Nutritious plantains Novel use of banana stalk Measuring Fusarium virulence Surveying nematodes in Kenya How do mycorrhizae empower bananas? Enhancing awareness for BXW control Vol. 16 No. 1 & 2 June & December 2007 The International Journal on Banana and Plantain InfoMusa - Vol. 16 No. 1-2, June & December 2007 1 INFOMUSA 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 ISSN 1023-0076 Editorial Office: INFOMUSA, Bioversity, Parc Scientifique Agropolis II, 34397 Montpellier Cedex 5, France. Telephone + 33-(0)4 67 61 13 02; Telefax: + 33-(0)4 67 61 03 34; E-mail: bioversity-france@cgiar.org Authors of articles submitted for publication which are not published in this latest issue of INFOMUSA are free to submit them for publication elsewhere. No manuscript will be returned unless solicited by authors. Unless accompanied by a copyright notice, articles appearing in INFOMUSA may be quoted or reproduced without charge, provided acknowledgement of the source is given. An electronic version is available at the following address: http://bananas.bioversityinternational.org/ content/view/31/48/lang,fr Views expressed in articles are those of the authors and do not necessarily reflect those of Bioversity. InfoMusa Vol. 16 No.1&2 Cover photo: Sale of bananas in a village inTamil Nadu, India (Inge van den Bergh, Bioversity) 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. Contents 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 2 Preparation of a cation exchanger containing carboxyl groups from banana stalk and its utilization as chelating agent T.S. Anirudhan and I.G. Shibi 7 Associative nitrogen fixation by Azospirillum and Bacillus spp. in bananas Md. Abdul Baset Mia, Z.H. Shamsuddin, Z. Wahab and M. Mahmood 11 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 16 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 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 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.promusa.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 20072 InfoMusa - Vol. 16 No. 1-2, June & December 2007 3 Micronutrient deficiencies are con-sidered to be the main nutritional and health problem affecting poor populations. Iron and zinc deficiencies are commonly reported in children and women of reproductive age in many developing countries (de Pee et al. 1996, Frossard et al. 2000, Gibson et al. 2000). Iron deficiency is the prevailing nutritional deficiency in the world and is the main cause of anaemia (UN ACC/SCN 1997, Halberg and Hulthén 2000). Dietary zinc deficiency leads to various growth and reproduction disorders and often results in death, especially in developing countries (FAO/WHO 1996, Brown et al. 2002). More than half of pregnant women and one third of children under five all over the world suffer from nutritional anemia and iron deficiency to various degrees (UN ACC/SCN 1997). Vitamin A deficiency is given priority among global health problems (UNICEF 1990, FAO/WHO 1992). It can be responsible for increased mortality among children and women in developing countries and is the leading cause of preventable blindness in children, while enhancing the risk of disease and death from severe infections (Sommer and West 1996, Mclaren and Frigg 2001, OMS/UNICEF 2002). Banana and plantain (Musa spp.) are major staple foods for over 100 million people in sub-Saharan Africa, where they contribute significantly to food security (Sharrock and Frison 1998, INIBAP 2002). Plantain is considered an excellent source of energy and nutrients: it is estimated that 100 g of the edible part of raw plantain provides 122 kcal, contains 1.30 g of protein, 0.37 g of fat, 0.6 mg of iron, 0.14 mg of zinc and 457 μg of β-carotene (USDA 2004). In Nigeria, banana and plantain have always been very important traditional staple foods for both rural and urban populations. Annually, 6.7 million tons of plantain and other cooking bananas are produced in the country, with an average consumption of 150 kg/person/year or 348 calories per day (IITA 2000). Plantain may, therefore, play a key role in meeting nutritional requirements for Nigerian populations (Ajayi and Aneke 2002). The present study aimed at determining the iron, zinc and β-carotene contents of the plantain-based foods commonly consumed in Southern Nigeria to assess the quantitative contribution of plantain consumption in meeting iron, zinc and vitamin A requirements of both children and their mothers. Materials and methods Household surveys: A survey was carried out in the Abia, Akwa-Ibom, Edo, and Ogun, States of Southern Nigeria, targeting their capital cities, i.e., Umuahia, Uyo, Benin City, and Abeokuta, respectively. In each state, sub-samples of 30 urban and 30 rural households were randomly taken, totalling 240 households. One third of each sub- sample of households had a child under five years old who was definitively weaned and whose mother was present in the household. Data collection was done in June and July 2005. A questionnaire was devised and tested to collect data on the following factors: types of banana and plantain varieties used, forms of plantain foods preferred and consumption period of these foods, consumption levels of plantain-based meals, daily food consumption of women and their children under five years of age. The daily consumption of plantain-based foods was assessed by a 24-hour recall technique applied over three consecutive days (Bingham et al. 1988). On each day and in each household, the mother recalled all the types and quantities of plantain-based meals eaten by her children and herself during the last 24 hours. Local measures used to estimate the level of consumption were later converted to grams. Sample composition analysis: Samples of plantain-based foods were collected from households in which they were available, but 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 Nutritious plantains InfoMusa - Vol. 16 No. 1-2, June & December 20072 InfoMusa - Vol. 16 No. 1-2, June & December 2007 3 most of the samples were purchased in local markets. They were packed in aluminum foil within polyethylene bags inside plastic pots with lids, and transferred to the laboratory. During transport, samples were kept in cool boxes with cooling elements. They were stored in a deep-freezer at -20ºC for one week before analysis. The concentrations of iron and zinc were determined by atomic absorption spectrophotometry in the laboratory of the Faculty of Agronomic Sciences at the University of Abomey-Calavi in Benin. β-carotene was determined at the Institute of Medical Research Noguchi of the University of Lagon, Ghana. Samples of 0.01 - 0.9 g were collected after homogenization; β-carotene was extracted by High Performance Liquid Chromatography (HPLC) using Tetra Hydro Furan, as described by Takyi (1999). Retinol activity equivalent was calculated using the following formula: [β- carotene value / 12]. All measurements were made in triplicate. Data analysis: The average daily consumption of each plantain-based food was evaluated for the mothers and children in the survey. Thus, for each type of plantain- based food, the daily consumption was obtained by calculating the average of three days’ consumption for this type of food. Iron, zinc and vitamin A supplies from the average daily consumption of plantain-based meals per child or mother were evaluated using the micronutrient composition of the samples. Throughout the three days of investigation six types of plantain-based foods were commonly consumed and two of them were often consumed daily by the surveyed populations. In this regard, the average daily supply of iron, zinc and vitamin A from plantain was obtained by taking a third of the total amount provided by all the six plantain- based food types eaten over the three days. For each surveyed subject (children and mothers), these average values were compared with the dietary recommended allowances for the corresponding micro- nutrients as defined by FAO (Latham 2001). Data were analysed using SPSS Software. All continuous data were expressed as means ± standard deviations. One-way analysis of variance (ANOVA) was used to determine how the area (rural or urban) and age (child or mother) affected their consumption and the nutritional importance of plantain-based food. The Least Significant Difference (LSD) test at 0.05 and 0.01 levels was used to compare means. Results and discussion Forms of plantain preferred in the households The variety of plantain mostly eaten in the surveyed households was the local variety “landrace Agbagba”. Households surveyed used various techniques for processing plantain at different ripening stages (Table 1). Some of these techniques have been found in South-east Asia (Sharrock 1996) and in Cameroon (Ngoh Newilah et al. 2005). Of these processing techniques, the most popular in all the households surveyed was frying. Boiled and roasted plantains were also popular in both urban and rural areas. However, most of the urban respondents preferred plantain chips. Ripe plantain or banana was preferred by 76% of the rural households surveyed, as against 43% in urban areas. Preferred times for eating plantain- based foods The different times of day when plantain- based foods are mostly eaten were the same everywhere. Most of the households surveyed ate fried or boiled plantain at breakfast (Table 2). Fried plantain was also preferred as an afternoon snack. Respondents preferred boiled plantain and pounded plantain for lunch; pounded plantain and plantain flour paste, often Table 1. Forms of plantain preferred in the households (% of N) Form of meals Rural area Urban area (N=120) (N=121) Fried plantain 100 100 Plantain chips 63 89 Boiled ripe or unripe plantain 88 71 Pounded plantain 49 35 Plantain flour paste 29 18 Ripe banana/plantain 76 43 Roasted plantain 96 83 Table 2. Preferred times for eating plantain foods in the households Form of meals Breakfast (%) Lunch (%) Snack (%) Dinner (%) Fried plantain 47 9 32 8 Plantain chips 6 3 30 2 Boiled plantain 30 27 5 17 Pounded plantain 2 21 2 22 Plantain flour paste 2 15 0 20 Ripe banana/plantain 2 6 9 2 Roasted plantain 5 9 19 7 InfoMusa - Vol. 16 No. 1-2, June & December 20074 InfoMusa - Vol. 16 No. 1-2, June & December 2007 5 associated with cassava or yam flour, were the most preferred for dinner. Generally, these eating habits were similar to those reported by Ajayi and Aneke (2002) in the town of Nsukka of Nigeria. Consumption level in rural/urban areas There is a significant difference between rural and urban consumers of plantain-based foods. The number of children consuming plantain chips and the daily amount consumed were higher in urban areas (P <0.05) than in rural areas (Table 3). The daily intake of roasted plantain by urban children was higher than that of their rural counterparts (P <0.05). A similar trend was also observed with their mothers; however, the difference between rural and urban mothers was not significant for the consumption of roasted plantain (Table 4). In contrast, the number of consumers of pounded plantain and the quantity consumed per capita were lower in urban than in rural zones (86 rural mothers versus 49 urban mothers; 82 rural children versus 61 urban children). However the difference was significant only for the quantity consumed by the children (P < 0.05). In both rural and urban zones, mothers’ consumption levels were at least twice those of children. Iron, zinc and β-carotene contents The iron, zinc and β-carotene contents of the plantain-based foods (including means and standard deviations) are given for 100g FW of edible food (Table 5). The values of β-carotene are lower than those previously reported for banana by Englberger (2004). The iron content of all plantain products analysed ranged from 0.71 mg to 1.30 mg/ 100g FW. It was higher in plantain chips than in fried plantain, which had the lowest iron content of all plantain