The International Magazine on Banana and Plantain

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INFOINFOMUSAMUSA The International Magazine on Banana and Plantain

Vol. 10 N° 1 June 2001 IN THIS ISSUE In situ mass propagation of FHIA-20 using benzylaminopurine Socioeconomic aspects of plantain cultivation in Colombia Production of plantain leaves for the agrifood industry The evolution of photosynthesis, transpiration and chlorophyll during the development of leaves of plantain Estimation of root development from shoot traits in Musa spp. Evaluation of cultural, chemical and biological control of vascular rot and wilt in plantain Evaluation of FHIA hybrids in comparison with local Musa clones in Peru Evaluation of Musa germplasm against banana weevil borers Distribution of Fusarium wilt of banana in Kenya and its impact on smallholder farmers VCG of the populations of Fusarium (Foc) in Vietnam Black Sigatoka disease in Mexico Effect of number of subcultures on in vitro multiplication of banana clones MusaNews The banana world loses two friends and colleagues INIBAP News Thesis 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) The mission of the International Network for the Improvement of Banana and Plan- tain 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 INFOMUSA and at the conservation and use of Musa diversity The International Magazine on Banana and Plantain Vol. 10 N° 1 • To promote and strengthen collaboration and partnerships in banana-related re- July 2001 IN THIS ISSUE In situ mass propagation of FHIA-20 using benzylaminopurine Socioeconomic aspects search activities at the national, regional and global levels of plantain cultivation in Colombia Production of plantain leaves for the agrifood industry The evolution of photosynthesis, transpiration and chlorophyll during the development of leaves • To strengthen the ability of NARS to conduct research and development activities of plantain Estimation of root development from shoot traits in Musa spp. Evaluation of cultural, chemical and biological on bananas and plantains control of vascular rot and wilt in plantain Evaluation of FHIA hybrids in comparison with local Musa clones in Peru Evaluation of Musa germplasm • To coordinate, facilitate and support the production, collection and exchange of against banana weevil borers Distribution of Fusarium wilt of banana in Kenya and its impact on smallholder farmers VCG of the populations of Fusarium (Foc) in Vietnam information and documentation related to banana and plantain. Black Sigatoka disease in Mexico Effect of number of subcultures on in vitro multiplication of banana clones MusaNews INIBAP is a programme of the International Plant Genetic Resources Institute The banana world loses two friends and colleagues INIBAP News Thesis Books etc. (IPGRI), a Future Harvest center. Announcements PROMUSA News

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

Vol. 10, N° 1 INFOMUSA Vol. 10, N° 1 Cover photo: Local sale of bananas in Bolivia. (L. Pocasangre, INIBAP) CONTENTS Publisher: International Network for the Impro- vement of Banana and Plantain In situ mass propagation of the FHIA-20 banana hybrid using benzylaminopurine...... 3 Managing editor: Claudine Picq Socioeconomic aspects of plantain cultivation in Colombia...... 4 Editorial Committee: Emile Frison, Jean-Vincent Escalant, The production of fire-softened plantain leaves for the agrifood industry...... 9 Suzanne Sharrock, Charlotte Lusty The evolution of photosynthesis, transpiration and chlorophyll during Printed in France the development of leaves of plantain (Musa AAB Simmonds)...... 12 ISSN 1023-0076 Editorial Office: Estimation of root development from shoot traits in plantain INFOMUSA, INIBAP, Parc Scientifique and banana (Musa spp.)...... 15 Agropolis II, 34397 Montpellier Cedex Evaluation of cultural, chemical and biological control of vascular rot 5, France. Telephone + 33-(0)4 67 61 and wilt in plantain (Musa AAB Simmonds) ...... 17 13 02; Telefax: + 33-(0)4 67 61 03 34; E-mail: inibap@cgiar.org Evaluation of FHIA hybrids in comparison with local Musa clones in a black http//:www.inibap.org Sigatoka-free area of eastern Peru...... 21 Subscriptions are free for developing countries readers. Article contributions Evaluation of Musa germplasm against banana weevil borers...... 26 and letters to the editor are welcomed. Articles accepted for publication may be Distribution of Fusarium wilt of banana in Kenya and its impact edited for length and clarity. INFOMUSA on smallholder farmers ...... 28 is not responsible for unsolicited mater- Vegetative compatibility groups of the populations of Fusarium oxysporum ial, however, every effort will be made to f.sp. cubense in Vietnam ...... 32 respond to queries. Please allow three months for replies. Unless accompanied Black Sigatoka disease (Mycosphaerella fijiensis Morelet) in Mexico ...... 33 by a copyright notice, articles appearing in INFOMUSA may be quoted or repro- Effect of number of subcultures on in vitro multiplication duced without charge, provided ac- of four banana clones ...... 38 knowledgement is given of the source. MusaNews ...... 40 French-language and Spanish-language editions of INFOMUSA are also pub- The banana world loses two friends and colleagues ...... 40 lished. To avoid missing issues of INFOMUSA, INIBAP News...... 42 notify the editorial office at least six Thesis ...... 47 weeks in advance of a change of address. Books etc...... 47 Views expressed in articles are those of the authors and do not nec- Announcements...... 50 essarily reflect those of INIBAP. PROMUSA News ...... I to XVI Agronomy Rapid propagation In situ mass propagation of the FHIA-20 banana hybrid using benzylaminopurine

D. Manzur Macias out in furrows. The plants were fertilized one month after planting accord- G1S ing to the results of soil analyses and ananas and plantains are giant nutritional requirements of FHIA-20 perennial grasses resulting from plant material. Bintra- and interspecific hy- Ten months after planting, each plant bridization of two diploid forest had developed 8 to 10 suckers, reaching species: Musa acuminata (banana) a height of 15-20 cm and a diameter of and M. balbisiana (plantain). They 15-20 cm at the collar of the rhizome. flourish in tropical regions and are the These are the first-generation suckers most important carbohydrate source (G1S) (Figure 1). in local economies (Stover and Sim- Using a knife disinfected with formol monds 1987). The most alarming phe- 2% before each operation, the pseu- nomenon for banana and plantain cul- dostem of each sucker was cut transver- tivation has been the appearance and sally 2 cm above the collar of the rhizome spread of diseases such as black Siga- and the apical meristem was removed at toka (Mycosphaerella fijiensis a depth of 4 cm, leaving a 2 cm-diameter Morelet) and those caused by the cavity in the rhizome (Figure 2A); the streak mosaic virus (BSV) and cucum- Figure 1. Differentiation of first-generation pseudostem fragment was then cut with ber mosaic virus (CMV). These prob- suckers (G1S). crosswise incisions, boring down to the lems have been tackled by breeding rhizome collar (Figure 2B). Once these programmes which have succeeded in G2S cuts had been made on each sucker, developing plantain varieties resistant A 4 ml of benzylaminopurine (BAP) at to black Sigatoka (Vuylsteke 1998), 40 mg/l distilled water were deposited with a high yield and high palatability. D in the cavity left by the removal of the The FHIA-20 hybrid bred by Dr Phil apical meristem (Figure 2C). The rhi- Rowe at Fundación Hondureña de In- C zomes were then covered with a mix- vestigación Agrícola (FHIA) is an ex- ture of equal parts of sandy loam and ample. poultry dung compost to 5 cm above Improved plantains are polyploid B ground level. The so-called second- and parthenocarpic and are thus prop- generation suckers (G2S) appeared agated vegetatively from daughter after 3 months on each treated sucker suckers obtained from mother-plants (Figure 2D). just before harvesting. The dormant Figure 2. Differentiation of second-generation When the suckers originating from buds located on the rhizome of the suckers (G2S). A. Extracted apical meristem. G2Ss had differentiated and reached a mother-plant are stimulated after B. Cross-shaped cutting. C. Apical meristem height of 20 to 30 cm, they were dis- bunch harvesting, by cutting the rhi- cavity. D. Second-generation sucker. sected again using the procedure de- zome into pieces or by removing the scribed above, the same quantity of base of the leaf sheaths and dissecting BAP was placed in the cavities and the the developed buds (Auboiron 1997). Materials and methods operation was completed in the same In vitro mass propagation, or micro- FHIA-20 hybrid tissue culture plants way (Figure 3A) until third-generation propagation, is performed routinely from FHIA were multiplied by micro- suckers (G3S) were obtained (Figure based on the proliferation of apical propagation in the tissue culture labo- 3B). meristems cultivated on Murashige & ratory of the Plant Technology Depart- Sixty days later, the G3Ss were Skoog medium enriched with cy- ment, using the protocols developed by treated in the same way as the preced- tokinins and vitamins (Krikorian and various authors (Ma and Shii 1972, ing generations to obtain fourth-genera- Cronauer 1984). One of the common- Hwang et al. 1984). The plantlets ob- tion suckers (G4S) that were left to est limiting factors for the extension of tained, after becoming acclimatized to grow (Figure 4A) for ulterior planting a plantation of plantain banana is the field conditions under intermittent and rooting in sterile soil under inter- difficulty in obtaining planting mater- misting, were transplanted to their mittent misting (Figure 4B). ial, owing to the very nature of the final location at Montelindo Farm plant, its limited sucker production (owned by Caldas University), located Results and slow development (Tézenas du 5°5N and 75°40’W at an elevation of This in situ mass propagation tech- Montcel 1985). 1050 m with average temperature of nique (extraction of apical meristem, The present study is aimed at evalu- 23°C and ‘Typic Distrandept’ soils, on a cross-shaped cutting and addition of ating an in situ multiplication tech- 25-plant plot surrounded by Dominico BAP) gives an average of four suckers nique for FHIA-20 plantain. hartón plantain with a 2 x 3 m plant lay- at the G1S and G2S stages; the same

INFOMUSA — Vol 10, N° 1 3 A G3S G4S B

Figure 3. Differentiation of third-generation Figure 4. Differentiation of fourth-generation suckers (G4S). suckers (G3S). A. Extracted apical meristem. A. Developing buds. B. Plantlet transplanted to a bag. B. Third-generation sucker.

technique applied to GS3 produces an mother-plant, which produces its bunch de matériel de plantation de bananiers et plan- average of 13 plantlets, which are very normally. It also makes it possible to tains. CRBP, Douala, Cameroon. 4pp. similar to those obtained in vitro. obtain, in 8 months, propagules that are Krikorian A.A. & S.S. Cronauer. 1984. Aseptic cul- Thus, a total of 156 plantlets are ob- practically free of pests and diseases; ture techniques for banana and plantain impro- tained from one treated sucker, from healthy plants can then be selected in vement. Economic Botany 38: 322-331. the first to the third generation the field for multiplication. Hwang S.C., C.L. Chen, J.C. Lin & H.L. Lin. 1984. [(4 + 4 + 4) x 13]. If five G1Ss were This technique is easy and conve- Cultivation of banana using plantlets from meris- selected on each FHIA-20 plant and nient for use in the field in case of tem culture. Hort Science 19: 231-233. used for this in situ mass propagation, shortage of planting material or for Ma S.S. & C.I. Shii. 1972. In vitro formation of ad- 780 plantlets (156 x 5) could be ob- mass multiplication of promising, high- ventitious buds in banana shoot apex following tained in 8 months. yielding varieties such as the FHIA-20 decapitation. Journal of the Chinese Society of hybrid. Horticultural Science 18: 135-142. Discussion When applied to FHIA-20 plants close Stover R.H. & N.W. Simmonds. 1987. Banana. 3rd An FHIA-20 hybrid rhizome possesses a to flowering, the technique facilitates ed. Longman, UK. 468pp. potential 14 to 16 buds when the bunch the breaking of bud dormancy due to Tézenas du Montcel H. 1985. Le bananier plantain. appears. Each of these produces 6 to 8 apical dominance. Maisonneuve & Larose, Paris. 143pp. axillary buds. When these are dissected, Vuylsteke D.R. 1998. Shoot–tip culture for the pro- the apical meristem removed, and BAP Acknowledgements pagation, conservation, and distribution of Musa applied, they develop 4 to 5 propagules The author thanks Jairo Castaño Z. and germplasm. IITA, Ibadan, Nigeria. 82pp. in G1S and G2S and as many as 13 in Manuel Aristizábal L. for having revised G3S. this paper.

It should be noted that the technique The author is Professor of tissue culture at Departa- is used when the suckers of the mother- References mento de Fitotecnía, Facultad de Ciencias Agropecua- plant are 30 cm above ground, without Auboiron E. 1997. La multiplication sur souche dé- rias, Apartado Aéreo 275, Manizales, Colombia. damaging the root system of the cortiquée. Fiche technique: propagation rapide E-mail: [email protected]

Agroeconomics Survey in Colombia Socioeconomic aspects of plantain cultivation in Colombia

J. L. Rodríguez Martínez nent of the diet. More than half of the from 17 to 35°C. Some 358 000 ha of and A. Rodríguez Saavedra cultivated area in Colombia belongs to plantain is cultivated producing an small farmers (Rodríguez Saavedra annual 2.5 million tonnes of fruits, of et al. 1999). which 95% is sold on the domestic lantain cultivation has become a In the agricultural sector, plantain is market and the rest is exported. The feature of great socioeconomic in fifth position after coffee, sugar cane, main production centres are in the Pimportance in Colombia from the potatoes and flowers. It accounts for coffee production zones in the An- point of view of food security and job 6.8% of the country’s total agricultural dean region where 231 000 ha is creation. In addition, plantain belongs production (CCI 2000). under plantain (64% of the total culti- to the traditional sector of the rural Plantain is grown in different vated area), accounting for 67% of na- economy, where it is used mainly to agroecological zones from sea level to tional production. Other important shade coffee and is an essential compo- 2000 m and at temperatures ranging natural regions for plantain are the

4 INFOMUSA — Vol 10, N° 1 Orinoco, the Pacific, the Caribbean to 1995. This type of product is still United States increased from 80 000 and Amazonia. aimed at communities of Latin Ameri- tonnes worth US$ CIF 28 million in Of the area cultivated, 87% is devoted can or African origin, but it is sought to 1992 to 109 000 tonnes worth US$ CIF to traditional cultivation with coffee, target consumers of Anglo-Saxon origin 40.4 million in 1999, representing 4.6% cocoa, yucca and fruit trees and the re- since they form the majority of the pop- growth in export volumes. maining 13% is used for mechanized ulation of North America, making it one Ecuador is the second exporting coun- monoculture (Rodríguez Saavedra et al. of the most sought-after markets for try after Colombia. Exports to the 1999). plantain exporters. The following com- United States have decreased consider- The central coffee zone supplies most panies account for 90% of the market: ably in the past eight years, with 7.3% of the main markets in the country. The Mariquita, Migrand Chips, Goya Foods average variation. The smallest volume ‘Dominico hartón’ clone is the variety and Chifles Chips (CCI 2000). was in 1999 when the total that had most commonly planted in this region. In the European Union, the Nether- been 57 000 tonnes worth US$ CIF 10.6 ‘Hartón’ is the main clone in other pro- lands, Belgium and Spain are the main million in 1992 dipped to 26 000 tonnes duction regions such as the Caribbean, importing countries and in turn export worth US$ CIF 7.5 million in 1999, rep- the Orinoco, the Pacific and Amazonia the produce to other EU members. The resenting a 10.6% negative growth rate. because it is more suitable and produc- European market for green plantain is The country supplied 13.1% of total US tive in zones below 1000 m (Rodríguez limited and fairly stable since demand imports in 1999. In contrast, exports to Saavedra et al. 1999). is only from Latin American, Caribbean the European Union increased from According to Corporación Colombia and African communities. The main 396 tonnes in 1995 to 546 tonnes in Internacional, the consumption of supplier countries are Colombia and 1998, representing an 11.3% growth rate. fresh plantain was estimated at 62 kg Costa Rica, although some African Venezuela is the third-largest supplier of per person per year in 1999, one of the countries also make a marginal contri- plantains to the North American market highest figures in the world. bution to market supplies (CCI 1998). with an average of 8.2% over the past eight years and a 13% contribution to US The present state of plantain supplies in 1999 equalling that cultivation Table 1. World plantain production in of Ecuador. The country has gradually 1999 (FAO 1999). increased its market share, with an in- In the world Region Area Yield Production crease from 16 000 tonnes in 1992 worth For agroclimatic reasons, plantain (103 ha) (t/ha) (103 t) US$ CIF 6.5 million to 26 000 tonnes in growing is concentrated in Africa, Latin Latin 1999 worth US$ CIF 17.2 million, i.e. a America America and the Caribbean. and the 6.8% growth rate. In contrast, its ship- Table 1 shows that in 1999 the world Caribbean 830.7 8.30 6 898.0 ments to the European Union decreased plantain area totalled 4.8 million Africa 3 966.5 5.72 22 706.7 from 33 tonnes in 1994 to 12 tonnes in hectares producing 30.6 million tonnes Asia 89.0 11.39 1013.3 1998, a 22.4% negative growth rate. of fruits. The regions with the largest Total 4 886.2 6.27 30 618.0 Costa Rica and Colombia profited from production are in Africa and Latin this to increase their market shares. America with respectively 74.2% and 22.5% of world production in compari- Importing countries International prices son with 3.3% in Asia. The United States, Europe and Japan The price of plantain has not in general The four main producer countries in are the main importers of plantain, pur- increased significantly in the North Africa are, in descending order, Uganda, chasing 80% of exports. The United American market in the past eight Rwanda, Ghana and Nigeria; the main States imports plantain only from Latin years. The Dominican Republic obtains producers in Latin America and the America and the Caribbean—from the highest price at US$ 0.58 per kg, fol- Caribbean are Colombia and Peru and, Colombia, Ecuador, Venezuela, Costa lowed by Venezuela with US$ 0.45, finally, Sri Lanka is the main producer Rica and the Dominican Republic Costa Rica and Colombia with US$ 0.39 in Asia. among others. Japan imports from the and finally Ecuador with US$ 0.19. Colombia accounts for 39.1% of pro- Philippines, China and South Africa Figure 1 shows that Venezuela holds duction in Latin America and the whereas the European Union imports the historic price record in the face of Caribbean and 8.8% of world produc- plantain from its former colonies and Colombia and Ecuador. This is because tion. These figures have been fairly sta- also from Latin America and the Venezuelan plantains are larger than ble for the past eight years. Peru fol- Caribbean. Europe also produces what fruits from Colombia and Ecuador and lows with 4.4% of world production and is commonly referred to as ‘community are much appreciated by the Latin 19.5% of that of Latin America and the plantain’ from Spain, Portugal, Greece American communities living in the Caribbean. and French overseas departments such United States and especially in Miami as Martinique and Guadeloupe (Ro- and New York, where most of the Latin World consumption dríguez Saavedra et al. 1999). Americans and West Indians who eat The greater part of world plantain pro- green plantain are to be found. duction is aimed mainly at covering the Exporting countries Plantain is more expensive on Euro- domestic requirements of the producer Colombia is considered as being the pean markets than in North America. countries. Only 1% is sold on interna- main plantain exporter to the United This is mainly the result of high trans- tional markets to meet the demand States and European Union markets, port costs and customs dues, without from consumers of Latin American ori- with slow growth in terms of the vol- forgetting that plantain is an exotic gin and, to a lesser extent, of African umes exported. In 1995, plantain ex- fruit on this type of market. Figure 2 origin (CCI 2000). ports totalled 105 000 tonnes worth US$ shows that the price of fresh plantain It is estimated that 10% of the plan- FOB 36 million. The figures in 1998 has varied between US$ 0.06 and 1.64 tains imported by the United States are were 121 000 tonnes worth US$ FOB per kg. Another feature is that the high- used for derived products whose con- 42.1 million, a growth rate of 4.9%. Ex- est price was obtained by an African sumption increased by 15% from 1991 port shipments from Colombia to the country, Ghana, with an average of

INFOMUSA — Vol 10, N° 1 5 0.70 and 9% of production, followed by 24% 0.66 of production and cultivated area in the 0.60 0.63 Caribbean regions, the Orinoco, Amazo- nia and the islands of San Andrés and 0.50 0.50 Providencia. The departments with the largest cul- 0.40 0.40 0.40 tivated areas and production at na- 0.40 0.35 0.36 0.33 0.37 0.37 tional level are Antioquia, Quindío and 0.36 0.34 Tolima with respectively 14%, 10% and 0.30 0.32 0.32 0.32 US$/kg/plantain 0.29 0.29 0.29 9% of the area under plantain. In pro- 0.25 0.19 duction, Quindío and Antioquia account 0.20 0.19 for 14% and Tolima 10%. Plantain is grown in mixed cropping 0.10 systems with coffee (81%), as a sole 0.01 0.01 crop (15%) and intercropped 4%. 0.00 1992 1993 1994 1995 1996 1997 1998 1999 Types of producers Years Four producer categories can be estab- Colombia Ecuador Venezuela lished (small, medium, large and industrial) (Table 3) on the basis of the num- Figure 1. Purchase CIF price by the United States in US$ per kg of plantain from Colombia, Ecuador and Venezuela, 1992–1999 (calculations by Corpoica Regional Nueve, Oficina de Planeación, using CCI ber of hectares cultivated and the type 1999 data). of holding, with the main cropping system being mixed cropping followed to a less extent by sole cropping (Rodríguez 1.80 Saavedra et al. 1999). 1.62 1.64 In all cases, production is marketed 1.60 1.64 1.46 locally, nationally or internationally ac- 1.40 1.34 cording to the volumes produced, with the exception of small producers who 1.31 1.20 1.18 reserve the crop for personal consump- 1.08 1.00 tion or for animal feed. 1.00 0.89 0.97 0.82 Industrial exploitations and some- 0.80 0.82 times the large producers use special- US$/kg/plantain 0.65 0.62 0.63 0.65 ized technical assistance, whereas the 0.60 0.59 0.63 0.60 0.60 majority of small and medium produc- 0.55 0.56 0.40 0.51 ers do not use this type of service (Ro- dríguez Saavedra et al. 1999). 0.20

0.06 0.00 National consumption 1994 1995 1996 1997 1998 Plantain is a crop of great strategic importance in the rural sector in Colom- Years bia. It also has a privileged position in Colombia Dominica urban food distribution. Plantain is Costa Rica Ghana Venezuela eaten both green and very ripe and is prepared using different recipes in the Figure 2. Purchase CIF price by the European Union in US$ per kg fresh plantain, 1994–1998 various regions of the country. It is also (calculation by Corpoica Regional Nueve, Oficina de Planeación, using CCI 1999 data). found in the form of meal, chips and snack foods but only a very small percentage of production is processed in- US$ 1.53 per kg over a 4-year period, Uraba region. Prices on re-exported dustrially. followed by Dominica (Lesser Antilles) plantain markets are distinctly higher. Over the past eight years, fresh plan- with US$ 0.99 per kg, Venezuela with Plantain is re-exported all the year tain consumption has decreased from US$ 0.75 per kg (the latter country dis- round on the French and British mar- 73.3 to 61.9 kg per person per year, i.e. played a 77.5% negative price trend kets and the best prices are obtained in negative growth of 2.4% from 1992 to from 1996 to 1998), Costa Rica at US$ Britain (CCI 1998, 2000). 1999. In contrast, the per capita con- 0.63 per kg and finally Colombia with sumption of processed plantain in- an average of US$ 0.58 per kg. Prices Plantain growing at the creased by 6% over the same period were stable for the last two years of the national scale (from 0.02 to 0.03 kg per person per period analyzed. year). This is explained by changes in It should be noted that Colombian Distribution of production zones eating habits, with the trend being to- produce fetched higher prices than that Table 2 shows production distribution wards processed products (CCI 2000). of Costa Rica in France and Great in 1999 according to the natural geo- It can be seen that the prospects for Britain in 1998. The price in Great graphic zones. The Andean region is the agri-industrial demand for the product Britain varied between US$ 0.4 and 1.7 most important production zone with are favourable. Indeed, consumption in- per kg. From February 1999 onwards, 64% of the cultivated area producing creased from 900 tonnes in 1992 to 2000 Colombian plantain fetched US$ 0.1 to 67% of total national production. Then, tonnes in 1999, a 12.1% growth rate. 0.5 less than Costa Rican produce be- in descending order, come the Pacific The processing industries consider that cause of a decrease in supplies from the region with 12% of the cultivated area this trend may continue during the

6 INFOMUSA — Vol 10, N° 1 coming five years if consumer interest Table 2. Cultivated areas, production and plantain yield in 1999 in different regions in this type of product does not weaken of Colombia (Carlos Humberto Gutiérrez, Minagricultura, June 2000). (CCI 2000). Region Cultivated area Production Yield % % cultivated Figure 3 shows that Bogotá is the (ha) (t/year) (t/ha/year) production area largest plantain consumer with 29% Caribbean consisting of 70% Hartón and 30% mis- Guajira 2 276 14 339 6.3 0.58 0.63 cellaneous clones such as Cachaco and Magdalena 1 780 11 715 6.6 0.47 0.50 Dominico hartón. Bogotá is followed by Cesar 3 381 23 905 7.1 0.97 0.94 Atlántico 418 3 201 7.7 0.13 0.12 Medellín and Cali with 17% and 14% re- Bolívar 5 417 35 980 6.6 1.46 1.51 spectively, consisting of 80% Dominico Sucre 1 027 4 886 4.8 0.20 0.29 hartón and 20% Hartón. Barranquilla Córdoba 25 101 169 496 6.8 6.87 7.00 comes last with 5% of national con- Sub-total 39 400 263 522 6.7 10.68 10.99 sumption, mainly Hartón. Nearly 20% of Pacific consumers at the Cali, Barranquilla and Choco 16 245 98 541 6.1 3.99 4.53 Bogotá markets and 32% of those in Cauca 5 576 34 937 6.3 1.42 1.56 Medellín prefer ripe plantain (CCI Nariño 20 561 88 681 4.3 3.60 5.74 2000). Sub-total 42 382 222 159 5.2 9.01 nd Andean and Inter-Andean Job creation Antioquia 49 594 340 041 6.9 13.78 13.83 The mechanized, traditional or inter- Valle del Cauca 11 985 127 283 10.6 5.16 3.34 crop cultivation cultivation of one Caldas 18 651 106 675 5.7 4.32 5.20 hectare of plantain generates respec- Risaralda 18 135 72 227 4.0 2.93 5.06 tively 1.68, 0.39 and 0.19 permanent di- Quindío 36 080 345 262 9.6 14.00 10.06 rect jobs per ha and per year. In the Tolima 32 972 234 581 7.1 9.51 9.20 light of this, it is estimated that one Cundinamarca 12 808 127 932 10.0 5.19 3.57 hectare of plantain generates an aver- Boyacá 3 305 39 413 11.9 1.60 0.92 age of 0.75 permanent job. When set Santander 8 530 70 842 8.3 2.87 2.38 against the national cultivated area, Norte Santander 12 475 89 223 7.2 3.62 3.48 this gives approximately 288,375 perma- Huila 26 638 95 310 3.6 3.86 7.43 nent jobs. This is the equivalent of Sub-total 231 173 1 648 789 7.1 66.84 64.48 58,000 families of five persons devoted Orinoco to growing plantain. Arauca 8 909 60 976 6.8 2.47 2.49 Casanare 2 367 19 439 8.2 0.79 0.66 National prices Vichada 157 1 413 9.0 0.06 0.04 Although plantain is a crop with perma- Meta 11 458 117 881 10.3 4.78 3.20 nent production, harvesting periods are Sub-total 22 891 199 709 8.7 8.10 6.39 influenced by external factors such as Amazonia coffee production and harvesting or by Amazonas 243 413 1.7 0.02 0.07 very cold seasons. These production Caquetá 10 094 61 629 6.1 2.50 2.82 movements or periods in turn cause up- Guainía 547 3 702 6.8 0.15 0.15 ward or downward price trends accord- Guaviare 4 252 21 718 5.1 0.88 1.19 ing to supply and demand volumes (Ro- Putumayo 7 033 41 333 5.9 1.68 1.96 dríguez Saavedra et al. 1999). Vaupés 476 3 630 7.6 0.15 0.13 It should be noted that the three Sub-total 22 645 132 425 5.8 5.37 6.32 main wholesale markets (Bogotá, Cali San Andrés y Prov. 14 152 10.9 0.01 0.00 and Medellín) display identical behav- TOTAL 358 505 2 466 756 6.9 100.00 100.00 iour in both supply and demand, even though plantains are harvested continu- ously (Rodríguez Saavedra et al. 1999). Seasonal variations in current prices on the three main wholesale markets second half of 1998 until the first half of Table 3. Types of producers, holding from 1992 to 1999 are shown in Figure 4. 1999. These phenomena had a direct ef- size and cropping system (Rodríguez It can be seen that these prices rise fect on production volumes and re- Saavedra et al. 1999). from January to April, with lower sulted in smaller supplies and high prices in Bogotá. In the second semes- prices. Type of Holding size Cropping ter, prices fall in Cali and Medellín and producer (ha) system remain very high in Bogotá until Sep- Marketing Small 0.1-5.0 Intercropping* Mixed** tember. The situation is then reversed Sole cropping and prices finally fall on the three Marketing channels Medium 5.1-15.0 Mixed large wholesale markets in November- The marketing of plantain is very diffi- Sole cropping December. cult because of the dispersal of the pro- Large 15.1- 30.0 Mixed A decrease in real incomes according duction zones, the lack or poor condi- Sole cropping to climatic features can be seen for tions of the lines of communication Industrial > 30.1 Mixed both producers and resellers as a result with urban consumption centres and Sole cropping of various factors including the effect of the irregular supplying of the market by «El Niño», which disturbed the climate wholesalers and middlemen who set the *Without uniform spatial distribution and that may include various cultivated plant species from March 1997 to the first half of prices. In addition, perishable produce **Distribution is according to planting systems defined in 1998 and that of «La Niña» from the like plantain suffers from continuous relation to the main crop in the mixed system.

INFOMUSA — Vol 10, N° 1 7 value added during the process (Ro- dríguez Saavedra et al. 1999). The traditional markets consisting of purchase centres, permanent and tem- Barranquilla Bucaramanga porary markets, a few supermarkets Santafé de Bogotá 5% 4% Cali and shops are characterized by control 30% 14% by middlemen. In order to define the conditions of negotiation—given the heterogeneity of the fruits—all the Cartagena 2% plantains must be presented at the site of the transaction (Rodríguez Saavedra Cúcuta et al. 1999). 2% The specialized market is characterized by an appropriate organization structure for selection, sorting and packing. Supermarket chains accept or Others Medellín not the batches presented by suppliers 26% 17% on condition that they have seen a sample of the produce, that their quality criteria are met and the supplies guar- anteed. A price range is generally set in this type of transaction in order to Figure 3. Distribution of plantain consumption in Colombia (CCI 2000). avoid fluctuations that are too sudden and require produce classification in conformity with the types usually sold (Rodríguez Saavedra et al. 1999). As everywhere, the national plantain market responds to the requirements of 1.10 supply and demand but lacks a regulat- ing body, which has contributed to the development of complex marketing 1.05 channels. In this context, five main channels leading to the consumer can be identified: collector>wholesaler> retailer; supplier>wholesaler>supermar- 1.00 ket; producer>supermarket; wholesaler>agri-industry; and producer>agri- Index industry (Rodríguez Saavedra et al. 1999, 0.95 CCI 2000).

On-farm post-harvest losses 0.90 Post-harvest fruit losses are estimated to be 10%. On the basis of national production of 2.5 million tonnes in 1999, 0.85 fruit losses are estimated at 250 000 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec tonnes, representing 62.5 thousand million colombian pesos (COP) (US$ 36 Bogota Cali Medellín million) assuming an average farm gate price of COP 250 per kg (1 US dollar Figure 4. Index of seasonal variations in plantain prices at the three main wholesale markets in = 1758.11 Colombian pesos in 1999). Colombia, 1992-1999 (Calculation by Corpoica Regional Nueve, Oficina de Planeación, using Cordicafé and CCI data for 1992-1999). These figures clearly show the need to develop a process that prevents economic losses and also generates value added for fresh plantain and avoids deterioration resulting from poor post- sumers on a large scale. As the whole- problems of pollution resulting from the harvest management. This aggravates salers are not numerous, information poor use of crop residues. loss of quality and quantity and thus af- about plantain moves between them The causes of losses consist mainly of fects the final price (Rodríguez Saave- rapidly, enabling them to agree, among the poor level of technology at cropping dra et al. 1999). other things, about prices and the level, poor harvesting, inefficient han- As plantain is a fruit that is generally quantities to be put on the market (Ro- dling of produce from production to eaten fresh and marketing is immedi- dríguez Saavedra et al. 1999, CCI 2000). consumption sites and poor product ate, it has the marketing characteris- Most plantain producers are small conformity. Packing and especially tics specific to all perishable foodstuffs growers who are very scattered and transport affect quality and the appear- whose production is complex and whose generally sell the fruit at the farm gate. ance of fruits since middlemen pay no distribution is difficult to organize ra- Middlemen therefore play an essential attention whatsoever to the improve- tionally. The process involves a large role in the coordination of purchases of ment of the packing system for fruit number of producers and a few whole- plantain, transport and sale, thus being transport. Bunches are generally trans- salers who distribute plantain to con- able to pocket a larger proportion of the ported in bulk, resulting in bruising and

8 INFOMUSA — Vol 10, N° 1 scarring and hence poor appearance References Rodríguez Martínez J.L., A. Rodríguez Saavedra & S. and decreased quality (Rodríguez Corporación Colombia Internacional (CCI). 2000. Belalcázar Carvajal. 1998. Importancia Socioe- Saavedra et al. 1999). http://www.cci.org.co conómica del Cultivo del Plátano en la Zona Cen- For specialized markets, the produce Corporación Colombia Internacional (CCI). 1999. tral Cafetera (Segunda Versión) Oficina Regional is packed and shipped in crates that Boletín CCI: SIM. Perfil de Producto Plátano de Planeación - Corpoica, Regional Nueve. Mani- protect the fruits during distribution No. 7. enero–marzo. 16pp. zales, marzo. operations and result in better product Corporación Colombia Internacional (CCI). 1998. Ministerio de Agricultura y Desarrollo Rural, Ofi- acceptation by the consumer (Ro- Inteligencia de Mercados. Precios Internacio- cina de Información y Estadística. 2000. Area Co- dríguez Saavedra et al. 1999). nales de Bananito (Musa acuminata). Boletín sechada, Producción y Rendimiento del Cultivo No. 4, octubre. del Plátano por Regiones Naturales en Colombia. Agri-industrial development Corporación Colombia Internacional (CCI). 1998. Información telefónica. Bogotá, D.C. Hartón and Dominico hartón clones cul- Inteligencia de Mercados. Precios Internacio- tivated in hot zones are easier to peel nales de Plátano Verde. Boletín No. 3, sep- and thus potentially easier to process. tiembre. The processing industries have set dis- Corporación Colombia Internacional (CCI). 1998. tinctions between the two clones. Sistema de Información de precios y volúmenes Hartón fruits are larger and have a transados. “SIPSA”. Precios mayoristas 3(42), oc- higher moisture content; Dominico tubre 10 al 16. hartón fruits have a higher soluble mat- Day B. 1987. Suculenta Fruta Tropical. Revista Se- ter content. It should be specified that lecciones: 76-80. José Luis Rodríguez Martínez is an Economist and no conclusive results allow such charac- FAO. 1999. http://www.fao.org Alfredo Rodríguez Saavedra is the Director of Ofi- terization of the clones and their advan- Rodríguez Saavedra A. & J.L. Rodríguez Martínez. cina de Planeación, Corporación Colombiana de In- tages for processing (CCI 2000). 1999. Aspectos Socioeconómicos del Cultivo del vestigación Agropecuaria (Corpoica) Regional 9, In Colombia, the preference is for Plátano en Colombia. Oficina Regional de Pla- Apartado 1287. PBX: (0968) 876197 Fax: (0968) fresh fruits and the taste for meal or neación - Corpoica, Regional Nueve. Manizales, 876204 Manizales, Caldas, Colombia. chips is minor. The agri-industrial de- abril. E-mail: [email protected] velopment of plantain processing in the central coffee growing zone is recent. An agri-food plant was established in early 2000 at Murrillo, in the Armenia municipal district, where ‘patacones’ (deep-fried crushed plantain portions) and slices frozen for distribution in supermarkets are prepared. Plantain is also packaged whole and frozen for certain factories that export the fruit in snack form, as meal or frozen for international markets. According to Day (1987), the pseudostem, leaves, flowers and roots can be used after the harvest for the production of meal, vinegar, paper, edible tor- tas, chipboard, animal feed, dyes, etc. Agroeconomics Production and use of leaves Expectations for future agri-industrial development are great in the central coffee-growing region. The production of fire-softened National and international market opportunities for plantain plantain leaves for the agrifood Colombia could increase plantain exports to North America, especially in industry the form of snack foods and baby food if the consumption of fresh and processed plantain increases in Latin American, African, Anglo-Saxon and European E. Echeverry Navarro leaves to be fire-softened for the agri- groups (CCI 2000). food industry. The clone has displayed Ministry of Agriculture forecasts for very good adaptability at an elevation of 2000 indicate that production will not The flat, hot zone in the central south- 400 m to difficult soil and climate con- cover domestic market demand, even ern part of Tolima department is inhab- ditions, characterized by degraded, low- though fresh consumption has de- ited almost entirely by descendants of fertility soils, a hot, dry climate, annual creased. This requires new areas for the the “Pijaos” tribe. Many of them, precipitation of 1000 to 1300 mm poorly crop or the establishment of technology grouped in native councils, work in distributed over the year and an aver- transfer to turn some holdings into in- farming and small-scale animal hus- age annual temperature of 25°C. tensive, mechanized operations to meet bandry. Within the zone studied (600 ha), unsatisfied demand. This would prevent One of the main crops in their subsis- 4500 to 5000 persons participate in the the increasing volume of plantain im- tence farming practices is the ‘Cachaco production of fire-softened leaves and ports from Ecuador and Venezuela (CCI común’ plantain clone (Musa ABB, live on the proceeds of their sale in 2000). Simmonds) that they use to produce bundles of 50. The production process

INFOMUSA — Vol 10, N° 1 9 is handled entirely by family groups leaf diseases. Up to now, defoliation or vation of 400 m. Annual precipitation is consisting of fathers and their sons, deleafing of plantain has been for phy- from 1000 to 1300 mm with irregular bi- whatever their age. tosanitary reasons and consists of re- modal distribution. Annual average The leaves of ‘Cachaco común’ are moving all the infected leaves or those temperature is 25°C. those most frequently used to wrap that have withered to more than 60%. 1997 was climatically atypical. Rain- cooked foods because they do not cause In general, the leaves of adult plan- fall was generally very scarce through- any modifications to the organoleptic tain are 70 à 100 cm wide and 150 to out the year as a result of the Pacific properties of foods and are fully steril- 400 cm long, with leaf indexes varying meteorological phenomenon ‘El Niño’. ized by exposure to flame to soften from 2 to 4 according to the clone and This drought caused the disappearance them. This is not the case of leaves of soil and climate conditions. Leaf thick- of numerous small plantain plantations plantains of other clones such as ‘Har- ness varies from 0.35 to 1 mm according and, from time to time, sharp price ton’ for example, which, among other to the portion of lamina considered and rises for leaves from COP1500 to 3500 things, gives tamales1 and cheeses a the polyploidy (Champion 1978, Belal- per bundle, a price considered very greenish colour when used to wrap cázar 1991). high by middlemen and wholesalers but them. Tamales have a particularly Under good conditions and in its envi- judged to be extremely satisfactory by pleasant smell when they are wrapped ronment, plantain emits a leaf every 8 the farmers. in ‘Cachaco común’ plantain leaf. to 10 days. In addition, the obtaining of The farm at which the study was con- No work previous to this is known on a good bunch of plantain requires at ducted has a loamy-sandy soil. With a plantations of ‘Cachaco común’ plan- least 7 to 8 functional leaves at flower- pH of 6.9, the soil has a small organic tain devoted exclusively to leaf producing (male flower emission) (Arcila et al. percentage (1.3%), an average sulphur tion for the agrifood industry. This ex- 1994, Belalcazar et al. 1996, Martinez level (6.0 ppm), large quantitites of plains why plant reactions to frequent, 1984). phosphorus (42.9 ppm), copper (1.13 severe defoliation are not known. It can It has been shown in other studies ppm), iron (18.4 ppm) and manganese be expected that the plant would in- that plantain requires 8 functional (37.02 ppm), small quantities of zinc crease its leaf emission rate and num- leaves for bunch size and weight not to (0.72 ppm) and boron (0.19 ppm), high ber, as reported by Belalcázar (1991), be reduced (Martinez 1984). Bunch calcium (18.43 meq/100 g soil) and unless, in contrast, the number de- weight is reduced by 50 and 40% respec- magnesium levels (4.03 meq./100 g creases, but this is less likely. tively when there are only 4 and 6 func- soil), a low potassium content (0.15 When a small grower of ‘Cachaco tional leaves during the vegetative meq/100 g soil) and a normal sodium común’ plantain decides to produce cycle. content (0.10 meq/100 g soil). leaves, we know in advance that he In addition, Belalcázar (1991) also ‘Cachaco común’ plantain was used. prefers to sell leaves every week or stressed that cutting the green leaves This has the best behaviour in the zone every fortnight rather than the fruits. In in addition to wilted leaves before thanks to its hardiness and drought tol- addition, any fruit bunches produced flowering leads to certain benefits, erance. It is also the clone most com- under these conditions are very small including: monly used for the production of fire- with poorly filled fingers for lack of • a strengthening of the plant physio- softened leaves for the agrifood leaves. logical processes leading to an in- industry, used for tamales, cheeses, en- Today, a bundle of 50 fire-softened crease in production; vueltos2 made with plantain, maize, plantain leaves fetches 2000 to 2500 • improved penetration by light to the rice, etc. No fertilizer or pesticide of Colombian pesos (COP), i.e. US$ 1 to foot of the plant, stimulating budding any kind was used. 1.25, roughly the equivalent of a bunch and sucker development; of plantains. In a year, a plant produces • enhanced aeration, reducing relative Methodology 150 to 175 leaves worth 6000 to 7500 humidity that is dangerously suitable A total of 96 suckers were planted in pesos against only a single marketable for the development of diseases; 30 cm x 30 cm x 30 cm holes with spac- bunch of ‘Cachaco’ plantains worth • more rapid decomposition of organic ing of 2 m x 2 m, in conformity with the hardly 2500 pesos. material; practices in the region. Leaf production is continuous and • a decrease in water loss by transpira- Soil analysis was performed but was steady – and can even increase – in tion during drought periods. not followed by fertilization. In a previ- subsequent years in the same planta- Research conducted by Belalcázar ous experiment, the application of inor- tion, whereas fruit bunch production (1991), Martinez (1984) and Merchán ganic fertilizers such as nitrogen (urea decreases in both quality and quantity (1994) among others confirmed that 46% N) and potassium (potassium chlo- after the first production cycle. plantain emits from some 36 to 39 ride 60% K2O) had caused the total leaves during the vegetative period, ex- blackening of leaves when exposed to Review of the literature cept under extreme climatic conditions flame and they could therefore not be According to the studies performed by that are difficult to handle. used in the agrifood industry. Martinez (1984), Arévalo (1986) and Belalcázar et al. (1998) consider that Weeding was performed by hand Belalcázar (1991), plantain can keep up of all triploid plantains with balbisiana three times during the experiment to 16 functional, erect, green, healthy dominance (ABB), ‘Cachaco común’ (15 months). Desuckering was not per- leaves. This corresponds to a growth would appear to be the hardiest and formed. Axillary buds were left to grow cycle of about 120 to 130 days per leaf most tolerant of drought and water around each mother plant as is the when the agri-meteorological condi- stress. custom in the region as the more tions (mainly soil, precipitation, tem- plants per mat, the more leaves can be perature, wind and relative humidity) Material harvested. are favourable for plant development The study was conducted for 15 months The treatment was fully random with and if there are no diseases – especially from November 1996 to January 1998 on four protocols with three repetitions in a farm at Agua Fría in Coyaima, in the central southern part of Tolima depart- 1 Tamales are a typical Colombian dish made with chicken, 2 Envueltos are flat cakes made with maize, potato or vegetables and maize flour steamed in plantain leaf. ment (Colombia). The farm is at an ele- plantain and wrapped in plantain leaf.

