The International Journal on and Plantain

Nutritious plantains Novel use of banana stalk Measuring virulence Surveying nematodes in How do mycorrhizae empower ? Enhancing awareness for BXW control

Vol. 16 No. 1 & 2 June & December 2007 InfoMusa Cover photo: Vol. 16 No.1&2 Sale of bananas in a village inTamil Nadu, India INFOMUSA (Inge van den Bergh, Bioversity) Vol. 16, No. 1 & 2

Publisher: International Network for the Improvement of Banana and Plantain

Publishing director: Claudine Picq

Editors: Claudine Picq, Vincent Johnson

Editorial Committee: Richard Markham, Inge van den Bergh, Nicolas Roux, Charles Staver

Layout: Crayon & Cie Printed in France Contents ISSN 1023-0076 Micronutrient value and contribution of plantain-derived Editorial Office: INFOMUSA, Bioversity, Parc Scientifique to daily intakes of iron, zinc, and β-carotene in Southern Agropolis II, 34397 Montpellier Cedex 5, F. Honfo, K. Hell, O. Coulibaly and A. Tenkouano 2 France. Telephone + 33-(0)4 67 61 13 02; Telefax: + 33-(0)4 67 61 03 34; E-mail: [email protected] Authors of articles submitted for publication Preparation of a cation exchanger containing carboxyl groups which are not published in this latest issue from banana stalk and its utilization as chelating agent of INFOMUSA are free to submit them for T.S. Anirudhan and I.G. Shibi 7 publication elsewhere. No manuscript will be returned unless solicited by authors. Unless accompanied by a copyright notice, articles appearing in INFOMUSA may Associative nitrogen fixation by Azospirillum and be quoted or reproduced without charge, Bacillus spp. in bananas provided acknowledgement of the source is given. Md. Abdul Baset Mia, Z.H. Shamsuddin, Z. Wahab and M. Mahmood 11 An electronic version is available at the following address: http://bananas.bioversityinternational.org/ Development of a suitable method for evaluating virulence of content/view/31/48/lang,fr Fusarium oxysporum f. sp. cubense race 1 (E.F. Smith) in banana Views expressed in articles are those T. Saravanan, M. Muthusamy, E.G. Ebenezar and R. Bhaskaran 16 of the authors and do not necessarily reflect those of Bioversity. Distribution of plant-parasitic nematodes on in Kenya K.V. Seshu Reddy, J.S. Prasad, P.R. Speijer, R.A. Sikora and D.L. Coyne 18

Assessment of the effect of commercial mycorrhizal fungi products on the growth of banana plants in the nursery A.S. Rodríguez-Romero and M.C. Jaizme-Vega 23

Focus on building knowledge for Banana Xanthomonas wilt control 28 Theses 32 MusaNews 38

Bioversity International, Bioversity for short, is the operating name of the International Plant Genetic Resources Institute (IPGRI) and the International Network for Improvement of Banana and Plantain (INIBAP). With our partners, we undertake research aimed at improving people’s lives through the use and conservation of agricultural biodiversity.

InfoMusa - Vol. 16 No. 1-2, June & December 2007 1 Editorial

Your last copy of InfoMusa… but let its spirit live on!

So here, at last, is the final volume of InfoMusa. It has taken somewhat longer to get to press than we had anticipated, mainly due to the large number and broad diversity of submitted papers that had accumulated. Inevitably, in making this final selection, we have had to put aside many potentially interesting articles. However, we hope that the authors will find alternative outlets to publish this work and that, among the range of articles that we have included, all our readers will find something of interest to them. Taken together, the articles and thesis summaries provide evidence of a dynamic banana research community that continues to find a balance between classical and novel approaches, as well as ‘upstream’ and ‘downstream’ work. The never-ending struggle with pests and diseases evidently continues to engage a great deal of our communal effort, with outbreaks of new diseases and new outbreaks of familiar ones being reported, as well as some new approaches to managing both old and new. Microbiology is becoming increasingly important in our understanding of managing healthy production systems. The molecular biologists are providing us with new insights into how our favourite crop copes with stresses of various kinds. And researchers interested in the social dimension are working on links to markets and the contribution that bananas can make to the nutrition of consumers. As promised, we have been busy establishing a new range of on-line resources, to help the banana research community to follow these developments and to debate the issues of the day more vigorously. If you have not already done so, we encourage you to visit: the all-new www..org “the website of the Musa R&D community.” There, you will find InfoMus@, on-line successor to this publication, with news and a ‘media watch’, feature articles and opinion pieces. The site also includes MusaTalk, a discussion forum where you can follow up on the opinion pieces and other current issues. Elsewhere, we are also developing various ‘resource platforms’, addressing major production and post-harvest issues, as well as a range of new resources relating to banana taxonomy and conservation. All of these can be accessed from the Bioversity banana section site (http://bananas.bioversityinternational.org); we also provide a brief guide to the new on-line resources at the end of this edition of InfoMusa. This last on-paper issue of InfoMusa does mark the end of an era and it is therefore a good moment to thank our editors (most recently Claudine Picq and Anne Vezina) and our reviewers, for their tireless efforts to raise publication standards, as well as our contributors and loyal readers. However, this is also a new beginning and in closing this editorial I urge you to contribute to the new on-line resources and take advantage of the new on-line technologies. Some of us will no doubt miss the satisfaction of holding a publication in our hands; but I hope that most will soon come to appreciate the timeliness of internet publication and the richness of material that can be captured, as well as the opportunities for lively and immediate debate. Please ‘take ownership’ of these new resources and help to develop them to serve your needs. Using these opportunities together, I believe we can help to bring new dynamism to the banana research effort in pursuit of better livelihoods for all.

Richard Markham, Director of the Commodity for Livehoods Programme

InfoMusa - Vol. 16 No. 1-2, June & December 2007 1 Nutritious plantains Micronutrient value and contribution of plantain- derived foods to daily intakes of iron, zinc, and β−carotene in Southern Nigeria F. Honfo, K. Hell, O. Coulibaly and A. Tenkouano

icronutrient deficiencies are con- (IITA 2000). Plantain may, therefore, play a sidered to be the main nutritional key role in meeting nutritional requirements Mand health problem affecting poor for Nigerian populations (Ajayi and Aneke populations. Iron and zinc deficiencies are 2002). commonly reported in children and women The present study aimed at determining of reproductive age in many developing the iron, zinc and β-carotene contents countries (de Pee et al. 1996, Frossard et of the plantain-based foods commonly al. 2000, Gibson et al. 2000). Iron deficiency consumed in Southern Nigeria to assess is the prevailing nutritional deficiency in the the quantitative contribution of plantain world and is the main cause of anaemia consumption in meeting iron, zinc and (UN ACC/SCN 1997, Halberg and Hulthén vitamin A requirements of both children and 2000). Dietary zinc deficiency leads to their mothers. various growth and reproduction disorders and often results in death, especially in Materials and methods developing countries (FAO/WHO 1996, Household surveys: A survey was carried Brown et al. 2002). More than half of out in the Abia, Akwa-Ibom, Edo, and Ogun, pregnant women and one third of children States of Southern Nigeria, targeting their under five all over the world suffer from capital cities, i.e., Umuahia, Uyo, Benin nutritional anemia and iron deficiency to City, and Abeokuta, respectively. In each various degrees (UN ACC/SCN 1997). state, sub-samples of 30 urban and 30 rural Vitamin A deficiency is given priority among households were randomly taken, totalling global health problems (UNICEF 1990, 240 households. One third of each sub- FAO/WHO 1992). It can be responsible sample of households had a child under five for increased mortality among children and years old who was definitively weaned and women in developing countries and is the whose mother was present in the household. leading cause of preventable blindness Data collection was done in June and in children, while enhancing the risk of July 2005. disease and death from severe infections A questionnaire was devised and tested (Sommer and West 1996, Mclaren and to collect data on the following factors: Frigg 2001, OMS/UNICEF 2002). types of banana and plantain varieties Banana and plantain (Musa spp.) are used, forms of plantain foods preferred major staple foods for over 100 million and consumption period of these foods, people in sub-Saharan Africa, where they consumption levels of plantain-based meals, contribute significantly to security daily food consumption of women and their (Sharrock and Frison 1998, INIBAP 2002). children under five years of age. The daily Plantain is considered an excellent source consumption of plantain-based foods was of energy and nutrients: it is estimated that assessed by a 24-hour recall technique 100 g of the edible part of raw plantain applied over three consecutive days provides 122 kcal, contains 1.30 g of protein, (Bingham et al. 1988). On each day and in 0.37 g of fat, 0.6 mg of iron, 0.14 mg of zinc each household, the mother recalled all the and 457 μg of β-carotene (USDA 2004). types and quantities of plantain-based meals In Nigeria, banana and plantain have eaten by her children and herself during always been very important traditional staple the last 24 hours. Local measures used to foods for both rural and urban populations. estimate the level of consumption were later Annually, 6.7 million tons of plantain and converted to grams. other cooking bananas are produced in the Sample composition analysis: Samples country, with an average consumption of of plantain-based foods were collected from 150 kg/person/year or 348 calories per day households in which they were available, but

2 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 3 most of the samples were purchased in local of plantain-based food. The Least Significant markets. They were packed in aluminum foil Difference (LSD) test at 0.05 and 0.01 levels within polyethylene bags inside plastic pots was used to compare means. with lids, and transferred to the laboratory. During transport, samples were kept in cool Results and discussion boxes with cooling elements. They were Forms of plantain preferred in the stored in a deep-freezer at -20ºC for one households week before analysis. The variety of plantain mostly eaten in the The concentrations of iron and zinc surveyed households was the local variety were determined by atomic absorption “landrace Agbagba”. Households surveyed spectrophotometry in the laboratory of used various techniques for processing the Faculty of Agronomic Sciences at the plantain at different ripening stages (Table 1). University of Abomey-Calavi in Benin. Some of these techniques have been found β-carotene was determined at the Institute of in South-east Asia (Sharrock 1996) and Medical Research Noguchi of the University in (Ngoh Newilah et al. 2005). of Lagon, . Samples of 0.01 - 0.9 g Of these processing techniques, the most were collected after homogenization; popular in all the households surveyed was -carotene was extracted by High β frying. Boiled and roasted plantains were Performance Liquid Chromatography (HPLC) also popular in both urban and rural areas. using Tetra Hydro Furan, as described by However, most of the urban respondents Takyi (1999). Retinol activity equivalent was preferred plantain chips. Ripe plantain or calculated using the following formula: [ - β banana was preferred by 76% of the rural carotene value / 12]. All measurements were households surveyed, as against 43% in made in triplicate. urban areas. Data analysis: The average daily consumption of each plantain-based food Preferred times for eating plantain- was evaluated for the mothers and children based foods in the survey. Thus, for each type of plantain- The different times of day when plantain- based food, the daily consumption was based foods are mostly eaten were the obtained by calculating the average of three same everywhere. Most of the households days’ consumption for this type of food. Iron, surveyed ate fried or boiled plantain at zinc and vitamin A supplies from the average (Table 2). was daily consumption of plantain-based meals also preferred as an afternoon . per child or mother were evaluated using the Respondents preferred boiled plantain micronutrient composition of the samples. and pounded plantain for lunch; pounded Throughout the three days of investigation plantain and plantain flour paste, often six types of plantain-based foods were commonly consumed and two of them Table 1. Forms of plantain preferred in the were often consumed daily by the surveyed households (% of N) populations. In this regard, the average Form of meals Rural area Urban area daily supply of iron, zinc and vitamin A from (N=120) (N=121) Fried plantain 100 100 plantain was obtained by taking a third of the Plantain chips 63 89 total amount provided by all the six plantain- Boiled ripe or unripe plantain 88 71 based food types eaten over the three Pounded plantain 49 35 days. For each surveyed subject (children Plantain flour paste 29 18 and mothers), these average values were Ripe banana/plantain 76 43 Roasted plantain 96 83 compared with the dietary recommended allowances for the corresponding micro- nutrients as defined by FAO (Latham 2001). Table 2. Preferred times for eating plantain foods in the households Data were analysed using SPSS Software. Form of meals Breakfast (%) Lunch (%) Snack (%) Dinner (%) All continuous data were expressed as Fried plantain 47 9 32 8 Plantain chips 6 3 30 2 means ± standard deviations. One-way Boiled plantain 30 27 5 17 analysis of variance (ANOVA) was used Pounded plantain 2 21 2 22 to determine how the area (rural or urban) Plantain flour paste 2 15 0 20 and age (child or mother) affected their Ripe banana/plantain 2 6 9 2 consumption and the nutritional importance Roasted plantain 5 9 19 7

2 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 3 associated with cassava or yam flour, were However the difference was significant only the most preferred for dinner. Generally, for the quantity consumed by the children these eating habits were similar to those (P < 0.05). In both rural and urban zones, reported by Ajayi and Aneke (2002) in the mothers’ consumption levels were at least town of Nsukka of Nigeria. twice those of children. Consumption level in rural/urban areas Iron, zinc and β-carotene contents There is a significant difference between The iron, zinc and β-carotene contents of rural and urban consumers of plantain-based the plantain-based foods (including means foods. and standard deviations) are given for 100g The number of children consuming plantain FW of edible food (Table 5). The values of chips and the daily amount consumed were β-carotene are lower than those previously higher in urban areas (P <0.05) than in rural reported for banana by Englberger (2004). areas (Table 3). The daily intake of roasted The iron content of all plantain products plantain by urban children was higher than analysed ranged from 0.71 mg to 1.30 mg/ that of their rural counterparts (P <0.05). 100g FW. It was higher in plantain chips than A similar trend was also observed with in fried plantain, which had the lowest iron their mothers; however, the difference content of all plantain products. Because between rural and urban mothers was not the cooking process for plantain chips was significant for the consumption of roasted similar to that of fried plantain, the difference plantain (Table 4). In contrast, the number observed in the two types could be attributed of consumers of pounded plantain and to the duration of cooking, which is longer for the quantity consumed per capita were chips than for fried plantain. It might also be lower in urban than in rural zones (86 rural due to the degree of ripening and maturity mothers versus 49 urban mothers; 82 of the plantains used; ripe plantain was used rural children versus 61 urban children). for frying and unripe fruit was used to make chips. Roasted plantain contains a moderate Table 3. Average size (mean ± standard deviation) of plantain meals eaten by the level of iron. children who ate the dishes The zinc content of the plantain-based Form of meals Rural area (g) Urban area (g) Significant level Fried plantain 32.9 ± 43.7 (112) 42.5 ± 68.9 (120) ns foods analysed ranged from 0.24 mg to Plantain chips 4.9 ± 39.7 (68) 24.2 ± 59.5 (114) * 0.37 mg/100 g FW. Unripe plantain and chips Boiled plantain 66.4 ± 93.3 (108) 52.2 ± 57.5 (68) ns contained the highest levels of zinc. Pounded plantain 59.9 ± 119.7 (82) 30.3 ± 85.0 (61) * The β-carotene content was higher in Ripe banana/plantain 34.9 ± 54.7 (109) 25.3 ± 45.7 (86) ns plantain-based products (203.0-695.12 g/ Roasted plantain 37.2 ± 78.0 (102) 81.0 ± 107.8 (109) * μ 100 g FW) compared to other staple foods, * = Significant difference at 5% ns = not significantly different at 5% level e.g. maize, cassava and yam, grown in Nigeria. The carotene levels generally Table 4. Average size (mean ± standard deviation) of plantain meals eaten by the increase with cooking: in cooked food, mothers who ate the dishes carotene is more efficiently extracted from Form of meals Rural area (g) Urban area (g) Significant level Fried plantain 56.4 ± 135.2 (107) 48.8 ± 297.0 (116) ns the food matrix by the solvent; however, this Plantain chips 23.1 ± 78.5 (46) 42.9 ± 142.5 (98) * may also depend on the method of cooking, Boiled plantain 72.3 ± 216.6 (108) 56.5 ± 191.2 (79) ns as a longer duration and higher temperatures Pounded plantain 102.8 ± 319.0 (86) 76.5 ± 287.4 (49) ns destroy carotenoids (Englberger 2004). In Ripe banana/plantain 71.9 ± 229.7 (111) 58.7 ± 264.8 (87) ns this study, and regardless of the plantain Roasted plantain 69.0 ± 204.4 (106) 79.4 ± 279.7 (109) ns variety used, -carotene levels generally * = Significant difference at 5% β s = not significantly different at 5% level Table 5. Iron, zinc, ß-carotene and vitamin A contents (mean ± standard deviation) in the samples Form of meal (n) Iron (mg/100g) Zinc (mg/100g) β-carotene (µg/100g) Vitamin A (RAE/ 100g) Boiled ripe plantain (4) 0.90 + 0.11 0.27 + 0.05 363.66 + 11.0 30.31 + 0.91 Boiled unripe plantain (3) 0.84 + 0.07 0.28 + 0.09 431.33 + 14.67 35.94 + 0.72 Flour of unripe plantain (2) 1.03 + 0.08 0.24 + 0.07 203.00 + 9.00 16.92 + 0.75 Fried plantain (4) 0.79 + 0.10 0.33 + 0.06 275.33 + 9.67 22.94 + 0.80 Plantain chips (4) 1.30 + 0.10 0.37 + 0.06 321.33 + 2.67 26.79 + 0.80 Pounded plantain (4) 0.71 + 0.10 0.28 + 0.08 400.00 + 9.00 33.34 + 0.79 Raw ripe plantain (3) 0.97 + 0.05 0.25 + 0.08 308.0 + 12.50 25.67 + 1.04 Roasted plantain (4) 1.13 + 0.08 0.33 + 0.08 695.12 + 10.00 57.93 + 0.81 Unripe plantain (3) 0.89 + 0.05 0.37 + 0.05 252.0 + 7.00 21.00 + 0.58

4 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 5 increased, but not in every case. Roasted supplied 30.9 μg Retinol Activity Equivalent plantain was particularly rich in β-carotene, (RAE) and 18.2 μg RAE of vitamin A in rural whereas lower β-carotene contents were and in urban areas respectively, accounting observed in unripe plantain and its flour. for 7.2% and 4.6% of the daily vitamin A This contrasts with the findings of several requirement for these children in rural and authors, including Isonfua and Omuaru urban zones respectively. The difference (1989), Barthkur and Arnold (1990) and observed between urban and rural zones Englberger (2004) who showed that the state for vitamin A contribution by plantain was of maturity affects carotenoid contents and significant (P < 0.05). that their levels in the fruit increase during For non-pregnant and non-lactating ripening. women, FAO recommends a daily intake of 48 mg of iron, 12 mg of zinc and 800 μg RE Contribution of plantain of vitamin A (Latham 2001). We found that in children’s and their the daily consumption of plantain-derived mothers’ diets foods provided approximately 1.80 mg of iron Based on the iron, zinc and β-carotene and 0.6 mg of zinc for mothers in both rural contents in plantain-based foods and their and urban zones (Table 8), representing 4% daily consumption levels by the surveyed of their average iron and zinc requirements children and their mothers, we calculated (Table 9). Plantain-derived foods could potentially provide as much as 46.7 g RAE the daily iron, zinc and vitamin A supplied by μ of vitamin A to rural mothers and 40 g RAE each plantain-based food to each subject μ to urban mothers daily. These values (Tables 6 and 7). For these micronutrients corresponded on average to 9.4% and 8% the dietary allocations recommended by of vitamin A requirements of mothers in FAO for children under five are 14 mg of rural and urban zones respectively. These iron, 10 mg of zinc and 400 μg of vitamin contributions to vitamin A requirements A (Latham 2001). Our results showed that could, however, reach up to 25% in both the daily consumption of plantain foods rural and urban zones. provided 0.98 mg and 0.77 mg of iron in rural and urban zones respectively, i.e. Conclusion 7% and 5.5% of the children’s daily iron This paper provided data on the iron, zinc requirement (Tables 8 and 9). Zinc provided and β-carotene contents in plantain-based daily by plantain-based foods in both rural foods commonly consumed in Southern and urban consumers’ children was 0.26 mg, Nigeria and their daily level of consumption representing almost 3% of the requirement. by children under five years old and their Daily consumption of plantain by the children mothers in both rural and urban areas.