products. Because the cooking process for plantain chips was similar to that of fried plantain, the difference observed in the two types could be attributed to the duration of cooking, which is longer for chips than for fried plantain. It might also be due to the degree of ripening and maturity of the plantains used; ripe plantain was used for frying and unripe fruit was used to make chips. Roasted plantain contains a moderate level of iron. The zinc content of the plantain-based foods analysed ranged from 0.24 mg to 0.37 mg/100 g FW. Unripe plantain and chips contained the highest levels of zinc. The β-carotene content was higher in plantain-based products (203.0-695.12 μg/ 100 g FW) compared to other staple foods, e.g. maize, cassava and yam, grown in Nigeria. The carotene levels generally increase with cooking: in cooked food, carotene is more efficiently extracted from the food matrix by the solvent; however, this may also depend on the method of cooking, as a longer duration and higher temperatures destroy carotenoids (Englberger 2004). In this study, and regardless of the plantain variety used, β-carotene levels generally Table 3. Average size (mean ± standard deviation) of plantain meals eaten by the children who ate the dishes Form of meals Rural area (g) Urban area (g) Significant level Fried plantain 32.9 ± 43.7 (112) 42.5 ± 68.9 (120) ns Plantain chips 4.9 ± 39.7 (68) 24.2 ± 59.5 (114) * Boiled plantain 66.4 ± 93.3 (108) 52.2 ± 57.5 (68) ns Pounded plantain 59.9 ± 119.7 (82) 30.3 ± 85.0 (61) * Ripe banana/plantain 34.9 ± 54.7 (109) 25.3 ± 45.7 (86) ns Roasted plantain 37.2 ± 78.0 (102) 81.0 ± 107.8 (109) * * = Significant difference at 5% ns = not significantly different at 5% level Table 4. Average size (mean ± standard deviation) of plantain meals eaten by the mothers who ate the dishes Form of meals Rural area (g) Urban area (g) Significant level Fried plantain 56.4 ± 135.2 (107) 48.8 ± 297.0 (116) ns Plantain chips 23.1 ± 78.5 (46) 42.9 ± 142.5 (98) * Boiled plantain 72.3 ± 216.6 (108) 56.5 ± 191.2 (79) ns Pounded plantain 102.8 ± 319.0 (86) 76.5 ± 287.4 (49) ns Ripe banana/plantain 71.9 ± 229.7 (111) 58.7 ± 264.8 (87) ns Roasted plantain 69.0 ± 204.4 (106) 79.4 ± 279.7 (109) ns * = Significant difference at 5% 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 InfoMusa - Vol. 16 No. 1-2, June & December 20074 InfoMusa - Vol. 16 No. 1-2, June & December 2007 5 increased, but not in every case. Roasted plantain was particularly rich in β-carotene, whereas lower β-carotene contents were observed in unripe plantain and its flour. This contrasts with the findings of several authors, including Isonfua and Omuaru (1989), Barthkur and Arnold (1990) and Englberger (2004) who showed that the state of maturity affects carotenoid contents and that their levels in the fruit increase during ripening. Contribution of plantain in children’s and their mothers’ diets Based on the iron, zinc and β-carotene contents in plantain-based foods and their daily consumption levels by the surveyed children and their mothers, we calculated the daily iron, zinc and vitamin A supplied by each plantain-based food to each subject (Tables 6 and 7). For these micronutrients the dietary allocations recommended by FAO for children under five are 14 mg of iron, 10 mg of zinc and 400 μg of vitamin A (Latham 2001). Our results showed that the daily consumption of plantain foods provided 0.98 mg and 0.77 mg of iron in rural and urban zones respectively, i.e. 7% and 5.5% of the children’s daily iron requirement (Tables 8 and 9). Zinc provided daily by plantain-based foods in both rural and urban consumers’ children was 0.26 mg, representing almost 3% of the requirement. Daily consumption of plantain by the children supplied 30.9 μg Retinol Activity Equivalent (RAE) and 18.2 μg RAE of vitamin A in rural and in urban areas respectively, accounting for 7.2% and 4.6% of the daily vitamin A requirement for these children in rural and urban zones respectively. The difference observed between urban and rural zones for vitamin A contribution by plantain was significant (P < 0.05). For non-pregnant and non-lactating women, FAO recommends a daily intake of 48 mg of iron, 12 mg of zinc and 800 μg RE of vitamin A (Latham 2001). We found that the daily consumption of plantain-derived foods provided approximately 1.80 mg of iron and 0.6 mg of zinc for mothers in both rural and urban zones (Table 8), representing 4% of their average iron and zinc requirements (Table 9). Plantain-derived foods could potentially provide as much as 46.7 μg RAE of vitamin A to rural mothers and 40 μg RAE to urban mothers daily. These values corresponded on average to 9.4% and 8% of vitamin A requirements of mothers in rural and urban zones respectively. These contributions to vitamin A requirements could, however, reach up to 25% in both rural and urban zones. Conclusion This paper provided data on the iron, zinc and β-carotene contents in plantain-based foods commonly consumed in Southern Nigeria and their daily level of consumption by children under five years old and their 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 InfoMusa - Vol. 16 No. 1-2, June & December 20076 InfoMusa - Vol. 16 No. 1-2, June & December 2007 7 Table 8. Average iron, zinc and vitamin A supply (mean ± standard deviation) by banana and plantain in children’s and their mothers’ diets Micronutrients Children Mothers Significant Rural area Urban area Rural area Urban area level Iron (mg) 0.98 ± 1.4 0.77 ± 1.2 1.95 ± 4.7 1.72 ± 5.0 ns Zinc ((mg) 0.29 ± 0.4 0.23 ± 0.4 0.49 ± 0.9 0.8 ± 1.1 ns Vitamin A (µg RAE) 30.9 ± 47.8 18.2 ± 38.9 46.7 ± 98.2 40.0 ± 103.9 * * = Significant difference at 5% for the children ns = not significantly different at 5% level Table 9. Contribution of plantain-based foods to the iron, zinc and vitamin A supply (mean ± standard deviation) to the children and their mothers Micronutrients Children Mothers Significant Rural area Urban area Rural area Urban area level Iron (%) 7.00 ± 10.2 5.50 ± 8.5 4.06 ± 9.7 3.58 ± 10.3 ns Zinc (%) 2.91 ± 4.4 2.30 ± 3.8 4.08 ± 7.5 3.17 ± 9.1 ns Vitamin A (%) 7.2 ± 11.9 4.6 ± 9.7 9.4 ± 15.7 8.0 ± 16.2 * * = Significant difference at 5% for the children ns = not significantly different at 5% level Some food items, e.g. roasted plantain, were identified as relatively rich in iron and β-carotene. The average daily level of consumption of plantain-based foods was higher for rural than for urban consumers. There was wide variation in consumption levels of plantain-based foods and hence of their contribution to micronutrient requirements. 