10 INFOMUSA — Vol 10, N° 1 each case. The protocols consisted of Table 1. Experimental protocols for the production of ‘Cachaco común’ plantain leaving 3, 4, 5 or 6 leaves on all the leaves for use as wrapping in the agrifood industry. Espinal 1999. plants in a mat after each harvest or leaf Protocol Description cutting operation as shown in Table 1. 1 3 leaves left on each plant after each leaf harvest Each protocol was applied to a row of 2 4 leaves left on each plant after each leaf harvest eight plants or eight mats each consist- 3 5 leaves left on each plant after each leaf harvest ing of a mother plant and its suckers. 4 6 leaves left on each plant after each leaf harvest The first three cuts were performed at 3-week intervals. Then, with no rainfall, cutting was performed every 4 weeks until a total of 8 leaf harvests Table 2. The effect of defoliation on the growth of ‘Cachaco común’ plantain. had been reached, considered to be a Espinal 1999. low figure for a 14-month period. A leaf Plant height Pseudostem girth forms every 8 days under normal rain- Protocol (average in cm) (average in cm fall conditions and every 10 to 12 days above ground level) during drought periods. 1 257.71 a* 34.92 a 2 264.17 a 40.00 a Results and discussion 3 258.13 a 38.29 a 4 258.83 a 39.88 a Growth parameters * Figures with the same letters are not significantly different. Table 2 presents the growth measurements performed during the eight leaf harvests of the experiment. Table 3. Total leaves harvested and average leaf weight for the eight harvests. The average values of the growth pa- Espinal 1999. rameters (height and girth) of Protocol Total leaves harvested Average leaf weight (g) ‘Cachaco’ plantain at Coyaima in 1 792 a* 277.08 Tolima department (Table 2) do not dis- 2 579 b 277.00 b play statistically significant differences, 3 520 b 271.39 b suggesting that different levels of sys- 4 338 c 298.24 tematic defoliation do not affect the * Figures with the same letters are not significantly different. vegetative development of the plants.

Leaf production parameters The two parameters measured immediately after leaf harvesting and which are used as indicators of the biomass produced in each protocol are shown in Table 3. was observed in protocol No. 2 at 206 1.25) depending on the time of year and The figures for total marketable cm long, not counting the petiole, and according to supply and demand. leaves harvested during the eight har- 69 cm wide in the central part, followed Purchases are completed on a cash vests performed during the experiment by a leaf 196 cm long and 67 cm wide in basis at mobile buying points at differ- clearly show that the best treatment protocol No. 4. ent places in the production zone and on was protocol No. 1, followed by Nos. 2 different days according to the time of and 3, which yielded respectively 72.6% Cutting period year, and especially on the occasion of and 65.2% of the amount in No. 1. No. 4 Unless there are diseases or serious the mid-year and end-of-year festivities. gave the smallest number of leaves at wind damage, the moment for cutting Leaves thus fetch the best prices dur- only 42.4% of the amount in No.1. ‘Cachaco común’ leaves depends mainly ing the St John’s day and St Peter’s day on the frequency of rainfall and market festivities in June and then in Decem- Leaf weight, length and width conditions. ber and January over Christmas, the The leaf weight parameter is neither Under the climatic conditions at New Year and Epiphany. The price of decisive nor essential for leaf produc- Coyaima, plants emit a leaf every 7 or leaves falls a little during the rainy sea- tion for the agrifood industry since it 8 days during the rainy season and son because production is substantial does not determine the price to be every 10 to 11 days during the dry sea- and supplies are plentiful. It also de- paid. The most important criteria are son. Cutting is performed when one, creases during the months in which length and width and above all the sani- two or three leaves are ready for fire- demand is weak. tary condition of the leaves. softening or, more specifically, when Leaf length and width are not very there is market demand. However, the Conclusions precise characteristics. A ‘Cachaco general custom is that of harvesting • Most of the saleable fire-softened común’ plantain leaf is considered to be every fortnight in the rainy season and ‘Cachaco común’ plantain leaves suitable for packing or containing every three weeks in the dry season. were obtained when at least three tamales, cheeses, envueltos and other leaves per plant were left after the food products when the lamina is more Economic parameters cutting of leaves for sale. This was than 1 metre long and 30 cm wide in After cutting, the leaves are exposed to performed without desuckering or the central portion. These two parame- a flame for softening on the spot. They fertilizer or pesticide application. ters were measured during the experi- are then folded and assembled in tied ment each time that leaves were cut or bundles of 50 leaves. The bundles are • Leaf production was largest with pro- harvested, but for reference purposes sold directly to middlemen/transporters tocol No. 1 in which three leaves were only. It is noted that the longest leaf who pay COP2000 to 2500 (US$1 to left on each plant after cutting. The

INFOMUSA — Vol 10, N° 1 11 smallest production was given with References rias. (M.J. Giraldo, S.L. Belalcázar, D.G. Cayón & protocol No. 4, in which six leaves Arcila M.I., S. Belalcázar, J.A. Valencia & G. Cayón. R.G. Botero, eds). Armenia, Quindío, Colombia. were left on each plant. 1994. Influencia del número de hojas en postflo- Belalcázar S., G. Cayon & M.I. Arcila. 1998. Manejo ración sobre el llenado de los frutos del clon de Agronómico. Manejo de plantaciones. Pp. 123- • The largest number of leaves in a sin- plátano Dominico Hartón, Musa AAB Simmonds. 136 in Mejoramiento de la producción del cultivo gle cut was 150 in protocol No. 1. Pp. 90-93 in Mejoramiento de la producción del de plátano (C.S. Belalcázar, O. Jaramillo, cultivo de plátano (C.S. Belalcázar, O. Jaramillo, J.A. Valencia, M.I. Arcila, H. Mejía & H. García, • Leaves are sold at mobile installa- J.A. Valencia, M.I. Arcila, H. Mejía & H. García, eds). ICA-CORPOICA, Colombia. tions in the production areas on days eds). ICA-CORPOICA, Colombia. Champion J. 1978. El plátano. Traducción de set beforehand with wholesalers who Arévalo E. 1986. Como manejar su platanera para Palenque. Impreso por Romagraf S.A. Barcelona. compete with each other. reducir el ataque de Sigatoka negra. Cartilla di- 247pp. vulgativa No. 00-4.4-33-086. ICA Regional No 5. Martínez G.A. 1984. Determinación del área foliar • Leaves are in bundles of 50 sold di- Pasto. 14pp. para el plátano en el trópico húmedo. Revista rectly for cash to middlemen/trans- Belalcázar S. 1991. El cultivo de plátano en el tró- ICA 19(2):183-186. porters at prices ranging from COP pico. Manual de asistencia técnica No. 50. Insti- Merchán, V.M. 1994. Comportamiento del clon de 1500 to 2500 per bundle. tuto Colombiano Agropecuario. ICA, Cali, Colom- plátano Dominico Hartón, Musa AAB Simmonds, bia. 376pp. en diferentes épocas de su ciclo vegetativo. • Producer prices are highest in June, Belalcázar S., J.A. Valencia, M.I. Arcila & G. Cayón. Pp. 168-179 in Mejoramiento de la producción December and the first fortnight in 1994. Efecto de la defoliación selectiva durante del cultivo de plátano (C.S. Belalcázar, January. This coincides with the fes- la floración sobre el llenado de los frutos del O. Jaramillo, J.A. Valencia, M.I. Arcila, H. Mejía tivities at those dates, when demand clon de plátano Dominico Hartón, Musa AAB & H. García, eds). ICA-CORPOICA, Colombia. for cooked food, and especially Simmonds. Pp. 104-111 in Mejoramiento de la tamales tolimenses, is at its highest. producción del cultivo de plátano (C.S. Belalcázar, O. Jaramillo, J.A. Valencia, M.I. Arcila, • The time for cutting leaves depends H. Mejía & H. García, eds). ICA-CORPOICA, mainly on the time of year (rainy sea- Colombia. son or dry season). Thus, leaves can Belalcázar S., G. Cayón, G.O. Jaramillo & L.C. Cor- be cut every fortnight or even every tez. 1996. Tecnología del Eje Cafetero para la week during the rainy season but the siembra y explotación rentable del cultivo de plá- interval is three weeks in the dry sea- tano. Comité departamental de Cafeteros del son and may even be four weeks in Quindío. Tercer informe técnico 1994-1996. Re- the most extreme cases. gional 9 CORPOICA, CRECED, Armenia, Quindío. 130pp. Acknowledgements Belalcázar S. M.I. Arcila & J.A. Valencia. 1998. Re- The author thanks Antonio María cursos généticos y métodos de caracterización. Caicedo, engineer at Centro de Investi- Conservación y evaluación de la colección Co- The author is a researcher at CORPOICA, C.I. Na- gaciones Nataima for his collaboration lombiana de Musáceas. Pp. 1-21 in Seminario in- taima, Apartado postal 064, Espinal, Tolima, Co- in the statistical analysis of the data. ternacional sobre producción de plátano. Memo- lombia.

Physiology Studies on the leaves The evolution of photosynthesis, transpiration and chlorophyll during the development of leaves of plantain (Musa AAB Simmonds)

G. Cayón S. ration and horizontal position of the ciency, plant growth and environmental leaf (Yoshida 1972). The angle of inser- adaptation. Kumar et al. (1972) demon- tion is very important for crop produc- strated the existence of a chlorophyll he growth and development of a tivity as this governs the exposure of gradient in sugar cane running from the cultivated plant depends essen- the leaves to sunlight and hence the tip to the base of individual leaves and Ttially on the progressive increase more even distribution of light through also between leaves of different ages. of its leaf area, which enables it to the plant cover, enabling more efficient Photosynthesis varies greatly according make more effective use of solar energy photosynthetic activity at the mid and to the age of the plant. As a leaf devel- during photosynthesis. The interception lower levels of the plant (Cayón 1992). ops and the chloroplasts become orga- of solar radiation by the leaf surface is The chlorophyll present in all green nized, photosynthetic activity increases influenced by the size, shape, age, angle plants is one of the pigments most rapidly to a peak level attained when of insertion on the stem, vertical sepa- closely related to photosynthetic effi- growth of the lamina is complete and

12 INFOMUSA — Vol 10, N° 1 then gradually decreases during leaf ageing. 14 It was long believed that the rates of 12 photosynthesis of perennial crops were y = -0.0006 x2+ 0.0242 x + 8.4694 R2= 0.80 lower than those of herbaceous plants, 10 but recent research has demonstrated mol/m2/s) that many trees and shrubs, including µ 8 certain conifers, display maximum pho- 6 tosynthetic rates that are very close to those of C3 plants (Catsky et al. 1987). 4

The highest photosynthetic rate is at- Photosynthesis ( tained in most leaves when the lamina 2 has fully unrolled; the rate then de- 0 creases with age. This decrease in pho- 20 40 60 80 100 120 140 160 tosynthetic capacity is typical of leaves -2 of perennial and short-cycle plants (Sil- Leaf age (days) veira 1987). The aim of this work was to deter- 4000 mine the behaviour and intensity of gas 3500 y = -0.3246 x2 + 23.966 x + 3215.1 exchange and of the chlorophyll synthe- R2 = 0.86 sis and degradation processes during 3000 the development of plantain leaves. mol/m2/s) 2500 µ Material and methods 2000 The experiment was conducted at the 1500 Palmira Research centre in the Valle 1000 del Cauca department, at 3º31’N and ( Transpiration 76º19’W, elevation 1001 m asl, average 500 annual temperature 24ºC, average relative humidity 75% and annual precipita- 0 tion 1000 mm. These climatic condi- 0 20 40 60 80 100 120 140 160 tions are those of dry tropical forest Leaf age (days) (df-T). The experimental plot had clay loam soil at pH 6.8 and contained 2.9% 12 organic matter. The clone Dominico hartón was planted at 3.0 m between y = -0.0007 x2 + 0.065 x + 6.6622 10 rows and 2.0 m between plants along R2 = 0,68 the rows with one sucker per planting 8 hole and overall density of 1666 plants ha-1. A fully randomized experimental 6 protocol was used with three repetitions and six plants per repetition. 4 When the plants had emitted 16 leaves

(five months after planting), the chlorophyll (mg/g m.s.) Total youngest fully unrolled leaf (leaf 1) was 2 identified on each one and the net pho- 0 tosynthetic rate, transpiration and 0 20 40 60 80 100 120 140 160 chlorophyll were measured every 20 days from complete unrolling of the half- Leaf age (days) lamina (day 0) to total leaf senescence (day 140). The photosynthetic and tran- Figure 1. Evolution of photosynthesis, transpiration and chlorophyll concentration during Dominico spiration rates were measured in the hartón plantain leaf development. central part of the leaf using an LI-6200 portable photosynthesis system (Licor). Chlorophyll was measured using the ethanol extraction method (Winter- mans et al. 1965) on 1.3 cm2 leaf discs taken from the same central leaf sec- Absorption at 649 and 665 nm were The results were subjected to analy- tion as that used for the measurement then read using a Spectronic 21 spec- ses of variance, correlation and regres- of gas exchanges. Extraction was per- trophotometer and the data were used sion using the MSTAT-C statistical pro- formed by cold maceration of each leaf to calculate chlorophyll a (Cla), b (Clb) gram (Michigan State University). disc in a mortar containing 4.0 ml of a and total chlorophyll (Clt), using the - solution (ethanol 98% + MgCO3 0.5 g l following formulas: Results and discussion 1), transfer of the extract to a test tube Cla = [(13.7 x A665) - (5.76 x A649)] x V / DW It is seen in Figure 1 that the evolu- and washing the mortar with 4.0 ml so- Clb = [(25.8 x A649) - (7.6 x A665)] x V / DW tion of gas exchange rates (photosyn- lution to complete the final volume to V = final volume of ethanol extract thesis and transpiration) and that of 8.0 ml. The extract was separated by (8.0 ml) chlorophyll synthesis follow a qua- centrifugation at 3000 x g for 5 minutes. DW = leaf disc dry weight (g) dratic regression pattern during leaf

INFOMUSA — Vol 10, N° 1 13 Table 1. Correlation matrix for leaf age, photosynthesis, transpiration and total lated with photosynthesis activity, also chlorophyll concentration. decreases during leaf senescence, in- Variables Leaf age Photosynthesis Transpiration Total chlorophyll dicating that the decrease in photo- Leaf age 1.000 - 0.783 ** - 0.688 ** - 0.634 ** synthesis is caused mainly by the func- Photosynthesis - 1.000 0.771 ** 0.842 ** tional degradation of the Transpiration - - 1.000 0.538 * photosynthetic systems (Camp et al. Total chlorophyll - - - 1.000 1982, Holloway et al. 1983, Kura-Hotta ** significant (P<0.01) * significant (P<0.05). et al. 1987). The lower concentrations of chlorophyll and other active photosynthetic pigments can limit the leaf photosynthetic process. This reduces life from day 0 to day 140. The photo- cal activity of banana and plantain photosynthesis if the concentration synthetic rate is low during the initial leaves during the vegetative growth falls below the optimum threshold leaf development stage and increases phase. They observed the highest (Gabrielsen 1948). rapidly to maximum (12.22 µmol CO2 photosynthetic and transpiration Photosynthesis, transpiration and m-2 s-1) attained 20 days after leaf un- rates in the youngest leaves (leaves 2, chlorophyll concentration are in- rolling (DAU); it then decreases 3, 4 and 5) and drastic decreases in versely correlated with leaf age slightly, remaining fairly steady until the oldest leaves (leaves 6, 7, 8 and (P<0.001), showing that they depend 80 DAU, and finally decreases consid- 9) (Robinson and Bower 1988, on the ontogenesis of the leaf and de- erably until the death of the leaf Kallarackal et al. 1990, Eckstein and crease as the latter advances (Table 1). blades (140 DAU). The lower leaf pho- Robinson 1995, Cayón et al. 1998). Photosynthesis is directly correlated tosynthetic level from the youngest The evolution of the chlorophyll con- with transpiration and chlorophyll stage (0 DAU) is explained by the fact centration is similar to that of photo- content at all leaf development stages, that the photosynthetic and enzymatic synthesis and transpiration; maximum showing that photosynthesis is func- systems are not fully formed. Chloro- pigment concentrations are observed tionally related to transpiration and phyll synthesis is in the initial stages, between 20 and 40 DAU and the level depends on the chlorophyll concentra- pigment concentration is not suffi- then decreases to minimum at com- tion in the leaf blade. cient to capture the solar energy re- plete senescence. The chlorophyll con- The leaf insertion angle is an impor- quired for photosynthesis and the centration is low during the leaf un- tant parameter for plantain productiv- stomata have not gained their full rolling period because the leaf is not ity as this governs the exposure of the physiological capacity. It is sufficient fully exposed to sunlight, and this gov- leaves to sunlight and the distribution to observe the pale green colour after erns the synthesis and accumulation of of photosynthetically active radiation the unrolling of the ‘cigar’. The maxi- chlorophyll. The chlorophyll level in- (PAR) in the plant. Photosynthesis mum photosynthetic rate (12.2 µmol creases markedly when leaf unrolling is takes place in the various superposed -2 -1 CO2 m s ) of each newly formed leaf complete and then remains constant leaf strata that shade each other in is conserved for a comparatively short during the intermediate period of leaf such a way that the incident PAR is ab- period of time (20 days), after which it life before decreasing at the onset of sorbed through the strata, with a max- decreases slightly and remains at be- senescence. imum in the best-exposed leaves. Pho- -2 -1 tween 5.53 and 7.12 µmol CO2 m s The photosynthetic and chlorophyll tosynthesis is thus greater in leaves in for 60 days before reaching a mini- concentration rates are proportional the medium stratum; the lower leaves mum at total senescence (140 DAU). during leaf development, with peaks receive less PAR and have lower photo- The drastic decrease in the photosyn- on 20 DAU when the concentration of synthesis rates. Each leaf emitted thetic rate during leaf senescence re- pigment for photosynthesis appears to changes position during plant growth, sults in a negative carbon balance be optimal. The photosynthesis which means that its photosynthetic since leaf respiration is constant process decreases considerably with a activity will only be at maximum while throughout the plant development limiting chlorophyll concentration. On it is well exposed to PAR. As the re- process. this subject, Cayón et al. (1994) ob- sults of this study show that the mini- Leaf transpiration is also low at the served that the maximum plantain leaf mum photosynthetic rate of young beginning of development and then photosynthetic rate depended on the leaves is conserved for a relatively increases to a maximum on 40 DAU chlorophyll content and that the high- short period of time (20 days) and that and finally decreases as the leaf ages. est chlorophyll concentration was the rate then decreases considerably It can be seen that leaf transpiration found in the central part of the leaf when they are shaded by new leaves, continues until 60 DAU, that is to say blade. Although loss of chlorophyll is a these new young leaves probably dis- for a longer period than photosynthe- typical symptom observed during leaf play physiological compensation by at- sis and this probably accelerates age- senescence, its disappearance is taining their highest photosynthetic ing. Plantain is phyllotaxic and con- nonetheless lower than that of the rate immediately after the preceding tinuously emits new leaves that other photosynthetic compounds leaves. In addition, the fact that pho- change position during plant develop- (Friedrich and Huffaker 1980, Hol- tosynthesis stabilizes at between 5.53 -2 -1 ment and hence change their exposi- loway et al. 1983, Kura-Hotta et al. and 7.12 µmol CO2 m s for the sub- tion to sunlight, and eventually 1987, Makino et al. 1983). sequent 60 days of development may become partially shaded. This con- Studies conducted to explain the be an essential contribution to the tributes to the gradual decrease in photosynthesis reduction mechanism physiological processes of the plant. photosynthetic and transpiration during leaf senescence show that the Indeed, from the strict point of view of rates, with repercussions on gas ex- phenomenon is caused by changes in production, it is very important that changes in the plant. These results the concentration and kinetics of the the functional leaves should maintain are in agreement with those of differ- Rubisco enzyme (Evans 1986, Makino a moderate and constant photosyn- ent authors who have studied interac- et al. 1985). The activity of the elec- thetic activity over as long a period as tions between the age and physiologi- tron transport chain, directly corre- possible.

14 INFOMUSA — Vol 10, N° 1 References coordination of ribulose-bisphosphate-carboxyla- nescence. Quantitative analysis by carboxyla- Camp P.J., S.C. Huber, J.J. Burke & D.E. Moreland. tion and electron-transport capacities. Planta tion/oxygenation and regeneration of ribulose 1,5 1982. Biochemical changes that occur during se- 167: 351-358. bisphosphate. Planta 166: 414-420. nescence of wheat leaves. I. Basis for the reduc- Friedrich J.W. & R.C. Huffaker. 1980. Photosynthe- Robinson J.C. & J.P. Bower. 1988. Transpiration tion of photosynthesis. Plant Physiol. 70: 1641- sis, leaf resistances and ribulose-1,5-bisphos- from banana leaves in the subtropics in response 1646. phate carboxylase degradation in senescing bar- to diurnal and seasonal factors and high evapora- Catsky J. & Z. Sestadk. 1987. Measurement of leaf ley leaves. Plant Physiol. 65: 1103-1107. tive demand. Scientia Horticulturae 37: 129-143. photosynthetic rate in trees. Pp. 29-46 in Tree Gabrielsen E.K. 1948. Effects of different chloro- Silveira M. 1987. Fotossintese no dossel das plantas crop physiology (M.R. Sethuraj & A.S. Raghaven- phyll concentrations on photosynthesis foliage cultivadas. Pp. 13-48 in Ecofisiologia da Produ- dra, eds.). Else, Amsterdam. leaves. Physiologia Plantarum 1: 5-37. çao Agricola. Associaçao Brasileira para Pes- Cayón G. 1992. Fotosíntesis y productividad de cul- Holloway P.J., D.J. Maclean & K.J. Scott. 1983. Rate- quisa da Potassa e do Fosfato, Piraciçaba, Brasil. tivos. Revista Comalfi 19 (2): 23-31. limiting steps of electron transport in chloro- Wintermans J.F.G.M. & A. De Mots. 1965. Spectro- Cayón G., J.E. Lozada & S. Belalcázar. 1994. Estu- plasts during ontogeny and senescence of barley. photometric characteristics of chlorophylls a and dios comparativos sobre la actividad fotosinté- Plant Physiol. 72: 795-801. b and their pheophytins in ethanol. Biochem. tica de clones de plátano (Musa AAB, ABB Sim- Kallarackal J., Milburn J.A. & D.A. Baker. 1990. Biophys. Acta 109: 448-453. monds) en Colombia. Pp. 549-558 in Memorias X Water relation of the Banana. III. Effects of Yoshida S. 1972. Physiological aspects of grain yield. reunión de ACORBAT. Octubre 1991, Tabasco, controlled water stress on water potential, trans- Ann. Rev. of Plant Physiol. 23: 437-464. México. (M.A. Contreras, J.A. Guzmán & L.R. piration, photosynthesis and leaf growth. Austra- Carrasco, eds). CORBANA, San José, Costa Rica. lian Journal of Plant Physiology 17: 79-90. Cayón G., S. Belalcázar & J.E. Lozada. 1998. Ecofi- Kumar A., Acin N.M. & A.G. Alexander 1972. Rela- siología del plátano (Musa AAB Simmonds). Pp. tionships of chlorophyll and enzyme gradients to 221-236 in Memorias. Seminario internacional sucrose contents leaves. Ann. Rept. Agric. Exp. sobre producción de plátano. (M.J. Giraldo, S.L. Sta. Univ. P.R. (1970-1971). Belalcázar, D.G. Cayón & R.G. Botero, eds). Ar- Kura-Hotta M., K. Satoh & S. Katoh. 1987. Relation- menia, Quindío, Colombia. CORPOICA, Universi- ship between photosynthesis and cholorophyll dad del Quindío, Comitecafé Quindío, Sena, IN- content during leaf senescence of rice seedlings. IBAP. Plant Cell Physiol. 28: 1321-1329. Eckstein K. & J.C. Robinson. 1995. Physiological Makino A., T. Mae & K. Ohira. 1983. Photosynthesis responses of banana (Musa AAA, Cavendish sub- and ribulose 1,5 bisphosphate-carboxylase in rice group) in the subtropics. I. Influence of internal leaves. Changes in photosynthesis and enzymes plant factors on gas exchange of banana leaves. involved in carbon assimilation from leaf deve- Journal of Horticultural Science 70: 147-156. lopment through senescence. Plant Physiol:

Evans J.R. 1986. The relationship between CO2-limi- 1002-1007. The author works at the Programa de Investigación en ted photosynthetic rate and ribulose-1,5 bisphos- Makino A., T. Mae & K. Ohira. 1985. Photosynthesis Plátano, Corporación Colombiana de Investigación Agro- phate-carboxylase content in two nuclearcyto- and ribulose 1,5 bisphosphate-carboxylase/oxyge- pecuaria, CORPOICA, Apartado aéreo 1807, Armenia, plasm substitution lines of wheat and nase in rice leaves from emergence through se- Colombia. E-mail: [email protected]

Physiology Root/shoot relationship Estimation of root development from shoot traits in plantain and banana (Musa spp.)

G. Blomme, R. Swennen, estimated from the number of leaves. shoot characteristics, across a wide A. Tenkouano, R. Ortiz Henderson et al. (1983) found that the range of Musa genotypes. and D. Vuylsteke extent of coarse root branching was very regular for Sitka spruce and could Materials and methods be estimated using the aboveground This study was carried out at the IITA he root system is the link be- stem diameter. Smith (1964) reported High Rainfall station at Onne in south- tween the plant and the soil. It is that root spread of several tree species eastern Nigeria. Its soil is an ultisol de- Tresponsible for the absorption of could be estimated from aboveground rived from coastal sediments, well water and nutrients, anchorage, syn- measurements. In the case of banana, drained but poor in nutrients and with thesis of some plant hormones and Swennen (1984), and Blomme and a pH of 4.3 in 1:1 H2O. The average an- storage (De Langhe et al. 1983, Martin Ortiz (1996) observed positive correla- nual rainfall is 2400 mm distributed Prével 1987, Stover and Simmonds tions between root system develop- monomodally from February to Novem- 1987, Lahav and Turner 1989, Price ment and aerial growth characteris- ber. Details of the site were described 1995). Root system development and tics, while Gousseland (1983) by Ortiz et al. (1997). Twenty-seven shoot growth are highly related estimated the number of cord roots of genotypes representing the various (Pearsall 1927, Broschat 1998, Fort the ‘Giant Cavendish’ dessert banana Musa genomic and ploidy groups were and Shaw 1998). Russell (1977) men- from the leaf area. The objective of assessed in this study. The planting ma- tioned that nodal root development in this study was to develop a method for terial was obtained through meristem winter wheat and pearl millet could be estimation of root development from culture using the methods of Vuylsteke

INFOMUSA — Vol 10, N° 1 15 Table 1. Correlation coefficients (P<0.05) between aerial growth and root system best shoot indicators of root growth characteristics at 20 WAP (weeks after planting) were leaf area, pseudostem circumfer- Trait LA PH PC LS ence and length of the tallest sucker DR 0.72*** 0.65*** 0.65*** -0.09 (Table 2). NR 0.46* 0.41* 0.29 0.16 These models suggest that reduced LR 0.64*** 0.54** 0.46* 0.08 leaf area, as may be caused by black AD 0.47* 0.51** 0.70*** -0.38* Sigatoka, will adversely affect root de- TL 0.41* 0.25 0.01 0.49* velopment. Conversely, increased leaf TD 0.65*** 0.53** 0.38 0.25 area as a result of fertilizer application LA: leaf area (cm2), PH: height of the plant (cm), PC: plant circumference (cm), LS: length of the tallest sucker (cm), DR: root dry weight (g), NR: number of cord roots, LR: cord root length (cm), AD: average diameter at the base of the cord roots (mm), TL: would stimulate root development. The total length of the cord roots of the mat (cm), TD: total dry weight of the roots of the mat (g). pseudostem is made up of leaf sheathes *, **, *** Significant at P<0.05, 0.01 and 0.001, respectively. and hence reflects the number of leaves and plant vigour. Plant pseudostem circumference will thus reflect shoot Table 2. Regression models to predict root system characteristics at 20 WAP using growth and is an important determi- aerial growth characteristics and ploidy level as independent variables. nant of root vigour in the regression models. The size of the tallest sucker Trait contributed positively to the extent of ^ 2 Trait LA PC LS PL R the mat root system. Most suckers ob- B+z10>DR 0.001628*** 0.596934** 0.93 served on 20 week-old plants were NR 0.001459*** 1.255633*** 0.93 peepers (i.e. small sucker with scale LR 0.066704*** 23.476717** 0.94 leaves) or sword suckers (i.e. larger AD 0.093835*** 0.681434*** 0.97 sucker with lanceolate leaves). These TL 0.099478*** 14.69139*** 0.92 suckers had their own root system, con- TD 0.002066*** 0.426590 0.171415* 0.93 for abbreviations see Table 1; PL: ploidy level. firming observations by Robin and ^: independent variables. Champion (1962) and Beugnon and *, **, *** Significant at P<0.05, 0.01 and 0.001, respectively. Champion (1966) who reported that sword suckers of the dessert banana (1989, 1998). The plantlets were accli- roots (NR), average diameter at the ‘Poyo’ had a well-developed root system. matized for six weeks in a greenhouse base of the cord roots (AD, mm), dry The observed positive effect of ploidy on nursery (Vuylsteke and Talengera 1998, weight of the roots (DR, g), length of cord root diameter confirms earlier ob- Vuylsteke 1998), prior to transplanta- the cord roots (LR, cm), total dry servations by Monnet and Charpentier tion in the field in June 1996. The ex- weight of the whole mat (i.e. plant crop (1965). perimental layout was a randomized and suckers) root system (TD, g) and Shoot to root ratios depend on the de- complete block design with two replica- total length of the cord roots of the velopmental stage of a plant (Brouwer tions of two plants per genotype. whole mat (TL, cm) were measured. 1966). In the case of banana, Gousse- The trial sites, which had been under The average diameter of the cord roots land (1983) estimated banana cord root grass fallow for eight years, were manu- was measured with a Vernier calliper, number from leaf area and reported an ally prepared with minimum soil distur- while the length of the cord roots was effect of plant developmental phase on bance. Planting was done using a spac- estimated according to the method of the accuracy of the regression model. ing of 2 m x 2 m to avoid overlapping of Newman (1966) and Tennant (1975). The author reported an underestima- adjacent root systems. The experimen- Statistical analysis was carried out on tion of the number of cord roots during tal area was treated with the nemati- the data set comprising 27 genotypes, the early vegetative phase. Hence, re- cide Nemacur (a.i. fenamiphos) at a using the SAS statistical package (SAS gression models will have to be fine- rate of 15 g per plant (three treatments 1989). Relationships between aerial tuned according to the developmental year-1) to reduce the nematode infesta- growth and root system characteristics phase of a plant. tion. Plants were fertilized at 300 N and were evaluated using correlation analy- In addition, shoot-root ratios are 450 K (kg·ha-1·year-1) split over six sis. In addition, multiple regression highly influenced by environmental equal applications during the rainy sea- analysis using stepwise selection was conditions (Brouwer and De Wit 1969, son (i.e. February-November). The carried out. The dependent variables, Squire 1993, Martinez Garnica 1997, fungicide Bayfidan (a.i. triadimenol) i.e. the root system characteristics, McMichael and Burke 1998). Therefore, was applied three times per year at a were regressed on shoot growth charac- fine-tuning of these models is needed rate of 3.6 ml per plant to control the teristics and ploidy level (PL). Both when growing plants under different leaf spot disease black Sigatoka correlation and regression analysis environmental conditions. (Mycosphaerella fijiensis Morelet). were carried out on 27 genotypes. This study shows that Musa root sys- Shoot and root traits were assessed tem development can be estimated during the mid-vegetative growth (i.e. Results and discussion from shoot growth characteristics. This 20 week-old plants). Shoot growth char- Significant correlations between aerial provides a means of relating above- acteristics included plant height (PH, growth and root system characteristics ground development to root growth, cm), circumference of the pseudostem were found during the vegetative devel- which may prove useful for non-destruc- at soil level (PC, cm) and leaf area (LA, opment (Table 1) and confirmed earlier tive assessment of root development. cm2). Leaf length and leaf widest width reports (Beugnon and Champion 1966, were measured and LA was calculated Gousseland 1983, Swennen 1984, Lavi- Acknowledgements according to Obiefuna and Ndubizu gne 1987, Blomme and Ortiz 1996). Financial support by VVOB (Vlaamse (1979). In addition, length of the tallest Regression analysis produced several Vereniging voor Ontwikkelingssamen- sucker (LS, cm) was measured. The equations, which attributed at least werking en Technische Bijstand, Flem- root system was completely dug out and 90% of the variation in root growth to ish Association for Development Coop- number of adventitious roots or cord variation in shoot development. The eration and Technical Assistance) and

16 INFOMUSA — Vol 10, N° 1 the Directorate General for Interna- Lavigne C. 1987. Contribution à l’étude du système Squire G.R. 1993. The leaf canopy and root system. tional Cooperation (DGIC, Belgium) is racinaire du bananier. Mise au point de rhizo- Pp. 33-70 in The Physiology of Tropical Crop Pro- gratefully acknowledged. The authors trons et premiers résultats. Fruits 42: 265-271. duction (G.R. Squire). CAB International, Wal- thank Ms Lynda Onyeukwu for helping Martinez Garnica A. 1997. Mineral nutrient defi- lingford, UK. with the data collection. ciency in plantain: Symptoms and disorders Stover R.H. & N.W. Simmonds. 1987. Bananas. 3rd under experimental and field conditions. Hohen- ed. Longman, New York. 180pp. heim Tropical Agricultural Series 4: 47-63. Swennen R. 1984. A physiological study of the suc- References Martin-Prével P. 1987. Banana. Pp. 637-670 in Plant kering behaviour in plantain (Musa cv. AAB). Beugnon M. & J. Champion. 1966. Etude sur les analysis as a guide to the nutrient requirements PhD Thesis. Dissertationes de Agricultura n°132, racines du bananier. Fruits 21: 309-327. of temperate and tropical crops (P. Martin-Pré- Faculty of Agriculture, Katholieke Universiteit Blomme G. & R. Ortiz. 1996. Preliminary assess- vel, J. Gagnard and P. Gautier, eds.). Lavoisier, Leuven. 180pp. ment of root systems in Musa (Abstract sub- Paris. Tennant D. 1975. A test of a modified line intersect mitted for presentation at the International McMichael B.L. & J.J. Burke. 1998. Soil tempera- method of estimating root length. Journal of Eco- Conference on Banana and Plantain for ture and root growth. HortScience 33(6): 947- logy 63: 995-1001. Africa, 14-18 Oct. 1996, Kampala). MusAfrica 950. Vuylsteke D. 1989. Shoot-tip culture for the propa- 10: 16. Monnet J. & J.M. Charpentier. 1965. Le diamètre gation, conservation, and exchange of Musa Broschat T.K. 1998. Root and shoot growth patterns des racines adventives primaires des bananiers germplasm. Practical manuals for handling crop in four palm species and their relationships with en fonction de leur degré de polyploidie. Fruits germplasm in vitro 2. International Board for air and soil temperatures. HortScience . 20: 171-173. Plant Genetic Resources, Rome, Italy. 56pp. Brouwer R. 1966. Root growth of grasses and ce- Newman E.I. 1966. A method for estimating the Vuylsteke D. 1998. Shoot-tip culture for the propa- reals. Pp. 153-166 in The growth of cereals and total length of root in a sample. Journal of Ap- gation, conservation, and distribution of Musa grasses (F.L. Milthorpe and J.D. Ivins, eds.). But- plied Ecology 3: 139-145. germplasm. International Institute of Tropical terworths, London. Obiefuna J.C. & T.O.C. Ndubizu. 1979. Estimating Agriculture, Ibadan, Nigeria. 82pp. Brouwer R. & C.T. De Wit. 1969. A simulation model leaf area of plantain. Scientia Horticulturae Vuylsteke D. & D. Talengera. 1998. Postflask mana- of plant growth with special attention to root 11: 31-36. gement of micropropagated bananas and plan- growth and its consequences. Pp. 224-244 in Ortiz R., P.D. Austin & D. Vuylsteke. 1997. IITA high tains. A manual on how to handle tissue-cultured Root Growth (W.J. Whittington, ed.). Butter- rainfall station: Twenty years of research for sus- banana and plantain plants. International Insti- worths, London. tainable agriculture in the West African Humid tute of Tropical Agriculture, Ibadan, Nigeria. De Langhe E., R. Swennen & G.F. Wilson. 1983. As- Forest. HortScience 32(6):969-972. 15pp. pects hormonaux du rejetonnage des bananiers Pearsall W.H. 1927. Growth studies. VI. On the rela- plantains. Fruits 38(4): 318-325. tive size of plant organs. Annals of Botany Fort S.B. & D.V. Shaw. 1998. Phenotypic correla- 41: 449-556. tions between root and shoot traits of strawberry Price N.S. 1995. Banana morphology. Part 1: Roots in fumigated and non-fumigated soils. HortS- and rhizomes. Pp. 190-205 in Bananas and plan- cience 33(2):222-224. tains (S. Gowen, ed.). Chapman & Hall, London. Gousseland J. 1983. Etude de l’enracinement et de Robin J. & J. Champion. 1962. Etudes des émissions l’émission racinaire du bananier ‘Giant Caven- des racines de la variété du bananier Poyo. G. Blomme*, A. Tenkouano and R. Ortiz work at dish’ (Musa acuminata AAA, sous-groupe Caven- Fruits 17: 93-94. the Crop Improvement Division, International Institute dish) dans les andosols de la Guadeloupe. Fruits Russell R.S. 1977. Plant root systems: their function of Tropical Agriculture (IITA), c/o L.W. Lambourn & 38: 611-623. and interaction with the soil. McGraw Hill, Co., Carolyn House, 26 Dingwall Road, Croydon CR9 Henderson R., E.D. Ford, E. Renshaw & J.D. Deans. London. 3EE, United Kingdom. *Guy Blomme is currently working in Kampala, Uganda as the INIBAP Assistant Re- 1983. Morphology of the structural root system of SAS. 1989. SAS/STAT user’s guide. Version 6, 4th gional Coordinator for East and Southern Africa. Sitka spruce: 1. Analysis and quantitative des- edition, Vol. 1. SAS Institute Inc., Cary, N.C. R. Swennen works at the Laboratory of Tropical cription. Forestry 56: 121-135. Smith J.H.G. 1964. Root spread can be estimated Crop Improvement, K.U. Leuven, Kasteelpark Aren- Lahav E., and D.W. Turner. 1989. Banana Nutrition. from crown width of Douglas fir, lodgepole pine berg 13, 3001 Leuven, Belgium. D. Vuylsteke (IITA) International Potash Institute, Berne, and other British Columbia tree species. Forestry died in a tragic air flight accident on 30 January Switzerland. Chronicle 40: 456-473. 2000.

Diseases Bacterial wilt Evaluation of cultural, chemical and biological control of vascular rot and wilt in plantain (Musa AAB Simmonds)

L.E. Gómez-Caicedo, E. Echeverry N. eastern part of Tolima department plantain (Musa ABB) but the disease and R. González S. (Colombia). The appearance of a dis- has recently attacked the ‘Dominico ease was reported at Icononzo in the hartón’ plantain clone (Musa AAB Sim- second half of 1996 and characteriza- monds), causing serious losses for anana and plantain are grown on tion of the symptoms showed that it was smallholders. some 25 000 hectares of land, a new problem in the region. The symp- Today, mechanized plantain growing Bmainly in the coffee zone, in the toms were first observed on ‘Cachaco’ in the study zone gives an average yield

INFOMUSA — Vol 10, N° 1 17 of 1000 bunches per hectare, represent- were not strongly developed. These bac- ing a commercial value of close to 3 mil- teria were antagonistic in vitro to lion pesos (US$1500). The disease isolates of E. chrysanthemi and could reduce production by nearly 70% E. carotovora. The morphological, phys- and cause serious problems in zones iological and biochemical characteris- with a rural economy (Echeverry, un- tics of the strains showed that they be- published data). longed to the genus Pseudomonas. The symptoms of the disease consist Belalcázar et al. (1991) pointed out of chlorosis in the lower leaves followed that the bacterium E. chrysanthemi by wilting at the petiole and then as- p.v. paradisiaca Victoria and Barros is cending wilt that finally affects all the endemic in the regions in which leaves of the plant (Figure 1). A cross- Musaceae are grown and that attacks section of an infected pseudostem are enhanced by drought and nutri- about 1 m above ground level reveals tional deficiencies in plantations. watery rot and an unpleasant smell Schneider’s observations (1991) of (Figure 2). In addition, the colour of the relation between mineral nutrition the inner leaf sheaths ranges from and host condition showed that all K, brown to dark brown (Figure 3). Ca and Mg applications limited the de- Unlike the symptoms of the pseu- velopment of certain types of wilt, and dostem soft rot observed by Guzmán especially those caused by Fusarium and Sandoval (1996) in FHIA-01 and sp. In the absence of KCl, the exudate FHIA-02 hybrids, the infection de- produced causes greater reduction of scribed at Icononzo moves from the rhi- Figure 1. Vascular rot is characterized by sugars and organic acids. KCl reduces zome towards the upper part of the chlorosis of the lower leaves followed by their the infection level. Mineral nutrition plant. wilting at petiole level and finally general therefore has a distinct effect on the upward wilting that affects all the leaves In a study on pseudostem soft rot in (photo: L.E. Gómez-Caicedo). nature of the exudate and on infection. FHIA hybrids with similar symptoms to Trichoderma can inhibit the those observed in ‘Dominico hartón’ pathogen via its antibiotic substances plantain, Guzmán and Sandoval (1996) or by breaking down bacterial cell walls isolated the Erwinia carotovora bac- by means of enzymes such as quiti- terium from plant tissues. The soft or nases, ß-1,3-glucanases, proteases, watery rots caused by Erwinia spp. mannases and hydrolases (Limón et al. bacteria are often observed in 1999). Musaceae in Latin America (Stover The relative importance of these two 1972). mechanisms in the antagonist process Stover (1972) noted that the bacteria depends specifically on host-pathogen E. chrysanthemi and E. carotovora af- interactions (Limón et al. 1999). Never- fect the corm and pseudostem of both theless, the combination of the action banana and plantain and observed that of hydrolytic enzymes and antibiotic cultivars of the Cavendish subgroup are substances in Trichoderma has demon- susceptible to Erwinia sp. but that strated its antifungal synergy (Schirm- the AAB and ABB genotypes are more böck et al. 1994). tolerant. Figure 2. A cross section of pseudostem infected Rivera and Ezavin (1989) observed by vascular rot reveals watery rot with yellow Materials and methods pathology characterized by corm dis- exudate, a foetid odour and an unpleasant The study was performed from 1997 to ease in Musa acuminata cultivars appearance characteristic of bacterial attack. 1999 in Icononzo in the Piedecuesta re- (AAA) in different banana zones in (photo: L.E. Gómez-Caicedo). gion in Tolima Department in Colombia. Venezuela. The pathogen was found to The experiment was conducted at San be the bacterium E. chrysanthemi Isidro at an elevation of 1380 m, with an- Burk. et al. nual average precipitation of 1500 to Cedeño et al. (1990) reported that 1700 mm and 80% average relative hu- the bacterium E. carotovora subsp. midity. The soil is slightly acid clay loam atroseptica is the pathogen of soft with a moderate percentage of organic pseudostem rot in ‘Hartón’ plantain matter and little potassium. (Musa AAB) in the southern Maracaibo Samples for laboratory analysis and lake area. identification of the pathogen were taken In his list of the main diseases of from the inner part of pseudostem of Musaceae crops in Zulia State in Dominico hartón plantain (Musa cv. Venezuela, Urdaneta (1994) mentions AAB) with symptoms of infection. E. carotovora as the causal agent of The samples were first washed in run- plantain pseudostem rot. ning water and then cut into small 2 cm Jiménez et al. (1994) reported that lengths and then disinfected with E. chrysanthemi Burk. et al. is the sodium hypochlorite 2.5% for 3 minutes causal agent of plantain corm necrosis. under constant agitation. They were The same authors isolated three strains washed in sterile distilled water to re- of bacteria in the rhizosphere of appar- move residues of hypochlorite. Figure 3. The inner leaf sheaths display colour ently healthy plantains from a field in ranging from brown to dark brown with yellow Once disinfected, the samples are which the symptoms of corm necrosis exudate (photo: L.E. Gómez-Caicedo). macerated in a mortar containing 1 ml

18 INFOMUSA — Vol 10, N° 1 Table 1. Summary of the results of the morphological and physicochemical tests and multiplied in the laboratory performed to characterize the bacterium isolated from pseudostems of ‘Dominico on the selective medium proposed hartón’ plantain (AAB). by Elad et al. (Chet 1987). Doses Test Result of 50 g/plant were placed in the Gram - soil at four points around the KOH 3% to confirm Gram test + pseudostem; odour foetid – control (T4). No substances were colour cream applied. consistency buttery Congo red rod-shaped Results and discussion fluorescence on King-B - catalase + Laboratory results levan - Morphological and physicochemical O-F test (Hugh-Leifson) + tests were performed to determine the Hydrolysis of gelatine + causal agent of vascular rot. The mor- NaCl 3% + phological Gram test revealed pink NaCl 4% + bacilli characteristic of Gram-negative tetracycline + bacteria. streptomycin + Congo red staining was also per- penicillin - formed to observe the rod-shaped bac- genus Erwinia terial cells. + = positive; - = negative. To confirm the Gram stain test, a fragment of bacterial growth was placed on a slide and a drop of KOH 3% was added. The formation of a vis- sterile distilled water. LB (Luria- the specified doses: urea (46%) cous, mucilaginous suspension was ob- Bertani) medium (yeast extract 5 g/l, 150 g/plant, KCl (60% K2O) served. This positive reaction con- tryptophan 10 g/l, NaCl 10 g/l, agar 200 g/plant, DAP (diammonium firmed that the bacterium is 20 g/l, pH 5.5-6.0) was added to 50 µl phosphate: P2O5 48%, N 18%) Gram-negative. of this macerated preparation. The 66 g/plant and “Micronfos” Among the physicochemical tests per- Petri dishes were then placed in an (Microfertiza,Colombia)200 g/plant; formed, growth on King-B medium to incubator (Precision Scientific Inc.) dolomitic limestone was applied characterize fluorescent bacteria gave a and kept at 28°C for 48 hours. The fol- at 300 g/plant to correct the pH; negative result. The catalase test con- lowing tests were performed to char- – biological treatment (T3). sisting of emulsifying a portion of bacte- acterize the pathogen: Gram stain, Kasumin 2% [kasugamycin: 3-0-(2- rial growth with hydrogen peroxide confirmation of Gram by KOH 3%, amino-4(1-carboxyformidoyle) (H2O2) 10% gave a positive result. The catalase, hydrolysis of gelatine, levan, amino 2, 3, 4, 6 tetra oxy-alpha-D- gelatine test was positive, indicating the King-B, OF test (Hugh-Leifson), toler- arabino hexapyranosyl)inositol] presence of proteolytic enzymes in the ance to NaCl 3% and 4%, antibiogram at 1 cc per L water was applied bacterium. The oxidation-fermentation with tetracycline, streptomycin and around the pseudostem; test (OF) on Hugh and Leifson medium penicillin. – control (T4). No substances were was positive and detected production of Three plots were marked out in a applied. acids by oxidation in aerobiosis. The 2600-m2 commercial holding planted • Phase II, from September 1998 to growth test on nutrient gelose with with 29-month Dominico hartón plan- February 1999 (6 months). Observa- NaCl 3% and 4% was positive, with the tain displaying symptoms of vascular tion of the results was followed by colonies growing normally. wilt; three planting layouts were used: modification of the three treatments Reaction to the antibiotics tetracy- 5, 4 and 3 metres between rows and applied in Phase I. The following cline, streptomycin and penicillin was 2.5 metres between plants. Four sub- treatments were applied and evalu- tested using antibiotic assay medium plots of 5 plants each with three repeti- ated in Phase II: No. 5 , pH 8.0 ± 0.1 (meat extract tions were laid out for each planting – chemical treatment (T1). Injec- 1.5 g/l, yeast extract 3.5 g/l, meat pep- distance. tion using a plastic syringe with a tone 6.0 g/l, agar-agar 15.0 g/l). Bacter- Four treatments were applied to each hypodermic needle of 5 cc Van- ial growth in saline solution (1 ml) was subplot. odine in four positions in the spread on the medium with a “rake” The experiment was conducted in pseudostem of each plant at 1 m type glass spreader before the discs of two phases: above ground level; antibiotics were placed. The reaction • Phase I, from November 1997 to Au- – cultural treatment (T2). Solely was positive for tetracycline and strep- gust 1998 (8 months), during which the application of potassium in tomycin and negative for penicillin. the following treatments were ap- KCl form at 200 g/plant every This means that the bacterium was very plied and evaluated: 30 days. Application was per- sensitive to the first two antibiotics, as – chemical treatment (T1). Monthly formed at three points around the shown by the transparent halo free of spraying of Vanodine 5 cc per l plant 50 cm from the base of the bacterial growth around the two discs water (composition: c/100 ml: sur- pseudostem; concerned. factant iodine compound; 2.5% – biological treatment (T3). At the Analysis of the results of these tests available iodine. Pfizer brand) beginning of the second vegeta- performed on bacterial samples from around the pseudostem of each tive cycle, a single inoculation of the pseudostems of plantains infected plant; the fungus Trichoderma spp., by bacterial rot showed that the – cultural treatment (T2). Applica- with the strain being isolated colonies consisted of Erwinia sp. tion of the following fertilizers at from soils from infected plants (Table 1).