Table 6. Daily iron, zinc and vitamin A supply by plantain foods for the children who ate the dishes Form of meal Iron (mg) Zinc (mg) Vitamin A (µg RAE) Rural area Urban area Rural area Urban area Rural area Urban area Fried plantain 0.26 (0.35) 0.34 (0.54) 0.11 (0.14) 0.14 (0.23) 7.54 (10.01) 9.75 (15.86) Plantain chips 0.06 (0.52) 0.31 (0.77) 0.02 (0.15) 0.09 (0.22) 1.31 (10.62) 6.48 (15.87) Boiled plantain 0.58 (0.81) 0.45 (0.50) 0.19 (0.26) 0.15 (0.16) 22.00 (29.43) 17.30 (19.01) Pounded plantain 0.43 (0.85) 0.22 (0.60) 0.17 (0.34) 0.08 (0.24) 31.94 (63.76) 16.16 (45.32) Ripe banana/plantain 0.34 (0.53) 0.25 (0.44) 0.09 (0.14) 0.06 (0.11) 8.95 (14.07) 6.49 (11.68) Roasted plantain 0.42 (0.88) 0.92 (1.22) 0.12 (0.26) 0.27 (0.32) 21.54 (45.06) 46.90 (61.71) Number in the brackets is standard deviation

Table 7. Daily iron, zinc and vitamin A supply by plantain foods among the mothers who consumed the dishes Form of meal Iron (mg) Zinc (mg) Vitamin A (µg RAE) Rural area Urban area Rural area Urban area Rural area Urban area Fried plantain 0.45 (1.07) 0.39 (2.35) 0.19 (0.45) 0.16 (0.98) 21.93 (31.0) 11.19 (48.10) Plantain chips 0.30 (1.02) 0.56 (1.85) 0.09 (0.29) 0.16 (0.53) 6.19 (19.03) 11.49 (36.18) Boiled plantain 0.63 (1.88) 0.49 (1.66) 0.20 (0.61) 0.16 (0.54) 23.96 (62.78) 18.72 (63.36) Pounded plantain 0.73 (2.26) 0.54 (2.04) 0.29 (0.89) 0.21 (0.80) 54.81 (102.09) 40.79 (133.24) Ripe banana/plantain 0.70 (2.23) 0.57 (2.51) 0.18 (0.57) 0.57 (0.66) 18.44 (48.92) 58.92 (67.92) Roasted plantain 0.78 (2.31) 0.90 (3.16) 0.23 (0.67) 0.26 (0.67) 39.95 (98.35) 45.97 (141.95) Number in the brackets is standard deviation

4 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 5 Table 8. Average iron, zinc and vitamin A supply (mean ± standard deviation) by banana FAO/WHO. 1992. World declaration on Nutrition: Plan and plantain in children’s and their mothers’ diets of Action for Nutrition. International Conference on Micronutrients Children Mothers Significant Nutrition, Rome. WHO/NUT/93.2. Geneva. Rural area Urban area Rural area Urban area level FAO/WHO/IAEA. 1996. Trace elements in human nutrition Iron (mg) 0.98 ± 1.4 0.77 ± 1.2 1.95 ± 4.7 1.72 ± 5.0 ns and health. Geneva: World Health Organization. Zinc ((mg) 0.29 ± 0.4 0.23 ± 0.4 0.49 ± 0.9 0.8 ± 1.1 ns Frossard E., J. Chunwongse & S.D. Tanksley. 2000. Vitamin A (µg RAE) 30.9 ± 47.8 18.2 ± 38.9 46.7 ± 98.2 40.0 ± 103.9 * Potential for increasing the content and bioavailability * = Significant difference at 5% for the children of Fe, Zn and Ca in plants for human nutrition. Journal ns = not significantly different at 5% level of the Science of Food and Agriculture 80:861-879. Gibson R.S., C. Hotz, L. Temple, F. Yeudall, B. Mtitimuni Table 9. Contribution of plantain-based foods to the iron, zinc and vitamin A supply & E. Ferguson. 2000. Dietary strategies to combat (mean ± standard deviation) to the children and their mothers deficiencies of iron, zinc, and vitamin A in developing Micronutrients Children Mothers Significant countries: development, implementation, monitoring, Rural area Urban area Rural area Urban area level and evaluation. Food Nutrition Bulletin 21:19-31. Iron (%) 7.00 ± 10.2 5.50 ± 8.5 4.06 ± 9.7 3.58 ± 10.3 ns Hallberg L. & L. Hulthén. 2000. Prediction of dietary iron Zinc (%) 2.91 ± 4.4 2.30 ± 3.8 4.08 ± 7.5 3.17 ± 9.1 ns absorption: an algorithm for calculating absorption Vitamin A (%) 7.2 ± 11.9 4.6 ± 9.7 9.4 ± 15.7 8.0 ± 16.2 * and bioavailability of dietary iron. American Journal of * = Significant difference at 5% for the children Clinical Nutrition 71:1147-1160. ns = not significantly different at 5% level INIBAP. 2002. Food and wealth. International Network for Some food items, e.g. roasted plantain, the Improvement of Banana and Plantain. were identified as relatively rich in iron IITA. 2000. Project2, Improving plantain and Banana and β-carotene. The average daily level of based systems. International Institute of Tropical consumption of plantain-based foods was Agriculture. Annual report. higher for rural than for urban consumers. Izonfua W.L. & V.O.T. Omuaru. 1988. Effect of ripening on the chemical composition of plantain peels and pulps There was wide variation in consumption (Musa paradisiaca). Journal of the Science of Food and levels of plantain-based foods and hence Agriculture 45:333-336. of their contribution to micronutrient Mclarean D.S. & M. Frigg. 2001. Sight and life manual on requirements. Nevertheless, the study vitamin A deficiency disorders (VADD). 2nd ed. Basel, Switzerland: Task Force Sight and Life. showed that plantain is a good source of Ngoh Newilah G., J. Tchango Tchango, E. Fokou & F.X. β-carotene and can make a substantial Etoa. 2005. Processing and food uses of banana and contribution to meeting micronutrient plantains in Cameroon. Fruits 60:245-253. requirements of children and mothers if Latham, M.C. 2001. La nutrition dans les pays en eaten in substantial quantities. développement. FAO, Rome. Olivares M., F. Pizarro, S. de Pablo, M. Araya & R. Uauy. 2004. Iron, zinc and copper: contents in common Acknowledgements Chilean foods and daily intakes in Santiago, Chile. We gratefully acknowledge financial support Nutrition 20(2): 205-212. from the Harvest Plus Challenge Programme OMS/UNICEF. 2002. Progrès en vue de l’élimination des through Contract # 5001 to the International troubles dus à la carence en vitamine A. rapport d’une consultation conjointe d’experts OMS/UNICEF. 36pp, Institute of Tropical Agriculture (IITA). We Geneva. also extend sincere thanks to all those who Platt B.S. 1979. Tables of representative values of foods participated in the data collection in Nigeria. commonly used in tropical countries. London: HMSO, Special Report Series. No 302. References de Pee S. & C.E. West. 1996. Dietary carotenoids and Ajayi A. R. & M.O. Aneke. 2002. Consumption and their role in combating vitamin A deficiency. A review of expenditure patterns of banana and plantain consumers the literature. Europeen Journal of Clinical Nutrition 50 in Nsukka Urban, Nigeria. InfoMusa 11(1):50-53. (suppl.): S38-53. Barthkur N.N. & N.P. Arnlod. 1990. Chemical evaluation Sharrock S & E. Frison. 1998. Musa production around Fernande Honfo, Kerstin of Musa « Bhimkol » as a baby food. Journal of the the world- trends, varieties and regional importance. Science of Food and Agriculture 53:497-504. Pp. 42–47 in INIBAP Annual Report. INIBAP, Hell and Ousmane Coulibaly Baiyeri K.P. & G.O. Unadike. 2001. Ripening stages Montpellier, France. work at the International and days after harvest influenced some biochemical Sommer A. & K.P. West. 1996. Vitamin A deficiency: Institute of Tropical Agriculture properties of two Nigerian plantains (Musa sp. AAB) health, survival, and vision. New York: Oxford (IITA-Bénin), 08BP0932 . Plant Product Research Journal 6:11-19. University Press. Bingham S.A., M. Nelson & P. Alison. 1988. Methods Takyi E.K. 1999. Children’s consumption of dark green Cotonou, Bénin and Abdou for data collection at an individual level. In: Manual leafy vegetables with added fat enhances serum on methodology for food consumption studies Oxford Tenkouano formerly based at retinol. Journal of Nutrition 129:1549-1554. IITA-Cameroun, works now for (Cameron M.E. & W.A. van Staveren, eds). Oxford University press. UN ACC/SCN. 1997. Third report on the world nutrition AVRDC in Tanzania Brown K.H., Peerson J.M., Rivera J. & Allen L. H. 2002. situation. United Nations Administrative Committee on Corresponding author: Effect of supplemental zinc on the growth and serum Coordination/Subcommittee on Nutrition Geneva. [email protected] zinc concentrations of prepubertal children: a meta- UNICEF. 1990. World declaration on the survival, [email protected] analysis of randomised trials. American Journal of protection and development of children. The World Clinical Nutrition 75:1062-1071. Summit for Children. New York. tel.: 229 21 35 01 88 Englberger L. 2004. Carotenoid-rich bananas in USDA. 2004. National Nutrient database for Standard fax: 229 21 35 05 56 Micronesia. InfoMusa 12(2):2-5. Reference, Release 17.

6 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 7 Preparation of a cation exchanger containing carboxyl Banana stalk as heavy-metal groups from banana stalk and its utilization as sponge chelating agent T.S. Anirudhan and I.G. Shibi

he ability of inexpensive agricultural 16,000,000 metric tonnes annually. After the residues to adsorb heavy metal ions use of banana fruits, the banana stalk (BS) Thas received considerable attention for are often under valued and considered as a the development of an efficient, clean and waste material, creating a disposal problem. cheap technology for wastewater treatment It is largely composed of cellulose, hemi (Montanher et al. 2005, Nasernejad et cellulose, lignin, tannin and pectin. The al. 2005). Numerous low cost agricultural main objective of this study is to investigate residues have been used as adsorbents the feasibility of using BS as a precursor for the removal of heavy metals, including material to develop a suitable adsorbent for saw dust, coconut husk, coconut coir pith, the removal of heavy metals from aqueous , bagasse fly ash, orange solutions. peel, moss peat and nut shells (Annadurai et al. 2002). Most of these adsorbents have Material and methods good adsorption capacity as compared All chemicals were analytical grade and used to activated carbons and commercial ion as purchased without further purification. The exchangers, but their use in their original aqueous solutions of various concentrations forms was found to be limited due to leaching of the heavy metals ions were prepared by of organic substances into the solutions. dissolving their chloride (E. Merck India Efforts are therefore needed to prevent Ltd.) in distilled water. organic substances from leaching during The carboxylate functionalized BS was the adsorption process without affecting the synthesized using polyacrylamide grafting in adsorption capacity. the presence of ferrous ammonium sulphate/ Chemical modifications of agricultural H2O2 redox initiator (Shibi and Anirudhan residues via esterification, cross linking 2002). Scheme I represents the general and grafting have been extensively studied procedure adopted for the preparation of (El-Sayed 1996). It has been reported polyacrylamide grafted BS (PGBS) having that the introduction of reactive functional –COOH groups (PGBS-COOH). In order groups into the backbone of cross linked to design the optimum conditions for lignocellulosics resulted in products that preparing large quantity of PGBS, a series were capable of removing heavy metals of experiments were conducted by varying from industrial waste waters (Khalil et al. the concentration of ferrous ammonium 1991). We have investigated the synthesis sulphate, H2O2, acrylamide, reaction time and of quality adsorbents and ion exchangers temperature. The results of these experiments from lignocellulosics for effluent treatment have been reported in our earlier publication preparation. Characterization of polymer (Shibi and Anirudhan 2002). For a typical grafted lignocellulosics based on saw dust synthesis, 10 g of BS was transferred into Scheme 1. Preparation of PGBS-COOH (Raji and Anirudhan 1998, Unnithan and 2+ Anirudhan 2001), coconut husk (Sreedhar Fe / H2O2 BS + CH2 CH CONH2 BS P CONH2 and Anirudhan 2000) coconut coir pith 70 �C (AAm ) (Unnithan et al. 2004) and banana stem (PGBS) (Noeline et al. 2005) and their application (en) 2 in removing heavy metals from aqueous BS P CONHCH 2CH 2NH2 and effluent liquids were examples of these 100 �C,Toluene studies. In international trade, bananas are the most commonly traded fruit after those BS P CONH- (CH2 )2-NH CO(CH 2)2 - CO OH of citrus family. India is the market leader pH 4.0 (PGB S-COOH) in the production of bananas, producing Scheme 1. Preparation of PGBS-COOH 6 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 7 a reaction flask equipped with a mechanical experiments, metal concentrations used for stirrer and thermometer, and then 80 mL adsorption ranged from 10 to 700 mg/L. of ferrous ammonium sulphate (0.02 M) The experimental conditions for the was added. The suspension was allowed desorption of metals from spent adsorbent to soak for 15 min (this time was found to were similar to those of the batch adsorption be sufficient for maximum impregnation of tests. Initially the PGBS-COOH was loaded Fe(II) ions onto BS), while the flask was with metal ions by applying the adsorption kept in a thermostatically controlled water process for 4 h at pH 6.5. The concentrations ° of Pb(II), Co (II), Hg(II) and Cd(II) in the loaded bath at 30 C. To the mixture 120 mL of H2O2 PGBS-COOH was 12.46, 12.41, 12.32 and (0.2 M) was added and then purified N2 was passed through the vessel for 5 min at room 10.94 mg/g respectively. Desorption was temperature (30°C). The polymerization was performed by transferring 0.1 g of spent started by adding 50 g acrylamide (AAm). PGBS-COOH to a flask containing 50 mL The reaction mixture was stirred at 70°C for of 0.2 M HCl. The flasks were shaken at ° 4 h. Graft copolymer (PGBS) was recovered 30 C for 4 hours. After attaining equilibrium, by filtration, washed and finally dried. The dry the supernatant was filtered and filtrate reaction product was soxhlet extracted with was analyzed for metal concentration using water for 6 h to remove homopolymer and atomic absorption spectrometry. Comparison dried at 80°C. of these values with those observed with To convert into cation exchanger, PGBS the initial adsorption step was used to was refluxed with 100 mL of ethylenediamine compute the percentage recovery values. (en) for 8 h.The product was then washed All experiments were performed in duplicate 2 and mean values are presented. Maximum with toluene and dried. One part by weight of deviation was 5%. the material was then refluxed with an equal ± part by weight of succinic anhydride in 1,4– Results and discussion dioxane at pH 4.0 for 6 h. The product was Sorption of all metals at pH 2.0 was washed with 1,4–dioxane to remove excess negligible, increasing with increase of pH, succinic anhydride and dried. The PGBS- attaining an optimum in the range of 5.5–8.0 COOH obtained was sieved and particles (Figure 1). The pH of the aqueous solution having an average diameter 0.096 mm were is an important controlling parameter in the used throughout the study. The physical and heavy metal adsorption process. The sharp surface properties of PGBS-COOH were increase in metal uptake from pH 2.0 to determined by scanning electron micrograph, 5.5 can be explained by ion exchange. The X-ray, TG and FTIR (Shibi and Anirudhan dependence of metal uptake on pH suggests 2002; Shibi and Anirudhan 2005). The that the weak acidic carboxyl groups characteristics of PGBS-COOH are: surface – R-COO (pKa range between 3.5–5.5) of 2 area; 110 m /g; porosity, 0.51 mL/g; pHzpc, 5.5; PGBS-COOH are the probable adsorption cation exchange capacity, 2.38 meq/g; sites. At a pH of less than 5.5 the predominant carboxylate content, 1.84 meq/g and apparent metal species [M2+ and M(OH)+] are density, 0.72 g/mL. positively charged and therefore the uptake Batch adsorption experiments were carried out in 100 mL stoppered flasks by transferring 120 Initial concentration : 50 mg/L 50 mL of an aqueous solution of heavy metal Sorbent dose : 2 g/L 100 and 0.1 g PGBS-COOH. The solution pH was Agitation speed : 200 rpm adjusted using 0.1M HCl and NaOH. The Temperature : 30 °C 80 flasks were shaken for 4 h at an agitation Time : 3h ° Pb speed of 200 rpm at 30 C. The contents of 60 Co the bottle were filtered and the residual metal

Removal (%) Hg concentration in the filtrate determined using 40 Cd atomic absorption spectrophotometry. The 20 PGBS-COOH–metal sorption suspension PGBS-COOH BS was equilibrated at pH 2-8 for determining the 0 0 1 2 3 4 5 6 7 8 9 10 effect of pH on sorption. A metal concentration pH of 50 mg/L and an adsorbent dose of 100 mg Figure 1. Effect of pH on the removal of metal ions by PGBS- per 50 mL were employed. For isotherm COOH and BS

8 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 9 8 of metals in the pH below 5.5 is a H+ – M2+/ Sorbent dose : 2 g/L M(OH)+ exchange process. (formula 1 7 Equilibrium time : 3 h and 2). Agitation speed : 200 rpm The maximum removal capacities at pH 6 pH : 6,5 6.5 were found to be 96.4, 95.1, 91.3 and 5 Pb

(g/L) Co 80.6 % for Pb(II), Co(II), Hg(II) and Cd(II) e /q

e 4 Hg respectively at an initial concentration of C Cd 50 mg/L. The sorbent surface becomes 3 more positively charged at lower pH, due 2 to the higher concentration of the hydrogen ions, thus reducing the attraction between 1 sorbent surface and the metal cations. The 0 pH of the PGBS-COOH was found to be 0 100 200 300 600 500 600 zpc Ce (mg/L) 5.5 and above this pH the sorbent surface Figure 2. Langmuir isotherm plots for the adsorption of metal ions became negatively charged, while the metal onto PGBS-COOH at 30°C species were still present as M(OH)+. Under this condition M(OH)+ ions were adsorbed be explained by the high stability of the through a favourable electrostatic attraction. complexes formed by the cationic species of Over the pH range of 2.0-8.0, the adsorption this metal with carboxylate in PGBS-COOH of the metal ions by BS was found to be very compared with those formed by Co(II), Hg(II) much less than that of PGBS-COOH, clearly and Cd(II) (Baes et al. 1996). The values of demonstrating the effectiveness of the latter the correlation coefficient (r) obtained in the in the metal removal process. present study vary between 0.982-0.990 for The saturation capacities of metals different metals and indicate the goodness for PGBS-COOH at 30°C were analyzed of fit of experimental data to the Langmuir using the Langmuir isotherm model. This model. Comparison of metal adsorption is valid for monolayer adsorption onto on PGBS-COOH with the data from the surfaces containing finite numbers of literature indicates that the adsorption identical sorption sites, a situation which capacity of PGBS- COOH is very much is described by the following equation greater than that of other adsorbents such (see formula 3) where q and Q0 are the e as activated carbons, ion exchange resin, observed and maximum uptake capacities beet pulp, shell, sawdust (mg/g adsorbent) respectively. C is the e and curdlan-activated carbon composite equilibrium concentration (mg/L solution) (El-Shafey et al. 2002; Rengaraj and Moon b is the equilibrium constant. The linear 2002, Reddad et al. 2002, Basso et al. 2002, plots of C /q vs C for all metals indicate e e e Moon and Lee 2005). the applicability of the Langmuir adsorption Table 1 summarizes the results of isotherm. To evaluate the isotherm constants desorption and regeneration of PGBS-COOH the experimental data were treated after for all metals. The percentage adsorption/ discarding the data that lay outside the 95 % desorption values were calculated in relation confidence interval, which was established with the original amount of the adsorbent. by a method of least squares for the linear The total desorbed amount was calculated function. The uncertainty of the parameters of the fitted straight line corresponds to the standard deviations. The values of Q0 and Formula 1 b (the slope and intercept of the plots) were 2 PGBS-COOH + M2+ (PGBS-COO) M + 2 H+ found to be (Q0) 185.34, 166.7, 137.89 and 2 65.88 mg/g and (b), 1.8 x 10-2, 1.4 x 10-2, 0.92 x 10-2 and 0.34 x 10-2 L/mg for Pb(II), Co(II), Formula 2 Hg(II) and Cd (II) respectively (Figure 2). The PGBS-COOH + M(OH)+ PGBS-COO M(OH) + H+ higher the value of b, the higher is the affinity of adsorbent for the metal adsorbed. The Formula 3 affinity of PGBS-COOH to sorb the metals Ce 1 Ce tested in this study followed a[pattern, ______= ______+ ______0 0 0 as was noted for Q (Pb>Co>Hg>Cd). qe Q b Q The relative preference for Pb(II) may

8 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 9 and compared to the initial sorbed amount. Desorption of Pb, Hg, Co and Cd The ability of HCl of 0.2 M concentration to from metal–laden PGBS-COOH in strip out most of the adsorbed metals may flasks with 0.2 M HCl established the be attributed to ion exchange. It has been chemical stability of the carboxylate postulated that the high concentration of groups and the regeneration ability of H+ ions at low pH is responsible for the PGBS-COOH. A small fraction of adsorbed displacement of adsorbed metal via the metals was not recoverable by regeneration. ion exchange mechanism. As shown in This fraction presumably represents the Table 1, with three adsorption/desorption metals which are bonded through stronger cycles the Pb(II) adsorption capacity of interaction and as a result, sorption efficiency the PGBS-COOH decreased slightly from is reduced in subsequent cycles. Recovery 99.7 % in the first cycle to 95.2 % in the third of metals from an adsorbent is of importance cycle. The recovery of Pb(II) in 0.2 M HCl for application of sorption technology in decreased from 97.8 % in the first cycle to the waste water industry. The results show 93.0 % in the third cycle. The variation of the that the spent adsorbent can be effectively adsorption capacity and recovery efficiency regenerated by 0.2 M HCl. in the adsorption-desorption cycles was also observed for Co(II), Hg(II) and Cd(II). After Conclusion two cycles, the adsorption capacity of PGBS- The present study shows that the PGBS- COOH for Cd(II) was reduced by 4.7 % and COOH prepared from banana stalk, an on the other hand recovery was decreased agro-based waste biomass, can be used from 84.3 % in the first cycle to 77.5 % in as an adsorbent for the removal of heavy the third cycle. It was also found that the metals [Pb(II), Co(II), Hg(II) and Cd(II)] from highest recovery efficiency was for Pb(II) their aqueous solutions. A pH range 5.5 – 8.0 and lowest for Cd(II). An explanation may was found to be effective for the maximum be found in the differences in the strength removal of metal ions. The carboxyl groups of solution complexation with chloride and were the main reaction sites responsible preferred coordination numbers. Pb(II) can for metal binding on PGBS-COOH. The form stable complexes with chloride due to equilibrium data fitted very well to a Langmuir its high coordination number, usually 6. On isotherm equation, confirming the monolayer the other hand, Cd(II) typically exhibits a low sorption capacity of metals onto PGBS- coordination number 4, which results in less COOH. The adsorbed metal ions were competition with chloride ions from solution desorbed quantitatively by 0.2 M HCl and the and reaction sites on the adsorbent. Detailed adsorbent can be reused successfully after study of the effect of chloride is necessary regeneration. Batch adsorption-desorption to understand the formation and strength studies illustrate that PGBS-COOH could be of chlorocomplexes of metals and their used to remove heavy metals from aqueous influences on the adsorption properties. solutions and industrial effluents.