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Europeen Journal of Clinical Nutrition 50 (suppl.): S38-53. Sharrock S & E. Frison. 1998. Musa production around the world- trends, varieties and regional importance. Pp. 42–47 in INIBAP Annual Report. INIBAP, Montpellier, France. Sommer A. & K.P. West. 1996. Vitamin A deficiency: health, survival, and vision. New York: Oxford University Press. Takyi E.K. 1999. Children’s consumption of dark green leafy vegetables with added fat enhances serum retinol. Journal of Nutrition 129:1549-1554. UN ACC/SCN. 1997. Third report on the world nutrition situation. United Nations Administrative Committee on Coordination/Subcommittee on Nutrition Geneva. UNICEF. 1990. World declaration on the survival, protection and development of children. The World Summit for Children. New York. USDA. 2004. National Nutrient database for Standard Reference, Release 17. Fernande Honfo, Kerstin Hell and Ousmane Coulibaly work at the International Institute of Tropical Agriculture (IITA-Bénin), 08BP0932 Cotonou, Bénin and Abdou Tenkouano formerly based at IITA-Cameroun, works now for AVRDC in Tanzania Corresponding author: f.honfo@cgiar.org fernandeh@yahoo.fr tel.: 229 21 35 01 88 fax: 229 21 35 05 56 Scheme 1. Preparation of PGBS-COOH + CH2 CONH2 70 �C Fe2+ / H2O2 CH (PGBS) (en) 2 100 �C,Toluene CONH2 pH 4.0 CONH- (CH2 )2-NH CO(CH 2)2 - CO OH (PGB S-COOH) BS CONHCH 2CH 2NH2 BS P BS P BS P (AAm ) InfoMusa - Vol. 16 No. 1-2, June & December 20076 InfoMusa - Vol. 16 No. 1-2, June & December 2007 7 The ability of inexpensive agricultural residues to adsorb heavy metal ions has received considerable attention for the development of an efficient, clean and cheap technology for wastewater treatment (Montanher et al. 2005, Nasernejad et al. 2005). Numerous low cost agricultural residues have been used as adsorbents for the removal of heavy metals, including saw dust, coconut husk, coconut coir pith, banana peel, bagasse fly ash, orange peel, moss peat and nut shells (Annadurai et al. 2002). Most of these adsorbents have good adsorption capacity as compared to activated carbons and commercial ion exchangers, but their use in their original forms was found to be limited due to leaching of organic substances into the solutions. Efforts are therefore needed to prevent organic substances from leaching during the adsorption process without affecting the adsorption capacity. Chemical modifications of agricultural residues via esterification, cross linking and grafting have been extensively studied (El-Sayed 1996). It has been reported that the introduction of reactive functional groups into the backbone of cross linked lignocellulosics resulted in products that were capable of removing heavy metals from industrial waste waters (Khalil et al. 1991). We have investigated the synthesis of quality adsorbents and ion exchangers from lignocellulosics for effluent treatment preparation. Characterization of polymer grafted lignocellulosics based on saw dust (Raji and Anirudhan 1998, Unnithan and Anirudhan 2001), coconut husk (Sreedhar and Anirudhan 2000) coconut coir pith (Unnithan et al. 2004) and banana stem (Noeline et al. 2005) and their application in removing heavy metals from aqueous and effluent liquids were examples of these studies. In international trade, bananas are the most commonly traded fruit after those of citrus family. India is the market leader in the production of bananas, producing 16,000,000 metric tonnes annually. After the use of banana fruits, the banana stalk (BS) are often under valued and considered as a waste material, creating a disposal problem. It is largely composed of cellulose, hemi cellulose, lignin, tannin and pectin. The main objective of this study is to investigate the feasibility of using BS as a precursor material to develop a suitable adsorbent for the removal of heavy metals from aqueous solutions. Material and methods All chemicals were analytical grade and used as purchased without further purification. The aqueous solutions of various concentrations of the heavy metals ions were prepared by dissolving their chloride salts (E. Merck India Ltd.) in distilled water. The carboxylate functionalized BS was synthesized using polyacrylamide grafting in the presence of ferrous ammonium sulphate/ H2O2 redox initiator (Shibi and Anirudhan 2002). Scheme I represents the general procedure adopted for the preparation of polyacrylamide grafted BS (PGBS) having –COOH groups (PGBS-COOH). In order to design the optimum conditions for preparing large quantity of PGBS, a series of experiments were conducted by varying the concentration of ferrous ammonium sulphate, H2O2, acrylamide, reaction time and temperature. The results of these experiments have been reported in our earlier publication (Shibi and Anirudhan 2002). For a typical synthesis, 10 g of BS was transferred into Preparation of a cation exchanger containing carboxyl groups from banana stalk and its utilization as chelating agent T.S. Anirudhan and I.G. Shibi Banana stalk as heavy-metal sponge Scheme 1. Preparation of PGBS-COOH 120 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 pH Re m ov al (% ) Pb Co Hg Cd Initial concentration : 50 mg/L Sorbent dose : 2 g/L Agitation speed : 200 rpm Temperature : 30 °C Time : 3h PGBS-COOH BS InfoMusa - Vol. 16 No. 1-2, June & December 20078 InfoMusa - Vol. 16 No. 1-2, June & December 2007 9 experiments, metal concentrations used for adsorption ranged from 10 to 700 mg/L. The experimental conditions for the desorption of metals from spent adsorbent were similar to those of the batch adsorption tests. Initially the PGBS-COOH was loaded with metal ions by applying the adsorption process for 4 h at pH 6.5. The concentrations of Pb(II), Co (II), Hg(II) and Cd(II) in the loaded PGBS-COOH was 12.46, 12.41, 12.32 and 10.94 mg/g respectively. Desorption was performed by transferring 0.1 g of spent PGBS-COOH to a flask containing 50 mL of 0.2 M HCl. The flasks were shaken at 30°C for 4 hours. After attaining equilibrium, the supernatant was filtered and filtrate was analyzed for metal concentration using atomic absorption spectrometry. Comparison of these values with those observed with the initial adsorption step was used to compute the percentage recovery values. All experiments were performed in duplicate and mean values are presented. Maximum deviation was ± 5%. Results and discussion Sorption of all metals at pH 2.0 was negligible, increasing with increase of pH, attaining an optimum in the range of 5.5–8.0 (Figure 1). The pH of the aqueous solution is an important controlling parameter in the heavy metal adsorption process. The sharp increase in metal uptake from pH 2.0 to 5.5 can be explained by ion exchange. The dependence of metal uptake on pH suggests that the weak acidic carboxyl groups R-COO– (pKa range between 3.5–5.5) of PGBS-COOH are the probable adsorption sites. At a pH of less than 5.5 the predominant metal species [M2+ and M(OH)+] are positively charged and therefore the uptake Figure 1. Effect of pH on the removal of metal ions by PGBS- COOH and BS a reaction flask equipped with a mechanical stirrer and thermometer, and then 80 mL of ferrous ammonium sulphate (0.02 M) was added. The suspension was allowed to soak for 15 min (this time was found to be sufficient for maximum impregnation of Fe(II) ions onto BS), while the flask was kept in a thermostatically controlled water bath at 30°C. To the mixture 120 mL of H2O2 (0.2 M) was added and then purified N2 was passed through the vessel for 5 min at room temperature (30°C). The polymerization was started by adding 50 g acrylamide (AAm). The reaction mixture was stirred at 70°C for 4 h. Graft copolymer (PGBS) was recovered by filtration, washed and finally dried. The dry reaction product was soxhlet extracted with water for 6 h to remove homopolymer and dried at 80°C. To convert into cation exchanger, PGBS was refluxed with 100 mL of ethylenediamine (en)2 for 8 h.The product was then washed with toluene and dried. One part by weight of the material was then refluxed with an equal part by weight of succinic anhydride in 1,4– dioxane at pH 4.0 for 6 h. The product was washed with 1,4–dioxane to remove excess succinic anhydride and dried. The PGBS- COOH obtained was sieved and particles having an average diameter 0.096 mm were used throughout the study. The physical and surface properties of PGBS-COOH were determined by scanning electron micrograph, X-ray, TG and FTIR (Shibi and Anirudhan 2002; Shibi and Anirudhan 2005). The characteristics of PGBS-COOH are: surface area; 110 m2/g; porosity, 0.51 mL/g; pHzpc, 5.5; cation exchange capacity, 2.38 meq/g; carboxylate content, 1.84 meq/g and apparent density, 0.72 g/mL. Batch adsorption experiments were carried out in 100 mL stoppered flasks by transferring 50 mL of an aqueous solution of heavy metal and 0.1 g PGBS-COOH. The solution pH was adjusted using 0.1M HCl and NaOH. The flasks were shaken for 4 h at an agitation speed of 200 rpm at 30°C. The contents of the bottle were filtered and the residual metal concentration in the filtrate determined using atomic absorption spectrophotometry. The PGBS-COOH–metal sorption suspension was equilibrated at pH 2-8 for determining the effect of pH on sorption. A metal concentration of 50 mg/L and an adsorbent dose of 100 mg per 50 mL were employed. For isotherm 87 6 5 4 3 2 1 0 0 100 200 300 600 500 600 Sorbent dose : 2 g/L Equilibrium time : 3 h Agitation speed : 200 rpm pH : 6,5 Pb Co Hg Cd Ce (mg/L) C e /q e ( g/ L) 2 PGBS-COOH + M2+ (PGBS-COO)2 M + 2 H + PGBS-COOH + M(OH)+ PGBS-COO M(OH) + H+ Ce 1 Ce ______ = ______ + ______ qe Q 0b Q0 Formula 1 Formula 3 Formula 2 InfoMusa - Vol. 16 No. 1-2, June & December 20078 InfoMusa - Vol. 16 No. 1-2, June & December 2007 9 of metals in the pH below 5.5 is a H+ – M2+/ M(OH)+ exchange process. (formula 1 and 2). The maximum removal capacities at pH 6.5 were found to be 96.4, 95.1, 91.3 and 80.6 % for Pb(II), Co(II), Hg(II) and Cd(II) respectively at an initial concentration of 50 mg/L. The sorbent surface becomes more positively charged at lower pH, due to the higher concentration of the hydrogen ions, thus reducing the attraction between sorbent surface and the metal cations. The pHzpc of the PGBS-COOH was found to be 5.5 and above this pH the sorbent surface became negatively charged, while the metal species were still present as M(OH)+. Under this condition M(OH)+ ions were adsorbed through a favourable electrostatic attraction. Over the pH range of 2.0-8.0, the adsorption of the metal ions by BS was found to be very much less than that of PGBS-COOH, clearly demonstrating the effectiveness of the latter in the metal removal process. The saturation capacities of metals for PGBS-COOH at 30°C were analyzed using the Langmuir isotherm model. This is valid for monolayer adsorption onto surfaces containing finite numbers of identical sorption sites, a situation which is described by the following equation (see formula 3) where qe and Q 0 are the observed and maximum uptake capacities (mg/g adsorbent) respectively. Ce is the equilibrium concentration (mg/L solution) b is the equilibrium constant. The linear plots of Ce/qe vs Ce for all metals indicate the applicability of the Langmuir