INFOMUSA — Vol 10, N° 1 19 Field results Table 2. Comparison of the average numbers of healthy and wilted leaves per plant It is important to note that there are no in ‘Dominico hartón’ plantain with three planting distances. Icononzo (Tol.). Phase I, significant differences between average 1997-1998; Phase II, 1998-1999. numbers of healthy leaves and average Planting Average number of Average number of numbers of wilted leaves in the main distances healthy leaves per plant wilted leaves per plant Phase I plots. This means that planta- Phase I Phase II Phase I Phase II tion densities are not a factor in en- 5 m x 2.5 m 3.1 A* 8.0 A* 0.77 A* 1.4 AB* hancing the upward wilting of leaves on 4 m x 2.5 m 2.3 A 7.3 A 0.70 A 1.6 A the plants (Table 2). The same trend 3 m x 2.5 m 3.1 A 8.0 A 0.82 A 1.2 AB with regard to average healthy leaves * no significant difference between figures in the same column marked with the same letter (P = 0.05). was observed in the main plots during Phase II. In contrast, significant differ- Table 3. The effect of four treatments on the average numbers of healthy and ences were observed in the average wilted leaves per plant in ‘Dominico hartón’ plantain. Icononzo (Tol.). Phase I, 1997- numbers of wilted leaves, as shown in 1998; Phase II, 1998-1999. Table 2. Treatments Average number of Average number of Although Phases I and II are statisti- healthy leaves per plant wilted leaves per plant cally similar, there is a marked differ- Phase I Phase II Phase I Phase II ence in the average numbers of healthy Chemical 3.0 AB* 7.9 AB* 0.81 A* 1.70 AB* and wilted leaves per plant. Whereas an Cultural 3.8 B 8.6 A 0.79 A 1.07 B average of 2.84 healthy leaves was ob- Biological 2.6 AB 7.7 B 0.78 A 1.61 A served in Phase I, the figure attained Control 2.0 B 6.9 C 0.67 A 1.33 AB 7.77 in Phase II. The same applies to * no significant difference between figures in the same column marked with the same letter (P = 0.05). wilted leaves with 0.76 in Phase I and 1.40 in Phase II. Table 4. Total number and average weight of bunches of ‘Dominico hartón’ This may be explained by the change plantain harvested per treatment during Phase I, 1998, and Phase II, 1999. Icononzo in fertilization from urea + DAP + “Mi- (Tol.). cronfos” to KCl alone. On this subject, Treatment Number of bunches harvested Average weight (kg) according to Jacob et al. (1961), the nu- Phase I Phase II Phase I Phase II trients taken up by plantain include cultural 33 30 15.2 15.8 very large amounts of potassium. chemical 19 10 12.8 14.1 The same author observed that since biological 19 11 11.8 12.6 it is an extremely potassium-hungry control 7 5 10.6 12.1 plant, certain considerations concern- ing the application of other nutrients such as Ca and Mg must be taken seri- than in Phase I leaves. The fungus This confirms Machado’s observation ously. Indeed, it has been demonstrated Trichoderma sp. has a positive effect quoted by Jacob et al. (1961) that the that excess potassium causes the physi- on weight, size and leaf and flower pro- greater part of potassium absorption ological disorder referred to as “blue duction (Chet 1987). (84%) takes place during the fruit for- disease” and yellow pulp, affecting fruit On this subject, Kleifel et al., quoted mation period. The same author calcu- quality. by Chet (1987), observed earlier germi- lated that a plantation of 1333 plants It is also important to note that the nation and increased leaf length, width per hectare absorbed approximately average number of wilted leaves was and dry weight in melon, tomato, cu- 3493 kg potassium in 14 months. lower in the control than in the other cumber, radishes and bean plants. There were no significant differ- treatments. This may be explained by It was observed that the cultural ences between the chemical and bio- the fact that the control plants did not treatment gave the best results in both logical treatments for the same “har- have a normal number of leaves, that Phase I and Phase II for the number vested bunch weight” variable but leaf emission was small and that and average weight of commercial there were differences with respect to practically all the leaves were infected bunches harvested (Table 4). the control. (Table 3). The same phenomenon was There were no significant differences The chemical and biological treat- observed in the response to Phase II in commercial bunch weights in the ments displayed very similar “harvested treatments. The cultural control treat- main plots during each phase of the ex- bunch weight” results in Phases 1 and ment was better than the biological and periment but the differences between II of the experiment. However, several control treatments. However, it should subplots were very highly significant. deviations might result from the num- be stressed that there is a difference of This demonstrates the effectiveness of ber of functional leaves at flower emis- 4.92 in the average number of healthy the treatments, and especially the cul- sion, a determinant parameter for fruit leaves in the two phases, that is to say tural treatment in the provision of nu- weight and quality (Belalcázar et al. that there were more healthy leaves in trients via fertilization. 1991). Phase II than in Phase I (Table 3). Furthermore, no significant differ- Various hypotheses might account for ences were found in the interaction be- Conclusions these differences in behaviour. The tween planting differences and treat- The causal agent of rot and vascular most acceptable concerns the synergy ments (PP*SP), as is shown by the wilt of plantain is the bacterium between the presence of the fungus results of analysis of variance in Table 5. Erwinia, and probably the species Trichoderma sp. and the ability of the The analysis shows that the cultural E. carotovora. plant to absorb nutrients from the soil. treatment displays significant differ- Planting densities have no effect on This was verified by observation of the ences at 5% in comparison with the the presence or not of vascular rot in leaf size and colour in ‘Dominico hartón’ chemical and biological treatments and the plantain crops. plantain; leaf size and the intensity of the control for the “harvested bunch Potassium-rich fertilization and the green were greater in Phase II leaves weight” variable. presence of the fungus Trichoderma sp.

20 INFOMUSA — Vol 10, N° 1 Table 5. Analysis of variance of the “harvested bunch weight” variable of Sait G. 1999. Trichoderma. A promising bio-fungi- ‘Dominico hartón’ plantain in Phase I, 1998, and Phase II, 1998-1999. Icononzo (Tol.). cide. Nutri-Tech Solutions (NTS), New Zealand. Phase I Phase II 4pp. Variation Degrees of Root mean P – F Degrees of Root mean P – F Schirmböck M., M. Lorito, M., Y. Wang, C.K. Hayes, freedom square freedom square I. Arisan-Atac, F. Scala, G. Harman & C. Kubicek. PP (plantation distance) 2 53.08 0.8992 ns 2 332.111 0.53 ns 1994. Parallel formation and synergism of hydro- SP (treatments) 3 3183.064 0.0001 ** 3 444.444 0.0001 ** lytic enzymes and peptaibol antibiotics, molecu- PP x SP 6 134.91 0.687 ns 6 235.37 0.376 ns lar mechanisms involved in the antagonistic ac- ** Highly significant (P = 0.01); ns = not significant. tion of Trichoderma harzianum against phytopathogenic fungi. Appl. Environ. Microbiol. 60: 4364-4370. Schneider R.W. 1991. Influence of mineral nutrition on Fusarium wilt: a proposed mechanism invol- help to maintain plant vigour and re- Innovative Approaches to Plant Diseases Control ving cell water relations. Pp. 83-91 in Fusarium duce the probability of attack by patho- (I. Chet, ed.). John Wiley & Sons, New York. wilt of banana (R.C. Ploetz, ed.). International genic microorganisms. Guzmán M. & J. Sandoval. 1996. Síntomas de la pu- Conference on Fusarial Wilt of Banana, Miami Although chemical (Vanodine) and drición suave del pseudotallo en los híbridos (USA), 27-30 Aug. 1989. APS, St Paul, USA. biological (Kasugamycine 2%) treat- FHIA-01 y FHIA-02. CORBANA 2(46): 145-150. Stover R. 1972. Banana, plantain and abaca ments do not contribute directly to the Jacob A. & H. von Uexküll. 1961. Fertilización. Nu- diseases. Commonwealth Mycological Institute/ control of plantain vascular rot, they trición y abonado de los cultivos tropicales y sub- Commonwealth Agricultural Bureaux, Kew, contribute to preventing the spread of tropicales (L. López Martínez de Alva, trad.). In- United Kingdom. 316pp. the problem. ternationale Handelmaatschappij voor The frequent use of sodium hypochlo- Meststoffen N.V., Amsterdam. 626pp. rite in commercial form (50% dilution Jiménez G. & N. Cordoves. 1992. Caracterización de in water) is an essential practice for bacterias de la rizosfera del plátano antagónicas disinfecting tools and should be a Erwinia chrysanthemi Burk. Protección de strongly encouraged. Plantas (Cuba) 2(2):67-75. Limón M.C., J.A. Pintor-Toro & T. Benitez. 1999. In- Acknowledgements creased antifungal activity of Trichoderma har- The authors thank Mr Alfonso Guerrero zianum transformants that overexpress a 33-kDa Gacha, the owner of San Isidro farm at chitinase. Phytopathology 89: 254-261. Icononzo, for all the facilities that he Rivera N. & M. Ezavin. 1989. Necrosis del cormo del The authors work at Centro de Investigación Na- placed at their disposal during the field plátano causada por Erwinia chrysanthemi. taima, CORPOICA, Apartado Postal 064, Espinal, To- experiment. They also thank Mr Anto- Ciencia y Técnica en la Agricultura 12(2): 59-70. lima, Colombia. nio María Caicedo, engineer at the CORPOICA Nataima center, for his collaboration in the statistical analysis of the results.

References Belalcázar S., V.M. Merchán & M. Mayorga. 1991. Germplasm evaluation Disease resistance Control de Enfermedades. Pp. 243-297 in El cultivo del plátano en el trópico. Comité Departa- mental de cafeteros del Quindío. ICA. CIID. IN- Evaluation of FHIA hybrids in IBAP. Armenia, Quindío, Colombia. Belalcázar S., C. Salazar, G. Cayón, J.E. Lozada, LE. comparison with local Musa Castillo & J.A. Valencia. 1991. Manejo de Planta- ciones. Pp. 149-239 in El cultivo del plátano en el clones in a black Sigatoka-free trópico. Comité Departamental de cafeteros del Quindío. ICA. CIID. INIBAP. Armenia, Quindío, area of eastern Peru Colombia. Cammue B.P.A., M.F.C. De Bolle, F.R.G. Terras & W.F. Broekaert. 1993. Fungal disease control in Musa: application of new antifungal proteins. Pp. U. Krauss, W. Soberanis hybrids and popular and/or high-yield- 221-225 in Breeding banana and plantain for re- and J. Jarra ing local clones. sistance to diseases and pests (J. Ganry, ed.). In- Diseases aggravated by almost com- ternational Symposium on Genetic Improvement plete lack of control measures are the of Bananas for Resistance to Diseases and Pests, he International Musa Testing most limiting factor for Musa produc- Montpellier, France, 7-9 Sept. 1992. CIRAD- Programme (IMTP) aims at com- tion in Peru. Black Sigatoka is present FLHOR, Montpellier, France. Tparing improved Musa germplasm, in part of the production area only Cedeño M., L.R; B.M. Nieves M. & E.L. Palacios. most notably FHIA hybrids, with popu- (Krauss et al. 1999). Elsewhere, yellow 1990. Erwinia carotovora subsp. atroseptica, lar clones in over 50 countries world- Sigatoka and Cordana leafspot are the causante de la pudrición blanda del plátano wide (Orjeda et al. 1999). Peru has not most important diseases. Neither has “Hartón” (Musa AAB) en Venezuela. Fitopato- participated in this effort. The limited received major attention from breed- logía Venezolana 3(1): 6-9. information available on Musa producers because black Sigatoka is of Chet I. 1987. Trichoderma - application, mode of tion in Peru was reviewed by Krauss et greater importance on an interna- action, and potential as a biocontrol agent of al. (1999) and the authors recom- tional scale. In fact, few recent evalua- soilborne plant pathogenic fungi. Pp. 137-160 in mended germplasm trials with FHIA tions on yellow Sigatoka resistance are

INFOMUSA — Vol 10, N° 1 21 Table 1. Musa hybrids and clones included for this study in a black Sigatoka-free area of eastern Peru. Disease reactions were summarized from Krauss et al. (1999) for Peru and, when not available, from Jones (2000). Hybrid or Clone Genomic make-up Subgroup (intl. clone) Reported disease reaction Black Sigatoka1 Yellow Sigatoka1 Cordana leafspot FHIA-01 AAAB Hybrid Resistant nd2 nd FHIA-03 AABB Hybrid Resistant nd nd Inguiri AAB French Plantain Susceptible Moderately Resistant Susceptible Bellaco AAB Horn Plantain (Hartón) Susceptible Moderately Resistant Susceptible Isla del Alto Huallaga 3 ABB? Pisang Awak? Moderately resistant Resistant Susceptible 1 Krauss et al. (1999) reported a wide range of disease reactions by local clones to black and yellow Sigatoka. The reaction listed here represents the conclusion reached in their article. 2 nd, no data. 3 According to Thierry Lescot (personal communication, 1999), Isla belongs to the Iholena group (AAB). Until the affiliation of Isla to a group is confirmed, we will tentatively maintain it as “Pisang Awak?” as generally believed in Peru (Krauss et al., 1999). available because black Sigatoka re- shown in Table 1. The trial sites and their Results places yellow Sigatoka except at high characteristics are given in Table 2. Table 3 shows the agronomic charac- altitudes and existing data are highly Fields were located in the “coca-belt” teristics of the Musa clones and hy- variable. Resistance to yellow Sigatoka of the upper Huallaga valley. All coop- brids in this study. Isla had the shortest is not correlated with black Sigatoka erating farmers actively expressed in- production cycle with significantly resistance (Jones 2000). We found no terest in participating in “alternative fewer days from planting to flowering comparative study on Cordana crops” activities and had years of ex- and harvest than the other varieties. leafspot. perience in Musa production in adja- In the ratoon crop, this phenomenon Yellow Sigatoka is caused by Myco- cent fields. Trials were designed in a was even more pronounced. FHIA-01 sphaerella musicola Leach and is dis- participatory manner: no attempt was had the second fastest harvest-to- tributed almost worldwide. In the ab- made to optimize or standardize agro- harvest cycle, FHIA-03 and Inguiri sence of black Sigatoka caused by nomic practices; these were left at the were intermediate, and Bellaco had Mycosphaerella fijiensis Morelet, yel- discretion of the farmer (Table 2). We the longest harvest-to-harvest interval. low Sigatoka can cause major losses, believe this to be the most informative Isla also had the highest leaf produc- especially on AAA bananas. Plantains approach to germplasm evaluation tion rate in the plant crop although (AAB) are resistant to yellow Sigatoka under local conditions because it en- this failed to reach statistical signifi- at sea level. At higher altitudes, how- compasses local crop husbandry prac- cance. Therefore, this parameter was ever, especially under poor growing tices. It also increases data variability; not analyzed for the ratoon crop. conditions, they are susceptible. ABB therefore, ten plants per plot were used All varieties were marginally taller clones, including Pisang Awak, are re- rather than the recommended four to at harvest than at flowering; all except garded to be resistant (Jones 2000). six. In other aspects, the INIBAP guide- FHIA-01 in the plant crop also showed Both the AAB and ABB groups are af- lines for germplasm evaluation (Or- a slight increase of pseudostem fected by yellow Sigatoka in Peru jeda 1998) were followed. Tissue-cul- perimeters from flowering to harvest. under poor management (Krauss et al. tured FHIA hybrids were the courtesy Bellaco was the tallest clone, followed 1999). of FHIA. After hardening in a green- by Inguiri. These clones also had the Cordana leafspot is caused by Cor- house, the plantlets were transplanted thickest pseudostems. The two FHIA dana musae (Zimmermann) Höhnel at a spacing of 3 m x 3 m as practised hybrids were in the same height range and is distributed worldwide. Although for local clones. Trials followed the at flowering and harvest. Isla was the it is of minor importance internation- randomized block design with one in- shortest clone with the thinnest pseu- ally, it can cause severe defoliation es- complete block: Bellaco was not dostem. Pseudostem circumference in- pecially on plantain. Wet weather and planted in the field in Marona. creased in the order Isla, FHIA-01, debilitated plants favour the disease. Shortly after installation, the plot in FHIA-03, Inguiri, Bellaco. This trend Both factors are prevalent in Peru, Cotomonillo was abandoned because became more pronounced with time where up to 4000 mm of rainfall in of terrorist activity and no data are (Table 3). some growing areas and the lack of available. Evaluation elsewhere took The number of functional leaves was drainage and sucker control as well as place in fortnightly intervals. For the very similar to the total number of pests weaken the plants. C. musae at- plant crop, key parameters were leaves. In the plant crop, varieties re- tacks several Musa spp. and Ensete recorded six months (182 days) after tained between 93% (Inguiri) and 100% glaucum; most Musa subgroups are planting, at flowering and at harvest. (FHIA-03) of leaves at flowering. All re- regarded to be susceptible (Jones The maximum leaf production rate tained over 98% at harvest (Table 3). 2000). was calculated from Gompertz-curves This apparent “increase” in plant The objective of this study was to constructed by logistic regression on health over time is due to the fact that compare the improved hybrids FHIA-01 Genstat 5. For the ratoon crop, key pa- deleafing was practised during the (AAAB) and FHIA-03 (AABB) with the rameters were recorded at flowering later stages of plant development only local clones Inguiri (AAB, French and harvest only. The second ratoon of (Table 2) so that non-functional leaves plantain), Bellaco (AAB, Horn plan- Isla was compared with the first ra- were removed more diligently closer to tain “Harton”) and Isla del Alto Hual- toon of the other cultivars because harvest. The more realistic indicator laga (ABB, Pisang Awak subgroup?)1 these coincided in terms of timing of disease progress is the decrease of in agronomic, pathological and eco- and, thus, the concomitant seasonal both total and functional leaves from nomical terms. fluctuations of climatic conditions, flowering to harvest. In the plant crop, disease pressure and marketing poten- this was most pronounced for Inguiri Materials and methods tial, whereas the first ratoon crop of which lost 23.6% of total leaves and The reported disease reactions of the Isla was already harvested before any 18.1% of functional leaves between germplasm used in this study are of the other varieties flowered. flowering and harvest. Bellaco lost

22 INFOMUSA — Vol 10, N° 1 17.3% and 16.2% and Isla 15.2% and for FHIA-01 and 5.8% and 5.8% for Leaf loss was directly related to 14.3%, respectively. The least loss oc- FHIA-03, of total and functional disease susceptibility (Table 4). curred in the FHIA hybrids: 8.3% and leaves, respectively. The highest leaf FHIA hybrids, especially FHIA-03, 7.5% for FHIA-01 and 7.2% and 9.0% for loss in the ratoon crop was encoun- were least susceptible to both yellow FHIA-03, of total and functional tered for Bellaco (11.8% and 12.9%), Sigatoka and Cordana leafspot leaves, respectively. In the ratoon followed by Isla (8.9% and 9.1%) whether measured as average sever- crop, FHIA hybrids, again, had the and Inguiri (6.5%, both values) ity or infection indices at different lowest leaf loss with 4.9% and 5.0% (Table 3). times. In the plant crop, Inguiri and

Table 2. Description of the farmer-managed germplasm trial plots. Appraisal according to Location Planting date Farmers Researchers Agronomic practices (days after planting) Cotomonillo 19/03/98 Good Musa soil. Fertile, alluvial soil, flood-prone; Not determined (Aucayacu) Wind damage 30 years under banana/plantain without fertilization, but with plant materials as mulch. Stem borer damage; Furadan used occasionally. Pendencia 19/03/98 Good Musa soil. Fairly fertile soil, flood-prone, 3rd year under Musa Weed control and deleafing (181, 234, 503) (Fondo Bazán) Wind damage without fertilization; plant materials used as mulch; Deleafing (292, 345, 651) previously cocoa. Stem borer damage; Borer control (Furadan) (234) Furadan used occasionally. Weed control, deleafing and borer control (471) Marona 23/3/98 Fair Musa soil. Fertile, alluvial soils; no risk of inundation. Weed control and deleafing (260, 281, 425) Stem borer Stem borer damage; Fusarium sp. Deleafing (325, 437, 589) “Seca seca” has yet to be confirmed in this field. (= Fusarium wilt) Application of lime in 1997 and Furadan in 1998. First year under banana following papaya. Pendencia 16/4/98 Good Musa soil. Fertile alluvial soils; lack of natural drainage, Weed control (22, 83, 338) (Fondo Magno) Stem borer flood-prone. Stem borer damage confirmed. Deleafing (464) First year of Musa. Weed control and deleafing (193, 263, 316, 542, 625)

Table 3. Agronomic characteristics of FHIA hybrids as compared with those of Peruvian clones FHIA-01 FHIA-03 Inguiri Bellaco Isla1 (AAAB Hybrid) (AABB Hybrid) (French Plantain) (Horn Plantain) (Pisang Awak?) Plant crop Days to flowering 268 b 279 b 279 b 284 b 198 a Days to harvest 390 b 411 b 403 b 410 b 299 a Height at flowering (cm) 239 b 233 b 268 c 311 d 201 a Height at harvest (cm) 243 b 238 b 287 c 320 d 203 a Circumference of pseudostem 77.3 b 88.7 b 91.7 b 109.8 c 59.4 a at flowering (cm) Circumference of pseudostem 76.5 b 93.7 c 101.2 c 114.1 d 62.5 a at harvest (cm) Total number of leaves at flowering 10.8 b 11.1 b 8.9 ab 8.1 a 7.9 a Total number of leaves at harvest 9.9 b 10.3 b 6.8 a 6.7 a 6.7 a Loss of total leaves from flowering 8.3 7.2 23.6 17.3 15.2 to harvest (%) Functional number of leaves at flowering 10.7 b 11.1 b 8.3 a 8.0 a 7.7 a Functional number of leaves at harvest 9.9 b 10.1 b 6.8 a 6.7 a 6.6 a Loss of functional leaves from 7.5 9.0 18.1 16.2 14.3 flowering to harvest (%) Maximum leave production rate 0.28 a 0.42 a 0.41 a 0.35 a 0.64 a (leaves per week) Ratoon crop Days from flowering to flowering 278 b 286 b 283 b 296 b 150 a Days from harvest to flowering 156 b 150 b 154 b 164 b 49 a Days from harvest to harvest 265 b 276 bc 276 bc 298 c 149 a Height at flowering (cm) 245 b 244 b 291 c 328 d 211 a Height at harvest (cm) 250 b 248 b 296 c 334 d 216 a Circumference of pseudostem at flowering (cm) 72.9 a 86.7 b 98.7 c 115.1 d 62.2 a Circumference of pseudostem at harvest (cm) 77.2 b 89.0 c 102.0 d 115.5 e 65.5 a Total number of leaves at flowering 10.2 b 10.4 b 7.7 a 8.5 a 7.9 a Total number of leaves at harvest 9.7 b 9.8 b 7.2 a 7.5 a 7.2 a Loss of total leaves from flowering 4.9 5.8 6.5 11.8 8.9 to harvest (%) Functional number of leaves at flowering 10.0 b 10.3 b 7.7 a 8.5 a 7.7 a Functional number of leaves at harvest >9.5 b 9.7 b 7.2 a 7.4 a 7.0 a Loss of functional leaves from flowering 5.0 5.8 6.5 12.9 9.1 to harvest (%) 1 The second ratoon of Isla was compared with the first ratoon of the other clones and hybrids. a, b, c, d Values within a row followed by the same letter do not differ at P = 0.05 (Tukey test).

INFOMUSA — Vol 10, N° 1 23 Table 4. Leafspot reaction of FHIA hybrids as compared with those of Peruvian clones. FHIA-01 FHIA-03 Inguiri Bellaco Isla1 (AAAB Hybrid) (AABB Hybrid) (French Plantain) (Horn Plantain) (Pisang Awak?) Plant crop Yellow Sigatoka Average severity (%)2 0.54 a 0.12 a 4.09 c 0.69 ab 2.57 bc Infection Index 6 months after 0.53 a 0.00 a 4.37 b 0.58 a 4.36 b planting 3 Infection Index at flowering 1.09 a 0.56 a 3.55 b 0.82 a 3.73 b Infection Index at harvest 1.67 ab 0.25 a 7.45 c 3.11 b 6.73 c Cordana leafspot Average severity (%) 1.21 a 0.95 a 2.00 ab 2.96 b 1.57 ab Infection Index 6 months after 1.73 a 1.26 a 2.37 ab 3.62 b 2.52 ab planting Infection Index at flowering 1.85 ab 0.92 a 3.01 b 2.67 b 3.00 b Infection Index at harvest 2.28 ab 1.95 a 3.55 b 5.88 c 5.04 bc Ratoon crop Yellow Sigatoka Average severity (%) 2.13 a 1.71 a 5.48 b 2.05 a 3.68 ab Infection Index at flowering 1.07 a 0.79 a 3.03 a 2.15 a 3.04 a Infection Index at harvest 1.86 a 1.27 a 2.61 a 1.07 a 3.37 a Cordana leafspot Average severity (%) 2.08 a 0.97 a 4.91 a 1.57 a 1.65 a Infection Index at flowering 0.64 a 0.11 a 1.50 a 1.51 a 1.60 a Infection Index at harvest 1.05 a 0.19 a 1.43 a 0.06 a 1.67 a 1 The second ratoon of Isla was compared with the first ratoon of the other clones and hybrids. 2 Calculated by multiplying percentage disease severity per leaf with percentage leaves infected and averaging this value over time. 3 Infection indices were calculated according to Orjeda (1998). a, b, c Values within a row followed by the same letter do not differ at P = 0.05 (Tukey test).

Isla were most affected by yellow Siga- the ratoon crop, Isla surpassed FHIA-03 worst in the overall performance. Nev- toka. Bellaco was most susceptible to in terms of economics due to a ertheless, the collaborating farmers Cordana leafspot, closely followed by remarkably fast ratooning and flower- were the more conscientious ones of Inguiri and Isla. In the ratoon crop, ing rate (Table 3). These characteris- that area with interest and experience all cultivars were affected more by tics compensated for its susceptibility in Musa production. We are aware that yellow Sigatoka than by Cordana to yellow Sigatoka and a somewhat crop management was above the re- leafspot but disease severity and in- lower price. FHIA-01 was consistently gional average. dices were more variable. Inguiri, fol- the third most economical hybrid be- None of the participating farmers lowed by Isla, exhibited the highest cause of its large bunches and disease recognized fungal diseases as limiting yellow Sigatoka severity. Other para- resistance. It fell into the same price- factors for production. Instead, they meters and Cordana leafspot did not class as Isla. Inguiri was less profitable. were regarded as normal leaf appear- reach statistical significance (Table Bellaco was least economical. The ance. All growers had problems with 4). In contrast to the plant crop in lower economic returns for these two toppling and attributed this to wind, which disease increased over time, in plantain clones were because of their which is negligible in the area, or wee- the ratoon crop, yellow Sigatoka in small bunches (Table 5) and high dis- vil attack, which is ubiquitous. Average Inguiri and, even more pronounced, ease susceptibility (Table 4) which out- farmers do not usually recognize this both diseases in Bellaco seem to de- weighed a high per-finger price. All va- pest. The complete lack of drains in crease from flowering to harvest. This rieties became more profitable in the flood-prone fields further contributed to can be attributed to deleafing just be- ratoon crop, although farm-gate prices weakened root systems. Nematodes fore these clones were harvested for all varieties, except Bellaco, had were no major problem as all fields ex- (Table 2) and is also in agreement dropped. cept the lost plot in Cotomonillo were with the high percentage leaf loss of only recently planted to Musa (Table 2). Bellaco from flowering to harvest One farmer used insecticide during the (Table 3). Discussion duration of the trial. All practised man- An economic comparison (Table 5) We deliberately chose a participatory ual weed control and deleafing. The lat- indicates that FHIA-03 was the overall approach to germplasm evaluation be- ter only commenced about six months most lucrative Musa variety during the cause we believe this to be the most re- after planting when flowering of Isla first production cycle after planting. It alistic representation of local growing had begun and resumed before the har- produced the most fingers per bunch conditions. Both agronomic practices vest peak of both production cycles and attracted a high price, which is cal- (or the lack of them) and personal (Tables 2 and 3). Little attention was culated on a per 1000 finger basis in preferences for a variety could be in- paid to the crop during early develop- eastern Peru. Large bunches combined cluded in the evaluation. Interestingly, ment stages, i.e. when the bunches dif- with a fast production cycle made Isla one farmer decided not to plant Bel- ferentiated and the number of fingers, the second most economical clone. In laco. This clone later proved to be the

24 INFOMUSA — Vol 10, N° 1 on which the pricing structure is based, (1999) concluded that Isla is resistant because of its small bunch sizes. were determined. and Inguiri and Bellaco are moderately FHIA-01 was less popular but also FHIA-01 was bred as a dessert ba- resistant to yellow Sigatoka but these has good potential. It fell into the nana substitute. It is the only hybrid resistance reactions are dependent on same price class as Isla, one of the with simultaneous resistance to black the growing conditions. All three most popular clones in Peru (Krauss Sigatoka, Panama disease and crown clones are reported as susceptible to et al. 1999). Only the most favoured rot. Furthermore, it is high-yielding Cordana leafspot (Krauss et al. 1999). local clones were included in this even under poor conditions including According to the plant crop data, Isla study, and it is a great achievement drought (FHIA 2000). FHIA-01 tends to and Inguiri are similarly susceptible to for a new variety to compete with any perform better in subtropical than trop- yellow Sigatoka, whereas Bellaco may of these on the market during the ical conditions, especially with respect be less susceptible to yellow Sigatoka first year of planting. Participating to fruit quality (Jones 2000). FHIA-03 but more so to Cordana leafspot. How- farmers also reported good markets was bred as Bluggoe substitute. It is re- ever, in the ratoon crop, all varieties for FHIA planting material. However, sistant to black Sigatoka and Moko dis- suffered more from yellow Sigatoka none of them was prepared to sell ease. It is a sturdy hybrid which yields than from Cordana leafspot. The high followers of FHIA-03 which produces well under adverse conditions such as variability of disease incidence in the fewer suckers than FHIA-1. Instead, poor soils and drought. Its main weak- ratoon crop, especially near harvest- farmers expanded their own area ness is the short greenlife. FHIA-03 is ing, may be due to increased phytosan- under FHIA-3. therefore recommended for homegar- itary pruning around that time. We can wholeheartedly recommend dens and local consumption (FHIA Isla has recently been incorporated the introduction of FHIA hybrids on 2000). Boiling time for cooking green into FHIA’s breeding programme a larger scale in Peru, especially in FHIA-03 is only half that required for (Phil Rowe, pers. comm., 2000). It view of the constant expansion of Bluggoe (Jones 2000). would be worthwhile to investigate black Sigatoka. Evaluations of FHIA In the black Sigatoka-free area whether aggressive pathotypes of M. hybrids in areas affected by black where this study was conducted, musicola have evolved in the Hual- Sigatoka are ongoing (Phil Rowe and FHIA-03 performed similar to the best laga valley and/or to corroborate the Raul Anguiz, pers. comm., 1999). local clone (Isla) and FHIA-01 also affinity of Isla clones with Pisang compared very well. The Huallaga val- Awak. Isla has also been suggested to ley is a highly tropical environment belong to the Iholena (AAB) sub- Acknowledgements and poor drainage rather than drought group (Thierry Lescot, pers. comm., This paper was prepared during a di- is a problem (Krauss et al. 1999). 1999). Furthermore, “Isla” is the col- versification project funded by the Under these circumstances, it is grati- lective term for five to seven distinct United States Department of Agricul- fying to see FHIA hybrids perform so clones within one group (Krauss et ture-Agricultural Research Service well and be accepted by local and met- al. 1999). It is conceivable that their (USDA-ARS) and managed by CABI ropolitan markets at the same time. disease reactions differ. Bioscience. During the first three Especially, the success of FHIA-03 is The present trials show that the months, additional funds were re- surprising. Bluggoe is not appreciated FHIA germplasm exhibits resistance ceived from the Organization of Ameri- in Peru (Krauss et al. 1999), but farm- to yellow Sigatoka and Cordana can States (Inter-American Drug ers reported multipurpose uses for leafspot under mediocre growing Abuse Control Commission, FHIA-03. conditions and management in east- IADACC/OAS). FHIA germplasm was The study also indicated that Isla del ern Peru. FHIA-03 was least suscepti- generously provided by Phil Rowe. The Alto Huallaga is moderately susceptible to diseases, produced the largest authors also wish to thank colleagues ble to yellow Sigatoka in eastern Peru. bunches, and was rated in the high- at CABI (Commonwealth Agricultural This finding contradicts Table 1, but est price class. This suggests that Bureau International, UK), CIRAD- may help to resolve a point of con- this clone has excellent marketing FLHOR (Centre de coopération inter- tention: Isla has been classified as sus- potential for Peruvian internal mar- nationale en recherche agronomique ceptible to highly resistant and Inguiri kets. Bellaco commanded a similar pour le développement-Département and Bellaco as susceptible to resistant price to FHIA-03 (for 1000 fingers), des productions fruitières et horti- by different authors. Krauss et al. but Bellaco was the least economical coles, France), FAO (Food and Agricul-

Table 5. Economic returns of FHIA hybrids as compared with those of Peruvian clones. FHIA-01 FHIA-03 Inguiri Bellaco Isla1 (AAAB Hybrid) (AABB Hybrid) (French Plantain) (Horn Plantain) (Pisang Awak?) Average number of fingers per bunch 120 150 84 33 110 Plant crop Farm-gate price (US$ per 1000 fingers) 30.03 39.04 36.04 39.04 30.03 Gross income (US$ ha-1 yr-1)2 3747 5778 3047 1,747 4,481 Ratoon crop Farm-gate price (US$ per 1000 fingers) 28.90 34.68 28.90 40.46 28.90 Gross income (US$ ha-1 yr-1)1 5307 7644 3567 1,817 8,653 1 Second ratoon of Isla was compared with the first ratoon of the other clones and hybrids. 2 Based on a planing density of 1111.11 plants per ha.

INFOMUSA — Vol 10, N° 1 25 ture Organization of the United Na- References Orjeda, G., J.-V. Escalant and N. Moore. 1999. The tions), UNAS (Universidad Nacional FHIA. 2000. Bananas and Plantains. . Phase II: Overview of final report and summary ROCK for helpful comments and dis- Jones D.R. (ed.). 2000. Diseases of banana, abacá of results. InfoMusa 8 (1): 3-10. cussion. We are particularly grateful to and enset. CABI Publishing, Wallingford, UK. Phil Rowe who reviewed this manu- Krauss U., R. Figueroa, A. Johanson, E. Arévalo, script. He is missed with deep sadness R. Anguiz, O. Cabezas & L. García. 1999. Musa Ulrike Krauss (author for correspondence) works at but will be remembered as the mag- clones in Peru: classification, uses, production CABI Bioscience, c/o CATIE, 7170 Turrialba, nanimous and always encouraging per- potential and constraints. InfoMusa Costa Rica, Fax: (+506) 556 0606, Email: [email protected]. Whilly Soberanis works at son I had the privilege to know. 8(2): 19-26. CABI Bioscience, c/o Universidad Nacional Agraria de Mentioning of trade-names Orjeda, G. 1998. Evaluation of Musa germplasm la Selva (UNAS), Apdo 156, Tingo María, Peru. José is this article is not to be understood for resistance to Sigatoka disease and Fusarium Jarra works at the IAHRC (Inter-American Human as recommendation of a particular wilt. INIBAP Technical Guidelines 3. INIBAP, Rights Court of the Organization of American States, product. Montpellier, France. OAS) in Tingo María, Peru.