Table 1. Regeneration data Adsorption (%) Desorption (%) No. of cycles Pb(II) Co(II) Hg(II) Cd(II) Pb(II) Co(II) Hg(II) Cd(II) 1 99.7 99.3 98.6 87.5 97.8 96.3 94.8 84.3 2 97.2 97.1 96.3 85.1 95.0 93.2 93.2 80.2 3 95.2 93.1 93.1 82.8 93.0 91.6 91.6 77.5

References Annadurai.G., R.S. Jueng & D.J. Lee. 2002. Adsorption Basso M.C., E.G. Cerrella & A.L. Cukierman. 2002. of heavy metals from water using banana and Lignocellulosic materials as potential biosorbents of orange peels. Water Sci. Technol. 47: 185-190. trace toxic metals from wastewater. Ind. Eng. Chem. Ecxhange and absorption of some heavy metals in a Res. 41: 185-190. modified coconut coir cation exchanger. Water Science and Technology 34:193-200. El-Sayed Z. 1996. Utilization of saw dust in the Baes A.U., S.J.P. Umali & R.L. Mercado 1996. Ion preparation of some ion exchange resins. PhD Thesis. exchange and absorption of some heavy metals in a Cairo University. Cairo. modified coconut coir cation exchanger. Water Science El-Shafey E.I., M. Cox, A.A. Pichugin & Q. Appleton. and Technology 34:193-200. 2002. Application of a carbon sorbent for the removal

10 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 11 of cadmium and other heavy metal ions from aqueous Reddad. Z, C. Gerente, Y. Andres & P.L. Cloirec. 2002. solution. J. Chem. Tech. Biotechnol. 77:429-436. Adsorption of several metal ions onto a low-cost Khalil M.I., A.Waly, S. Frag & A. Hebeish. 1991. Preparation biosorbent: kinetic and equilibrium studies. Environ. of cationic– exchange containing phosphoric Sci. Technol. 36: 2067-2073. acid groups. J. Appl. Polym. Sci. 43:2303-2309. Shibi I.G. & T.S. Anirudhan. 2002. Synthesis, charac- Montanher S.F., E.A. Oliveira & M.C. Rollenberg. 2005. terization and application as a mercury(II) sorbent Removal of metal ions from aqueous solutions by of banana stalk (Musa paradisiaca)-polyacrylamide sorption onto rice bran. J. Hazard. Mater. B117:207-211. grafted copolymer bearing carboxyl groups. Ind. Eng. Moon C.J & J.H. Lee. 2005. Curdlan and activated Chem. Res. 41:5341-5352. carbon composed adsorbents for heavy metal removal. Shibi I.G. & T.S. Anirudhan 2005. Adsorption of Co(II) by Process Biochem. 40: 1279-1283. a carboxylate– functionalized polyacrylamide grafted Nasernejad B., T.E. Zalah, B.B Pou, M.E. Bygi & A. lignocellulosics. Chemosphere 58:1117-1126. T.S. Anirudhan works at the Zamani. 2005. Comparison for biosorption modeling Sreedhar M.K. & T.S. Anirudhan. 2000. Preparation of an Department of Chemistry, of heavy metals [Cr(III), Cu(II), Zn(II)] adsorption from adsorbent by graft polymerization of acrylamide onto University of Kerala, wastewater by carrot residues. Process Biochem. 40: coconut husk for mercury(II) removal from aqueous 1319-1322. solution and chlor-alkali industry wastewater. J. Appl. Thiruvananthapuram, 695 Noeline B.F, D.M. Manohar & T.S. Anirudhan. 2005. Kinetic Polym. Sci. 75:1261-1269. 581, Kerala, India (Email: and equilibrium modelling of lead(II) sorption from water Unnithan M.R. & T.S Anirudhan. 2001. The Kinetics and [email protected]) and and wastewater by polymerized banana stem in a batch thermodynamics of sorption of Chromium(III) onto to reactor. Sep. pur. ;Technol. 45(2):131-140. the iron(III) complex of a carboxylated polyacrylamide I.G. Shibi works at the Raji C. & T.S. Anirudhan. 1998. Batch Cr(VI) removal grafted saw dust. Ind. Eng. Chem. Res. 40:2693-2701. Department of Chemistry, S.N. by polyacrylamide-grafted saw dust: Kinetics and Unnithan M.R., V.P. Vinod & T.S Anirudhan. 2004. College, Chempazhanthy, thermodynamics. Water Res. 32:3772-3280. Synthesis, characterization and application as Thiruvananthapuram, 695 587, Rengaraj S & S.H. Moon. 2002. Kinetics of adsorption a chromium(VI) adsorbent of amine-modified of Co(II) removal from water and wastewater by ion polyacrylamide-grafted coconut coir pith. Ind. Eng. Kerala, India exchange resin. Water Res. 36:1783-1793. Chem. Res. 43:2247-2235. (Email: [email protected]).

Associative nitrogen fixation by Azospirillum and Bacillus spp. in bananas Md. Abdul Baset Mia, Z.H. Shamsuddin, Z. Wahab and M. Mahmood

ssociative N fixation and plant by PGPR in association with banana roots, 2 Bacteria enhance N use growth promotion by rhizobacteria are which has not been investigated. Therefore, Aimportant in achieving a sustainable the present study was conducted to quantify agricultural system. Crops rely mainly on the amount of N2 fixation by Azospirillum the N2 fixation process by associative, strain Sp7 and Bacillus strain UPMB10 and symbiotic and free living bacteria in the in association with banana roots at different rhizosphere (Cocking 2000). Graminaceous levels of fertilizer-N. plants potentially are capable of establishing associations with diazotrophic bacteria where Materials and methods Azospirillum provide the host with a source Plants were grown hydroponically with a of N (Brimecombe et al. 2001). Plant growth modified Cooper (1979) nutrient solution promoting rhizobacteria (PGPR) inoculation in small pots (4.0L). The experiment was can provide 31% N requirement in maize completely randomized with two factors: and 40% of the N requirement for oil palm inoculation - uninoculated, inoculated with seedlings through BNF under glasshouse Azospirillum brasilense strain Sp7, isolated condition (Amir et al. 2001, El-Komy et al. from Digitaria grass, originally provided by 1998). In field condition, rice can obtain 20% EMBRAPA, Brazil, and Bacillus sphaericus of its N and sugar cane 70% of its total N strain UPMB10, isolated from oil palm roots (Shamsuddin and Roslina 1995) - and requirement through N2 fixation (Boddey et al. 1995, Shrersta and Ladha 1996). fertilizer-N 3.2, 8, 20 and 50 mg/kg) with six

In general, N2 fixation is increased with replications. modest levels of soil or fertilizer-N, but A 1-ml sample of bacterial stock suspension declines at high N levels because mineral- was transferred into nutrient broth (Okon

N depresses N2 fixation (Marschner 1995). et al. 1977) in 250 ml Erlenmeyer flasks A lower but specific level of fertilizer-N containing 100 or 300 ml nutrient media,

might be required for optimum N2 fixation respectively, that were agitated on a rotary 10 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 11 shaker at 125 rpm, 30± 2°C for 48 h. The distillates were collected and concentrated 15 optical density (OD600) was taken using a for N analysis by Emission Spectrometer spectrophotometer (Ultrascope III). Uniform (NOI-6PC) at the Malaysian Institute for 10 to 11 cm height tissue-cultured banana Nuclear Technology Research (MINT). The plantlets cv. ‘Berangan’ (Musa spp., AA 15N abundance found in the plant tissue was type) with 3-4 leaves and were used in the corrected for the % 15N excess (a.e.) experiments. The plantlets were acclimatized present in the atmosphere (0.3663% 15N with tap water for 3 days and the existing roots a.e.) by using the formula of Warembourg were gently removed aseptically from the corm (1993) where: using a sterile blade before transplanting. %15N atom excess (a. e.) in each plant part The N supply, labelled with 15N as = %15N abundance – 0.3663% 15N (a.e.) 15 Quantification of fixation based on {( NH4)2SO4, 10% a.e.)} was applied after the establishment of seedlings (3 days dilution was calculated by the formula of after transplanting) (Malik and Zafar 1985). Fried and Middelboe (1977), see formula (1), A 40-ml broth cultures of Sp7 or UPMB10 where fs: fixing system, nfs: non fixing were applied to the respective pot after system. establishment of seedlings in the system The 15N enrichment of the plant tissue was (3 days) and same volume of killed bacterial corrected according to the formula of Jensen broth cultures (autoclaved at 121°C for et al. (1985), see formula (2). 15 minutes) were applied to the control The average 15N enrichment present pots as in the procedure by Malik and Zafar in different plant parts at the end of the (1985). The pots were aerated with an air experiment was calculated using the pump for 6 hours at 6-hourly intervals to formula (3). ensure an uninhibited root respiration and The collected data were analyzed bacterial growth. statistically using the Statistical Analysis The banana plantlets were harvested at System (SAS version 6.12, 1989). Following 45 DAI, separated into root, stem and leaves the analysis of variance procedure (ANOVA), and oven dried for 48 hours at 70°C. The differences among treatment means were dried samples were weighed and ground to determined using Duncan’s New Multiple 390 µm. Samples (500 mg) of this ground Range Test (DMRT) comparison method material were analyzed for total N by semi- (whenever applicable) at 5% level of micro Kjeldahl method (Bremner 1996). significance. The digested sample was distilled within Results the minimum distillation period of 4 minutes using Buchi TM K314 distillation unit. The Dry matter production liberated cooled ammonia was received in Plants inoculated with PGPR strains, an Erlenmeyer flask containing 10 ml 0.1N especially with UPMB10, significantly HCL+Tashiro indicator solution for titrimetric increased the total dry matter (TDM) + determination of NH4 . A back titration was production when compared to the control performed to determine the amount of acid (Table 1). Plants inoculated with UPMB10 consumed by ammonia and to calculate the produced more TDM at all the fertilizer-N amount of total N in sample using 0.1N NaOH levels. These inoculated with Sp7 produced (tNaOH=1.000). Finally the total 14N and 15N a significant effect at 3.2 and 8 mg/kg,

(15N atom % excess) Formula (1) % N fixed= 1_ fs x 100 (15N atom % excess)

N x % 15N (a. e) Formula (2) % 15N a.e. corr.= N plantlets (D harvest) - N plantlets (D planting)

(%15N a.e. root x N root) + (%15N a.e. stem x N stem) + (%15N a.e. leaf x N leaf) Formula (3) Average % 15N a.e. = (N root + N stem + N leaf)

12 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 13 but not at 20 and 50 mg kg-1N levels, in Table 1. Dry matter in root, pseudostem and leaves of banana plantlets inoculated with root, pseudostem and leaves dry matter PGPR strains Sp7 and UPMB10 and supplied with various levels of fertilizer-N grown production was significantly increased due under hydroponics condition Dry matter (g/plant) to fertilizer-N application and inoculation Treatments Fertilizer-N levels (mg/kg) process (Table 1). In uninoculated plants, 3.2 8 20 50 dry matter of root, pseudostem and leaves TDM Control 1.8 b 3.4 c 6.4 b 9.3 b Sp7 2.7 a 3.9 b 7.1 b 10.2 ab increased with fertilizer-N. Similarly, plants UPMB10 2.7 a 4.5 a 8.4 a 12.0 a inoculated with UPMB10 also increased Root Control 0.96 b 1.57 c 2.52 b 2.0 c their root dry matter at 3.2, 8, and 20 mg/kg, Sp7 1.47 a 1.94 b 2.52 b 2.29 b UPMB10 1.39 a 2.32 a 3.43 a 2.93 a pseudostem dry matter at 3.2, 20, and 50 Pseudostem Control 0.26 b 0.78 a 1.64 b 3.21 b mg/kg, and leaf dry matter at 3.2 mg/kg. Sp7 0.49 a 0.82 a 1.91 ab 3.69 b Plants inoculated with Sp7 did not show UPMB10 0.50 a 0.94 a 2.21 a 4.68 a Leaf Control 0.58 b 1.05 a 2.24 a 4.09 a the similar trend as control or UPMB10. Sp7 0.74 ab 1.14 a 2.67 a 4.22 a Plants inoculated with UPMB10 produced UPMB10 0.81 a 1.24 a 2.76 a 4.39 a significantly higher root dry matter at 3.2 and 8 mg/kg, but not at 20 mg/kg and Table 2. Nitrogen yield in root, pseudostem and leaves of banana plantlets inoculated

pseudostem at 3.2 mg/kg but not at higher N with PGPR strains Sp7 and UPMB10 and supplied with various levels of fertilizer-N fertilizer levels. No difference between Sp7 grown under hydroponics condition and the control was observed at any N level Total N (mg/plant) Treatments Fertilizer-N levels (mg/kg) for leaf tissue. 3.2 8 20 50 Nitrogen yield Root Control 6.5 b 13.1 b 21.9 b 41.2 a Sp7 9.4 a 16.3 a 23.6 b 32.7 a Plants inoculated with Sp7 or UPMB10 UPMB10 8.8 a 16.6 a 28.7 a 37.6 a and supplied with 3.2, 8.0 or 20 mg/kg Pseudostem Control 2.2 b 4.0 b 9.1 b 30.4 a fertilizer-N yielded more total N. Plants Sp7 3.6 ab 6.1 a 10.2 ab 39.5 a UPMB10 4.4 a 5.4 ab 12.3 a 38.4 a supplied with 50 mg/kg fertilizer-N showed Leaf Control 9.0 b 14.0 b 37.5 b 104 a no significant difference between inoculated Sp7 11.4 a 19.2 a 49.0 a 105 a and uninoculated plants (Table 2). Total N UPMB10 11.1 ab 21.1 a 45.8 a 107 a TDM Control 17.7 b 31.1 b 68.5 b 176 a yield of inoculated and uninoculated plants Sp7 24.4 a 41.6 a 82.8 a 177 a increased with increasing fertilizer-N in all UPMB10 24.3 a 43.1 a 86.8 a 183 a treatments until at the highest fertilizer-N Means having same letter (s) in a column do not differ at 0.05 significant level by DMRT level (50 mg/kg). Generally the N yield in root, pseudostem, Table 3. Distribution of % 15N atom excess in root, pseudostem and leaves of PGPR and leaves of inoculated plants was inoculated plants at various levels of fertilizer-N significantly higher compared to control % Atom excess until 50 mg kg-1N-fertilization; at 50 mg kg-1 Treatments Fertilizer-N levels (mg/kg) fertilizer-N there was no significant difference 3.2 8 20 50 Root Control 5.85 a 7.55 a 8.48 a 9.02 a between inoculated and uninoculated plants. Sp7 3.71 b 5.81 b 7.38 b 8.49 b Results were similar to those observed UPMB10 3.63 b 6.08 b 7.55 b 8.45 b for dry matter, except more significant Pseudostem Control 9.29 a 9.47 a 9.31 a 9.52 a Sp7 5.32 b 7.02 b 8.24 b 8.58 b differences were observed for leaf N than for UPMB10 4.35 b 7.20 b 8.01 b 8.84 b leaf dry matter. Leaf Control 6.36 a 8.08 a 8.75 a 9.12 a Sp7 4.10 b 6.11 b 7.66 b 8.82 b 15 N isotope dilution UPMB10 3.91 b 6.29 b 7.60 b 8.71 b Plant growth promoting rhizobacterial Means having same letter (s) in a column of a parameter do not differ significantly at 0.05 significant levels by DMRT inoculation significantly diluted and lowered the plant 15N concentration, which is an of atom excess in inoculated and non

indication of N2 fixation. The percentage of inoculated plant parts ranged from 3.63 15N atom excess (a.e.) of inoculated plant to 9.52. The dilution capacity between the parts was significantly less than those of strains was not significantly different. reference plants (control; nfs) (Table 3). The highest dilution (3.63-5.32% a.e.) was Nitrogen fixation found in plants using the lowest level of PGPR inoculation could potentially fix N2 fertilizer-N and the lowest (8.45-8.84% a.e.) in association with the roots of banana

in the highest level of fertilizer-N. Root of plantlets. Estimation of N2 fixed (% Ndfa) inoculated plants with UPMB10 showed on a total plant basis by the PGPR strains the lowest dilution (3.63%). The percentage Sp7 and UPMB10 were in the range of 5.0 to

12 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 13 39.3 % Ndfa (Table 4). As expected, higher activity of banana leaves as observed in % Ndfa (37.0 to 39.3%) was recorded under the previous studies. The higher dry matter a low fertilizer-N regime (3.2 mg/kg) by both production in the PGPR inoculated plants, strains Sp7 and UPMB10 and lowest (5.0- however, resulted in greater N yield of these 5.3%) under higher fertilizer-N (50 mg/kg) plants, which corresponded positively to the

condition. However, the total amount of increased N2 fixation and nutrient uptake. The N2 fixed (9.03 to 9.55 mg/plant) at the low results of the current experiment indicated level (3.2 mg/kg) was not significantly higher that bacterial inoculation in combination with

compared to N2 fixed at higher (50 mg/kg) minimal fertilizer-N (20 mg/kg) increased N fertilizer-N level (8.85 to 9.69 mg/plant) yields through a highly synergistic effect. (Table 5). Similarly, Marschner (1995) concluded that

contribution of N2 fixation to the total N per Table 4. Percentage of N derived from atmosphere (%Ndfa) in banana plantlets (total plant is increased by moderate level of soil plant basis) inoculated with PGPR strains Sp7 and UPMB10 and supplied with various or fertilizer-N but declines at high levels in fertilizer-N levels grown under hydroponic conditions % Ndfa legumes. %Ndfa Fertilizer-N levels (mg/kg) The higher N yield of the inoculated plants Treatments 3.2 8 20 50 was due to effective N2 fixation because Control 0 b 0 b 0 b 0 b the PGPR association with roots of banana Sp7 37.0 a 24.3 a 12.4 a 5.0 a plantlets successfully contributed 37-39% UPMB10 39.3 a 22.1 a 12.5 a 5.3 a fixed-N to the host plant as estimated by Means having same letter (s) in a column do not differ significantly at 0.05 level by DMRT 15N isotope dilution technique. The results of this experiment are in agreement with the Table 5. Total N2-fixed in banana plantlets inoculated with PGPR strains Sp7 and UPMB10 and supplied with various levels of fertilizer-N grown under hydroponic findings of several investigators (Dobereiner conditions and Baldani 1998, Amir et al. 2001) who N2-fixed (mg/plant) reported that Azospirillum and rhizobacterial Fertilizer-N levels (mg/kg) inoculation could contribute a substantial Treatments 3.2 8 20 50 Control 0 b (x) 0 b (x) 0 b (x) 0 b (x) amount (25-50% in oil palm; 40% in non- Sp7 9.03 a (x) 10.11 a (x) 10.26 a (x) 8.85 a (x) legumes) of the total plant N requirement UPMB10 9.55 a (x) 9.53 a (x) 10.85 a (x) 9.69 a (x) through BNF process. Malik et al. (1997) Means having same letter (s) in a column do not differ significantly at 0.05 level by DMRT have also reported that N2 fixation ability by Azoracus in association with kallar grass Discussion contributed 26% of the plant-N content. The evidence of N fixation was further Bacterial inoculation combined with fertilizer-N 2 supported by nitrogenase activity of application significantly increased dry matter inoculated roots as estimated by acetylene production in root, pseudostem and leaves of reduction assay (ARA). Rhizobacteria could banana plantlets and consequently the total persist, multiply and had the nitrogenase dry matter yield grown under hydroponics activity at the end of experimental period. condition for 45 days. In general, the higher Both the PGPR strains used produced dry matter production resulted in increased higher ARA values compared to the N yield in the inoculated plants. The N yield uninoculated controls. Higher ARA activity is the final product of fixed N in the nitrogen 2 could be attributed to the presence of more nutrition of crop plants. Plants inoculated bacterial cells on the root. The study on root with bacteria produced higher N yield colonization supported this finding that PGPR when supplied with fertilizer-N from 3.2 to strains Sp7 and UPMB10 could effectively 20 mg/kg; but did not show any increment at colonize roots of banana plantlets. 50 mg/kg. This difference is likely because of The present study clearly shows that a lower contribution of BNF in the presence rhizobacterial inoculation combined with of higher inorganic-N concentration in the minimal fertilizer-N (50 mg/kg) could fix nutrient solution. Nitrogen distribution to 8.85 to 9.69 mg N/plant (5.0-5.3% Ndfa). different plant parts were also increased The total amount of N2 fixed was further by the inoculation process. The leaves increased (11.0 mg/plant; 12.5% Ndfa) produced the highest amount of N due by lowering the fertilizer-N (20 mg/kg). to a higher demand for photosynthesis Similarly, Galal et al. (2000) found negative and other physiological functions. PGPR effect of using high fertilizer-N on BNF by inoculation stimulated the photosynthetic Azospirillum in association with wheat roots

14 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 15 Cocking E.C. 2000. Helping plants get more nitrogen from where grain utilized most of the fixed-N2. The results suggested that PGPR inoculation air. European Review 8(2):193-200. Cooper A. 1979. The ABC of NFT. Grower Books. London, and fertilizer-N (20 mg/kg) application have 181pp.

