-1-9 bananas and plantains in the area of the USA, 17-20 July 2001 CORPOICA could certainly be proud for genetic transformation, allowing the pro- ISBN: 2-910810-48-08 the effort they made recently to identify, duction of transgenic banana plants. The Global Musa Genomics Consortium analyse and compile the existing infor- The study of the diversity of the Musa was launched at a meeting held in balbisiana genome, using both morpho- Arlington, Virginia, which was critical for logical and molecular characterization was the first recommendation from this session. It was also recommended, that in anticipation of the needs of genetic improvement programmes, the T and S genomes, Musa textilis and Musa schizo- carpa respectively, should also be screened. Mutation induction techniques should no longer be seen as an independent genetic improvement strategy but more mation on plantain research and technol- as a tool that can contribute to cross- ogy transfer in Colombia under the title breeding programmes by increasing ‘Resúmenes Analíticos de la genetic diversity of parental lines. Investigación sobre Plátano en Mutants could also help in understanding Colombia’. This information product is a the mechanisms of resistance (functional laying a solid foundation for future col- unique inventory of the major part of the genomics). laboration in Musa genomics and allowed scientific literature published on this the first important steps to be taken topic including grey literature. It Session 5. Integrated disease towards the development of a coherent includes 792 abstracts and authors- and management strategy for Musa genomics. thematic indexes which make the search Yellow and black Sigatoka control strate- This document provides further back- easier for the reader. gies on banana can, according to the ground information about the establish- This important document in Spanish is country and the scale of production, ment of the Consortium and its aims and available in both electronic (database on include not only chemical and cultural objectives. It also provides a review of the CD-Rom) and printed (400 pages) for- practices but also the use of mixed crops current status of Musa genomics mats and will certainly be highly appreci- or resistant clones. The important research and provides details of the ated by all those working on plantain at inhibitory effect of some natural sub- nature and scale of the work to be carried international level. stances derived from microorganisms out by the Consortium members. The document is available on request antagonistic to fungi, have also been Information is provided on an incremen- at CORPOICA, Apartado Aéreo No 1087, reported as effective in reducing the tal strategy developed by the Consortium Av Bolivar Sector Regivit 28 Norte, development of M. fijiensis in vitro. to achieve its goals and the proposed Armenia, Quindío, Colombia – Fax: (57- The integration of various or specialists modus operandi, as agreed during the 6) 7496331 and at the INIBAP Office for from different disciplines has been rec- Arlington meeting. Further details are Latin America and the Caribbean, C/o ommended to facilitate the development provided in the Annexes. CATIE, 6170 Turrialba, Costa Rica. of an achievable integrated pest manage- Copies are available from INIBAP ment (IPM) approach for banana leaf Headquarters. Banana varieties: The ACIAR years spot diseases. The participants in the 1987-1996 workshop also recommended to investi- Addendum to the ‘Descriptors for J.W. Daniells and N.J. Bryde gate the potential of natural or synthetic Banana (Musa spp.)’ Information series Q101013 substances able to promote or activate To complete the revised version of the ISBN 0727-6273 systemic acquired resistance in its broad- ‘Descriptors for Banana, Musa spp.’ and During the period 1987-1996, the est sense. responding to demand from East Africa, Queensland Department of Primary The proceedings of the workshop will additional characters specific to the East Industries (QDPI) was the lead agency of be published shortly by INIBAP. This pub- African Highland bananas were incorpo- the ACIAR project ‘Banana improvement lication will include the full papers of all rated as an addendum in 2001. The in the South Pacific’. In the course of the presentations and a summary of the dis- descriptors for Musa are unique in project, banana varieties were collected cussions and recommendations. It is including a colour chart. This helps to from all around the world. One hundred

INFOMUSA — Vol 11, N° 1 65 and six varieties are reported in this pub- Announcements with support of INIBAP, the Food and lication which represent significant Agriculture Organization of the United cross-section of those available for evalu- Nations (FAO), Department of Agriculture ation. They include some hybrids from 3rd International symposium on and Cooperation, Government of India and conventional breeding programmes, the molecular and cellular biology Indian Council of Agricultural Research, is selections originating as offtypes from tis- on Musa organizing a global conference on banana sue culture propagation, existing culti- 9-11 September 2002, Leuven, Belgium and plantain focusing on ‘Banana produc- vars and wild species. The first symposium, held in March 1999 at tion for nutrition and livelihood security’. The report put together the agronomic Cornell University in Ithaca, USA, was This Conference is being organized information collected along with colour organized to open a forum where all those with the following objectives: photographs of bunches which permit a interested in molecular and cellular biol- •To bring together global players in good appreciation of each variety. Many ogy had the opportunity to meet and banana research, development and readers will also find these photographs exchange ideas about their research activi- trade for deliberation and discussion useful for identification purposes. ties. The meeting was a resounding suc- on various issues for sustainable devel- cess. It was therefore suggested that this opment of banana. Clean & green bananas – Where to concept be continued under the auspices •To deliberate upon the opportunities from here? of PROMUSA. After the 2nd International for Indian banana and banana products J.W. Daniells symposium on the Molecular and cellular in international trade. Information series Q101014 biology of bananas held in October 2001 in •To involve national and international ISBN 0727-6273 Byron Bay, Australia, INIBAP and experts to develop and recommend pol- Current sales for clean & green/organic KULeuven are pleased to announce the 3rd icy initiatives for nutritional and liveli- bananas are very limited in Australia. International symposium on the Molecular hood security through banana produc- Organic export is risky and uncertain. and cellular biology of bananas, organized tion. However, the market is shifting in this by INIBAP and KULeuven, to be held in The Conference deliberations will focus direction and in the longer term, the Leuven, Belgium from the 9th to the 12th on the following topics: organic niche will probably eventually September 2002. • Genetic resource management and grow to 10-15% of the market. Major sales The scientific programme includes five crop improvement; of both products will be facilitated by sessions. During the event, more than • Biotechnological advances; supermarket participation and the cur- 30 papers and 31 posters will be presented. • Strategies in production technology; rent price of the existing organic product Twelve keynote lectures will also be • Organic production of banana; will need to come down. Efficiencies of given by outstanding speakers: • Integrated disease and pest manage- production must be improved by specific Prof. Francis Quétier, Dr Xavier Draye and ment; research on limiting factors such as soil Prof. Guido Volkaert (Session 1: Genomics), • Post harvest management, product fertility management, leaf disease con- Drs A. De Picker and D. Inzé (Session 2: diversification and value addition; trol and greater development/exten- Gene expression and transformation), • Policy support and programmes; sion/adoption of existing technology. Prof. G. Gheseyn, Drs Johan Nayts and • National and international trade; Good progress has been made by the D. Van Der Straeten (Session 3: Molecular • International cooperation. banana industry to reduce pesticides use plant pathology and disease/pest resis- but it is now necessary to pull together tance), Drs Isabel Roland-Ruitz and Peter Important dates the body of knowledge and develop an Breyne (Session 4: Biodiversity characteri- Deadline for submission of abstracts - ECO-OK type system implemented on zation and conservation) and Drs Emma 31st July 2002.Deadline for submission of commercial farms which complies to Schofield and W. Peumans (Session 5: full papers - 30th September 2002 auditable standards and market develop- Biochemistry and physiology). ment so that producers are rewarded for A workshop on Intellectual property Registration fees their efforts. and genetically modified organisms will The two publications mentioned above also be led by Dr Victoria Henson. Before 30 August 2002 are available on request at Department A scientific visit to KULeuven and the Member of AIPUB: 50 US$ of Primary Industries, GPO Box 46, INIBAP Transit Centre facilities will be No member: 75 US$ Brisbane QLD 2001, Australia. organized. Corporate: 100 US$ To know more about the symposium, visit the INIBAP web site at: After 30 August 2002 http://www.inibap.org/actualites/actu Member of AIPUB: 100 US$ alites_eng.htm No member: 125 US$ Corporate: 150 US$ Global conference on banana and Registration form along with the plantain fees should reach the Conference Grand Ashok Hotel, Kumara Krupa, Secretariate: High Grounds, Bangalore, India Bagwani Bhavan, 47 Janakpuri October 28 to 31, 2002 Institutional Area, Pankha Road, New To address the new emerging agenda Delhi-110058; Tel (91-11) 5622150/5531211; for growth and development of the Fax (91-11)-5531211/33849780. banana industry, the Association for the For more information and registra- Improvement in Production and tion, visit the web site: Utilization of Banana (AIPUB), India, http://www.aipub.org/conferences.htm

66 INFOMUSA — Vol 11, N° 1 INIBAP addresses

• Headquarters C/o CATIE Associate Scientist, Technology transfer Parc Scientifique Agropolis II Apdo 60-7170 Turrialba, COSTA RICA Guy BLOMME 34397 Montpellier Cedex 5 – FRANCE Tel/Fax: (506) 556 2431 PO Box 24384 e-mail: [email protected] e-mail: [email protected] Kampala http://www.inibap.org UGANDA Director • Regional Office for Asia and the Fax: (256-41) 28 69 49 Dr Emile FRISON Pacific e-mail: [email protected] e-mail: [email protected] Regional Coordinator Plant Genetic Resources Scientist Dr Agustín MOLINA • INIBAP Transit Center Dr Jean-Vincent ESCALANT C/o IRRI Collaborators Center (ITC) e-mail: [email protected] 3rd Floor, Los Baños, Officer in charge Germplasm Conservation Scientist Laguna 4031 Ms Ines VAN DEN HOUWE Ms Suzanne SHARROCK PHILIPPINES Katholieke Universiteit Leuven e-mail: [email protected] Fax: (63-49) 536 05 32 Laboratory of Tropical Crop Head Information/Communications e-mail: [email protected] Improvement Ms Claudine PICQ Kasteelpark Arenberg 13, e-mail: [email protected] • Regional Office for West and B-3001 Leuven Officer in charge MGIS Central Africa BELGIUM Ms Elizabeth ARNAUD Regional Coordinator Fax: (32-16) 32 19 93 e-mail: [email protected] Dr Ekow AKYEAMPONG e-mail: Financial Manager Associate Scientist, Technology transfer [email protected] Mr Thomas THORNTON Kim JACOBSEN e-mail: [email protected] C/o CRBP – BP 12438 • Associate Expert, Nematology Douala, CAMEROON Mr Thomas MOENS • Regional Office for Latin America Fax: (237) 342 91 56 C/o CORBANA and the Caribbean e-mail: [email protected] La Rita Research Station Regional Coordinator Apdo 390-7210 Dr Franklin E. ROSALES • Regional Office for Eastern and Guápiles, Associate Scientist, Southern Africa COSTA RICA Musa technology transfer Regional Coordinator Fax: (506) 763 30 55 Luis POCASANGRE Dr Eldad KARAMURA e-mail: [email protected]

Instructions to authors

Typescripts should be prepared in text, followed by the acronym in ber in the text. Each table should English, French or Spanish and submit- parenthesis. include a title. ted in duplicate to the Managing Editor. • References: All literature references Illustrations: These should be numbered They should be double-spaced through- made in the text should be referred to consecutively and referred to by these out. All pages (including tables figures, by author(s) and year of publication numbers in the text. Each illustration legends and references) should be num- (e.g.: Sarah et al. 1992, Rowe 1995). A should include a clear and simple caption. bered consecutively. Include the full list of references, in alphabetical Graphs: provide the corresponding raw name of all the authors of the paper, order, should be provided at the end of data with the graphs. together with the addresses of the the text. Drawings: provide originals if this is authors at the time of the work reported Please follow the style shown below: possible. in the paper. Indicate also the author Periodicals: Sarah J.L., C. Blavignac & Black and white photographs: provide nominated to receive correspondence M. Boisseau. 1992. Une méthode de them on bright paper and with good con- regarding the paper. laboratoire pour le criblage variétal trast. If the typescript was prepared on a des bananiers vis-à-vis de la résistance Colour photographs: provide good qual- computer, please send a copy on diskette aux nématodes. Fruits 47(5): 559-564. ity proofs and films or original slides. (or by e-mail) along with the printed Books: Stover R.H. & N.W. Simmonds. Note: When plant material used for the ones, indicating the name and version of 1987. Bananas (3rd edition). Longman, experiments reported originates or is reg- the wordprocessor used. London, United Kingdom. istered in the INIBAP genebank, its acces- • Abstracts: An abstract not exceeding Articles (or chapters) in books: Bakry F. sion number (ITC code) should be indi- 200-250 words should be sent in the & J.P. Horry. 1994. Musa breeding at cated within the text or in a tabular form. same language as the typescript, as CIRAD-FLHOR. Pp. 169-175 in The well as translations (including the Improvement and Testing of Musa: a title) into the two other languages, if Global Partnership (D.R. Jones, ed.). Thank you in advance for following this is possible. INIBAP, Montpellier, France. these instructions • Acronyms: These should be written in Tables: These should be numbered con- This will facilitate and accelerate full the first time they appear in the secutively and referred to by these num- the editing work. PROMUSA N° 9

Contents 3rd meeting of the PROMUSA Sigatoka working group ...... p.I PROMUSA: banana weevil working group inauguration ...... p.VI Abstract of presentations ...... p.VIII 4th and final FAO/IAEA research coordination meeting on Cellular biology and biotechnology including A global Programme for Musa Improvment mutation techniques for creation of new useful banana genotypes . . . .p.XIV A summary report ...... p.XIV Abstract of presentations ...... p.XV 3rd meeting of the PROMUSA Sigatoka working group 24-25th May 2002, EARTH, Costa Rica

The meeting started with a brief presentation M. fijiensis and M. eumusae occur are neces- by each participant of their research capaci- sary. The name of the banana clone affected, ty, in terms of human resources, research an indicator of the severity of the leaf spot and What is PROMUSA ? facilities, and future and ongoing projects local environmental data would be useful as The Global Programme for Musa related to Mycosphaerella leaf spot dis- this may help explain distribution. IMTP trials Improvement (PROMUSA) is a broad-based eases. Participants also commented on their are seen as ideal locations for assessing the programme which aims at involving all the participation in PROMUSA, defining their reaction of different clones to the different leaf major players in Musa improvement. It was areas of interest where they would like to spot pathogens. The collection and diagnosis developed as a means to link the work carried out towards addressing the problems develop partnerships with other participants. of specimens of leaf spot from IMTP trials sites of export banana producers, with those The participants identified various needs to be continued. The cooperation and initiatives directed towards improving banana research priorities and defined the main collaboration of scientists in south and south- and plantain production at the subsistence activities that should be carried out. east Asia is viewed as essential. Identification and smallholder level. The global programme tools should be provided to enable diagnoses builds upon existing achievements and is to be undertaken locally ‘. based upon ongoing research initiatives. Recommendations PROMUSA is therefore a mechanism to further maximize the outputs and accelerate Development of a detailed Development of national collections the impact of the overall Musa improvement of the different Mycosphaerella effort. The programme is an innovative understanding of the population leaf spot pathogens mechanism to bring together research carried structures of M. musicola, The PROMUSA Sigatoka working group rat- out both within and outside the CGIAR, M. fijiensis and M. eumusae ified the recommendation made during the creating new partnerships between National nd Agricultural Research Systems (NARS) and “2 International workshop on Mycos- research institutes in both developing and Survey of geographical distribution phaerella leaf spot diseases of bananas”: A developed countries. The formation of such of the three Mycosphaerella species reliable, rapid test to distinguish M. musico- partnerships will also contribute to The survey of the distribution of the different la, M. fijiensis, M. eumusae and possible strengthening the capacity of NARS to species requires wide sampling at the national other Mycosphaerella pathogens/sapro- conduct Musa-related research. level of the different agroecological areas phytes needs to be developed to aid identifi- The major thrust of PROMUSA is to develop a wide range of improved banana varieties where Musa is found, and the morphological cation. Information on how to distinguish the from which growers worldwide can select characterization of the species through the 3 pathogens on morphological characteris- those most suited to their needs. The observation of the anamorph stage (conidia), tics also needs to be produced and circulat- programme brings together conventional including molecular characterization using ed to banana scientists. INIBAP was asked breeding based on hybridization techniques PCR diagnostics. to address this need”. with genetic engineering and The PROMUSA Sigatoka working group The creation of a national collection of dif- biotechnological breeding approaches. This broad-based genetic improvement effort is ratified the recommendation made during the ferent strains from different Mycosphaerella nd supported by research being carried out on “2 International workshop on Mycosphaerella leaf spot pathogens on Musa is of special specific pests and diseases within the leaf spot diseases of bananas” held from 20 to relevance in the understanding of the popu- various PROMUSA working groups. An 23 May 2002 in San Jose, Costa Rica: ‘The lation structure. The collection must be efficient mechanism for evaluating new exact distribution of M. eumusae needs to be based on single-ascospore cultures with an varieties produced within the framework of known. Further surveys in south and south- in vitro characterization of the anamorph PROMUSA is also an essential component of the programme. east Asia to determine where M. musicola, stage (in vitro sporulation of conidia). It has

INFOMUSA — Vol 11, N° 1 PROMUSA I been recommended to provide the partici- tion already published on these different required (collection and sampling, mono- pants with a protocol to sample, establish methods. ascosoporing cultures and molecular and maintain the collection. INIBAP was markers). mandated to collaborate with CIRAD in the Identification of new sources development and distribution of the technical of resistance Durability of the resistance information required. The establishment of a Significant changes in the levels of resist- national collection should be promoted and The need of new sources of resistance to ance to Sigatoka and black Sigatoka dis- facilitated through the organization of a train- Mycosphaerella leaf spot diseases has eases have been reported in Australia, India ing course; especially for those countries been identified several times in the past. and Cuba. However these may exist just that develop breeding programmes, but also Collecting missions in Indonesia, north because of high inoculum loads. Thus, where disease resistant hybrids of banana India and Vietnam have already taken changes in pathogen populations should be are used on an industrial scale, and where place but information has only been pro- distinguished from particular epidemiological the high diversity of Musa would have origi- vided on the characterization of the resist- effects. Therefore, the group recommended nated similar diversity in the pathogens. ance of the different materials collected. studying the changes in pathogen popula- The PROMUSA working group recom- Genetic population structure tions in response to selection pressure from mended that INIBAP help to gather any The study of the genetic population structures new banana genotypes resistant to Sigatoka information already available. The group of Mycosphaerella leaf spot diseases is diseases. It is essential to monitor changes also recommended to stimulate the char- already on going at national, regional and in pathogen populations in areas where new acterization of existing collections where international levels. However, the group rec- resistant hybrids are being grown on a large M. eumusae has already been reported ommends increasing the number of countries scale. A special recommendation towards together with other Mycosphaerella spp. involved at the national level, which will allow the development of specific trials in Cuba e.g. MARDI, in Malaysia. In order to facil- refinement of both regional and international has been made. Two different aspects of itate the screening, the group suggested studies. Both biological (morphological) and the durability of the resistance need to be using the ‘severity index’ as the unique molecular determinations have been recom- addressed: the genetic drift of the pathogen parameter to detect any source of resist- mended to improve the understanding of the resistance and the selection effect within the ance. This information should allow the different population structures. The sampling pathogen population. Participants in PRO- definition of the different reference clones protocol and methodology should be stan- MUSA Sigatoka working group recom- needed to evaluate the resistance to dardized and the recognition of the different mended: Eumusae leaf spot disease. species facilitated through the development of • the selection of areas where resistant The ‘severity index’ will also be used to a technical factsheet to be widely distributed. hybrids have been grown for a long time evaluate the PROMUSA segregating popu- INIBAP and CIRAD agreed to work together (e.g. Cuba) and to follow the evolution of lations hosted at CORBANA. in the preparation of this information which the pathogen populations, isolating should include several detailed illustrations of Mycosphaerella strains on resistant and the different pathogens and their anamorph Diagnostics susceptible cultivars or hybrids, stages. This information will also become Several leaf fungal diseases have been • the development of molecular markers part of the IMTP guidelines. The develop- reported on Musa and other related species. linked to the pathogenicity of the fungal ment of more molecular markers as SSR and The group recommended the development of strains (molecular markers will inform on CAPS should allow the study of the different specific diagnostic tools according to the the genetic drift when pathogenic evalua- populations to be refined. The recommenda- three main species of Mycosphaerella tion will be related to the selection effect), tion to include partners from south and south- pathogen on Musa: M. fijiensis, M. musicola • the quantification of the selection pressure east Asia made during the last global meeting and M. eumusae. The achievement of these over the time, and of PROMUSA in Bangkok was reiterated by diagnostic tools will remain within the devel- • the study the breakdown of resistance by in the participants who strongly suggested that opment of a worldwide collection of vitro testing. the INIBAP regional office for Asia and Pacific Mycosphaerella isolates; the morphological strengthen and facilitate any exchange description of all the different Mycosphaerella Dispersal of Mycosphaerella between Asian partners and the rest of the subspecies associated with banana leaves leaf spot diseases on Musa PROMUSA community. and the development of primers species-spe- M. eumusae is currently limited in extent cific as microsatellites and ITS-sequences Pathogenic characterization throughout most of Asia, although there is and their test on the worldwide collection of The pathogenicity of the different strains some evidence that the pathogen may have Mycosphaerella isolates. should be approached using either the in reached Africa. The dynamics of the dis- vitro or in vivo inoculation systems. It is therefore suggested: ease are not fully understood. Some projec- However, it is recommended that the differ- • to develop diagnostic tools to distinguish tions indicate that this disease will become ent methodologies that currently exist be the main pathogens and assess currently more important than black Sigatoka. In standardized. The methodology for the in available molecular methods for speci- order to prepare adequate disease control vitro inoculation on leaf fragments devel- ficity, strategies, a detailed knowledge of the epi- oped at CIRAD should be distributed, • to develop a manual with descriptions of demiology of this pathogen is urgently together with the methodology used to iso- symptoms and morphological characters, required. To address the epidemiology of the late, cultivate and produce the inoculum of • to develop protocols for collection and different Mycosphaerella spp. pathogens on the different pathogens. INIBAP and CIRAD analysis of samples, Musa, the group recommended: have been requested to compile, in a single • to transfer to and train PROMUSA partici- • the collection of disease incidence data technical document, all the different informa- pants on the different technologies from the field and literature,