Germplasm evaluation Resistance to weevils

Evaluation of Musa germplasm against banana weevil borers

B. Padmanaban, P. Sundararaju, K.C. Velayudhan and S. Sathiamoorthy

ananas and plantains constitute the fourth most important crop of Bthe developing world and India is the largest producer in the world. Of the 40 million tonnes of fruits produced in India, banana occupies the top position with an annual output of 13.5 MT from an area of 400 000 ha. Among the insect pests of banana, the banana rhizome weevil, Cosmopolites sordidus (Germ.) and banana stem weevil, Odoiporus longicollis (Oliv.) are the key pests limiting the production and productivity of bananas and plantains (Figure 1) (Ostmark 1974). Both types of weevil borers have been a threat to banana under garden land cultivation and their occurrence in non-traditional areas of Tamil Nadu has been reported (Padmanaban and Sundararaju 1999). Bionomics and chemical control of the pests have been studied (Dutt and 12 3 Maiti 1972, Reghunath et al. 1992, Mathew et al. 1997). Figure 1. Banana stem weevil (BSW) infestation. (1) BSW-infested plant showing sheath rotting and drying. The reaction of banana clones to (2) BSW-infested plant with the outer leaf sheath removed to expose the damage. major biotic stresses has been studied (3) Adult weevil and grubs tunnelling the sheath. by Anitha et al. (1996). This study did not however include the banana pseu- weevil borers: banana stem weevil rhizome weevil (Pavis and Minost dostem weevil O. longicollis. Indeed, (BSW, Odoiporus longicollis), and ba- 1993). Ortiz et al. (1995) indicated that only a limited number of banana culti- nana rhizome weevil (BRW, Cosmopo- investigations of resistance mecha- vars have been evaluated against ba- lites sordidus). nisms in the corm should consider pres- nana stem weevil (Charles et al. A negative correlation between corm ence of antifeedants or absence of es- 1996). This paper reports on field hardness and infestation rate led to the sential nutrients. Attraction of the screening of diverse Musa germplasm hypothesis of mechanical resistance to pseudostem to adults was not used as a under natural infestation of banana oviposition or larval development of the criterion of resistance to weevil be-

26 INFOMUSA — Vol 10, N° 1 cause no correlation was found be- Table 1. Germplasm free from banana weevil attacks. In the ABB group, Ja- tween attraction and infestation (Pavis stem weevil (BSW, O. longicollis) mani was tolerant to the pest, while infestation. and Minost 1993). Malaimonthan and Peykunnan have IC No. Local name Genome moderate levels of resistance to BRW. Materials and methods TCR 7 Sannachenkadali AA Host plant resistance introduced Banana germplasm available at the 84809 Karumpoovan AAB through breeding offers a safe and long- National Bureau of Plant Genetic Re- TCR 22 Nattuvazhai ABB term control strategy for the banana sources (NBPGR) Regional Station, TCR 29 Sivakositu ABB weevil (Seshu Reddy and Lubega 1998). Vellanikkara, Kerala was evaluated 84833 Sakkai (Chakkiya) ABB Observations on the germplasm evalu- under field conditions against the ba- 84863 Poozhachendu AAB ated against the banana weevil borers nana weevil borers during 1999-2000. 84889 Senkadali AAA indicate that weevils have a preference The crop was raised during 1995 with TCR 78 Koombillakai AAB for AAB and ABB genomic groups. Stud- a row-to-row and plant-to-plant dis- TCR 133 Morris AAA ies conducted by various authors else- tance of 2.8 x 2.8 m. The normal pack- 127933 Kadali AA where also indicated a similar trend age of practice was followed and the 127936 Tongat AA (Haddad et al. 1979, Mesquita et al. crop was managed under rainfed con- 127938 Namrai AAB 1984, CRBP 1992, Simmonds 1966). ditions. Stem girth near crown as well 127940 Sannachenkadali AA The BSW shows a high degree of host as the base and percentage of infesta- 127941 Karivazha AAA plant preference. When all the commer- tion were recorded. Highly infested 127943 Bodles Alta Fort AAAA cial cultivars like Nendran, Robusta, plants were cut open to record the 127944 Hybrid sawai ABBB Rasthali, Red Banana, Pisang Awak are number of adult weevils and grubs in- 127946 Elavazhai BB available in the same vicinity, the stem 127947 Kunnan AB side the plant. From the harvested weevil recognizes and infests only plan- 127952 Padalimoongil AB plants the rhizome was uprooted and tain cultivars. The ability of the weevil 127958 Radjasree AAB the damage assessed. 127963 Vannan AAB to distinguish an acceptable host plant 127974 Karibale AAB from others may be aided by the pres- Results and discussions 127978 Velipadathi AAB ence of an array of sensory chemore- Field evaluation of banana germplasm 127980 Peyan ABB ceptors on the antenna and mouth against banana stem weevil indicated 127981 Ashy Bathesa ABB parts (Nahif et al. 1994, Nahif et al. that out of the 229 accessions, 62 acces- 127984 Octoman ABB 2000). Further studies are needed at sions belonging to AAB, ABB, AB, AAA, 127986 Kalibow AAB the laboratory level with the identified BB and ABBB genomic groups had BSW 127987 Boodithabontha bath ABB resistant accessions to single out the infestation. Maximum infestation was TCR 195 Padathi AAB most promising. noticed in the AAB genome, and 37 ac- 127994 Ennabenian ABB cessions belonging to the following 127996 Cheenabale AAB Acknowledgements genome: ABB, AAB, AA, BB, AB, AAA, TCR 216 Boothibale ABB Thanks are due to Dr Z. Abraham, Sci- ABBB and AAAA were found free from TCR 221 Morris AAA entist-in-charge, NBPGR, Thrissur for BSW infestation (Table 1). TCR 241 Padalimoongil AB providing necessary facilities and Ms C. Incidence of BSW was recorded in 84776 Njalipoovan AB Rajalakshmy, Technical Officer, for 5.50% of the accessions in 1999 and 84760 Madavazha ABB technical assistance. there was a four-fold increase (21.36%) TCR 300 M. balbisiana BB during 2000. References Due to the BSW infestation, there Anitha N., L. Rajamony & T.C. Radhakrishnan. 1996. was 50-86% reduction in the stem girth Reaction of banana clones against the major bio- near the crown region. In the infested Table 2. Germplasm free from banana tic stresses. The Planter 72: 315-321. plants, 2-15 adult weevils, 10-15 grubs rhizome weevil (BRW, C. sordidus) Charles J.S.K., M.J. Thomas, R. Menon, T. Premala- and 5-8 pupal cases were found. Dutt infestation. tha & S.J. Pillai. 1996. Field susceptibility of ba- and Maiti (1972) have reported that the IC No. Local name Genome nana to pseudostem borer, Odoiporus longicollis portions of the banana pseudostem TCR 7 Sannachenkadali AA (Oliv.). P. 32 in Symposium on Technological ad- with circumference ranging from 25 to 84809 Karumpoovan AAB vancement in banana/plantain production and 50 cm and up to a height of 125 cm in 84863 Poozhachendu AAB processing - India - International, 20-24 Aug. tall varieties like Martaman (AAB), 84866 Sakkai ABB 1996. Abstracts of papers. (N.K. Nayar & T.E. Champa (AAB) and Kanchekela (ABB), 84889 Senkadali AAA George, eds.). Kerala Agricultural University, and up to a height of 100 cm in dwarf 127946 Elavazha BB Mannuthy, India. varieties like Kabuli (AAA), are the pre- 127949 Njalipoovan AB CRBP. 1992. Rapport de synthèse. Recherches 1991 ferred sites for oviposition. Our studies TCR 216 Borthibale ABB [Synthesis report. 1991 research]. Centre Régio- indicated that there was no relation be- TCR 261 Njalipoovan AB nal Bananiers et Plantains (CRBP), Douala, Ca- tween infestation, stem girth and plant meroon. height, with even smaller plants being Dutt N. & B.B. Maiti. 1972. Occurrence of non-sex li- infested. Anitha et al. (1996) reported that 87 mited variations in conspecific sympatric phena Field evaluation of banana germplasm clones representing various genomic of Odoiporus longicollis (Oliv.). Sci. & Cult. against banana rhizome weevil, C. sor- groups (AA, AB, AAA, AAB and ABB) 37: 572-574. didus, was carried out on 143 accessions. were screened under natural condi- Haddad O., J.R. Surya & M. Wagner. 1979. Relacion Of these, 134 were from the following ge- tions. Among the AA clones, San- de la composicion genomica de las musaceas con nomic groups: ABB, AAB, AAA, AA, BB and nachenkadali was tolerant to rhizome el grado de attrecion de adultos y larvas de Cos- AB and ABBB were infested while nine ac- weevil. In the AB group, Njalipoovan, mopolites sordidus (Germ.) (Coleoptera : Curcu- cessions from the groups: ABB, AAB, AAA, Kunnan and Poomkalli were resistant lionidae). Agronomia Tropical 29: 429-438. AA, BB, AB were free from rhizome weevil to the pest. Among AAB clones, Mysore Mathew M.P., S.R. Nair & S. Sivaraman. 1997. Mana- under field conditions (Table 2). Poovan was highly tolerant to rhizome gement of pseudostem borer of banana, Odoipo-

INFOMUSA — Vol 10, N° 1 27 rus longicollis (Oliv.). Indian J. Entomology Padmanaban B. & P. Sundararaju. 1999. Occurrence Seshu Reddy K.V. & M.C. Lubega. 1993. Evaluation 59: 269-273. of banana weevil borers (Curculionidae : Coleop- of banana cultivars for resistance/tolerance of Mesquita A.L.M., I.J. Alves & R.C. Caldas. 1994. Re- tera) in Tamil Nadu. Insect Environment 5: 135. the weevil C. sordidus (Germar.). Pp. 143-148 in sistance of banana cultivars to C. sordidus (Ger- Pavis C. & C. Minost. 1993. Banana resistance to the Breeding bananas and plantain for resistance to mar.). Fruits 39: 254-257. banana weevil borer Cosmopolites sordidus Ger- diseases and roots. International symposium on Nahif A.A., A. Koppenhofer & G. Madel. 1994. Mor- mar (Coleoptera : Curculionidae) : Role of pseu- genetic improvement of bananas for resistance phologie, Biologie und Bedeutung von Cosmopo- dostem attractivity and physical properties of the to diseases and pests, Montpellier, France, 7-9 lites sordidus Ger. Z. Angew. Zool. 4: 435-447. rhizome. Pp. 129-142 in Breeding bananas and Sept. 1992 (J. Ganry, ed.). CIRAD-FLHOR, Mont- Nahif A.A., B. Padmanaban & P. Sundararaju. 2000. plantain for resistance to diseases and roots. In- pellier, France. Ultrastructure of the banana pseudostem weevil, ternational symposium on genetic improvement Simmonds N.W. 1966. Bananas. Longman, London. Odioporus longicollis (Coleoptera : Curculioni- of bananas for resistance to diseases and pests, dae). Poster presented at the Entocongress 2000 Montpellier, France, 7-9 Sept. 1992 (J. Ganry, held at the University of Kerala, Trivandrum, 5-8 ed.). CIRAD-FLHOR, Montpellier, France. B. Padmanaban, P. Sundararaju and S. Sathia- November 2000. Reghunath P., A. Visalakshi, T. Biju Mathew, N. Mo- moorthy work at the National Research Centre on handas, S. Naseema Beevi & K.S. Remamoni. Ortiz R., D. Vuylsteke, B. Dumpe & R.S.B. Ferris. Banana, #17, Ramalinga Nagar South Extn., Vayalur 1995. Banana weevil resistance and corm hard- 1992. Insecticidal management of the pseudos- Road, Tiruchirapalli - 620 017, India. K.C. Velayud- ness in Musa germplasm. Euphytica 86:95-102. tem borer Odoiporus longicollis Oliver (Coleop- han is based at NBPGR (National Bureau of Plant Ostmark H.E. 1974. Economic insect pests of bana- tera : Curculionidae) in banana. Entomon Genetic Resources) Regional Station, Vellanikkara, nas. Ann. Rev. Entomol. 19: 161-176. 17(1-2): 113-115. Thrissur - 680 654, India.

Country report Fusarium wilt

Distribution of Fusarium wilt of banana in Kenya and its impact on smallholder farmers

J.N. Kung’u, M.A. Rutherford and effects of climate, Wardlaw (1972) sug- erage annual temperature of a region P. Jeffries gested that the effects of soil type (Sombroek et al. 1982). A moisture should also be considered. Earlier ob- availability zone of an ACZ was deter- servations showed that the spread of mined as the ratio of measured annual anana (Musa spp.) is becoming Fusarium wilt in Central America was rainfall (r) and computed average an- an important crop in the Kenyan faster in some regions than in others nual evaporation (Eo). Beconomy, which is predomi- (Stotzky and Martin 1963), leading to The objectives of this study were to nantly agriculture-based. For the last the classification of soils in banana determine the distribution of Fusar- 20 years banana production has in- areas of the region on the basis of “ef- ium wilt in Kenya, identify cultivars af- creased significantly, partly due to a fective banana-producing life” (short, fected by the disease and determine reduction in income from coffee, intermediate and long). Subsequently, whether there is any apparent correla- which has forced farmers to adopt ba- attempts were made to correlate effec- tion between disease distribution and nana as an alternative cash crop for tive banana-producing life with spe- AEZ, ACZ or specific soil factors. This local markets. Bananas are mainly cific soil properties such as texture, information is of importance to dis- grown by smallholder farmers, and the pH, cation-exchange capacity, total ease management, as it will facilitate crop integrates well into other agricul- soluble salts, available nutrients, or- deployment of tolerant/resistant culti- tural enterprises. For instance, har- ganic matter and drainage (Stotzky vars in ‘disease epidemic zones’, while vested pseudostems are an important and Martin 1963). Among these prop- susceptible cultivars (which currently fodder for dairy animals, particularly erties clay mineralogy was found to cannot be replaced by other banana during drought, thus contributing to correlate very closely with the effec- types) may only be grown in ‘non-epi- milk production (another important tive banana-producing life. demic’ or disease-free zones. source of income to smallholder farm- Kenya has a diverse ecology, being ers) and manure which is recycled partitioned into a number of agroeco- Materials and methods back to the farm. Although the crop logical zones (AEZ) (Jaetzold and has potential as an export commodity Schmidts 1982a, b, c) and agroclimatic Survey areas where samples were for Kenya, banana diseases and pests zones (ACZ) (Sombroek et al. 1982) collected are major limiting factors in its pro- within which there are also diverse Areas surveyed covered all major ba- duction (Kung’u 1995). Fusarium wilt soil types. It is of importance to con- nana-growing districts of Kenya except is currently the most important ba- sider either or both of these zones Lamu district. The survey area was di- nana disease in Kenya (Kung’u 1995, when studying plant disease epi- vided into three main regions (Figure 1), 1998). demics. AEZ were established by FAO the Coast area (Kilifi, Kwale The effects of climate and rainfall (1978) based on climatic yield poten- and Taita-Taveta districts), the on distribution, incidence and severity tial of the main leading crops within a Central/Eastern area (Murang’a, of Fusarium wilt of banana are com- region. ACZs on the other hand were Kirinyaga, Nyeri, Embu and Meru dis- plex (Wardlaw 1972). In studying the based on moisture availability and av- tricts) and the Western area (Kisii,

28 INFOMUSA — Vol 10, N° 1 then overlaid onto AEZ, ACZ, soil and elevation maps of the study areas to ° 34°E 38 E 42°E create thematic maps depicting disease distribution in relation to cultivar, AEZ, ACZ, soil type (including physicochemi-

SUDAN cal properties) and elevation. E T H I O P I A Lake 4°N Results and discussion

Distribution of Fusarium wilt and Turkana cultivars affected Fusarium wilt of banana was observed in all of the districts surveyed with the exception of Nyeri district (Figure 1).

Coast survey area

U G N D A A In Kwale and Kilifi districts bananas are grown along the coastal strip. Be- tween Vanga in the South and Malindi S O M A L I A I L A M S O 1 in the North the predominant cultivar, 3 2 1 2 Bluggoe (ABB), was found to be af- 0° Kisumu fected by Fusarium wilt. Tall and dwarf 4 13 9 Bluggoe types were grown, the former 5 1 0 1 1 7 8 being the most common, and both ex- 6 Lake Victoria hibited symptoms. It is suspected that Nairobi this cultivar has gradually been deci- mated by a number of factors, including TANZANIA Fusarium wilt, that had not been recog- 1 7 Lamu nized by farmers. Cultivars Wang’ae (= Ney Poovan, AB), Mshale (AA) and Mbuu (probably silk, AAB), which are not cultivated by many farmers in the 0 100 200 1 6 Malindi Taveta O C E AN area, were also affected. Dwarf Kilometers 1 4 Cavendish (AAA) was also commonly ° Mombasa 4 S grown but was not found to be affected 1 5 D I A N by the disease even on farms where it

I N was intercropped with severely affected Bluggoe plants. Gros Michel (AAA) was not found in the coastal strip.

Figure 1. Map of Kenya showing the major banana-growing Districts of the country that were Central and Eastern survey areas surveyed: 1. Busia, 2. Siaya, 3. Kakamega, 4. Kisumu, 5. Homa Bay, 6. Migori, 7. Kisii, 8. Murang’a, In the Central and Eastern areas Gros 9. Nyeri, 10. Kirinyaga, 11. Embu, 12. Meru, 13. Nithi, 14. Taita-Taveta, 15. Kwale, 16. Kilifi. District 17 (Lamu) was not surveyed. Michel (AAA), Wang’ae (AB), Muraru (AA?), Mbuu (AAB) and Mugithi (ploidy unknown) were found to be affected by Fusarium wilt. Muraru, proba- Homa Bay, Migori, Kisumu, Siaya, by Booth (1971) and Nelson et al. bly an East African Highland banana Kakamega and Busia districts). Farms (1983). Monoconidial cultures were (EA-AAA), seemed to be tolerant of the and plants to be sampled were taken raised from the SNA cultures for further disease, and was only found to be af- at random (Kung’u 1998). characterization and preserved in ster- fected on some farms in Murang’a dis- ile soil (Smith and Onions 1983) for the trict. Gros Michel, mostly grown in Mu- Isolation and identification of duration of the study. Confirmation of rang’a district but also found in F. oxysporum f.sp. cubense (Foc) strains Foc was through pathogenicity tests as significant numbers in Embu, Kirinyaga On return to the laboratory, individual detailed earlier (Kung’u 1998). and Meru districts, was also affected. It leaf-sheaths were peeled away from the was not common in Nyeri district pseudostem pieces collected from the Data processing and mapping where, despite intensive surveys, wilt field, the outer surface wiped with 70% Maps of the area surveyed were pre- was not observed (including on (v:v) alcohol and the material briefly pared from a base map of Kenya (scale Wang’ae). The extent of wilt has forced flamed. Using a sterile scalpel, small 1:1000 000) using MapInfo for Profes- some farmers in Murang’a and Embu pieces of discoloured vascular bundles sionals™ (Version 4.1). Similarly, maps districts to completely replace Gros were excised from the flamed leaf for ACZ, AEZ, soil types and elevation Michel with Lacatan (AAA). sheaths and placed onto 2% (w:v) tap were also prepared from the base maps. Fusarium wilt was not found on any water agar (TWA). Emerging fungal Data on the geographical origin of iso- of the Cavendish bananas types (e.g. colonies were subcultured onto potato lates and host cultivars were geocoded Lacatan and Valery) grown in the Cen- sucrose agar (PSA) and synthetic nutri- (assigned geographic coordinates) to tral or Eastern areas, nor on East ent agar (SNA: Nirenberg 1976). Iso- provide accurate maps of Fusarium wilt African Highland bananas (EA-AAA) lates were identified to species level distribution within the study areas. such as Kiganda, Mutika and Mutore or based on morphological keys described These disease distribution maps were Mutahato (probably also AAA).

INFOMUSA — Vol 10, N° 1 29 Table 1. Number of plant samples per susceptible cultivar collected in districts surveyed for Fusarium wilt in Kenya (numbers include only those samples from which isolates tentatively identified as F. oxysporum were obtained). Cultivar and number of samples collected District Bluggoe Gros Michel Muraru Wang’ae Mbuu Total Kwale 10 0 1 2 0 13 Kilifi 9 0 0 2 1 12 T/Taveta 10 7 0 6 1 24 Murang’a 0 6 5 9 0 20 Kirinyaga 0 3 0 6 0 9 Meru 0 8 0 7 4 19 Embu 0 13 1 7 0 21 Kisii 0 0 0 10 0 10 Migori 0 1 0 4 1 6 Homa Bay 0 0 0 11 0 11 Kisumu 2 0 0 11 2 15 Siaya 2 0 0 1 4 7 Busia 6 0 0 3 4 13 Kakamega 0 0 0 14 0 14 Total 39 38 7 93 17 194

Western survey area Wang’ae production due to a disease Wang’ae, and Mbuu (Odhigo) in Fusarium wilt was found in all dis- that they did not understand but that, Kisumu, Siaya and Busia in Kakamega tricts surveyed in the Western survey from their descriptions, appears to be district the disease was found primar- area. In Kisii district, the disease was Fusarium wilt. Incidence of the dis- ily on Wang’ae. Although only a few restricted to cultivar Wang’ae (known ease was almost 100% on some farms Bluggoe plants were observed in locally as Egesukari). Other cultivars where pure stands of Wang’ae were Kakamega district, these were disease- in Kisii, which were mainly East grown. free. African Highland types, showed no In Homa Bay and Migori districts Isolation and identification of symptoms of wilt. Farmers interviewed Fusarium wilt was found on Wang’ae F. oxysporum strains from sympto- in the district stated that they had and Mbuu (Odhigo), the predominant matic plant material generally noticed a drastic decline in cultivars in the area, and on Bluggoe, A total of 204 samples were collected from the three survey areas, of which 194 yielded isolates that were identified as Fusarium oxysporum 38°E (Table 1).

Correlation between Fusarium wilt distribution and ecological factors IV2 From the survey observations, the dis- III3 tribution of Fusarium wilt appears to correlate with ACZ, and specifically M E R U I5 temperature zone (Figures 2 and 3), III4 II3 IV6 III2 but not with AEZ or soil type. The dis- III6 ease was generally restricted to temper- E quator IV5 0°N ature zones 1, 2, and 3 (0-1500 m asl) I4 II2 but was most prevalent in temperature Mt Kenya III6 I6 zone 3 (20-22°C, 1200 to 1500 m asl). I5 Temperature zone 1 (24-30°C) is the N I T H I predominant zone in the coastal strip, III6IV5 III5 II3 III2 I6 I6 I5 except at Taita hills (temperature zone II6 IV4 E 2). The coast region of Kenya generally II4 II3 IV1 has hot and humid conditions, which do I6 N Y R I District E M B U seem to favour development of the dis- I4 III4 boundary IV2 ease. In the Central and Eastern Provinces the disease was also found in I6 M U R A N G A K I R I N Y A G A IV3 Fusarium wilt zones I4 temperature zone 2 (22-24°C, 900-1200 Temperature m asl). The temperature zones 1, 2, and III3 zone 3 3 are also characterized by at least oc- III4 casional, if not frequent, water stress, IV3 Temperature 0 15 30 zone 2 particularly during the months of July, Kilometers August and September. It is during these periods that bananas appear to be worst affected by Fusarium wilt, ei- Figure 2. Correlation between Fusarium wilt distribution and agroclimatic zones in the ther because infestation is more likely Central/Eastern region of Kenya (the Roman numerals represent the moisture zones while the Arabic numbers indicate the temperature zones). The disease appeared most severe within the temperature and more rapid or simply because zone 3 but was also occasionally found in temperature zone 2. symptoms are more pronounced. Wilt

30 INFOMUSA — Vol 10, N° 1 was not observed in temperature zones

4 (18–20°C), 5 (16-18°C), or 6 (14- 34

16°C) where bananas also grow fairly ° well. E I4 II5 There are many reasons, direct and II4 indirect, why temperature may have an I3 effect of wilt development. These in- I3 clude the ability of the host plant to I3 I5 produce gels and tyloses that help to 0 I4 occlude vascular vessels and hence re- I3 strict movement of the pathogen within them. At temperatures most conducive III3 I3 to disease development (27-28°C), for I4 0° N example, gel structures in susceptible II3 II3 I4 hosts are much weaker than in resis- III3 II3 tant hosts, thus permitting systemic II4 colonization in the former (Beckman II5 1990). Although the mean annual tem- III3 perature for zone 3 for example is 22-24°C, the maximum temperature for III4 District Boundary this zone during the months of Decem- II4 II4 ber to March ranges between III3 26.4-30.4°C which is within the temper- II4 F usarium wilt zones atures conducive to disease develop- L a k e V i c ot r i a II3 I3 I5 ment in susceptible hosts. I4 I4 Temperature In addition to the absence of disease III3 I3 I3 zone 3 on the East African Highland bananas, I3 the disease was not found on the III3 I4 II3 Cavendish group either. The Cavendish 0 20 40 is obviously a suitable and available al- Kilometers ternative to the susceptible dessert bananas. II4 Although precise dates are unavail- able, it is known that Bluggoe and II5 Wang’ae were introduced into Kenya earlier than Gros Michel. Given that Figure 3. Correlation between Fusarium wilt distribution and agroclimatic zones in the Western region of Kenya (the Roman numerals represent the moisture zones while the Arabic numbers the first report of Fusarium wilt in indicate the temperature zones). The disease was restricted within the temperature zone 3. Kenya was in the Coast Province (possibly on Bluggoe) in 1952 and in the same year in Central Province (possibly on Wang’ae), it can be spec- ulated that Fusarium wilt of banana originated with these cultivars in Kenya. holder farmers. From the 1960s to the crop and switched to banana produc- middle of the 1970s, coffee production tion. Dessert cultivars, such as the wilt Impact of the disease on the livelihoods of on smallholder farms provided farmers susceptible Gros Michel, Wang’ae, and unemployed smallholder farmers with a substantial income (Turner et Muraru, were the most popular in The impact of Fusarium wilt of banana al. 1997). Farmers increased coffee urban centre markets and were in Kenya is considerable. At present it production areas at the expense of planted en masse. Without nurseries directly affects the livelihood of more other crops as coffee could generate to obtain suckers, farmers multiplied than one million people in the Coastal sufficient income to enable them to plantlets from existing material and in survey area, more than three million meet all of their basic necessities, in- this way no doubt exacerbated prob- in the Central and Eastern survey cluding food purchase. During this pe- lems caused by wilt. Gros Michel, lo- areas and more than over five and a riod coffee was the major earner of cally known as ‘Kampala’ in some re- half million in the Western survey area foreign exchange for Kenya and was gions, was initially introduced by a few (figures based on 1989 Kenya’s popula- referred to as ‘black gold’. However, farmers as a few suckers from a neigh- tion census and not including con- the 1978-1979 global oil crisis caused a bouring country in the late 1960s. sumers of bananas based in urban cen- significant increase in the price of pe- From this original source the cultivar, tres and other areas). Since the first troleum and led to a rise in the cost of which has a very narrow genetic base, report in 1952, the status of the dis- coffee production due to increased has gradually been distributed ease in Kenya was unclear for nearly costs of oil-dependent inputs. Between throughout many of the main banana- 40 years. The immense socioeconomic 1980 and 1990 real international growing regions, particularly in Cen- impact resulting from the devastating prices for Africa’s coffee exports fell tral and Eastern Kenya. Although the disease outbreaks in the 1980s, partic- by 70% (Turner et al. 1997). Earnings imported material may have been ularly in the Central and Eastern re- from coffee production fell far below pathogen-free, distribution of highly gions, was probably based largely on those for food crops such as bananas, susceptible planting material in this the declining status of coffee as a maize, and beans and, by 1986, most way may well explain the rapid, wide- major source of income for small- farmers had abandoned coffee as a spread and devastating spread

INFOMUSA — Vol 10, N° 1 31 of Fusarium wilt on this particular Kung’u J.N. 1998. Ecology, distribution and popula- Sombroek W.G., H.M.H. Braun & B.J.A. van der cultivar. tion structure of Fusarium oxysporum f.sp. cu- Pouw. 1982. Exploratory soil map and agroclima- bense in Kenya. PhD Thesis, University of Kent at tic zone map of Kenya, 1980 (Scale 1:1000000). Acknowledgements Canterbury, United Kingdom. Kenya Soil Survey, National Agricultural Labora- The authors would like to thank the UK Kung’u J.N. 1995. Fusarium wilt and other banana tories, Ministry of Agriculture, Republic of Department for International Develop- diseases in Kenya. InfoMusa 4: 14-16. Kenya. ment (DfID) for funding this work Kung’u J.N., L.K. Njuguna, J.M. Ong’aro & S. Mako- Stotzky G. & R.T. Martin. 1963. Soil mineralogy in through the Kenya Agricultural Re- kha. 1995. Investigations on Fusarium wilt of ba- relation to the spread of Fusarium wilt of bana- search Institute (KARI)/DfID Crop Pro- nana, nematodes, weevils and other factors asso- nas in central America. Plant and Soil tection Project. ciated wilt disease in Murang’a District, Kenya. 18: 317-337. KARI/ODA Crop Protection Project Interim Turner T.E., W.M. Kaara & L.S. Brownhill. 1997. So- References Report. cial reconstruction in rural Africa: a gendered Beckman C.H. 1990. Host responses to the patho- Kung’u J.N. & R.W. Gatumbi. 1989. Banana diseases class analysis of women’s resistance to export gen. Pp. 93-105 in Fusarium wilt of banana (R.C. in Kenya. Kenya Farmer No. 17, November 1989. crop production in Kenya. Canadian Journal of Ploetz, ed.). American Phytopathological Society, Nelson P.E., T.A. Toussoun & W.F.O. Marasas. 1983. Development Studies 18: 213-238. St Paul, Minnesota. Fusarium species. An illustrated manual for Wardlaw C.W. 1972. Banana diseases including Booth C. 1971. The Genus Fusarium. Common- identification. The Pennsylvania State University plantain and abaca. 2nd ed. Longman, London. wealth Mycological Institute, Kew, Surrey, Uni- Press, University Park and London. 193pp. 878 pp. ted Kingdom. Nirenberg H. 1976. Untersuchungen über die mor- FAO. 1978. Report on the agroecological zones pro- phologische und biologische Differenzierung in ject. Vol. 1: Methodology and results for Africa. der Fusarium Sektion Liseola [Research on the World Soil Resources Report no. 48. FAO, Rome. morphological and biological differentiation in Jaetzold R. & H. Schmidt (eds.). 1982a. Farm mana- the Fusarium Section Liseola]. Mitteilunngen gement handbook of Kenya. Volume II/A: West aus der Biologischen Bundesantsalt für Land- Kenya. 360pp. und Forstwirtschaft, Berlin-Dahlem 169: 1-117. J.N. Kung’u currently works at the Plant Pathology Jaetzold R. & H. Schmidt. (eds.) 1982b. Farm mana- Ploetz R.C., D.R. Jones, K. Sebasigari & W.K. Tushe- Section, National Agricultural Research Laboratories, Kenya Agricultural Research Institute, PO Box 14733, gement handbook of Kenya. Volume II/B: Central mereirwe. 1995. Panama disease on the East Nairobi, Kenya. M.A. Rutherford works at the Uni- Kenya. 686pp. African Highland bananas. Fruits 49: 253-260. versity of Kent at Canterbury, Kent CT2 6NJ, United Jaetzold R. & H. Schmidt (eds.) 1982c. Farm mana- Smith D. and A.H.S. Onions. 1983. The preservation Kingdom. P. Jeffries works at CABI Bioscience, UK gement handbook of Kenya. Volume II/C: East and maintenance of living fungi. Commonwealth Centre, Bakeham Lane, Egham, Surrey TW20 9TY, Kenya. 328pp. Mycological Institute, Kew. United Kingdom.

Country report Study of Fusarium diversity Vegetative compatibility groups of the populations of Fusarium oxysporum f.sp. cubense in Vietnam

Do Nang Vinh, Nguyen Van Khiem, fungal populations. The exchange of ge- Spores isolated from discoloured vascu- Chu Ba Phuc and Le Huy Ham netic information in an asexual popula- lar strands dissected out of banana tion is limited to the individuals that plants affected by Fusarium wilt were can form a viable heterokaryon. The maintained on sterile filter paper as de- usarium wilt (Panama wilt) dis- loci that govern heterokaryon incom- scribed by Correll et al. (1986). Nitrate ease caused by Fusarium oxyspo- patibility are referred to as het, tal, vc non-utilizing (nit) mutants were gener- Frum Schlecht. f.sp. cubense (E.F. and vic (Leslie 1990). The het loci be- ated by transferring pieces of colonized Smith) W.C. Snyd. & H.N. Hans (Snyder have as if they were part of a recogni- filter paper onto potato dextrose agar et al. 1940), is regarded as one of the tion system that enables individuals to (PDA) amended with 1.5% KClO3 and most significant threats to banana identify each other and to differentiate incubating for 7-14 days at 25°C. Chlo- (Musa spp.) production worldwide themselves from each other. The het rate-resistant mutants were transferred (Persley et al. 1987), including Vietnam loci can delimit the pathotypes of asex- to minimal medium (Puhalla 1995) and (Vakili 1968). ual phytopathogenic fungi, as occurs in were assigned to phenotypic classes as Fusarium oxysporum f.sp. cubense the genus Fusarium (Correll et al. described by Correll et al. (1987). Any (Foc) affects species of Musa and Heli- 1987, Ploetz 1990). nit 1 or nit 3 mutants were paired with conia, and strains have been classified Our objective in this study was to tester nit M mutants of four known into four physiological races based on characterize isolates from different VCGs (VCG 0123, 0124, 0124/5 and their pathogenicity to host cultivars: provinces of North Vietnam using vege- 0125) on minimal media with nitrate as race 1: Gros Michel (AAA), Lady Finger tative compatibility. the sole nitrogen source. The develop- (AAB); race 2: Bluggoe and closely re- ment of dense aerial mycelium at the lated clones (ABB); race 3: Heliconia Material and methods point of contact of the two nit mutants sp.; and race 4: Cavendish cultivars and In order to determine the VCGs to was an indication of complementation. all cultivars susceptible to race 1 and which belong the Vietnamese Foc popu- race 2 (Persley et al. 1987). lations, banana plants bearing symp- Results and discussion Vegetative compatibility groups toms of Fusarium wilt were sampled in Forty-two isolates of Foc were collected (VCGs) are a natural way of subdividing different provinces in North Vietnam. and tested from 11 districts of

32 INFOMUSA — Vol 10, N° 1 7 provinces in North Vietnam (Hanoi, (Silk AAB) were attacked by Foc race 1 f.sp. cubense, the incitant of Fusarium wilt of ba- Hatay, Hungyen, Vinhphuc, Phutho, (VCGs 0124, 0124/5, 0125, VCG 0124/ nana (Musa sp.). Australian Journal of Agricultu- Bacninh, Thuathienhue). Twenty-one 5-0125, VCG 0124-0124/5-0125). Infec- ral Research 41: 863-870. isolates from all seven provinces be- tion by race 4 on the Cavendish group Corell J.C., J.E. Puhalla & R.W. Schneider. 1986. longed to VCG 0124; 4 isolates from (AAA) has not yet been detected in Identification of Fusarium oxysporum f.sp apii Hanoi and Hungyen provinces belonged Vietnam. on the basis of colony size, virulence and vegeta- to VCG 0124/5; 2 isolates from Hanoi This study demonstrates the value of tive compatibility. Phytopathology 76: 396-400. and Hungyen provinces belonged to using vegetative compatibility analysis Correll J.C., C.J.R. Klittich & J.F. Leslie. 1987. Ni- VCG 0125; 2 isolates from Hungyen to assess variability in populations of trate non-utilising mutants of Fusarium oxyspo- province were vegetatively compatible Foc. It also gives an indication of the rum and their use in vegetative compatibility with both VCG 0124/5 and 0125; 13 iso- dissemination potential of strains of tests. Phytopathology 77: 1640-1646. lates from Hanoi, Hungyen and Bacninh this pathogen and contributes to the Leslie J.F. 1990. Genetic exchange within sexual provinces were vegetatively compatible more effective deployment of resistant and asexual populations of the genus Fusarium. with VCGs 0124, 0124/5 and 0125. All of banana cultivars. Pp. 37-38 in Fusarium wilt of banana (R.C. these Foc isolates are in race 1. It is important to select and create Ploetz, ed.). APS, St Paul, USA. Isolates that were cross-compatible new cultivars with resistance to Foc to Mai Van Tri. 1997. Vegetative Compatibility of iso- were recovered in this study, forming a replace infected Chuoi Tay and Com La. lates of Fusarium oxysporum f.sp. cubense in bridge between VCGs 0124, 0124/5, and It may be possible to use Cavendish cul- the MeKong Delta. Journal of Agriculture and 0125. They may represent a stage in the tivars to replant these varieties where Food Industry 6: 255-256. divergence and formation of the ‘new Foc is present. The results of our study Persley G.J. & E.A. De Langhe. 1987. Summary of VCG’. Similar results were obtained also indicate that it is essential to apply discussions and recommendations. Pp. 9-17 in with isolates studied in Australia by quarantine procedures to prevent race 4 Banana and Plantain Breeding Strategies (G.J. Brake et al. (1990). from being introduced to Vietnam from Persley & E.A. De Langhe, eds.). ACIAR Procee- Results of analysis showed that other countries. dings no. 21. ACIAR, Canberra, Australia. VCG 0124 and ‘VCG 0124-0124/5-0125’ Ploetz R. 1990. Population biology of Fusarium were widespread, being detected in Acknowledgements oxysporum f.sp. cubense. Pp. 63-76 in Fusarium North Vietnam. No isolate was identi- The authors wish to acknowledge the fi- wilt of banana (R.C. Ploetz, ed.). APS, St Paul, fied as belonging to VCG 0123 in North nancial support of the World Bank for USA. Vietnam, whereas Mai Van Tri (1997) this study. We are also grateful to Dr Na- Puhalla J.E. 1985. Classification of strains of Fusa- collected and analyzed 8 isolates from talie Moore, Mr Ken Pegg and Mr Bob rium oxysporum on the basis of vegetative 6 districts of 4 provinces in South Viet- Davis, QDPI (Department of Primary compatibility. Canadian Journal of Botany nam and showed that 5 isolates be- Industries, Queensland, Australia) who 63: 179-183. longed to VCG 0123, and 3 isolates be- supervised the VCG techniques and pro- Snyder W.C. & H.N. Hansen. 1940. The species longed to VCG 0124/5. This can indicate vided the testers. concept in Fusarium. Am. J. Bot. 27:64-67. that VCGs 0123 and 0124/5 were wide- Vakili N., L.V. Thai & V.N. Dinh. 1968. Chuoi Viet spread in South Vietnam. In 1998, Bentley References Nam - Phuong phap trong tia, cai thien. Vien et al. analyzed 21 isolates from Bentley S., K.G. Pegg, N.Y. Moore, R.D. Davis & I.W. khao nghien cuu Nong nghiep Saigon. 7 provinces in North, Centre, and South Buddenhagen. 1998. Genetic variation among ve- Vietnam. They showed that 5 isolates getative compatibility groups of Fusarium oxy- belong to VCG 0124/5, 11 isolates be- sporum f.sp. cubense by DNA fingerprinting. long to VCG 0123, and 5 isolates belong Ecology and Population Biology 88(12): 1283- to VCG 0124-0125. 1293. Results of analysis indicated that Brake V.M., K.G. Pegg, J.A.G. Irwin & P.W. Langg- Chuoi Tay (Pisang Awak ABB), Chuoi don. 1990. Vegetative compatibility groups within The authors work at the Institute of Agricultural Ge- Ngop (Bluggoe ABB) and Chuoi Com La Australian populations of Fusarium oxysporum netics, Tu liem, Hanoi, Vietnam.

Country report Focus on… Black Sigatoka disease (Mycosphaerella fijiensis Morelet) in Mexico

M. Orozco-Santos, gions in the world (Fullerton 1994, Fuller- The presence of the disease in Mex- J. Farías-Larios, ton and Stover 1990, Mourichon and ico has caused serious losses in all the G. Manzo-Sánchez and Fullerton 1990). Black Sigatoka was iden- banana production regions by disturb- S. Guzmán-González tified in America for the first time in 1972 ing plantation management, in particu- (Stover and Dickson 1976) in Honduras, lar with regard to fungicide spray pro- from where it spread to all the countries in grammes. This has resulted in lack Sigatoka, caused by the as- Central America, to Mexico and to part of increased production costs. Today, con- comycete fungus Mycosphaerella South America (Fullerton and Stover 1990, trol of the disease in Mexican planta- Bfijiensis Morelet (teleomorphic) or Stover 1980). In Mexico, it was first identi- tions depends mainly on chemicals Paracercospora fijiensis (Morelet) fied in the south-east part of the country whose effects are completed by certain Deighton (anamorphic), is the most seri- in Chiapas and Tabasco states (Contreras cultural practices. The work presented ous disease affecting the commercial pro- 1983) and it is currently found in all the here is aimed at providing information duction of banana and plantain banana and plantain production regions about the current situation with regard (Musa spp.) in most of the production re- (Orozco-Santos 1998). to the disease in the banana and plan-

INFOMUSA — Vol 10, N° 1 33 for reasons of non-productivity and the UNITED STATES OF AMERICA losses were estimated to be 50 000 tonnes of fruits. Abandoned areas totalled 5000 ha in March 1991, a 50% de- Veracruz crease in the cultivated area (Orozco- Oaxaca Santos et al. 1996). Today, only 4700 ha is cultivated in Colima state (Orozco- Romero et al. 1998). The appearance of black Sigatoka in Tabasco Mexico has resulted in changes in plantation management and especially in GOLF OF fungicide spray programmes. Prior to Nayarit MEXICO the 1980s, yellow Sigatoka was the most serious phytosanitary problem for the Jalisco Chiapas foliage of cultivated species but did not require strict fungicide spray pro- Colima grammes. The arrival of black Sigatoka Michoacán considerably changed the control pro- CENTRAL PACIFIC SOUTH PACIFIC grammes, requiring the use of more powerful fungicides at shorter intervals. It is estimated that measures to control Figure 1. Localization of banana production zones in Mexico. Black Sigatoka represent 35 to 45% of total production costs. In parallel, changes involved greater cultural tech- tain production regions in Mexico and spread to all the other production re- nical skills (mineral nutrition, popula- to review various aspects of epidemiol- gions in the country (Stover 1962). It is tion density, deleafing, desuckering, ogy, treatments and research. now being pushed back by black Siga- pest, disease and weed control), im- toka. This is probably the result of the proving fruit quality and per-hectare The importance of bananas greater aggression and better adapt- yields (Orozco-Santos 1998). in Mexico ability of M. fijiensis in tropical regions Today, chemical methods form the Banana and plantain are grown in Mex- whose elevation does not exceed 500 m, most reliable alternative for controlling ico on 72 700 ha with production of as reported by Mouliom-Pefoura and the disease. However, this has in- 2.2 million tonnes of fruits, 95% of Mourichon (1990) and by Mouliom-Pe- creased production costs and has also which is for the domestic market foura et al. (1996). The first official no- led to problems of environmental pollu- (Orozco-Romero et al. 1998). The pro- tifications of outbreaks of M. fijiensis tion, public health and resistance to duction zones are located in the tropi- were made in Chiapas and Tabasco fungicides caused by residues of chemical regions around the Gulf of Mexico states in 1981. However, the disease had cals and protective substances (citro- and on the Pacific coast. The main pro- been observed for the first time in the line). Nearly 370 million pesos (US$ 43 ducer states are Chiapas, Veracruz, Tapachula zone (Chiapas) at the end of million) is spent in Mexico each year on Tabasco, Nayarit, Colima, Michoacan, 1980 (Contreras 1983). Black Sigatoka the control of black Sigatoka. Some Oaxaca, Jalisco and Guerrero. These has since continued to spread rapidly 430 000 kg of active substances was ap- form three main production regions: and reached Veracruz and Oaxaca plied until 1995, consisting mainly of the Gulf of Mexico with 4.6% of the cul- states in 1985 (Robles et al. 1988). systemic fungicides, and nearly 13 mil- tivated area, Central Pacific with 24.4% In the Central Pacific region, the dis- lion litres of citroline, i.e. an average of and Southern Pacific with 30.1% (Fig- ease was first detected in Colima state 184 litres per hectare per year. Today, ure 1). The taxonomic groups most in 1989 and a year later had reached control programmes using protective strongly represented in Mexico are AAA neighbouring Michoacan, Jalisco and fungicides have significantly reduced (‘Gran Enano’ and ‘Valery’, Cavendish Guerrero states. The disease was ob- the amounts of citroline or spray oil. subgroup), AAB (‘Macho’ or ‘False served in Nayarit state in November Nevertheless, the quantity of fungicide Horn’ and ‘Dominico’, Plantain sub- 1994 (Orozco-Santos et al. 1996). This active ingredients per unit area has in- group), AAB (‘Manzano’ or ‘Silk’), ABB makes it possible to affirm that black creased and the total has reached (‘Pera’ or ‘Cuadrado’) and AA (‘Dátil’). Sigatoka is now found in practically all 7 million kg per year in Mexico as a Information about production regions, the Musaceae production zones in Mex- whole (Orozco-Santos 1998). taxonomic groups and cultivated areas ico (Orozco-Santos 1998). Little research has yet been con- in Mexico is grouped in Table 1. ducted on the environmental impact The main climate and elevation char- Impacts of the disease and and the health problems resulting from acteristics of the production regions in chemical control the continuous application of fungi- Mexico are shown in Table 2. Black Sigatoka has had a devastating cides and citroline in banana planta- effect on the banana zones of Mexico. tions. However, we know that certain The distribution of black Sigatoka The first epidemic caused losses of 50 fungicides and bactericides are highly in America to 100% of fruit production and a con- toxic and act as molecular inducers of For many years, the disease called siderable decrease in cultivated areas. the cellular activity responsible for the ‘chamusco’ or yellow Sigatoka, caused by The disease caused the disappearance endocrine functions that regulate the the fungus Mycosphaerella musicola of some 2000 ha of bananas in Tabasco hormonal control of reproduction, sex Leach, was the most serious disease of state in the early 1980s. In Colima differentiation and the proliferation of banana and plantain leaves in Mexico. It state, where it was detected eight immuno-competent cells (Chambers first arrived in 1936 in the south-eastern months later in September 1989, over and Yarbrough 1982). Both human be- states (Chiapas and Tabasco) and then 3000 ha of plantations were grubbed up ings and fauna are exposed to fungi-

34 INFOMUSA — Vol 10, N° 1 cides and bactericides as a result of Table 1. Banana and plantain production regions in Mexico. aerial spraying, contaminated food- Region (states) Taxonomic groups Cultivated areas (ha) stuffs and polluted potable water sup- Gulf of Mexico plies. Aerial spraying is certainly a Tabasco AAA, AABp*, AA 12 900 rapid technique for applying chemicals Veracruz AAA, AABp 14 200 to very large areas. However, water run- Oaxaca AAA, AABp, AAB 3 900 ning from storage sites, runways and Central Pacific sprayed zones can pollute the neigh- Colima AAA 4 700 bouring aquatic and land environments Michoacán AAA 4 700 (Henriques et al. 1997). Jalisco AAA 1 800 The fungicide propiconazole was used Nayarit AAA, AAB, AABp, ABB 6 600 in Mexico for 20 years to control black Southern Pacific Sigatoka and high concentrations are Chiapas AAA, AABp 21 900 found in drainage water next to banana Other AAA, AAB, AABp, ABB 2 500 plantations, as demonstrated in Costa National 72 900 Rica, where concentration of 24.2 µg * AABp = plantain subgroup. Source: Orozco-Romero et al. (1998). per L water have been observed (Mortensen et al. 1998). Mancozeb be- Table 2. Climate and elevation of the production regions in Mexico. came the key fungicide in control pro- Region Climate Temperature Precipitation Number of Elevation grammes from 1995 onwards. In Costa (mm) dry months (m) Rica, measurement in channels after Gulf of Mexico hot and humid 24-27 °C 1700 to 3900 0 to 2 10 to 80 one spray revealed mancozeb residues Central Pacific hot and dry 26-28 °C 700 to 1100 7 to 8 10 to 500 3 ranging from 0.77 to 2.38 µg/cm Southern Pacific hot and subhumid 26-27 °C 1500 to 2500 4 to 5 20 to 80 (Mortensen et al. 1998). Chlorothalonil has been found to be toxic for aquatic invertebrates and fish, while mancozeb is carcinogenic and benomyl is terato- spread throughout Mexico in spite of chemical control, the incubation period genic (Lacher et al. 1997). the long distances (over 1000 km) and running from infection to the early Furthermore, the intensive use of sev- the natural barriers (mountain ranges) stage (2 on Fouré’s scale) lasts for 24 to eral systemic fungicides has resulted in between banana zones or regions. In 39 days and the spot stage (4 on Fouré’s the appearance of resistance in the fun- only 14 years, the disease spread to all scale) from 33 to 58 days. In the dry gus M. fijiensis (Castro et al. 1995, the states in which banana and plan- season (December to May), incubation Romero and Sutton 1997, 1998). This is tain are grown. The spread of the time is 48 to 87 days until the early because some types of systemic fungi- pathogen can be ascribed to move- stage and 84 to 141 days until spotting cides (benzimidazoles and triazoles) ments of infected plant material (dried stage. The most serious damage is display strong activity at small doses foliage) during transport of fruit caused to the most recent leaves. The and act on a single site in the pathogen (Orozco-Santos et al. 1996), of infected leaves emitted from June to October (Russell 1995). Problems of resistance plants or rhizomes and wind. M. fijien- are totally destroyed over a period of 82 mean that the control of black Sigatoka sis ascospores are the main source of to 120 days, whereas those emitted has become more complex and more ex- inoculation and dispersion for long dis- from November to May resist for 135 to pensive since the loss of susceptibility tances in a given zone (Burt et al. 1997, 200 days. The most marked severity is to fungicides means more sprays. Stover 1980). closely linked with the rainy season (June to October) and that of dew for- Actions undertaken to prevent The behaviour of black Sigatoka mation on leaves (November to Janu- the spread of the disease Gulf of Mexico. Several epidemiologi- ary). These results show that under dry The presence of black Sigatoka in the cal studies were performed in the tropical conditions black Sigatoka dis- banana-growing regions in south-east Tabasco region (Avila et al. 1994). Re- plays an epidemic phase induced by Mexico led the Dirección General de search on the disease is fairly rare in rainfall and a low-severity phase in the Sanidad Vegetal to impose permanent the other Gulf of Mexico production dry season (Orozco-Santos 1998). domestic quarantine No. 18. The main zones (San Rafael, Veracruz and Tuxte- Southern Pacific. Information gath- aim of this measure is to prevent or pec, Oaxaca). Early symptoms (1 and 2 ered in a banana plantation with inade- delay the arrival of the disease in ba- on Fouré’s scale) in banana plantations quate chemical control showed that the nana zones or regions that are still free with no chemical control appear 18 to most serious damage (severity 12 to of it. Respect of the following points 32 days after contamination and leaf 25%) occurs from June to December, was recommended in the campaign: spots appear after 34 to 73 days. Full the season with the heaviest rainfall. 1. restriction of movements of plant ma- development of the symptoms can take During this period, 25 to 58% of in- terial from infected zones; from 50 to 115 days. The longest incuba- fected leaves 4 to 6 are at the spotted 2. the establishment of quarantine station is seen during the driest period of stage. The period of least severity (Jan- tions; the year. The disease is endemic and its uary to May) coincides with that of the 3. forbidding the use of leaves to protect severity varies with the climatic condi- lowest rainfall, during which spotting is fruits in vehicles during transport; tions. The most severe period is during observed on 7 to 25% of infected leaves 4. disinfection of vehicles; the most rainy weather and reaches 15 7 to 9 (Escudero, unpublished data). 5. inspection of banana plantations; to 25% from July to December. The dis- 6. application of fungicides; ease is less aggressive from January to Treatment of black Sigatoka 7.grubbing up the most severely af- March with average severity of 5 to 10% Treatment of the disease in banana plan- fected plantations. (Ramírez and Rodríguez 1996). tations is strongly dependent on fungi- This quarantine did not have the Central Pacific. From June to Novem- cides. Their action is completed by a hoped-for effects and the disease ber in banana plantations with no number of cultural practices (deleafing,