a synergistic effect on N2 fixation in banana Dobereiner J. & V.L.D. Baldani. 1998. Biological nitrogen plantlets grown under hydroponic conditions fixation by endophytic diazotrophs in non-legume crops in the tropics. Pp3-7 in Nitrogen Fixation with Non- in small pots (4.0 l) for 45 days. legumes. (Malik K.A., Sajjad Mirza M.and Ladha J.K., eds). Kluwer Academic Publishers, London. Conclusion El-Komy, H.M.A., T.M.M. Moharram & M.S.A. Safwat. Plant growth promoting rhizobacterial strain 1998. Effects of Azospirillum inoculation on growth and N2 fixation of maize subjected to different levels of Sp7 and UPMB10 inoculation in combination FYM using 15N-dilution method. Pp. 49-59 in Nitrogen with minimal fertilizer-N increased the N Fixation with Non-legumes. (Malik K.A., Sajjad Mirza yield in banana plantlets. Inoculated plants M.and Ladha J.K., eds). Kluwer Academic Publishers, together with the least fertilizer-N supply (3.2 London. Fried M. & Middelboe V. 1977. Measurement of amount mg/kg, 2.13% of the total N requirement) of nitrogen fixed by a legume crop. Plant and Soil 47: produced the highest Ndfa (37-39%) while 713-715. those with higher inorganic-N (50 mg/kg, Galal Y.G.M., L.A. El Ghandour & S.S. Aly. 2000. Non- isotopic method for the quantification of biological 33% of the total N requirement) showed nitrogen fixation and wheat production under field the lowest Ndfa (5%). However, PGPR conditions. Biology and Fertility of Soils 32:47-51. inoculation with 20 mg/kg fertilizer-N (13% Jensen E.S., A.J. Andersen & J.D. Thomsen. 1985. The influence of seed-borne N in 15N isotope dilution of total plant N) produced a synergistic studies with legumes. Acta Agriculture of Scandinavian effect on N2 fixation with higher amounts 35:438-443. Malik K.A., B. Rakhshanda, S. Mehnaz, G. Rasul, M.S. of N2 fixed (11.0 mg/plant; 12.5 % Ndfa). The results suggest that PGPR strains Sp7 Mirza & S. Ali. 1997. Association of nitrogen-fixing plant-growth promoting rhizobacteria (PGPR) with and UPMB10 are effective as biofertilizer kallar grass and rice. Plant and Soil 194:37-44. for banana plantlets when applied together Malik K.A. & Y. Zafar. 1985. Quantification of root- with 20 mg/kg (13% of total requirement) associated nitrogen fixation as estimated by 15N Md. Abdul Baset Mia isotopic dilution. Pp 161-171 in Nitrogen and the fertilizer-N supply. is Associate Professor Environment (Malik K.A., Bhatti N.A. and Kausar F., Department of Crop Botany, eds). Nuclear Institute for Agriculture and Biology, References Faisalabad, Pakistan. Bangabandhu Sheikh Mujibur Amir H.G., Z.H. Shamsuddin, M.S. Halimi, M. F.Ramlan Marschner H. 1995. Functions of Mineral Nutrients: Rahman Agricultural University & M. Marziah. 2001. Effects of Azospirillum inoculation Macronutrients. Pp. 889 in Mineral Nutrition of Higher Gazipur 1706, Bangladesh, on N fixation and growth of oil palm plantlets at nursery Plants. Academic Press, London. 2 e-mail: [email protected], stage. Journal of Oil Palm Research 13(1):42-49. Okon Y., S.L. Albrecht & R.H. Burris. 1977. Methods for Boddey R.M., O.C. Oliveira, S. Urquiaga, V. M. Reis, growing Spirillum lipoferum and for counting it in pure Zulkifli H. Shamsuddin F.L. Olovares, V.L.D. Baldani & J. Dobereiner. 1995. culture and different solution in association with plants. and Zakaria Wahab work Biological nitrogen fixation associated with sugar cane Applied and Environmental Microbiology 33:85-88. respectively at the Department and rice: contribution and prospects for improvements. SAS. 1989. SAS/STAT. Guide to Personal Computers. Plant and Soil 174:195-209. Version 6.12. SAS Institute Inc., Cary, North Carolina. of Land Management and Bremner J.M. 1996. Nitrogen-Total. pp. 1085-1121 in Shamsuddin Z.H. & A.W. Roslina. 1995. Isolation of Department of Crop Science, Methods of Soil Analysis (Part 3). Chemical Methods. Azospirillum from oil palm roots. 18th Microbiology Faculty of Agriculture, and (Spark D.L., Page A.L., Helmke P.A., Loeppert R.H., Symposium, Kuching, Sarawak. Marziah Mahmood at the Sultanpour P.N., Tabatabi M.A., Johnsto C.T. and Shrestha R.K. & J.K. Ladha. 1996. Genotypic variation Sumner M.E., eds). American Society of Agronomy, in promotion of rice nitrogen fixation as determined Department of Wisconsin. by Nitrogen-15dilution. Soil Science Society America and Microbiology, Faculty of Brimecombe, J.M., F.A. De Leij & J.M. Lynch. 2001. Journal 60(6):1815-1821. Science and Environmental The effect of root exudates on rhizosphere microbial Warembourg F. R. 1993. Nitrogen fixation in soil and plant Studies, Universiti Putra population. Pp 95-140 in The Rhizosphere. (Pinton R. system. Pp 127-155 in Nitrogen Isotope Technique Varanini Z. and Nannipieri P., eds). Marcel Dekker Inc. (Knowles R. and Blackburn T. H., eds). Academic , 43400 UPM, New York, USA. Press, London. Serdang, Selangor, Malaysia.

14 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 15 Measuring Fusarium virulence Development of a suitable method for evaluating virulence of Fusarium oxysporum f. sp. cubense race 1 (E.F. Smith) in banana T. Saravanan, M. Muthusamy, E.G. Ebenezar and R. Bhaskaran

usarium wilt disease is a serious were mixed at a ratio of 19:1 and sterilized at constraint in banana production 1.4 kg/cm2 pressure for 2 hours. The sterilized Fworldwide (Stover 1962). Its causal sand medium was inoculated by transferring agent, Fusarium oxysporum f. sp. cubense a disc (9mm) of the pathogenic culture grown (Foc) is a genetically stable soil-borne on PDA plate into sterilized sand medium and fungus. The identification of wilt disease- shaken for 1 min in order to spread over the producing strains is a big challenge scientists medium. All these were carried out under are facing because suitable methods for aseptic condition. They were incubated for testing the virulence of the disease in a period of 3 weeks at room temperature greenhouse conditions are still not efficient. (28 ± 2ºC). The fully grown inoculum of However, Sun and Su (1984) developed a Foc was used for the virulence tests. Spore method using tissue-cultured plantlets to suspension was prepared by adding sterile study virulence, but the availability of such distilled water to fungus grown in PDA slants plantlets for all cultivars is a problem with this at 10 ml per slant. The slant was gently shaken method. Hence, the aim of this research was for 5 min and suspension was filtered through to develop a suitable method to measure the a muslin cloth. The filtered suspension was virulence of Foc race 1 in banana. used as spore suspension for corm injection Materials and methods and sucker dipping in the study. Seven treatments were included in the The experiment was carried out at the study: Department of Plant Pathology, Agricultural College and Research Institute, Madurai, T1 Sand inoculum of the isolate at 10% w/v India. Fusarium wilt (race 1) affected banana of tank soil, plants (cv Rasthali) were collected from T2 Sand inoculum of the isolate at 15% w/v banana growing areas of southern Tamil of tank soil, Nadu. Suckers showing brown discoloration T3 corm injection of the isolate at 3 ml/plant were used for isolation of Foc. The suckers + Sand inoculum of the isolate at 10% w/v showing typical symptoms of the disease of tank soil, were washed in water and cut into small T4 sucker dipping for 30 min (106 colony pieces using a sterilized scalpel and surface forming unit of the isolate per ml - cfu/ml) sterilized in 0.1 % mercuric chloride for 30 sec + Sand inoculum of the isolate at 10% w/v followed by washing in several changes of tank soil, of sterile distilled water. Sterilized T5 sucker dipping for 30 min (106 cfu/ml), dextrose agar (PDA) medium amended T6 infected plant parts at 10% w/v of tank with 100 ppm of strephtomycin sulphate (to soil and avoid bacterial contamination) was poured T7 infected soil (105 cfu/g of soil) at 10% w/v into sterile Petri plates at 15 ml per plate and of soil. 3 surface sterilized plant pieces were placed Corm injection was done by injecting 3 ml of at equidistance in each plate. All these were spore suspension (106 cfu/ml) of the isolate carried out under aseptic condition. The at the base of pseudostem of 3 month-old plates were incubated at room temperatures suckers of cv Rasthali using banana injector (28± 2°C) for 7 days and observed for the TNAU model. The suspension was injected fungal growth. The fungus was purified by a into corm at an angle of 45°. single spore isolation technique, transferring Infected soil was collected from banana a single spore into PDA slant and incubated field where natural infection of the Fusarium at 28°C for five days. The fungus was then wilt was observed as more than 80%. The multiplied in a sand medium (Ricker and soil was taken from rhizosphere regions of Ricker 1936). Fine sand and maize flour the infected plants.

16 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 17 The infected banana plants including completely clean, no vascular discoloration, pseudostem and corm were taken from the 2: Isolated points of discoloration in naturally infected field. They were then cut vascular tissue, 3: Discoloration of up to into small pieces and used in the study. 1/3 of vascular tissue, 4: Discolouration Three month-old suckers of cv Rasthali of between 1/3 and 2/3 of vascular tissue, were planted in tanks (90 x 60 x 45 cm). 5: Discoloration greater than 2/3 of vascular Each tank contains 10 suckers and was tissue, 6: Total discoloration of vascular treated as a replicate, of which there were tissue. The per cent vascular discoloration 4 per treatment- representing 40 suckers per index was worked out using Mc Kinney’s treatment- arranged in randomized blocks. (1923) formula described above. The tanks were kept in a screened house, regularly watered and constantly observed Results and discussion for disease symptoms. Five isolates of Foc were isolated from Three months after the initiation of the diseased suckers and used for this study. experiment, the incidence of wilt and After three months, corm injection (T3) had vascular discoloration were assessed. The induced the highest per cent wilt index, wilt incidence was assessed using the scale followed by sucker dipping (T4) (Table 1). described by Ploetz et al. (1999): The highest vascular discoloration incidence 1: Healthy, 2: Slight cholorosis and was also recorded in T3 and T4 (Table 2). wilting with no petiole buckling, 3: Moderate Of the isolates, Foc 3 produced the highest chlorosis and wilting with some petiole wilt and vascular discoloration index in the buckling and or splitting of leaf bases, plants. Earlier, Stover (1959) found that 4: Severe chlorosis, severe wilting, petiole banana plants showed wilting after three buckling and dwarfing of the newly emerged months of pathogen inoculations. Hadi et al. leaf, 5: dead. (1987) observed that inoculation of banana The per cent wilt index was worked out roots with Foc induced the lesions after one using Mc Kinney’s (1923) formula: week and mechanical punctures allowed the pathogen to colonize the cells in the cortex Total sum of numerical ratings x 100 causing reddish brown lesions. Sivamani and Total number of plants observed x Gnanamanickam (1998) reported that a 10% Maximum category value in the scale rice/sand mixture inoculum of Foc showed severe internal discoloration in corm after The plant was pulled out and vascular three to four weeks of pathogen inoculation. discoloration was identified by cutting of In our study, corm injection of the pathogen suckers horizontally. The percentage of the may have facilitated internal colonization, vascular discoloration was worked out by the and soil application of the pathogen created scale described by Orjeda (1998): 1: Corm initial establishment which permitted the

Table 1. Virulence of F. oxysporum f.sp. cubense isolates assesed on per cent wilt index. Treatments Per cent wilt index of isolate* Foc 1 Foc 2 Foc 3 Foc 4 Foc 5 T1 Sand/maize inoculum of the isolate at 10% w/v of tank soi 26.67(31.09) 35.00(36.27) 51.67(45.96) 40.00(39.23) 33.33(25.26) T2 Sand/maize inoculum of the isolate at 15% w/v of tank soil 33.33(35.26) 41.67(40.21) 54.67(45.96) 36.67(37.27) 35.00(36.27) T3 Corm injection of the isolate at 3 ml/plant + sand/ maize inoculum of the isolate at 10% w/v of tank soil 43.33(41.16) 45.00(42.13) 60.00(50.76) 55.00(47.87) 45.00(42.13) T4 Sucker dipping for 30 min (106 cfu/ml) + sand/maize inoculum of the isolate at 10% w/v of tank soil 31.67(34.24) 41.67(40.21) 52.06(46.18) 51.60(45.91) 43.33(41.16) T5 Sucker dipping for 30 min (106 cfu/ml) 38.33(35.28) 35.00(36.27) 41.67(40.19) 35.00(36.27) 35.00(36.27) T6 Infected plant parts at 10% w/v of tank soil 33.35(35.27) 38.75(38.49) 43.33(41.17 30.00(33.21) 34.43(32.27) T7 Infected soil (105 cfu/g of soil) at 10% w/v of soil 36.67(37.27) 27.50(31.63) 40.00(39.23) 31.67(34.24) 35.06(36.27) Control 20.00(26.56) 20.00(26.56) 20.00(26.56) 20.00(26.56) 20.00(26.56)

* Mean of four replications, each replication contains ten plants Values in parenthesis are arc sine transformed values Critical difference value Methods: 1.42 Isolates: 1.87 Methods x Isolates: 1.65

16 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 17 Table 2. Virulence of F. oxysporum f.sp. cubense isolates assesed on per cent vascular discoloration index Treatments Per cent vascular discoloration index in isolate * Foc 1 Foc 2 Foc 3 Foc 4 Foc 5 T1 Sand/maize inoculum of the isolate at 10% w/v of tank soil 21.60(27.69) 18.97(25.81) 26.13(30.74) 20.94(27.23) 26.68(31.10) T2 Sand/maize inoculum of the isolate at 15% w/v of tank soil 25.71(30.51) 29.37(32.82) 32.00(34.45) 32.23(34.58) 25.83(30.55) T3 Corm injection of the isolate at 3 ml/plant + sand/maize inoculum of the isolate at 10% w/v of tank soil 81.85(64.79) 69.09(56.23) 88.25(70.07) 63.61(52.90) 62.46(52.22) T4 Sucker dipping for 30 min (106 cfu/ml) + sand/maize inoculum of the isolate at 10% w/v of tank soil 63.20(52.60) 59.35(50.40) 66.32(54.53) 55.00(47.87) 55.92(48.40) T5 Sucker dipping for 30 min (106 cfu/ml) 54.96(47.85) 57.43(49.28) 41.24(39.95) 40.34(39.43) 37.57(37.67) T6 Infected plant parts at 10% w/v of tank soil 36.50(37.19) 30.17(33.32) 35.75(36.72) 30.17(33.32) 29.18(32.69) T7 Infected soil (105 cfu/g of soil) at 10 % w/v of soil 27.05(31.34) 26.38(30.90) 29.68(33.01) 25.49(30.32) 25.98(30.65) Control 16.67(24.09) 16.67(24.09) 16.67(24.09) 16.67(24.09) 16.67(24.09) * Mean of four replications, each replication contains ten plants Values in parenthesis are arc sine transformed values Critical difference value Methods: 1.56 Isolates 2.05 Methods x Isolates: 1.98 pathogen to colonize inside the cell system. Ploetz R.C., Haynes & A. Vazquez. 1999. Responses of new banana accessions in South Florida to Panama Hence, corm injection of spore suspension disease. Crop Protection 18(7):445-449. of the isolate at 3 ml/plant + soil application Ricker A.J. & R.S. Ricker. 1936. Introduction to research of sand inoculum of the isolate at 10% w/v of on plant diseases. Johns Swift Co. Mc., New York. 117pp. tank soil should prove to be a rapid method Sivamani E. & S.S. Gnanamanickam. 1988. Biological T. Saravanan works at the for testing the virulence. control of Fusarium oxysporum f. sp. cubense in Agricultural Research Station, banana by inoculation with Pseudomonas fluorescens. References Plant Soil 107(1): 3-9. Kovilpatti – 628 501, Hadi M.A.A., F. Fadel & A.I. Ghorab. 1987. Root rot of Stover R.H. 1959. A rapid and simple pathogenecity test Tamil Nadu, India, e-mail: bananas and its control in Egypt. Pp. 161: 171 in for detecting virulent clones of Fusarium oxysporum f. sp. cubense using seedlings of . [email protected] Proceedings of the Conference of the Agricultural Nature 184:1591-1592. Development Research, Cairo, Egypt. M. Muthusamy, E.G. Ebenezar Stover R.H. 1962. Fusarial wilt () and R. Bhaskaran work at the Orjeda G. 1998. Evaluation of Musa germplasm for of bananas and Musa species. Common Wealth Department of Plant Pathology, resistance to Sigatoka disease and Fusarium wilt. Mycological Institute, Kew, Surrey, UK, p 117. INIBAP Technical Guidelines 3. Inibap, Montpellier, Agricultural College and Sun E.J. & H.J. Su. 1984. Rapid method for determining France. p. 29. differential virulence of Fusarium oxysporum f.sp. Research Institute, Madurai Mc Kinney H.H. 1923. A new system of grading plant cubense using banana plantlets. Tropical Agriculture – 625 104, India diseases, Journal of Agricultural Research 26:195-218 (Trinidad) 61(1):7-8.

Musa nematodes in Kenya Distribution of plant-parasitic nematodes on Musa in Kenya K.V. Seshu Reddy, J.S. Prasad, P.R. Speijer, R.A. Sikora and D.L. Coyne

ananas (Musa spp.) are grown Michel’, which are an important component in many districts of Kenya. They of diets and household incomes (INIBAP Bcurrently cover an area of 40 000 1986). Banana production is faced by a ha, producing 510 000 tonnes (FAOstat number of constraints, although a complex 2006). In the higher western regions of of plant-parasitic nematodes, banana Kenya, the East African highland bananas weevil, poor agronomic practices, Fusarium (EAHB) (Musa AAA, ‘Matooke’ and ‘Mbidde’ wilt, black Sigatoka disease and poor soil cultivars) are most common, while in the fertility combine to adversely affect yields in central and eastern highlands and coastal Kenya (Nguthi 1996, Inzaule et al. 2005). areas, cultivars are the most The plant-parasitic nematodes previously popular, especially ‘Cavendish’ and ‘Gros reported from Musa in Kenya include

18 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 19 Pratylenchus goodeyi, Radopholus similis Materials and methods and Helicotylenchus multicinctus (Gichure In 1990 observations were made on the and Ondieki 1977, Inzaule et al. 2005). In nematode species profile on bananas in neighbouring , severe production Oyugis district. The country-wide survey was losses on EAHB, as a result of R. similis conducted in 1993 across thirteen primary and H. multicinctus infection, have been Musa growing districts (see Table 1). documented (Speijer et al. 1994, Speijer et Oyugis district survey al. 1999, Gowen et al. 2005). Pratylenchus In Oyugis district, six farms, each with a goodeyi is also common on Musa at minimum of ten mats/plant, aged between altitudes above 1400 masl in Uganda, 2-3 years, of each of the two cultivars while R. similis tends to be more important ‘Nakyetengu’ (Musa AAA, Matooke) and below 1400 masl (Kashaija et al. 1994). ‘Sukali Ndizi’ (Musa AB), were selected. H. multicinctus is highly prevalent across On each farm, five mats per were Uganda, but R. similis would appear to sampled at the base of the most advanced be responsible for the majority of the plant in each mat. All the roots from an damage caused by nematodes to Musa. excavation of 20 cm x 20 cm x 20 cm, made using a spade, were bulked by cultivar for With interest in Musa production increasing each farm and transported in a plastic bag in Kenya, surveys to establish nematode in a cool-box to ICIPE’s Mbita Point Field species profiles on Musa throughout the Station for processing. Samples were stored country were undertaken between 1990 overnight at 4°C in a fridge before being and 1993. Details of the cropping systems processed the following day. Roots were cut and management practices relating to the into ca. 1 cm segments, thoroughly mixed, farms visited during the current study have and 5 x 5 g sub-samples were randomly been presented previously, and included removed for nematode extraction. Sub- details of the distribution of Musa insect samples were macerated for 2 s using a pests and preliminary information on kitchen blender prior to extraction using a nematode observations (Prasad and Seshu modified Baermann method for 48 h (Hooper et al. 2005). The recovered nematodes were Reddy 2000). The current report, however, identified and counted in 3 x 2 ml aliquots provides specific details concerning removed from a 25 ml suspension for each nematodes, including from the preliminary sub-sample. In the case of high nematode (pre-survey) studies undertaken within densities, the suspension was diluted to farms in Oyugis district, in addition to data 50 ml or 75 ml. Mean nematode densities from the nationwide survey assessed by per farm per cultivar were determined and Musa group. presented per 100 g root fresh weight. In

Table 1. Plant-parasitic nematode densities on East African Highland bananas (Musa AAA, Matooke group) and exotic bananas (Musa AAA and ABB) in Kenya in 1993. Site Alt AAA-EA Exotic Hm Pg Pc Rs Roty Tr Mel Hm Pg Pc Rs Roty Tr Mel Western region Bungoma 1 575 2 988 12 050 0 0 0 0 600 3 750 4 667 0 0 0 0 0 Kakamega 1 495 0 39 000 0 0 0 0 0 0 39 000 0 0 0 0 0 Kisii 1 480 0 9 667 0 0 0 0 0 0 41 000 0 0 0 0 0 Vihiga 1 460 0 18 500 0 0 0 0 0 1 000 12 000 250 0 0 0 600 Siaya 1 400 15 000 3 000 0 0 0 0 0 15 000 0 0 0 0 0 8 000 Oyugis 1 400 390 55 680 50 195 103 4 470 0 0 66 0 0 983 Busia 1 160 15 000 0 0 0 5 000 0 10 500 250 3 000 0 0 5 000 0 0 Homa Bay 1 120 2 500 190 000 0 0 0 0 0 20 000 81 250 0 15 000 1 100 15 000 3 500 Central region Nyeri 1 640 0 40 125 0 0 0 0 0 2 000 21 083 250 0 0 0 400 Embu 1 480 0 18 750 0 0 0 0 0 0 0 0 0 0 0 0 Muranga 1 203 0 3 333 59 000 0 0 0 0 0 11 111 20 000 0 0 0 15 000 Coastal region Kwale 168 0 0 0 0 0 0 0 8 250 15 000 0 0 0 0 0 Kilifi 15 0 15 000 0 0 0 0 0 18 888 2 733 0 0 0 0 48 050 Hm: Helicotylenchus multicinctus, Pg: Pratylenchus goodeyi, Pc: Pratylenchus coffeae, Rs: Radopholus similis, Roty: Rotylenchus clavicuadatus, Tr: Trophurus n.sp. Mel: Meloidogyne sp.