adsorption isotherm. To evaluate the isotherm constants the experimental data were treated after discarding the data that lay outside the 95 % confidence interval, which was established by a method of least squares for the linear function. The uncertainty of the parameters of the fitted straight line corresponds to the standard deviations. The values of Q0 and b (the slope and intercept of the plots) were found to be (Q0) 185.34, 166.7, 137.89 and 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), Hg(II) and Cd (II) respectively (Figure 2). The higher the value of b, the higher is the affinity of adsorbent for the metal adsorbed. The affinity of PGBS-COOH to sorb the metals tested in this study followed a[pattern, as was noted for Q0 (Pb>Co>Hg>Cd). The relative preference for Pb(II) may be explained by the high stability of the complexes formed by the cationic species of this metal with carboxylate in PGBS-COOH compared with those formed by Co(II), Hg(II) and Cd(II) (Baes et al. 1996). The values of the correlation coefficient (r) obtained in the present study vary between 0.982-0.990 for different metals and indicate the goodness of fit of experimental data to the Langmuir model. Comparison of metal adsorption on PGBS-COOH with the data from the literature indicates that the adsorption capacity of PGBS- COOH is very much greater than that of other adsorbents such as activated carbons, ion exchange resin, sugar beet pulp, Brazil nut shell, sawdust and curdlan-activated carbon composite (El-Shafey et al. 2002; Rengaraj and Moon 2002, Reddad et al. 2002, Basso et al. 2002, Moon and Lee 2005). Table 1 summarizes the results of desorption and regeneration of PGBS-COOH for all metals. The percentage adsorption/ desorption values were calculated in relation with the original amount of the adsorbent. The total desorbed amount was calculated Figure 2. Langmuir isotherm plots for the adsorption of metal ions onto PGBS-COOH at 30°C InfoMusa - Vol. 16 No. 1-2, June & December 200710 InfoMusa - Vol. 16 No. 1-2, June & December 2007 11 Desorption of Pb, Hg, Co and Cd from metal–laden PGBS-COOH in flasks with 0.2 M HCl established the chemical stability of the carboxylate groups and the regeneration ability of PGBS-COOH. A small fraction of adsorbed metals was not recoverable by regeneration. This fraction presumably represents the metals which are bonded through stronger interaction and as a result, sorption efficiency is reduced in subsequent cycles. Recovery of metals from an adsorbent is of importance for application of sorption technology in the waste water industry. The results show that the spent adsorbent can be effectively regenerated by 0.2 M HCl. Conclusion The present study shows that the PGBS- COOH prepared from banana stalk, an agro-based waste biomass, can be used as an adsorbent for the removal of heavy metals [Pb(II), Co(II), Hg(II) and Cd(II)] from their aqueous solutions. A pH range 5.5 – 8.0 was found to be effective for the maximum removal of metal ions. The carboxyl groups were the main reaction sites responsible for metal binding on PGBS-COOH. The equilibrium data fitted very well to a Langmuir isotherm equation, confirming the monolayer sorption capacity of metals onto PGBS- COOH. The adsorbed metal ions were desorbed quantitatively by 0.2 M HCl and the adsorbent can be reused successfully after regeneration. Batch adsorption-desorption studies illustrate that PGBS-COOH could be used to remove heavy metals from aqueous solutions and industrial effluents. References Annadurai.G., R.S. Jueng & D.J. Lee. 2002. Adsorption of heavy metals from water using banana and orange peels. Water Sci. Technol. 47: 185-190. Ecxhange and absorption of some heavy metals in a modified coconut coir cation exchanger. Water Science and Technology 34:193-200. Baes A.U., S.J.P. Umali & R.L. Mercado 1996. Ion exchange and absorption of some heavy metals in a modified coconut coir cation exchanger. Water Science and Technology 34:193-200. and compared to the initial sorbed amount. The ability of HCl of 0.2 M concentration to strip out most of the adsorbed metals may be attributed to ion exchange. It has been postulated that the high concentration of H+ ions at low pH is responsible for the displacement of adsorbed metal via the ion exchange mechanism. As shown in Table 1, with three adsorption/desorption cycles the Pb(II) adsorption capacity of the PGBS-COOH decreased slightly from 99.7 % in the first cycle to 95.2 % in the third cycle. The recovery of Pb(II) in 0.2 M HCl decreased from 97.8 % in the first cycle to 93.0 % in the third cycle. The variation of the adsorption capacity and recovery efficiency in the adsorption-desorption cycles was also observed for Co(II), Hg(II) and Cd(II). After two cycles, the adsorption capacity of PGBS- COOH for Cd(II) was reduced by 4.7 % and on the other hand recovery was decreased from 84.3 % in the first cycle to 77.5 % in the third cycle. It was also found that the highest recovery efficiency was for Pb(II) and lowest for Cd(II). An explanation may be found in the differences in the strength of solution complexation with chloride and preferred coordination numbers. Pb(II) can form stable complexes with chloride due to its high coordination number, usually 6. On the other hand, Cd(II) typically exhibits a low coordination number 4, which results in less competition with chloride ions from solution and reaction sites on the adsorbent. Detailed study of the effect of chloride is necessary to understand the formation and strength of chlorocomplexes of metals and their influences on the adsorption properties. 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 Basso M.C., E.G. Cerrella & A.L. Cukierman. 2002. Lignocellulosic materials as potential biosorbents of trace toxic metals from wastewater. Ind. Eng. Chem. Res. 41: 185-190. El-Sayed Z. 1996. Utilization of saw dust in the preparation of some ion exchange resins. PhD Thesis. Cairo University. Cairo. El-Shafey E.I., M. Cox, A.A. Pichugin & Q. Appleton. 