II PROMUSA INFOMUSA — Vol 11, N° 1 • the development of methodologies to Institutional interest in the activities of PROMUSA understand the mechanisms of spore Research topics Institution willing to participate release and spore survival in the air at lab- Population structure oratory level, and • Survey of the geographic distribution in Asia • the clarification of laboratory data at plan- • Nigeria IITA tation level and to assess the potential for • National collections INISAV, CORBANA, CORPOICA, FHIA, EMBRAPA, FABI, windborne dispersal (as opposed to further • QDPI, CIRAD spread of the disease by the transfer of • Genetic structure of populations INISAV, FABI, CATIE, CIRAD, CORPOICA, CARBAP inoculum). • Pathogenic characterization CORBANA, CATIE, CIRAD, FHIA, EMBRAPA, CORPOI• CA, CARBAP, IBP Evaluation of segregating population CORBANA, EMBRAPA Host-pathogen interactions Diagnostics CRCTPP, CIRAD, CBS, FABI The genetic approach has been shown to be Durability of resistance INISAV, CIRAD, CATIE, EMBRAPA, CARBAP and FHIA extremely powerful when studying host- Dispersal pathogen interactions in some pathosys- • M. eumusae , Asia NRI tems (such as Magnaporthe grisea). This • M. fijiensis , Caribbean NRI, CIRAD, WIBDECO, CRCTPP Host-pathogen interactions FABI, CIRAD, IBP, CARBAP and CICY approach does not require the identification • Mechanism of pathogenicity of pathogenicity factors a priori and includes • Mechanism of resistance the study of gene expression during infection (differential display, DNA chip, SSH, etc.), production of pathogenicity mutants, comparative genomic and gene function validation techniques. Here again, the PROMUSA Sigatoka working group ratified the recommendation made in the framework of the “2nd International workshop on Mycosphaerella leaf spot diseases of bananas” to study: ”the development of genetic and molecular biology tools for M. fijiensis in collaboration with M. graminicola groups as well as to launch a genomic initiative to access to genomic tools (EST collection, physical map, genome sequence) and set up a genomic-wide comparison of M. fijiensis to M. graminicola”.

International core collection host the international collection using a sim- The group recommended to develop an ilar mechanism as INIBAP developed with international core collection of M. fijiensis, M. KULeuven to host the international Musa musicola and M. eumusae. The different germplasm at the INIBAP Transit Center. strains should be conserved as fungal INIBAP was asked to address this need in mycelia and DNA. CIRAD was suggested to collaboration with CIRAD.

Institutional contribution to PROMUSA Institution, Country Research facilities Human resources Research topics On-going activities Contact CORBANA, Costa Rica Tissue culture lab. • Field evaluation on new clones • IMTP phase III J.A. Sandoval, Plant pathology lab. • Inoculation of M. fijiensis at greenhouse • Evaluation of Musa segregating R. Vargas Germplasm field bank level populations. M. Guzman Experimental fields • Evaluation of M. fijiensis segregating populations INISAV, Cuba Plant pathology labs • Epidemiology of M. fijiensis in hybrids • Population survey of M. fijiensis L. Pérez Vicente Experimental stations resistant populations (variability and distribution) Coll. with CIGB • IMTP phase III • Durability of the resistance • Diversity and distribution of M. fijiensis of FHIA hybrids to M. fijiensis (molecular and morphological characterization) CRCTPP, QDPI, Plant pathology labs Principal plant pathologist (1) • Mycosphaerella leaf spot diseases • Molecular diagnosis of Oceanic R. Peterson (QDPI) Australia Glasshouse Technical officers (2) • Black leaf streak: Sigatoka isolates J. Henderson Research stations Technician lab. (1) Cultivar evaluation & diagnostics • Isolate collection & preliminary ID (CRCTPP) • Sigatoka: Diagnostics & epidemiology by symptomatology and morphology K. Grice (QDPI) • Molecular diagnosis of black leaf • Sequence analysis of ITS regions streak and Sigatoka diseases of Oceanic isolates • If needed, sequence of other suitable genes will be undertaken

INFOMUSA — Vol 11, N° 1 PROMUSA III Institution, Country Research facilities Human resources Research topics On-going activities Contact

CORPOICA, Colombia Molecular laboratory • Mycosphaerella leaf spot diseases • Characterization of the Colombian A. Gutierrez Rojas Plant pathology lab. • Population structure and diversity: population of Mycosphaerella leaf spot S. Aponte Field stations (different altitudes) • Morphological and molecular pathogens characterization • Morphological and molecular • Evaluation of germplasm characterization of germplasm CICY, Mexico Biotechnology labs Researchers (4) • Genetic improvement using • Construction and characterization of 2 genetic A. James Biochemistry & molecular Technicians (3) biotechnology BIBAC libraries of 2 diploid bananas, and D. Kaemmer biology of plants MSc students( 4) development of a transformation protocol using L. Conde Equipment facilities: PhD students (2) vacuum infiltration mediated by Agrobacterium, L. Peraza CHEF MAPPER Undergraduates (13) CONACYT, 3 years (Resp.: Dr A. James) Capillar Sequencer • Genetic and physical mapping of M. fijiensis, CHEF DRII CONACYT; 3 years (Resp.: Dr D. Kaemmer) Screening of Calcutta IV BAC library for Resistance genes (submitted) (Resp.: Dr D. Kaemmer) • Construction of a BAC library of M. fijiensis (submitted) (Resp.: Dr A. James) • Agronomic evaluation of newly introduced banana and plantain cultivars in Mexico, and of mutant-induced plants (submitted) (Resp. : Dr A. James) CIRAD, Montpellier Phytopathology labs Researchers ( 2) • Taxonomy and identification of J. Carlier Mycosphaerella spp. C. Abadie Greenhouse & climatic chamber Geneticist (1) • Collection In vitro evaluation facilities Technicians (3) • Molecular diagnostic Access to in vitro laboratory Students (2-3) • Distribution of Mycosphaerella spp. in Asia Collection of about 2500 isolates • Pathogen populations structure at different scales Laboratory of molecular biology • Collection (CAPS & microsatellites, sequencers, • Molecular markers genomic) • Pathogenicity • Population structure of Mycosphaerella leaf spot pathogens in Asia • Efficacy and durability of partial resistance • Characterization of new resistance sources • Genetic of resistance • Evolution of pathogen population on resistant cultivars (large area of culture) • Other • Genomic and host-pathogen interaction studies • Collection (core collection, network? data base?) CATIE, Costa Rica Molecular biology Lab. Researchers (2) • Biological control and resistance On-going: A. S. Riveros Plant pathology Lab. Technician (2) induction • Studies of population structure of M. fijiensis G. Rivas Biologic control Lab. • Bank of antagonistic bacteria and fungi • Biotechnology of Musa: tissue culture, Tissue culture lab. • Microbiological products with bombardment protocols Particles bombardment antifungal potential or elicitor molecule Submitted: Shade house • Screening of plants with antifungal or • Population structure of M. fijiensis resistance induction in Dominican Republic. Tools offered: • Botanical products with antifungal or • Presented by T. Polanco (IDIAF) to IAEA in M. fijiensis: Isolating protocols, elicitor molecule January 2002. Partners: CATIE (Costa Rica) DNA extraction, DNA amplification, • Adaptation/development of a rapid and CIRAD (France). DNA electrophoresis and efficient screening method to • Population structure of M. fijiensis in Use of molecular markers evaluate black Sigatoka resistance. Honduras and Dominican Republic. for studies of populations • Genetic transformation of Musa to Presented by G. Rivas (CATIE) to FINNIDA. Induction of resistance introduce resistance to black leaf May 2002. Partners: FHIA (Honduras), IDIAF Pathogenesis tests streak disease (Dominican Republic) and CIRAD (France). Musa tissue culture protocols • Transformation protocol in Musa Studies of population structure of M. fijiensis Bombardment protocols • Biotechnology of Musa: tissue culture, bombardment protocols Population structure of M. fijiensis in Honduras. Project supported by INIBAP/FHIA. Partners: CATIE (Costa Rica) and CIRAD (France).

IV PROMUSA INFOMUSA — Vol 11, N° 1 Institution, Country Research facilities Human resources Research topics On-going activities Contact EMBRAPA, Brazil Cooperative network of 3 EMBRAPA Researchers (9) • Integrated control of Mycosphaerella On-going: Zilton Cordeiro research centres including their leaf spot diseases in Brazil including • Evaluation of genetic and pathogenic variability Maria de Jesus B. laboratories and fields breeding in M. musicola Calvacante • Evaluation of resistance to black and yellow leaf streak diseases • Epidemiology New proposal: • Creation and evaluation of segregating population of Musa acuminata (AA) for resistance to black and yellow leaf streak diseases. FHIA, Honduras Tissue culture lab. Plant breeder (1) • Breeding On-going: M. Rivera Shade house Research assistants (3) • Field evaluation of new clones • IMTP phase III J.F. Aguilar Experimental fields Plant pathologists (2) • Epidemiology • CFC Musa hybrid testing project Conventional plant pathology lab. Agronomist (1) • Banana breeding ELISA capacity installed Technician (1) Perspective: PCR capacity (soon) • Population structure of M. fijiensis in Honduras. Project supported by INIBAP/FHIA. Partners: CATIE (Costa Rica) and CIRAD (France) IBP, Cuba Plant tissue culture lab. Biotechnologist • Early screening On-going Y. Alvarado Molecular biology lab. Plant breeders • Evaluation of germplasm • Mutagenesis /breeding program Phytopathology lab. Plant pathologist (2) in screen-house • Standardized methods for early screening Commercial plant tissue culture lab. Molecular biologist • Genetic transformation • Evaluation in greenhouse Greenhouse/ Shade house Microbiologist (plant/pathogen) • M. fijiensis transformation Experimental field Agronomist • Plant transformation Perspective: • Use of similar test for pathogenesis evaluation • Development of methods to identify virulent and avirulent isolates Plant Pathology Unit, Pathology lab. • Biological control against fungal P. Lepoivre University of Gembloux, Virology lab. diseases J.-P. Busogoro Belgium Greenhouse • Selection for resistance to plant diseases Molecular biology • Study on resistance mechanism and serology facilities NRI, University Plant pathology and virology Plant pathologist (3) • Epidemiology Perspective: P. Burt of Greenwich, UK laboratories Mycologist (1) • Airborne spread of M. fijiensis. CT rooms and environmental cabinets Technicians (3) Electron microscopy Biometeorologist (1) Greenhouse Library FABI, University Pre-graduate Plant Pathologist (2) • Fungal taxonomy • Morphological and molecular identification A. Viljoen of Pretoria, Post-graduate Mycologist (1) • Population genetics of Mycosphaerella spp. of Pretoria, BIOTECHNOLOGY: • Molecular biology • Population genetics of Mycosphaerella spp. South Africa Microarray facility • Disease management Sequencer Light cycler MYCOLOGY: Electron and light microscopes Culture maintenance Genetic and molecular analysis of fungi PLANT FACILITIES: Tissue culture laboratories Transformation and GMO facilities Quarantine facilities BTI, Cornell University, • Molecular genetic tools to study host- USA Phytopathology labs. pathogen interaction Greenhouse & climatic chamber • Genomics approaches to identify fungal and In vitro evaluation facilities plant genes expression Access to in vitro lab. • Development of high throughput screening Laboratory of molecular biology methodologies for evaluating pathogenicity (CAPS & microsatellites, sequencers, and virulence in plant. genomic) Genetic transformation Asian institutions, • It has been recommended to take the • Incidence/severity of Mycosphaerella represented by INIBAP opportunity of the next BAPNET species on Musa in Asia (morphological) Coordinator for Asia meeting to have a workshop with Asian • Evaluation and characterization of various represented by INIBAP participants to the PROMUSA Sigatoka germplasm collections in Asia against and Pacific and working group to define their M. musicola, M. fijiensis and M. eumusae. Executive Secretary programme and activities. • Epidemiological studies on M. eumusae. of BAPNET. • Population structures of M. eumusae using molecular tools and corresponding pathogenicity • Yield loss assessment due to the different Mycosphaerella leaf spot pathogen on Musa.

INFOMUSA — Vol 11, N° 1 PROMUSA V Project proposals Development of diagnostic tools Mauricio Rivera - FHIA Luis Pocasangre – INIBAP The group also worked on the development for Mycosphaerella species on Luis Pérez Vicente – INISAV of different concept notes to achieve the dif- banana David Jones - Consultant, UK ferent recommendations made. Potential partners Jean Carlier and Catherine Abadie - CIRAD Determination of the pathogenic Airborne dispersal Pedro Crous - CBS of Mycosphaerella pathogens variability of M. fijiensis and M. Altus Viljoen - FABI musicola populations of Musa - Monitoring of airborne Juliane Henderson and Elizabeth Aitken - spread of M. fijiensis into CRCTPP Potential partners uninfected areas of the Caribbean Kathy Grice and Ron Peterson - QDPI Yelenis Alvarado - IBP Ronald Vargas - CORBANA Potential partners Investigation of the durability of Laura Conde - CICY Catherine Abadie - CIRAD the resistance of banana hybrids Sergio Aponte - CORPOICA Peter Burt – NRI to M. fijiensis Zilton Cordeiro - EMBRAPA Henry Fagan - WIBDECO Mauricio Guzmán - CORBANA Juliane Henderson - CRCTPP Potential partners Galileo Rivas - CATIE Ronald Vargas – CORBANA Catherine Abadie -CIRAD

Name Institution Country Name Institution Country Alba .S. Riveros CATIE Costa Rica Juliane Henderson CRCTPP Australia Alice Churchill BTI, Ithaca USA Kathy Grice QDPI Australia Altus Viljoen FABI South Africa Laura Conde CICY Mexico Andres Gutierrez Rojas CORPOICA Colombia Leticia Peraza CICY Mexico Andrew James CICY Mexico Lorna Herradura BPI Philippines Aristoteles Pires de Matos EMBRAPA Brazil Luis Perez Vicente INIVIT Cuba Bob Fullerton Hort Research New Zealand Luis Pocasangre INIBAP-CATIE Costa Rica Catherine Abadie CIRAD-FLHOR France Mauricio Guzman CORBANA Costa Rica David Jones Consultant UK Mauricio Rivera FHIA Honduras Dieter Kaemmer CICY Mexico Moses Buregyeya NARO Uganda Elizabeth Aitken CRCTPP Australia Peter Balint-Kurti DNA Plant Technologies USA Eric Foure CIRAD/ CARBAP Cameroon Peter Burt NRI UK Fritz Elango EARTH Costa Rica Philippe Lepoivre Univ. Gembloux Belgium Galileo Rivas CATIE Costa Rica R. Selvarajan NRCB, Trichy India Gus Molina INIBAP Philippines Ron Peterson QDPI Australia Indra Ariyarathne ARS Sri Lanka Ronald Vargas CORBANA Costa Rica Jean Carlier CIRAD-AMIS France Sergio Aponte CORPOICA Colombia Jean-Vincent Escalant INIBAP France W. Tushemereirwe NARO Uganda Jorge Sandoval CORBANA Costa Rica Yelenis Alvarado IBP, Santa Clara Cuba José G. Garcia Lopez INIFAP Mexico Zilton Cordeiro EMBRAPA Brazil

PROMUSA: Banana weevil working group inauguration 2 March 2002, Tenerife, Canary Islands, Spain

Origin of PROMUSA production, particularly for small-scale Agenda and outputs farmers who account for over 80% of the As an introduction, Eldad Karamura, INI- global production. Consequently, inter- of the meeting BAP regional coordinator for eastern disciplinary working groups have and southern Africa, briefed participants been created to generate complementa- The meeting agreed on the following dis- on PROMUSA. He explained that PRO- ry information needed by the genetic cussion agenda/outputs: MUSA was born out of the realization improvement working group. It was • Structure, membership and financing of that genetic improvement is the most clarified that participating in the PRO- working groups, sustainable strategy for addressing the MUSA research agenda did not preclude • Research priorities for the Weevil working majority of constraints limiting banana other research activities on the same topics. group (inputs required for genetic improve-

VI PROMUSA INFOMUSA — Vol 11, N° 1 ment from the Weevil working group), • To compile and exchange information on other crop protection research activities, e.g. • Management of the Weevil working group. methods and checks. Standardizing sam- pheromones and entomopathogens, are pling methods is a prerequisite for develop- less important. These should be addressed ing screening methods, more efficiently as researchers benefit from Output 1: Membership • To have standard protocols for screening the multidisciplinary dynamics created by • Agreement on the actual membership in germplasm and for identifying sources of PROMUSA. other working groups resistance, • Core group: scientists with active proj- • To compile information on mechanisms of ects on aspects of genetic improve- resistance, Way forward ment, • To assess the possibility of site specific dif- • General membership: people working ferences regarding resistance, Formation of the core group – This group on weevil biology, including policy mak- • To develop research priorities that address should include scientists who actively con- ers and those involved in technology the compatibility of genetic improvement tribute to genetic improvement, e.g. breed- transfer. with other management practices. ers and scientists working on host plant • Convenor’s role: it was agreed that the • To consider developing IPM research pri- resistance, mechanisms of resistance, convenor is responsible for convening orities that contribute to the genetic hybrids resistant to weevil, sources of resist- meetings, information exchange and liais- improvement of the banana, ance, relevant genetic studies, conventional ing with PROMUSA secretariat. Certain institutions whose research breeding, biotechnological methods and interests go beyond genetic ameliora- screening methods. Question 1. Who funds the convenor’s run- tion, raised the possibility of establishing It was not felt necessary to split this group ning costs e.g. meetings? the working group as a separate entity into scientists working on plantains, bananas The working group in collaboration with from PROMUSA for fear of being or other banana types. For the time being, INIBAP, through PROMUSA, looks for funds restricted by the latter’s mission. In the the group can include anyone working on for the operation of the core group. Usually, end, everybody agreed there would be a any aspect of crop improvement of all PROMUSA working group meetings are core working group on activities related bananas and plantains. held back-to-back with other international to genetic improvement, but that the It was suggested that the group adopt the meetings, a cost-effective way of organizing general membership would include poli- same procedures for forming this group as meetings. cy makers and all those working on those used by other working groups. banana weevil (including its biology and It was agreed that: Output 2: Research priorities status as a pest, control methods and • Members who were present at this meeting for genetic improvement technology transfer). It was also sug- form the working group, gested to create a list-server to facilitate • A convenor should be elected to take • Identify sources of resistance, information exchange on all aspects of charge of the working group, • Develop screening methods and protocols, banana weevil research. • The convenor should organize a meeting • Agree on references/checks. The fact that PROMUSA focuses on within the coming year to work out the way The following suggestions were made: genetic improvement does not mean that forward.