18 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 19 order to assess within-site variability, three Results adjacent mats per cultivar were selected and sampled from a single farm, Silpa Odak, Oyugis District where root samples were collected and Considerable variation in nematode densities processed separately for each mat. between farms within Oyugis district was observed with P. goodeyi present at higher Nationwide survey densities than other nematodes (Table 2). In the country-wide survey, one farm per Radopholus similis was observed on three banana-producing district was selected of the six farms on ‘Nakyetengu’, but at randomly for sampling where both EAHB none on ‘Sukali Ndizi’, while Meloidogyne and exotic cultivars were present. Three spp. (second stage juveniles) were observed on ‘Sukali Ndizi’ on all farms, but on only plants per Musa type were randomly three farms on ‘Nakyetengu’. At the farm selected on each farm and assessed for of Silpa Odak nematode root densities nematode incidence. Mats were sampled differed (P<0.05) between mats for the same as in Oyugis district. Detached roots were cultivar, despite the fact that mats of similar bulked per cultivar per farm and transported age were sampled (Table 3). Although not to the laboratory at Mbita Point Field Station, subjected to analysis, the nematode species where they were held at 4°C in the fridge profile between cultivars appeared to vary until being processed within a few days. considerably, especially for P. goodeyi and Roots of each sample were chopped finely H. multicinctus. This apparent large variation (ca. 2-5 mm) and thoroughly mixed. therefore provided a basis for the separate Nematodes were extracted from a 10 g root sampling of exotic and EAHB cultivars in the sub-sample, removed from the whole sample country-wide survey. and placed directly on a modified Baermann Nationwide survey dish for ca. 48 h. Nematodes were identified The lesion nematode, P. goodeyi, was and counted as above. widespread on EAHB in most districts Nematode data were subjected to ANOVA surveyed, except Busia near the Ugandan using Ln(x+1) transformed densities with border and at Kwale at the coast (Table 1). In the SAS statistical package. Data were both the low altitude coastal districts of Kwale presented as nematodes per 100 g root prior and Kilifi however, P. goodeyi was observed to transformation. on exotic bananas. The presence of R. similis

Table 2. Plant-parasitic nematode densities occurring on the cultivars ‘Nakyetengu’ (Musa AAA, Matooke group) and ‘Sukali Ndizi’ (Musa AB) at six neighbouring farmer fields in Oyugis district, Kenya. ‘Nakyetengu’ ‘Sukali Ndizi’ Farmer Rs Hm Pg Mel Rs Hm Pg Mel Silpa Odak 381a 1174a 255b 84a 0a 651a 119b 618b Ms. Ongewach 254a 540ab 529b 116a 0a 0b 115b 2398a Daniel Koyi 253a 540ab 529b 115a 0a 0b 115b 2503a Joseph Ogembo 0 b 0b 166144a 39a 0a 2b 125b 1344ab Rose Adel 0b 83b 139743a 82a 0a 620a 23588a 21b Daniel Ogembo 0b 0b 26906a 735a 0a 0b 2864a 42b Rs: Radopholus similis, Hm: Helicotylenchus multicinctus, Pg: Pratylenchus goodeyi, Mel: Meloidogyne sp.; Numbers in a column followed by the same letter are not significantly different (P < 0.05) following analysis using ln(x+1) transformed nematode data; pre-transformed data presented; n=5 per cultivar per farm

Table 3. Nematode species densities per 100 g root on the cultivars ‘Nakyetengu’ (Musa AAA, Matooke group) and ‘Sukali Ndizi’ (Musa AB) on adjacent mats in a single farmer’s field (Silpa Odaka) in Oyugis district, Kenya. ‘Nakyetengu’ ‘Sukali Ndizi’ Rs Hm Pg Mel Rs Hm Pg Mel mat 1 41 412a 1457 b 123a 115 a 0a 0 c 2503 a mat 2 530 254ab 540 b 116a 0 b 0a 585 a 48 b mat 3 207 46 b 10107 a 230a 0 b 0a 125 b 35 b Pg: Pratylenchus goodeyi, Rs: Radopholus similis, Hm: Helicotylenchus multicinctus, Mel: Meloidogyne sp.; numbers in a column followed by the same letter are not significantly different (P < 0.05) following analysis using ln(x+1) transformed nematode data; pre-transformed data presented.

20 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 21 was limited to Oyugis and Homa Bay districts Given the damage potential observed from only. In Kwale and Kilifi H. multicinctus and studies on plantain in (Speijer in Kilifi Meloidogyne spp., were recorded et al. 2001, Brentu et al. 2004), P. coffeae only from exotic banana cultivars and not appearance on Musa in the region is a cause EAHB. In Muranga district P. coffeae was for concern, even though ‘P. coffeae’ appears recovered extensively from both EAHB and to be composed of a number of closely exotic banana and was also recorded on related species which may differ in their exotic bananas in Nyeri and Vihiga districts. pathogenic potential and host specificity (e.g. The nematodes Rotylenchus clavicaudatus, Duncan et al. 1999). Currently the effect of Scutellonema spp., Cricomema spp., the nematode on EAHB and dessert banana Xiphinema spp, Hemicycliophora spp. and is not known. Of additional interest is the an undescribed species of Trophurus were observation of P. goodeyi at the low altitude also observed infrequently. sites on the Kenyan coast. Pratylenchus goodeyi is usually found at higher altitudes Discussion (above 1400 masl in mainland Africa) where The most widespread nematode observed conditions are cooler (which it is less able attacking EAHB and exotic bananas in to survive) than at lower altitudes (Bridge Kenya in 1993 was P. goodeyi. It was et al. 1995). Its occurrence therefore on also found at the highest densities. the relatively hot coastal areas of Kenya is H. multicinctus was also prevalent, while unusual. With the exception of its occurrence R. similis, which is prevalent in neighbouring in the (Price 2000) where it is Uganda and Tanzania, was observed at a problem on commercial dessert bananas, relatively low densities and infrequently. this would appear to be a unique observation The relatively high density at which some of on mainland Africa, and perhaps an the nematode species was observed would indication of a heat-tolerant strain. likely correspond to levels suggesting they It is not known whether the P. coffeae in are causing production losses (Gowen et Muranga district is native or was introduced al. 2005). However, while there are good through imported material. Its presence data establishing the adverse effect of R. is of potential concern, however, while similis on Musa, the relation between P. information on its current distribution and goodeyi, Meloidogyne spp., H. multicinctus pathogenicity would be useful to the Kenyan and Musa is less clear, although information Musa industry. Similarly, the presence of relating to their damage potential is gradually P. goodeyi at the coast may possibly be a increasing (Walker et al. 1984, Barekye et al. result of introduction, and may indicate that 1999, Bridge 2000, Ssango et al. 2004, sampling in the current study may have taken Moens et al. 2005). There also appears place within a relatively short period after its to be a strong Musa genotype influence introduction on planting material, before when considering the association of these the nematode died out. It would therefore nematodes (Pinochet and Rowe 1978, be similarly useful to establish the current Hartman J., IITA, unpublished), although situation and, if necessary, take avoiding the highly pathogenic nature of P. coffeae action. The infrequent occurrence of other on Musa is becoming increasingly apparent nematode species is not viewed as a cause (Speijer et al. 2000, Sundararaju 2001, for any concern, although other species, Brentu et al. 2004). Of particular interest from such as Rotylenchus spp. are recognized the current study therefore, are the relatively as serious pests in some Musa production high densities of P. coffeae in Muranga, and areas (De Waele et al. 2000). its occurrence in Nyeri and Vihiga districts. The results of the current study reinforce the Previously P.coffeae was not observed on need for the assessment of introduced Musa Musa in Kenya (Gichure and Ondieki 1977), material against key pests such as P. coffeae but appears to be increasingly observed in as well as R. similis. It also reinforces the , albeit erratically. In North West need to restrict the movement of (potentially) Tanzania, for example, it was not recorded in infected material, and / or to improve the 1984 (Walker et al. 1984), but was soon after promotion and dissemination of information (Sikora et al. 1989), while it was not detected on the use of Musa sanitation practices and in Zanzibar in 1992 (Maas 1992), but occurred use of healthy planting material. The current frequently more recently (Rajab et al. 1999). study was undertaken some years ago,

20 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 21 and its publication has unfortunately been FAOstat. 2006. Food and Agriculture Organization of the delayed due to unforeseen circumstances. United Nations, Rome. Gichure E. & J.J. Ondieki. 1977. A survey of banana It remains however highly relevant, providing nematodes in Kenya. Zeitshchrift fur Pflanzenkrankeiten an outline of the plant-parasitic nematode und Pflanzenschutz 84:724-728. distribution on Musa in Kenya upon which to Gowen S.C., P. Quénéhervé & R. Fogain. 2005. Nematode parasites of bananas and plantains. Pp. 611-643 in guide future activities and studies to benefit Plant parasitic nematodes in subtropical and tropical the industry. agriculture, 2nd ed. (M. Luc, R.A. Sikora and J. Bridge, In our study, the high level of variation eds). CAB International, Wallingford, U.K. Hooper D.J., J. Hallman & S. Subbotin. 2005. Methods in nematode species’ profiles between for extraction, processing and detection of plant and sites and cultivars creates difficulty in soil nematodes. Pp. 53-86 in Plant parasitic nematodes assessing their importance to Musa in in subtropical and tropical agriculture, 2nd ed. (M. Luc, Kenya. However, with the relatively high R.A. Sikora and J. Bridge, eds). CAB International, Wallingford, U.K. densities observed and the wide variation INIBAP. 1986. Banana research in Eastern Africa. Proposal of nematode species, including some which for a regional research and development network. are associated with severe production loss INIBAP/86/3/001 rev. INIBAP, Montpellier, France. 106pp. to bananas in other countries, considerable Inzaule S.S.S., F. Kimani, S. Mwatuni & M. Makokha. 2005. losses due to nematodes are to be expected. Status of banana pests and diseases in Western Kenya. Screening of popular cultivars or those to be African Crop Science Proceedings 7:309-312. released in Kenya against such nematodes Kashaija I.N., P.R. Speijer, C.S. Gold & S.R. Gowen. 1994. Occurrence, distribution and abundance of plant parasitic is recommended. nematodes on bananas in Uganda. Preliminary results of a diagnostic survey. African Journal of Crop Science Acknowledgments 2:99-104. Dedicated to the memory of Paul Speijer. Maas P.W. 1992. Observations on plant parasitic nematodes in bananas and some other crops The authors wish to thank the Federal in Zanzibar. 12-26 July, 1992. IPO-DLO Report Ministry of Economic Cooperation (BMZ), No. 92-14, DLO. Research Institute for Plant Protection, K.V. Seshu Reddy – formerly Germany for its financial support to conduct Wageningen, The Netherlands. Moens T., M. Araya, R. Swennen & D. De Waele. researcher at the International this research. 2005. Screening of Musa cultivars for resistance to Centre of Insect Physiology Helicotylenchus multicinctus, Meloidogyne incognita, and Ecology (ICIPE) - P.O. References Pratylenchus coffeae and Radopholus similis. Barekye A., I.M. Kashaija, E. Adipala & W.K. Australasian Plant Pathology 34:299-309. Box 30772, Nairobi, Kenya, Tushemereirwe. 1999. Pathogenicity of Radopholus Nguthi F. 1996. Banana production and research in Kenya. his current address is 202 similis and Helicotylenchus multicinctus on bananas MusAfrica 12. K.K. Heights, Masab Tank, in Uganda. Pp. 319–326 in Mobilizing IPM for Pinochet J. & P.R. Rowe. 1978. Reaction of two banana Hyderabad-500 028, India. sustainable banana production in Africa, Proceedings cultivars to three different nematodes. Plant Disease of a workshop on banana IPM, Nelspruit, South Africa Reporter 152:727-729. J.S. Prasad - formerly 23-28 November 1998 (E.A. Frison, C.S.Gold, E.B. Prasad J.S. & K.V. Seshu Reddy. 2000. Pest problems of researcher at ICIPE, can Karamura and R.A. Sikora, eds). INIBAP, Montpelier, banana in Kenya: a survey report. Pest Management in now be contacted at the France. Horticultural Ecosystems 6:50-54. Brentu C.F., P.R. Speijer, K.R. Green, B.M.S. Hemeng, D. Directorate of Rice Research, Price N.S. 2000. The Biogeography of the Banana De Waele & D.L. Coyne. 2004. Micro-plot evaluation Nematodes Radopholus similis and Pratylenchus Rajendranagar, Hyderabad-500 of the yield reduction potential of Pratylenchus coffeae, goodeyi in Proceedings of the First International 030, India. Helicotylenchus multicinctus and Meloidogyne javanica Conference on Banana and Plantain for Africa, R.A. Sikora is a professor on plantain cv. Apantu-pa (Musa spp., AAB-group) in Kampala, Uganda, 14 -18 October 1996. (K. Craenen, Ghana. Nematology 6:455-462. R. Ortiz, E.B. Karamura & D.R. Vuylsteke, eds). Acta at the University of Bonn, Bridge J. 2000. Nematodes of bananas and plantains in Horticulturae 540:431-440. Nussalee 9, 5300 Bonn, Africa: research trends and management strategies Rajab K.A., S.S. Salim & P.R. Speijer. 1999. Plant- Germany. relating to the small-scale farmer. In Proceedings of the parasitic nematodes associated with Musa in Zanzibar. first international conference on banana and plantain D.L. Coyne (corresponding African Plant Protection 5:105-110. for Africa. Acta Horticulturae 540: (Craenen, K., R. Sikora R.A., N.D. Bafokazara, A.S.S. Mbwana, G.W. Oloo, author, e-mail: Ortiz, E.B. Karamura & D.R. Vuylsteke, eds). Acta B. Uruno & K.V. Seshu Reddy. 1989. Interrelationships [email protected]) works at Horticulturae 540:391-408. between banana weevil, root lesion nematode and the International Institute of Bridge J., N.S. Price & P. Kofi. 1995. Plant parasitic agronomic practices and their importance for banana nematodes of plantain and other crops in Cameroon, decline in Tanzania. FAO Plant Protection Bulletin 37(4): Tropical Agriculture (IITA) – P.O. West Africa. Fundamental and Applied Nematology 18: 151-157. Box 7878, Kampala, Uganda 251-260. Speijer P.R., C.S. Gold, E.B. Karamura & I.N. Kashaija. - correspondence address: De Waele D., S.R. Gowen, M. Tessera & A.J. Quimio. 1994. Assessment of nematode damage in East African c/o Lambourn & Co., Carolyn 2000. Other Nematodes. Pp. 314–315 in Diseases Highland banana systems. Pp. 191-203 in Banana of bananas, abacá and enset (D.R. Jones, ed.). CAB nematodes and weevil borers in Asia and the Pacific: House, 26 Dingwall Road, International, Wallingford, UK. Proceedings of a conference/workshop on nematodes Croydon CR9 3EE . Duncan L.W., R.N. Inserra, W.K. Thomas, D. Dunn, I. and weevil borer affecting banana in Asia and the Pacific, Paul Speijer also worked Mustika, M.L. Frisee, L.M. Mendes, K. Morris & D.T. Kuala Lumpur, Malaysia, April 18-22, 1994, Los Baños, Kaplan. 1999. Molecular and morphological analysis Laguna, Philippines, (R.V. Valmayor, R.G. Davide, J.M. at IITA-Uganda. of isolates of Pratylenchus coffeae and closely related Stanton, N.L. Treverrow and V.N. Roa, eds). INIBAP/ He died on 30 January 2000. species. Nematropica 29:61-80. ASPNET, Los Baños, Philippines.

22 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 23 Speijer P.R., C. Kajumba & F. Ssango. 1999. East African Karamura and D.R. Vuylsteke, eds). Acta Horticulturae highland banana production as influenced by nematodes 540:225-232. and crop management in Uganda. International Journal Ssango F., P.R. Speijer, D.L. Coyne & D. De Waele, of Pest Management 45:41-49. 2004. Path Analysis: a novel approach to determine Speijer P.R., M.O. Rotimi & D. De Waele. 2001. Plant the contribution of nematode damage to East African parasitic nematodes associated with plantain (Musa Highland banana (Musa spp., AAA) yield loss under two crop management practices in Uganda. Field spp., AAB-group) in Southern Nigeria and their relative Crops Research 90:177-187. importance compared to other biotic constraints. Sundararaju P. 2001. Research on nematodes at Nematology 3:423-436. the National Research Centre for banana in India. Speijer P.R., F. Ssango & D. Vuylsteke. 2000. Evaluation of INFOMUSA 10:16-18. host plant response to nematodes in Musa germplasm Walker P.Y., M.J. Hebblethwaite & J. Bridge, 1984. Project in Uganda in Proceedings of the First International for Banana Pest Control and Improvement in Tanzania. Conference on Banana and Plantain for Africa, Kampala, EEC Report for the Government of Tanzania. London. Uganda, 14-18 October 1996 (Craenen K., R. Ortiz, E.B. TDRI. 129pp.

Assessment of the effect of commercial mycorrhizal Fungi promote growth fungi products on the growth of banana plants of bananas in the nursery A.S. Rodríguez-Romero and M.C. Jaizme-Vega

rbuscular mycorrhizal fungi (AMF) represents the biggest difficulty facing the are endophytic and biotrophic necessary transfer of this biotechnology Amutualistic symbionts that colonize from the scientific field to the agricultural most crops’ roots. Mycorrhization increases sector. The increasing number of small the capacity to absorb certain mineral and medium-sized businesses producing nutrients and is particularly efficient in the inoculants bears witness to the high assimilation of phosphorus. The mycorrhizal demand of this kind of product. Its publicity infection causes physical, biochemical and is based on conceiving AMF as a guarantee physiological changes in the colonized of healthy and beneficial products of natural roots that lead to improvements in the origin (von Alten et al. 2002). Aspects such general condition of the plant and help as the content of nutrients of the inoculant, to alleviate biotic and/or abiotic stresses its medium, pH and recommended doses (Barea et al. 2004). The benefits from are part of the information that must be inoculating micropropagated banana plants provided to consumers (von Alten et al. with AMF during the first stages of growth 2002). However, the density of propagules have already been clearly proved (Yano- (quantification of infective units) or the Melo et al. 1999). The beneficial effects of concentration of spores constitute quality early mycorrhization of this crop have even criteria for these products. However, the been detected one year after inoculation in quality control of AMF-based products is plants that have been in the field for nine so far carried out only by the companies months (Jaizme-Vega et al. 2002). themselves, there being no public Since AMF are obligate symbionts, it regulations on such checks (von Alten et is impossible to multiply and reproduce al. 2002). them in the absence of live roots, so This experiment was undertaken with the this has been a serious limitation to the purpose of comparing the effectiveness production of inocula on a large scale. of three AMF-based products. At the first Nevertheless, over the last few years, stage, the optimum dose of each product several substrata and methods have been was determined on the basis of growth- described for the production of inocula for related and economic criteria (amount of horticulture, fruitgrowing and forestry (von product consumed). Once the optimum Alten et al. 2002). Today, the optimization dose for each formulation was established, and commercialization of arbuscular a comparative study was done to determine mycorrhizal inoculants on a large scale the effectiveness of the products.

22 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 23 Materials and methods was also a control treatment (no application), The experiment was carried out in the Plant also consisting of 10 replications. The multi-pot trays were put into the protection laboratory of the Instituto Canario greenhouse under black mesh and plastic, de Investigaciones Agrarias in La Laguna, and at controlled temperature (25ºC) and Tenerife, Canary Islands during 2005. The humidity (90%). The plants were irrigated material used was micropropagated Musa with distilled water (50-75 ml/plant, acuminata Colla (AAA) cv. Petite Naine depending on their needs). The stage of (Dwarf Cavendish) and cv. Gruesa (a acclimatization ended eight weeks after commercial cultivar widely used in Canary the trial setup. At this time, the plants were Islands). The plants, which were 7 1 cm ± moved into individual 2-liter pots of black tall and had 2-3 fully grown leaves, were polyethylene with a steam sterilized medium received on an agar medium in a glass consisting of equal parts of soil, volcanic bottle under aseptic conditions. They were ash and peat (TKS1Instant). Each pot was carefully taken out of the bottles to wash the fertilized with 0.5 g of Osmocote 17-10-10, roots and eliminate the remains of the agar. a slow-release mineral fertilizer. The roots were cut, leaving 0.5-1 cm long The test lasted for another eight weeks ends. Then, the plants were transplanted to after transplanting into the individual-pot black polyethylene multi-pot trays, each of stage, after which time the plants were which contained 15 cells of 250 ml. The cells taken out of the pots and analysed to were filled with a steam-sterilized substrate determine the percentage of mycorrhizal consisting of a mix of equal parts of soil, colonization of their roots (Koske and volcanic ash and peat (TKS1 Instant, Gemma 1989) and the following variables Floragard, Germany). The AMF products associated with growth response: fresh were applied at the time of transplanting. weight of the roots and aerial parts (g), The Table 1 below shows the characteristics pseudostem and root length (cm) and leaf of the products and the doses. area, which was measured with a Li-3100 The inoculation was carried out by adding area meter (LI-COR, USA). the amount of product volume determined Since the inoculants and doses were for each dose right underneath the plants. evaluated in the same test, the data The decision on the doses to apply was were grouped in two different ways: a) made after consulting the manufacturers. comparison between different doses of The intermediate dose of each formulation the same inoculant and selection of the was that recommended by the growers. optimum dose of each; b) comparison This was the starting dose, which was then between the three inoculants. The data increased or decreased, depending on were statistically analysed through ANOVA the experimental conditions, with special and the averages were compared through attention being paid to the volume of Tukey’s multiple range test (P≤0.050). All substrate. Each treatment (dose and type this was performed with Systat 7.0 software of inoculum) was replicated 10 times. There (SPSS Inc., Chicago, USA).