2002. Application of a carbon sorbent for the removal InfoMusa - Vol. 16 No. 1-2, June & December 200710 InfoMusa - Vol. 16 No. 1-2, June & December 2007 11 Reddad. Z, C. Gerente, Y. Andres & P.L. Cloirec. 2002. Adsorption of several metal ions onto a low-cost biosorbent: kinetic and equilibrium studies. Environ. Sci. Technol. 36: 2067-2073. Shibi I.G. & T.S. Anirudhan. 2002. Synthesis, charac- terization and application as a mercury(II) sorbent of banana stalk (Musa paradisiaca)-polyacrylamide grafted copolymer bearing carboxyl groups. Ind. Eng. Chem. Res. 41:5341-5352. Shibi I.G. & T.S. Anirudhan 2005. Adsorption of Co(II) by a carboxylate– functionalized polyacrylamide grafted lignocellulosics. Chemosphere 58:1117-1126. Sreedhar M.K. & T.S. Anirudhan. 2000. Preparation of an adsorbent by graft polymerization of acrylamide onto coconut husk for mercury(II) removal from aqueous solution and chlor-alkali industry wastewater. J. Appl. Polym. Sci. 75:1261-1269. Unnithan M.R. & T.S Anirudhan. 2001. The Kinetics and thermodynamics of sorption of Chromium(III) onto to the iron(III) complex of a carboxylated polyacrylamide grafted saw dust. Ind. Eng. Chem. Res. 40:2693-2701. Unnithan M.R., V.P. Vinod & T.S Anirudhan. 2004. Synthesis, characterization and application as a chromium(VI) adsorbent of amine-modified polyacrylamide-grafted coconut coir pith. Ind. Eng. Chem. Res. 43:2247-2235. T.S. Anirudhan works at the Department of Chemistry, University of Kerala, Thiruvananthapuram, 695 581, Kerala, India (Email: tsani@rediffmail.com) and I.G. Shibi works at the Department of Chemistry, S.N. College, Chempazhanthy, Thiruvananthapuram, 695 587, Kerala, India (Email: shibiig@sify.com). Associative nitrogen fixation by Azospirillum and Bacillus spp. in bananas Md. Abdul Baset Mia, Z.H. Shamsuddin, Z. Wahab and M. Mahmood Associative N2 fixation and plant growth promotion by rhizobacteria are important in achieving a sustainable agricultural system. Crops rely mainly on the N2 fixation process by associative, symbiotic and free living bacteria in the rhizosphere (Cocking 2000). Graminaceous plants potentially are capable of establishing associations with diazotrophic bacteria where Azospirillum provide the host with a source of N (Brimecombe et al. 2001). Plant growth promoting rhizobacteria (PGPR) inoculation can provide 31% N requirement in maize and 40% of the N requirement for oil palm seedlings through BNF under glasshouse condition (Amir et al. 2001, El-Komy et al. 1998). In field condition, rice can obtain 20% of its N and sugar cane 70% of its total N requirement through N2 fixation (Boddey et al. 1995, Shrersta and Ladha 1996). In general, N2 fixation is increased with modest levels of soil or fertilizer-N, but declines at high N levels because mineral- N depresses N2 fixation (Marschner 1995). A lower but specific level of fertilizer-N might be required for optimum N2 fixation by PGPR in association with banana roots, which has not been investigated. Therefore, the present study was conducted to quantify the amount of N2 fixation by Azospirillum strain Sp7 and Bacillus strain UPMB10 and in association with banana roots at different levels of fertilizer-N. Materials and methods Plants were grown hydroponically with a modified Cooper (1979) nutrient solution in small pots (4.0L). The experiment was completely randomized with two factors: inoculation - uninoculated, inoculated with Azospirillum brasilense strain Sp7, isolated from Digitaria grass, originally provided by EMBRAPA, Brazil, and Bacillus sphaericus strain UPMB10, isolated from oil palm roots (Shamsuddin and Roslina 1995) - and fertilizer-N 3.2, 8, 20 and 50 mg/kg) with six replications. A 1-ml sample of bacterial stock suspension was transferred into nutrient broth (Okon et al. 1977) in 250 ml Erlenmeyer flasks containing 100 or 300 ml nutrient media, respectively, that were agitated on a rotary Bacteria enhance N use of cadmium and other heavy metal ions from aqueous solution. J. Chem. Tech. Biotechnol. 77:429-436. Khalil M.I., A.Waly, S. Frag & A. Hebeish. 1991. Preparation of cationic– exchange starches containing phosphoric acid groups. J. Appl. Polym. Sci. 43:2303-2309. Montanher S.F., E.A. Oliveira & M.C. Rollenberg. 2005. Removal of metal ions from aqueous solutions by sorption onto rice bran. J. Hazard. Mater. B117:207-211. Moon C.J & J.H. Lee. 2005. Curdlan and activated carbon composed adsorbents for heavy metal removal. Process Biochem. 40: 1279-1283. Nasernejad B., T.E. Zalah, B.B Pou, M.E. Bygi & A. Zamani. 2005. Comparison for biosorption modeling of heavy metals [Cr(III), Cu(II), Zn(II)] adsorption from wastewater by carrot residues. Process Biochem. 40: 1319-1322. Noeline B.F, D.M. Manohar & T.S. Anirudhan. 2005. Kinetic and equilibrium modelling of lead(II) sorption from water and wastewater by polymerized banana stem in a batch reactor. Sep. pur. ;Technol. 45(2):131-140. Raji C. & T.S. Anirudhan. 1998. Batch Cr(VI) removal by polyacrylamide-grafted saw dust: Kinetics and thermodynamics. Water Res. 32:3772-3280. Rengaraj S & S.H. Moon. 2002. Kinetics of adsorption of Co(II) removal from water and wastewater by ion exchange resin. Water Res. 36:1783-1793. InfoMusa - Vol. 16 No. 1-2, June & December 200712 InfoMusa - Vol. 16 No. 1-2, June & December 2007 13 shaker at 125 rpm, 30± 2°C for 48 h. The optical density (OD600) was taken using a spectrophotometer (Ultrascope III). Uniform 10 to 11 cm height tissue-cultured banana plantlets cv. ‘Berangan’ (Musa spp., AA type) with 3-4 leaves and were used in the experiments. The plantlets were acclimatized with tap water for 3 days and the existing roots were gently removed aseptically from the corm using a sterile blade before transplanting. The N supply, labelled with 15N as {(15NH4)2SO4, 10% a.e.)