List of participants

Name Research focus Cliff Gold – IITA IPM, microbial control, screening, resistance mechanisms, collaboration with breeders. Roger Fogain - CARBAP Integrated Management of the weevil (screening, resistance, biological control). Consuelo Castrillon - CORPOICA IPM, screening. Stijn Messiaen - KUL IPM, screening. Aurelio Carnero - ICIA IPM, genetic resistance. Gloria Lobs – ICID Protease inhibitors, post-harvest evaluation of genetically modified varieties. Schalk Schoeman - ARC-ITSC IPM of weevil, screening for ‘Cavendish’ sub-types. Douglas Cubillo - CORBANA IPM, screening. Thierry Lescot – CIRAD-FLHOR IPM application in diversified systems. Links between research and development. Fernando Garcia del Pino – Univ. Autónoma Barcelona Entomopathogenic nematodes for biological control. Angeles Padilla - ICIA Entomopathogenic nematodes for biological control, artificial diets. Dennis Alpizar - Costa Rica IPM in plantain, pheromones. Vincent Ochieng - ICIPE Use of genetics in banana weevil biotyping in relation to control and quarantine. Prem Govender - FABI IPM in commercial banana plantations, active plant pathology group, especially with biotechnology. Felix Ortego – CSIC Activity/ insecticidal protein in insects. Miguel Montesdeosca - ICIA Activity/ insecticidal proteases in banana weevil, pheromones. Pedro Castañera -CSIC Activity/ insecticidal proteins for insect control. Caroline Nankinga - NARO/IITA IPM, entomopathogenic fungi for biological control of weevil, on-farm screening. Andrew Kiggundu - NARO Use of foreign genes for resistance to weevil, protease inhibitors.

INFOMUSA — Vol 11, N° 1 PROMUSA VII Contributions EMBRAPA, Brazil, was not represented very closely with NARO and banana net- of the various partners but may be interested in conventional bree- works in East and West Africa. IITA is inter- ding, biotechnology and screening for local ested in mechanisms of resistance, as well CIRAD, Guadeloupe, expressed the desi- stress. Needs to be contacted. as conventional and biotechnological re to participate in agronomy and biotech- FHIA, Honduras and EMBRAPA will be methods for developing resistance (Cliff nology related activities and to coordinate contacted to find out their interests (Marline Gold). activities in Guadeloupe and Montpellier. Fancelli). Spanish organizations will also provide ITSC, South Africa, proposed to screen Election of the convenor support to the working group. new banana varieties, especially CORPOICA, Colombia, will contribute to ‘Cavendish’ (Schalk Schoeman). The It was proposed that Dr Cliff Gold of IITA be the screening of cultivars for resistance to University of Pretoria will supervise stu- selected as convenor. He has worked exten- banana weevil and nematode. CORPOICA dents conducting research in banana bio- sively on banana weevil, speaks both English and Spanish and has access to communica- will provide support for screening methods technology. tion facilities. He can easily coordinate the and could also provide a weevil biotechno- CARBAP will screen for resistance to preliminary activities. Therefore Dr Cliff Gold logy specialist (Consuelo Castrillon). weevils, nematodes and black Sigatoka was nominated and unanimously approved. CORBANA, Costa Rica, will provide sup- (Roger Fogain). port for screening methods and evaluation IITA has a breeding programme on high- of germplasm. land bananas and plantains. IITA works

Abstracts of papers presented during the meeting and plantain producing areas showed that the uct is still the only efficient insecticide used in Session 1. Status percentage of occurrence of C. sordidus in commercial banana farms. However, its con- of Cosmopolites sordidus Cameroon varies between 50 and 90%, and tinuous application will probably produce that 82.5% of the farmers are aware of the resistant weevil populations in the near future. in the world problem and capable of recognizing weevil Therefore, it is recommended to use it alter- Studies on the banana borer damage (Ngamo and Fogain 1998). Research nately with nematicides, like Counter (ter- on the population dynamics of the weevil in two buphos) and Furadan (carbofuran), that have weevil in Cameroon of the most important production zones indi- insecticidal activity and can be used when cates that higher populations are observed populations are relatively low. In the Moungo R. Fogain, S. Messiaen & E. Fouré beween August and September. However, this department, the threshold for treatment in CARBAP (Centre africain de recherches sur bananiers et result need confirmation. industrial banana plantations is when 5% of plantains) P.O.Box 832, Douala, Cameroon the 20 sampled mats per hectare are Control methods attacked, based on the method proposed by In Cameroon, bananas and plantains are a Vilardebo (1973). Other insecticides with inter- major staple food for a large proportion of the In commercial banana plantations, chemical esting properties are: tebupyrimphos, athi- population. A total of 1.7 million tonnes are control, the use of clean planting material, and amethoxam, cartap and imidacloprid. produced annually. These crops are threat- weevil habitat management are the most common methods for controlling weevil popula- Timely chemical control is an efficient way of ened by a wide range of pests and diseases knocking down adult weevil populations in tions. Development of alternative control among which the banana borer weevil commercial farms, but is too expensive for the measures and of an integrated pest manage- (Cosmopolites sordidus) is the major insect majority of resource limited farmers and has ment strategy are highly recommended for pest. For more than six decades, investigations unfavourable side effects on beneficial non- have been carried out on this pest but empha- resource limited small-scale farmers, the target organisms. According to a survey con- sis was given to testing insecticides that satisfy major producers of plantain. ducted in southwest Cameroon, 57% of small- the needs of large-scale commercial banana Chemical control holder farmers said they did not use pesticides plantations. It is only recently that studies on (Chantelot 1993). Forty-three percent, mostly integrated control options were initiated in At the beginning of the 70s, weevil populations in mixed plantain-cocoa plantations, treated order to develop control strategies that could were efficiently controlled with Kepone suckers before planting and 87% used insecti- also be used by resource limited farmers. (Chlordecone) in Cameroon’s commercial cides generally referred to as ‘gabaline’, insec- This report presents the activities carried out banana farms. Between 1975 and 1983, the ticides used against timber or cocoa pests in Cameroon over the past ten years on the withdrawal of the product from the market and which include lindane (HCH), Dursban banana borer weevil. caused a significant increase in weevil popula- (chloropyrifos-ethyl) and methylparathion. tions because of its replacement with HCH Three percent of the farmers who treat suck- and other less effective insecticides, like ers before planting use a nematicide with Distribution and population dynamics Dursban (chloropyrifos-ethyl) and Primicide insecticidal activity, such as Mocap Four weevil species are found in the banana (pyrimiphos-ethyl) (Kehe 1985). The rapid (Ethoprophos), and 10% use other products and plantain producing areas of Cameroon: decline of banana production was halted by (Chantelot 1993). Another survey in west, Cosmopolites sordidus (Germar), Polytus the arrival on the market of Curlone southwest, central and south Cameroon mellerborgi (Boheman), Metamasius hemi- (Chlordecone) in the early 90s. With one or revealed that only 11% of smallholders use pterus sericeus (Olivier) and M. hemipterus two applications per year, weevil populations pesticides, 57% do not use anything and more (L.). C. sordidus seems to be the only weevil of where effectively controlled. But the product than 32% use ashes because they believe it economic importance in banana and plantain was soon withdrawn from the market because controls weevils (Ngamo and Fogain 1998). plantations (Fogain 1994, Ysenbrandt et al. of its limited degradability. Regent (fipronil) 2000). The insect is found in all banana and later came on the market, allowing efficient Cultural control plantain producing areas in Cameroon (Fogain control of weevil populations with two or three It is important to plant an uninfested field with 2001). A survey carried out in all the banana applications per year. In Cameroon, this prod- clean planting material which can be obtained

VIII PROMUSA INFOMUSA — Vol 11, N° 1 from weevil-free plantations or tissue culture from soil samples collected in Cameroon southwest Cameroon), investigations are need- facilities. Ninety-five percent of smallholders using C. sordidus larvae. ed for other provinces, such as the major plan- practice paring of suckers before planting tain producing areas of the centre and south (Chantelot 1993), but since the availability of Use of botanicals (Anonymous 2000). The insect was found to be good planting material is a major limitation in Dipping suckers in a 20% neem (Azadirachta present everywhere in the country where Cameroon, infested suckers of minor quality indica) seed solution at planting protects the bananas and plantains are produced. A larger are often planted. young suckers from weevil attack for several spectrum of insecticides is available, but mainly Residual corms in the soil should be months, but a crown application three times a to commercial growers. Several insecticides destroyed and post harvest residues slashed year is not effective in reducing damage from different chemical groups are now avail- in order to prevent the multiplication of wee- (Fogain and Ysenbrandt, 1998). It achieves able and can therefore be used in rotation to vils. Weeding should be done regularly in this result by reducing oviposition, through its avoid the development of resistance to C. sor- order to avoid development of a favourable repellent effect on adult weevils, and by didus. As for botanical and biological control humid weevil habitat. In smallholder farms, blocking egg hatching (Messiaen 1999). agents, neem (A. indica) and the entomopatho- habitat management is neglected because genic fungus B. bassiana show great potential labour is limited or rented labour is not produc- Trapping for weevil control. But field trials are needed to confirm greenhouse results. Sources of resist- tive enough. Weeding is minimal (two to three Trapping using pseudostem traps does not ance to the weevil have been identified and times a year) and herbicide application is rare. always reduce weevil populations, depending breeding programmes can now use them to A minority of farmers prop with bamboo even on the cropping system, the level of weevil develop genotypes resistant to the insect. though the practice can give good results with immigration from neighbouring infested plots, Research on other non-chemical control meth- minimal investments. In a survey carried out the number of traps placed and the initial pop- ods, such as cultural control and the use of between February 1997 and March 1998 of ulation. In Cameroon, trapping does not seem pheromones, continues. 240 plantain plants in eight smallholder farms, to be a viable control option in smallholder plant losses due to nematodes, weevils, and farms because of the unrealistic amount of References water and nutrient stress represented 60% of pseudostem and labour needed, and because Anonymous 2000. Annuaire des statistiques du sec- the losses, of which 37% were due to toppling of weevil immigration from adjacent plots. teur agricole, Agri-Stat No. 006, Direction des Etudes et Projets Agricoles, Ministère de or falldown (mainly at the beginning of the Testing of a mass trapping system using ramp rainy season, due to violent winds) and 29% l’Agriculture, Cameroon. traps baited with sordidin, an aggregation Anonymous. 1998. L’observatoire des problèmes phy- were due to breakage of the pseudostem pheromone, indicated that the traps were not tosanitaires. Plantinfo (34):9-11. (mainly at the end of the dry season due to attractive enough to constitute a viable control Chantelot E. 1993. Enquête diagnostic plantain dans la water stress) (Anonymous 1998). option in industrial banana plantations, but province du sud-ouest du Cameroun: description de More than 30 % of smallholder farmers use additional research is needed to assess l’échantillon de parcelles. Document CRBP, 40 pp. household ashes at planting because they whether the attractiveness can be improved Fogain R. 1994. Les ravageurs des bananiers au with another type of trap and kairomones. In Cameroun. INFOMUSA 3(1):19-20 believe it reduces damage to the corm Fogain R. 2001. Nematodes and weevil of bananas (Ngamo and Fogain 1998). It is not clear smallholder farms, pheromone mass trapping and plantains in Cameroon: occurrence and host whether ashes have an insecticide or merely a does not seem to be a viable option for con- susceptibility. International Journal of Pest fertilizer effect. Under laboratory conditions, trolling weevil populations because of prob- Management 47(3):201-205 ashes have a repellent effect on adult C. sor- lems of storage and costs (Messiaen 2000a). Fogain R. & N.S. Price. 1994. Varietal screening of didus, but the toxicity to adults is quite low some Musa cultivars for susceptibility to the banana (Messiaen 1999). Host plant resistance borer weevil. Fruits 49(4): 247-251 Fogain R. & H. Ysenbrandt. 1998. Utilisation du neem Commercial banana plantations are renewed Screening for resistance to the banana borer (Azadirahta indica) et du champignon Beauveria every five to six years. During the fallow peri- weevil at CARBAP stated in the 1994 with the bassiana contre le charançon noir des bananiers et od, residual corms are usually destroyed by discovery of the field resistant ‘Yangambi km5’ plantains. Pp. 223-229 in Proceedings of the Vth local women who use the fallow field for food and of the highly susceptible clones of the Annual Conference Bioscience and Food Security. crop production. Tissue cultured plants are plantain subgroup (Musa AAB) compared to Kehe M. 1985. Le charançon du bananier treated with Regent5G (fipronil) or the ‘Cavendish’ (Musa AAA) (Fogain and (Cosmopolites sordidus Germar) en culture de Price 1994). Since then, techniques for early bananiers et plantains. Proceedings of the annual Counter10G (terbufos) at planting and two or reunion of WARCORP, 2-6 December, Douala, three times a year. Crop hygiene (weeding, screening in the field and under controlled Cameroon. 9pp. herbicide application, slashing of residual conditions have been refined. In a recent Messiaen S. 1999. Neem (Azadirachta indica), wood pseudostems and toppled mats) as well as screening, more than 80 varieties were tested. ashes, coffee husk and hot pepper (Capsicum spp.) propping and guying are commonly practised. Several varieties, including CARBAP hybrids, for controlling the banana weevil (Cosmopolites sor- have been selected for enhanced screening in didus): investigations into their effect and mode of Biological control the field. Results of preliminary screening action. Technical report, CRBP, Cameroon. Messiaen S. 2000a. Evaluation of a pheromone baited At CARBAP, research on biological control show a large variety of responses to weevil attack between and within genomic subgroups mass trapping system of C. sordidus with B. bassia- using the entomophagous fungus Beauveria na. Technical report, CRBP, Cameroon bassiana started in 1994 with the discovery of (Messiaen 2000b). No genotypes are more Messiaen S. 2000b. Early varietal screening of Musa local strains in Cameroon (Fogain 1994). susceptible to weevil attack than the ones in varieties for sensibility to the banana weevil: preli- Since then, studies have been carried out the plantain subgroup. Preliminary results minary results. Technical report, CRBP, Cameroon. Ngamo L. & R. Fogain. 1998. Perception paysanne under controlled conditions to test the efficien- point to differences in larval development. If the results are confirmed in the field, it will be des problèmes phytosanitaires en culture de bana- cy of the strains and the possibility of mass niers au Cameroun. Document CRBP, 10pp. production for field trials. Three strains of possible in the short or medium term to develop hybrids partially resistant to C. sordidus. Vilardebo A. 1973. Le coefficient d’infestation, critère Beauveria bassiana, isolated from infected d’évaluation du degré d’attaque des bananeraies weevils caused 92% mortality after nine days par Cosmopolites sordidus Germar, le charançon under laboratory conditions. Research is Conclusions noir du bananier. Fruits 28(6): 417-426. presently carried out on the maintenance of Significant information has been gathered over Ysenbrandt H., R. Fogain, S. Messiaen & P. Sama Lang. 2000. Infestation levels of weevil species on viability with regards to delivery systems and the past ten years on the distribution and popu- Musa cultivars Grande naine (AAA) and French the feasibility of mass production for farmers lation dynamics of the weevil. Despite gains in sombre (AAB) and subsequent plant mortality in or economic agents in Cameroon. Entomo- knowledge on the dynamics of C. sordidus in southwest Cameroon. African Plant Protection pathogenous nematodes have been isolated the Fako and Mungo divisions (littoral and 6(1):21-24.

INFOMUSA — Vol 11, N° 1 PROMUSA IX established fields, weevil damage can result in Although its presence has been known for The biology and management of reduced bunch weight, plant loss, mat die-out some time, the banana weevil, Cosmopolites the banana weevil, Cosmopolites and shortened stand life. In Uganda and sordidus,is not a major source of problems. sordidus in South Africa Tanzania, it has been implicated as a primary The first identification dates back to the 19th factor in the decline and disappearance of century. 1 2 P. Govender and A. Viljoen cooking bananas in traditional growing areas. In 1992, started the installation of spot irrigation 1 Department of Zoology & Entomology; 2 Department of Uganda has ranked banana weevil as the systems and the use of varieties produced in Microbiology & Plant Pathology, Forestry and Agricultural most important biotic constraint on highland vitro. In both instances, this has been accompa- Biotechnology Institute (FABI), University of Pretoria, Pretoria, banana production. 0002, South Africa nied by outbreaks of C. sordidus. In one case Salient features of the weevil’s biology are (spot irrigation), outbreaks increased because The banana weevil, Cosmopolites sordidus, restricted host range (Musa and Ensete), long farmers stopped doing certain things like bury- introduced in South Africa about 30 years ago, life span (up to 4 years), low fecundity (1-3 ing plant residues, a practice which helped con- is the most important insect pest of banana, eggs/week), 1:1 sex ratio, nocturnal activity, trol the pest. In the other case, available data causing economic losses in the Mpumalanga uncommon flight and limited dispersal capabil- suggest that C. sordidus prefers plants pro- and the south coast of Kwazulu-Natal regions. ity. The weevil most commonly enters new duced in vitro. Even though they occur in all Collectively this area represents about 78% of fields through infested planting material. plantations, these problems are more frequent the total 12 078 hectares under commercial Because of populations building up over time, in the above-mentioned situations. banana production in the subtropical pockets pest problems are more pronounced in older For some years, banana growers applied to the of South Africa. The weevil has a limited plantations. In one trial in Uganda, yield loss base of the pseudostem an insecticide potential to migrate from its current distribution increased from 5% in the plant crop to 47% in (Baytion) which has foxime as an active ingredi- area unless it is transferred with infected the third ratoon. This loss was attributed ent. They also used a pirimifos-ethyl based planting material. Information on the life cycle equally to plant loss and bunch weight reduc- product (Bullit) but stopped when these insecti- appears to be consistent with published litera- tions. By the 7th cycle of the second trial, 35% cides were banned. They currently apply eto- ture; the total developmental period being of the mats had died out in weevil-infested profos (Mocap 10G) directly on the soil. about 33 days. Adults emerge during spring plots compared to 2% in controls. Overall A recent innovation is the use of pheromone and late summer, and their nocturnal activity yield losses were 50% for the trial. increases during or after rainfall. Females traps, produced by N.P.P. Calliope (France), to Although pesticides can be an effective control generally lay one egg per week from late fight C. sordidus. The first results are promising. August to February but this number can method, in Uganda the weevil has developed increase during optimal environmental condi- resistance to one chemical. IITA and the Detection of Cosmopolites Ugandan National Banana Research tions and low pest densities. Adults have a sordidus Germar on Tenerife life span of about two years. Although weevil Programme work closely together on cultural numbers are low in the winter months (May to and biological controls, and on host plant resist- Island July), they increase rapidly in spring and early ance. Clean planting material is an important Ruth Torres del Castillo and Clemente summer (August to November). Weevils are means of keeping weevils out of new planta- Méndez Hernández monitored using pseudostem traps at a densi- tions, but the effect normally disappears within ICIA, Tenerife, Canary Islands, Spain ty of 50 traps/ha. Economic threshold values a few crop cycles. A one-year trapping study showed some positive effects on population are 1-2 adults/trap/week and 10 or more larval The agricultural service of the government of reduction but this control is beyond the tunnels/corm. Weevils are strongly attracted Tenerife Island (Cabildo) did a series of sur- resources of most Ugandan growers. Current to the ‘Williams’ and ‘Chinese Cavendish’ cul- veys to determine the extent and severity of emphasis are on the use of neem, endemic tivars. A tentative recommendation algorithm damages caused by Cosmopolites sordidus ants, microbial control (i.e. Beauveria bassiana was developed for the management of C. sor- Germar. Carried out between 1996 and 2001, and endophytes) and host plant resistance. didus in South Africa. It integrates standard the surveys were repeated every three years, Available data indicate that all highland banana cultural control, trapping, biocontrol and chem- depending on the study area. Peeled off pseu- ical control. Two local entomopathogenic clones are susceptible to weevil. However dostem double-disc traps and those with a fungi (Aspergillus flavus and Beauveria screening trials suggest that many resistant horizontal section of the corm were highly cor- bassiana) have been isolated and tested in Musa clones do exist and that antibiosis is the related (R2=0.93). The distribution of C. sor- South Africa but their host specificity and path- predominant means of resistance in these didus in relation to the type of irrigation, the ogenicity requires further investigation. clones. altitude of the plantation and the variety used Various nematicides with insecticidal proper- (‘Pequeña enana’ or ‘Gran enana’) was also ties have been tested in limited field trials and Cosmopolites sordidus in the studied. Time trends were done to see the currently only aldicarb 15% GR, at a rate of autonomous region of Madeira evolution in the distribution of this pest and the 2.03 to 3.00 g a.i./planting station, is regis- damages it causes. tered for use against the banana weevil. Luís Nuno and V. P. Ribeiro Direcção Regional de Agricultura da Regional Autónoma da Management of banana Madeira – Portugal Overview of banana weevil and plantain weevil borer Direcção de Serviços de Produção Agrícola/Divisão de research in Uganda Bananicultura, Centro de Bananicultura – Lugar de Baixo – in Costa Rica 9360-119 Ponta do Sol, Portugal C.S. Gold1 and W.K. Tushemereirwe2 Douglas Cubillo y Mauricio Guzmán Located between 32°38’ north and 16°54’ 1IITA-ESARC, P.O. Box 7878, Kampala, Uganda; 2Uganda west, the island of Madeira lies about 800 km Sección de Fitopatología y Entomología, Dirección de NBRP, P.O. Box 7065 Kampala, Uganda from the coast of Morocco. Banana planta- Investigaciones de CORBANA S.A., Costa Rica The banana weevil is an important constraint tions occupy nearly 850 hectares of the island Chemical control and cultural practices are the on the production of East African highland and are exposed to a subtropical climate. base for the management of banana weevil in bananas and plantains. The larvae attack the In Madeira, the culture of banana started in plantain and banana plantations in Costa corm, reducing nutrient uptake and weakening the 18th century. The most widely grown vari- Rica. Usually the damage of the pest is more the stability of the plant. Attack in newly plant- ety on the island is the ‘Pequeña enana’, intro- important in plantains (Musa AAB) than in ed banana stands can lead to crop failure. In duced in 1842. bananas (Musa AAA), due to differences in