Table 1. Relation between products and doses used in the test. Manufacturer Product Composition Substrate Dose Code Comercial (mL) Biorize Endorize Mix Glomus Tuslane & Tuslane sp.1 Granular 5 EM I (France) Glomus Tuslane & Tuslane sp. 2 inoculant, sand, 10 EM II Glomus intraradices Schenck & Smith zeolite and clay 15 EM III Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe Plants Works Terra Vital* GlomusTuslane & Tuslane spp. Granular 12 TV I (United Kingdom) Ectomicorrizas inoculant 25 TV II Bioaditivos (algae, Mg) TV II 40 Tritón TRI TON* Glomus etunicatum Becker & Expanded clay 10 T I (Germany) Gedermann 15 T II Glomus fasciculatum (Thaxter) 20 T III Gerd. & Trappe emend. Walker & Koske Glomus intraradices Schenck & Smith * Note from the editor: The trade name of these two products changed recently. ‘Terra Vital’ is currently known as ‘Rootgrow’ and ‘TRI TON’ MYCOSYM-TRI TON’.

24 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 25 Results production of the plants inoculated with dose I outperformed that of those plants Selection of the optimum dose for that received 25 and 40 ml. However, each product the differences between doses were not Effects on plant growth significant in statistical terms, in spite of Each inoculant behaved in a different which dose I was selected as the optimum way depending on the dose used, there one, since the highest values were recorded being no evidence of a direct relationship with it for most of the variables studied. between dose and effect. Although all the Furthermore, economic criteria were products and doses tested resulted in a considered in the selection of such dose. better performance than that of the control The study of TRI TON revealed that there plants, such improvement was not always were no significant differences between significant for each and every one of the doses for 4 out of the 5 variables of growth growth variables measured. (Table 4). Despite that, dose I (10 ml) seems The positive effect of Endorize Mix was to be the most effective and economically significant for dose II (Table 2). The highest attractive. values of the five variables measured were Mycorrhizal colonization recorded with this dose. Doses I and III did The mycorrhizal colonization of the root not cause a significant a priori increase in turned out to be another indicator of growth. This fact, considered together with the differing behaviours of the products the amount of product used, led us to select (Figure 1). In the case of Endorize Mix, the dose II as the optimum one. percentage of colonization increase did not In the case of Terra Vital, and evaluating surpass 35%, there being a direct correlation all the variables combined, it was the lowest between dose and colonization. Terra Vital dose (dose I = 12ml) that had the maximum showed the most variable results: whereas effect on the plants (Table 3). The biomass the highest dose gave rise to an increase

Table 2. Effect of three doses of Endorize Mix on micropropagated banana 16 weeks after application. Treatment Fresh weight Length Leaf area (g) (cm) (cm2) Aerial part Root Pseudostem Root Control 68,61b* 33,32 b 19,70b 24,05b 974,08b EM I (5mL) 92,70a 37,11 ab 22,30a 22,95ab 1352,79a EM II (10mL) 96,41a 41,34 a 22,65a 26,15a 1352,15a EM III (15mL) 81,53ab 35,25 a 22,15a 20,05ab 1186,37ab * N=10. The differences between the figures of a column followed by the same letter are not statistically significant according to Turkey mutiple range test (P≤0,050).

Table 3. Effect of three doses of Terra Vital on micropropagated banana 16 weeks after application. Treatment Fresh weight Length Leaf area (g) (cm) (cm2) Aerial part Root Pseudostem Root Control 68,61b* 33,32 b 19,70b 24,05a 974,08c TV I (12mL) 89,08a 37,33 a 23,25a 19,90b 1241,99ab TV II (25mL) 75,54ab 33,19 b 20,60b 23,45ab 1084,90bc TV III (40mL) 85,93a 38,42 a 21,10ab 23,35ab 1317,96a * N=10. The differences between the figures of a column followed by the same letter are not statistically significant according to Turkey mutiple range test

Table 4. Effect of three doses of TRI TON on micropropagated banana 16 weeks after application. Treatment Fresh weight Length Leaf area (g) (cm) (cm2) Aerial part Root Pseudostem Root Control 68,61b* 33,32 b 19,70b 24,05a 974,08c T I (10mL) 79,42a 40,03 a 21,10a 20,95b 1086,54a T II (15mL) 74,46a 32,58 a 19,00a 21,95b 1208,97a T III (20mL) 75,29a 32,14 a 19,80a 26,12a 1117,60a * N=10. The differences between the figures of a column followed by the same letter are not statistically significant according to Turkey mutiple range test (P≤0,050).

24 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 25 2 % more than the control plants (974.08 cm ), 100 whereas Terra Vital produced an increase of 27%, with 1241.98 cm2. Although TRI TON generated an increase in comparison 80 to the control plants, the increase was not significant. The root colonization by AMF at 60 this second stage of the test is represented in Figure 4. Despite the difference in absolute values between products, the percentage 40 increases in mycorrhizal colonization were not significantly different in statistical terms. 20 g cm 150 25 0 EM I EM II EM III TV I TV II TV III T I T II T III 120 20

90 15 Figure 1. Percentage of mycorrhizal colonization of up to 63%, doses I and II generated for each product and dose at the end of the increases of 29 and 23%, respectively. In 60 10 experiment. this case, the data turned out clearly divided 30 5 EM I: Endorize Mix (5 mL) into two statistical groups. As for TRI TON, EM II: Endorize Mix (10 mL) 0 0 Control Endorize Mix Terra Vita I TRITON EM III: Endorize Mix (15 mL) it showed a similar behaviour to that of Endorize, with a direct relation between root Fresh weight Plant TV I: Terra Vital (12 mL) of aerial part height TV II: Terra Vital (25 mL) colonization and doses, as well as significant TV III: Terra Vital (40 mL) differences between levels I and III (23 and Figure 2. Effect of the products on plant growth: fresh weight of aerial part and length of pseudostem. T I: TRITON (10 mL) 48%, respectively). The combined results T II: TRITON (15 mL) concerning mycorrhizal colonization and T III: TRITON (20 mL) cm2 plant growth made it possible to confirm 1600 the selection of the optimum dose for each product. The doses selected for the next 1200 stage of the study were 10 ml for Endorize Mix and TRI TON and 12 ml for Terra Vital. 800 Comparative assessment of the three products 400 The data obtained show that Endorize Mix 0 had a significant advantage over the other Control Endorize Mix Terra Vita I TRITON two products for most of the growth variables Figure 3. Effect of the products on plant growth: leaf area. measured. Thus, the plants treated with it had higher growth of aerial biomass, which, with 96.41 g, outweighed the control group (68.61 g) by 40%. Terra Vital generated the % second most significant growth, with 89.08 g, 30 that is, higher than the control group by 30%. As for TRI TON, it only increased the aerial 20 biomass by 15%. The growth estimated in terms of plant height repeated this trend, 10 with some variations (Figure 2). Here, Terra 0 Vital proved to be the most effective, with EM II TV I T I a significant increase in height of 18% in Figure 4. Percentage of mycorrhizal colonization for the three comparison to the control plants. Endorize products at the optimum dose selected. Mix increased the plant height by 15%. Unlike TRI TON, these two treatments were statistically grouped together. Regarding Discussion impact on the leaf area, this variable showed Although references on the use of similar results (Figure 3). Here, Endorize Mix commercial mycorrhizal inoculants for was the most effective, with 1352.15 cm2, 39% nursery production of plants of agronomic

26 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 27 interest are not abundant, they generally The results of this experiment demonstrate describe positive results in terms of increases the benefits from the inoculation of banana in growth and biomass (Lovato et al. 1992, during the post vitro stage. The use of Niemira et al. 1995, Busch and Lelley commercial mycorrhizal products could help 1997, Bourrain et al. 1999, Corkidi et al. to simplify and optimize the utilization of this 2005) and/or the rates of survival during the biotechnology in nurseries, with favourable acclimatization of micropropagated material effects on the quality of the vegetal material. (Bourrain et al. 1999). Furthermore, field However, an initial assessment of the testing of ecological production of strawberry products should be made in order to adjust showed an increase in the harvest after the application of diverse mycorrhizal inoculants, the doses and types of inoculants to the even though the yield within the categories of specific conditions of each situation. commercially produced fruit was not affected References (Bull et al. 2005). Some of these findings Barea J.M., R. Azcon & C. Azcon-Aguilar. 2004. Mycorrhizal indicate – as does this test – the variability fungi and plant growth promoting rhizobacteria. Pp. of the responses, which depend on both 351-372 in Plant Surface Microbiology (A. Varma, L. the product and the cultivar (Bull et al. Abbott, D. Werner, R. Hampp, eds). Springer-Verlag, 2005, Corkidi et al. 2005). Therefore, most Germany. authors propose conducting preliminary Bourrain L., J.C. Navatel, C.B. Blal & J. Parat. 1999. Les mycorrhizes: des champignons symbiotiques en aide tests that make it possible to select the aux pépinièristes. Infos Paris 154: 36-39. most appropriate inoculant for each specific Bull C.T., J. Muramoto, S.T. Koike, J. Leap, C. Shennan situation, this way ensuring a successful & P. Goldman. 2005. Strawberry cultivars and application (Busch and Lelley 1997, Bull et mycorrhizal inoculants evaluated in California organic al. 2005, Corkidi et al. 2005). However, the production fields. Crop Management doi:10.1094/CM- 2005-0527-02-RS. lack of correlation between infectivity and Busch E. & J.L. Lelley. 1997. Use of endomycorrhizal effectiveness of the fungi inoculated – as fungi for plant cultivation on buildings. Angewandte found in this experiment - was also detected Botanik 71: 50-53. in some of these studies (Bull et al. 2005). Corkidi, L., E.B. Allen, D. Merhaut, M.F. Allen, J. Downer, The results contributed by this experiment J. Bohn & M. Evans. 2005. Effectiveness of commercial allow us to confirm without any room for mycorrhizal inoculants on the growth of Liquidambar styraciflua in plant nursery conditions. Journal of doubt – even with the variability of response Environmental Horticulture 23 (2): 72-76 recorded – the positive effect of the products Jaizme-Vega M.C., M. Esquivel Delamo, P. Tenoury tested on the banana cultivar Petite Naine and Domínguez & A.S. Rodríguez-Romero. 2002. Effects the infective capacity of the fungi that these of mycorrhization on the development of two cultivars products contain. The species of mycorrhiza of micropropagated banana. INFOMUSA 11(1): 25-28. present in each product and their physico- Koske R.E. & J.H. Gemma. 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycological chemical support substrates influence this Research 92: 486-505. behavioural variability. The good results of Lovato P., J.P. Guillemin & S. Gianinazzi. 1992. Applications this test support the possibility of using AMF of commercial arbuscular endomycorrhizal fungal commercial products on a large scale during inoculants to the establishment of micropropagated the post vitro acclimatization of banana in grapevine rootstock and pineapple plants. Agronomie 12 (10): 873-880. nurseries, as indicated for other crops by Niemira B.A., G.R. Safir, V. Hammerschmidt & G.W. other authors (Bull et al. 2005, Corkidi et Bird. 1995. Production of prenuclear minitubers of al. 2005). This assertion is also supported potato with peat based arbuscular mycorrhizal fungal by the small amount of inoculant needed inoculum. Agronomy Journal 87 (5): 942-946. to increase growth and lower the average Von Alten H., B. Blal, J.C. Dodd, F. Feldmann & time of stay in the nursery, which directly M. Vosatka. 2002. Quality control of arbuscular mycorrhizal fungi inoculum in Europe. Pp. 281- Ana Sue Rodríguez- influences the commercial profitability of 296 in Mycorrhizal Technology in Agriculture: from Romero and María del the inoculants analysed. By publicising Genes to Bioproducts (S. Gianinazzi, H. Schüepp, Carmen Jaizme-Vega* work the guarantee of health and natural origin J.M. Barea & K. Haselwandter, eds). Birkhäuser at the Dpto. de Protección benefits for plants, the commercial brands Verlag, Switzerland. Vegetal, Instituto Canario de have serious possibilities of occupying a Yano-Melo A.M., J.S. Jr. Orivaldo, J.M. Lima-Filho, Investigaciones Agrarias, Apdo. N.F. Melo & L.C. Maia. 1999. Effect of arbuscular market in need of such products and still mycorrhizal fungi on the acclimatization of 60, 38200 La Laguna, Tenerife, lacking exhaustive regulations by the public micropropagated banana plantlets. Mycorrhiza 9 Spain authorities (von Alten et al. 2002). (2): 119-123 *E-mail: [email protected]

26 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 27 Focus on building knowledge An effective strategy for combating outbreaks of for BXW control banana Xanthomonas wilt Compiled by E. Karamura and V. Johnson

trengthening the capacity of the epidemic and restore productivity in the regional National Agricultural banana-based farming systems. “SResearch Systems (NARS) to The spread of the disease can be sustainably manage the outbreak of banana significantly arrested by increasing farmers’ Xanthomonas wilt in East and Central and other stakeholders’ knowledge and Africa” - a brief project report. skills for diagnosis, spread mechanisms The in East and Central and control measures (Molina 2006). Low Africa is a major contributor to regional food cost management technologies developed and income security. Since 2001, a new wave by research institutions needed to be of Xanthomonas wilt epidemics in bananas transferred to the affected farmers. There has become a serious threat to regional food was a need to involve more stakeholders security. Entire crop holdings have been along the production-consumption chains wiped out in areas where highly susceptible to help manage the disease in a coordinated banana cultivars were dominating farming regional strategy (Karamura et al. 2006). systems. The Banana Research Network The aims of the regional strategy were for Eastern and Southern Africa (BARNESA) boosted when the Catholic Relief Services appealed for resources to search for a (CRS) and the International Institute of solution to the problem. A Ugandan impact Tropical Agriculture (IITA) secured funds from assessment study estimated that up to USAID to execute the Crop Crisis Control US$ 4 billion could be lost by 2010 if no Project (C3P). This is an 18-month initiative measures were taken to arrest the epidemic to facilitate and intensify a coordinated fight (Karamura 2006).Subsequently, the Inter- against Xanthomonas wilt of bananas and national Network for the Improvement Cassava Mosaic Disease in six countries of of Banana and Plantains (INIBAP) (now Central and East Africa: Burundi; Democratic Bioversity International), in collaboration Republic of Congo (DRC); Kenya; Rwanda; with the Food and Agriculture Organization Tanzania and Uganda (See figure 1). (FAO) convened a regional stakeholders’ The CRS sub-contracted Bioversity to meeting in February, 2005 and developed strengthen the capacity of regional NARS to a strategy that envisioned multi-disciplinary, sustainably manage banana Xanthomonas multi-sectoral approaches in a coherent wilt in the countries participating in the C3P. regional response. This took account of the Bioversity, IITA and the NARS used the countries/regions that were Xanthomonas regional strategy developed in the framework wilt-free but threatened; the frontline areas of the BARNESA and the Association for This article is a synthesis of where the disease had just arrived; and Strengthening Agricultural Research in the results of the Crop Crisis the endemic areas where the disease was East and Central Africa (ASARECA) to Control Project (C3P) funded already established. It also sought to raise develop, test and disseminate Xanthomonas by USAID and executed by the awareness of all stakeholders along wilt diagnostic and management tools to the Catholic Relief Services the production-consumption chains and stakeholders along the banana production- together with the International to provide them with knowledge and skills consumption chains. Institute of Tropical Agriculture. for the diagnosis and management of the The specific objectives were: CRS subcontracted Bioversity disease. Importantly, it was realised that • To equip stakeholders with skills know- to strengthen the capacity phenologically wilt-resistant banana cultivars ledge/tools for sustainable management of of the regional NARS could still be infected via contaminated Xanthomonas wilt at the farm level; to sustainably manage pruning tools. • To raise public awareness of the disease the outbreak of Banana Hence, awareness-raising and associated threat and appropriate control measures Xanthomonas wilt in the C3P training were seen as an integral component • To develop, evaluate, and disseminate participating countries. of the intervention strategy to control the information materials to stakeholders

28 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 29 Central Africa Republic Cameroon

Uganda Congo Kenya Gabon Rwanda Dem. Republic of Congo Burundi Tanzania Project area

Angola Mozambique Zambia Malawi

Figure 1. C3P project area

• To mainstream information on the ecology project managers (CPMs) collaborated in and management of Xanthomonas wilt of this capacity building initiative bananas in field demonstrations; The ‘-peel‘ training concept adopted • To establish an early warning/surveillance a level of technical content appropriate system to facilitate timely response/actions to each successive training tier, with the against the Xanthomonas wilt epidemic; regional trainings having the most technical • To strengthen farmer capacity to introduce content. At regional and national level, and manage clean banana planting Training of Trainers (ToT) participants were material of farmer-selected varieties; given electronic and hard copies of all the • To evaluate on-farm the effectiveness tools used in the training, so they could of the Xanthomonas wilt of bananas produce or modify training materials as Diagnostic and Management tools; need arose. The farmer-trainings were more • To facilitate the drawing up of national visual demonstrations of symptoms and frameworks (Nation Action Plans) for the management techniques than theoretical control and management of Xanthomonas disease-management presentations. wilt of bananas and other banana pests Research and extension team leading and diseases. national level campaigns against • To strengthen NARS linkages with regional Xanthomonas wilt, were given specific (CIALCA, BARNESA) and international training aimed at developing responses platforms (Bioversity International, IITA, appropriate to the stage of the epidemic ARIs). (disease-free, frontline and endemic Trainers were trained at regional and regions) relative to crop phenology. country levels and community level Training accommodated for the needs trainings were back-stopped. The sub- and roles of farmers, extension providers project also deployed novel approaches and researchers and covered the use and for communicating its messages to target management of ‘clean’ planting materials. groups aimed at mass mobilization against Each country team had different frame- the disease. Diagnostic and management works for training, developed according tools and sensitization materials were to disease pressure and existing levels of developed, tested and disseminated. knowledge. In general, national level training National level training produced trainers of research, extension and policy managers for community-based trainers who then involved four components: trained farmers in disease recognition and 1. An overview of pest and diseases in management. banana-based cropping systems, of their The sub-project also developed com- impact on the banana industry and of the munication and public awareness tools, concomitant potential for improvement in and organized appropriate exchange visits regional livelihoods. for farming communities and/or extension 2. A description of Xanthomonas wilt in teams to gain field experiences and exploit bananas, its diagnosis, mechanisms of synergies between NARS. spread and its control, and a review of Finally, sub-project effectiveness and strategies to raise public awareness of the impact were evaluated. The IITA and country disease. Disease management strategies

28 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 29 targeted the eradication of disease pockets symptoms and control measures. It also and longer-term disease management. provided opportunity for cross-fertilization 3. Planning skills for sustainable integrated of ideas and experiences from other pest and disease control strategies, countries with similar activities. At the end including developing national action plans of the workshop, the back-stopping scientist and establishing farm demonstrations and facilitated a workshop review so that trainees other farmer-learning approaches. Field were equipped to address the needs of the visits included demonstrations of symp- next level. toms and control measures, and a At the start, different NARS had different participatory critique. levels of capacity to manage Xanthomonas 4. Visits to research centres working against wilt. To address capacity-gaps, the project wilt, and to private sector laboratories facilitated sharing cross-border information and technologies via research/extension/ involved in the production and distribution farmer teams exchange visits. Appropriate of banana planting materials. This approaches were designed for the different strengthened linkages and generated contexts: the Rwandese/Congolese teams information and technology updates. visited Uganda targeting farmer-empowering At the end of each tier training, trainees approaches; the Ugandan teams visited developed and presented a training Tanzania to learn about stakeholder mobi- programme for their respective target areas lization and to target policy makers; the and subsequently published their training Kenyans visited Uganda to learn skills for schedules to enable IITA and Bioversity raising public awareness. to backstop the country level training. The Communities including researchers, back-stopping support included providing extension, local councils, universities and Figure 2. BXW awareness-raising poster teaching tools, field demonstration of NGOs/CBOs, and their resources were mobilised to arrest the spread of any outbreak, by delivering information in form of seminars, meetings/workshops/barazas and as radio and television talk shows, in the quest to reach as many people as possible. Public awareness of the disease was raised via posters, pamphlets, brochures, talk shows, drama, workshops/seminars video documentaries and field days. The aim was also to attract more partners and extra resources, and to exploit comparative advantages of participating partners. In order to track progress and assess impact and effectiveness, participatory monitoring and evaluation tools (see below) were developed through Caritas-led farmer- workshops: • The two workshop locations were characterised by: representative banana cultivars and overall higher-capacity. • A manual was produced in English, French and other vernacular languages. • Posters (see Figure 2) and brochures were tested, suggested modifications incorporated into final versions and then translated. Each country received 1000 posters (Francophones in French and Anglophones in English), along with the respective electronic versions. This enabled countries to translate the tools into languages of the target sites.