INFOMUSA — Vol 10, N° 1 35 desuckering, drainage, weed manage- Table 3. Lines of research on black Sigatoka in Mexico. ment and mineral nutrition) aimed at Production regions reducing the sources of inoculation and Lines of research Gulf of Mexico Central Pacific Southern Pacific preventing the coinciding of conditions (Tabasco) (Tolima) (Chiapas) favourable for pathogen development Biology of the fungus X X (Marín and Romero 1992). Until 1995, Epidemiology X X X chemical control was performed using Cultural practices X X X systemic fungicides belonging to the tri- Chemical control X X X azole group (tebuconazole, propicona- Biological warning X zole, bitertanol and hexaconazole), the Biological control X pyrimidines (fenarimol), the benzimi- Evaluation of germplasm X X X dazoles (benomyl, carbendazim and Susceptibility to fungicides X X thiophanate-methyl) and morpholines Genetic diversity1 XX 1Studies performed by the Centro de Investigaciones Científicas de Yucatán (A. James, personal communication), Universidad de (tridemorph). The strobilurines (azox- Colima and the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. istrobin) and other triazoles (fenbu- Note: The genetic transformation studies are carried out by the Centro de Investigaciones Avanzadas (CINVESTAV) of the Instituto conazole) have been added more re- Politécnico Nacional (Gómez-Lim 1998). cently (Orozco-Santos 1998). Contact fungicides (chlorotalonil and mancozeb) were also included in the spray fungicides such as mancozeb, spraying the fungus (Marín and Romero 1992, programmes. Today, the use of protec- is required weekly during the rainy Stover 1990, Wielemaker 1990). tive fungicides is increasing in all pro- season and every 10 to 14 days during duction zones (Escudero and Rendón the dry season, making a total of 30 to Research on black Sigatoka 1996) with applications at 7 to 12-day 35 sprays during the year. in Mexico intervals. In the Southern Pacific region, up to Research on the disease has been ori- The traditional systemic fungicide/pro- 35 sprays were required each year in ented towards the biology of the tective substances programme in the the traditional systemic fungicide/pro- pathogen, epidemiology, the evaluation Gulf of Mexico required 20 to 25 sprays tective substances programme, with of plant material, chemical control, bio- in the San Rafael zone in Veracruz and systemics sprayed every 10 to 14 days logical warning and, more recently, re- 30 to 35 in the Tabasco zone. During the during the rainy season and an alterna- sistance to fungicides, genetic diversity rainy season, systemic fungicides were tion of systemic and protective fungi- of the pathogen and genetic transfor- used alone or mixed every 10 to 12 days cides during the dry season. In this re- mation, the latter being carried out out- and contact fungicides were sprayed gion, as around the Gulf of Mexico, only side of the banana and plantain produc- every 14 days during the dry season protective fungicides are used (mainly tion zones (Table 3). (Ramírez et Rodríguez 1996). Spray chlorotalonil) (Escudero and Rendón programmes have been introduced re- 1996). Applications are performed Conclusions and prospects cently that consist solely of protective weekly during the rainy season and Since its appearance in Mexico in 1980, fungicides (mainly mancozeb) to avoid every 10 to 14 days during the dry black Sigatoka has become the main the use of citroline. Spray intervals vary season. phytosanitary problem in all the banana from 7 to 12 days according to the time At the world scale, chemical control of and plantain production zones. The dis- of year. Protective programmes result in black Sigatoka is considered to be a ease has adapted to various environ- 40 to 52 sprays per year. high-risk activity because of the prob- mental conditions and the pathogen In the Central Pacific region, the lems of the resistance developed by the has become more aggressive, making number of sprays of systemic and pro- fungus to several fungicide groups. There farming more difficult and increasing tective fungicides varies from 15 to 20. have been numerous publications on the production costs. Incidence is less The disease is controlled during the loss of susceptibility of M. fijiensis to marked in the dry tropical region (Cen- rainy season (June to October) and the benzimidazoles (Romero and Sutton tral Pacific) than in the humid tropical dew season (November to January) by 1998, Stover 1979) and more recently to regions (Gulf of Mexico and Southern spraying systemic fungicides every 14 triazoles (Castro et al. 1995, Romero Pacific) because of differences in pre- to 21 days, whereas protective or sys- and Marín 1990, Romero and Sutton cipitation depth and distribution. In temic fungicides are sprayed every 25 1997). The evaluation of new fungicide 20 years, the disease has spread to all to 40 days in the dry season (January substances with little or no harmful ef- the banana production zones, where to May) (Orozco-Santos 1998, Orozco- fects on the environment and health is chemical control is the most commonly Santos et al. 1996). Recent studies becoming a priority in the search for used response. However, as time passes, have shown that with the help of the new ways of managing the disease. This it seems that fungicide application is biological warning system proposed by group includes azoxistrobin, which is not a suitable solution because of the Marín and Romero (1992), only 10 to safe from the environmental point of complex nature of the pathogen (type 12 sprays are needed during the rainy view. In addition, a new substance of reproduction, pathogenicity and and dew seasons and no sprays are called acibenzolar-S-methyl has come spread among other features) and the necessary during the dry season on to the market (Madrigal et al. 1998); host characteristics (genetic unifor- (Orozco-Santos 1995). Control of the this is reported to activate natural mity, extensive planting, etc.), which disease has been found to be inade- plant defences, a phenomenon known have facilitated a very close host-para- quate in plantations intercropped with as acquired systemic resistance site relationship. Research should be coconut. Indeed, the trees mean that (Sticher et al. 1997). A small number of concentrated on sustainable manage- the aircraft must be flown at an alti- systemic fungicides are used in the con- ment of the disease aimed at reducing tude of 35 to 40 m, causing a loss of trol of black Sigatoka today and it is ur- the risks of environmental pollution part of the emulsion as it is deposited gent that they should be used properly and of danger to health and enable the on the palms (Orozco-Santos et al. to ensure them a longer useful life and conservation of natural resources. The 1996). In programmes using protective maintain their efficacy with regard to evaluation of genetic material with re-

36 INFOMUSA — Vol 10, N° 1 sistance to the disease (Orozco-Romero Henriques W., R.D. Jeffers, T.E. Jr. Leacher & R.J. Robles H.E., M.F. Velázquez, M. Ulloa & S.S. Del- et al. 1998) and genetic transformation Kendall. 1997. Agrochemical use on banana plan- gado. 1988. La Sigatoka negra del plátano en (Goméz-Lim 1998) are priority chal- tations in Latin America: perspectives on ecolo- México (Monografía). SARH, Dir. Gen. de San. y lenges to be taken up in the medium gical risk. Environmental Toxicology and Chemis- Prot. Agrop. y For. Dirección de Sanidad Vegetal. and long term in the research pro- try 16(1): 91-99. México. 60pp. gramme on Musaceae in Mexico. In Lacher T.E. Jr., S.R. Mortensen, K.A. Johnson & R.J. Romero R.A. & D. Marín. 1990. Observations on the the short term, it is important to con- Kendall. 1997. Environmental aspects of pesti- sensitivity of the Mycosphaerella fijiensis moni- tinue research on Cavendish subgroup cide use on banana plantations. Pesticide Out- toring method to triazole fungicides. Pp. 100-106 bananas (‘Gran Enano’ and ‘Valery’) look, December 24-28. in Sigatoka leaf spot diseases of bananas (Fuller- and on plantain cultivars in order to Madrigal A. 1998. CGA Z45704, a new plant activa- ton, R. A. and Stover, R. H. (eds.). Proceeding of improve management of the disease. tor to improve natural resistance of banana an international workshop held at San José, Studies on cultural control, biological against black Sigatoka (Mycosphaerella Costa Rica, March 28 – April 1, 1989. INIBAP, warning and the evaluation of fungi- fijiensis. Pp. 266-274 in Proceeding XIII ACOR- Montpellier, France. cide spray programmes according to BAT Meeting, Guayaquil, Ecuador. Romero R. A. & T.B. Sutton. 1997. Sersitivity of My- their impact on the environment will Marín V.D. y C.R. Romero. 1992. El combate de la cosphaerella fijiensis causal agent of black Siga- make it possible to reduce the number Sigatoka negra en banano. Corporación Bana- toka of bananas, to propiconazole. Phytopatho- of spray cycles. In parallel, it is very nera Nacional. Costa Rica. Boletín No. 4. 22 pp. logy 87: 96-100. important to conduct specific research Mortensen S.R., K.A. Johnson, C.P. Weisskopf, M.J. Romero R. A. & T.B. Sutton.. 1998. Characterization on the pathogen (genetic diversity and Hooper, T.E. Lacher & R.J. Kendall. 1998. Avian of benomyl resistence in Mycosphaerella fijien- pathogenic variability, epidemiology exposure to pesticides in Costa Rican banana sis, cause of black Sigatoka of banana, in Costa and susceptibility to fungicides) in plantations. Bulletin of Environmental Contami- Rica. Plant Disease 82: 931-934. order to develop strategies to control nation and Toxicology 60: 562-568. Russell P. E. 1995. Fungicide resistance: ocurrence the disease. Mouliom-Pefoura A. & X. Mourichon. 1990. Dévelop- and management. Journal of Agricultural pement de Mycosphaerella musicola (maladie Science, Cambridge 124: 317-323. References de Sigatoka) et M. fijiensis Morelet (maladie des Sticher L., B. Mauch-Mani & J.P. Métraux. 1997. Avila A.C., M. de E.M. Contreras & O.D. Teliz. 1994. raies noires) sur les bananiers et plantains. Systemic acquired resistance. Annual Review of Epidemiología de la Sigatoka negra en planta- Etude du cas particulier des productions d’alti- Phytopathology 35: 235-270. ciones comerciales de banano (Musa AAA, sub- tude. Fruits 45(1): 17-24. Stover R.H. 1962. Intercontinental spread of banana grupo Cavendish) en Tabasco, México. Pp. 257- Mouliom-Pefoura A., A. Lassoudiére, J. Foko & D.A spot (Mycosphaerella musicola Leach). Tropical 263 in Memorias de la X Reunión ACORBAT Fontem. 1996. Comparison of development of Agriculture (Trinidad) 39:327-338. (Contreras M.A.,Guzmán J.A. y Carrasco L.R., Mycosphaerella fijiensis and Mycosphaerella Stover R.H. 1979. Field observations on benomyl to- eds.). CORBANA, San José, Costa Rica. musicola on banana and plantain in the various lerance in ascospores of Mycosphaerella fijien- Burt P.J.A., J. Rutter & H. Gonzalez. 1997. Short dis- ecological zones in Cameroon. Plant Disease sis var. difformis. Trans. Br. Mycol. Soc. 69:500- tance windborne dispersal of the fungal patho- 80: 950-954. 502. gens causing Sigatoka diseases in banana and Mourichon X. & R.A. Fullerton. 1990. Geographical Stover R.H. 1980. Sigatoka leaf spot of bananas and plantain. Plant Pathology 46: 451-458. distribution of the two species Mycosphaerella plantains. Plant Disease 64: 750-755. Castro O., A. Wang & L.F. Campos. 1995. Análisis in musicola Leach (Cercospora musae) and M. fi- Stover R.H. 1990. Sigatoka leaf spots: Thirty years of vitro de la sensibilidad de Mycosphaerella fijiensis Morelet (C. fijiensis), respectively agents changing control strategies: 1959-1989. Pp. 66-74 jiensis a los fungicidas fenarimol, tridemorph y of Sigatoka disease and black leaf streak disease in Sigatoka leaf spot diseases of bananas (Fuller- propiconazole. Phytopathology 85: 382. in bananas and plantains. Fruits 45(3): 213-218. ton, R. A. and Stover, R. H. (eds.). Proceeding of Chambers J.E. & J.D. Yarbrough. 1982. Effects of Orozco-Romero J., G. Ramírez-Sandoval & V. Váz- an international workshop held at San José, chronic exposure to pesticides on animal sys- quez-Valdivia. 1998. Comportamiento del banano Costa Rica, March 28 – April 1, 1989. INIBAP, tems. Raven, New York, NY, USA. FHIA-01 y plátano FHIA-21 en México. Pp.112- Montpellier, France. Contreras M. de E.M. 1983. El chamusco negro (Si- 121 in Memorias del primer Simposium Interna- Stover R.H. & J.D. Dickson. 1976. Banana leaf spot gatoka) una nueva enfermedad de la hoja de los cional sobre Sigatoka Negra. SAGAR, INIFAP. caused by Mycosphaerella musicola and plátanos. Universidad Autónoma de Chapingo. Manzanillo, Colima, México. M. fijiensis var. difformis: a comparison of the México. Revista de Geografía Agrícola 4: 61-102. Orozco-Santos M. 1998. Manejo integrado de la Si- first Central American epidemics. FAO Plant Escudero M.C. & E.C. Rendón. 1996. Integrated ma- gatoka negra del plátano. SAGAR, INIFAP, Protection Bulletin 24: 36-42. nagement experiences with black Sigatoka (My- CIPAC. Campo Experimental Tecomán. Te- Wielemaker F. 1990. Practical notes on black Siga- cosphaerella fijiensis) and non-systemic fungi- comán, Colima, México. Folleto técnico No. 1. Di- toka control. Pp. 107-114 in Sigatoka leaf spot di- cides in Soconusco, Chiapas, Mexico. P. 53 in XII visión Agrícola. 95pp. seases of bananas (Fullerton, R. A. and Stover, R. Meeting ACORBAT. Santo Domingo, Republica Orozco-Santos M. 1995. Control químico de la Siga- H. (eds.). Proceeding of an international work- Dominicana (Abstract). toka negra (Mycosphaerella fijiensis) del plá- shop held at San José, Costa Rica, March 28 – Fullerton R.A. 1994. Sigatoka leaf diseases. Pp. 12- tano mediante el sistema de preaviso biológico. April 1, 1989. INIBAP, Montpellier, France. 14 in Compendium of tropical fruit diseases. XXII Congreso Nacional de la Sociedad Mexicana (Ploetz, R.C., Zentmeyer, G.A., Nishijima, W.T., de Fitopatología. Guadalajara, Jalisco, México. Rohrbach, K.G., and Ohr, H.D., eds.). APS Press. Resumen No. 19. St. Paul, Minnesota, USA. Orozco-Santos M., J. Orozco-Romero, J. Farias-La- Fullerton R.A. & R.H. Stover (eds.). 1990. Sigatoka rios & V.Vazquez. 1996. La cercosporiose noire leaf spot diseases of bananas: Proceeding of an (Mycosphaerella fijiensis) dans l’ouest du international workshop held at San José, Costa Mexique. Black Sigatoka (Mycosphaerella Mario Orozco-Santos works at Instituto Nacional de Rica, March 28 – April 1, 1989. INIBAP, Montpel- fijiensis) of bananas in the West of Mexico. Investigaciones Forestales Agrícolas y Pecuarias, Campo Experimental Tecomán. Apartado postal 88. lier, France. 374 pp. INFOMUSA 5(1): 23-24. Tecomán, Colima, Mexico 28100. E-mail: ctecoman Gómez-Lim M.A. 1998. Genetic transformation of Ramírez S.G. & C.J.C. Rodríguez. 1996. Manual de @volcan.ucol.mx , Javier Farías-Larios, Gilberto bananas: strategies to control sigatoka disease. producción de plátano para Tabasco y Norte de Manzo-Sánchez and Salvador Guzmán-González Pp.122-125 in Memorias del primer Simposio In- Chiapas. INIFAP. CIRGOC. Campo Experimental work at Facultad de Ciencias Biológicas y Agropecua- ternacional sobre Sigatoka Negra. SAGAR, IN- Huimanguillo. Tabasco, México. Folleto técnico rias, Universidad de Colima, Apartado postal 36. Te- IFAP. Manzanillo, Colima, México. No. 13. 80pp. comán, Colima, Mexico.

INFOMUSA — Vol 10, N° 1 37 Short communication Effect of number of subcultures on in vitro multiplication of four banana clones

N.D. Jambhale, ber of subcultures on micropropagation and rooted on MS media + 3 mg/l NAA, S.C. Patil, A.S. Jadhav, of banana clones. solidified with agar (8 g/l). Five S.V. Pawar and clumps, each with 3 shoots, were B.D. Waghmode Materials and methods transferred to fresh medium in jam Shoot tip explants of four clones: Bas- jars every 3 weeks for multiplication. rai (AAA), Nendran (AAB), Lal Kela Observations were recorded on in issue culture-propagated plants of (AAA) and Safed Velchi (AB) were es- vitro multiplication after the 8th, 10th, banana have been extensively tablished in vitro, multiplied on MS 12th and l4th subcultures. Plantlets Tplanted in India in recent years, medium + 6 mg/l BAP + 1 mg/l IBA hardened after every subculture were mainly to achieve a healthy, early and synchronously maturing crop. However, instances of occurrence of abnormal plants with changed morphology and Table 1. Multiple shoot formation rate of different clones according to subculture reduced vigour have been observed in cycle. some populations. This could be due to Clone Average number of multiple shoots/ bottle after the repeated subculture of the in vitro 8th subculture 10th subculture 12th subculture 14th subculture cultures. Potential hazards of tissue Basrai (AAA) 12.33 10.91 7.62 7.10 culture have been reported (Daniells Nendran (AAB) 8.63 6.22 5.47 5.92 1997). A study was therefore under- Lal Kela (AAA) 10.72 7.91 6.62 7.12 taken to investigate the effect of num- Safed Velchi (AB) 8.63 8.09 7.24 6.33

Table 2. Growth response of different clones, using plantlets derived after different numbers of subcultures. Clone Characters Growth response of four months old hardened plantlets taken from 8th subculture 10th subculture 12th subculture 14th subculture Basrai (AAA) Stem height (cm) 49.09 45.00 31.60 19.70 Stem girth (cm) 6.50 6.00 4.10 3.50 No. of leaves 12.00 12.00 9.00 7.00 Leaf breadth (cm) 10.50 9.80 6.20 4.90 Leaf length (cm) 20.50 18.90 15.70 11.60 Leaf colour Dark green Light green, slight Pale green waxy, Yellowish, waxy, leathery leaves leathery leaves leathery leaves Vigour Normal Slightly stunted Medium stunted Highly stunted Nendran (AAB) Stem height (cm) 55.0 50.3 36.7 27.2 Stem girth (cm) 7.0 6.2 5.1 4.4 No. of leaves 10.0 8.0 7.0 6.0 Leaf breadth (cm) 7.5 8.2 4.23 3.3 Leaf length (cm) 27.0 23.4 17.4 12.33 Leaf colour Dark green Light green Pale green, Highly leathery leathery leaves leaves Vigour Normal Slightly stunted Medium stunted Highly stunted Lal Kela (AAA) Stem height (cm) 59.0 53.0 39.2 29.3 Stem girth (cm) 5.1 4.3 3.8 2.7 No. of leaves 8.0 6.0 6.0 5.0 Leaf breadth (cm) 7.8 6.3 5.1 4.7 Leaf length (cm) 18.2 15.2 13.3 11.7 Leaf colour Dark green Pale green Pale green, Yellowish, waxy, leathery leaves leathery leaves Vigour Normal Slîghtly stunted Medium stunted Highly stunted Safed Velchi (AB) Stem height (cm) 52.7 52.2 40.3 35.1 Stem girth (cm) 6.2 6.0 5.0 4.6 No. of leaves 9.0 8.0 8.0 6.0 Leaf breadth (cm) 6.6 6.5 5.8 5.2 Leaf length (cm) 20.4 18.2 18.2 16.0 Leaf colour Dark green Pale green Pale green, Pale green, leathery leaves leathery leaves Vigour Normal Slightly stunted slightly stunted Medium stunted

38 INFOMUSA — Vol 10, N° 1 Table 3. Spectrum of variants clones observed in tissue-cultured plants of different banana. Clone Variant Description Basrai Variant 1 Tall vigorous plantlet, stem pale green, broad long leaves with faint blotching and red margin to peduncle pale green. Variant 2 Tall lanky plantlet with long pale green peduncle, stem pale green, long narrow leaves without blotching. Nendran Variant 1 Dwarf, light red stem and peduncle, broad short leaves with red margin and light blotching. Variant 2 Dwarf, light red stem and peduncle, narrow long leaves with short peduncle, green leaf margin. Variant 3 Dwarf, light red stem and peduncle, narrow long leaves with short peduncle and red leaf margin. Variant 4 Tall purplish green stem and peduncle, broad and long leaves with red leaf margin Lal Kela Variant 1 Dwarf, light red stem and peduncle, broad and long leaves with light red leaf margin Safed Velchi - NIL-

Table 4. Frequency of occurrence of variants in different subcultures of the banana clones. Name of clone No. of plants and frequency of variants after 8th subculture 10th subculture 12th subculture 14th subculture Basrai —- Variant 1: 3 (1.0%) Variant 1: 11 (1.89%) Variant 1: 31 (3.44%) —- —- Variant 2: 9 (1.54%) Variant 2: 19 (2.11%) Nendran —- Variant 1: 18 (7.14%) Variant 1: 35 (11.20%) Variant 1: 46 (15.43%) —- Variant 2: 22 (8.73%) Variant 3: 48 (15.38%) Variant 2: 30 (10.06%) —- —- —- Variant 3: 30 (10.06%) Lal Kela —- Variant 1: 7 (3.00%) Variant 1: 12 (4.8%) Variant 1: 28 (7.20%) Safed Velchi 0.00 0.00 0.00 observed for their growth in the green- Some plantlets which were conspicu- growth and vigour of the hardened plants house. ously distinct from the parental clones and the increased number of somaclonal were observed in the populations of variations observed after 8th subculture, Results and discussion hardened plants after the 8th subcul- it may be that for some clones, the num- Multiple shoot formation rate varied ture (Table 3). The percentage of vari- ber of subcultures in micropropagation with the clones (Table 1). Average num- ants varied in the different genotypes should be restricted to eight. ber of multiple shoots/bottle after the under study. Gomez and Garcia (1997) 14th subculture was maximum in Basrai also reported similar results. Variations References (12.33) followed by Lal Kela (10.72) with regards to stature, pigmentation, Daniells J. 1997. Beware the potential hazards of and minimum in Nendran and Safed growth, peduncle and leaf size etc. were tissue culture. INFOMUSA 6(2): 17-18. Velchi (8.63). Multiple shoot formation seen in the 10th, 12th and 14th subcul- Daniells J.W. & M.K. Smith. 1993. Somatic muta- rate declined eventually with increase tures of all the clones except Safed tions of bananas -their stability and potential. in the number of subcultures in all four Velchi. Nendran exhibited maximum Pp. 162-171 in International symposium on re- clones. The multiple shoots/bottle after variants in the 10th (15.87%), 12th cent developments in banana cultivation (R.V. the l4th subculture were 7.10 in Basrai, (26.58%) and 14th (36.49%) subcultures. Valmayor et al., eds) 1NIBAP/ASPNET, Los 7.12 in Lal Kela, 6.33 in Safed Velchi Basrai exhibited variant frequency of Baños, Philippines. and 5.92 in Nendran (Table 1). 1 to 5.55%, while percentage variation Gomez I.H. & E.G. Garcia. 1997. Agronomic study of The growth of the clones as measured was 15.87 to 36.49% in Nendran and 3 to Cavendish bananas grown by in vitro culture. by stem height, girth, number of leaves 7.20% in Lal Kela (Table 4). INFOMUSA 6(2): 23-26 and leaf size declined after the 8th sub- The percentage of variants in tissue culture, with some plants exhibiting culture-propagated plants as high as The authors work at the Plant Tissue Culture Labo- th very stunted growth after the 14 sub- 91 % has been reported previously ratory, Department of Agricultural Botany, Mahatma culture (Table 2). Safed Velchi was (Daniells and Smith 1993). Considering Phule Krishi Vidyapeeth, Rahuri 413-722, Dist. Ah- found to be less affected. the reduced multiplication rate, reduced mednagar, Maharashtra, India.

Errata in previous issues of INFOMUSA Screening Musa hybrids for resistance to Radopholus similis In Dochez et al. (p. 3-4), reference is made to TMP2x-47 and INFOMUSA 9(2) – December 2000 TMP2x-50. These hybrids should be referred to as TMP2x2521S-47 and TMP2x2521S-50, respectively. Distribution of Blood disease Consumer acceptability of introduced bananas in Uganda In the PROMUSA section (p IX), it was reported that blood disease In Nowakunda et al. (p. 22-25), it should read TMPx5511-2 instead had spread from Indonesia into West PNG. This should in fact have of TMPx5511/2 (table 1). The correct identification of the genome been reported as West Papua (Irian Jaya) and not PNG. Blood of the hybrid TMPx5511-2 is AAAB instead of AABB (see tables 1, 2, disease has not been reported from PNG. 3 and 5). Methodological considerations in the evaluation of banana bunch trimming (Musa AAA, cv. ‘Valery’) INFOMUSA 9(1) – June 2000 In Vargas and Blanco’s article (p.19), part ‘Material and methods’, In the Musanews section (p. 34-35), the name and contact address 3rd paragraph, it should read: “The following treatments were per- of the author of the research on ‘Medicinal weeds in banana formed on bunches with eight, nine and ten true hands at flower- orchards in Chhattisgarh, India’ was involuntarily omitted in the ing: 1) removal of two true hands and 2) removal of three true English version. It is as follows: P. Oudhia, Department of Agronomy, hands.“ Indira Ghandi Agricultural University, Raipur-492001, India.

INFOMUSA — Vol 10, N° 1 39 Screening of the germplasm collection complemented by in vitro breeding Musanews has revealed a number of resistant strategies, which include the creation Black Sigatoka outbreak diploid clones which are being used in of variability through mutagenesis and in Australia the hybridization programme. A num- the use of anti-mitotic agents to in- An outbreak of black Sigatoka has re- ber of promising synthetic diploids have crease ploidy levels. cently been reported from Queensland, been developed and these are being Australia. The area in question is near used in further crosses with cultivated Tully, in far north Queensland, approxi- diploids and triploids. Some of the Further information about the banana improvement mately 140 km south of Cairns. North- newly evolved synthetic hybrids appear programme of Tamil Nadu Agricultural University are available from K. Soorianathasundaram ern Queensland is the major producer to have good levels of resistance to and N. Kumar, Dept. of Pomology, Horticultural of bananas in Australia and the disease nematodes and leaf spot diseases and College and Research Institute, Tamil Nadu is deemed a significant threat to the re- acceptable agronomic characters. The Agricultural University, Coimbatore, India 561003. gion’s $200 million banana industry. conventional breeding programme is email: [email protected] There is concern that the outbreak could trigger a very significant increase The banana world loses two friends and colleagues in the cost of production of bananas in Australia. While far north Queensland has recorded eight outbreaks of black Ren Gonsalves Phil was born and schooled in Sigatoka in feral bananas within the INIBAP regrets to have to announce the Arkansas. After he graduated from past ten years, this is the first time the death of Reynold Gonsalves who died of Michigan State University, he moved disease has been detected in a com- cancer on February 15, 2001 at the age with his wife, Jeannette, to Honduras to mercial production area. In the past of 72. take up a post in what was then the quarantine authorities have used the Born in Cuba in 1928, Reynold Gon- United Fruit Company. He quickly be- presence of black Sigatoka in many salves, of Jamaican nationality, gradu- came responsible for the banana breed- central American countries as a reason ated from Howard University, Washing- ing programme and has continued to to reject import applications. If this ton with a Bachelor of Science Degree, lead the research ever since, seeing outbreak is not eradicated, it is ex- majoring in biology. through the transition from a private pected that there will be renewed pres- In 1952 he became Senior Station enterprise to a government research in- sure for banana imports to be allowed Master at the Banana Breeding Station, stitute, the Fundación Hondureña de into Australia. Jamaica and Senior Plant Breeder in Investigación Agrícola (FHIA). 1969. Ren was particularly known in the The exceptional FHIA hybrids, which Banana breeding in India international Musa community for his Phil developed, are some of the best Banana breeding was initiated at the research on breeding bananas with re- performing banana varieties in the Central Banana Research Station, sistance to Panama disease and to both world. Eight varieties have been made yellow and black Sigatoka diseases. He available for trials in the International was awarded the order of distinction in Musa Testing Programme. They have the rank of commander for his contri- proved resistant to multiple diseases bution to agriculture. and pests, highly yielding and consis- Reynold’s work made a considerable tent in performance over wide ranging contribution to international Musa environmental conditions. Indeed on breeding and he participated in numer- the strength of their results, these various international conferences and eties have been selected for adoption meetings. He was a prominent figure in and are gradually being taken up in the INIBAP Regional Network for Latin many banana-producing areas. They America and the Caribbean and his will particularly benefit the smallholder participation and input was much producers, farming in marginal areas appreciated. without pesticides and fertilizers. Reynold Gonsalves was Managing Where the hybrids have been intro- Director of the Jamaican Banana Board duced, they have been taken up with since 1996. He was married and has 4 alacrity by farmers. Ongoing projects sons and 1 daughter. His other great in- distributing the varieties to small-scale terest in life was horseracing and he producers in Nicaragua and Tanzania, was Chairman of the Jamaica Racing have resulted in preliminary increases Commission. in yields by a third. However, Cuba, having adopted the FHIA hybrids on the Phil Rowe widest scale, provides the most enlight- On Sunday, 25 March 2001, Dr Phillip R. ening example. Increased yields with- Aduthurai, Tamil Nadu, in 1949. This Rowe died in La Lima, Honduras at the out the use of pesticides have had an was one of the first systematic banana age of 62 years. For over thirty years he immediate and impressive impact on improvement efforts in India. The dedicated his career to breeding ba- farmers’ incomes. breeding work initiated here has been nanas and plantain. He developed a Phil’s conscientious dedication to his continued since 1971 at the Tamil Nadu number of important improved varieties work will continue to bring benefits to Agricultural University at Coimbatore. that are now being distributed around millions of people worldwide. His gen- The University maintains a collection of the world, from Florida to Uganda, erosity, humour and compassion will, 127 distinct accessions and work is fo- where they are bringing substantial re- no doubt, be remembered by a more cused on hybridisation and selection of lief from the effects of banana pests intimate circle of friends, colleagues offspring with resistance to nematodes, and diseases, most notably black Siga- and people who benefited from his leaf diseases and Fusarium wilt. toka and Fusarium wilt. kindness. He leaves behind him a wife,

40 INFOMUSA — Vol 10, N° 1 two sons and a grandson. In the follow- and never promised more than he could ing text, his long time colleague and expect or predict from the proposed friend, Franklin Rosales, INIBAP re- work. He did not ask even for a new ve- gional coordinator for Latin America hicle, when we were riding a very old and the Caribbean, describes some of and beaten up car. Mentioning his ma- his memories of Phil and celebrates terial desires, I can tell you that he his life. owned only two cars during his profes- sional life at Honduras (more than 30 In Memoriam years!): an old Chevy and a red Toyota Talking about a friend who has already car that Jeanette, his wife, had for left for a better place, is difficult to do. shopping in San Pedro Sula. The first One tries to summarize in a few lines one, he sold to a Missioner for about all the good things he has done, and $300 and he used to laugh when telling quickly finds out that it is not an easy the story. He said “I have never seen a task. Talking about Phil Rowe as a happier face than the one of the Mis- friend, scientist, father, brother, hus- sioner when I told him that the price band, adviser, counsellor, etc., is even for the Old Chevy was $300”. He was harder since he was so good at all of never worried about money or material them. Whatever one says about him it things, not because he was a rich fellow will be too little that the intention of but because he had a simple taste for highlighting his influence on this earthy life. He always said: “the ones with the life will always be insufficient. simple taste will survive and live more He was a “Good Samaritan”; better happily than the rest”. He could live than the one mentioned in the Bible, happily under a camping tent eating because he cared and loved people, not red beans and tortillas. He used to just once but every single day of his life. laugh when talking about his experi- Every day at the Breeding Station gate ence in the Stock Market where he at Guarumas, La Lima, or in the dirt tried once to bet on Silver. He said, road just close to the station, there “Franklin, I am so happy that I did not would be a long line of people waiting make any money because to tell you the to see him to ask for support. Support truth I would not know how to spend it that they always got without hesitation anyway”. from Phil’s side. More than one young Phil was a positive and enthusiastic boy or girl received “scholarships” from person, not just in the breeding work Phil for primary or secondary school. that was “his life and love” but also in Just how many - only Phil and God will all other activities. Bad times did not ever know since he tried very hard to exist for Phil; he always had hope in any prevent people from “discovering” the situation, no matter how bad it looked extent of his Charity Ministry. Para- to other people. He also had excellent plegics, widows, old timers, sick people and happy humour with a joke ready in were among those on his daily list of any situation. He used to start his protected ones. As his older son Mark breeding presentations with a joke and says “Something that we will never for- he was the first to laugh at it. get is my Dad’s interest in helping the He was a very quiet fellow. He with- poor. There was no one that came to our stood stormy times without fighting door that left without money, advice or back, even if those involved were being food. He grew up in a very humble fam- improper. He was a “Gandhi”, having a ily and that’s why he always wanted to “monk’s patience”, trying to solve all help the most needy ones”. problems in a peaceful manner. He I worked with Phil for more than 10 “fought” for his crew, to obtain better years and I can testify that these things working conditions and that was re- just touch the surface of Phil’s testi- flected every year when the Adminis- mony as a Christian, in the full and true tration gave him the chance to evalu- sense of that word. I will never forget ate his people: They always got the that happy smile on his face, no matter highest marks of all FHIA’s staff. He the size of the problem we were con- was very proud and dedicated to those fronted with and his open hand to give working with him, even if could result and assist any one asking for help. in a reprimand. He also tried in all pos- He was humble, including when we sible arenas to convince people that were reporting to donors. He always “traditional breeding” was the best al- said ”I like to see the donors’ eyes when ternative for the banana and plantain they come to see me and ask me where industry. Few people understood the the money went or what we are getting message or were willing to express from those funds”. When asked about views sharing or supporting Phil’s what he wanted for the breeding pro- dreams. In fact, very few people appre- gramme, his answer was quick and al- ciate the magnitude of his work and ways the same: “more pollinators”. He what it will mean for the world in the never asked for anything for himself; years to come. Among the few who un-

INFOMUSA — Vol 10, N° 1 41 to limit nematode damage on bananas, carrying out her studies both on-farm and in the laboratory. She will also be dedicating an equal amount of time to developing aspects of technology transfer and assisting in the West and Cen- tral Africa office. Mr Kamulindwa has joined IPGRI- INIBAP as Project Administrator for the Uganda Banana Biotechnology Pro- ject. He reported for duty on May 3, 2001. Before joining INIBAP, Mr Ka- mulindwa worked with the Ministry of Finance of Uganda, CRRE Interna- tional, CIAT-Africa and Heifer Project International, and comes to INIBAP with a wealthy experience in project management. He will be based at NARO-KARI at Kawanda for 75% of his time and 25% at INIBAP regional office in Kampala.

Asian Agriculture Congress Phil Rowe (center) with two Cuban farmers who obtained the world weight record A scientific conference, coorganized by for a bunch of FHIA-03: 84.5 kg! the Asian Crop Science Association (ACSA), the Society for the Advance- derstood and appreciated Phil’s work than later. I just hope that, one day, I ment of Breeding Research in Asia and are the Cubans. We travelled together can go to same place in heaven where Oceania (SABRAO) and the Federation to Cuba and visited all the plots con- he is now. of Crop Science Societies of the Philip- taining FHIA’s hybrids; we went from Franklin E. Rosales pines (FCSSP) on “Food Security and Havana all the way to Guantanamo. Environment Protection in the New The gratitude expressed by the Cuban Millennium” took place in Manila, people at all levels was the best he Philippines, on 24-27 April. INIBAP News ever got and I am pretty sure that he INIBAP and IPGRI shared a stall, appreciated it and kept it very deep in New recruitments which they used to illustrate how ba- his heart. As Jose Manuel Alvarez from Kim Jacobsen is joining INIBAP as as- nana germplasm is distributed world- Cuba expressed in his condolence for sociate scientist in the West and Cen- wide, as well as to display panels and Phil “In Cuba we will always remember tral Africa office. Her position, finan- posters with information on INIBAP’s him with admiration, love and respect; cially supported by Vlaamse and IPGRI’s networking activities and and all those feelings will be material- Vereniging voor Ontwikkelingsamen- the importance of bananas and other ized in the farms around the Island werking en Technische Bijstand plant genetic resources to food security. where today the fruits of his work are (VVOB), is specifically focussed There was also a hands-on demonstra- flowering”. When we came back to on technology de- tion of MUSADOC 2000 and the multi- Honduras he kept a wide and happy velopment and media CD-ROM on bananas. About 500 smile for many days and Jeanette told transfer, and ne- agricultural scientists and policy him “Phil, I do not know what you did matology. She makers from the region and beyond in Cuba but I will have to send you has studied at attended. back there any time I want you with a Ghent University Other international centres that pre- happy smile on your face”. in Belgium off sented exhibitions included the Inter- As I mentioned at the beginning, and on for seven national Rice Research Institute writing about Phil is difficult, because years in zoology (IRRI), International Livestock Re- it will never be enough. I would re- and nematode search Institute (ILRI), and Interna- member him as a very dear friend and embryology, com- tional Service for the Acquisition of boss, as a unique, dedicated and suc- pleting a Masters Agri-biotech Applications (ISAAA). cessful banana breeder, but most of all thesis and part of the way towards a as a person with a great sensibility for Ph.D. Taking up her INIBAP position Report on fourth MUSACO the social and human aspects of life. on 1 May, Kim is spending her first Steering Committee meeting He was humble as all the great scien- three months in Uganda, shadowing The fourth Steering Committee meet- tists are, he was modest, simple, noble, Guy Blomme and learning about the ing of the Musa Research Network for timid and always good. He served the Integrated Pest Management (IPM) West and Central Africa, MUSACO, poor in a silent but abundant way. His project taking place in East and South- took place in Accra, Ghana, on 2-4 passion was the development of a bet- ern Africa. She will then be posted April 2001. Ghana’s Minister of the En- ter banana or plantain that could be firstly at the International Institute of vironment, Science and Technology de- used all over the world to feed the peo- Tropical Agriculture (IITA) then at the livered the opening address. While en- ple who depend almost exclusively on Centre régional de recherches sur ba- couraging the researchers present to this crop. I am pretty sure that Phil’s naniers et plantains CRBP in continue to develop technologies to dream of seeing his banana hybrids all Cameroon. A large part of her time will augment production of plantain and over the world will be fulfilled sooner be devoted to researching IPM options banana, he lamented the absence of

42 INFOMUSA — Vol 10, N° 1 farmers at the meeting. Professor Walter Alhassan, Director General of the Council for Scientific and industrial research gave the welcoming address and Dr Marcel Nwalozie announced that the West and Central African Council for Agricultural Research and Development (WECARD/CORAF) will provide funds to MUSACO to complete the on-going collection of Musa baseline information in West and Central Africa. Unlike previous meetings where country reports formed the basis of discussions, this year’s meeting was structured around on-going projects; on periurban banana production, germplasm evaluation and the collection of Musa baseline information, and the presentations of the plantain research team of Ghana. Members were also updated with news from representatives of IITA, INIBAP and WECARD/CORAF. Scientists from the University of Ghana, Crops Research Institute, the Kwame Nkrumah University of Science and Technology and the Ministry of Food and Agriculture gave brief reports on the various Musa research and developmental activities taking place in Ghana, ranging from nematology and virology to sucker multiplication. For example, at farmer field schools organised by the national integrated pest management project, Ghanaian plantain farmers have been trained to use clean suckers to establish new plots. Farmers have been selected to take part in the periurban project in Ghana and Benin, and nursery and hardening facilities have been constructed in both countries. In Ghana, the project being implemented by the Crops Research Institute, the Ministry of Food and Agriculture and World Vision Interna- Views of INIBAP’stand at the Asian Agricultural Congress.

Participats of the fourth MUSACO Steering Committee meeting.