X PROMUSA INFOMUSA — Vol 11, N° 1 varietal susceptibility and in crop manage- bassiana cultures collected from the field and Xenorhabdus, respectively. Nematode ment. We are carrying out studies on the traps in the first five weeks after application dauer juveniles, harbouring cells of their specif- ecology and biology of the pest, and on meth- were highly infective causing 60-100% wee- ic bacteria in their intestine, search for insects ods for chemical, biological, ethological and vil mortality in 14 days, but the infectivity of in the soil. After penetration in the host insect, cultural control. The combination of some of the fungus was significantly reduced in the they release their symbionts. The bacteria mul- these methods could be the best alternative wet season; likely due to contamination by tiply and produce suitable conditions for nema- for the management of the pest. other soil micro-organisms. The maize cul- tode reproduction in the dead insect. After ture formulation (2 x 1015 conidial/ha) and about two weeks, dauer juveniles emigrate from maize-soil based formulation (2 x1014 coni- the cadaver and search for a new host. dia/ha) applied at the base of the mats Session 2. Control of the The use of entomopathogenic nematodes reduced the adult banana weevil popula- for the control of Cosmopolites sordidus banana borer weevil tions by 30-50% and kept them at lower lev- Cosmopolites sordidus els than in the untreated plots. The treated should now be economically feasible. plant also showed reduced weevil damage Production and formulation techniques have been improved to provide these Overview of Beauveria bassiana and up to 16% B. bassiana disease infection was observed in dead weevils in the nematodes to growers at a cost equivalent for microbial control of the field. to or lower than the one of chemical insecti- banana weevil in Uganda cides. Application of entomopathogenic Previous studies have demonstrated that nematodes requires less labour than insec- good potential exists for the use of B. C.M. Nankinga1, C.S. Gold1, ticides, avoids the problems of insecticide bassiana for microbial control of the banana W. Tushemereirwe2 resistance and has little or no adverse weevil. The collaborative team, composed effects on the environment. However, for 1International Institute of Tropical Agriculture, Eastern and of scientists from the International Institute Southern Africa Regional Centre, P.O. Box 7878, Kampala, of Tropical Agriculture (IITA), the National reliable results, it is necessary to improve 2 Uganda; National Banana Research Programme, Kawanda Banana Research Programme of the the application technique, and to select the Agricultural Research Institute, P.O. Box 7065, Kampala appropriate species and strains of ento- Uganda Uganda National Agricultural Research Organization (NARO), CABI Biosciences UK mopathogenic nematodes. Finally, various Collaborative research is being conducted and University of Reading is undertaking fur- strategies for the control off Cosmopolites in Uganda to assess the microbial control ther research into the mass production and sordidus using entomopathogenic nema- potential of Beauveria bassiana (Balsamo) formulation of B. bassiana and exploring todes are discussed. Vuillemin (Hyphomycetes) for the banana other delivery systems of B. bassiana for weevil, Cosmopolites sordidus (Germar), integration with other control measures (Coleoptera: Curculionidae). Since the under farmers’ conditions. Research is early 1990s, isolation, characterization and The use of two insecticide- pathogenicity studies have come up with a geared to developing economically viable nematicides to control the weevil mass production, formulation and delivery selection of indigenous isolates of B. Cosmopolites sordidus and the bassiana that have good growth and pro- systems that will overcome the problems duction traits, causing 50-100% weevil mor- associated with field fungal efficiency, per- nematode Radopholus similis tality within 10-21 days after inoculation, sistence and transmission. Further research and their effect on some on the ecological relationships between depending on the isolate. In small-scale production variables of the field trials conducted at Kawanda banana weevils and entomopathogens will Agricultural Research Institute, one B. also be undertaken to understand the condi- banana ‘Gran enana’ in relation bassiana isolate (code G41), which showed tions under which B. bassiana is likely to be to the environmental conditions most effective in controlling this pest. This high pathogenicity to C. sordidus as well as encountered in Costa Rica superior growth and sporulation compared include studies on the behaviour of the wee- to other isolates, was tested. Three meth- vil, which might influence the likelihood of Dennis Alpízar M. ods of delivering B. bassiana, namely (i) the insect contacting the pathogen; the bio- types in the Cosmopolites sordidus species, Estación Experimental Los Diamantes. Ministerio de application of the fungus on topsoil around Agricultura y Ganadería. Guápiles, Limón, Costa Rica the base of the banana mat (ii) application which might exhibit different susceptible lev- of the fungus with pseudostem and disc-on els to the pathogen; and pathogen viability In Costa Rica, insecticide-nematicides are com- stump traps and (iii) application of the fun- and virulence under aerobic (ordinarly pseu- monly used to control Cosmopolites sordidus. gus to banana planting suckers, were evalu- dostem traps) and anaerobic conditions Pseudostem or corm traps are also used while ated. Treating banana suckers with a B. such as in the semio-chemical-based trap- the utilisation of the aggregation pheromone bassiana dry maize culture formulation and ping systems. We acknowledge funding Cosmolure®, introduced at the end of the 90s from the Rockefeller Foundation, DFID and a soil-maize based formulation (2.3 x 1012 to control the banana weevil, is still a new agri- BMZ in support of this work. conidia/planting hole), reduced weevil dam- cultural practice in the country’s banana and age by 20-30% within a period of eight plantain plantations. weeks after planting in holes dug in a 2 to The objective of this study is to compare the 3-year-old banana field of local EAAH cook- Entomopathogenic nematodes effect of using or not using, under the same ing cultivar. Dead C. sordidus adults and for the control of insect pests. conditions over two years, two insecticide- larvae with B. bassiana fungal growth were nematicides: terbufos (four applications) and observed in the treated suckers indicating The outlook for the control etoprofos (one application). immature stage infection. When the maize of Cosmopolites sordidus After two years the results show, using the and soil-based formulations of B. bassiana Fernando García del Pino were applied beneath the pseudostem and Universidad Autónoma, Barcelona, Spain Fisher t-test (0.05), that the ‘weight of the disc-on stump traps, it was observed that bunch’ was slightly but significantly higher in the moist conditions under the traps, in Entomopathogenic nematodes (Heterorhabditis the non-treated plots while the ‘number of addition to attracting weevils, also provided spp. and Steinernema spp.) are used in biocon- nematodes (Radopholus similis) in the func- a favourable environment for extra sporula- trol against different insect pests in soil and tional root of the sucker’ was slightly lower in tion of B. bassiana and this enabled the fun- cryptic habitats. They are symbiotically associ- the treated plots. The other variables were gus to remain potentially infective. B. ated with bacteria of the genera Photorhabdus not statistically different.

INFOMUSA — Vol 11, N° 1 PROMUSA XI The cost of applying the insecticide-nemati- can survive and maintain itself in the soil dur- cide was $US 1200 per hectare for the two- ing at least three months. During bioessays, Session 3. Molecular biology year duration of the study. the mixture infected a pest of palm trees (Carpophilus dimidiatus) similar to the weevil. Resistance of diploid banana Alternatives to the control Moreover, B. bassiana can colonize the peti- genotypes to Cosmopolites of Cosmopolites sordidus oles of P. dactylifera. We think that such endo- sordidus (Germ. 1824) phytic behaviour is useful in controlling pests (coleoptera: curculionidae) (Germar) in banana plantain like the weevil. plantations M. Fancelli, A. Souza Do Nascimento, Research on the Musa borer N. Fritzons Sanches, R. Correa Caldas and A. Padilla Cubas, F. García del Pino, S. De Oliveira E Silva weevil (Cosmopolites sordidus) L.V. López Llorca and A. Carnero Hernández EMBRAPA, Mandioca y fruticultura, Rua EMBRAPA s/n, Caixa ICIA, Apartado aéreo 60, 38080 La Laguna, Tenerife, Islas at CARBAP Postal 007, 44380-000 Cruz das Almas, Bahia, Brazil Canarias, Espaa Eric Fouré1, S. Messiaen1, Roger Fogain1, Plant resistance to insects is considered a In the Canary Islands, Cosmopolites sordidus Thierry Lescot2 secure and durable strategy for the control of (Germar) is the most important pest in planta- Cosmopolites sordidus, especially in plantations of banana plantain. Given the results of 1CARBAP, B.P. 832, Douala, Cameroon; 2CIRAD-FLHOR tions where investments are low. Despite the chemical treatments, alternatives are sought to BPA, Boulevard de la Lironde, TA50/PS4, 34 398 Montpellier existence of a large number of varieties, the Cedex 5, France control the weevil. We therefore sampled soils, number of cultivars used in Brazil is small, cultivated and not cultivated, in the province of The banana borer weevil Cosmospolites sor- hence the importance of evaluating the new Santa Cruz de Tenerife looking for parasitized didus is the most important pest of banana genotypes introduced and/or generated in organisms. Using ‘Galleria mellonella’ traps, we and plantain in African plantations. The genetic improvement programmes. Although especially searched for entomophagous nema- African Center of Banana and Plantain (CAR- in the fields all varieties are infested, some todes and fungi. studies show differences among genotypes BAP) carries out researches on C. sordidus, Entomophagous nematodes were found in two with respect to development, survival and with a focus on integrated pest management, sampling points from which we isolated attractiveness for oviposition. Given the cur- Heterothabditis spp. and Steinernema spp. As namely: rent expansion of banana plantations in Brazil for entomophagous fungi, we isolated • genetic resistance, and the development of methodologies for the Aspergillus flavus, Beauveria bassiana, • population dynamics, production of in vitro plantlets, there is a grow- Metarhizium anisopliae and Paecilomyces ing interest for improved varieties, including spp. Verticillium lecanii was isolated form • biological (bio-insecticides and pheromones) resistant ones. The tetraploid hybrids of white flies collected in different localities. and chemical control. banana (AAAB) are obtained by crossing We characterized the morphology of the fungi Results are presented on selection for weevil diploid (AA) genotypes with triploid (AAB) cul- and studied their germination and sporulation resistance; weevil evolution; effects on plan- tivars of types Prata (Silver) and Maçã capacity, their production of biomass and their tain production in southwest Cameroon; best (Pomme). A genetic improvement programme behaviour under various naturally occurring parameters for field infestation representation; of diploid genotypes is carried out, with the conditions of humidity, temperature and pH. efficiency and limits of chemical insecticides; aim of increasing yield and resistance to We also studied their enzymatic activity: quiti- use of pheromones in trapping system; effi- pests, one more reason for a close partner- no, amylo, proteo, lipo and pectinolytic. ciency of Beauveria bassiana in southwest ship between breeders and geneticists. We also conducted biological assays using Cameroon; and use of insecticide plants The objectives of the present work are: ‘Galleria mellonella’ and, based on the results, against weevils. • To evaluate diploid hybrids of banana in rela- we inoculated C. sordidus using two methods. tion to Cosmopolites sordidus. Finally, we evaluated the interactions between • To study the mechanisms of resistance to these isolates and Fusarium oxysporum, the Research on the Musa borer the weevil in diploid genotypes. main pest of banana. weevil (Cosmopolites sordidus) at CIRAD Behaviour of Methodology The following genotypes are being studied: entomophagous fungi Christian Chabrier1, Thierry Lescot2 0304-02; 0337-02; 0323-03; 1318-01; 2803- on vegetal tissue 1CIRAD-FLHOR BPA, B.P. 153, 97202 Fort de France, 01; 4223-03; 5012-02; 4215-02; 4279-13 and L.V. Lopez-Llorca Martinique, France, 2see above 4252-03. These materials are diploid hybrids generated by the Banana Genetic Departamento de Ciencias Ambientales y Recursos Naturales, The Banana, Plantain and Pineapple pro- Universidad de Alicante, Aptdo. Correos 99, 03080 Alicante, Improvement Programme, most of them pre- Spain gramme (BPA) of CIRAD-FLHOR carries out senting resistance to black Sigatoka. Plantlets research in the French West Indies and in the from these genotypes were placed in We studied the use of ornamental plant vege- central laboratories of Montpellier, France, as screened planting holes in the field and infest- tal residues from nurseries to produce inocula well as collaborations with CARBAP in ed with adults of the banana weevil using the of antagonistic fungi, including ento- Cameroon and applications in Indian Ocean methodology adopted by Seshu-Reddy and mophagous fungi. islands. Lubega (1993). Plants without the insect are Seeds of Phoenix dactylifera turned out to be The research focuses on integrated pest man- kept under the same conditions to get informa- excellent for the production of Beauveria agement along the same lines as CARBAP. tion on the injuries caused by the pest. The bassiana. Scanning electron microscopy genotype Terra is being used as susceptibility revealed a very porous substrate, a feature The results presented focus on the combina- pattern. The variables analyzed are: coeffi- which facilitates fungal development and tion of two types of synthetic pheromones; the cient of infestation, number of insects present sporulation. A soil-based formulation of B. use of antomopathogenous bacteria and in the galleries, plant height, pseudostem bassiana can sporulate and overcome fungis- nematodes; and the efficiency and limits of diameter, time period until inflorescence emis- tasis. Thanks to this type of seed, the fungus chemical insecticides. sion, yield, bunch weight, number of hands,