30 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 31 For each of the six countries: • Two disease-stricken districts were selected in similar ecological and / or 100 94.4 equivalent geographical zones (12 districts 82.8 85.0* in all). • From each district, 2 X 25 km2 were 66.7 69.4* randomly selected (24 sites in all) • From each site, 4 farms were randomly selected.(96 farms in all) 50 38.9 All the sites selected had been exposed

to Xanthomonas wilt sensitization, training % with BXW tools and associated messages. Data were collected by extension staff manning the district and/or the sites as specified in table 1, and analyzed using 0 Yes No Yes No Yes No SPSS and means separated using a chi- De-budding Disinfecting tools Removing infected mats square test. Recommended BXW control measure * not statistically significantly different The training reached 51 400 people (7 times the original target size). This was a problem. This was attributed to the Figure 3. Effect of C3P - enhanced awareness partly because bananas and this threatening constraints of flaming in fire where building on BXW control levels disease are important to regional livelihoods, fires is restricted or the expense and scarity and partly due to the C3P mobilization. of bacterial products. Conversely, it was also observed that in a few sites farmers did not readily adopt In conclusion, strengthening the capacity recommendations that included high short- of stakeholders, especially at the grass-root term losses via plant destruction. level, significantly reduced Xanthomonas Training evaluation showed that farm wilt disease incidence on farm by 4-6 times. competencies gained were very high (60- Those applying the recommendations had 100%) for disease symptoms and control significantly higher levels of disease-free measures, but that farmers could not explain bananas than those that were not (see the various disease spread mechanisms. figure 3). Similarly in all countries, farmers were For effective control, all management effectively applying two of the first-line-of- options (cultural-biological and institutional defence recommendations (de-budding mechanisms) need to be applied at the and destruction of infected material) but same time. De-budding, destruction of sterilization of infected tools remained infected materials and the disinfection of

Table 1. Data collection in C3P project. Data type Data characteristics Capacity to distinguish between Xanthomonas wilt (XW) and Fusarium wilt Farmer’s ability • male-bud to recognise • bunch Xanthomonas wilt • within plant (sectioned banana fingers & pseudo- symptoms stems) • development (how XW enters and develops within Competence levels to recognise plants) and manage the disease • spread (plant to plant, mat to mat, location to Farmers’ knowledge location) on disease • management (de-budding; disinfection of tools and destruction of infected materials; setting up an efficient surveillance system, use of clean planting material and other phytosanitary measures. • XW task forces formed at different levels Numbers of: • participants trained • organizations participating in the fight against XW

Baseline data of disease incidence • When sites were established & distribution levels • 4-6 months later (on disease incidence on farm)

30 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 31 tools, as well as mobilization of stakeholders subsequently to local languages by the and monitoring and evaluating the NARS participating in the project implementation of control options were Two monitoring and review meetings were seen as the first lines of defence within the conducted in Kigali, Rwanda (July 2007) to overall management strategy against the evaluate management and diagnostic tools, Xanthomonas wilt of bananas (and Enset). and in Bujumbura, Burundi (December 2007) As a result of C3P project activities, the rate to discuss sub-projects outputs). The following of reported disease outbreaks in all countries recommendations arose: has gone down by about 20-40%, except in • to develop the best approaches for managing the northern part of the western rift valley, the disease more research should be which brings together DR Congo, Uganda, conducted on: and Rwanda. From here it is threatening to • host-vector-pathogen interactions; expand southwards into Burundi and western • developing user-friendly methods for Tanzanian region of Kigoma. More resources disinfecting tools at farm level; will be required to keep the disease out of the role of other pests and diseases in the northern Tanzania, central Kenya, and the • Indian Ocean islands and coastal regions. transmission of the wilt In order to raise awareness in those disease • pathogen longevity in soil and on -free but threatened banana-growing agro- decaying plant debris under field ecologies, the sub-project has prepared both conditions. electronic and hard-copy information tools, • To create a regional Xanthomonas key among which are: wilt coordination center where further BXW diagnostic and management manual developing mechanisms for regional that outlines the approaches that were surveillance and information exchange tested and used in the subproject to control would be facilitated. This would provide the disease, available both as hard copies for data management as a basis for and on the web (http://platforms.inibap.org/ consolidating national surveillance xanthomonaswilt/). methods and the formulation of regional Pest risk assessment (PRA) that was strategies against the disease. published with the collaboration of Central References Eldad Karamura is the Science Laboratory (UK) and which provides all available information on the disease and Karamura E., Kayobyo G., Blomme G., Benin S., Eden- Regional coordinator of the Green S.J. and Markham R. 2006. Impacts of XW banana and plantain research assesses the risks to the banana industry epidemic on the livelihoods of rural communities in group of Bioversity International in the region. The document is available as Uganda. Proceedings of the 4th International Bacterial for East and central Africa hard copies and on the web. Wilt Symposium. Central Science Laboratory, UK. in Kampala, Uganda and Management tools including posters, Molina A. 2006. Managing bacterial wilt/fruit rot disease pamphlets, brochures and audiovisual of banana in . Pp 26-31 in Developing Vincent Johnson works as a regional strategy to address the outbreak of banana scientific editor at Bioversity in material were produced and disseminated. Xanthomonas wilt in East and Central Africa. INIBAP, Montpellier. These were translated into French and Montpellier, France.

Thesis An Improved Agrobacterium-mediated transformation method for banana and plantain (Musa spp.) Geofrey Arinaitwe

PhD thesis submitted in February 2008* to production is continuously declining in Uganda the Katholieke Universiteit Leuven, Leuven, due to pests and diseases. A leaf defoliating Belgium. disease called black Sigatoka (BSD), is most Banana (commonly known as Matooke) destructive and reduces banana yield by 50% provides food for over eight million Ugandans. in susceptible cultivars. Resistance breeding More than half of Uganda’s population is for BSD, using conventional methods, is employed in its production process. Banana complicated by the crop’s biology and genetics.

32 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 33 Through the use of modern biotechnological clones was demonstrated. Transformation and tools, genes with potential to control BSD have integration of rice chitinase genes Cht-2 and been isolated. Cell and tissue culture techniques Cht-3 in banana are presented. Chitinases are also available. are enzymes that hydrolyze chitin, a major In the current study we compared an component of most fungi. We also evaluated existing Agrobacterium-mediated transfor- the possibility of introducing more than one mation method of banana with the particle gene and developed a fast method to detect bombardment-mediated transformation method of banana. Then we improved the multiple genes. Effects of the use of single Agrobacterium-mediated transformation versus two different selectable marker genes method by changing infection length, age are presented. This study is on-going as and volume of the embryogenic suspension. chitinase activity is analysed in the generated The effects of polyamine spermidine on transgenic lines and 26 lines are field tested in regenerability of transformed embryogenic cell Uganda.

Characterization and isolation of T-DNA tagged banana Thesis promoters active during in vitro regeneration and low temperature stress Efrén Germán Santos Ordóñez

PhD thesis submitted in April 2008* to the were analyzed for the T-DNA copy number Katholieke Universiteit Leuven, Leuven, by Southern hybridization. T-DNA inserts Belgium averaged 3.6 (range from 1 to 5) in 10 independent lines tested. DNA sequences A genome-wide T-DNA tagging strategy flanking the right and left T-DNA borders was pursued for the characterization and were isolated via TAIL-PCR and inverse isolation of novel banana promoters. PCR. Continuity of sequence between the Banana embryogenic cell suspensions were corresponding right and left border flanking transformed via Agrobacterium tumefaciens sequences was revealed by linking PCR containing a promoterless, codon-optimized using flanking sequence specific primers. luciferase (luc+) gene next to the right T-DNA RT-PCR analysis was performed in lines border. Approximately 89,000 transgenic with multiple inserts to identify and confirm banana cell colonies were screened for the sequence that activated luciferase luminescence two to three months after expression. Four candidate promoter transformation using a sophisticated camera sequences which were transcriptionally system in a light-tight box. Screening occurred fused to the luc+ were cloned upstream of a under controlled temperature conditions in uidAINT reporter gene and back-transformed which luminescence was monitored in real- to banana, which confirmed promoter activity time at 26°C followed by a gradual decrease for two sequences in in vitro cultures. to different low temperatures (LT) including Promoter activity in mature banana plants 18°C, 16°C, 12°C and 8°C. The frequency remains to be investigated. To summarize, of cell colonies showing luminescence at a high-throughput T-DNA tagging system 26°C and the different LT treatments ranged has enabled us to characterize and isolate from 0.17% to 2.69%. Transgenic cell novel banana promoters with potential colonies responsive to 8°C (i.e. showing an enhanced or repressed luciferase expression for a number of downstream applications pattern) were regenerated to plantlets and including banana improvement via genetic luminescence was monitored at different in modification. vitro developmental stages. With increasing differentiation the number of lines with LT * Note from the editor: The delayed printing of this last issue of INFOMUSA provided us the opportunity to add the up-regulated LUC expression decreased. abstracts of two theses submitted in 2008 that may be of great Banana plants showing luminescence interest for our readers.

32 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 33 Thesis Optimized proteomic methods to unravel biochemical processes in banana meristems during in vitro osmotic stress acclimation S. Carpentier

PhD thesis submitted in February 2007 separation via two-dimensional gel to the Katholieke Universiteit Leuven, electrophoresis and protein identification Leuven, Belgium. via tandem mass spectrometry (MS/MS) The Laboratory of Tropical Crop is the most informative approach for Improvement (KULeuven, Belgium) a poorly characterized organism like hosts, under the authority of Bioversity banana. Proteins are relatively well International, the global in vitro collection conserved, making the identification of of banana varieties (Musa and Ensete non-model gene products in comparison spp.). The aim of this international to well-known orthologous proteins quite genebank is to conserve all banana and efficient. Intact proteins (separated via plantain genetic resources safely and 2DE) are essential for a good and reliable to supply the germplasm to any bona identification. fide users. However, in vitro preserved Protein sample preparation is a germplasm needs regular sub-culturing, critical step and is absolutely essential creating the possibility of losses due to for obtaining good results. Most plant contamination and human error. Moreover, tissues are not a ready source for protein an important problem associated with extraction and need specific precautions. in vitro culture is the occurrence of The cell wall and the vacuole make up the somaclonal variation. KULeuven was one majority of the cell mass, with the cytosol of the pioneers to explore the possibilities representing only 1 to 2 % of the total of storing plant material in liquid nitrogen cell volume. Subsequently, plant tissues (at -196°C). However, for successful have a relatively low protein content storage at -196°C, cells need to survive compared to bacterial or animal tissues. first a severe dehydration process prior Banana is a good representative of a to freezing and then to regenerate recalcitrant plant species since it contains after thawing. In banana, meristems numerous interfering compounds. are the most suitable structures to be Banana contains extremely high levels of cryopreserved. Although meristems have oxidative enzymes (polyphenol oxidase), the potential to regenerate, they still need phenolic compounds (simple phenols to survive the dehydration associated with (dopa), flavonoids, condensed tannins, cryopreservation. In general, dehydration lignin), carbohydrates, terpenes, suberin, tolerance is achieved by a sucrose- and waxes. The majority of the plant mediated osmotic stress acclimation. protocols introduce a precipitation step to However, more than half of the collection concentrate the proteins and to separate consists of varieties that have a non- them from the interfering compounds. A existent or low cryopreservation survival phenol extraction protocol was optimized rate. Hence, there is a need to understand for small amounts of banana plant tissue the mechanisms behind acclimation (down to 50 mg fresh weight (FW)) and during the pre-culture of meristems on different sample preparation methods sucrose and to obtain insight into the were evaluated. Only TCA/acetone genotype-specific diversity. precipitation and phenol extraction Understanding gene function and gene methanol/ammonium acetate precipitation expression profiling can be approached are useful as standard methods for via several techniques. A concise recalcitrant plant tissue. The optimized overview of the existing technologies is phenol extraction was most efficient in given with the emphasis on proteomics removing interfering substances and and proteomic technologies. Protein resulted in the highest quality gels. This

34 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 35 method proved also to be useful for other an osmoprotective intracellular sucrose plant species like apple, pear and potato. concentration, the enhanced expression of Proteins can be identified by comparing particular genes of the energy-conserving the proteins of interest to orthologous glycolysis and the conservation of proteins of species that are well the cell wall integrity are essential to characterised. In one approach, peptide maintain homeostasis, to acclimate and mass fingerprints (PMF) and peptide to survive dehydration. By comparing the sequence information were combined to dehydration tolerant variety (ABB) with search and to correlate the un-interpreted a dehydration sensitive variety (AAAh), spectra with the theoretical product it was possible to distinguish several ion spectra from different databases genotype specific proteins (isoforms) in an automated way. However, and to associate the dehydration-tolerant some orthologous genes retain a low variety with proteins involved in energy percentage of identity and the chance of (e.g. phosphoglycerate finding significant and conserved peptides kinase, phosphoglucomutase, UGPase) decreases, so a PMF-MS/MS approach and proteins that are associated with might fail. A method that extracts stress adaptation (e.g. OSR40-like protein-identifying information directly protein, ASR). This work proves that from spectra without being dependent on proteome analysis is successful to existing databases is extremely useful. perform quantitative difference analysis However, deriving sequences from MS/ and to characterize genetic variations in a MS spectra is not straightforward. MALDI recalcitrant crop. TOF/TOF ion spectra contain in general To get a better insight into the incomplete ion series typically rich in acclimation process and to correlate various ion types. Attempts to simplify acclimation with cryopreservation, both the de novo identification rely mainly on a dynamic study (screening over time) the simplification of the fragmentation and a comparison between varieties pattern. The addition of a sulfonic acid were performed, with the focus on the group to the N-termini of tryptic peptides validation of candidate proteins. Although facilitates de novo peptide sequencing. the sucrose pre-treatment and the The strong acidic group at the N-terminus concomitant high intracellular sucrose is deprotonated and therefore neutralizes concentration are required for a low the positive charge of the b-ion by its mortality rate after cryopreservation, it also negative charge. This results in simple induces negative stress effects. Sucrose spectra displaying mainly y-ions series. pre-culture is correlated to high relative In this chapter, an innovative approach abundance of phosphoglycerate kinase, in which the extracted tryptic peptides UGPase and OSR40 and a low relative are N-terminal sulfonated without any abundance of SuSy, lectin and chitinase further sample purification is reported and and abscisic stress, ripening protein-like successfully applied to banana. It was protein (ASR). We hypothesise that a low demonstrated that this approach results mortality rate after cyopreservation can in a high and reliable identification rate be achieved by a low relative abundance and that it is adequate to identify different of SuSy and high relative abundance isoforms resulting from allelic variations, of phosphoglycerate kinase, OSR 40, or from different gene loci. UGPase, ASR and storage proteins such Banana is a unique model for the study as lectin and chitinase. A screening of of meristems thanks to the development of candidate proteins links several proteins the shoot meristem tip culture technique. and specific isoforms to the tolerant For the first time, a large scale study of a B genome, which opens perspectives meristematic proteome is reported. The to apply proteomics for taxonomical results suggest that the maintenance of characterization and classification.

34 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 35 Thesis Market access and agricultural production: The case of banana production in Uganda F. Bagamba

PhD thesis submitted in 2007 to and Sigatoka contribute to the low banana Wageningen University, The Netherlands. production in the central region. Farm size is positively related to farm The study analyses smallholder household productivity. However, production is more response to production constraints (crop efficient on smaller plots (decreasing returns pests and diseases, soil constraints) and the to scale). The low productivity on small development of product markets and off-farm farms leads to question the sustainability employment opportunities. It was carried out of smallholder agriculture, given the in the central region, Masaka and southwest, imperfections in labour and food markets which have different production constraints and limited access to purchased inputs. and opportunities. Findings from labour supply analysis show Results for the cost-benefit analysis show that farmers are responsive to economic that banana is the most profitable of all incentives. Access to off-farm opportunities the crops grown in terms of gross margin. takes away the most productive labour from However, imperfections in labour and farm production. Education and road access food markets cause farmers in the central tend to increase the time allocated to off- region to allocate more land and labour farm activities while farm size is negatively to the less profitable annual crops (sweet correlated to hours worked on off-farm potatoes, maize and cassava) which are activities. more satisfactory in terms of household The study reveals that policies that food requirements. High food prices and promote income diversification into off- limited off-farm opportunities induce farmers farm activities can contribute to sustained to rely on their own farm production for development in the rural sector. In particular, their household food needs. Results from policies that reduce transaction costs are the technical efficiency analysis show that likely to improve productivity and efficiency banana farmers in Uganda are technically in both the off-farm sector and farm sector. inefficient, and output can be increased Investment in road infrastructure, education by 30% in the southwest and 58% in the and financial institutions that are suited to central region. Improved roads, formal smallholder production needs could help in education, access to credit and extension alleviating the bottlenecks in the labour, food work are some of the factors that improve and financial markets, and improve resource productivity and technical efficiency. Results allocation between the farm and non-farm confirm that infestation by the banana weevil sectors.

Thesis Study of fungal endophytes as resistance inductors for black Sigatoka (Mycosphaerella fijiensis) control in plantain M. Barrios Murillo

MSc thesis submitted in 2006 to the banana and plantain crops around the world, Tropical Agricultural Research and causing enormous production losses and Higher Education Center - CATIE, requiring large agrochemical investments Turrialba, Costa Rica for its control. It is very important to develop Black Sigatoka is a foliar disease caused by alternative control methods to reduce Mycosphaerella fijiensis fungus. It attacks economic and environmental costs. The

36 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 37 objective of this study was to evaluate relationship), showed a slight unbalance colonization, growth promotion and black in the treatments evaluated (except the Sigatoka control in plantain tissue culture control), probably due to FE soil nutrient plantlets, inoculated with four Fungal uptake. In relation to black Sigatoka control, Endophytes (FE) isolate under controlled a series of comparative measurements using greenhouse conditions. Results showed the Fouré (1985) and Romero (2005) scales, highest FE colonization percentage in the T4 (Fusarium oxysporum isolate) was the roots, followed by the corm, pseudostem only treatment showing a significant delay and leaf. Trichoderma atroviride isolates (five days) in the development of disease lost their colonization power over time, symptoms compared to the control. There while Fusarium oxysporum strains gradually are already literature reports on resistance increased theirs. induction using FE inoculated into roots to Significant differences were found for control foliar diseases. Although the results growth promotion of leaf area (p<0.0001) obtained from this study do not demonstrate and pseudostem diameter (p<0.0093); the expected response with enough certainty, combined FE strain treatments had a positive it is recommended to continue working in this effect, attributed to synergic action by the direction, since this is the first research effort combined strains. Leaf analysis (Ca-Mg-K with FE conducted in plantain.

Evaluation of endophytic fungi and botanical extracts Thesis for black Sigatoka (Mycosphaerella fijiensis Morelet) control in banana G. Osorio

M.Sc thesis submitted in 2006 to the Sigatoka severity and incidence were Tropical Agricultural Research and evaluated using Fouré (1985) and Gauhl Higher Education Center - CATIE, (1989) scales and following Marin and Turrialba, Costa Rica Romero (1998) methodology. During the vegetative period, the following variables Black Sigatoka (Mycosphaerella fijiensis were recorded: State (ES), Morelet) causes important losses to Foliar Emission Rythm (FER), Total Leaves banana production. The use of fungicides (TL), Youngest Diseased Leaf (YDL) and has been the most efficient mean to control Infection Index (IND). At flowering, only this foliar disease. The development of innovative strategies to diminish one reading was done for TLF, YDLF and conventional chemical control dependency INDF, along with the following phenological is a permanent challenge for a sustainable variables: Period from Planting to Flowering and environmentally respectful agriculture. (PPF), Plant Height at Flowering (PHF) and The objective of this research was to Pseudostem Circumference at Flowering evaluate mutualist endophytic fungi and (PCF). Parallel to this, phytochemical botanical extracts to control black Sigatoka analysis of B1 and B2 were conducted to in the field. Banana (cv. Grand naine) determine main secondary metabolites. An vitroplants and corms inoculated separately ANOVA and a Duncan test were run using with Trichoderma atroviride strains (E1 the SAS system. E2) were utilized. These materials were The Area under the progress curve of planted in bags and acclimatized for three the disease (ABCPE, Spanish acronym) months before their establishment in the expressed in the ES and IND was field, under a completely randomized block calculated. Results at root level reveal design in split plots. Foliar applications of statistically significant differences (p<0.05) botanical extracts, Momordica charantia for ES and IND variables, outstanding E2 (B1) and Senna reticulata (B2) started six after Q, with low values in comparison months after planting in the field. Black to remaining treatments. Nevertheless,

36 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 37 there was no evidence of positive effects (polyphenols, coumarins, quinones, on the other analyzed variables. At foliar saponins, triterpenes, flavonoids, among level, there were statistically significant other), some of which have been reported differences (p<0.05) favoring B2 with a in the literature for their antifungal or less ABCPE for ES and less IND infection resistance induction activity. This research percentage. However, the chemical allowed to select B2 as “highly promising” treatment (chlorothalonil) was the most to be proposed as a candidate for banana effective control. Comparing the planting black Sigatoka integrated management due material type (“seeds”), vitroplants to its protectant action. However, it is not showed the best behavior regarding known which molecule exerts the disease severity, TL, YDL and APF. Significant control effect. If an opportunity to continue differences (p<0.05) were observed for some interactions, standing out TL and YDL with this research exists, it is recommended variables in the tri-factorial relationship: to isolate the active compound. Regarding seeds with roots treatment and the endophytic fungi, it is necessary to conduct chemical application at foliar level. In this more studies to optimize their efficiency case, seed with nematicide and fungicide with subsequent reinforcement application, interaction showed the best behavior for extending and inducing molecules disease protection. Phytochemical analysis liberation, which are intermediate signs of indicated a variety of secondary metabolites a possible systemic resistance.