INFOMUSA — Vol 10, N° 1 43 tional is taking place around Kumasi tivities at IITA and INIBAP, MusAfrica work for Latin America and the and Sekondi-Takoradi, respectively, is now being co-published by the two Caribbean, MUSALAC. second and third largest cities in the institutions. Members were invited Researchers and extension person- country. The periurban areas of Coto- to inform colleagues to send contribu- nel from the Dominican Republic nou and Abomey Calavi are the sites for tions to IITA or INIBAP. Information on joined in the two days of lectures and the project in Benin, where the imple- MUSACO network activities is posted at discussions led by Dr Sylvio Belalcázar, mentors are the Institut national de the INIBAP and WECARD/CORAF web a Colombian scientist and the brain recherche agricole du Bénin (INRAB) sites. WECARD/CORAF offered to host behind the technologies. The group vis- and CARDER-Atlantique. Project staff electronic discussions on their server. ited a plantain farmer in Moca, in the from the two countries have been The President of MUSACO, Mrs Espallat province of the Dominican trained in weaning and hardening of Adèle Sambo from Gabon was re- Republic, who is practising high den- tissue culture plantlets. elected and it was decided that the 5th sity planting and harvests 110,000 fin- The germplasm evaluation trials will MUSACO meeting would be held in gers of plantain per hectare per year be entirely planted out by the end of Cotonou, Benin. instead of the average 27,200 obtained this year. The weaning and hardening by farmers who rely on traditional of the tissue culture plants has caused West African scientists visit practices. some delay in some of the nine coun- the Dominican Republic The study group, including scientists tries involved. The meeting recom- and Costa Rica from the Dominican Republic, moved mended that a training course be orga- Plantains are an important staple food to Costa Rica, where they joined with a nized for scientists and technicians to and cash crop in the lowland humid leader of a plantain marketing cooper- learn how to handle tissue culture zones, of West and Central Africa. ative, an extension officer and a re- plants. International Institute of Tropi- WECARD/CORAF, the sub-regional searcher from the Corporación cal Agriculture (IITA), INIBAP and the body coordinating agricultural re- Bananera Nacional (CORBANA). Ani- network will together look for funds to search and development in West and mated discussions took place with sev- stage such a course. Central Africa recognised their impor- eral farmers in the Talamanca region Baseline data collection on Musa is tance by selecting plantain as one of of Costa Rica. Bunch yields on these taking place in nine out of the 12 mem- the priority crops in the sub-region. farms have risen from 9-12 kg to 15 to ber countries but only about four have However, average plantain yields in 20 kg since high density planting was completed their work. A young profes- West and Central Africa are below adopted and farm incomes have risen sional officer seconded by the Food 10 t/ha, considerably lower than yields sharply as a result. and Agriculture Organization of the in Latin America and the Caribbean, The basic elements of the techno- United Nations (FAO) to the MUSACO where improved technologies have logy are: secretariat will assist in this work and been adopted. 1. Planting false horn plantains at high funds will permit the collection of data For 10 days in April 2001, two farmers, densities (2500 to 5000 plants through surveys. four scientists and two extension offi- per ha); The coordinator of the International cers from Benin, Cameroon, Ghana, 2. Using uniform planting materials and Musa Testing Programme (IMTP) of Côte d’Ivoire and Guinea (Conakry) ac- 3.Applying fertilizers, fungicides and INIBAP, Dr Jean-Vincent Escalant in- companied by the INIBAP Regional Co- pesticides at critical developmental vited countries to participate in either ordinator for West and Central Africa stages of the crop. the ‘in-depth’ or the ‘performance eval- and the Head of the Department of 4.For maximum yield on a piece of uation’ trials. Countries hoping to con- Seminars and Studies at CTA attended land, the crop is re-planted after each duct IMTP trials were asked to nomi- a course on plantain production tech- harvest nate candidates for the training course nologies, which took place first in the For the technology to succeed in West on leaf spot diseases and data collec- Dominican Republic and then in Costa and Central Africa farmers should have tion planned for June 2001 in Asia. Rica. The Technical Center for Agricul- access to credit to purchase the neces- Dr Adiko Amoncho, the West and tural and Rural Cooperation (CTA) sary inputs, which equally must be Central Africa representative on the and INIBAP funded the study tour and available at affordable prices. Farmers steering committee of PROMUSA, re- the Centro para el Desarrollo ought to be able to irrigate areas where ported briefly on the PROMUSA meet- Agropecuario y Forestal (CEDAF) and rainfall is insufficient in order to sup- ing that was held in Thailand. He the INIBAP regional office in Latin port the high evapo-transpirational noted the low level of representation of America and the Caribbean provided needs of the high density planting. Mar- scientists from the region on the work- logistical support. keting is also very important. The par- ing groups. He exhorted country repre- The specific objectives of the study ticipants were unanimous in their sentatives to nominate scientists to tour were to: desire to see the technology adopted by one of the five working groups. • Study the different plantain produc- farmers in West and Central Africa. A special delegation from the tion systems being used in the Do- They agreed to develop a proposal to Cameroonian Ministry of scientific and minican Republic and Costa Rica and look for funds to conduct farmer parti- technical research was present to ex- to compare and contrast them to the cipatory trails to render the technology plain the creation of Centre africain situation in West and Central Africa; applicable under the bio-physical and de recherche régionale sur bananiers • Exchange information on plantain socio-economic conditions of West and et plantains (CARBAP). The creation production technologies with Domini- Central Africa. Each has also decided to of CARBAP demonstrates the willing- can Republic and Costa Rican scien- establish his own high density plantain ness of the Cameroonian Government tists, extension personnel, and farm- demonstration plot. to give a regional dimension to this ers; center which takes over from CRBP. • Establish links with plantain re- Report of the sixth BARNESA Participants were informed that as searchers in Latin America and the Steering Committee meeting part of the common Musa programme Caribbean through the framework of The sixth meeting of the Steering Com- for sub-Saharan Africa, which links ac- the INIBAP-coordinated banana net- mittee of the Banana Research Network

44 INFOMUSA — Vol 10, N° 1 for Eastern and Southern Africa was Charles Eledu, the scientist in charge of Biotechnology project held in Zanzibar, Tanzania from 22–23 this Rockefeller Foundation funded Significant advances have been made in February, 2001. The meeting was project. Six countries are participating the Ugandan government-funded pro- opened by the Hon. Deputy Minister of in the project, which is being imple- ject on the ‘Novel approaches to the im- Agriculture and a welcoming speech mented in collaboration with IITA and provement of the banana production in was provided by the Hon. Minister of NARO, Uganda. It is planned that the Eastern Africa – the application of Health. New members to the Commit- project will link closely with the NARO biotechnological methodologies’. The tee were welcomed from Sudan and soils programme, which is also funded project is bringing together the exper- Eritrea. by Rockefeller Foundation. Equipment tise of the International Institute of for this project, including GIS hardware Tropical Agriculture (IITA), National BARNESA strategic plan and software are now in place, and it is Agricultural Research Organization The BARNESA Coordinator reported hoped that the project will be able to (NARO), Makerere University, Centre that the evaluation of the ASARECA make rapid progress in assembling and de coopération internationale en networks had been completed by the analysing available banana research recherche agronomique pour le consultants engaged by the European and production information. développement (Cirad), Katholieke Union. As a result, BARNESA has been Following a presentation on the In- Universiteit Leuven (KUL) and INIBAP classified as an emerging network and ternational Musa Testing Programme to improve the production of East will receive limited funding from the by INIBAP’s Germplasm Conservation African Highland banana varieties by EU from July 2001. However continued Scientist, BARNESA members were enhancing their resistance to black funding will be dependant on the re- asked to consider participating in the Sigatoka, nematodes and weevils. The alignment of the BARNESA strategy to- programme by hosting either ‘perfor- supervisor of the Tissue Culture Labora- wards the ‘market-orientated’ approach mance’ or ‘in depth’ trial sites. Several tory at NARO, the administrator and of ASARECA. In order to address this countries expressed an interest in be- four technicians have been selected to issue, the meeting agreed that a Select coming part of the IMTP. work on the project. The laboratory is Committee should be appointed to work The BARNESA Chairperson reported being equipped and measures are being on the finalization of the BARNESA on her participation in the PROMUSA put in place to ensure that all electrical Strategic Plan. Terms of reference and meeting in Thailand in November 2000, equipment runs consistently. The ‘Cof- a time frame for the work of the Com- which she attended as the representa- fee building’ at NARO research station mittee were developed. tive of the NARS of Eastern and South- will also be developed to host a molecu- The Steering Committee also agreed ern Africa. She noted that the prime lar biology laboratory, which will help that the BARNESA priorities as identi- focus of PROMUSA is genetic improve- to enhance on-going studies and build fied in the original strategic plan are ment and that previously weevils had on molecular biology capacities. still valid but will need to be placed in not been included in the programme. A regular supply of male flower buds the context of market oriented re- However, during the meeting it was is being established as starting material search. In this respect, it was noted recommended that initial steps be for the establishment of embryogenic that marketing experience is lacking in taken towards the formation of a Wee- cell suspensions (ECS). Farmers are the present Steering Committee, which vil working group. This is welcome currently providing the plant material, is composed exclusively of biological news for the BARNESA region where but sufficient supplies will soon come scientists. This was recognized as a gap weevils are one of the main constraints from plants grown at NARO research in relation to market-oriented research. to production. station in Kawanda. Currently, 450 It therefore resolved that the Select plants representing 4 different cultivars Committee should be requested to ad- INIBAP/IITA joint programme for Africa are established. Careful management is vise on possible new members who In order to strengthen their collabo- planned to ensure adequate control of could help to fill the recognized gaps in ration on bananas INIBAP and IITA black Sigatoka, nematodes, weevils and the Committee. have agreed to merge their banana re- viruses. search agenda for Africa. This means A tissue culture training course for Ongoing activities that the planning and implementation project personnel took place on 19-25 An update on the banana integrated of activities in both West and East April 2001 at NARO, involving three pest management project (IPM) which Africa will be jointly executed. It is trainers from KUL and Cirad-Départe- is funded by DFID, UK and is being car- hoped this will improve the delivery of ment des productions fruitières et hor- ried out in Kenya, Tanzania and Uganda results and facilitate collaboration ticoles (Cirad-Flhor). Methods were was provided by Guy Blomme, the Assis- with NARS. It was noted that the publi- taught for inoculating different starting tant Coordinator for East and Southern cation MusAfrica is now being co-pub- materials, obtaining embryogenic cul- Africa. A number of stakeholder meetings lished by the two institutions. Mem- tures and establishing ECS. More than have been held at regional and local bers were invited to inform colleagues 200 male buds from 4 different vari- levels to introduce the project and its to send contributions to IITA or eties, suckers from 6 different cultivars, aims and objectives. In each country INIBAP. as well as scalps from another 6 differ- a project site has been selected and As the tenure for the Chairperson of ent banana varieties from the INIBAP baseline data is being collected. IPM BARNESA lasts for two terms, Mary Musa Germplasm Collection have been options to be tested in the project are Wabule of KARI, Kenya will continues inoculated as a starting point. The su- being determined in collaboration with as Chairperson for 2001-2002. It was pervisor of the Tissue Culture Labora- participating farmers and all trials will agreed that the next meeting could be tory, Ms Priver Namanya, will receive be conducted on-farm. A number of in- held in Ethiopia. The Coordinator sug- further training on the cell suspension teresting technologies are being consi- gested and it was agreed that the meet- methods at KUL and Cirad. dered, including the use of botanicals, ing be held in parallel with a national Finally, contacts have been estab- ash and cow urine as natural pesticides. stakeholders meeting to enable com- lished with potential partners in the An overview of the banana baseline mittee members to interact with local United Kingdom, including John Innes information project was provided by banana stakeholders. Centre, University of Leeds and DFID.

INFOMUSA — Vol 10, N° 1 45 The John Innes Centre have developed medium for the exchange of informa- and submitted to DFID a complemen- tion on genetic resources with col- tary project proposal on the genetic leagues not only in India but also transformation of East African High- throughout the region and beyond. This land Banana. The Cooperation and De- training course brings the number of velopment Department at the French curators trained in the use of MGIS to Embassy in Kampala also provided a 40. MGIS presently contains 4122 positive reception to a presentation of records and INIBAP is now working to- the project. wards making this valuable database freely available for consultation IPM training course through the Internet. Within the framework of the DFID The MGIS training course was fol- funded banana IPM project, a training lowed by a workshop on Names and syn- course was conducted for field techni- onyms of banana varieties for India. cians and scientists, from 3 till 10 May, This proved to be an excellent comple- 2001. The venue was at NARO, Kawanda ment to the MGIS training course, pro- Agricultural Research Institute, in viding an opportunity for germplasm cu- Uganda. The training focussed on Musa rators from all over India to discuss Trainees from India during the MGIS course. pests and diseases, IPM technologies, cultivar diversity, farmer participatory research methodologies, socio-economics and farming systems. The course covered broad overviews and a practical training on the use of pest and disease assessment protocols. A farm visit was organised, to pay special attention to both socio-economic aspects of on farm participatory research and to the assessment of pest and disease distribution and incidence. The course was attended by 15 trainees, comprising of extension offi- cers, NARS research technicians, scientists, NGO representatives, representatives from FAO, Farmer Field School projects, curators of banana collections, a participant from the KCDP project in Kagera, Tanzania and Kim Jacobsen, a VVOB associate expert, who is on a three month orientation visit to both INIBAP and IITA programs in Uganda. The acquired knowledge will facilitate Practicizing MGIS in the banana fields. the execution of project activities and the dissemination of IPM technologies in the three project countries and beyond.

MGIS training course and workshop of Names and synonyms in India A national Musa Germplasm Informa- tion System (MGIS) training course was held in India from 21 to 24th of May, at Tiruchirapally, Tamil Nadu. The course was co-organized with the National Re- search Centre on Banana (NRCB) under the supervision of Dr Sathi- amoorthy and Dr S. Uma. Twelve genebank curators, coming from the major growing banana regions of India: Andrah Pradesh, Karnataka, Kerala, Tamil Nadu, West Bengal and Andaman and Nicobar Islands participated in the course. The curators were very enthusiastic about MGIS and its value as a tool for the management of germplasm data. MGIS was also considered a very useful Participants of the workshop on Names and synonyms.

46 INFOMUSA — Vol 10, N° 1 issues related to Indian banana diver- Thesis sity. A large number of synonyms are recognised for bananas in India, but a number of accessions unique to specific Partial resistance assessment areas were also identified. The important role that MGIS can play in assist- of bananas against the black leaf ing with the clarification of names and synonyms became evident during the streak disease and evaluation two courses. of the aggressiveness variability Books etc. of the causal agent, Strategies for utilization of genetic variation in plantain Mycosphaerella fijiensis improvement Dirk R. Vuylsteke PhD thesis submitted at Faculté Universitaire des Sciences Agronomiques, Gembloux, Thesis submitted for the posthumous Belgium, 2000. PhD award in Agricultural and Applied Biological Sciences, Katholieke Universiteit Leuven (K.U. Leuven), Belgium Abdelbasset El Hadrami resistant cultivars for some infectious life-cycle sequences either in natural than controlled conditions. Thus, some On 30 January 2000, Dirk R. Vuylsteke, Banana improvement programmes aim resistance components were suggested. an outstanding Musa scientist and hu- at the creation of new varieties partially The experimental device used in the manitarian, died resistant to the black leaf streak dis- field did not, however, make it possible in a tragic air ease. The principal objective of this to judge the epidemiologic weight of flight accident. study was to characterize the compo- some of them. Elsewhere, a low vari- His family, partic- nents of this resistance using the infec- ability of M. fijiensis aggressiveness ularly his wife tious life-cycle parameters, to judge was detected and no ‘cultivar x isolate’ Kathelyne and their epidemiologic weight and to eval- specific interaction was highlighted. their children uate the variability of the aggressive- These results have an implication for Sarah and Yan- ness of the pathogen. Significant differ- the selection of effective and durable nick, with the ences were detected between partially partial resistances. support of their friends, decided after Dirk’s death to make available to Musa scientists world-wide the most Dirk’s PhD thesis provides a testi- new phase of Musa evolution.” This relevant parts of his two decades of re- mony of his innovative ideas and dedi- PhD thesis, as well as Dirk’s many other search at IITA through the publication cation for improving the Musa crop, journal articles and book chapters, will of his PhD thesis. Dirk wrote the Gen- particularly for African smallholder be always a source of inspiration to his eral Introduction and Synthesis chap- farmers. As pointed out in the tribute colleagues and the new generation of ters during August 1997 while at Copen- written by his academic mentor Prof. scientists involved in germplasm en- hagen, and also selected nine of his De Langhe, “Particularly striking is the hancement of research-neglected tropi- manuscripts (published either in inter- very last paragraph of his thesis with a cal crops. national refereed journals or as book prophetic character in which his credo chapters) which are included as the re- is formulated in a few sentences, a Summary search chapters of this PhD Thesis. All credo that is currently proved to be Plantain has long been considered in- materials included in his thesis were based on a sound and scientifically ma- tractable in terms of genetic improve- retrieved from his desktop computer ture insight.” In Dirk’s words: “A broad- ment, as only landraces are cultivated (file name: Magnum Opus) at his office based, improved Musa germplasm despite years of breeding endeavours. in Namulonge and collated at his home with pest/disease resistance will be a Recent advances in several Musa in Kampala during April 2000. This the- major component to achieve sustain- breeding programmes have demon- sis, whose promotor at K.U. Leuven was able production of this vegetatively strated that development of improved Prof. Em. Edmond De Langhe, consists propagated, perennial crop. Such plantain and banana germplasm of four parts: I. General Introduction, germplasm can be produced through through conventional cross-breeding II. Somaclonal Variation in Plantain conventional cross-breeding, enhanced may eventually result in new cultivars (which includes 3 research journal arti- by the utilization of innovative meth- for local consumption and commercial cles and 1 review book chapter), III. ods for the introduction of additional production. The recent interest in Cross-breeding of Plantain (comprising genetic variation. Also, the increased Musa breeding was mainly sparked by 1 research journal article, 3 germplasm use of molecular markers will acceler- the black Sigatoka epidemic, to which registrations and 1 review article), and ate the process of recurrent selection of resistance is now readily available. IV. Strategies for Plantain Improve- improved Musa germplasm and, hence, Other major production constraints, ment. His thesis was submitted orally facilitate the development of new hy- particularly nematodes, Fusarium and by Prof. Rony L. Swennen in an acade- brids. The prospects of banana and viruses, are now receiving increased at- mic ceremony at K.U.Leuven on 29 plantain breeding are unlimited and tention from breeders. Further progress March 2001. increased efforts will at once initiate a in breeding may help to make Musa a

INFOMUSA — Vol 10, N° 1 47 modern crop. Seed set rates are work- vides descriptions and photographs of International Research Centre for able in many Musa subgroups. Insight species and varieties covering most of Agricultural Sciences). Detailed proto- into combining abilities, heterotic cols are given for all steps to follow from groups, and the genetics of qualitative the preparation of the plant material to and quantitative traits has been gained the recovery and regeneration of whole and is being applied to make breeding plants. These methodologies are spe- more efficient. A wide array of breed- cific to the type of banana tissue to be ing schemes is being explored, combin- cryopreserved: individual meristems, ing conventional and innovative ap- meristem clumps (cauliflower-like proaches, and producing potential structures), embryogenic cell suspen- cultivars from primary tetraploids, sec- sions, and zygotic embryos. Each ondary triploids and other populations. method is illustrated with figures and A number of improved genotypes are colour photographs. Advantages and undergoing multilocational evaluation, limitations of each method are high- from which knowledge on genotype-by- lighted, and current perspectives for environment interaction and stability optimizing the methodologies presented of important traits is acquired. Though are discussed. It is hoped that these some important Musa subgroups guidelines will facilitate the adoption (Cavendish, False Horn plantain) re- and use of the methodologies described. main recalcitrant to conventional the diversity in the genus Musa. The The publication includes bibliographical breeding, biotechnology holds promise catalogue is divided into two parts. The references and some useful practical infor their improvement. However, so- first part focuses on the wild species, formation: composition of media and maclonal variation through micropropa- covering the sections Australimusa, solutions, and list of the basic equip- gation has a limited use in plantain im- Callimusa, Eumusa and Rhodochlamys, ment needed. Available also in French provement as it mostly mimics naturally while the second part provides informa- and Spanish. occurring variation along with the ob- tion on cultivated varieties. Each entry Copies of this publication are avail- served poor horticultural performance in the catalogue is represented by a able from INIBAP Headquarters. of somaclonal variants. The purpose of photograph and a morpho-taxonomic the research reported in this thesis was description of the plant in the field. The Banana Fusarium wilt to foster an increasingly scientific, and publication is based on information management: towards not merely empirical, approach to plan- provided to INIBAP through the Musa sustainable cultivation tain (and banana) improvement. It was Germplasm Information System (MGIS) Edited by A.B. Molina, also hoped by the author, that his thesis by germplasm curators from Guade- N.H. Nik Masdek and K.W. liew may contribute, be it indirectly, to the loupe, Cameroon, Australia and ISBN: 971-91751-14-1 much needed transformation of the tra- Uganda. Fusarium wilt is one of the most devas- ditional African agriculture to a mod- Copies of Musalogue II are available tating banana diseases at the global ern, science- and technology-based, yet from INIBAP Headquarters. level and is the number one constraint sustainable, system. to banana production in Asia. A first In- Cryopreservation ternational Conference on Fusarium Availability of Musa germplasm wilt was held in Miami in 1989, and this A copy of this PhD thesis may Bart Panis and Nguyen Tien Thinh resulted in the publication of a book en- be obtained from: INIBAP Technical Guidelines 5 titled ‘Fusarium wilt of Banana’. Since Prof. R.L. Swennen, Edited by J.V. Escalant then, many developments have taken K.U.Leuven, and S. Sharrock Laboratory of Tropical Crop ISBN: 2-910810-45-3 Improvement This publication describes cryopreserva- Kasteelpark Arenberg 13 tion methods developed for Musa tissue B-3001 Leuven, Belgium Tel: (32-16) 32 14 20 Fax: (32-16) 32 19 93 Or INIBAP Headquarters, Parc Scientifique Agropolis 2, 34397 Montpellier Cedex 5, France Tel: (33) 4 67 61 13 02 Fax: (33) 4 67 61 03 34

Musalogue II – Diversity in the genus Musa J. Daniells, C. Jenny, D. Karamura and K. Tomekpe place. Research has made significant Edited by E. Arnaud progress, especially in the development and S. Sharrock and release of resistant varieties, and in ISBN: 2-910810-42-9 the biochemical and molecular charac- INIBAP has just published the second teriszation of Fusarium strains. A sec- edition of Musalogue - a catalogue of at KUL (Katholieke Universiteit Leu- ond International workshop was there- Musa diversity. This publication proven, Belgium) and JIRCAS (Japanese fore organized in 1999 in order to

48 INFOMUSA — Vol 10, N° 1 review the current status of Fusarium Musa species as well as for the main IAEA-312.D2.RC.579.3) the full version wilt disease on bananas and identify sub-groups of cultivated bananas. In ad- of papers presented during the third priorities for future research. The pro- dition to the classification key, a list of FAO/IAEA research coordination meet- ceedings of this workshop include sci- synonyms is provided for each distinct ing on “Cellular biology and biotechnol- entific papers from the banana world’s cultivar. The publication includes a ogy...“ Abstracts of these papers were Fusarium wilt specialists covering the large number of colour plates, which il- published in PROMUSA 4 pp. VI-XVI themes of pathogen diversity; monitor- lustrate the diversity of bananas in (INFOMUSA vol. 8, No. 2). ing and screening methodology; varietal India as well as providing clear descrip- improvement; and disease manage- tions of the taxonomic characters used Available soon ment. The publication also contains in the key. This publication is required Two new Musa disease factsheets are country reports from Asia, the Pacific, reading for anyone interested in Musa presently in press. Africa and Latin America. diversity in India. The Disease factsheet No. 9 on Copies of this publication are avail- Copies of this publication are avail- “False Panama disorder on banana“ able from the INIBAP Regional Office able from: The Director, National Re- was prepared by Zaag de Beer, Julio M. in the Philippines. search centre for Banana (ICAR) #17 Ramalinganagar South extension, Vay- A tentative key for identification alur Road, Tiruchirapalli, 620 017, and classification of Indian Tamil Nadu, India. Email: nrcb- bananas [email protected]; [email protected] H.P. Singh, S. Uma and S. Sathiamoorthy There is a large diversity of bananas in Organic/environmentally friendly India, with more than 90 distinct clones banana production having been identified in different Edited by F.E. Rosales, S.C. Tripon genebanks spread across the subconti- and J. Cerna nent. However the systematic identifi- ISBN : 2-910810-99-2 cation of individual cultivars from one The English version of the proceedings location to another is severely ham- of the workshop “Producción de banano pered by the large number of synonyms orgánico y, o, ambientalmente amiga- ble” held in Costa Rica in July 1998 is now available. Request for copies can be addressed to INIBAP regional office in Costa Rica. Hernández and Sonia Sabadel. The Cellular biology and factsheet No. 10 authored by Africano biotechnology, including Kangire and Mike Rutherford focuses mutation techniques on “Wilt-like disorder of bananas in for the creation of new useful banana genotypes IAEA reproduced in a working docu- ment with limited distribution (ref :

(mainly vernacular names) which are used. For example, the well-known cultivar ‘Poovan’ has no less than 27 synonyms in India. This publication provides a key for the classification of Indian banana cultivars, based on the genome classification system of Sim- Uganda“. Both factsheets will be avail- monds and Shepherd. Detailed descrip- able in July in English, French and tions are provided for a number of wild Spanish.

INFOMUSA — Vol 10, N° 1 49 Announcements

FHIA is seeking a plant breeder

The Honduran Foundation for Agricultural Research (Fun- breeding. Fluency in English and Spanish languages is de- dación Hondureña de Investigación Agrícola – FHIA) is sired. A competitive salary, based on qualifications and expe- seeking an experienced plant breeder to direct and play an rience, plus benefits is offered. Interested parties please con- active breeding role in its internationally recognized banana tact Dr Dale T. Krigsvold, Director of Research at and plantain breeding programme located in La Lima, Hon- [email protected]; Telephone: (504) 668-2809; Fax (504) duras, Central America. The successful candidate will have 668-2313 or send applications with résumés to Recursos Hu- an advanced degree in plant breeding, experience in Musa manos, FHIA, Apartado Postal 2067, San Pedro Sula, Cortés, breeding, experience in research administration, and knowl- Honduras 21105 or by E-mail at [email protected]. Applica- edge and experience in modern techniques used in plant tions will be received until a suitable candidate is found.

Vavilov-Frankel Fellowships 2002

The International Plant Genetic Resources Institute plant breeding or molecular characterization will not be se- (IPGRI) has established the Vavilov-Frankel Fellowship lected. Fellows are encouraged to present the results of Fund to commemorate the unique contributions to plant their research at an international conference. This can take science by Academician Nikolai Ivanovich Vavilov and Sir place within one year of termination of the Fellowship. Otto Frankel. Applications for the year 2002 are invited from develop- The Fund aims to encourage the conservation and use ing-country nationals, aged 35 or under, holding a masters of plant genetic resources in developing countries degree (or equivalent) and/or doctorate in a relevant sub- through awarding Fellowships to outstanding young reject area. Application forms in English, French and Spanish searchers. may be obtained from: Vavilov-Frankel Fellowships, IPGRI, The Fellowships will enable the applicants to carry out Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), relevant, innovative research outside their own country for a Rome, Italy; Fax: (39)0661979661 or e-mail: period of between three months to one year. The research [email protected] or URL: http://www.ipgri.cgiar.org/train- should have a clear benefit to the home country, preferably ing/vavilov.htm and should be returned to IPGRI, Rome. Ap- in areas of the applicant’s future research. Awards can be plications can be sent by mail, fax or email. Applications held concurrently with other sources of support. must be received at IPGRI by 16 November 2001. In 2002, a total of US$50,000 will be made available for Applications must be in English, French or Spanish and awards. The maximum award per Fellow will be US$25,000 should include a covering letter, completed application which is intended to cover travel, stipend, bench fees, equip- form, full curriculum vitae, research proposal (maximum ment, conference participation or any other appropriate use. 1000 words which should include a clear statement of objec- Such research should be linked to innovative topics related tives, feasibility, methodology, materials, justification of the to the conservation and use of plant genetic resources such relevance to plant genetic resources, and possible outcomes as new conservation technologies and strategies, socioeco- or impacts), a letter of acceptance from the proposed host nomic and human aspects of conservation and use, institute and a letter of support from the home institute. germplasm management, forest genetic resources, policy de- The successful applicants will be informed by 31 March 2002 velopment, genetic erosion assessment and mitigation and and are required to take up their Fellowships before 31 conservation and utilization of specific crops. Work solely on December 2002.

VIth International Symposium of Plant Biotechnology

(1rst announcement) isms; Microbial contamination in in vitro tissue culture; IPB, Cuba, 17-21 June 2002 Obtaining of secondary metabolites; Information, commerce This symposium in organized by the Institute of Plant and intellectual property in plant biotechnology Biotechnology (IBP) and Central University “Marta Abreu” For further information please contact: of Las Villas, Villa Clara, Cuba. Lic. Orlando Gregorio Chaviano Institute of Plant Topics include: Genetic transformation and Molecular Biotechnology, Carretera a Camajuaní km. 5.5, Santa Clara, Biology; Tissue culture; Somatic embryogenesis and artifi- Villa Clara, Cuba. cial seed; Massive propagation; Plant improvement through E-mail: [email protected] mutagenesis, somaclonal variation and in vitro selection; More details and registration form are also available from: Decontamination and diagnosis of pathogen microorgan- http://www.inibap.org/actualites/villaclara/indexevenin.htm

50 INFOMUSA — Vol 10, N° 1 INIBAP addresses

• Headquarters: Apdo 60-7170 Turrialba, COSTA RICA Katalima Road Parc Scientifique Agropolis II Tel/Fax: (506) 556 2431 Naguru 34397 Montpellier Cedex 5 – FRANCE e-mail: [email protected] Kampala, UGANDA e-mail: [email protected] Fax: (256-41) 28 69 49 http://www.inibap.org • Regional Office for Asia and the Pacific e-mail: [email protected] Director Regional Coordinator Dr Emile FRISON Dr Agustín MOLINA • INIBAP Transit Center (ITC) e-mail: [email protected] C/o PCARRD Officer in charge Genetic Resources Scientist Los Baños, Laguna 4030 Ms Ines VAN DEN HOUWE Dr Jean-Vincent ESCALANT PHILIPPINES Katholieke Universiteit Leuven e-mail: [email protected] Fax: (63 49) 536 05 78 Laboratory of Tropical Crop Germplasm Conservation Scientist e-mail: [email protected] Improvement Ms Suzanne SHARROCK Kasteelpark Arenberg 13, e-mail: [email protected] • Regional Office for West and Central B-3001 Leuven, BELGIUM Head Information/Communications Africa Fax: (32 16) 32 19 93 Ms Claudine PICQ Regional Coordinator e-mail: e-mail: [email protected] Dr Ekow AKYEAMPONG [email protected] Officer in charge MGIS Associate expert entomology: Ms Elizabeth ARNAUD Stjin MESSIAEN • Associate Experts, Nematology e-mail: [email protected] C/o CRBP – BP 12438 Ms Inge VAN DEN BERGH Financial Manager Douala, CAMEROON C/o VASI Mr Thomas THORNTON Fax: (237) 42 91 56 Van Diem, Than Tri e-mail: [email protected] e-mail: [email protected] Hanoi, VIETNAM Fax: (84) 4 861 39 37 • Regional Office for Latin America • Regional Office for Eastern and e-mail: [email protected] and the Caribbean Southern Africa Mr Thomas MOENS Regional Coordinator Regional Coordinator C/o CORBANA Dr Franklin E. ROSALES Dr Eldad KARAMURA La Rita Research Station Associate Scientist : Associate Scientist: Technology transfer Apdo 390-7210 Musa technology transfer Guy BLOMME Guápiles, COSTA RICA Luis POCASANGRE PO Box 24384 Fax: (506) 763 30 55 C/o CATIE Plot 106 e-mail: [email protected]

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

INFOMUSA — Vol 10, N° 1 51 Publications from INIBAP

www.inibap.org

The following publications are available from headquarters: INIBAP/ACIAR 1997. E. Arnaud & J.P. Horry (eds). Musalogue, a catalogue of Musa germplasm: Papua New Guinea collecting missions 1988-1989. INIBAP/CTA/CIRAD 2001. J. Daniells, C. Jenny, D. Karamura & K. Tomekpe. Musalogue II – Diversity in the genus Musa (E. Arnaud & S. Sharrock, compil.). INIBAP/CTA/FHIA/NRI/ODA 1997. B.K. Dadzie & J.E. Orchard. Post-harvest Routine INIBAP/CTA 2001. B. Panis & N.T. Thinh. Cryopreservation of Musa germplasm (J.V. Screening of Banana and Plantain Hybrids: Criteria and Methods. INIBAP Technical Escalant et S. Sharrock, eds). INIBAP Technical Guidelines 5. Guidelines 2. INIBAP 2001. Annual Report 2000. INIBAP/CTA 1997. P.R. Speijer & D. De Waele. Screening of Musa Germplasm for Resistance and Tolerance to Nematodes. INIBAP Technical Guidelines 1. INIBAP 2000. M. Holderness, S. Sharrock, E. Frison & M. Kairo (eds). Organic banana 2000: Towards an organic banana initiative in the Caribbean. Report of the international INIBAP/The World Bank 1997. E.A. Frison, G. Orjeda & S. Sharrock (eds). PROMUSA: A workshop on the production and marketing of organic bananas by smallholder farmers. Global Programme for Musa Improvement. Proceedings of a meeting held in Gosier, 31 October-4 November 1999, Santo Domingo, Dominican Republic. Guadeloupe, March 5 and 9, 1997. CIRAD/INIBAP 2000. Bananas. INIBAP-IPGRI/CIRAD. 1996. Descriptors for Banana (Musa spp.). INIBAP 2000. G. Orjeda (compil.). Evaluating bananas: a global partnership. Results of IMTP Phase II. The following publications are available from Asia and the Pacific office: INIBAP/EARTH/IDRC 1999. F.E. Rosales, S.C. Tripon & J. Cerna (eds). Organic/environmentally friendly banana production. Proceedings of a workshop held at INIBAP-ASPNET/MARDI 2001. A.B. Molina, N.H. Nik Masdek & K.W. Liew (eds). Banana EARTH, Guácimo, Costa Rica, 27-29 July 1998. Fusarium wilt management: towards sustainable cultivation. Porceedings of the international workshop on the management of Fusarium wilt disease held in Genting, INIBAP/CRBP/CTA/CF 1999. C. Picq, E. Fouré & E.A. Frison (eds). Bananas and food Malaysia, 18-20 October 1999. security/Les productions bananières: un enjeu économique majeur pour la sécurité alimentaire. Proceedings of an International Symposium held in Douala, Cameroon, 10- INIBAP 2000. R.V. Valmayor, S.H. Jamaluddin, B. Silayoi, S. Kusumo, L.D. Danh, O.C. Pascua 14 November 1998. & R.R.C. Espino. Banana cultivar names and synonyms in Southeast Asia. INIBAP/FHIA 1999. F.E. Rosales, E. Arnaud & J. Coto (eds). A tribute to the work of Paul H. INIBAP-ASPNET 2000. A.B. Molina & V.N. Roa (eds). Advancing banana and plantain R & D Allen: a catalogue of wild and cultivated bananas. in Asia and the Pacific. Proceedings of the 9th INIBAP-ASPNET Regional Advisory INIBAP/RF/SDC 1999. E.A. Frison, C.S. Gold, E.B. Karamura & R.A. Sikora (eds). Mobilizing Committee meeting held at South China Agricultural University, Guangzhou, China, 2-5 IPM for sustainable banana production in Africa. Proceedings of a workshop on banana IPM November 1999. 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 the Joven (eds). Managing banana and citrus diseases. Proceedings of a regional workshop second Steering Committee meeting held at Douala, Cameroon, 15-16 November 1998. on disease management of banana and citrus through the use of disease-free planting INIBAP 1999. K. Shepherd. Cytogenetics of the genus Musa. materials held in Davao City, Philippines, 14-16 October 1998. INIBAP 1998. E. Akyeampong (ed.). Musa Network for West and Central Africa. Report of INIBAP/ASPNET 1999. V.N. Roa & A.B. Molina (eds). Minutes: Eighth meeting of the first Steering Committee meeting held at Douala, Cameroon, 8-10 December 1997. INIBAP/ASPNET Regional Advisory Committee (RAC) hosted by the Queensland INIBAP 1998. E.A. Frison & S.L. Sharrock (eds). Banana streak virus: a unique virus-Musa Horticulture Institute (DPI) in Brisbane, Australia, 21-23 October 1998. interaction? Proceedings of a workshop of the PROMUSAvirology working group held in INIBAP/ASPNET 1998. Minutes: Seventh meeting of INIBAP/ASPNET Regional Advisory Montpellier, France, 19-21 January 1998. Committee (RAC) hosted by the Vietnam Agricultural Science Institute (VASI) in Hanoi, INIBAP 1998. C. Picq (ed.). Segundo seminario/taller de la Red regional de información sobre Vietnam, 21-23 October 1997. banano y plátano de America Latina y el Caribe. San José, Costa Rica, 10-11 July 1997. INIBAP/ASPNET 1997. V. N. Roa & R. V. Valmayor (eds). Minutes: Sixth meeting of INIBAP 1998. B.K. Dadzie. Post-harvest characteristics of black Sigatoka resistant banana, INIBAP/ASPNET Regional Advisory Committee (RAC) hosted by National Research cooking banana and plants hybrids. INIBAP Technical Guidelines 4. Center on Banana (ICAR) in Tiruchirapalli, India, 26-28 September 1996. INIBAP 1998. G. Orjeda in collaboration with the PROMUSA working groups on Sigatoka INIBAP/ASPNET 1996. R. V. Valmayor, V. N. Roa & V. F. Cabangbang (eds). Regional and Fusarium. Evaluation of Musa germplasm for resistance to Sigatoka diseases and Information System for Banana and Plantain - Asia and the Pacific (RISBAP): 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). PROMUSA N° 7

Contents

First meeting of the PROMUSA working groups’ convenors ...... p. I

2nd International Symposium on the Molecular and Cellular Biology of Banana ...... p. II PROMUSA Abstracts of presentations . . . . .p. II

• Genomics ...... p. II A global Programme for Musa Improvement

• Gene expression in transgenic plants ...... p. V

• Intellectual property and genetically modified organisms ...... p. VII First meeting of the PROMUSA working groups’ • Plant pathology and disease convenors resistance ...... p. VII

• Biodiversity and evolution ...... p. XIII

• Biochemistry and fruit ripening . . .p. XV A first meeting of the convenors of the PRO- • Materials, tools and methods available for MUSA working groups was held in Montpellier distribution What is PROMUSA? on 18-20 April. Up-to-date news of the activities • Availability of biological materials and con- of each of the five groups were shared and it ditions to obtain them The Global Programme for Musa was agreed that the Genetic improvement • Information about current collaborative ac- Improvement (PROMUSA) is a broad-based working group should continue to operate tivities and novel collaborative areas programme which aims at involving all the major players in Musa improvement. It was through two subgroups and not divide into two • Current training activities, and also areas of developed as a means to link the work independent groups as had been proposed. expertise and facilities for training. carried out towards addressing the problems A formulation for two levels of participation in Changes were suggested for the PRO- of export banana producers, with those the working groups has been conceived: MUSA Web site. Each working group will have initiatives directed towards improving banana • those who are interested in receiving infor- its own page containing information on: and plantain production at the subsistence mation in order to develop research in gen- • Members (with a link to the proposed data- and smallholder level. The global programme eral, and base above) builds upon existing achievements and is based upon ongoing research initiatives. • those whose participation is more proactive • Research priorities PROMUSA is therefore a mechanism to and involved in the development of priority • Any relevant databases on aspects of re- further maximize the outputs and accelerate areas of research in the group. search (e.g. Foc database) the impact of the overall Musa improvement The convenors will have the responsibility • Protocols and methodologies available (with effort. The programme is an innovative to familiarize themselves with the work of par- contact details) mechanism to bring together research carried ticipants and identify those who are most ac- • Useful publications: fact sheets, technical out both within and outside the CGIAR, tive, and to stimulate information-sharing and guidelines, handouts (Word or PDF ver- creating new partnerships between National the use of the list server. Working group mem- sions) Agricultural Research Systems (NARS) and research institutes in both developing and bers will be encouraged to send regular up- • Links to other relevant homepages developed countries. The formation of such dates on publications, meetings, training It was also proposed that posters be prepared partnerships will also contribute to events and to collaborate in writing project for scientific meetings, both on PROMUSA in strengthening the capacity of NARS to proposals. The PROMUSA secretariat will as- general and on the work of the different work- conduct Musa-related research. sist in proposal-writing by making available in- ing groups. The individual benefits of the The major thrust of PROMUSA is to develop formation on donors, proposal-writing guide- global PROMUSA meetings and working a wide range of improved banana varieties lines, background information on banana and group meetings were discussed. Future global from which growers worldwide can select plantain production and by assisting in editing meetings should invariably be scheduled those most suited to their needs. The programme brings together conventional and English, if necessary. The responsibilities back-to-back with another major scientific breeding based on hybridization techniques of INIBAP’s regional coordinators in stimulat- meeting. The following schedule has been with genetic engineering and ing participation from all banana-growing re- tentatively suggested: biotechnological breeding approaches. This gions was emphasized, and the responsibili- • Nematology working group (24-25 May broad-based genetic improvement effort is ties of the secretariat in assisting the 2001) after the International Symposium on supported by research being carried out on convenors were also consolidated. Nematology meeting in South Africa (21-23 specific pests and diseases within the A database of PROMUSA participants will May 2001) various PROMUSA working groups. An efficient mechanism for evaluating new be set up, using and linking to the INIBAP data- • Sigatoka working group (March 2002) in varieties produced within the framework of bases, BRIS and MUSALIT. The scope of the Latin America back-to-back with an Interna- PROMUSA is also an essential component of database will be relatively broad and partici- tional symposium on banana leaf spot dis- the programme. pants will be asked to provide information on: eases

INFOMUSA — Vol 10, N° 1 PROMUSA I • Genetic improvement working group + • Fusarium working group meeting is to be more time for working groups to meet inde- Breeding strategies meeting on banana decided - suggestions welcome pendently and make significant progress. after the 3rd International Symposium on • Virology working groups meetingnis to be The next PROMUSA meeting would there- Molecular and Cellular Biology on Banana defined - suggestions welcome fore take place in 2003 and may possibly be in Leuven, Belgium (September/Octo- It is also suggested that global meetings held back-to-back with an International Musa ber 2002) should take place every three years, to allow Congress.