XII PROMUSA INFOMUSA — Vol 11, N° 1 finger diameter and number of fingers/hand. have limited conventional breeding efforts on The beauty of genetic engineering is that genes Under laboratory conditions the development Musa. from several sources can be exploited and that of the insect and its non-preference for feed- The literature on weevil resistance in Musa these can be transformed using a gene pyra- ing and oviposition will be studied for the reviewed by Pavis and Lemaire (1997), miding strategy. However, a great deal of infor- same genotypes in order to identify the types Kiggundu et al. (1999) and Kiggundu (2000) mation about the complex nature of weevil of resistance involved in the interaction suggests that antibiosis is the key resistance resistance is missing and molecular marker between the weevil and the banana plant. The mechanism. Sources of resistance have also analysis can assist in genetic analysis and duration and viability of the larval and pupa been found in the Musa germplasm tested by mapping. Opportunities also exist for quickly phases, the weight of the pupa after 24 hours Fogain and Price 1994, Lemaire 1996, and developing weevil resistance through genetic and the number of adults with defects, will be Kiggundu et al. (in press). transformation. noted. Tests for attractiveness and consump- From a screening trial conducted over four tion will be carried out. Analyzes will be done References to identify the presence of attractive/repellent crop cycles at the International Institute of Tropical Agriculture-East and Southern Carozzi N. & M. Koziel (eds). 1997. Advances in insect substances, as well as phago stimulants control the role of transgenic plants. Taylor and and/or phago deterrents. Rhizome hardness Regional Center (IITA-ESARC), in Uganda, we Francis, London. 301pp. will be evaluated using a penetrometer. found that East African highland bananas Fogain R. and N.S. Price. 1994. Varietal screening of (EAHB) (AAA-EA) and plantains (AAB) were some Musa cultivars for susceptibility to the banana the most susceptible. These were followed by weevil, Cosmopolites sordidus (Coleoptera: Reference Curculionidae). Fruits 49(4):247-251. Seshu-Reddy K.V & M.C Lubega. 1993. Evaluation of ABB bananas (cvs ‘Pisang awak’ and ‘Bluggoe’), diploid banana derived hybrids, AB Gold C.S. 1998. Banana weevil: ecology pest status banana cultivars for resistance to tolerance of the and prospects for integrated control with emphasis weevil Cosmopolites sordidus Germar. Pp. 143-148 bananas (cvs ‘Ndiizi’ and ‘Kisubi’), AAA on East Africa. Pp. 49-74 in Proceedings of a in Breeding banana and plantain for resistance to bananas (cvs ‘Yangambi km5’, ‘Cavendish’ and Symposium on Biological Control in Tropical diseases and pests. (J. Ganry, ed.), CIRAD/INIBAP, ‘Gros Michel’) and the wild AA type ‘Calcutta 4’ Habitats: Third International Conference on Tropical Montpellier, France. being the most resistant. Several factors were Entomology 30 October – 4 November 1994. (S.K. shown to contribute to weevil resistance. Dry Saini, ed.). ICIPE, Nairobi, Kenya. matter content (representing corm hardness), INIBAP 1986. Banana research in East Africa. Proposal for a regional research development net- Aspects of banana weevil resin/sap production of the corm and suckering work. INIBAP, Montpellier, France. resistance in Musa and prospects ability were important for weevil resistance in all Kiggundu A., D. Vuylsteke & C. Gold. 1999. Recent accessions. Corm diameter (size) was also advances in host plant resistance to banana weevil, for genetic engineering against important in the EAHBs. Preliminary investiga- Cosmopolites sordidus (Germar). Pp. 87-96 in the banana weevil tions into the chemical basis of resistance, Mobilizing IPM for sustainable banana production in using high-performance liquid chromatography Africa. (E. A. Frison, C.S. Gold, E.B. Karamura and Andrew Kiggundu1 and Clifford S. Gold2 profiles of corm extracts, indicated that com- R.A. Sikora, eds.). INIBAP, Montpellier, France. Kiggundu A. 2000. Host-plant interactions and resis- 1National Banana Research Programme, Kawanda Agricultural pounds present in some resistant cultivars tance mechanisms to banana weevil Cosmopolites Research Institute, P.O. Box 7065, Kampala, Uganda and the (especially those with a B genome) negatively sordidus (germar) in Ugandan Musa germplasm. Forestry and Agricultural Biotechnology Institute, University of correlated with weevil damage. These com- M.Sc. Thesis. University of the Orange Free State, Pretoria, 74 Lunnon Road, Hillcrest, Pretoria, 0002 South pounds were absent in most of the susceptible Bloemfontain, South Africa. Africa; 2International Institute of Tropical Agriculture (IITA). East cultivars tested. Lemaire L. 1996. Les relations sémiochimiques chez and Southern Africa Regional Centre (ESARC) P. O. Box 7878, le charançon du bananier Cosmopolites sordidus Kampala Uganda Ortiz et al. (1995) studied the genetic inheri- Germar (Coleoptera: Curculionidae) et la résistance The banana weevil (Cosmopolites sordidus tance of banana weevil resistance and found it de sa plante-hôte, le bananier. Thèse de doctorat, Germar) is probably the most important pest to be under the control of more than one gene Université de Montpellier II, France. affecting the production of banana and plan- with partial dominance towards susceptibility. Ortiz R., D. Vuylsteke, B. Dumpe & R.S.B. Ferris. They found significant additive effects and mod- 1995. Banana weevil resistance and corm hardness tain. Attack by the weevil results in severe in Musa germplasm. Euphytica 86:95-102. crop losses from plant toppling, snapping, ifier genes plus dosage effects of susceptibility Pavis C. & L. Lemaire. 1997. Resistance of Musa death and reduced bunch weights (INIBAP genes, causing higher susceptibility at higher germplasm to the banana borer weevil, 1986). Pesticides are effective, but uneco- ploidy levels. Cosmopolites sordidus Germar (Coleoptera: nomical for small-scale subsistence farmers. Conventional crossbreeding in combination with Curculionidae) INFOMUSA 5(2):3-9. In addition, the weevil is resistant to a wide molecular biotechnology techniques, like mark- Seshu-Reddy K.V & M.C Lubega. 1993. Evaluation of range of insecticides and although cultural banana cultivars for resistance to tolerance of the er assisted selection (MAS) and genetic trans- weevil Cosmopolites sordidus Germar. Pp. 143-148 controls may help, labour and material formation, appears to be an attractive option to in Breeding banana and plantain for resistance to requirements often limit their adoption (Gold further understand resistance to weevil and diseases and pests. (J. Ganry, ed.), CIRAD/INIBAP, 1998). simultaneously develop resistant cultivars. Montpellier, France. Host plant resistance shows potential for the Sources of transgenes can include the Musa long-term control of the banana weevil on genome itself and others of plant or animal ori- small-scale farms, within an integrated pest gin (Carozzi and Koziel 1997). The National management perspective (Seshu-Reddy and Banana Research Programme at Kawanda Genetic biodiversity in banana Lubega 1993). However, the development of Agricultural Research Institute, Uganda, in col- weevil (Cosmopolites sordidus) weevil resistance in banana is still in its infan- laboration with IITA-ESARC, is interested in cy and breeding programmes have only developing a MAS programme to identify mark- from different banana growing recently included weevil resistance as a crite- ers and possibly resistance genes. At the regions ria for introgression into cultivated Musa. The Forestry and Agricultural Biotechnology cumbersome nature of resistance screening Institute (FABI), South Africa, we are involved Vincent Ochieng International Center for Insect Physiology and Ecology methods has made work on weevil difficult, in studies to identify differentially expressed (ICIPE), PO Box 30772, Nairobi, Kenya slow and at times expensive. Lack of under- genes during weevil infestation of resistant ver- standing of resistance mechanisms and their sus susceptible Musa. Efforts are also expand- Genetic variability in banana weevil popula- associated genes, coupled with long genera- ed to investigate the potential of using protease tions was analyzed in a large number of tion times, triploid sterility of most edible culti- and/or alpha-amylase inhibitors in the trans- samples obtained from 15 banana growing vars and poor seed set due to incompatibility, genic control of banana weevil. tropical countries using random amplified

INFOMUSA — Vol 11, N° 1 PROMUSA XIII polymorphism DNA (RAPD). The study variability in relation to biological control; 4) to included 46 RAPD markers. Poly- Alternatives to control the identify and study the behaviour of the ento- morphisms, as measured by percentage of banana borer weevil, mophagous organisms attacking the weevil. polymorphic RAPDs, varied between Cosmopolites sordidus Germar Our participation in this project consists in deter- 78.3% and 97.8%. Genetic variability was (Coleoptera: Curculionidae), on mining weevil inter and intra-population variabili- measured using Shannon’s information ty among the populations affecting the banana index and partitioned into inter and intra- banana plantations of Tenerife, Gomera and La Palma, population components. Overall, genetic P. Castañera, F. Ortego, in order to provide information which could help variation between C. sordidus populations M. Montesdeoca Montesdeoca in controlling this pest, such as the origin and was 25.3% ((Hsp-Hpop)/Hsp). The genetic the dispersal patterns of these weevils. and A. Carnero Hernández Detecting DNA polymorphism will be done using diversity for the species (Hsp) was 83.4%. Consejo Superior de Investigaciones Científicas (CSIC), The mean of intra-population values (Hpop) Madrid, Spain the randomly amplified polymorphic DNA tech- for all populations was 62.3%. The total nique, following the methodology used to study genetic diversity was explained by high The banana borer weevil, Cosmopolites sor- the population genetic of another species of variation between populations (mean didus Germ. is considered the main pest of Curculionidae, a pest of sugarbeet in Andalusia Gst=0.213), which is consistent with low banana in the world. In the Canary Islands, it (Taberner et al. 1997, J. Mol. Evol. 45:24-31). gene flow between populations was first noticed in 1945 and eradicated. It We also plan to identify and characterize the (Nm+0.811). The high inter-population reappeared in 1987, but despite attempts to digestive enzymes of the weevil, and to con- genetic variation observed in this study control it by chemical and cultural means, the duct bioassays with protease inhibitors in order could be explained by limited migration, pest has spread causing increasingly severe to determine their effect on the survival and given to the restricted mobility and damages. development of this pest. This information is monophagy of banana weevils. In a paral- A project (RTA 02-100-C3) was recently initi- necessary for the eventual production of transgenic banana plants expressing defence pro- lel study, PCR-RFLP analysis was used to ated to develop an integrated pest manage- teins, a possibility which would offer new ment strategy against the weevil. Its aim is to differentiate the CO1 mtDNA of 19 banana means of controlling the weevil. Preliminary maintain the profitability of the culture by pro- weevil populations. The PCR-RFLP studies suggest that larvae and adults possess ducing an environmentally friendly banana method produced unambigous profiles that a complex proteolytic system which includes differentiated certain populations from oth- that should have a competitive edge on aspartyl, cysteine and serine endoproteases as ers. In another study, the genetic variabili- European markets over bananas from other well as amino and carboxypeptidases. Once ty in weevil populations was analysed in producing countries. the characterization of digestive proteases is samples obtained from 15 sites in banana The objectives of the project are: 1) to optimize finished, we will study their interactions with growing regions of Uganda. Although the the use of pheromone to control the weevil; 2) to specific inhibitors in order to determine which weevil populations in this case were very evaluate the relation between the extent of the inhibitors, or combination of inhibitors, could be similar genetically, some variability was damages caused by the weevil and yield losses; incorporated by genetic manipulation in poten- observed. 3) to determine weevil inter and intra-population tially resistant banana plants.

Fourth and final FAO/IAEA research coordination meeting on Cellular biology and biotechnology including mutation techniques for creation of new useful banana genotypes

Summary report the conventional breeding of banana to “Banana improvement: Cellular and molecu- induce desirable variation such as disease lar biology, and induced mutations”. By S. Mohan Jain resistance, dwarfism and earliness, and Overall achievements also to promote the development of meth- FAO/IAEA Joint Division, International Atomic Energy Agency, Research tools were developed for A-1400, Box 100, Wagramerstrasse 5, Vienna, Austria. Email: ods for large-scale and rapid multiplication germplasm characterization and improve- [email protected] of the mutants/segregants through somatic ment through induced mutations, cryop- Bananas and plantains are grown in over embryogenesis and micropropagation. The reservation, somatic embryogenesis, 100 countries throughout the world with Belgium Government decided to fund this somaclonal variation and genetic engineer- annual production around 88 millions metric CRP in 1996. Since than, three Research ing. Some of the existing cultivars have tons. Banana fruit production is severely Coordinated Meetings (RCM) were held in been improved for disease tolerance and limited by several diseases and pests such different countries including Vienna, important agronomic traits. Collaborations as banana bunchy top virus, burrowing Malaysia and Sri Lanka. The fourth and among participating laboratories were nematodes (Radopholus similis), Moko dis- final RCM of this banana CRP was held at established, including exchange of staff, ease (Ralstonia solanacaearum), black the Katholieke Universiteit Leuven training and technology transfer. Sigatoka or black leaf streak (Mycos- (KULeuven), Leuven, Belgium, 24-28 phaerella fijiensis), Fusarium wilt (Fusarium September 2001. A total of ten participants Practical achievements oxysporum f.sp. cubense). FAO/IAEA start- attended it including from Austria Research contract holders J. Lopez Torres ed banana Coordinated Research Project (FAO/IAEA), Belgium, Cuba, Czech (Cuba), Mak Chai (Malaysia), A. James (CRP) in 1994 with the general aim to inte- Republic, Germany, Israel, Mexico, (Mexico), and J. Dolezel (Czech Republic) did grate radiation induced mutations in vitro Philippines, and Sri Lanka. The results of exceedingly well in this CRP. Nicolas Roux culture and molecular genetics methods into CRP will be published in a book entitled (FAO/IAEA) has been instrumental in dissoci-

XIV PROMUSA INFOMUSA — Vol 11, N° 1 ation of chimerism and developing flow 5. Virus indexing procedures were transfor gene tagging, using maize Ac element, cytometry protocol. ferred to Sri Lanka for indexing local in banana genome. A substantial number Several young students benefited from this banana virus strains. of distinct mutants were generated and CRP in completing their master and PhD pro- 6. An early screening technique was devel- characterized. grammes in Israel, Czech Republic, and oped for Fusarium wilt using tissue culture- 11. Fluorescence in situ hybridization Belgium. Some of the participants presented derived plants in a double-tray system. (FISH) protocol was developed for Musa for their results in international conferences. A 7. A selection system was developed against detailed studying of karyotypes, providing total of 51 research papers have been pub- black Sigatoka disease by using Mycosphaerella distinct chromosome landmarks, gene local- lished in conference proceedings and interna- fijiensis crude extract, the semi-purified, and one ization, analysis of long-range chromosome tional refereed journals. purified fraction (juglone). structure, and linking to physical and genet- Many international trainees received 8. Screening techniques for nematode ic maps. training on several aspects of banana tis- resistance were developed in Musa under 12. A total of 28 allele-specific simple sue culture, molecular cytogenetics and shade-house and field conditions. Aseptic sequence repeat (SSR) markers were gen- molecular markers at KULeuven and cultures of Radopholus similis and erated for Musa and used to detect: poly- Faculté universitaire des sciences Pratylenchus coffeae were established morphisms between the A and B genomes, agronomiques, Gembloux (FUSAGx), using alfalfa calli, and their pathogenicity identify hybrids, and trace back the B Belgium; Institute of Experimental Botany was confirmed after greenhouse tests. genome in hybrids. These markers are now (IEB); and University of Frankfurt, 9. DNA flow cytometry was used for detec- used within the CRP and worldwide. A total Germany. The trainees came from China, tion of polyploidy, monitoring of cytochimera of 24 locus-specific, highly polymorphic Cuba, Egypt, Mexico, and Rwanda. The dissociation, and analysis of karyological SSR markers were also produced for outcome of these trainings was very suc- stability of ECS. Mycosphaerella fijiensis to discriminate cessful. For example, a Cuban trainee 10. Transposon mutagenesis was explored them from other species. was successful in establishing new somatic embryogenic banana cell suspensions from Cuban plant material. In addition, he successfully irradiated plant material in Cuba. In Sri Lanka, 20 persons from coun- th tryside were given training in tissue culture Abstracts of papers presented during the 4 and final technology for mass production of banana. FAO/IAEA research coordination meeting Post-graduate training on indexing of banana viruses was organized. plants micropropagated from the male inflo- Flow cytometry facilities were estab- Detection of DNA methylation changes in micropropagated rescence with 14 (3%) and the lowest in lished at the International Institute for plants micropropagated from the sucker with Tropical Agriculture (IITA, Nigeria) and banana plants (Musa AAA cv. 8 (1.7%). These differences were not statisti- The Malaysian Institute for Nuclear ‘Grande naine’) using cally significant. In leaf tissue of convention- Technology MINT, Malaysia). The transfer the methylation-sensitive ally propagated plants DNA methylation polymorphisms were not detected. involved staff training in the Institute of amplification polymorphism Experimental Botany (IEB, Czech Micropropagated plants were relatively hyper- Republic) and expert visit. (MSAP) technique methylated in comparison to conventionally A. James, S. Peraza-Echeverria, propagated plants, with some bands being methylated in all micropropagated plants but Specific achievements V. Herrera-Valencia non-methylated in all conventionally propagat- 1. Detection of DNA methylation polymor- and L. Peraza- Echeverria ed plants. These results demonstrated the phism in banana micropropagated plants Unidad de Biotecnología, Centro de Investigación Científica usefulness of MSAP to detect DNA methyla- with amplified fragment length polymor- de Yucatán, Mérida, Yucatán, Mexico. tion events in micropropagated banana plants phism (AFLP). and indicate that DNA methylation changes The extent of DNA methylation polymor- 2. Somatic embryogenic cell suspension cul- are associated with micropropagation. phisms was evaluated in leaf tissue of micro- tures (ECS) were developed for several propagated banana (Musa AAA cv. ‘Grande banana cultivars including plantains (AAB). naine’) derived either from the vegetative Discovery of functional genes Three cryopreservation techniques were apex of the sucker or the floral apex of the in the Musa genome developed for long-term conservation of male inflorescence using the methylation-sen- E. Khayat1, A. Ilan1, I. Regev2, S. Gepstein2, meristems. An INIBAP technical guideline sitive amplification polymorphism (MSAP) L. Sagi3, R. Swennen3 and H. Schoofs3 for cryopreservation of banana was pub- technique, which utilizes the restriction lished in English, French and Spanish. isoschizomer pair Msp I and Hpa Il, whose 1Rahan Meristem, Dept of R&D, Kibbutz Rosh, Hanikra 22825 ability to cleave at the sequence 5’-CCGG-3’ Israel 3. Induced mutations generated a series of 2 is affected by the methylation state of the Technion, Israel Institute of Technology, Faculty of Biology, improved clones that were screened for dif- Haifa 32000, Israel cytosines. In all, 465 fragments, each repre- 3 ferent traits such as early flowering, reduced Laboratory of Tropical Crop Improvement, KULeuven, senting a recognition site cleaved by either or Kasteelpark Arenberg 13, B-3001 Leuven, Belgium height, large fruit size, and tolerance to both of the isoschizomers were amplified Fusarium. using eight combinations of primers. A total Development of stable and reproducible 4. Both Agrobacterium-mediated transfor- of 107 sites (23%) were found to be methylat- transformation and regeneration technologies mation and particle bombardment methods ed at cytosine in the genome of micropropa- opened new horizons in banana and plantain were used for banana transformation, and gated plants. The highest number of DNA breeding. Several transformation strategies transformation rate was cultivar dependent. methylation polymorphisms was detected in have been published in the last five years by

INFOMUSA — Vol 11, N° 1 PROMUSA XV different banana biotechnologists. Disease demonstrates that it is possible to perform flow resistance and improvement of fruit quality Analysis of Musa genome cytometric ploidy analysis in distant laborato- have been the focal points of most Musa using flow cytometry ries. breeders. However, despite growing interest and molecular cytogenetics In attempt to characterize DNA sequences in banana biotechnology, the pool of Musa contributing to structure and evolution of Musa genes in public databases is relatively small J. Dolezel1, M. Valárik1, J. Vrána1, J. Safár1, 1 1 chromosomes, we have constructed partial (of the approximately 300 accessions placed E. Hribová , N. Gasmanová , I. Van den genomic DNA libraries in M. acuminata and M. 2 1 2 in the NCBI database less than 25% are anno- houwe , M. Dolezelová , R. Swennen and balbisiana and screened them for clones car- tated cDNA’s). Our laboratory is currently H. Simková1 rying highly repeated sequences, and employing several approaches for the identifi- 1Institute of Experimental Botany, Laboratory of Molecular sequences carrying rDNA. Isolated clones cation of functional genes in the Musa Cytogenetics and Cytometry, Olomouc, Czech Republic were characterised in terms of copy number, genome. These include transposon tagging, 2Laboratory of Tropical Crop Improvement, KULeuven, genomic distribution in M. acuminata and M. ‘high throughput’ random mutation by Kasteelpark Arenberg 13, B-3001 Leuven, Belgium balbisiana, and sequence similarity to known ribozyme cleavage of mRNA, suppression This project focuses on the analysis of Musa DNA sequences (Table 1). In contrast to subtractive hybridization (SSH) and bioinfor- many plant species where mobile elements matic annotation of clustered EST’s. genome at nuclear and chromosomal level with the aim to understand long-range organi- and their remnants contribute most of the We have introduced the maize Ac transpos- zation of Musa chromosomes and to charac- nucleotide content, our observations indicate able element into the Musa genome and fol- terize changes of chromosome structure dur- that these elements do not represent a major lowed excision and insertion of the element in ing speciation and evolution of cultivated fraction of the Musa genome. All repetitive numerous transgenic lines. The goal was to clones. sequences were more abundant in M. acumi- investigate the frequency of transposition and nata. As the genome of M. acuminata is larg- We have used flow cytometry to determine distribution of insertions along chromosomes. er compared to M. balbisiana, the present ploidy levels of Musa accessions held at the The constructs we have used include an Ac results demonstrate that the increase in INIBAP Transit Center (KULeuven). Flow element fused to GUS reporter under the 35S genome size of M. acuminata was due to mul- cytometric ploidy assay involved preparation promoter. PCR analysis of a variety of tiplication of some repetitive sequences. The of suspensions of intact nuclei from small mutants revealed that most carried a chimeric findings of this study improve the knowledge amounts of leaf tissue and the analysis of fluo- pattern with regard to expression of the for- of long-range organization of chromosomes in eign genes. Consequently, only a few trans- rescence intensity after staining with DAPI. Chicken red blood cell (CRBC) nuclei were Musa. The availability of homologous probes genic lines (tissue cultured siblings) showed for fluorescence in situ hybridization (FISH) detectable differences in the banding pattern included in every sample as an internal reference standard (Figure 1). From the 890 will allow more specific mapping of rDNA on Southern hybridization blots. Attempts sequences. were made to stabilize the Ac element follow- accessions analysed so far, 8.4% were classi- A novel protocol for isolation of high-molecu- ing a limited number of transpositions, by fied for the first time, and 7.6% accessions lar-weight DNA in Musa has been developed silencing the gene encoding the transposase exhibited other ploidy then reported previous- and the work is in progress to construct bacte- enzyme after excision. ly. In 2% of the accessions, plants of mixed ploidy were detected. A reliable and high- rial artificial chromosome (BAC) library for the Differentially expressed genes, which are acti- throughput system for ploidy screening in B genome of Musa. Availability of the BAC vated in the post climacteric phase of fruit Musa is an important outcome of the study. library will permit isolation of clones containing development, were analyzed in the peel and The use of CRBC nuclei, allowed high-resolu- low proportion of repetitive DNA. Such clones pulp of banana fruit. Using suppression sub- tion analysis, and the results obtained so far will be localized using FISH and will be used tractive hybridization (SSH) we have isolated indicated suitability of this system for rapid to increase the number of already existing over 200 partial cDNA’s encoding genes detection of aneuploidy. As the materials for chromosome landmarks. Furthermore, BAC which are expressed during the final stages of analysis were sent by express mail, this work library will be screened for clones, which con- fruit development (senescence). High throughput screening by membrane hybridiza- Figure 1. Examples of histogrammes of relative DNA content, which were obtained during ploidy screening of ITC accessions using flow cytometry. The ploidy of individual plants was estimated tion was employed for preliminary selection of based on the ratio of positions of peaks corresponding to G1 nuclei of Musa and CRBC. While the candidate genes involved in regulation of the analysis confirmed ploidy classification for M. acuminata ssp. banksii (ITC 0885), ‘Lakatan’ (ITC 0573), and TMPx 2637-49 (ITC 1196), the classification was not confirmed for ‘Cavendish 901’ (ITC 0738), onset of senescence. Sequence analysis and which was found to be hexaploid. blasts against GeneBank databases revealed approximately eighty non-redundant clones, 250 which were up regulated in the post-climac- M. acuminata ssp. banskii CRBC teric phase. Most, but not all of these genes 200 were up regulated, after exposure of green CRBC fruit to 1000 ppm ethylene for 24 hours. The 150 Peak ratio: 0.534 Peak ratio: 0.813 Lakatan sequenced pool of up-regulated cDNA’s fall 100 Diploid (2x) Triploid (3x) into one of three major categories: Genes involved in metabolic processes, main- 50 ly carbohydrates and lipid components. 0 Genes involved in cellular regulation (protein CRBC CRBC kinases, transcription factors etc.). 200 TMPx 2637-49 Number of events Cavendish 901 Genes involved in protection from pathogens 150 and environmental stress conditions - metal- lothionein like protein, super oxide dismutase, 100 Peak ratio: 1.089 Peak ratio: 1.620 osmotin-like protein, pathogen related proteins Tetraploid (4x) Hexaploid (6x) etc. 50