MusaNews Banana bunchy top disease in Mozambique and Zambia W.T. Gondwe, B.M.L. Mwenebanda, E. Natha and P. Mutale

Banana bunchy top disease (BBTD) is Symptoms of the disease include dark considered as one of the most serious virus green spots along the leaf veins, midrib and disease affecting bananas worldwide (Thomas petiole, upright leaves with wavy margins, et al. 1994). Banana bunchy top virus (BBTV) stunted growth, more erect leaves giving the is disseminated through infected planting plant a rosetted or ‘bunchy top’ appearance (Jones 2000). materials. Within the field, the virus is transmitted BBTV symptoms on cv. Williams at Vila The disease has been reported and Ulongwe government banana nursery block. by a banana aphid, Pentalonia nigronervosa. confirmed in several Asian and Pacific islands as well as in African countries. In Africa, it has been reported in Egypt, Gabon, Congo, Democratic Republic of Congo, Rwanda, Burundi, Central African Republic, Malawi (Tushemereirwe and Bagabe 1998, Thomas and Iskra-Caruana 2000) and more recently in Angola (Pillay et al. 2005.). Recently symptoms of bunchy top disease were observed in the government banana nursery block at Vila Ulongwe (1239 masl, 14°32’ S and 33°41’ E) in Angonia district, Tete Province, western Mozambique and on a commercial banana farm at Chipata town (1108 masl, 13°37’ S and 32°37’ E) in Chipata district, eastern Zambia. The symptoms were observed in both giant and dwarf Cavendish cultivars, ‘Williams’ and ‘Kabuthu’ respectively. In Zambia,

38 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 39 both ‘Bluggoe’ and ‘’ which potential source of partial resistance to surrounded the farm BBTV. were not affected. This is the first reported case of BBTV in Cultivars of the Cavendish subgroup have been reported to be highly both Mozambique and Zambia. susceptible to BBTV (Thomas and Iskra- Caruana 2000). In BBTV hot spots in Malawi, Cavendish References bananas, other AAA bananas and some Geering Andrew. 2003. Banana bunchy top virus plantains (AAB) were first wiped out - screening for virus resistance. 3rd meeting of the before ABB cultivars. There was a slow PROMUSA Virology working group. PROMUSA symptoms appearance in the ABB cvs Newsletter10:8. such as ‘Bluggoe’ and ‘Pisang awak’ Jones D. 2000. Diseases of banana, abacá and enset. (Mwenebanda 1998, pers. observation). In Angola, the disease was spotted on CAB International, Wallingford, Oxon, UK. False horn plantains (AAB) and Cavendish Pillay, M., Blomme, G., Rodrigues, E. & A.L. Ferreira. bananas (Pillay et al. 2005). 2005. Presence of banana bunchy top virus in Angola. Tushemereirwe and Bagabe (1998) InfoMusa 14(1):44–45 reported that no study to establish yield Thomas J.E & M-L. Iskra-Caruana. 2000. Diseases losses due to BBTV has been reported caused by Viruses. Pp 241-253 in Diseases of banana, in Africa. However, it was reported abacá and enset (D.R. Jones, ed.). CAB international, that severely infected plants of highly Wallingford, Oxon, UK. susceptible cultivars fail to produce Thomas J.E, M-L. Iskra-Caruana & D.R. Jones. 1994. W.T. Gondwe and B.M.L. bunches resulting into 100% yield loss. Banana Bunchy Top Disease. Musa Disease Fact Sheet Mwenebanda work at Geering (2003) reported that there No. 4. International Network for the Improvement of IITA/SARRNET, Chitedze was partial resistance to BBTV in Banana and Plantain, Montpellier, France. Agricultural Research Station, various banana genotypes. The same P.O. Box 30258, Lilongwe Tushemereirwe W.K. & M. Bagabe. 1998. Review of reporter cited ‘Gros Michel’ as having a 3, Malawi, E. Natha at IITA/ low susceptibility to BBTV with delayed disease distribution and pest status in Africa Pp. 139- SARRNET, c/o Total Land symptom expression. He also stated that 147 in Mobilizing IPM for Sustainable banana production Care, Vila Ulongwe, Angonia, ‘Highgate’, a member of the ‘Gros Michel’ in Africa. Proceedings of a workshop on banana IPM Mozambique and P. Mufale at banana subgroup, is used by the breeding held in Nelspruit, South Africa- 23 – 28 November 1998. IITA/SARRNET, c/o Msekera programme of the Fundación Hondureña International Network for the Improvement of Banana and Agricultural Research Station, de Investigación Agrícola (FHIA) as a Plantain, Montpellier, France. Chipata, Zambia.

INIFAT 02: a new cooking-banana hybrid is born MusaNews in A. Rodríguez Nodals, A. Rodríguez Manzano and A. Rodríguez Manzano

In recent years, geneticists have produced were selected as parental material (Table 1), more disease-resistant banana and plantain and among them, Burro CEMSA ( ) and hybrids that have contributed to worldwide Pelipita (ABB) ( ) produced 508 seeds food security. This was favoured by the and (after discarding abnormals) in turn introduction of clones from the Fundación 84 seedlings for evaluation. The promising Hondureña de Investigación Agrícola (FHIA). clone INIFAT 02 was successfully produced In Cuba, important results have also been achieved, such as the clone Burro CEMSA from this cross, despite the triploid condition (ABB), highly resistant to Fusarium wilt and of both parents, due to their residual fertility to yellow Sigatoka and, to some extent, to and to the Musa phenomenon of unreduced black leaf streak (black Sigatoka). For a meiosis – or reduced though irregular further breeding programme, several clones meiosis

38 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 39 At the beginning of flowering, male flowers INIFAT 02 was found to contain two were selected from those clones where genomes of Musa balbisiana Colla and one good levels of fertility had been reported of Colla. (Simmonds 1966, Rowe and Rosales 1992, The following are some of INIFAT 02’s Román et al. 1990). The flowers were main agronomically valuable characteristics: rubbed against the stigma of the female • Shorter cycle than those of its parents flowers which opened in the morning. This under similar conditions. This clone was process was continually repeated as more harvested 270 days after planting, whereas flowers opened. Burro CEMSA and Pelipita were harvested Eleven types of crosses were performed at 300 and 395 days, respectively. whose pulp was extracted from ripe • Tall plants (3-5 m): height depends on such factors as climate, soil and management. Table 1. Names and genomic groups of the This clone is intermediate between its parents. parents under equal conditions. On a Parents typical red ferralitic soil, the average Paka (AA) Highgate (AAA) height was 3.05 m during the first harvest SH-3362 (AA) Burro CEMSA (ABB) Pelipita (ABB) Congo (AAA) cycle, whereas Burro CEMSA and Pelipita Pelipita (ABB) reached, on average, 3.50 and 2.90 m, Gros Michel (AAA) respectively. • Assessments eight months after planting bunches. Hybrids displaying undesirable showed that the average INIFAT 02 characteristics were eliminated (e.g. tiller-number was five per plant, whereas albino plants, broader-than-long or very Burro CEMSA and Pelipita produced, on thick leaves, or dwarfism (expressed as average, seven and three suckers per resetting). Bunches were selected with plant, respectively. notable agronomic value (e.g. appropriate • The bunches consist of 5-9 hands. Figure 1 finger length, high bunch weight, short or shows a clone grown in an urban patio. Finger-size was intermediate between medium cycle, and tolerance/resistance to • those of both parents (Figure 2). In the first black leaf streak). The selected clones’ hand, central finger average length was genomic groups were determined using 16 cm, whereas Burro CEMSA and Pelipita Simmonds and Shepherd’s principle (1955), averaged 13 and 20 cm, respectively. quoted by Simmonds (1966). From a genetic point of view it is interesting Physical characteristics and yield com- that the above characteristics show a type of ponents were described with international “intermediate” inheritability, except for the descriptors for the genus Musa. The triploid harvest precocity. genetic constitution was determined according • Like its parents, INIFAT 02 is highly to Simmonds’ morphogenetic principle. resistant to black leaf streak. It has shown Triploidy, common to most bananas and no wilting caused by Fusarium, which was plantains, confers some vigour which greatly, contributes to sterility (Tomekpé et al. 2004), therefore constraining varietal improvements via crossbreeding. Pelipita has lower female fertility than Burro CEMSA, generating the so-called “maternal effect.” Five clones were selected (INIFAT 01-05) for their good bunch constitution and other agronomic characteristics (see next column). INIFAT 02 was selected for displaying intermediate characteristics between its parents in most aspects of agronomic and culinary interest, and because of its elimination of Pelipita defects such as slow suckering, fruit hardness (when cooked Figure 1. Bunch of ‘INIFAT 02’ cultivated in urban patios in La green) and long growth cycle. Habana.

40 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 41 tested in nearby plantations of Cavendish and Plantain subgroups, both of which are highly susceptible to this fungus. • In culinary terms, it is suitable for being consumed green, half-ripe or ripe. In a test involving 50 tasters, 58%, 24% and 18% preferred, respectively, the “cooked hard-ripe”, “cooked green” and “cooked ripe” options. Once cooled, “cooked green” fruits are still soft and do not show the characteristic hardness of Pelipita. • The experimental yield (preliminary) was 18-27 and 21-33 kg per bunch in the first and second cycle, respectively, on a sample of 20 plants harvested. Conclusion behaviour of INIFAT 02 in those municipalities Figure 2. Size and format of the fingers A total of 84 seedlings were obtained of Cuba most affected by drought. of ‘INIFAT 02’. by the crossbreeding of Burro CEMSA x Pelipita; 28 of them were transplanted for References field testing, and 5 promising clones were Román M., M. J. Manzano & A. Rodríguez Nodals. 1990. selected. INIFAT 02 was the clone that Determinación de la fertilidad masculina en clones de plátano. Ciencia y Técnica en la Agricultura, Serie featured the best agronomic characteristics. Viandas, Hortalizas y Granos, La Habana, Cuba. INIFAT 02 is an ABB triploid and a hybrid Rowe P. & F. Rosales. 1992. Genetic improvement of between the subgroups Bluggoe and bananas, plantains and cooking bananas in FHIA, Honduras. Pp.243-266 in Breeding bananas and Pelipita, both of which are ABB. plantains: proceedings of an International Symposium INIFAT 02 features better culinary on Genetic Improvement of Bananas for their Resistance to Diseases and Pests (J. Ganry, ed.). properties than Pelipita, and is more Cirad-Flhor, Montpellier, France. precocious and gives a similar yield. Simmonds N.W. 1966. Bananas. 2nd ed. Longmans, For the first time a successful 3x/3x London. crossbreeding has been reported, which was Simmonds N.W. & K. Shepherd. 1955. The taxonomy and origins of the cultivated bananas. J. Linnean Soc. of possible thanks to the female fertility of the London, Botany 55:302-312 selected parent, to the low though sufficient Tomekpé K., P. Rowe, H. Tezenas du Montcel & D. fertility of Pelipita pollen and to the genetic Vuylsteke. 1995. Plantain and Popoulou/Maia Maoli breeding: Current approaches and future opportunities. phenomena of meiosis. Workshop INIBAP/MARDI, Serdang, Malaysia. Owing to the success achieved in the trials carried out in different zones of Cuba, the clone INIFAT 02 was included in the Catálogo Oficial de Variedades de Cuba-2006 (2006 Official Catalogue of Cuba’s Varieties). For more information, contact Adolfo It is recommended that further ecological- Rodríguez Nodals, Arlene Rodríguez zone studies of this clone be carried out to Manzano or Adolfo Rodríguez Manzano, assist its launching as a new commercial Instituto de Investigaciones Fundamentales hybrid. With regard to most of the significant en Agricultura Tropical (INIFAT), Calle 1, agronomic characteristics, it stands out for esq 2. Santiago de las Vegas, Ciudad de La combining the virtues of both parents and Habana, Cuba. is more precocious at harvest than either E-mail: [email protected] ; of them. Research should be done on the [email protected]

40 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 41 MusaNews Progress in conserving banana diversity

For the past several years, Bioversity has accessions that are at risk from disease been working with its partners around the or other hazards, and to provide a ‘safety world to achieve a consensus on how best back-up’ of unique accessions in vitro – on to conserve the diversity of bananas. It site, if the facilities are available, and at the was a tribute to the effectiveness of this International Transit Centre in Belgium. concerted effort that the Global Strategy The Trust is also providing additional for Conservation Musa was one of the first support for cryopreservation work, crop-specific conservation strategies to previously supported by the World Bank be approved and published by the Global and Gatsby Charitable Foundation, which Trust. The conservation effort seeks to assure the long-term security of received further encouragement when, at Musa diversity by placing a copy of the the end of 2007, banana was among the first international collection (and a ‘black-box’, crops to receive a long-term grant, under a additional back-up) in liquid nitrogen. This partnership agreement between the Trust time around, the National Bureau of Plant and Bioversity. Genetic Resources in India will share the Now there are yet more developments workload with the Katholieke Universiteit that will provide additional impetus to the Leuven in Belgium. conservation effort. As this issue goes to The international in vitro collection, alth- press, many key banana collections around ough it has existed and has been growing the world are beginning to receive grants steadily for over twenty years, has previously from the Trust, via Bioversity, to regenerate been funded on competitive grants of limited duration. The Belgian government has now decided to put the maintenance of the international banana genebank on a more secure footing by providing a special grant for this purpose, outside the competitive grant system. With so much progress on ex situ conservation of cultivated banana diversity, this leaves one area that requires desperately urgent attention: the conservation of wild species in the forests of Asia, in the face of rampant deforestation. Bioversity and its partners will now be making a special effort to save these beautiful plants, along with the potentially invaluable genes they contain. Banana wild relative Musa acuminata burmannicoides, popularly known as Calcutta 4, For more information, visit the website used as a source of disease resistance in many breeding programmes, survives now only in ex dedicated to the conservation and use of the situ collections, such as this one of bananas at www.musa- at Solok in net.org.

In memory of Dr Bakelana Congo loses banana champion

On March 7, 2007, the banana community obtaining a PhD from Ohio State University lost an important African agronomist, in 1992. Dr Antoine Bakelana Ba-Kifumfutu. From 1994, he headed the Fruits and Born in the Democratic Republic of Congo Banana programme of the Institut national in 1947, Dr Bakelana started to study pour l’étude et la recherche agronomique agronomy in his country and then in the USA, (INERA) in M’Vuazi and became Director

42 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 43 of the INERA M’Vuazi research center in Those who knew and worked with Dr 1998. Bakelana will remember him for his good Dr Bakelana dedicated most of his life to humour, even under difficult conditions, for his bananas, and was an active member of the dedication to banana research, and his ability to African banana research networks MUSACO get along with his fellow banana scientists. and BARNESA. He was the president of MUSACO at the time of his death and was not able to realize his dream of hosting MUSACO Steering Committee meeting in Kinshasa. From 2001 to 2007, he was coordinator of the “Farmer-participatory evaluation and dissemination of improved Musa germplasm” project for DRCongo, a project funded by the Common Fund for Commodities and implemented in seven countries around the world by the banana research group of Dr Bakelana (on the left) is happy to see the Bioversity. future harvest from a plot of the CFC project.

Le bananier et sa culture Books A. Lassoudière

2007, 384 pages, 12 x 19 cm Édition Quæ, Collection: Savoir-faire ISBN : 978-2-7592-0046-7 Réf : 02045 This book presents the technical components of a sustainable banana production intended for the international market. After a reminder of knowledge about the plant, parasitic constraints and recent economic developments of this product, the author introduces the concepts of reasoned culture and organic banana. Taking into account recent developments in phytosanitary and agricultural areas as well as fertilization and irrigation, the author develops every aspect of the production process: best planting conditions to reduce inputs and hence pollution, best practices to get a satisfactory productivity; needed packaging operations to ensure the quality of fruit; technical decision support for reasoning cultivation practices. The author André Lassoudière, an engineer at CIRAD If you are interested to buy one or more since 1967, has worked as a researcher copies, please contact : within the banana production chain, but also Éditions Quæ, c/o Inra, RD 10, as an expert with banana producers and 78026 Versailles Cedex, France professional bodies in this sector. In addition Tel. + 33 1 30 83 34 06 to his long stays in Côte d’Ivoire, Cameroon Fax + 33 1 30 83 34 49 and the French West Indies, he has made email : [email protected] numerous expert missions in all the world. www.quae.com

42 InfoMusa - Vol. 16 No. 1-2, June & December 2007 InfoMusa - Vol. 16 No. 1-2, June & December 2007 43 A bunch of web resources Musa web ressources

As explained in the editorial, we have been Using these technologies, we hope to help to very busy establishing a new range of on- bring new dynamism to the banana research line resources. We urge you to visit these community and facilitate knowledge sharing. resources using the links indicated below. The new technologies used to develop these All these websites can be accessed through web resources allow all those interested to our institutional web site (http://bananas.biov easily contribute and enrich them through ersityinternational.org/) with a single click or constructive debates and knowledge through their own address. sharing.

http://platforms.inibap.org

The resource center is structured by thematic platforms featuring the most up-to-date information organized in a user-friendly manner; tools for learning and for training; literature; images; fora; links to additional resources, etc. Some of the platforms are available in various languages. The resource center is enriched with more themes as the need arises

http://www.promusa.org

The website of the Musa R&D community. On it, you will find InfoMus@, the on-line successor to INFOMUSA, featuring news, articles, opinion pieces and a ‘media watch’ section. The site also offers MusaTalk, a discussion forum where ProMusa members can exchange views.

http://www.musa-net.org

MusaNet is the network of scientists, genebank curators and other experts who are working together to ensure the banana’s survival. The MusaNet website will be keeping track of their progress in establishing a shared taxonomy of Musa, assessing the threats faced by wild and cultivated bananas, collecting the diversity not represented in genebanks, and conserving the Musa genepool.

http://banana-diversity.org/mgis/

This site gives you access to the Musa Germplasm Information System (MGIS), a user- friendly database containing detailed information on the accessions stored in different Musa genebanks around the world.

44 InfoMusa - Vol. 16 No. 1-2, June & December 2007 International ISHS/ProMusa Symposium Global Perspectives on Asian Challenges Guang Dong, China, September 14-18, 2009

ProMusa, in collaboration with the Guang Dong Academy for Agricultural Sciences (GDAAS), the International Society for Horticultural Science (ISHS) and Bioversity’s Banana and Plantain Regional Network for Asia and the Pacific (BAPNET), has the pleasure to announce the “International Banana Symposium: Global Perspectives on Asian Challenges” to be held in Guang Dong, China, September 14-18, 2009. The symposium will focus on two major themes: Session 1: Novel approaches to understanding, conserving and using banana genetic diversity This session will include such topics as Musa diversity studies, characterization and evaluation of germplasm, wild species, conservation issues, conventional breeding and non-conventional approaches, genomics and other -omics, host reaction to biotic and abiotic stresses, … Session 2: Integrated approaches to managing Fusarium wilt and other emerging disease threats This session will report on progress in the area of Fusarium wilt research, including surveys and mapping, alternative management options, the identification and use of sources of resistance, the evaluation of improved materials, etc. Other important diseases in Asia, like e.g. BBTV, will also be part of the programme. Special attention will be given to the role of tissue culture in the management of important diseases. Each session will be opened by a one or two keynote speakers, followed by 10-15 oral presentations and a comprehensive poster session, and be concluded by a discussion forum. Submission of abstracts: Deadline for abstract submission is 31 December, 2008. Registration Early registration: Until 31 December, 2008 Late registration: From 01 January 2009

Fees: Participants Early Registration Late Registration From LDC* and China + students US$325 US$375 Other US$375 US$425

ISHS members receive a reduction of: US$65 The registration fee will cover: conference package, lunches, dinners, field trip, proceedings, ISHS membership fee

For more information, please go to the symposium website: http://www.promusa.org/symposium_2009/home.html *Least developed countries

44 InfoMusa - Vol. 16 No. 1-2, June & December 2007 Publications Identification and characterization guide for FHIA banana and plantain hybrids. J.M. Alvarez and F.E. Rosales. 2008. This 15 page-guide is a tool that will allow the field identification of all hybrids released by FHIA in a simple and precise manner. It is written in a concise and easy-to-use way, illustrated with photographs of the most distinctive characteristics of these otherwise very similar hybrids. English and Spanish versions are provided in the same document. An analysis of the risk from Xanthomonas campestris pv. musacearum to banana cultivation in Eastern Central and Southern Africa JJ. Smith, DR. Jones, E. Karamura, G. Blomme and FL. Turyagyenda. 2008. 32pp. The authors, scientists from the Central Science Laboratory in the UK and Bioversity International in Uganda, discuss the distribution and epidemiology of the causal agent of the banana Xanthomonas wilt (BXW) and the measures farmers can use to protect their plants from contracting this deadly disease. They also analyse the impact of the measures deployed to date and make recommendations to reduce the risk of the disease spreading to other countries. Also available on the same topic: - The Banana Bacterial Wilt Resource CD (Version 2) includes the most recent information about the disease; printable handouts and factsheets on how to recognize it and how to stop its spread; reports; selected literature; a flipbook; videos and pictures, and a searchable database. - The Xanthomonas wilt web platform ("http://platforms.inibap.org/xanthomonaswilt/"), available in French and English. Catalogue of introduced and local banana cultivars in the Philippines. F.S. Dela Cruz L.S. Gueco, O.P. Damasco, V.C. Huelgas, I.G. Banasihan, R.V. Lladones, I. Van den Bergh and A.B. Molina. 2007. This 59 page catalogue provides information on the morphological and yield characteristics of 19 introduced and 8 local banana cultivars grown at the demonstration plot of the Institute of Plant Breeding, University of the Philippines, Los Baños. It is intended to serve as a guide to help in identifying and selecting cultivars for further evaluation by researchers and planting by interested growers. MusaDoc 2008. The 8th edition of the MusaDoc CD-RoM is now available. The CD-RoM includes the updates of MusaLit, the bibliographic database on Musa and BRIS, the banana research information system as well as recent Bioversity publications on bananas and plantains and a virtual library of more than 1000 documents searchable through MusaLit.

To obtain a complete list of our publications, consult our website or contact Leila Er-rachiq at Bioversity in Montpellier. E-mail : "mailto:[email protected]"

Bioversity-France Parc Scientifique Agropolis II 34397 Montpellier Cedex 5 - FRANCE e-mail: [email protected]

http://bananas.bioversityinternational.org Fax: (33) 467 61 03 34