2nd International Symposium on the Molecular and Cellular Biology of Banana

The inaugural Symposium on the Molecular and (Queensland Department of Primary Indus- “Genomics and banana” (Colin Bird, Zeneca) Cellular Biology of Banana held in March 1999 in tries). The local organizing committee also re- and “Intellectual property and GMOs” (Dianne Ithaca, New York, USA, was organized by the ceived major assistance internationally from IN- Nicoll, University of Tasmania). Participants Boyce Thompson Institute for Plant Research. IBAP, Zeneca and DNAP (DNA Plant from the CSIRO (Commonwealth Scientific and The concept was to open a forum for all people Technology Corporation, USA). This second Industrial Research Organization) Plant Indus- involved in molecular and cellular biology to have symposium allowed participants from both de- try also delivered keynotes introducing the ses- an opportunity to meet and exchange information veloping and developed countries to present sions on gene expression in transgenic plants about their research activities. The meeting was their research activities, covering a broad (Peter Waterhouse), plant pathology and dis- a resounding success, and it was therefore sug- range of subject areas. ease resistance (Jeff Ellis), and biochemistry gested to continue the concept under the aus- The symposium was structured around the and fruit ripening (Simon Robinson). pices of PROMUSA. following sessions: genomics; gene expression With 50 papers presented and 60 partici- The 2nd International symposium on the Mol- in transgenic plants; plant pathology and dis- pants from 17 countries attending the sympo- ecular and Cellular Biology of Banana held 29 ease resistance; intellectual property and ge- sium, this event ranks among the most impor- October-3 November 2000 in Byron Bay, Aus- netically modified organisms; biodiversity and tant scientific fora on Musa. tralia, was organized by the Queensland Uni- evolution; and biochemistry and fruit ripening. As an additional contribution, INIBAP pub- versity of Technology (QUT) with the local col- Thanks to the support received from the partici- lishes hereunder a special PROMUSA supple- laboration of CRCTPP (Cooperative Research pating institutions, international scientists were ment containing the abstracts of presentations Center for Tropical Plant Pathology) and QDPI invited to attend and addressed keynotes on made at the symposium.

ity of the method to detect aneuploidy in differences in ploidy levels between leaf tissue Abstracts of presentations Musa. Chicken red blood cell (CRBC) nuclei and root tissue of the same plant were de- were used as internal reference standard and tected. Aneuploids have been particularly use- the DNA index was calculated by comparing ful in genetic studies of many plant species Genomics peak positions of CRBC nuclei and nuclei of such as maize, tomato, tobacco and wheat the sample. At a triploid level, the minimal dif- (Khush 1973). Following the work of Sears, Induction, detection and use ference between euploid (3x) and aneuploid the collection of aneuploid lines has been of aneuploids for genetic studies plant (3x ± 1) should be approximately 3%. made possible to define the relationship be- in Musa spp. Thus, all plants with DNA index differing by tween chromosomes of hexaploid wheat in N.S. Roux1, A. Toloza1, J. Dolezel2 more than 1.5 % from the index established terms of their origin and function (Law et al. and F.J. Zapata-Arias1 for control (3x) plants were considered aneu- 1987). In Musa spp., aneuploids are relatively 1Plant Breeding Unit, FAO/IAEA Agriculture and Biotechnology ploid. The results obtained by flow cytometry frequent and viable in triploid clones. Being Laboratory, Seibersdorf, Austria; 2Laboratory of Molecular were verified by chromosome counting in sterile, their value for genetic analyses is lim- Cytogenetics and Cytometry, Institute of Experimental Botany, meristem root-tip cells (Dolezel et al. 1998). ited. Nevertheless, they could be very useful Olomouc, Czech Republic. The results indicated that flow cytometry was for physical mapping and to link genetic and Polyploid and aneuploid banana plants were sensitive enough to detect aneuploidy in physical maps using already available molecu- obtained after gamma radiation and colchicine Musa. However, detection of aneuploidy with lar markers. treatments. Variation in chromosome number ± 1 chromosome accuracy required high-reso- was also observed in plants regenerated via lution analyses with coefficient of variation of References organogenesis or somatic embryogenesis DNA peaks lower than 2%. The advantage of Dolezel J., M. Lysak, I. Van den Houwe, M. Dolezelova from tissue cultures, which were not exposed flow cytometric assay was that abnormalities & N. Roux. 1997. Use of flow cytometry for rapid to any mutagenic treatment. Regenerated off- in DNA content could be detected at an early ploidy determination in Musa species. InfoMusa type plants were analyzed by flow cytometry stage of plant growth, and also during in vitro 6:6-9. as described by Dolezel et al. (1997) to esti- culture. Moreover, flow cytometry enabled de- Dolezel J., M. Dolezelova, N. Roux & I. Van den mate their ploidy levels and to check sensitiv- tection of mixoploidy. Thus, in several cases Houwe. 1998. A novel method to prepare slides for

II PROMUSA INFOMUSA — Vol 10, N° 1 high resolution chromosome studies in Musa spp. quences, including ribosomal RNA genes, Department, Forestry and Agricultural Biotechnology Institute, 3 InfoMusa 7:3-4. retrotransposon and BSV sequences have al- University of Pretoria, Pretoria 0002, South Africa; African Biotechnologies (PTY) LTD, Tzaneen 0850, South Africa. Khush G.S. 1973. Cytogenetics of aneuploids. ready been localized to Musa chromosomes Academic Press, New York, USA. (Balint-Kurti et al. 2000, Dolezelová et al. Somaclonal variation has long been recog- Law C.N., J.W. Snape & A.J. Worland. 1987. 1998, Harper et al. 1999). More DNA se- nized as a by-product of the propagation of Aneuploidy in wheat and its uses in genetic analysis. quences need to be isolated and mapped to plant cells through one or more cycles of dis- Pp. 71-108 in Wheat breeding: its scientific basis unravel the molecular structure of chromo- organized cell growth. Most of the transforma- (F.G.H. Lupton, ed.). Chapman & Hall, London. somes and to establish mechanisms of tion processes used in the generation of trans- genome differentiation in Musa. Identification genic plants include at least one step where Acknowledgements of individual chromosomes using physically cultured cells are grown and then plants are We thank Ms Ines Van den Houwe (INIBAP) mapped DNA sequences will allow analysis subsequently regenerated. Therefore all of the for providing the vegetative clones of Musa of their behaviour and segregation during individuals that are transgenic and have arisen and Mr Rony Swennen (K.U. Leuven) for pro- evolution and in breeding programmes. Phys- by this method have the potential to contain viding Musa embryogenic cell suspensions. ically mapped single- and low-copy DNA se- some of this variation, even in the absence of This work was supported by a Joint quences will provide anchor sites needed to any visible mutation. Many genomic alter- FAO/IAEA/GDIC (Belgian General Direction integrate physical and genetic maps. ations in transgenic plants have already been for International Cooperation) Coordinated Re- demonstrated using RAPDs and AFLPs. In search Project. The study was undertaken as References spite of the observation that similar polymor- part of the Global Programme for Musa Im- Balint-Kurti P.J., S.K. Clendennen, M. Dolezelová, phisms repeatedly arise, none of the variants provement (PROMUSA). M. Valárik, J. Dolezel, P.R. Beetham & G.D. May. have proved useful as predictors of the level 2000. Identification and chromosomal localization of genomic variation that has taken place. The Molecular cytogenetic and cytometric of the monkey retrotransposon in Musa sp. Mol. well-documented off-types arising from tissue analysis of Musa genomes Gen. Genet. 263:908-915. culture of bananas have been used as a D’Hont A., A. Paget-Goy, J. Escoute & F. Carreel. model system to identify the regions of the J. Dolezel, M. Valárik, J. Vrána, M. Dolezelová, 2000. The interspecific genome structure of genome that may be especially susceptible to J. Safár, M. Lysák, and H. Simková cultivated banana, Musa spp. revealed by genomic change and to develop markers to determine Laboratory of Molecular Cytogenetics and Cytometry, DNA in situ hybridization. Theor. Appl. Genet. the extent of that change. Representational Institute of Experimental Botany, Olomouc, Czech Republic. 100:177-183. difference analysis was used to isolate ge- The application of flow cytometry and molec- Dolezel J., M. Dolezelová & F.J. Novák. 1994. Flow nomic differences between two sets of normal ular cytogenetics stimulated progress in un- cytometric estimation of nuclear DNA amount in and variant banana cultivars – between derstanding of Musa genome at nuclear and diploid bananas (Musa acuminata and Williams and a masada/chlorotic off-type, and chromosomal level. Flow cytometric analysis M. balbisiana). Biol. Plant. 36:351-357. a normal Curare Eno individual and a dwarf was found a convenient method for estima- Dolezel J., M. Dolezelová, N. Roux & I. Van den off-type (the latter pair supplied by Dr R. tion of nuclear DNA content in Musa (Dolezel Houwe. 1998. A novel method to prepare slides for Swennen). In both instances difference clones et al. 1994) and has been used for ploidy ver- high resolution chromosome studies in Musa spp. were identified. Many of the sequences were ification in existing germplasm collections, InfoMusa 7:3-4. common to both sets of difference products, in characterization of newly collected materials, Dolezelová M., M. Valárik, R. Swennen, J.P. Horry & spite of the fact that they were different aber- and evaluation of karyological stability in J. Dolezel. 1998. Physical mapping of the rant phenotypes. One of the difference prod- vitro. Due to its high throughput, the method 18S-25S and 5S ribosomal RNA genes in diploid ucts identified was a minisatellite sequence may be easily incorporated into existing bananas. Biol. Plant. 41:497-505. that also appeared to be labile in date palms. breeding programmes. Samples can be sent Harper G., J. Osuji, J.S.P. Heslop-Harrison & R. Hull. These results add more evidence for the pres- to laboratories equipped with a flow cytome- 1999. Integration of banana streak badnavirus into ence of a labile segment of the genome that is ter, as only a small amount of plant tissue is the Musa genome: Molecular and cytogenetic preferentially modified during the generation of needed. The method also permits determina- evidence. Virology 255:207-213. somaclonal variants. These difference prod- tion of the size of nuclear genome. It was Lysák M.A., M. Dolezelová, J.P. Horry, R. Swennen & ucts are being further characterized with a found that Musa genomes are small with the J. Dolezel. 1999. Flow cytometric analysis of view to developing a series of markers that B genome being smaller compared to the A nuclear DNA content in Musa. Theor. Appl. Genet. can be used to identify early genomic changes genome (Lysák et al. 1999). The develop- 98:1344-1350. and also as diagnostics for specific pheno- ment of procedures for reliable and rapid de- types arising in the tissue culture process. tection of aneuploidy and for chromosome Acknowledgements flow sorting remains a major challenge. The study was undertaken as a part of the Identification of AFLP and ISSR Given the small size and poor morphological Global Programme for Musa Improvement markers associated with dwarf differentiation of Musa chromosomes (PROMUSA) and was supported by Research somaclonal variants (Dolezel et al. 1998), molecular cytogenetics Contract No. 8145/RB from the International in Cavendish banana holds major promise for karyotype analysis Atomic Energy Agency. T.R. Benatti1, S.A.C.D. Souza1, J.A. Scarpare2 and the study of chromosome organization. Filho, P.C. Santos3; A. Tulmann Neto1, While genomic in situ hybridization is suitable Markers for determining genomic E.A. Kido1 and A. Figueira1 integrity: somaclonal variants for determination of genomic constitution in 1Centro de Energia Nuclear na Agricultura, Universidade hybrids (D’Hont et al. 2000), fluorescent in in bananas as a model system de São Paulo, CP 96, Piracicaba, SP, 13400-970, Brazil; situ hybridization (FISH) permits physical C.A. CuIlis1, K. Kunert2 and B. Okole3 2ESALQ-USP (Escola Superior de Agricultura «Luiz de Queiroz», Universidad de São Paulo), Brazil; 3UNESP mapping of DNA sequences to chromo- 1Case Western Reserve University and NovoMark (Universidade Nacional do Estado de São Paulo), Ilha Solteira, somes. Several classes of repetitive DNA se- Technologies LLC, Cleveland, Ohio 44106, USA; 2Botany Brazil. E-mail: [email protected]

INFOMUSA — Vol 10, N° 1 PROMUSA III Somaclonal variation is a common feature of from these explants were micropropagated for methylation-sensitive amplification polymorphism some micropropagated banana cultivars, five subcultures. As controls for MSAP analy- technique. Mol. Gen. Genet. 261:439-466. caused by undetermined reasons. Early de- sis (Xiong et al. 1999), equivalent leaf tissue tection of variants is desirable for commercial was taken from ten conventionally propagated Banana streak badnavirus sequences micropropagation or to establish methods to plants. Ten combinations of primers were in Musa increase variability for breeding. Molecular used for AFLP analysis and eight primers for 1 2 2 markers offer a great potential to detect and the MSAP analysis. No significant differences G. Harper , T. Schwarzacher , C. Hansen , 2 1 to disclose causes of somaclonal variation. were found between either kind of explants P. Heslop-Harrison and R. Hull The objective of this study was to test Ampli- using AFLP or in MSAP in leaf tissue of plants 1John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom; 2Department of Biology, University of Leicester, fied Fragment Length Polymorphism (AFLP) derived from conventional propagation. How- Leicester LE1 7RH, United Kingdom. and Inter-Simple Sequence Repeat (ISSR) ever, when compared to the explants, the mi- assays, using polyacrylamide gels and silver cropropagated plants derived from them had Molecular and cytogenetic data show unequiv- staining, comparing a Cavendish cultivar significantly more DNA polymorphisms. In ad- ocal evidence of the integration of banana “Nanicão Jangada” with its somaclonal dwarf dition, we found that the explant source had a streak badnavirus (BSV) sequences into the variant. Twelve ISSR primers were tested, significant influence on the extent of AFLP genome of Musa plantain Obino l’Ewai (AAB) and two (16.6%) presented 3 polymorphic DNA polymorphisms in regenerants. Inflores- and these sequences are essentially identical fragment present only in the dwarf variant. All cence-derived regenerants gave the highest to that of an episomal virus causing infection AFLP primer combinations from kit AFLP variation of 6.36% compared to sucker-de- in Musa (Harper et al. 1999, Ndowora et al. System I (Life Technologies, Rockville, MD, rived regenerants which gave 3.96% polymor- 1999). There are two loci, differing in copy USA) were tested, amplifying a total of 1665 phisms. number of BSV sequence in Obino l’Ewai and, bands. Each primer combination amplified an A total of 107 (23%) out of 465 bands were for at least one of them, the integrated se- average of 26.4 fragments, ranging from 7 to found to be cytosine-methylated in microprop- quence structure is rearranged with respect to 44 bands. Forty-three polymorphic fragments agated plants, whilst in conventionally propa- the virus sequence. Significant BSV infections (2.6%) were identified, with 19 (1.1%) pre- gated plants 18% of the bands were found to are detected in certain B genome containing sent only in the dwarf variant. Polymorphic be cytosine-methylated. There was no signifi- Musa germplasm during meiosis or tissue cul- fragments were stable between assays. cant difference in the extent of DNA methyla- ture and the circumstantial evidence points to Methylation-sensitive AFLP assay, based on tion polymorphisms between inflorescence-de- episomal BSV infection arising from the acti- the differential ability of a pair of rived micropropagated plants (3%) and vation or mobilization of integrated BSV se- isoschizomers to restrict methylated cytosine, sucker-derived plants (1.7%). Most of the quences. A model involving recombination has was also tested. A combination of 24 primers polymorphic bands were of high molecular been proposed that links integrated sequence were used to amplify DNA from both geno- weight (above 700 bp) and were hyper-methy- to the generation of replicative forms of the types. An average of 24.8 fragments were lated. This was also the case for most of the virus (Ndowora et al. 1999). This phenomenon amplified from HpaII-treated DNAs and 22.1 hyper-methylated bands common to all micro- has major implications for Musa pathology, im- from MspI-treated, comparable to the regular propagated plants but which were not methy- provement, germplasm movement and quar- AFLP. Twelve polymorphic bands (2.1%) lated in conventionally propagated plants. A antine. were present only in “Nanicão Jangada” in correlation was found between some plants The BSV integration phenomenon has par- HpaII-digested, while eight fragments (1.6%) with AFLP polymorphisms and plants with allels in two other cases, the pararetroviruses were polymorphic for MspI-treated. Only methylation polymorphisms. Petunia vein-clearing virus (PVCV) (Richert- three polymorphisms (0.5%) might have de- Thus the banana micropropagation process Pöggeler and Shepherd 1997) and Tobacco rived from differences in methylation. Other was found to generate significant genetic and vein-clearing virus (TVCV) (Lockhart et al. dwarf variants are being tested using the possibly epigenetic changes in micropropa- 2000). Episomal PVCV is found in Petunia hy- same primer combinations, and polymorphic gated ‘Grande Naine’ banana plants. The brida and appears following environmental fragments will be cloned and sequenced. question as to whether the hypermethylation stress such as nutrient deficiency and episo- found in all regenerants is developmentally-re- mal TVCV is found in the hybrid Nicotiana ed- Application of the amplified fragment lated or a consequence of the tissue culture wardsonii after changes in daylength. Inte- length polymorphism (AFLP) environment per se remains to be answered. grated viral sequences essentially identical to and the methylation-sensitive amplified The correlations found between AFLP and the episomal virus sequences are found at polymorphism (MSAP) techniques MSAP polymorphisms provide indirect evi- high copy number in both the hybrid species. for the detection of DNA polymorphisms and changes in DNA dence that hyper-methylation may induce As is the case for Musa and BSV, the virus se- methylation in micropropagated base changes, perhaps by deamination quences are integrated into only one of the bananas (Kaeppler et al. 2000). All the regenerants are parental genomes of the hybrid, although epi- presently being grown to maturity in our exper- somal virus is not detectable in that parent. A. James, V. Herrera, L. Peraza and S. Peraza imental plantation in Yucatan so that a pheno- This suggests that the other parental genome Centro de Investigación Científica de Yucatán, Calle 43 #130, typic characterization will be possible. plays a part in the “activation” of the virus se- Colonia Churburna de Hidalgo, CP 97200, Mérida, Yucatán, Mexico. quences in the hybrid. References Fragments of a tobacco pararetrovirus-like The effect of the explant source on DNA poly- Kaeppler S., H.F. Kaeppler & Y. Rhee. 2000. (TPVL) sequence have been found in genomic morphisms and changes in methylation in the Epigenetic aspects of somaclonal variation in plants. DNA of Nicotiana sp. (Jakowitsch et al. 1999). leaves of micropropagated Musa AAA Plant Mol. Biol. 43:179-188. We have shown that pararetrovirus sequences ´Grande Naine´ was investigated. Explants Xiong L.Z., C.G. Xu, S. Maroof & Q. Zhang. 1999. probably comprise an important and wide- were derived from either the young male floral Patterns of cytosine methylation in an elite rice spread component of plant genomes including apices or suckers, and shoot cultures induced hybrid and its parental lines, detected by a Gymnosperms and Angiosperms. Their pres-

IV PROMUSA INFOMUSA — Vol 10, N° 1 ence may have consequences for gene silenc- land. These new hybrids, originating from the DNA from two diploid B bananas, as well as ing and genome evolution. As yet there is CIRAD Musa breeding programme, were from cvs. Obino l’Ewai, Goldfinger and Pisang no evidence that these sequences give rise to under evaluation for resistance to Fusarium Ceylan, suggesting that sequence of BSV-GF novel viral symptoms, as suggested for related oxysporum f.sp. cubense for 12-18 months is also integrated. No hybridization was ob- integrated pararetroviral sequences. prior to symptom expression. These plants served between either the BSV-Mys or BSV- We are examining the nature and genomic tested negative for BSV-Onne by immuno- GF probes and DNA from a range of AA and context of integrated BSV sequences in Obino capture- (IC-) PCR. However, IRFA 909 and AAA cultivars, suggesting that the integrated l’Ewai and in other Musa. A moderately re- 910 did test positive for BSV-Goldfinger by DNA is linked to B genome of cultivated Musa. peated sequence, which flanks the integrated IC-PCR. The badnavirus from IRFA 914 was BSV sequence in Obino l’Ewai (MusaOL) is unlike any previously examined. We have Reference concentrated with varying copy number near named this virus isolate BSV-IM. Using de- Geering A.D.W., L.A. McMichael, R.G. Dietzgen & the centromeres of most chromosomes of generate PCR primers, we amplified DNA of J.E. Thomas. 2000. Genetic diversity among both the A and B genome of Musa. The low BSV-IM, and using sequence of the DNA Banana streak virus isolates from Australia. numbers of BSV-related integrans per fragment, designed virus-specific primers. Phytopathology 90:921-927. genome indicates that BSV integration oc- Using this new PCR assay, we have shown curred after the amplification and distribution that IRFA 909 and 910 were infected with Gene expression of MusaOL sequences and hence is most both BSV-Goldfinger and BSV-IM. In re- in transgenic plants likely a recent event. peated assays over time, IRFA 914 has only ever tested positive for BSV-IM, and not Agrobacterium-mediated References BSV-GF. We have also found BSV-IM infect- transformation for the generation Harper G., J.O. Osuji, J.S. Heslop-Harrison & R. Hull. ing an IRFA 914 plant in New Caledonia. of transgenic banana (Musa spp.) 1999. Integration of banana streak badnavirus into We purified virus from IRFA 910, and have the Musa genome: molecular and cytogenetic obtained DNA clones representing the whole 1* 1 evidence. Virology 255:207-213. genome of BSV-IM. We have completed se- J.B. Pérez Hernández , R. Swennen , V. Galán 2 1 Ndowora T., G. Dahal, D. LaFleur, G. Harper, R. Hull, quencing this virus and initial sequence Saúco and L. Sági 1 N. Olszewski & B. Lockhart. 1999. Evidence that analyses suggest that the BSV-IM is a dis- Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven, Belgium; 2Department of Tropical Fruits, badnavirus infection in Musa can originate from tinct virus species. When proteins encoded Instituto Canario de Investigaciones Agrarias, La Laguna, Spain. integrated pararetroviral sequences. Virology by ORFs I, II and III of BSV-OL (GenBank (*Current address: Department of Tropical Fruits, Instituto 255:214-220. Accession AJ002234) and BSV-IM were Canario de Investigaciones Agrarias, La Laguna, Spain). Richert-Pöggeler K.R. & R.J. Shepherd. 1997. Petunia compared, the sequence identities were 60.5, A systematic evaluation of the successive vein clearing virus: a plant pararetrovirus with the 42.3 and 64.3%, respectively. We have con- steps in the natural Agrobacterium-plant in- core sequences for an integrase function. Virology sidered the possibility that BSV-IM has arisen teraction resulted in the elaboration of an effi- 236:137-146. from integrated virus sequences. Our virus cient transformation protocol for banana. Lockhart B.E., J. Menke, G. Dahal & N. E. Olszewski. clones hybridized to EcoRI and HindIII di- Chemotaxis and physical attachment of bac- 2000. Characterization and genomic analysis of gested DNA of two diploid B parents of the terial cells were observed in different cells tobacco vein-clearing virus, a plant pararetrovirus that IRFA hybrid lines, but failed to hybridize to and tissues of various banana cultivars is transmitted vertically and related to sequences similarly digested DNA from cvs. Obino (Pérez Hernández et al. 1999). Transient re- integrated in the host genome. J. Gen. Virol. 81:1579- L’Ewai Calcutta 4 and several AAA cultivars. porter gene expression was demonstrated in 1585. The virus clones also hybridized to uncut ge- several tissues cocultivated with vir-induced Jakowitsch J., M.F. Mette, J. van der Winden, nomic DNA of both diploid B parents. Both Agrobacterium and the highest frequencies M.A. Matzke and A.J.M. Matzke. 1999. Integrated diploid B parents have never shown symp- were found in embryogenic cell suspension pararetroviral sequences define a unique class of toms of BSV infection, and have tested nega- cultures. Stable transformation was obtained dispersed repetitive DNA in plants. Proceedings of tive for BSV infection by immunosorbent after selection on geneticin- or Basta-contain- the National Academy of Sciences USA electron microscopy of concentrated leaf ex- ing medium. In total, more than 600 trans- 96(23):13241-13246. tracts. The hybridization patterns observed genic plants were regenerated in five inde- are not consistent with those expected with pendent experiments, and more than 90% of episomal virus DNA. These results suggest Is banana streak virus strain OL them expressed the introduced genes (gfp or the only activateable virus integrant that BSV-IM has arisen via activation of inte- gusA). Molecular characterization revealed a in the Musa genome? grated sequences. simple integration pattern in most transgenic We have also examined the possibility that A.D.W. Geering1, J.N. Parry1, L. Zhang2, plants. Transgenic plants containing the gene other strains of BSV are also integrated into N.E. Olszewski3, B.E.L. Lockhart2 encoding the Ace-AMP1 antimicrobial pep- the Musa genome. Using probes to the com- and J.E. Thomas1 tide (Cammue et al.) were screened with a plete genome of BSV-Mys (Geering et al. leaf disc bioassay and candidate plants with 1Queensland Horticulture Institute, Department of Primary 2000), we observed complex hybridization increased fungus tolerance were identified Industries, 80 Meiers Road, Indooroopilly, QLD 4068, patterns with EcoRI and HindIII digested DNA Australia; 2,3 Departments of Plant Pathology and Plant (Pérez Hernández 2000). Biology, respectively, University of Minnesota, St. Paul, from three diploid B bananas, as well as from Minnesota 55108, USA. cvs. Obino l’Ewai (AAB group), Goldfinger References (AAAB group) and Pisang Ceylan (AAB Pérez Hernández J. B., S. Remy, V. Galán Saúco, R. In 1999, there were severe outbreaks of ba- group), suggesting that sequence of BSV-Mys Swennen & L. Sági. 1999. Chemotactic movement nana streak virus (BSV) in plantings of the is integrated. Likewise, when probed with a and attachment of Agrobacterium tumefaciens to IRFA 909, 910 and 914 hybrids at separate 1.3 kb BSV-GF probe (Geering et al. 2000), a single cells and tissues of banana. Journal of Plant locations in New South Wales and Queens- ca. 20 kb HindIII fragment was detected in Physiology 155:245-250.

INFOMUSA — Vol 10, N° 1 PROMUSA V Cammue B.P.A., K. Thevissen, M. Hendricks, K. transformation of Cavendish banana (Musa spp. tive generations in the field. Two of these pro- Eggermont, I.J. Goderis, P. Proost, J. Van Damme, AAA group) cv “Grand Nain” via microprojectile moters have also been used in experiments to R.W. Osborn, F. Guerbette, J.-C. Kader & W.F. bombardment. Plant Cell Reports 19:229-234. delay fruit ripening by inhibiting fruit-specific Broekaert. 1995. A potent antimicrobial protein from Pérez Hernández J.B. 2000. Development and ethylene synthesis using sense suppression. onion (Allium cepa L.) seeds showing sequence application of Agrobacterium-mediated genetic Transgenic plants have been assessed in field homology to plant lipid transfer proteins. Plant transformation to increase fungus-resistance in trials in Costa Rica and southern Mexico and Physiology 109:445-455. banana (Musa spp.). PhD Thesis. Katholieke several lines have been shown to have signifi- Pérez Hernández J.B. 2000. Development and Universiteit Leuven, Belgium. cant delays in fruit ripening over multiple gen- application of Agrobacterium-mediated genetic erations. A ~23 base RNA fragment diagnostic transformation to increase fungus-resistance in Virus and plant-derived promoters of the gene silencing phenomenon has been banana (Musa spp.). PhD Thesis. Katholieke for transgene expression identified in these suppressed lines. Universiteit Leuven, in banana Transgenic lines expressing five putative Belgium. S.R. Hermann, B. Dugdale, disease resistance genes are currently under- O.K. Becker, R.M. Harding and J.L. Dale going field-testing in Costa Rica. Transfor- A novel PCR-based method Centre for Molecular Biotechnology, Queensland mants expressing 11 more putative disease for the characterization of transgene University of Technology, GPO Box 2434, Brisbane QLD resistance genes or combinations of genes insertion in transgenic plants 4001, Australia. are at various stages of preparation. We are * also using a leaf piece assay to assess some J.B. Pérez Hernández , R. Swennen and L. Sági Promoter regions derived from banana bunchy of our transgenics in house. The symptoms Laboratory of Tropical Crop Improvement, Katholieke Universiteit top virus (BBTV) satellite components (S1 and Leuven, Belgium. (*Current address: Department of Tropical produced in this assay are similar to those S2) and banana actin genes have been iso- Fruits, Instituto Canario de Investigaciones Agrarias, La Laguna, seen in the field in terms of appearance, tim- Spain). lated and characterized in transgenic banana ing and cultivar specificity. plants. The BBTV SI and S2 promoters di- An anchored-PCR (APCR) method was devel- rected vascular-associated reporter gene ex- oped which allows a fast molecular characteri- pression in both dicots and monocots. In ba- Biotechnological approaches zation of transgenic plants generated via for banana improvement nana, the activity of these promoters was Agrobacterium-mediated transformation. Ge- significantly increased by the inclusion of T.R. Ganapathi, P. Suprasanna, nomic DNA fragments obtained by digestion monocot-derived introns. Actin gene candi- V.M. Kulkarni, Apratim Chakrabarti with restriction enzymes are specifically ampli- dates and their associated 5’ upstream se- and V.A. Bapat fied with a T-DNA-specific primer in combina- quences were isolated from a variety of plant Plant Cell Culture Technology Section, Nuclear Agriculture and tion with an adaptor-specific primer. The incor- sources, including banana, using a novel liga- Biotechnology Division, Bhabha Atomic Research Centre, poration of suppression PCR conditions Trombay, Mumbai 400 085, India. tion-mediated PCR approach for amplifying (Siebert et al. 1995) resulted in a significant flanking sequences. Expression levels and the improvement and allowed the one-step ampli- Bananas and plantains are the fourth most tissue specificity of one particular banana fication of specific APCR fragments. Southern important food crop and staple food crop for actin gene (ACT1) were further characterized. hybridization of T-DNA border-specific probes millions of people in the developing world. Northern analysis suggested banana ACT1 is to the APCR fragments revealed that they India is the largest producer of banana in the expressed in both reproductive and vegetative were indeed correctly amplified from the trans- world. In this country, banana is the second tissues. In transgenic banana plants, the gene(s). The APCR analysis of a tester set of most important fruit crop and is grown ° 0.4 ACT1 promoter directed strong reporter gene 20 transgenic banana plants demonstrated million ha with a production of 10 million tons. expression in both leaves and roots. Trunca- that about 70% of them contained one or two Conventional method of breeding is compli- tions of the ACT1 promoter indicated all the transgene insertions, which compares cated due to the triploid nature and only a few necessary regulatory elements required for favourably with the transgene insertion pattern diploid clones produce viable pollen. Improve- high level (2-fold greater than CaMV 35S) in plants obtained via microparticle bombardment for disease resistance and productivity near constitutive expression are located within ment (Becker et al. 2000). The technique also requires the use of biotechnological tools. Our 1.2 kb of the ACT1 ATG. allowed the fine structure of the integrated group is engaged in tissue culture, somatic transgene(s) to be revealed: correct as well as Better bananas - the biotech way embryogenesis, synthetic seeds, in vitro mu- truncated insertions were observed, and tagenesis and selection, DNA fingerprinting plants containing vector backbone sequences P. Balint-Kurti, E. Firoozabady, Y. Moy, J. and Agrobacterium-mediated gene transfer. could be identified. In addition, transgenic Mercier, R. Fong, L. Wong and N. Gutterson Thirty cultivars/wild species have been con- plants representing identical transformation DNAP (DNA Plant Technology Corporation), 6701 San Pablo served and propagated in vitro. Tissue cul- Avenue, Oakland, Ca. 94608-1239, USA. E-mail: balint- events were easily recognized. Finally, nu- [email protected] ture-raised plants planted at multilocations ex- cleotide sequence analysis of cloned APCR hibited increased yield, early maturity and fragments fully confirmed the above findings At DNAP, our efforts in banana are focused on more uniform production cycle. In vitro cul- (Pérez Hernández 2000). black Sigatoka resistance, with emphasis at tures were gamma-irradiated and field evalua- the early stages of variety development on un- tion of the irradiated population resulted in References derstanding expression characteristics of can- certain promising variants. Isolated variants Siebert P.D., A. Chenchik, D.E. Kellogg, didate gene expression signals. Using the and parent cultivars were analyzed in the K.A. Lukyanov & S.A. Lukyanov. 1995. An improved chimeric uidA gene constructs to assess pro- field, and at molecular level using RAPDs. PCR method for walking in uncloned genomic DNA. moter function, we have been able to identify Protocols have been developed for somatic Nucleic Acids Research 23:1087-1088. several promoters with relatively strong activ- embryogenesis using shoot tip sections in cv. Becker D.K., B. Dugdale., M.K. Smith, ity in leaf, fruit and root tissue. These activities Rasthali (AAB) and male flower buds in cv. R.M. Harding & J.L. Dale. 2000. Genetic seem to be maintained over several vegeta- Shrimanti (AAA). Embryogenic cell cultures

VI PROMUSA INFOMUSA — Vol 10, N° 1 have been successfully established and main- humans or animals; b) plants and animals, ing banana. The disease is found in almost all tained by regular subcultures for the past two but not microorganisms; c) biological banana-growing regions of the world except years (in Rasthali). High frequency conversion processes for producing plants and ani- the Caribbean and the Americas. In the of somatic embryos to plants has been mals, but not technical processes. Plant va- 1920s, bunchy top was the major limiting fac- achieved and the somatic embryo-derived rieties must be protected in one way or an- tor to banana production in Australia. The dis- plants are being field-evaluated. other. ease has since been controlled in Australia Agrobacterium-mediated transformation (For full text of the Agreement, see WTO through the implementation of strict phytosani- using embryogenic cell cultures of cv. Website:http://www.wto.org/english/ tary control measures backed up by strict gov- Rasthali has been standardized and is now tratop_e/trips_e/t_agm3c_e.htm) ernment legislation. Our group has been char- routinely used for gene transfer. Currently, The patentability of living organisms was acterizing this virus for the past 10 years in an we are working with an anti-microbial pep- uncertain prior to the decision of the US effort to develop transgenic virus resistance tide, msi99 (a synthetic homologue of Maga- Supreme Court in Diamond v Chakrabarty 447 and to further exploit the virus. inin). Studies have shown that this peptide US 303 (1980). The Court decided that living BBTV was initially thought to be caused by effectively inhibits the growth of Fusarium organisms could be patented by a slim major- a luteovirus based on symptoms, persistent oxysporum, the causal agent of Fusarium ity (5 to 4). If the decision had gone the other aphid transmission and dsRNA profiles. How- wilt. Rasthali, a highly susceptible cultivar, way it may well have led to a decrease in in- ever, it is now known that BBTV is an isomet- has been transformed with msi99 and trans- vestment in the biotechnology industry. ric virus with a genome comprising at least six genic plants have been regenerated. Case law in the USA and in Europe indi- different components of circular single- cates that the limitations on patentability of stranded DNA (BBTV DNA-1 to -6) ranging in biotechnological inventions are not yet fully size from 1018 to 1111 nucleotides. Each Intellectual property defined. DNA component shares a common genome and genetically modified 1. Courts have interpreted patent legislation organization including (i) one major gene in organisms to include living organisms. the virion sense (except DNA-1 which con- 2. Ordre public/morality arguments are only tains two genes) with an associated Keynote likely to succeed in the most extreme polyadenylation signal, (ii) a conserved major cases. common region (CR-M) and stem-loop region D. Nicoll 3. US Courts are attempting to meet some (CR-SL), and (iii) a potential TATA box located Centre for Law and Genetics, Law School, University of of the issues associated with broad patent 3’ of the stem-loop. The CR-M is located 5’ of Tasmania, GPO Box 252-89, Hobart, Tas 7001, Australia. claims. the CR-SL and comprises approximately 92 nt It is probably fair to say that patenting will now Article 27 of TRIPs allows member coun- with at least 72% homology amongst the DNA usually be in the mind of the genetics re- tries some flexibility in deciding which types of components (except for DNA-1 which has a searcher for a number of reasons, including: biotechnological inventions should be 26 nt deletion). The CR-M is believed to be in- 1. the changing nature of academic science, patentable. This, together with the scope for volved in replication, where it is thought to act in particular the need for accountability in varied interpretation of national IP legislation as a binding site for an endogenous ~80 nt economic terms; by national courts, gives individual countries DNA primer. The CR-SL comprises 69 nt with 2. the nature of biotechnological research: ex- some leeway to provide the level of IP protec- at least 62% homology between components. pensive, time-consuming and easily copied; tion that they deem to be acceptable, within It incorporates a stem-loop structure which and the framework of their own cultural, moral and contains a 10 bp stem (14 nt conserved) and 3. the increasing involvement of the private legal norms (trade barriers aside). an 11 nt loop (9 nt conserved). Based on the sector in the research phase. Institutions and global programmes, like sequence analysis of DNA-1, -3 and -5, there The most influential international treaty on INIBAP and PROMUSA, have an important are two distinct groups of BBTV isolates, the intellectual property (IP) rights is the Agree- role to play in IP management. In particular, South Pacific group (Australia, Burundi, Egypt, ment on Trade Related Aspects of Intellectual they have the capacity to influence decisions Fiji, India, Tonga and Samoa) and the Asian Property Rights, or TRIPs, which is an annex- about the acquisition of material that may be group (Philippines, Taiwan, Vietnam). These ure to the WTO Agreement. If a country wants used to create patentable inventions and the two groups differ by ~10% over the entire nu- to trade it has to have TRIPs-compatible IP transfer of technology using that material. cleotide sequence and by ~30% within the law. Article 27 sets out the following patent CR-M. law requirements: The major gene of DNA-1 contains motifs • 27.1: Patents are mandatory for any inven- Plant pathology associated with rolling circle replication and tions in all fields of technology. The ele- and disease resistance dNTP binding and encodes a replication initi- ments of novelty, inventive step (non-obvi- ation (Rep) protein. This Rep protein has ousness) and industrial applicability (utility) The molecular biology of banana been shown to possess site-specific nickase must be satisfied. bunchy top virus and ligase activity (cleaves between nt 7 and • 27.2: Inventions may be excluded to pre- R.M. Harding, B. Dugdale, G.J. Hafner, C.L. 8 of the stem-loop). The function of the inter- vent commercial exploitation of the inven- Horser*, R. Wanitchakorn and J.L. Dale nal gene of DNA-1 is currently unknown. tion to protect ordre public or morality, in- Centre for Molecular Biotechnology, Queensland University DNA-3 encodes the coat protein while the cluding protecting human, animal or plant of Technology, GPO Box 2434, Brisbane QLD 4001, Australia. gene product of DNA-5 has been shown to life or health and avoiding serious prejudice (*Current address: CSIRO Plant Industry, Canberra, ACT, possess retinoblastoma-binding activity and 2601, Australia). to the environment. is thought to be a cell-cycle protein responsi- • 27.3: Other inventions that may be ex- Banana bunchy top disease, caused by ba- ble for switching infected cells into S-phase cluded include: a) diagnostic, therapeutic nana bunchy top nanovirus (BBTV), is consid- to facilitate virus replication. DNA-4 and -6 and surgical methods for the treatment of ered the most important virus disease affect- appear to encode proteins associated with

INFOMUSA — Vol 10, N° 1 PROMUSA VII cell-to-cell movement and nuclear shuttling, The epidemiology of banana bunchy fibre crop Musa textilis. Six viruses have been respectively. The function of DNA-2 remains top virus in Vietnam characterized from Musa to date (Jones unclear. K. Bell1, P.A. Revill2, H.V. Cuong3, 2000), but additional, uncharacterized viruses BBTV has recently been classified in the V.T. Man4 and J.L.Dale2 have also been recognized. genus Nanovirus – viruses with isometric viri- 1Seowon Building, 4th Floor, 57 Garak-Dong, Songpa-Gu, Banana bunchy top virus (BBTV) has ons which are phloem limited and possess a Seoul, South Korea 138-160; 2Centre for Molecular 18-20 nm isometric virions and a multicompo- multicomponent, circular, single-stranded DNA Biotechnology, Queensland University of Technology, GPO nent ssDNA genome. It is transmitted in the Box 2434, Brisbane QLD 4001, Australia; 3Department of Plant genome. Other members of this genus include Pathology, Hanoi Agricultural University, Gia Lam, Hanoi, persistent manner by the banana aphid Pen- subterranean clover stunt virus (SCSV), faba Vietnam. talonia nigronervosa and has a scattered dis- bean necrotic yellows virus (FBNYV), milk tribution in Africa and the Asia-Pacific region. Banana bunchy top virus (BBTV) causes the vetch dwarf virus (MDV) and possible coconut Cucumber mosaic virus (CMV) has 29 nm most serious viral disease of bananas world- foliar decay virus (CFDV). isometric virions and a tripartite ssRNA wide. Banana bunchy top disease almost de- BBTV DNA-1 to -6 are considered integral genome. It is transmitted in the non-persistent stroyed the banana industry in Australia in the to the BBTV genome since these components manner by a number of aphid species, and early 1920s, and similar epidemics have oc- are consistently associated with all BBTV in- has a widespread international distribution. curred in other countries throughout the world. fections worldwide. Several additional BBTV- Banana bract mosaic virus (BBrMV) and BBTV was first identified in Vietnam in 1968, associated DNA components have also been Abaca mosaic virus (AbaMV) both have fila- and is endemic throughout the country. How- isolated from various BBTV infections. Like mentous virions, a ssRNA genome and are ever the epidemiology of BBTV in Vietnam ap- BBTV DNA-1, these additional components transmitted in the non-persistent manner by a pears to be quite different to that observed in appear to encode Rep proteins. However, number of aphid species. AbaMV has only other countries, as it does not cause serious they differ from BBTV DNA-1 in several re- been recorded from the Philippines, while epidemics, and appears to move more slowly spects, including: BBrMV has a scattered distribution in the through a crop. BBTV is transmitted by the • genome organization – in general, the CR-M Asia-Pacific region. Banana streak virus aphid Pentalonia nigronervosa, or through in- and CR-SL are absent, and the TATA box (BSV) has bacilliform virions (30 x 130 nm) fected plant suckers and corms, and typically is located 5’ of the stem-loop; and containing a dsDNA genome, and has a moves rapidly through a crop. However, in • they have a limited geographical distribution worldwide distribution. Vietnam it is not unusual to find older BBTV-in- – they are almost exclusively restricted to The filamentous virions of Banana mild mo- fected plants adjacent to healthy plants, with the Asian group of BBTV. saic virus (BanMMV) contain a ssRNA banana aphids feeding on all plants. In addi- We have been examining the replication of genome of 7353 nt, encoding five ORFs. Al- tion, we have not observed typical BBTV BBTV to determine (i) the integral compo- though related to carlaviruses, foveaviruses symptoms on the local cultivar Chuoi tay. It is nents of the BBTV genome, (ii) which com- and potexviruses, the genome organization unknown whether Chuoi tay is a host for ponent encodes the “master” Rep, and (iii) and phylogenetic relationships of BanMMV BBTV, or whether it is resistant to BBTV infec- the role of the BBTV DNA-1 internal gene. place it apart from all previously described tion. To improve our understanding of BBTV These studies have involved the bombard- virus taxa (Gambley and Thomas, in press). epidemiology in Vietnam, we investigated a ment of Bluggoe embryogenic cell suspen- The virus occurs in a wide range of Musa number of factors: (1) we investigated the level sions with cloned 1.1mers of the different genotypes and has a worldwide distribution. of sequence variability of DNA-1, the master BBTV DNA components either singly or in The virus often occurs as symptomless infec- rep-encoding component, and showed that se- combination. DNA was extracted from the tions and mixed infections with other viruses, quence variability of BBTV in Vietnam is higher cells at 0, 4 and 8 days post-bombardment though its mode of transmission is not known. than previously recorded in Asia. We also ob- and analyzed with component-specific Its economic impact is unknown. served that sequences separated into northern probes for replicative intermediates. These Serological and PCR-based diagnostic as- and southern Vietnamese isolates, depending studies have shown that DNA-1 encodes the says are available for all the characterized on their origin in Vietnam; (2) we identified a “master” viral Rep protein and represents the viruses of Musa, but BSV still presents chal- new putative satellite DNA component en- minimal replicative unit of BBTV since this lenges. With BSV, symptoms can be promi- demic to Vietnam. Finally, we screened Chuoi component, and not the additional Rep- nent, but occur sporadically. Considerable se- tay plants from throughout Vietnam for BBTV, encoding components, is capable of self- quence diversity has been found in BSV, and but did not detect virus in any plants using replication as well as directing the replication five of these isolates (BSV-OL, BSV-Mys, PCR and/or Southern hybridization. This sug- of the other integral BBTV genomic compo- BSV-GF, BSV-IM and BSV-Lac) are probably gests that Chuoi tay may be resistant to BBTV nents. We also showed that the internal gene sufficiently distinct to be considered separate in Vietnam, which could be one of the factors of DNA-1 is not essential for replication but viruses (Geering et al. 2000, A.D.W. Geering, influencing the epidemiology of banana enhances replication in cis (possibly N.E. Olszewski, B.E.L. Lockhart and J.E. bunchy top disease in Vietnam. analagous to the REn protein of bego- Thomas, unpublished). Immunocapture (IC) moviruses). Finally, we have identified poten- Viruses and Musa germplasm assays are required to differentiate episomal tial Rep-binding sites (iterons) on the BBTV and integrated BSV sequences. IC-PCR with genome which appear to be similar to those J.E. Thomas, C.F. Gambley, A.D.W. Geering, microplate detection has been developed for of the begomoviruses. The results of this L.A. McMichael, J.N. Parry and M. Sharman all the characterized viruses of banana. A study have suggested the possibility of two Queensland Horticulture Institute, Department of Primary multiplex assay for BBrMV, BBTV and CMV Industries, 80 Meiers Road, Indooroopilly, QLD 4068, groups of nanoviruses: (i) BBTV – which in- Australia. has been published (Sharman et al. 2000). fects monocots and contains an internal gene Assays for BanMMV and all known strains of in the “master” Rep, and (ii) FBNYV, MDV Commercially important Musa spp. include ed- BSV (multiplex) have also been developed and SCSV – all infect dicots and do not pos- ible bananas and plantains (mostly hybrids of (M. Sharman, A.D.W. Geering, J.N. Parry and sess an internal gene in the “master” Rep. M. acuminata and/or M. balbisiana) and the J.E. Thomas, unpublished). These assays are

VIII PROMUSA INFOMUSA — Vol 10, N° 1 used in conjunction with ELISA and ISEM for enset. CABI Publishing, Wallingford, UK/New York, preservation was performed on meristematic routine virus indexing. USA. clumps excised from highly proliferating All viruses of Musa are transmitted through Sharman M., J.E. Thomas & R.G. Dietzgen. 2000. meristem cultures by the vitrification proce- vegetative propagules, including in vitro Development of a multiplex immunocapture PCR dure using the PVS-2 solution (Sakaï plantlets, and this has implications for the with colourimetric detection for viruses of banana. et al. 1990). health of planting material, the conduct of Journal of Virological Methods 89:75-88. Our results show that eradication rates after breeding and transformation programmes Thomas J.E., M.K. Smith, Kessling, A.F. & S.D. Hamill. cryopreservation of highly proliferating meris- and the transfer of germplasm. Virus-free 1995. Inconsistent transmission of banana bunchy tems reach 39% (32 plants out of 83 tested planting material is a major factor in field con- top virus in micropropagated bananas and its plants) and 94% (31 plants out of 33 tested trol of these pathogens and, additionally, sev- implication for germplasm screening. Australian plants) for CMV and BSV respectively. In com- eral of these viruses have limited distribu- Journal of Agricultural Research 46:663-671. parison, eradication rates obtained by culture tions. Few studies have been conducted on of meristems excised from highly proliferating the transmission of banana viruses through Elimination of banana and plantain meristems reached 11% and 63% for CMV tissue culture. Several studies have shown (Musa spp.) viral diseases and BSV respectively. that, through normal subculturing, a propor- by cryopreservation Ultrastructural study of highly proliferating tion of virus-free meristems arise from initially B. Helliot1, B. Panis2, A. Locicero1, K. Reyniers2, meristems performed after a 1-week in vitro BBTV-infected clones. This process appears R. Swennen2 and P. Lepoivre1 culture following cryopreservation showed that to be accelerated somewhat at elevated tem- 1Plant Pathology Unit, Gembloux Agricultural University, 5030 cryotherapy acts as a micro-scalpel. Small peratures, and plants derived from these Gembloux, Belgium. Email: [email protected]; 2Laboratory areas of living cells located in the meristematic meristems remain virus-free (Thomas et al. of Tropical Crop Improvement, Katholieke Universiteit Leuven, dome and at the base of the primordia survive 3001 Leuven, Belgium. 1995, and references therein). Recently, the the cryopreservation procedure, while more reverse situation has occurred with BSV. Cryopreservation is becoming a routine in vitro differentiated cells, distant to the apical dome Virus infections have been detected in prog- technique which overcomes the serious limita- are killed. Associated with an uneven distribu- eny of hybrids from breeding programmes, tions encountered by the traditional tion of viral particles in the meristem, this where there was no evidence of virus infec- germplasm conservation strategies used in could explain the efficiency of cryopreservation in the parent lines. This has been shown field, seed and in vitro culture collections. The tion. The specific localization of viral particles to be due to «activation» or «release» of BSV conservation at ultra-low temperatures, usu- within the meristem is now under investiga- sequences that are integrated into the Musa ally at –196°C which is the temperature of liq- tion. We hope to gain a better understanding genome (Hull et al. 2000). Recent evidence uid nitrogen, allows a long-term and contami- of the variations in observed eradication rates suggests that several additional strains of nation-free storage of plant genetic resources. according to the virus and according to the BSV may be integrated into different compo- Recently, Brison and collaborators (1997) therapy. nents of the hybrid Musa genome (A.D.W. have demonstrated that cryopreservation Geering, N.E. Olszewski, B.E.L. Lockhart and could be used, in addition to germplasm con- References J.E. Thomas, unpublished). servation, to eliminate viruses from in vitro Brison M., M.T. de Boucaud, A. Pierronnet & F. Dosba. The INIBAP Transit Centre at K.U. Leuven plum shoots infected with plum pox virus with 1997. Effect of cryopreservation on the sanitary houses the world’s largest in vitro Musa an eradication rate of up to 50%. The possibil- state of a cv Prunus rootstock experimentally germplasm collection comprising over 1100 ity of applying a short (few hours) cryopreser- contaminated with Plum Pox Potyvirus. Plant accessions. These accessions are being in- vation treatment instead of a long (few weeks) Science 123(1-2):189-196. dexed for viruses at three international Virus heat treatment would be highly promising. Panis B., H. Schoofs, N.T. Thinh & R. Swennen. 2000. Indexing Centres (CIRAD, Montpellier, PPRI, We previously reported on the successful Cryopreservation of proliferating meristem cultures Pretoria, and QDPI, Brisbane), and only ac- cryopreservation of proliferating meristems of of banana. Pp. 238-243 in Cryopreservation of cessions testing negative for known viruses different accessions of banana, one of the tropical plant germplasm. Current research progress are released. BanMMV and BSV are the most most important staple food crops of the world and applications (F. Engelmann & H. Takagi, eds.). frequently detected viruses, probably due to (Panis et al. 2000). Bananas, which belong to Japanese International Research Center for frequent latent infection, and the additional the Musa genus, are found in about 120 Agricultural Sciences, Tsukuba, Japan / factor of BSV integration. BBTV and BBrMV countries, mainly tropical and subtropical, on International Plant Genetic Resources Institute, have not been detected in the collection. five continents and provide subsistence to Rome, Italy. millions of people. However, banana plants Sakai A., S. Kobayashi & I. Oiyama. 1990. References are threatened by different biotic agents such Cryopreservation of nucellar cells of navel orange Gambley C.F. & J.E. Thomas. 2001. Molecular as bacteria, fungi or viruses, like cucumber (Citrus sinensis Osb. var. brasiliensis Tanaka) by characterisation of Banana mild mosaic virus, a new mosaic virus (CMV), banana bunchy top virus vitrification. Plant Cell Rep. 9:30-33. filamentous virus in Musa spp. Archives of Virology (BBTV), banana streak virus (BSV), banana (in press). bract mosaic virus (BBrMV) and banana mild A DNA-based diagnostic test for Geering A.D.W., L.A. McMichael, R.G. Dietzgen & J.E. mosaic virus (BaMMV). ‘tropical’ race 4 of Fusarium wilt Thomas. 2000. Genetic diversity among Banana In the framework of an INIBAP project enti- of banana streak virus isolates from Australia. Phytopathology tled “Development of in vitro culture tech- S. Bentley, N. Moore, J. Pattemore, 90:921-927. niques for the elimination of banana and J. Anderson and K. Pegg Hull R., G. Harper & B. Lockhart. 2000. Viral plantain (Musa spp.) viral diseases”, we CRCTPP (Cooperative Research Center for Tropical Plant sequences integrated into plant genomes. Trends in aimed to evaluate the effect of cryotherapy Pathology), University of Queensland, Level 5, John Hines Plant Science 5(9):362-365. on the sanitary state of plant material in com- Bldg St Lucia, Brisbane QLD 4072, Australia. Jones D.R. (ed.) 2000. Diseases caused by viruses. parison with traditional methods such as Fusarium wilt of banana is a significant prob- Pp. 241-293 in Diseases of banana, abacá and meristem culture. For this purpose, cryo- lem to the Australian banana industry. The