A significant number of sequences showed no 0 substantial homology to functional genes in 050100 150 200 0 50 100 150 200 250 the GeneBank. Relative nuclear DNA content (channel number)

XVI PROMUSA INFOMUSA — Vol 11, N° 1 Table 1. Results of homology search of newly isolated repetitive DNA sequences. 1 Plant Breeding Unit, FAO/lAEA Agriculture and Biotechnology Laboratory, International Atomic Energy Agency Laboratories, Homology to known DNA sequences A -2444 Seibersdorf, Austria 2 Musa repetitive DNA sequence Genebank Smallest Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovska 6, CZ-77200 DNA clone accession number sum probability Olomouc, Czech Republic Radka2 Hordeum murinum 5S ribosomal RNA gene AF096721 3x10-07 3 Laboratory of Tropical Crop Improvement, KULeuven, Radka1 Rice 26S ribosomal RNA gene M11585 0.0 Kasteelpark Arenberg 13, B-3001 Leuven, Belgium 4 Radka7 Rice 26S ribosomal RNA gene M11585 0.0 Plant Pathology Unit, Faculté universitaire des sciences agronomiques de Gembloux, Passage des déportés 2, B-5030 Radka14 — — — Gembloux, Belgium Radka5 M. acuminata repetitive element Y10144 6x10-16 Radka8 M. acuminata retrotransposon monkey AF143332 3x10-21 Induced mutation techniques are particularly Radka9 M. acuminata retrotransposon monkey AF143332 3x10-21 important for bananas and plantains (Musa Radka3 — — — species) where there is limited sexual repro- Radka6 — — — duction that could generate genetic variation, Radka12 — — — the basis for selection. Even though spontaneous mutations have contributed to the Radka10 MUSA1 clone (cacao swollen shoot badnavirus) AF106947 10-108 genetic diversity of Musa and significantly Radka4 — — — increased the variation used to breed Musa spp. their occurrence is too low. The use of in vitro cultures for induced mutations in Musa tain molecular markers and/or genes of inter- variation in the related Musa species, M. tex- spp. could be a method of choice if several est. This strategy should result in effective tilis, by utilizing some of the results of the DNA steps of the mutation induction process could integration of genetic and physical maps. marker techniques generated for banana. The be optimized. The following aspects were Once developed, physically mapped molecu- highlights of our accomplishments are: lar markers will facilitate map-based cloning of investigated: the possibility to detect genetic • Advanced generations of induced mutants of instability in DNA content, the determination of genes of interest including those induced by two Philippine banana cultivars ‘Lakatan’ irradiation and chemical mutagenesis. an optimal mutagenic dose, the elimination of (AAA) and ‘Latundan’ (AAB) with promising chimerism and the application of an early Ploidy screening of Musa germplasm was traits were obtained from irradiation using mass screening for the selection of useful supported in part by INIBAP. 40Gy gamma ray and 3Gy fast neutron and mutants. subsequent in vitro culture manipulation and field evaluation. With the increased use of embryogenic cul- Analyses of induced mutants tures in micropropagation of banana, • DNA marker techniques such as RAPD, somaclonal variation occurs among regenerat- of Philippine bananas (Musa SSR and AFLP were successfully used to ed plantlets. This variation may interfere with acuminata, cvs ‘Lakatan’ (AAA) characterize genomic differences between mutations, which could be obtained through and ‘Latundan’ (AAB)) and the two banana cultivars used. However, mutation techniques. Although the causes of only RAPD and AFLP techniques were able germplasm collection of Abaca this chromosome instability are poorly under- to detect genomic alterations between non- stood, chromosome instability itself is believed (Musa textilis Nee) using irradiated and induced mutants in the two to be one of the most common causes of tis- morphological, RAPD, SSR cultivars. Due to better reproducibility and sue culture-induced variation. Using flow higher multiplex ratio, AFLP technique is pre- and AFLP markers cytometry, variation in chromosome number ferred over RAPD technique. Hence, this could be detected among plants regenerated D.M. Hautea1, N.O. Bituin1, H.F. Galvez1, technique was used to detect polymorphism via somatic embryogenesis from tissue cul- C. Caspillo1, C.H. Balatero1, R.B. Quilloy1, between the original mutated clones and ture. The results obtained by flow cytometry derived suckers. Silver staining procedure E. Perez1, G.C. Molina1, J. Torrion1, were verified by chromosome counting in for SSR and AFLP analyses were routinely A.J. Jamiri2, R.P. Laude3 and L. Gonzal4 meristem root-tip cells. After standardization used. of the method, the results indicated that flow 1 Institute of Plant Breeding, CA, University of the Philippines • RAPD, SSR and AFLP techniques devel- cytometry was sensitive enough to detect ane- Los Baños, Philippines oped for banana through this CRP project 2College of Science and Mathematics, U.P. Mindanao, uploidy in Musa with ± 1 chromosome accura- Philippines were found to be highly applicable to abaca. cy. Abnormalities in DNA content could be 3lnstitute of Biological Sciences, University of the Philippines A number of SSR primers developed for detected at an early stage, during in vitro cul- Los Baños, Philippines banana gave amplification products using ture. For the first time, a banana embryogenic 4National Abaca Research Center, Visayas State College of abaca DNA. With complimentary funds pro- Agriculture, Philippines cell suspensions (ECS) with five chromo- vided by a grant from the Philippine govern- somes missing was reported. ment, RAPD, SSR and AFLP analyses were Banana (Musa acuminata) and abaca (M. tex- To irradiate ECS, several preliminary studies successfully conducted to evaluate the tilis Nee) are two of the most economically were performed. The first radio sensitivity genetic variation in the abaca germplasm important Musa species cultivated in the tests of Musa ECS were performed and it has collection of the Philippines. Comparison Philippines for fruit and fibre, respectively. been found that cell suspensions from Musa These crops are both difficult to breed by con- between morphological and molecular analyses was also conducted. can tolerate up to 200 Gy. At 100 Gy the ventional means and they share a number of growth curve is only affected at 50% com- common diseases. This report summarizes pared to the control. our efforts during the past five years to gener- Usefulness of embryogenic When irradiating cell suspensions, large popu- ate useful induced mutants of Philippine cell suspensions for the lations can be handled under controlled condi- banana cultivars and to evaluate the useful- induction and selection tions and if embryos are of single cell origin, ness of DNA markers in characterizing of mutants in Musa spp. they overcome the problem of chimerism. We genomic alterations in the advanced genera- simulated this by treating ECS with colchicine 1 1 2 tions of induced mutants. Furthermore, this N. Roux , A. Toloza , J. Dolezel , and determined the ploidy of the regenerated report highlights the valuable contribution R. Swennen3 , P. Lepoivre4 and F.J. Zapata- plants by flow cytometric analysis. Colchicine made by this CRP project in analyzing genetic Arias1 treatment induced polyploidy and mixoploidy

INFOMUSA — Vol 11, N° 1 PROMUSA XVII (chimerism) if embryos are not of single cell markers, STMS) was developed for each interested in whether Mycosphaerella origin. To date no mixoploid regenerated pathogen, using a microsatellite enrichment pathogens of banana have the potential to plants from colchicine treated ECS were strategy. Microsatellites of the (CAA)n, exhibit differential resistance to plant defence detected. (GAA)n, (GA)n, (CA)n and (TA)n type were responses, such as the generation of reactive An early mass screening method based on the specifically captured, cloned, positive clones oxygen species (ROS), or to themselves pro- use of the toxin Juglone (5-Hydroxy-1, 4-naph- identified by hybridization, sequenced, and duce ROS-generating toxins. We determined thoquinone), the main toxin to be responsible microsatellite-flanking primers designed with that M. musicola produces a singlet oxygen- of the global effect of the fungus Primer 3. Together with selective amplifica- generating anthraquinone toxin and exhibits Mycosphaerella fijiensis, was used to screen tion of microsatellite polymorphic loci (SAMPL) significantly greater resistance to a wide range for resistance to the black Sigatoka disease. and DNA amplification fingerprinting (DAF) of light-activated, singlet oxygen-generating The test was applied when the acclimatized markers, the STMS were used to study the dyes than does M. fijiensis. These results plants reached the six leaves stage. The dose genetic variability of both pathogens in popula- suggest possible differences in the way the of 25 ppm permitted to differentiate between tions from two continents, four Latin American two fungi interact with banana. In M. musico- the tolerant variety ‘Fougamou’ and the sus- countries and five different locations in la, the correlation between production of a ceptible variety ‘Grande naine’. To date, from Colombia. First, all marker types generally light-activated toxin, high self-resistance to around 4000 irradiated ‘Grande naine’ plants detected a high degree of polymorphism in exogenous photoactivated singlet oxygen- screened, eight putative mutants were select- both species. Second, only few markers of generating toxins, and increased disease ed for their tolerance to 25 ppm of Juglone. any type were sufficient to discriminate two development in the presence of light is intrigu- These plants are now being evaluated for their isolates unequivocally. Third, distinct popula- ing and will be examined further. Such a cor- tolerance to the inoculation of the fungus. tions can be detected and differentiated from relation is not evident in the M. fijiensis host- each other. For example, the number of pathogen interaction. STMS haplotypes turned out to be higher in Towards basic genomics of Nigerian as compared to Mexican M. fijiensis, Mycosphaerella fijiensis and M. and the M. musicola populations generally are Banana improvement through musicola: DNA markers for much more clearly separated from each other gamma irradiation and testing genetic diversity, population than the M. fijiensis populations, pointing to less gene flow. Fourth, usually isolates from for banana bract mosaic structure and genetic mapping one region grouped together. Fifth, about 10% virus (BBrMV) in Sri Lanka of the banana pathogens of the primers designed for one pathogen could also be used for the other pathogen. W.K. Hirimburegama, W.K.G. Dias C.M. Molina1,2 and G. Kahl1 The three marker techniques are used to con- and K. Hirimburegama 1 Plant Molecular Biology, Biocentre, University of struct and saturate a first genetic map of M. Department of Botany, University of Colombo, Colombo 03, Frankfurt/Main, Germany. fijiensis and to tag a fungicide resistance Sri Lanka. 2 Corporación Colombiana de Investigación Agropecuaria (CORPOICA), Santafé de Bogotá, D.C., Colombia gene. Banana is the most widely consumed fruit in Sri Lanka, and is an attractive perennial fruit Though much research is presently devoted to Genetic and phenotypic crop for small-scale farmers. This is due to its banana genomics, its two most severe fungal high economic value throughout the year. diseases black Sigatoka (causal agent: variability in Mycosphaerella Thus, lowland rice fields in some areas are Mycosphaerella fijiensis) and yellow Sigatoka pathogens of banana being converted for banana cultivation. (causal agent: M. musicola) are definitely A.C.L. Churchill Among the local cultivars, ‘Embul’ (Mysore, under researched. Yet both diseases led, AAB) has the highest demand for cultivation. lead, and will lead to major yield losses, espe- Boyce Thompson Institute for Plant Research, The annual production of banana is around cially in banana and plantain plantations, but Ithaca, NY, USA 450 000 metric tonnes. Until recently, banana also on smallholders fields. M. musicola, first We are using fungal molecular biology cultivation was limited to small plots, but large reported in Java in 1902, spread all over the approaches to study the interactions between fields are now being established. More and major production areas in Asia, Africa and Mycosphaerella pathogens and their banana more rice farmers are changing over to South America during the first half of the last hosts. We have developed a DNA-mediated banana cultivation as the net profit is about century. It was regionally displaced by the transformation system for M. fijiensis, M. musi- four times higher than for rice, and less labour more aggressive M. fijiensis, first reported on cola, and M. eumusae in which genetically and other inputs are required. During the last the Fiji islands in 1962. Both pathogens stable transformants constitutively express six years, 2500 ha have been converted to forced to develop control measures, which in green fluorescent protein in vitro and in vivo. banana cultivation (Anon. 2001, 2002). In essence increased fungicide doses drastically Stable transformation is a first step in develop- addition, it was noted that the nutritional level and reduced the time interval between appli- ing tools for the genetic manipulation of these in farmers’ families has improved due to the cations, but also relied on the introduction of pathogens, which will lead to the identification habit of consuming more fruit. Usually, farm- new, partly resistant host varieties. In of pathogenicity and virulence factors required ers do not use pesticides for banana cultiva- response, the pathogen populations became for plant infection and symptom development. tion and this has benefits for human health more aggressive and partly resistant to the Significant phenotypic variability exists in most and the environment. prevalent fungicides. Mycosphaerella banana pathogens. When Since 1990, research on banana micropropa- In spite of the economic importance of the cultured on agar-based media, single colonies gation through shoot-tip culture, induced fungi, systematic research on the pathway of are typically light to dark grey or black in mutation through gamma irradiation, cell sus- infection, the crosstalk between the pathogen colour. Stable spontaneous mutants altered in pension cultures and somatic embryogenesis, and the host plant, the molecules encountered pigmentation are not uncommon. We have and ploidy analysis for variation detection has and the genes involved remained largely elu- isolated pigment mutants that appear to be been conducted by the University of Colombo. sive. Therefore we started a more basic study deficient in melanin production and have From 1995, both ‘Embul’ and ‘Cavendish’ cul- on both pathogens with the aim to understand cloned a gene fragment with high similarity to tivars were included in the mutation breeding the molecular basis of the interaction(s). fungal genes involved in melanin biosynthesis. programme. After irradiation of in vitro shoot A set of elite, highly polymorphic microsatellite Further characterizations of the pigment tips with 45 Gy, two selections were made markers (sequence-tagged microsatellite site mutants are in progress. Additionally, we are with shorter height and early fruiting, six

XVIII PROMUSA INFOMUSA — Vol 11, N° 1 month after planting (Hirimburegama et al. A study was done with the objective of devel- regeneration system, and to induce genetic 1997). Micropropagated plants of these selec- oping a low-cost DAS-ELISA detection kit. variability for mutant selection. However, the tions were tested for stability of the characters Anti-serum for BBrMV (of Queensland success in obtaining desirable mutants is until the second generation. The technology Department of Primary Industries - QDPI, restricted to a few allele combinations and was perfected and is being transferred to the Australia) was tested as the coating antibody thus, more efficient alternatives are needed for farmers (Laksiri and Hirimburegama 1999). to replace the relevant component of the an early mutant selection. Earlier, clones Early fruiting and harvesting of micropropagat- Agdia commercial kit. Results showed a rela- obtained by gamma irradiation of meristematic ed banana plants saves at least one month in tively high efficiency with the QDPI antibody. apices, LD50 dose obtained in Seibersdorf comparison to traditionally grown plants that Work is also in progress to make the alkaline (Austria) and the Centro de Estudios usually need eight months to flower. Thus, phosphatase enzyme conjugated antibody to Aplicados al Desarrollo de la Energia Nuclear the number of ratoons in two years becomes substitute the one of the test kit. Once the (CEADEN) in Cuba, were tested in field condi- three instead of the usual two, thereby local antiserum is produced, it is expected that tions. Somatic embryogenic cell suspension increasing the income of farmers with 25% an effective and low cost local diagnostic kit cultures were established and the somatic (equivalent to about US$350 per ha per year). will be developed for the routine indexing of embryo formation is being further modified to Mass production of plants is in progress. banana plants for BBrMV. Purification of the enhance further plant regeneration rate, espe- Thus, indexing/testing of plants for viruses, i.e. virus extract (Thomas et al. 1997) is a limiting cially of AAB genotypes in collaboration with banana bract mosaic (BBrMV) and banana factor for obtaining the antigen for the anti- KULeuven. Preliminary evaluations were car- streak virus (BSV) has become essential, body production process. ried out to study the action of crude extract of since virus-free, indexed mother stocks are M. fijiensis on cell suspension cultures of required for micropropagation. The causative References ‘Navolean’ in a solid ZZ medium. Filter paper agent of the banana bract mosaic disease was Anon. 2000. Annual Reports, Mahaweli Authority of Sri discs were used to test different concentra- confirmed in the widely cultivated local variety Lanka, Colombo, Sri Lanka. tions of crude extract on cell culture growth for ‘Embul’ from Sri Lanka by Thomas et al. Anon. 2001. Annual Reports, Mahaweli Authority of Sri selecting toxin tolerant cell cultures. Effect of (1996, 1997). This disease is more prevalent Lanka, Colombo, Sri Lanka. fungal crude extract on oxygen uptake studies in cultivations, which are not properly man- Hirimburegama K. & N. Gamage. 1997. Cultivar speci- were made in ‘CEMSA ?’ clone, as well as in aged, but the impact on the yield appears to ficity with respect to in vitro micropropagation of vitroplants of different cultivars used in the IMTP for black Sigatoka studies. The vitro- be significant. Musa spp. (bananas & plantains). J. of Hort. Sci. plant leaves were treated with different con- 72(2):205-211. centrations of crude extract solution of ethyl Laksiri B.D.P. & K. Hirimburegama. 1999. Banana acetate by placing them on different sizes of improvement in Sri Lanka through radiation induced disks, e.g. starting from 0.28 cm2 size disks. mutation and tissue culture. INFOMUSA 8(2):PRO- Oxygen uptake was measured by Warburg’s MUSA 4:XII. manometers. Based on our results, a short Thomas J.E. & L.V. Magnaye. 1996. Banana bract stature mutant (‘Parecido al Rey’ 6.44) was mosaic disease. Musa Disease Fact Sheet No. 7. obtained. Few more mutants were selected, International Network for the Improvement of which were tolerant to black Sigatoka with increased yield, in some cases (‘Parecido al Banana and Plantain. Montpellier, France. Rey’ 6.32 and ‘Gran Enano’ 6.44 mutants and Thomas J.E., A. Geering, C.F. Gambley, A.F. Kessling III-2). However, these characters were unsta- & M. White. 1997. Purification properties and diag- ble, probably affected by the environment, or nosis of banana bract mosaic potyvirus and its dis- in vitro culture conditions. Therefore, we tinction from abaca mosaic potyvirus. looked for other alternative approaches for Phytopathology 87(7):698-705. improving induced mutation rates, e.g. somatic cell suspension cultures and later mass of somatic embryogenic cells were established. From each batch of somatic embryogenic cell Advances and perspectives suspension mass cultures, 3250 to 6625 for the genetic improvement somatic embryos were obtained with 20.7% germination and 95% plant survival in ex vitro of banana (Musa spp.) conditions. Their genetic stability is presently via biotechnological being tested in field conditions. The action of and nuclear techniques M. fijiensis extract on cell suspension culture at INIVIT resulted in large number of oxidized cells at higher extract concentrations. Damaged cells Jorge López Torres were characterized by a compact cytoplasmic content with a dark colour in its centre and Instituto de Investigaciones en Viandas Tropicales, leaving an empty space between it and the All the currently reported disease symptoms Biotechnology Laboratory, Apdo 116 CP 53000 Villa Clara, were observed in infected plants in the field Santo Domingo, Cuba cellular wall. Forty-five days after incubation, but in varying degrees. Spindle shaped purple more than 60% cells were found in the previ- or dark red coloured streak patterns on the Bananas and plantains constitute an important ous described conditions due to toxin diffusion pseudostem in addition to dark spindle shaped carbohydrate source in the Cuban diet. This is from discs. Oxygen uptake valves had streaks on bracts were the most common mainly due to their food habits and banana decreased normally in non treated control of symptoms of infection. Mature plants with production all the year round. There is a great all IMTP cultivars, and the maximum decrease inflorescence having black or reddish brown need to develop new banana cultivars due to observed in treated plants in comparison with streaks on the outer surface of the open poor yield and susceptibility to diseases non-treated plants was: ‘Yangambi km5’ bracts also had streaks on the pseudostem. (mainly black Sigatoka caused by the fungus (38.7%); ‘Calcutta 4’ (27.0%); ‘Pisang berlin’ The splitting of the base of young suckers Mycosphaerella fijiensis). The application of (13.4%); ‘Pisang lilin’ (20.9%) while oxygen could also be due to other banana virus dis- both biotechnological and nuclear techniques uptake was increased by 70% in ‘CEMSA ?’ eases. has enabled to develop an efficient plant clone. The cultivars with higher resistance to

INFOMUSA — Vol 11, N° 1 PROMUSA XIX black Sigatoka showed a tendency to Laboratory of Tropical Crop Improvement, Kasteelpark 1 decreased oxygen uptake; this is probably due Recent developments in early Arenberg 13 and Laboratory for Phytopathology and Plant in vitro screening for resistance Protection, Willem De Croylaan 42, KULeuven, B-3001 to the damaged tissues, instead of a toxicity Leuven, Belgium effect of toxin treatment. At present, the first against migratory endoparasitic somatic embryos obtained from irradiated cel- nematodes in Musa In the vast majority of African countries where lular suspensions are in the germination starchy bananas and plantains are grown, Musa phase and somatic embryogenesis in geno- A. Elsen and D. De Waele spp. are important staple food crops, often exclusively grown by small scale farmers. This types from the IMTP is being developed. Laboratory of Tropical Crop Improvement, KULeuven, especially applies to the highland cooking and Kasteelpark Arenberg 13, B-3001 Leuven, Belgium brewing bananas of East and Central Africa. Agrobacterium-mediated Over the last two decades, however, banana Among nematodes parasitizing bananas yields in the East African region have been cotransformation of banana throughout the world, Radopholus similis and steadily declining partly due to migratory or (Musa spp.) Pratylenchus coffeae are important migratory sedentary endoparasites: the burrowing nema- nematodes, causing severe yield losses in tode (Radopholus similis), the root-lesion nema- K.Z. Ahmed*, S. Remy, R. Swennen commercial and local consumption cultivars. todes (Pratylenchus coffeae) and root-knot Chemical control is currently the most used and L. Sági nematodes (Meloidogyne spp.). With the method to manage the nematodes although advent of transgenic methodologies, an attrac- Laboratory of Tropical Crop Improvement, KULeuven, nematicides are dangerous, toxic, and expen- tive method to control these nematodes is the Kasteelpark Arenberg 13, B-3001 Leuven, Belgium sive. Therefore, nematode control through transfer into target Musa spp. of lectin encoding *Permanent address: Department of Genetics, Faculty of genetic improvement of banana is widely genes. Prior to application in banana, the Agriculture. Minia University. El-Minia, ET-61517, Egypt encouraged. Many Musa cultivars have been nematicidal effect of a number of lectins or screened to find resistance against these root The introduction of foreign genes into the plant lectin-related proteins is currently tested for their pathogens. Screening research is time con- effectiveness against banana nematodes in genome is a basic technique to study gene suming because it most be carried out both transgenic Arabidopsis thaliana and/or tobacco. expression and physiological processes in under field and greenhouse conditions. In The different in vitro test systems will be dis- plants and for breeding programmes. vitro screening could facilitate and hasten cussed, together with the initial results. Improving the agronomic value of major crops incorporation of genetic nematode control into is likely to involve the introduction of multiple bananas. However, an in vitro screening method requires aseptic nematode cultures. genes, many of which will not provide directly Lectin binding to the plant screenable phenotypes among the initial prod- In this paper, the development of aseptic cultures of R. similis and P. coffeae and an in ucts of transformation. Major restrictions of parasitic nematode Radopholus vitro screening method are discussed. current transformation techniques are that similis and its effect on host Alfalfa callus on modified White’s medium has only a few genes can be transferred at the finding proved to be a good aseptic culture system for same time and that selectable marker genes both R. similis and P. coffeae. Although the N. Wuyts, A. Elsen, E. Van Damme1, have to be used, which frequently results in reproduction is significantly lower compared to D. De Waele, R. Swennen and L. Sági transgenic plants containing undesirable carrot disc cultures, this system has many antibiotic resistance genes. Laboratory of Tropical Crop Improvement, Kasteelpark advantages. The nematodes are not only cul- Arenberg 13 and 1Laboratory for Phytopathology and Plant The objective of the present study is to deter- tured under complete aseptic conditions but Protection, Willem De Croylaan 42, KULeuven, B-3001 mine the efficiency of cotransformation with this system is also less labour intensive and Leuven, Belgium visually scorable marker genes using offers a more continuous inoculum production. In addition, culturing on alfalfa callus did not The effect of lectins on the plant parasitic Agrobacterium tumefaciens and banana alter the pathogenicity of R. similis and P. cof- nematode Radopholus similis was studied in a (Musa spp.). Cell suspension culture of four feae. series of experiments. FITC- or colloidal gold- cultivars were infected with two different A. Both R. similis and P. coffeae could infect and labelled lectins of Canavalia ensiformis tumefaciens strains each carrying a distinct reproduce on the roots of in vitro grown (ConA), wheat (WGA) and Helix pomatia disarmed T-DNA containing one of three ‘Grande naine’ plants. For both nematodes (UPA) were found to bind the nematode in the reporter genes [Luciferase (LUC), ß- necrotic lesions were observed in the roots head region, at the excretion pore, the pores Glucuronidase (GUS) or Green Fluorescent within 2-3 weeks after inoculation. In a last of the reproduction system and those of the Protein (GFP)] as well as the neo selectable experiment, the reproduction of R. similis was phasmids. The viability and the chemotactic marker gene. Multicellular structures express- tested in vitro on six different Musa cultivars response towards plant roots, after treatment of nematodes with lectins, were examined in ing multiple genes were recovered, and with a known host response to R. similis. Except for ‘Yangambi km5’, their host vitro by analyzing movement tracks left on cotransformation frequencies were measured. agar plates. The assay included six plant The cotransformation frequency was less than response under in vitro conditions correspond- ed to their host response under greenhouse or lectins of five different classes and the banana the sum of the frequency of each single trans- field conditions. The susceptible status of thaumatin-like protein. A 1% concentration of formation. Negative correlation was found ‘Grande naine’, ‘Gros Michel’ and ‘Cachaco’ Phaseolus vulgaris agglutinin (PHA) had a between the transient expression of two visual was confirmed as well as the resistant status toxic effect on R. similis females: 68% showed marker genes introduced together for cotrans- of ‘Pisang jari buaya’ and ‘SH-3142’. no or very little movement after inoculation formation. Significant differences in (co- compared to an average of 30% for other )transformation frequency were detected lectins and 5% for the control treatment. A between the banana cultivars tested. 0.05% concentration of PHA still reduced the Evaluation for nematode control viability of R. similis females by 75%. ConA We anticipate that the simultaneous use of of transgenic plants expressing and WGA did not alter the chemotactic multiple reporter genes will provide a conven- different types of lectins response towards plant roots, despite of the ient method for the accurate determination of demonstrated binding of both lectins to R. sim- 1 cotransformation and will contribute to a strat- K. Carlens, A. Elsen, E. Van Damme , ilis. In contrast, Galanthus nivalis agglutinin egy for multigene transformation. L. Sági and R. Swennen (GNA) reduced orientated movement of R.

XX PROMUSA INFOMUSA — Vol 11, N° 1 similis females towards plant roots. Finally, tion enzymes will increase the chance of finding has only resulted in the development of single the secretions of R. similis were stained with more dwarf-related sequences. shoots and roots, whereas MT resulted in the Coomassie Brilliant Blue R. These secretions formation of watery callus and proliferation. TDZ appear at the amphids, the excretion pore, the mainly induced swelling of the explants. TDZ vulva, the spicules and the phasmids. Establishment of embryogenic proved to be better cytokinin over MT in the Moreover, nematodes treated with GNA pro- cell suspension cultures from induction and maintenance of good quality duced less abundant secretions. Indian banana cultivars scalps. These are currently under evaluation for embryogenic induction. The established ECS P. Suprasanna*, B. Panis, L. Sági will be characterized and used in cryopreserva- Early detection of dwarf off-types and R. Swennen tion and genetic transformation experiments. of banana using AFLP, TE-AFLP Laboratory of Tropical Crop Improvement, KULeuven, and MSAP analysis Kasteelpark Arenberg 13, B-3001 Leuven, Belgium *Permanent address: Nuclear Agriculture & Biotechnology RAPD, SSR and AFLP Division, Bhabha Atomic Research Centre, Trombay, Bombay I. Engelborghs, L. Sági and R. Swennen 400 085, India techniques in detecting Laboratory of Tropical Crop Improvement, KULeuven, polymorphism between Kasteelpark Arenberg 13, B-3001 Leuven, Belgium Banana (Musa spp.) is an important and major fruit crop in India, which is the largest produc- irradiated parents AFLP and several variant techniques were er of bananas in the world. However, dis- and suckers in two edible performed on banana in order to characterize eases and pests such as black Sigatoka and Philippine banana cultivars different dwarf phenotypes. The dwarf AAB Panama disease, bunchy top virus and nema- plantain ‘Curare enano’ and its normal-sized in todes remain as major threats to production. D.M. Hautea, G.C. Molina, vitro generated off-type were analyzed by Genetic modification using embryogenic cell N.B. Coronado, H.F. Galvez, C.H. Balatero AFLP, TE-AFLP, MSAP, cDNA-AFLP, and suspensions (ECS) appears to be a suitable and R.B. Quilloy cDNA-TE-AFLP. AFLP and TE-AFLP were approach for integrated genetic improvement. also performed on four pairs of naturally Institute of Plant Breeding, College of Agriculture, University of Progress has been made in the development the Philippines Los Baños, 4031 College, Laguna, The occurring dwarf/normal banana cultivars. of protocols for the establishment of ECS, and Philippines Differential AFLP patterns were obtained and up immature male flowers as well as proliferating to 25% polymorphism was observed depending in vitro cultures have mainly been used. This report summarizes the results of our on the primer combination and the cultivar. TE- Especially in vitro proliferating meristems efforts to evaluate the usefulness of DNA AFLP analysis generated shorter and a lower (‘scalps’) are an ideal starting material, marker techniques, such as random amplified number of fragments resulting in only relatively because they can be generated all round the polymorphic DNA (RAPD), microsatellites or few polymorphisms between the dwarf and nor- year from most cultivars. The method simple sequence repeat (SSR) and amplified mal-sized cultivars. The somaclonal variants includes a preculture of the proliferating meris- fragment length polymorphism (AFLP), to obtained in vitro from the dwarf ‘Curare enano’ tems on high cytokinin medium which provides characterize the genomic alterations in might have been caused by methylation induced embryogenic competence. induced mutants of the two Philippine banana by in vitro conditions. MSAP analysis, based on Several important Indian cultivars [(‘Robusta’ cultivars. Mutants were induced in the two the methylation (in)sensitivity of a pair of isoschi- (AAA), ‘Basrai’ (AAA), ‘Shrimanthi’ (AAA), most popular Philippine edible banana culti- someric restriction enzymes, appeared to be a ‘Karpoora valli’ (ABB)] have been employed in vars by gamma and fast neutron irradiation valuable tool in revealing differential cytosine this study for the induction of good quality scalps from in vitro shoot-tip cultures. Promising methylation. Cloning and sequencing differen- and embryogenic callus. Of these, ‘Robusta’ clones were selected and evaluated further tial fragments did not reveal significantly homol- have shown the formation of embryogenic cal- using molecular markers. In banana, several ogous matches in public databases. cDNA- lus, which subsequently has been used for the DNA marker techniques were used to investi- AFLP analysis between the dwarf and normal establishment of ECS. In addition, the effect of gate genetic relationships between Musa ‘Curare enano’, revealed a normal-specific frag- alternative cytokinins such as meta-topoline accessions and to determine differences in ment, while cDNA-TE-AFLP analysis resulted in (MT, thidiazuron (TDZ) and N-chloro-4-pyridyl- somaclonal variants and radiation induced a dwarf-specific fragment. AFLP and the variant N’-phenylurea (CPPU) has been studied on iso- mutants. In this study, RAPD, SSR, and AFLP techniques have shown the potential to differen- lated meristems of ‘Cachaco’ (ABB), ‘Williams’ techniques were successfully used under local tiate between closely related genotypes. More (AAA) as well as on scalps of ‘Robusta’. On iso- conditions and found useful in characterizing primer combinations and/or alteration of restric- lated meristems (‘Williams’, ‘Cachaco’), CPPU irradiated and non-irradiated banana clones. RAPD, SSR and AFLP markers also showed sufficient polymorphism to differentiate between the two cultivars used. However, Table 2. Summary of polymorphism detected in AFLP markers used in the analysis of irradiated parent SSR and AFLP markers were found to be clones, first cycle suckers and non-irradiated clones of cultivars Lakatan and Latundan more highly reproducible. One of the most Selective No. of bands Number of % Polymorphism significant accomplishments of this CRP proj- primer pairs polymorphic bands ect is the development of a high quality, non- E-ACG/M-CTC 35 11 31.4 radioactive, silver-staining technique for AFLP E-ACG/M-CTG 32 20 62.5 analysis, which can be easily adopted under E-ACG/M-CTT 18 14 77.8 laboratory conditions in developing countries. E-AGC/M-CAA 40 20 50.0 AFLP markers showed both high reproducibili- E-AGC/M-CAC 34 14 41.2 ty and discriminating capacity. Polymorphic AFLP markers were identified (Table 2) and E-AGC/M-CAG 29 14 48.3 found useful in fingerprinting bananas and E-AGC/M-CAT 31 14 45.2 other Musa species. AFLP was the only E-AGC/M-CTA 21 7 33.3 marker technique tested, which was able to E-AGC/M-CTC 29 19 65.5 detect variation in DNA profiles of induced E-AGC/M-CTG 32 18 56.2 mutant clones, their first cycle suckers and Mean 34 15.1 51.1 non-irradiated control clones (Figure 2), which

INFOMUSA — Vol 10, N° 2 PROMUSA XXI held at the international Musa germplasm Rat feeding tests of transgenic collection of INIBAP (based at KULeuven, banana expressing an Belgium) is tested by different virus indexing antifungal peptide from onion centres (South Africa, Australia and France). Currently about 25% of the collection com- S. Remy, G. Flo2, I. Deconinck, S. Lievens2. prising a significant number of potentially M. Cokelaere2, E. Decuypere1, L. Sági important and improved varieties is infected and R. Swennen with viruses. The most prevalent viruses in Laboratory of Tropical Crop lmprovement and 1 Laboratory of this infected germplasm are BSV (banana Physiology and Immunology of Domestic Animals, KULeuven, streak virus) but also with CMV (cucumber Kasteelpark Arenberg 13, B-3001 Leuven, Belgium mosaic virus), BBTV (banana bunchy top 2 Subfaculteit Geneeskunde KULAK, Sabbelaan 53, Kortrijk, virus), BanMMV (banana mild mosaic virus) The Netherlands and BBrMV (banana bract mosaic virus). Transgenic plants of the dessert banana A programme of virus elimination is therefore ‘Williams’ containing a gene encoding the carried out by the Plant Pathology Unit Ace-AMP1 antifungal peptide of onion (Allium (FUSAGx, Belgium) in collaboration with the cepa) were produced by particle bombard- Laboratory of Tropical Crop Improvement ment for enhanced tolerance against attack by (KULeuven, Belgium). Different in vitro tech- the fungal pathogen Mycosphaerella fijiensis. niques, such thermotherapy, chemotherapy, ELISA assays on extracts from lyophilized meristem culture and cryotherapy are tested. banana pulp showed that the concentration of Banana plants of the cultivar ‘Williams BSJ’ the Ace-AMP1 peptide reached up to 0.0316% (AAA) were either naturally infected with BSV of the total amount of soluble proteins or six or mechanically infected with CMV or BBTV. times above the background signal measured Proliferating in vitro cultures were produced in non-transformed banana pulp. We tested from this material. Excised meristematic whether this expression level had an effect on clumps were cryopreserved through vitrifica- rats fed on a diet containing transgenic tion using the PVS-2 solution. The health banana pulp. status of regenerated material was first While energetic content was comparable in checked on in vitro plants through ELISA. transgenic and control pulp, dry matter and Then, the putative virus-free material was protein content were lower and higher in trans- tested again after greenhouse acclimatiza- genic pulp than in control pulp respectively. tion. The virus eradication rates after cryop- Twenty per cent of lyophilized meal from con- reservation for CMV and BSV reached 30% trol or transgenic bananas were incorporated and 90% respectively. In comparison, the in regular rodent food and supplied to male frequency of virus-free plants regenerated and female Wistar rats. Feeding of the trans- directly from highly proliferating meristems, Figure 2. AFLP fingerprints of irridiated banana genic meal during six weeks did not cause any which reflects spontaneous eradication, clones (M), first cycle suckers (S) and non-irra- difference in food intake, growth rate and diated clones (N) on ‘Latundan’ (LT-3) and reached 0% and 52% for CMV and BSV ‘Lakatan’ (LK-40). weight of internal organs in comparison to respectively. The conventional meristem cul- feeding on control diet. Also, a complex blood ture resulted in 0% CMV-free plants and 76% analysis did not show any effect in rats con- BSV-free plants. suming the transgenic banana meal. are otherwise morphologically indistinguish- In conclusion, cryopreservation seems to be a able. The results indicate that AFLP is very very promising technique for virus eradication ideal and useful for fingerprinting purposes from Musa germplasm enabling to faster dis- compared with other marker systems because Cryopreservation tribute germplasm of interest. of its high multiplex ratio i.e. more bands (=loci) per gel can be resolved. While more for the elimination of cucumber primer combinations need to be tested, these mosaic and banana streak results suggest the potential usefulness of this diseases in banana An ultrasensitive luminescent technique in detecting genome variation (Musa spp.) detection system hi banana between cultivars and in detecting genome biotechnology: from promoter alterations in induced mutants of banana 1 2 2 B. Helliot B. Panis R. Swennen and P. tagging to Southern hybridization including those showing very similar pheno- Lepoivre1 types. The detected variation between the 1 S. Remy, G. De Weerdt, I. Deconinck, irradiated parent clones and suckers suggests Plant Pathology Unit, Gembloux Agricultural University, 5030 Gembloux, Belgium R. Swennen and L. Sági that the number of vegetative propagation 2 Laboratory of Tropical Crop Improvement, KULeuven, cycles for the shoot-tip technique used (Novak Kasteelpark Arenberg 13, B-3001 Leuven, Belgium Laboratory of Tropical Crop Improvement, KULeuven, et al. 1989) may not be sufficient to completely Kasteelpark Arenberg 13, B-3001 Leuven, Belgium eliminate chimeras in the mutated populations. Bananas and plantains (Musa spp.) are threatened by various pests and diseases, including Digital imaging using a cooled CCD camera is The results obtained could provide a sound becoming an increasingly versatile tool in basis for more successful application of muta- important viral diseases which constrain banana production and cross-border biotechnology research. An ultrasensitive tion and molecular marker techniques for camera system consisting of a liquid nitrogen improvement of banana in the Philippines. germplasm movement. This delays dramati- cally the distribution of high yielding and newly cooled slow-scan CCD camera connected to powerful image analysis software is able to Reference bred varieties to small farmers. detect very low levels of light emission from Novak F.J., R. Afza, M. Van Duren & M.S. Omar. INIBAP therefore established a system for 1989. Mutation induction by gamma irradiation of in several signal sources and is used for the vitro cultured shoot-tips of banana and plantain the safe international movement of Musa recording of results for different applications in (Musa cvs). Trop. Agric. (Trinidad) 67 (1):21-28. germplasm. This involves that all germplasm banana biotechnology.