INFOMUSA — Vol 10, N° 1 PROMUSA IX fungus which causes the disease, F. oxyspo- also be a useful research tool to study the bi- new desirable traits that are reflected in al- rum f.sp. cubense (Foc), is a highly diverse ology and ecology of Foc. tered phenotypes, for example enhanced dis- pathogen. At present, only a limited portion of ease resistance. Regulatory sequences or the global diversity of Foc has been found in Isolation of potential disease promoters are required to drive efficient ex- Australia. Thirty-three different vegetative resistance genes from banana pression of the introduced gene in transgenic compatibility groups (VCGs) and genotypes K.M. Taylor, J.A. McMahon, plants. Viral promoters, such as the 35S pro- of Foc have been identified globally, of which R.M. Harding and J.L. Dale moter of cauliflower mosaic virus, CaMV (Kay nine occur in Australia. Nearly all of the diver- et al. 1987), have been frequently used for Centre for Molecular Biotechnology, Queensland University sity within Foc has been identified in Asia, of Technology, GPO Box 2434, Brisbane QLD 4001, Australia. constitutive expression of transgenes in and our proximity to Southeast Asia presents E-mail: K0.taylor@ gut.edu.au many crops. To obtain strong promoters that considerable risk of introduction of new are suitable for high-level gene expression in strains of Foc, and in particular further intro- Bananas are susceptible to a wide range of transgenic banana, we have analyzed three ductions of the Cavendish-competent strain diseases, of which Fusarium wilt and black and novel promoter sequences from Australian ‘tropical’ race 4. ‘Tropical’ race 4 is wide- yellow Sigatoka are among the most devastat- banana streak badnavirus (BSV) isolates. spread throughout Indonesia and Malaysia, ing. Although most commercially grown dessert These were evaluated in different transient and has recently been detected in Irian Jaya. bananas are susceptible to these fungal and stable transformation assays using re- Several outbreaks of the ‘tropical’ race 4 pathogens, resistance has been identified in porter genes encoding the green fluorescent strain of Fusarium wilt have already occurred wild banana cultivars. A novel approach to protein (GFP) and B-glucuronidase (GUS) re- in the Northern Territory, and so far these identify the resistance genes (R genes) which porter enzymes (Schenk et al. 2001). In have been contained by quarantine mea- confer these resistance traits is to amplify ge- these experiments, 1322 bp (Cv), 2105 bp sures. This strain of Fusarium wilt poses a nomic banana DNA using degenerate primers (My) and 1297 bp (Go) DNA fragments sur- threat to the major Cavendish production designed to class 3 R genes. This approach rounding the transcription initiation site of the areas in north Queensland, which are has been used successfully on lettuce, soy- Cavendish, Mysore and Goldfinger BSV iso- presently free from all Cavendish-competent bean, rice and maize but to date no banana R lates (Geering et al. 2000) were analyzed for strains of the pathogen. gene candidates (RGCs) have been published. transcription-promoting activity. We are currently developing a DNA-based We have used degenerate primers to am- Using transient expression assays, the Cv, diagnostic test that is specific for the ‘tropical’ plify five independent RGC sequences from My and Go fragments were all shown to have race 4 strain of Foc. We have thoroughly an- banana, all of which show homology to previ- promoter activity in a wide range of plant alyzed genetic diversity within Foc from the ously characterized R genes. The five se- species including monocots (banana, maize, genus to the strain-specific taxon levels using quences were isolated from both resistant barley, millet, sorghum), dicots (tobacco, total genomic fingerprinting methods such as and susceptible cultivars and were present in canola, sunflower, Nicotiana benthamiana, DNA Amplification Fingerprinting (DAF) and low copy numbers. In addition, all five se- tipu tree), gymnosperm (Pinus radiata) and other PCR-based methods such as restriction quences were amplified from RNA, indicating fern (Nephrolepis cordifolia) (table 1). fragment length polymorphism (RFLP) and that they were transcribed. When the DNA GUS reporter enzyme activity was analyzed sequence analysis of the ribosomal (r) DNA. and RNA sequences from resistant and sus- in transgenic in vitro-grown banana plants We have identified DNA sequence informa- ceptible cultivars were compared, variability (cultivar Three Hand Planty) transformed with tion that is unique to the ‘tropical’ race 4 was observed between the five RGC se- the Cv or My promoter constructs. Longitudi- strain of Foc and designed PCR primers that quences (<53% homology) and within each nal and cross sections of roots, corms, pseu- specifically amplify DNA only from the ‘tropi- RGC (97-100% homology). Amplification of dostems and leaves revealed blue staining in cal’ race 4 strain. Database searches of DNA RGC flanking sequences revealed a 5’ all cell types analyzed (for colour photos, sequence information published in Genbank leucine zipper (LZ) domain and a 3’ leucine visit http://www.uq.edu.au/~uqtreman). The have indicated that there are no matches for rich repeat (LRR) domain, which is consistent strongest expression was observed in the these primers with any other organism, but with class 3 R genes. corm and the vascular tissue. In roots, a high we are currently completing the laboratory staining intensity was observed in vascular screening of the specificity of these primers. Banana streak virus promoters tissue and emerging side roots. Quantitative We will then adapt our laboratory PCR condi- are highly active in transgenic banana GUS activity levels for plants containing the tions for amplification of Foc DNA directly and other monocot and dicot plants My promoter constructs were higher in leaf, from infected plants and infested soil. The di- root and corm tissue compared to plants har- T. Remans1, L. Sági4, A.R. Elliott5, agnostic test will then require validation and bouring maize ubiquitin promoter constructs R.G. Dietzgen3, R. Swennen4, P. Ebert1, field-testing, prior to release to industry (Table 1). In glasshouse grown banana C.P.L. Grof5, J.M. Manners2,5 and P.M. Schenk2,3 and/or commercial laboratories. plants, the My promoter showed higher activi- 1Department of Biochemistry, The University of Queensland, We are also developing a DNA-based iden- Brisbane QLD 4072 Australia; 2CRCTPP (Cooperative ties than the maize ubiquitin and cauliflower tification system that will allow the accurate Research Center for Tropical Plant Pathology), University of mosaic virus 35S promoters (Table 1). The characterization of all the strains of Foc that Queensland, Level 5, John Hines Bldg St Lucia, Brisbane QLD Cv promoter showed activities that were simi- 4072, Australia; 3QDPI, Queensland Agricultural Biotechnology occur in Australia. This diagnostic system will Centre, The University of Queensland, St. Lucia, QLD 4072, lar to (root and corm) or higher than (leaf) allow the detection and identification of Foc di- Australia; 4Laboratory of Tropical Crop Improvement, those of the maize ubiquitin promoter in in 5 rectly from banana planting material and soil. Katholieke Universiteit Leuven, Belgium; CSIRO Plant vitro-grown banana plants, but which were Industries, Long Pocket Laboratories, 120 Meiers Road, This system will be useful for screening fields Indooroopilly, QLD 4068, Australia. E-mail for correspondence: significantly reduced in larger glasshouse- for the presence of Foc races prior to planting, [email protected] grown plants (Table 1). This may be related screening rhizomes or suckers used for plant- to silencing associated with the integrated ing material, identifying isolates of Foc from in- Genetic engineering of plants has proven to BSV sequence (Ndowora et al. 1999, Harper fected plant tissue or infested soil, and will be a useful method for the introduction of et al. 1999) in Three Hand Planty (AAB

X PROMUSA INFOMUSA — Vol 10, N° 1 genome) plants. As the integrated BSV se- References active for transgene expression in monocot and dicot quence is thought to be associated with the Geering A.D.W., L.A. McMichael, R.G. Dietzgen & plants. Plant Molecular Biology (submitted). B-genome, it would be interesting to see if J.E. Thomas. 2000. Genetic diversity among the Cv promoter is more active in AAA-type Banana streak virus isolates from Australia. “CIEN BTA-03”, a new somaclonal banana plants. Phytopathology 90:921-927. variant resistant to yellow Sigatoka: GFP levels in leaves and stems of trans- Harper G., J.O. Osuji, J.S. Heslop-Harrison & biochemical, genetic and molecular characterization and agronomic studies genic sugarcane plants harbouring a Cv pro- R. Hull. 1999. Integration of banana streak moter/GFP gene fusion were fluorometrically badnavirus into the Musa genome: E. de García1, C. Giménez1, M. del Carmen quantified (Remans et al. 1999) and found to molecular and cytogenetic evidence. Virology Vidal1, G. Palacios1 and O. Haddad2 be comparable to GFP levels in plants har- 255:207-213. 1Laboratorio de Biotecnología Vegetal, Universidad Central de bouring a maize ubiquitin promoter construct Kay R., A. Chan, M. Daly & J. McPherson. 1987. Venezuela, Apartado 80970, Caracas 1080, Venezuela (E- 2 (Table 1). The expression of both the Cv and Duplication of CaMV 35S promoter sequences mail: [email protected]); Instituto de Agronomía, Facultad de Agronomía, Universidad Central de Venezuela, Maracay, maize ubiquitin promoter remained also high creates a strong enhancer for plant genes. Venezuela. in ratoon plants of sugarcane. The My pro- Science 236:1299-1302. moter was active in young plants but GFP ex- Ndowora T., G. Dahal, D. LaFleur, G. Harper, In 1996, Trujillo and de García obtained a so- pression was not observed in mature plants. R. Hull, N.E. Olszewski & B. Lockhart. maclonal variant resistant to yellow Sigatoka Strong Go promoter activity was observed in 1999. Evidence that badnavirus infection in by induction of adventitious shoots from the transgenic sugarcane callus, but there was Musa can originate from integrated triploid clone Williams, subgroup Cavendish, no GFP expression in the regenerated pararetroviral sequences. locally named ‘Brasilero’, which is susceptible shoots. The Cv and My promoters were also Virology 255:214-220. to the disease (Trujillo and de García 1996, active in transgenic in vitro-grown tobacco Remans T., P.M. Schenk, J.M. Manners, C.P.L. Trujillo et al. 1999). This somaclonal variant is plants, but this activity was lost when these Grof & A.R. Elliott. 1999. A protocol for the not only resistant to the disease, but also dis- plants were grown to adult stage in the fluorometric quantification of mGFP5-ER plays a series of morphological and anatomi- glasshouse (Table 1). and sGFP(S65T) in transgenic plants. cal characteristics that distinguish it from The promoters from banana streak virus Plant Molecular Biology Reporter triploid clones: a) leaf blade 1.4 times thicker represent useful tools for the high-level ex- 17(4):385-395. than that of clone Williams (Hermoso et al. pression of foreign genes in both monocot Schenk P.M., T. Remans, L. Sági, A.R. Elliott, 1997, Trujillo et al. 1997); b) lower number of and dicot transgenic plants that could be R.G. Dietzgen, R. Swennen, P. Ebert, C.P.L. stomata per mm2 in both upper and lower epi- used interchangeably with the CaMV 35S or Grof & J.M. Manners. 2001. Promoters for dermis (Hermoso et al. 1997, Trujillo et al. maize ubiquitin promoters. pregenomic RNA of banana streak badnavirus are 1997); and c) higher phenol content. This clone was called CIEN BTA-03 (Figure 1). The aim of this work is to report the data of the biochemical, genetic and molecular characterization of CIEN BTA-03, as well as refer- ring to the evaluation of the resistant behaviour of the variant in the field. The biochemical studies based on the Table 1. Overview of BSV Cv, My and Go promoter activities compared to the CaMV 35S and maize analysis of the proteins by electrophoresis in ubiquitin promoters in different plant species. Values representing the highest expressing plant: GUS enzymatic activity (MU) in nmol MU/h/mg protein and GFP accumulation in mg GFP/mg protein. denaturing acrylamide SDS-PAGE gels, stained with coomassie blue and scanned in Cv My Go CaMV 35S Maize ubiquitin an Imaging Densitometer model GS-690 Transgenic plants (Bio-Rad) demonstrated the presence of two Banana (leaf in vitro) 1076 MU 6299 MU nt nt 214 MU polypeptides (14 and 17 kDa) in the Williams Banana (root+corm in vitro) 2502 MU 10650 MU nt nt 2571 MU clone that are neither observed in the CIEN Banana (leaf glasshouse) 0 MU 1658 MU nt 430 MU 418 MU BTA-03 clone, nor in the Fragro 7 (AAAA), Sugarcane (leaf glasshouse) 13.1 GFP < 0.05 GFP nt nt 11.6 GFP both resistant to yellow Sigatoka (Giménez Sugarcane (stem glasshouse) 5.57 GFP nt nt nt 0.80 GFP 1998). Tobacco (leaf in vitro) 0.68 GFP 1.35 GFP nt 1.68 GFP nt Cytogenetic analysis showed that both Tobacco (leaf glasshouse) < 0.06 GFP < 0.06 GFP nt 0.29 GFP nt clones presented mosaic tissues, but with a Transient assays different chromosome number distribution; Maize (sweet corn) +++ +++ +++ + +++ 22% of the cells of clone Williams have more Barley +++ +++ nt + nt Banana +++ +++ nt nt nt than 33 chromosomes and 78% have less Millet +++ +++ nt nt +++ than 33 chromosomes. On the contrary, 65% Sorghum +++ +++ nt + +++ of the cells in the resistant somaclonal variant Canola ++ ++ ++ +++ nt CIEN BTA-03 have more than 33 chromo- Tobacco ++ ++ nt +++ nt somes and 35% have less than 33 (Giménez Sunflower ++ ++ nt +++ nt 1998; Giménez et al. 2000). N. benthamiana ++ ++ nt +++ nt The flow cytometry analysis demonstrated Tipu tree +++ +++ nt +++ nt that somaclone CIEN BTA-03 presents a Pine tree ++ ++ nt ++ nt DNA content similar to or higher than that of Fishbone fern ++ ++ nt ++ nt clone Fagro 7 (Figure 2). The values obtained nt = not tested, +++ = strong expression, ++ = moderate to strong expression, + = moderate to weak expression. in the banana/rice mean ratio (B/R index)

INFOMUSA — Vol 10, N° 1 PROMUSA XI CIEN BTA-03 250 5

200 4

150 3

100 2 Rainfall (mm) 50 1 Degree of infection Degree

0 0 J A S O N D Jan F M A M J J

Months

Rainfall (mm) PISANG MAS GRAN NAIN WILLIAMS CIEN-BTA-03 YANGAMBI KM5

Degree of infection (Yellow Sigatoka): 0 No visible sign 1 Very low Yangambi Km 5 >>>>>CIEN BTA-03 FHIA-02 Prata Ana Gros Michel Pisang 2 Low "Saba" FHIA-03 Cavendish Mas 3 Intermediate 4 High 5 Very high Figure 3. Evaluation of the incidence of yellow Sigatoka on five banana clones growing in a dry forest at 450 m above sea level. Experimental Figure 1. Somaclonal variant CIEN BTA-03. Station Samán Mocho, Carabobo, Venezuela (1999-2000).

600 250 5 100 Plant (shoots) Cells/ml Total count Rice 60 608 5 064 Titiaro 41 738 5 119 200 4 500 Brasilero 27 632 5 126 Tetraploid 6 815 CIEN BTA-03 53 081 5 056 150 3 400

100 2

300 Rainfall (mm) 50 1 Count Degree of infection Degree

200 0 0 J A S O N D Jan F M A M J J

100 Months

Rainfall (mm) PISANG MAS GRAN NAIN WILLIAMS CIEN-BTA-03 YANGAMBI KM5 0 0 50 100 150 200 250 300 350 400 450 500 FL1 Degree of infection (Yellow Sigatoka): Partec 0 No visible sign Speed 0.50µl/s 1 Very low 2 Low Yangambi Km 5 Lamp (h) 113 >>>Gran NainPisang Mas Williams 3 Intermediate CIEN BTA - 03 Par Gain FL1 400 4 High 5 Very high Figure 2. Flow cytometry analysis of four banana clones. Figure 4. Evaluation of the incidence of black Sigatoka on five banana clones growing in a dry forest at 450 m above sea level. Experimental Station Samán Mocho, Carabobo, Venezuela (1999-2000).

range between 2.92 and 2.99, similar to the cultivar Yangambi km5, based on its resis- We concluded that we have a new clone re- tetraploid clone. tance to yellow Sigatoka (Figure 3). This so- sistant to yellow Sigatoka, with a high proba- Cluster analysis was done using the data maclone has proved to be also resistant to bility of being also resistant to black Sigatoka, obtained by random amplified polymorphic black Sigatoka (Figure 4). with good agronomic characteristics. It pro- DNA (RAPD) markers for CIEN BTA-03 and The efficiency and productivity indexes of duces a bunch of 34.53 kg and has a produc- 16 different genotypes of Musa spp. CIEN BTA-03 were compared with the FHIA- tivity index of 0.28 kg per day. (Giménez 1998, Giménez et al. 2000, Vidal 01, FHIA-02 and FHIA-03 indexes (García et and de García 2000). Fifty-six polymorphic al. 2000). CIEN BTA-03 indexes are very simi- Acknowledgements bands were used for the cluster analyses lar in value to FHIA-02 and FHIA-03 indexes This research was supported by a grant under using Ward’s Unweighted Pair-Group Aver- (Table 1). contract G-97000700 from the Consejo Na- age (UPGA), and Weighted Pair-Group Aver- age (WPGA) to calculate City-Block (Manhat- tan) distances. The dendrograms generated by the different methods were identical and Table 1. Comparison of the efficiency index and productivity index of four banana clones during showed that CIEN BTA-03 grouped with the second harvest cycle. Experimental Station Samán Mocho, Carabobo, Venezuela. FHIA-02 (AAAB) and is not closely related to Clone/Cultivar Genome Flowering to Bunch weight Efficiency Productivity the Cavendish subgroup, to which the parent harvest time (kg) index index cultivar William belongs (AAA) (Giménez days (days/kg) (Kg/days) 1998, Giménez et al. 2000). FHIA-01 AAAB 121.67 26.67 4.61 0.22 Field evaluation of the resistant character FHIA-02 AAAB 124.77 31.27 3.99 0.25 of CIEN BTA-03 (García et al. 2000) shows FHIA-03 AABB 126.90 36.85 3.47 0.29 that this somaclone can be grouped with the CIEN BTA-03 AAAA 121.07 34.53 3.52 0.28

XII PROMUSA INFOMUSA — Vol 10, N° 1 cional de Investigaciones Científicas y Tec- the genebank is to conserve Musa diversity for method transferable to the tropics. Electrophoresis nológicas of Venezuela (CONICIT) to Dr Eva the benefit of the international community and 19:152-157. de García. The authors are very grateful to Mr to distribute Musa species and cultivars for the Lagoda P.J.L., J-L. Noyer, D. Dambier, F-C. Baurens, Nicolas Roux (Plant Breeding Unit, FAO/IAEA, purpose of research and development. A. Grapin & C. Lanaud. 1998b. Sequence tagged Seibersdorf, Austria) for the flow cytometry The objective of this project is to obtain a microsatellite site (STMS) markers in the Musaceae. analysis. molecular characterization of this germplasm Molecular Ecology 7:657-666. in order to facilitate the classification and man- References agement of the genebank. Every year since Molecular studies on Musa acuminata de García E., O. Haddad, M. Dagert & 1998, about 200 individuals have been char- ssp. malaccensis and selected local R. Campagnone. 2000. Segundo informe de acterized at CIRAD-FLHOR in Guadeloupe Malaysian species avance. Proyecto CONICIT G-97000700. 269pp. with molecular markers. Yasmin Othman1, Norzulaani Khalid1, Giménez C. 1998. Características genéticas y Among the different methods available, the Asif Javed1, Mak Chai1 and Tan Siang Hee2 moleculares del variante somaclonal de banano choice of sequence-tagged microsatellite site 1Institute of Biological Sciences, University of Malaya, 50603 (CIEN BTA-03) asociadas al mecanismos de markers (STMS) is justified by numerous ad- Kuala Lumpur, Malaysia; 2Genome Centre, Institute resistencia a la Sigatoka amarilla y su vantages: these PCR highly polymorph Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. E-mail for correspondence: estabilidad genética. Tesis Doctoral. Facultad de codominant markers useable on in vitro [email protected] Ciencias, Universidad Central de Venezuela, plantlets are available, and the patterns can Caracas, Venezuela. 114pp. be interpreted in terms of genotypes, thus al- Banana is currently the second largest fruit Giménez C., E. de García, N. Xena de Enrench & I. lowing the detection of species-specific alle- crop in Peninsular Malaysia and contributes Blanca. 2001. Somaclonal variation in banana: les or identification of similarities. STMS poly- more than RM20 million in export earnings cytogenetic and molecular characterization of morphism was assayed by non radioactive (Jamaluddin 1998). the somaclonal variant CIEN-BTA-03. In Vitro urea-polyacrylamide gel electrophoresis, a However, widespread disease problems re- Plant 37(2). simple transferable methodless costly than main a major constraint to the industry, and Hermoso L., H. Lindorf & E. de García. 1997. most other molecular techniques (Lagoda et require that intensive efforts be made to intro- Anatomía foliar del variante somaclonal CIEN al. 1998a). Patterns and migration proce- duce new resistant cultivars. BTA-03 (Musa spp.), resistente a la Sigatoka dures on small and large gels were devel- The banana programme at the University of Amarilla. Anales de Botánica Agrícola. 4:63-66. oped and applied depending on the differenti- Malaya and Universiti Putra Malaysia has re- Trujillo I. & E. de García. 1996. Strategies for ation required among clones. The 10 STMS cently established a molecular breeding obtaining somaclonal variants resistant to yellow markers used have a high discrimination po- group, which will focus on local indigenous Sigatoka (Mycosphaerella musicola). InfoMusa tential and are localized independently on the species with a major emphasis on the wild ba- 5(2):12-13. different linkage groups (Lagoda et al. nana Musa acuminata ssp. malaccensis. The Trujillo I., L. Hermoso & E. de García. 1997. 1998b). At least 18 alleles have been identi- programme currently includes an expressed Caracterización estructural de clones de fied for each STMS. Some alleles specific to sequence tag (EST) project, STMS analysis, banano: resistentes y no resistentes a la the schizocarpa, balbisiana and Australimusa retrotransposon analysis, analysis of potential Sigatoka Amarilla. Anales de Botánica Agrícola. genomes were identified which allow the disease resistance genes and taxonomical 4:59-62. identification of interspecific clones. Most studies based on flow cytometry and cytology. Trujillo I., E. de García & J.L. Berroterán. 1999. clones revealed different patterns except for A cDNA library, constructed in a phage vec- Evaluación de banano obtenidas “in vitro”. clones within subgroups, such as Cavendish. tor ltrip1ex2, has been established for EST Anales de Botánica Agrícola. 6:29-35. The classification of clones was checked. analysis of Musa acuminata ssp. malaccensis Vidal M.C. & E. de García. 2000. Analysis of a Over 464 clones were studied, 34 classifica- genes. Clones from the library are being ran- Musa spp. somaclonal variant resistant to yellow tion errors were identified, classification of 23 domly sequenced and analyzed as part of a Sigatoka. Plant Molecular Biology Reports clones was completed and 31 non-classified long-term banana genomics project. Similarity 18:23-31. clones were assigned to a group and when searches against known sequences deposited possible to a subgroup. in the databases have already revealed identi- Biodiversity and evolution These data help to complete the germplasm ties with genes of known function and with morphological database (INIBAP-MGIS), to- other EST clones. All the sequences obtained Characterization of Musa germplasm gether with the data on ploidy level analysis will be used to generate a Musa EST data- held at INIBAP genebank using through flow cytometry (see Dolezel et al., base to be used for the further understanding STMS-PCR markers above) and eventually the data on chromo- and potential exploitation of banana genes. some genomic characterization through GISH Retrotransposon analysis has identified 1 2 F. Carreel , A. Duarte Vilarinhos , (genomic in situ hybridization) (D’Hont et al. Ty 1-copia-like elements in 10 varieties of ba- 3 4 I. Van den Houwe and S. Sharrock 2000). nana. A database search showed nucleotide 1CIRAD-FLHOR Neufchâteau, Sainte Marie, 97130 identities ranging between 85-97% and pre- Capesterre Belle Eau, Guadeloupe (E-mail: References [email protected]); 2CNPMF/EMBRAPA, Cx Postal 007, dicted aminoacid identities of between 57-82% CEP44380000 Cruz das Almas, Brazil; 3Katholieke D’Hont A., A. Paget-Goy, J. Escoute & F. Carreel. when compared to known RT genes of Ty 1- Universiteit Leuven, ITC, Kasteelpark Arenberg 13, 3001 2000. The interspecific genome structure of copia-like retrotransposons. The sequences Leuven, Belgium; 4INIBAP, Parc Scientifique Agropolis II, 34397 Montpellier cedex 5, France. cultivated banana, Musa spp. revealed by genomic were subdivided into eight distinct groups sim- DNA in situ hybridization. Theor. Appl. Genet. ilar to the Ty 1-copia retrotransposons found The International Musa Germplasm Collection 100:177-183. in other plant species such as Tto1 in Nico- maintained by INIBAP and hosted by the Lagoda P.J.L., D. Dambier, A. Grapin, F.-C. Baurens, tiana tabacum (Hirochika and Hirochika 1993). Catholic University of Leuven (KUL), holds C. Lanaud & J.-L. Noyer. 1998a. Nonradioactive Ty 3-gypsy-like retrotransposons have also more than 1100 accessions. The objective of sequence-tagged microsatellite site analyses: a been isolated with identities ranging from

INFOMUSA — Vol 10, N° 1 PROMUSA XIII 55-80% when compared to similar elements in mostly by small growers. The breeding pro- but supposed durable resistance should be the database. The ubiquity and heterogeneity gramme of EMBRAPA Mandioca Fruticultura, preferentially used in breeding programmes. of the Ty 1-copia like and Ty 3-gypsy-like Cruz das Almas, Bahia, Brazil, has devel- The main objectives of this work were to de- retrotransposons make them a suitable oped tetraploid hybrids based on a limited scribe the genetic structure of M. fijiensis pop- marker for the determination of biodiversity of number of triploid commercial selections and ulations at continental and local scales and to banana species in Malaysia. wild diploids. Identical cultivars with distinct evaluate the efficiency and the durability of In a separate project, flow cytometry names (synonymous) and distinct genotypes partial resistance. (Dolezel et al. 1991) was used to study ploidy with similar names (homonymous) might be a To study the population structure of a sin- and nuclear genome size variation in Musa common phenomenon, and somatic muta- gle pathogenic species, we have first to dis- species indigenous to Malaysia, i.e. Musa tions tend to accumulate in banana. The ob- tinguish this species from close relatives and acuminata subspecies, Musa balbisiana, jectives of this work were to characterize 33 to determine its distribution. Such a survey, Musa violascens and Musa textilis. No varia- triploid commercial cultivars and tetraploid conducted in South and Southeast Asia, led tion was observed in ploidy level, whereas a hybrids, plus 49 wild diploid genotypes from to the discovery of the previously unde- large amount of variation in the genome size EMBRAPA’s breeding programme using mi- scribed fungus, Mycosphaerella eumusae was observed among the different Musa crosatellite markers. Primers were purchased (anamorph Septoria eumusae, Carlier et al. species analyzed. Less variability was ob- from Research Genetics Inc. (Huntsville, AL, 2000). From a taxonomic and phylogenic served at the intraspecific level within the USA), and amplified fragments were scored study of the ribosomal DNA, we showed that species Musa acuminata. Statistical and clus- on denaturing polyacrylamide gels stained at least nine species, belonging to Mycos- ter analysis of data on genome size related in with silver nitrate. Based on cluster analysis, phaerella or related anamorph genera, can a grouping agreed well with the generally ac- triploid and tetraploid cultivars grouped ac- be isolated from banana leaves (Carlier et al. cepted taxonomic classification of Musa. cording to genome composition (presence of unpublished). Considering the presence of all Studies on disease resistance focus on re- B genome) and to subgroup classification. No these species, the primers defined in the ITS sistance of local wild bananas to Fusarium difference was detected among cultivars from region (Johanson and Jegger 1993) are not oxysporum, the major pathogen of banana in subgroups “Cavendish” and “Pome”. Culti- strictly specific to either M. fijiensis or M. mu- Malaysia. The ultimate aim will be the intro- vars with erroneous subgroup classification sicola. These results show that a good knowl- gression of resistance genes from the wild were identified. Tetraploid selections from the edge of the fungal species complex is neces- species into cultivated varieties using ap- same cross were not identical, and presented sary to develop diagnostic tools. From the proaches integrating genomics and marker- expected similarity with maternal triploids. phylogenic study we developed another tool assisted selection. Diploids were highly diverse, with the main based on a restriction assay of the ITS region The overall integrated approach of the pro- parental diploid lines employed to develop and began looking for new specific primers. gramme, with close links to transformation and tetraploid hybrids being very distinct. Some Such tools should be useful to determine the breeding groups in the country, hopes to con- primers amplified more than one locus sug- distribution and the importance of the differ- tribute to Musa improvement programmes gesting that loci duplication might be com- ent species. both locally and globally. mon in banana, as previously described in The population structure of M. fijiensis at the literature. Genetic distances may be used continental and local scales was analyzed References for selecting future crosses. from samples collected in Latin American, Dolezel J. 1991. Flowcytometric analysis of nuclear Caribbean and African countries using eight DNA contents in higher plants. Phytochem. Studies of Mycosphaerella fijiensis cleaved amplified polymorphic sequences or Analysis 2:143-154. populations structure and of partial CAPS, as molecular markers (Zapater et al. Hirochika H. & R. Hirochika. 1993. Ty 1-copia group resistance of bananas unpublished). Within local populations, we retrotransposons as ubiquitous components of found that most of the genetic variability is 1 2 3 plant genomes. Jpn. J. Genet. 68:35-46. C. Abadie , G.-G. Rivas ,A. El Hadrami , M.-F. distributed on a small scale corresponding to 3 3 Jamaluddin S.H. 1999. Commercial exploitation of Zapater and J. Carlier the plant scale. In Latin America and the 1 banana diversity in Malaysia. Pp. 45-51 in CRBP (Centre régional de recherches sur bananiers et Caribbean zone, the genetic diversity of M. plantains), BP 832, Douala, Cameroon; 2CATIE (Centro Proceedings of the First National Banana Seminar, Agronómico Tropical de Investigacíon y Enseñanza), 7170, fijiensis in Honduras and Costa Rica are rela- 23-25 Nov. 1998, Genting (Z. Wahab et al., eds.). Turrialba, Costa Rica; 3CIRAD (Centre de coopération tively high compared to the populations else- internationale en recherche agronomique pour le where, suggesting that the pathogen first en- développement), TA 40/02, avenue d’Agropolis, 34398 Genetic characterization of Brazilian Montpellier, France. E-mail: [email protected] tered the zone here. Within both Latin commercial triploid and tetraploid America/Caribbean and African areas, a high cultivars and wild diploid genotypes The ascomycete fungus Mycosphaerella fijien- level of genetic differentiation was detected using microsatellites sis (anamorph Paracercospora fijiensis) between most of the populations analyzed in- causes black leaf streak disease (BLSD), the dicating that gene flow is limited (Rivas et al. 2 1 1 S.A.C.D. Souza , A. Figueira , A. Tulmann Neto most destructive leaf spot disease of bananas and Carlier et al. unpublished). It is likely that 3 and S.O. Silva (Jones 2000). Knowledge of the extent and the disease, therefore, has spread in the re- 1Centro de Energia Nuclear na Agricultura, Universidade de distribution of variability within M. fijiensis is gions through infected plants and/or through Sao Paulo, CP 96 Piracicaba, SP, 13400-970, Brazil; 2ESALQ-USP (Escola Superior de Agricultura «Luiz de necessary for breeding and management of restricted dispersion of ascospores. Contin- Queiroz», Universidad de Sao Paulo), Brazil;3EMBRAPA BLSD resistance. A study of the genetic struc- ued research at a country level will help to (Empresa Brasiliera de Pesquisa Agropecuaria) Mandioca ture of M. fijiensis populations on a global specify the relative importance of both means Fruticultura, Cruz das Almas, BA, Brazil. E-mail: [email protected] scale showed that individual populations can of transmission. Aggressiveness variability maintain a high level of genetic diversity and was evaluated in two samples collected in In Brazil, banana cultivars from subgroups that recombination plays an important role in Cameroon and the Philippines by inoculation “Pome” and “Silk” (AAB) are widely cultivated, this pathogen (Carlier et al. 1996). Thus partial on five partially resistant cultivars using a leaf

XIV PROMUSA INFOMUSA — Vol 10, N° 1 pieces assay (El Hadrami et al. 1998). This New cytological methods to study old for germplasm maintenance, exchange and variability was similar for both countries al- problems in Musa L. also transportation. In vitro culture of vegeta- though the level of genetic diversity observed M. Pillay, M.T.V. Adeleke and A. Tenkouano tive meristems or floral apices is the most in the Philippines is much higher (Carlier et Crop Improvement Division, Plantain and Banana promising method for mass propagation. The al. 1996). No specific isolate ¥ cultivar inter- Improvement Project, International Institute of Tropical production of synthetic seeds by encapsulat- Agriculture, PMB 008 Nchia-Eleme, Port-Harcourt, Nigeria. actions were detected. Since only susceptible ing somatic embryos and vegetative propag- hosts are cultivated in these countries, these Musa breeding is hampered by a number of ules is rapidly becoming an applied technique results might be explained by the absence of constraints including a lack of knowledge of with potential for mass propagation of elite host selection. The potential of pathogen chromosome structure, ploidy and causes of plant species. Synthetic seed technology will populations to adapt to partial resistance sterility. There are no established karyotypes have a significant impact on crop production, should be analyzed by following their evolu- in Musa because of its poorly staining small, in both vegetatively propagated and seed- tion over time in plots of resistant banana uniform chromosomes and difficulties in ob- propagated crops. For the vegetatively prop- genotypes. taining good spreads. Defining correct ploidy agated plants, synthetic seeds would allow To evaluate the efficiency and the durability levels and establishing techniques for causes direct planting of clonal varieties and may of partial resistance, three complementary ap- of sterility are necessary in Musa breeding. provide a means for maintenance of elite proaches were used: characterization of partial This study describes (i) the use of silver nitrate germplasm. resistance components under controlled condi- as a staining agent for Musa chromosomes, (ii) Synthetic seeds have been prepared by tions, evaluation of the efficiency of these com- a new procedure to examine meiotic chromo- encapsulating shoot tips and somatic em- ponents in the field and population structure somes in Musa, (iii) ploidy variation in Musa bryos and their conversion into plantlets has analysis of the pathogen. Significant differ- germplasm, and (iv) pollen tube growth in been studied. Shoot tips of cv. Basrai encap- ences between 10 banana genotypes were ob- Musa. Acetocarmine, the most common stain sulated in sodium alginate containing differ- served at all stages of the infectious cycle used in Musa cytology, is effective for con- ent gel matrices regenerated in vitro on vari- using a leaf pieces assay (El Hadrami 2000). densed chromosomes such as those in ous substrates. Use of Whites medium Thus, different components of partial resis- metaphase, but is ineffective for prophase resulted in high conversion of encapsulated tance occur at these stages. Epidemiological chromosomes. Silver nitrate is shown to be a shoot tips into plantlets. Somatic embryos de- roles of selected resistance components are useful alternative stain for Musa chromo- rived from embryogenic cell cultures of cv. currently being studied under field conditions somes. An improved method to examine meio- Rasthali were also employed for the prepara- on different plots comprising each a single basis in Musa is described. The procedure in- tion of synthetic seeds. The encapsulated nana genotype. We are also comparing volves dissection of microsporocytes from the embryos converted into plants with varying pathogen population structure between these anthers, centrifugation to obtain a large num- frequencies on different gel matrices and plots in space and time. ber of microsporocytes, digestion with en- substrates. Plantlets developed from syn- zymes and treatment of cells with ethanol- thetic seeds have been successfully trans- References acetic acid. Although Giemsa and Leishrnan’s ferred to soil. Synthetic seeds offer a useful Carlier J., M.H. Lebrun, M.F. Zapater, C. Dubois & stain were effective for Musa chromosomes, tool since they can be handled like seeds and X. Mourichon. 1996. Genetic structure of the global silver staining was most effective for the less can be useful for storage, delivery and trans- population of Banana black leaf streak fungus contracted prophase chromosomes. This tech- portation of banana germplasm. Mycosphaerella fijiensis. Molecular Ecology 5:499- nique will be useful to develop pachytene kary- 510. otypes, characterize new hybrids, and identify Evaluation of regeneration Carlier J., M.F. Zapater, F. Lapeyre, D.R. Jones & nuclear restitution mechanisms (FDR or SDR). and transformation systems in Musa X. Mourichon. 2000. Septoria leaf spot of banana: a Ploidy and genome composition in some of our acuminata var. Pisang Mas (AA) and Pisang Berangan (AAA) newly discovered disease caused by Mycosphaerella Musa germplasm showed differences from that eumusae (anamorph Septoria eumusae). of existing data showing the need to better Norzulaani Khalid, Yasmin Othman, Phytopathology 90: 884-890. characterize existing germplasm. Finally, a Wirakarnain Sani, Mahanom Jalil El Hadrami A., M.F. Zapater, F. Lapeyre, C. Abadie & method to observe pollen tube growth in the and Noraziah Juli J. Carlier. 1998. A leaf disk assay to assess partial styles of Musa hybrids will be described. Institute of Biological Sciences, Faculty of Science, resistance of banana germplasm and aggressiveness University Malaya, 50603 Kuala Lumpur, Malaysia. of Mycophaerella fijiensis, the causal agent of black Biochemistry and fruit ripening leaf streak disease. 7th International Congress of Fusarium wilt of banana (Panama disease) is Plant Pathology, Edinburgh, Scotland. BSPP Vol. 2, Synthetic seeds in banana: native to peninsular Malaysia and has been p.1.1.24. a novel propagation and delivery recorded as a serious threat to the local indus- El Hadrami A. 2000. Caractérisation de la résistance system try (Thompson and Johnston 1953). However, partielle des bananiers à la maladie des raies noires attempts at improvement by conventional et évaluation de la variabilité de l’agressivité de T.R. Ganapathi, P. Suprasanna, L. Srinivas methods have been impeded by the infertile l’agent causal, Mycosphaerella fijiensis. Thèse and V.A. Bapat nature of cultivated bananas. For this reason, d’Université. Faculté Universitaire des Sciences Plant Cell Culture Technology Section, Nuclear Agriculture we are developing tissue culture and transfor- Agronomiques de Gembloux, Belgique. 153pp. and Biotechnology Division, Bhabha Atomic Research mation protocols for use on our local banana Johanson A. & M.J. Jegger. 1993. Use of PCR for Centre, Trombay, Mumbai 400 085, lndia. varieties, Musa acuminata var. Pisang Mas detection of Mycosphaerella fijiensis and M. musicola, Edible bananas are vegetatively propagated (AA) and Pisang Berangan (AAA) in our labo- the causal agents of Sigatoka leaf spots in banana by suckers as viable seeds are not usually ratory. Regeneration methods have been at- and plantain. Mycological Research 97:670-674. produced. New and effective means of prop- tempted from single and naked meristems Jones D.R. 2000. Diseases of banana, Abaca and Enset. agating bananas would be advantageous (scalps), meristematic globules and embryo- CABI Publishing, CAB International, UK. 544pp. over the conventional use of sucker material, genic calli. Embryogenic calli were derived

INFOMUSA — Vol 10, N° 1 PROMUSA XV from meristems (Novak et al. 1989) and male We are also isolating the antifungal gene and quality control, and provide the necessary inflorescences (Escalant et al.). The highest from wild banana, Musa acuminata ssp. production data. number of plants regenerated are from scalps. malaccensis. According to published data, this We are now planting these regenerated plants species is known to be resistant to Fusarium References in the field to test for somaclonal variation. We wilt races 1 and 4 (Vakili 1965). Escalant J.V., C. Teisson, A. Grapin & F. Côte. 1994. have observed that the regeneration frequency Innovations for the commercial production Somatic embryogenesis of banana and plaintain is higher in Pisang Berangan (AAA) than in of tissue culture plants are also being devel- from young flowers. InfoMusa 3(2):4-6. Pisang Mas (AA). Cell suspensions of both va- oped. We have developed a chamber which Novak F.J., R. Afza, M. Van Duren, M. Perea-Dallos, rieties are also being established. Cell suspen- we call a «steriponics chamber» which B.V. Conger & Tang Xiolang. 1989. sions from male inflorescences developed at a merges the principles of tissue culture and Somatic embryogenesis and plant regeneration faster rate than those from apical meristems. aeroponics. The advantages of this chamber in suspension cultures of dessert (AA and AAA) Plant transformation using both biolistic include faster plant production, minimum con- and cooking (ABB) banana (Musa spp.). and Agrobacterium methods were attempted. tamination risk and less dependency on Biotech. 7:154-159. Scalps and embryogenic calli were shown to labour. This chamber could also be used for Thompson A. and A. Johnston 1953. A host list be most responsive in the transformation ex- physiological and pathogen assessment ex- of plant diseases in Malaya. periments. Histochemical assays were used periments. Mycological papers no. 52. CMI, Kew, to optimize transformation parameters and A data tracking system has also been de- Surrey, England. identify suitable explants. Cell suspensions of veloped to monitor plant production using a Vakili N.G. 1965. Fusarium wilt resistance in both varieties will be used for transformation bar coding system. The use of this system seedlings and mature plants of Musa species. in future. would allow monitoring of virus-indexed plants Phytopathology 55:135-140.

XVI PROMUSA INFOMUSA — Vol 10, N° 1