XXII PROMUSA INFOMUSA — Vol 11, N° 1 The most sensitive reporter system, biolumi- eration. Molecular and biochemical analysis nescent luciferase (LUC) is used in T-DNA will be performed to confirm integration and Oxidative events induced mediated promoter tagging. Since integration expression of the transgenes in the different by Mycosphaerella fijiensis of the promoterless luc gene is random, the cultivars. metabolites in banana (Musa level of LUC expression is suboptimal in most spp.) black leaf streak disease transformants to be screened requiring a highly sensitive detection. Preliminary results on and analysis of early selection in vivo screening of LUC expression in hun- Cryopreservation of embryogenic feasibility for resistance to this dreds of putative promoter tagged cell cultures cell suspensions of banana disease will be presented. to support banana improvement 1 2 3 Chemiluminescence has been for several B. Panis, H. Strosse, S. Remy, L. Sági J.P. Busogoro , B. Panis , J. Messiaen , years the method of choice in our laboratory 3 2 and R. Swennen P. Van Cutsem , R. Swennen for non-radioactive hybridization analysis. and P. Lepoivre1 Although exposure to and development of an Laboratory of Tropical Crop Improvement, KULeuven, 1 X-ray film is a sensitive technique, it is time Kasteelpark Arenberg 13, B-3001 Leuven, Belgium Unité de Phytopathologie, Faculté Universitaire des Sciences consuming and costly because multiple expo- Agronomiques de Gembloux, Passage des Déportés 2, 5030 The initiation of embryogenic cell suspension Gembloux, Belgium sures are needed for the evaluation of results. 2 Laboratory of Tropical Crop Improvement, KULeuven, In addition to increased flexibility and higher cultures of banana is still difficult and time- Kasteelpark Arenberg 13, 3001 Leuven, Belgium sensitivity of detection, signals can be cap- consuming, irrespective of the starting materi- 3 Unité de recherche en Biologie Végétale Cellulaire, Facultés tured faster with the CCD camera than with al used (immature male flowers, immature Universitaires Notre Dame de la Paix, Rue de Bruxelles 61, film. Good results can be obtained with a sin- zygotic embryos or proliferating in vitro meris- 5000 Namur, Belgium gle exposure by adjusting the gray scale of the tems). The embryogenic response is very low The mechanisms of action of M. fijiensis toxins captured image as will be demonstrated. and slow. Indeed for most cultivars less than in black leaf streak (BLS) disease were stud- In addition to luminescence the camera can 1% of initial explants give rise to an embryo- ied. The ethyl acetate crude extract (EaCE) also detect fluorescent signals, which is genic callus suitable for cell suspension initia- from the pathogen culture filtrates and juglone demonstrated by the ability to monitor green tion and 9- 26 months are needed before such (5-hydroxy-1,4-naphthoquinone), which is a fluorescent protein expression in transgenic embryogenic cell suspension is established. purified metabolite from EaCE, were injected banana cultures. Quantification of light inten- Moreover, once established, these cell sus- in the leaves of two banana cultivars. The cul- sity by software analysis will be demonstrated. pensions are subject to somaclonal variation tivars ‘Grande naine’ and ‘Fougamou’ served and microbial contamination and a prolonged as a susceptible reference and partially resist- Agrobacterium- and particle culture period may result in a lower and even- ant reference respectively. These bioassays tually a total loss of morphogenic capacity. induced necrosis and showed decrease of the bombardment transformation of vitality index determined according to chloro- a wide range of banana cultivars Up to now, transformation protocols of banana phyll fluorescence data (Lichtenthaler et al. rely on embryogenic cell suspensions. 1986). The ‘Grande naine’ cultivar was more G. Arinaitwe*, S. Remy, H. Strosse, Particle bombardment as well as sensitive than Fougamou whatever the bioas- R. Swennen, and L. Sági Agrobactenum-based protocols resulted in say (induction of necrosis or chlorophyll fluo- transgenic banana plants. Somatic hybridiza- Laboratory of Tropical Crop Improvement, KULeuven, rescence) taken into account. The light- Kasteelpark Arenberg 13, B-3001 Leuven, Belgium tion and protoplast electroporation are also dependence of the toxicity revealed by these *Permanent address: Makerere University, Faculty of both depending on the isolation of regenerable tests, the early effect on chlorophyll fluores- Agriculture and Forestry, Department of Crop Science, P.O. protoplasts from embryogenic cell suspen- cence (Harelimana et al. 1997) and the Box 7062 Kampala, Uganda sions. Finally, embryogenic cell suspensions swelling of ‘Grande naine’ chloroplasts after injection with EaCE, are indicative that chloro- Genetic transformation of banana (Musa spp.) can be used for mass propagation as an alter- plasts could be a potential target site for M. by particle bombardment and Agrobacterium native to shoot-tip cultures. fijiensis toxins. is established only in a few laboratories world- The safe preservation of this valuable sus- wide. In general, transformation frequencies pensions through cryopreservation is thus of A mechanical protocol (Leegood and Malkin are reported to be cultivar dependent. Thus, outermost importance. A cryopreservation 1986) to isolate physiologically intact chloro- there is a need to optimize established trans- technique was developed which involves cry- plasts from banana leaves was developed. A formation protocols for any particular type of oprotection with 7.5% DMSO (dimethyl new bioassay based on the addition of juglone banana. In this study, the two transformation sulphoxide) and 180 g/L sucrose, followed by to banana chloroplast suspensions was used methods were compared and the effect of slow freezing at 1°C/min to -40°C and plung- to analyse the impact of M. fijiensis metabolites. By performing the Hill reaction (Allen physical parameters on transformation fre- ing into liquid nitrogen. Currently, 651 cry- and Holmes 1986) with the so treated suspen- quency was investigated in four banana culti- otubes containing embryogenic cell suspension to measure the ability of chloroplasts to vars: ‘Grande naine’ (AAA), ‘Obino l’ewai’ sions belonging to 48 independent cell lines (AAB), ‘Orishele’ (AAB), and ‘Three hand transfer electrons, a direct inhibiting effect of and 14 different cultivars are safely stored in juglone on this physiological activity was clear- planty’ (AAB). DNA transfer frequency was liquid nitrogen for the long term. Recently, measured by monitoring expression of the b?- ly demonstrated. Moreover, this effect was banana cell suspensions were recovered glucuronidase and the green fluorescent pro- again higher with ‘Grande naine’ chloroplasts tein gene. The results indicate major differ- after five years storage in liquid nitrogen. than with those of ‘Fougamou’. ences between the two transformation The ability to produce somatic embryos Since chloroplasts constitute one of the sites systems. Significantly higher transient and remained intact. Also their competence of active oxygen species production in plants stable gene expression, in all banana culti- towards Agrobacterium-mediated transforma- (Sutherland 1991, Foyer et al. 1997), their vars, were obtained with the Agrobacterium- tion was screened and compared to a non- direct interactions with juglone in bananas led based method. The effects of age and volume cryopreserved cell suspension of the same to a new hypothesis. Hence, oxidative events of cell suspensions as well as the length of cell line. The transient expression of the were suspected to be at the origin of the phys- infection were optimized. The cultivars were introduced marker gene as well as the regen- iological damages in the isolated chloroplasts. categorized on the basis of their competence eration efficiency of transgenic plantlets was In fact, involvement of fungal naphthoquinone for transformation and their capacity for regen- comparable. metabolites in oxidative process in not uncom-

INFOMUSA — Vol 11, N° 1 PROMUSA XXIII mon (Medentsev and Akimento 1998). Their mechanisms affected by the pathogen Beyer W., J. Imlay & I. Fridovich. 1991. Superoxide auto-oxidative property responsible of the oxi- metabolites during BLS development. dation of NADH and NADPH leads to the dismutase. Prog. Nucl. Acid Res. 40:221-53. Finally, a first in vitro selection assay for BLS Foyer C., G. Noctor & J.F. Morot-Gaudry. 1997. removal of these molecules from the oxidative resistance was also performed. Therefore, L’oxygène: bienfait ou danger pour les plantes? phosphorylation system as potential sources different juglone concentrations were mixed of reduction equivalents for the respiratory with embryogenic cell suspensions of two Biofutur 169:27-29. chain. lines of THP (‘Three hand planty’ cultivar) as Harelimana G., P. Lepoivre, H. Jijakli & X. Mourichon. In the case of BLS disease, assessment of well as with suspensions containing somatic 1997. Use of Mycosphaerella fijiensis toxins for the this hypothesis was performed by considering embryos of the same cell lines. After an selection of banana cultivars resistant to black leaf possible interactions between juglone and overnight incubation, the material was trans- streak. Euphytica 96:125-128. banana antioxidant systems. We observed ferred onto juglone-free fresh media. In gener- Leegood R.C. & R. Malkin. 1986. Isolation of sub-cellu- that juglone causes an in vitro oxidation of al, both plant materials necrosed and did not lar Photosynthetic Systems. Pp. 9-26 in ascorbic acid, the most abundant antioxidant show any weight increase during the period of Photosynthesis energy transduction. A practical in plants (Smirnoff 2000). The occurrence of incubation following the treatment. However, approach. (M.F. Hipkins and N.R. Baker, eds). IRL oxidative phenomena induced by this metabo- from somatic embryos of one treated line, lite in bananas was also assessed by some plants were obtained from tissues that Press, Washington, USA. analysing the superoxide dismutases (SOD) did not become necrotic with 50 ppm of Lichtenthaler H.K., Buschmann C., Rinderle U. & patterns at several intervals of time following juglone. These regenerated plants are going Schmuck G. (1986) Application of chlorophyll fluo- juglone injection into leaves of the two refer- to be evaluated for their eventual BLS resist- rescence in ecophysiology. Radiation and ence cultivars. In fact, superoxide dismutases ance with the abovementioned bioassays as Environemental Biophysics 25: 297-308. are assumed to play a central role in the well as by artificial inoculation of the pathogen. Medentsev A.G. & V.K. Akimenko. 1998. defence against oxidative stress (Beyer et al. These plant regenerates will constitute a pre- Naphthoquinone metabolites of the fungi. 1991, Scandalias 1993). Our preliminary cious material to further analyse the role Phytochemistry 47:935-959 observations showed that there was a repres- played by M. fijiensis toxins in development of Scandalias J. G. 1993. Oxygen stress and superoxide sive effect on one SOD isoform in ‘Grande BLS disease. naine’ while a stimulating effect on another dismutase. Plant Physiol. 101:7-12. SOD isoform in ‘Fougamou’ seemed to occur. Smirnoff N. 2000. Ascorbic acid: metabolism and func- On the base of the results obtained with the References tions of a multi-facetted molecule. Current Opinion two antioxidant systems analysed previously, Allen J.F. & N.G. Holmes. 1986. Electron transport and in Plant Biology 3:229-235. juglone could be supposed to deprive Redox Titration. Pp. 103-141 in Photosynthesis Sutherland M.W. 1991. The generation of oxygen radi- bananas partly of their antioxidant capacity. energy transduction. A practical approach (M.F. cals during host plant responses to infection. Further investigations have to be done in this Hipkins and N.R. Baker, eds). IRL Press, Physiological and Molecular Plant Pathology area in order to determine exactly all the Washington, USA. 398:79-93.

XXIV PROMUSA INFOMUSA — Vol 11, N° 1 Publications from INIBAP

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The following publications are available from INIBAP/ACIAR 1997. E. Arnaud & J.P. Horry (eds). Musalogue, a catalogue of Musa headquarters: germplasm: Papua New Guinea collecting missions 1988-1989. INIBAP/CTA/FHIA/NRI/ODA 1997. B.K. Dadzie & J.E. Orchard. Post-harvest Routine INIBAP 2002. Networking bananas and plantains: Annual Report 2001. Screening of Banana and Plantain Hybrids: Criteria and Methods. INIBAP Technical INIBAP 2002. The Global Musa Genomics Consortium. Strategy for the Global Musa Guidelines 2. Genomics Consortium: Report of a meeting held in Arlington, USA, 17-20 July 2001. INIBAP/CTA 1997. P.R. Speijer & D. De Waele. Screening of Musa Germplasm for INIBAP/CTA/CIRAD 2001. J. Daniells, C. Jenny, D. Karamura & K. Tomekpe. Musalogue: a Resistance and Tolerance to Nematodes. INIBAP Technical Guidelines 1. catalogue of Musa germplasm. Diversity in the genus Musa (E. Arnaud & S. Sharrock, INIBAP/The World Bank 1997. E.A. Frison, G. Orjeda & S. Sharrock (eds). PROMUSA: compil.). A Global Programme for Musa Improvement. Proceedings of a meeting held in Gosier, INIBAP/CTA 2001. B. Panis & N.T. Thinh. Cryopreservation of Musa germplasm Guadeloupe, March 5 and 9, 1997. (J.V. Escalant et S. Sharrock, eds). INIBAP Technical Guidelines 5. INIBAP-IPGRI/CIRAD. 1996. Descriptors for Banana (Musa spp.). INIBAP 2001. Networking bananas and plantains: Annual Report 2000. The following publications are available from Asia CIRAD/INIBAP 2000. Bananas. and the Pacific office: INIBAP 2000. G. Orjeda (compil.). Evaluating bananas: a global partnership. Results of IMTP Phase II. INIBAP-ASPNET 2001. A.B. Molina, V.N. Roa & M.A.G. Maghuyop (eds). Advancing banana INIBAP/EARTH/IDRC 1999. F.E. Rosales, S.C. Tripon & J. Cerna (eds). Organic/environ- and plantain R & D in Asia and the Pacific Vol. 10. Proceeding of the 10th INIBAP-ASP- mentally friendly banana production. Proceedings of a workshop held at EARTH, NET Regional Advisory Committee (RAC) meeting held at Bangkok, Thailand, 10-11 Guácimo, Costa Rica, 27-29 July 1998. November 2000. INIBAP/CRBP/CTA/CF 1999. C. Picq, E. Fouré & E.A. Frison (eds). Bananas and food INIBAP-ASPNET/MARDI 2001. A.B. Molina, N.H. Nik Masdek & K.W. Liew (eds). Banana security/Les productions bananières: un enjeu économique majeur pour la sécurité ali- Fusarium wilt management: towards sustainable cultivation. Proceedings of the inter- mentaire. Proceedings of an International Symposium held in Douala, Cameroon, 10- national workshop on the management of Fusarium wilt disease held in Genting, 14 November 1998. Malaysia, 18-20 October 1999. INIBAP/FHIA 1999. F.E. Rosales, E. Arnaud & J. Coto (eds). A tribute to the work of Paul INIBAP-ASPNET 2000. A.B. Molina & V.N. Roa (eds). Advancing banana and plantain H. Allen: a catalogue of wild and cultivated bananas. R & D in Asia and the Pacific. Proceedings of the 9th INIBAP-ASPNET Regional INIBAP/RF/SDC 1999. E.A. Frison, C.S. Gold, E.B. Karamura & R.A. Sikora (eds). Mobilizing Advisory Committee meeting held at South China Agricultural University, Guangzhou, IPM for sustainable banana production in Africa. Proceedings of a workshop on banana China, 2-5 November 1999. IPM held in Nelspruit, South Africa, 23-28 November 1998. INIBAP-ASPNET/FFTC 2000. A.B. Molina, V.N. Roa, J. Bay-Petersen, A.T. Carpio & J.E.A. INIBAP 1999. E. Akyeampong (ed.). Musa Network for West and Central Africa. Report of Joven (eds). Managing banana and citrus diseases. Proceedings of a regional workshop the second Steering Committee meeting held at Douala, Cameroon, 15-16 November on disease management of banana and citrus through the use of disease-free planting 1998. materials held in Davao City, Philippines, 14-16 October 1998. INIBAP 1999. K. Shepherd. Cytogenetics of the genus Musa. INIBAP-ASPNET 2000. R.V. Valmayor, S.H. Jamaluddin, B. Silayoi, S. Kusumo, L.D. Danh, INIBAP 1998. E. Akyeampong (ed.). Musa Network for West and Central Africa. Report of O.C. Pascua & R.R.C. Espino. Banana cultivar names and synonyms in Southeast Asia. the first Steering Committee meeting held at Douala, Cameroon, 8-10 December 1997. INIBAP-ASPNET 1999. V.N. Roa & A.B. Molina (eds). Minutes: Eighth meeting of INIBAP- INIBAP 1998. E.A. Frison & S.L. Sharrock (eds). Banana streak virus: a unique virus-Musa ASPNET Regional Advisory Committee (RAC) hosted by the Queensland Horticulture interaction? Proceedings of a workshop of the PROMUSAvirology working group held Institute (DPI) in Brisbane, Australia, 21-23 October 1998. in Montpellier, France, 19-21 January 1998. INIBAP-ASPNET 1998. Minutes: Seventh meeting of INIBAP/ASPNET Regional Advisory INIBAP 1998. C. Picq (ed.). Segundo seminario/taller de la Red regional de información sobre Committee (RAC) hosted by the Vietnam Agricultural Science Institute (VASI) in banano y plátano de America Latina y el Caribe. San José, Costa Rica, 10-11 July 1997. Hanoi, Vietnam, 21-23 October 1997. INIBAP/CTA/FHIA/NRI/DFID 1998. B.K. Dadzie. Post-harvest characteristics of black INIBAP-ASPNET 1997. V. N. Roa & R. V. Valmayor (eds). Minutes: Sixth meeting of Sigatoka resistant banana, cooking banana and plants hybrids. INIBAP Technical INIBAP-ASPNET Regional Advisory Committee (RAC) hosted by National Research Guidelines 4. Center on Banana (ICAR) in Tiruchirapalli, India, 26-28 September 1996. INIBAP/CTA 1998. G. Orjeda in collaboration with the PROMUSA working groups INIBAP-ASPNET 1996. R. V. Valmayor, V. N. Roa & V. F. Cabangbang (eds). Regional on Sigatoka and Fusarium. Evaluation of Musa germplasm for resistance to Sigatoka Information System for Banana and Plantain - Asia and the Pacific (RISBAP): diseases and Fusarium wilt. INIBAP Technical Guidelines 3. Proceedings of a consultation/workshop held at Los Baños, Philippines, 1-3 April 1996. CIRAD/INIBAP 1998. Les bananes. (ASPNET Book Series No. 6).