} was applied after the establishment of seedlings (3 days after transplanting) (Malik and Zafar 1985). A 40-ml broth cultures of Sp7 or UPMB10 were applied to the respective pot after establishment of seedlings in the system (3 days) and same volume of killed bacterial broth cultures (autoclaved at 121°C for 15 minutes) were applied to the control pots as in the procedure by Malik and Zafar (1985). The pots were aerated with an air pump for 6 hours at 6-hourly intervals to ensure an uninhibited root respiration and bacterial growth. The banana plantlets were harvested at 45 DAI, separated into root, stem and leaves and oven dried for 48 hours at 70°C. The dried samples were weighed and ground to 390 µm. Samples (500 mg) of this ground material were analyzed for total N by semi- micro Kjeldahl method (Bremner 1996). The digested sample was distilled within the minimum distillation period of 4 minutes using Buchi TM K314 distillation unit. The liberated cooled ammonia was received in an Erlenmeyer flask containing 10 ml 0.1N HCL+Tashiro indicator solution for titrimetric determination of NH4 +. A back titration was performed to determine the amount of acid consumed by ammonia and to calculate the amount of total N in sample using 0.1N NaOH (tNaOH=1.000). Finally the total 14N and 15N distillates were collected and concentrated for 15N analysis by Emission Spectrometer (NOI-6PC) at the Malaysian Institute for Nuclear Technology Research (MINT). The 15N abundance found in the plant tissue was corrected for the % 15N atom excess (a.e.) present in the atmosphere (0.3663% 15N a.e.) by using the formula of Warembourg (1993) where: %15N atom excess (a. e.) in each plant part = %15N abundance – 0.3663% 15N (a.e.) Quantification of fixation based on isotope dilution was calculated by the formula of Fried and Middelboe (1977), see formula (1), where fs: fixing system, nfs: non fixing system. The 15N enrichment of the plant tissue was corrected according to the formula of Jensen et al. (1985), see formula (2). The average 15N enrichment present in different plant parts at the end of the experiment was calculated using the formula (3). The collected data were analyzed statistically using the Statistical Analysis System (SAS version 6.12, 1989). Following the analysis of variance procedure (ANOVA), differences among treatment means were determined using Duncan’s New Multiple Range Test (DMRT) comparison method (whenever applicable) at 5% level of significance. Results Dry matter production Plants inoculated with PGPR strains, especially with UPMB10, significantly increased the total dry matter (TDM) production when compared to the control (Table 1). Plants inoculated with UPMB10 produced more TDM at all the fertilizer-N levels. These inoculated with Sp7 produced a significant effect at 3.2 and 8 mg/kg, % N fixed= 1_ (15N atom % excess) fs x 100 (15N atom % excess) % 15N a.e. corr.= N x % 15N (a. e) N plantlets (D harvest) - N plantlets (D planting) Average % 15N a.e. = (%15N a.e. root x N root) + (%15N a.e. stem x N stem) + (%15N a.e. leaf x N leaf) (N root + N stem + N leaf) Formula (1) Formula (2) Formula (3) InfoMusa - Vol. 16 No. 1-2, June & December 200712 InfoMusa - Vol. 16 No. 1-2, June & December 2007 13 but not at 20 and 50 mg kg-1N levels, in root, pseudostem and leaves dry matter production was significantly increased due to fertilizer-N application and inoculation process (Table 1). In uninoculated plants, dry matter of root, pseudostem and leaves increased with fertilizer-N. Similarly, plants inoculated with UPMB10 also increased their root dry matter at 3.2, 8, and 20 mg/kg, pseudostem dry matter at 3.2, 20, and 50 mg/kg, and leaf dry matter at 3.2 mg/kg. Plants inoculated with Sp7 did not show the similar trend as control or UPMB10. Plants inoculated with UPMB10 produced significantly higher root dry matter at 3.2 and 8 mg/kg, but not at 20 mg/kg and pseudostem at 3.2 mg/kg but not at higher N fertilizer levels. No difference between Sp7 and the control was observed at any N level for leaf tissue. Nitrogen yield Plants inoculated with Sp7 or UPMB10 and supplied with 3.2, 8.0 or 20 mg/kg fertilizer-N yielded more total N. Plants supplied with 50 mg/kg fertilizer-N showed no significant difference between inoculated and uninoculated plants (Table 2). Total N yield of inoculated and uninoculated plants increased with increasing fertilizer-N in all treatments until at the highest fertilizer-N level (50 mg/kg). Generally the N yield in root, pseudostem, and leaves of inoculated plants was significantly higher compared to control until 50 mg kg-1N-fertilization; at 50 mg kg-1 fertilizer-N there was no significant difference between inoculated and uninoculated plants. Results were similar to those observed for dry matter, except more significant differences were observed for leaf N than for leaf dry matter. 15N isotope dilution Plant growth promoting rhizobacterial inoculation significantly diluted and lowered the plant 15N concentration, which is an indication of N2 fixation. The percentage of 15N atom excess (a.e.) of inoculated plant parts was significantly less than those of reference plants (control; nfs) (Table 3). The highest dilution (3.63-5.32% a.e.) was found in plants using the lowest level of fertilizer-N and the lowest (8.45-8.84% a.e.) in the highest level of fertilizer-N. Root of inoculated plants with UPMB10 showed the lowest dilution (3.63%). The percentage of atom excess in inoculated and non inoculated plant parts ranged from 3.63 to 9.52. The dilution capacity between the strains was not significantly different. Nitrogen fixation PGPR inoculation could potentially fix N2 in association with the roots of banana plantlets. Estimation of N2 fixed (% Ndfa) on a total plant basis by the PGPR strains Sp7 and UPMB10 were in the range of 5.0 to Table 2. Nitrogen yield in root, pseudostem and leaves of banana plantlets inoculated with PGPR strains Sp7 and UPMB10 and supplied with various levels of fertilizer-N grown under hydroponics condition Total N (mg/plant) Treatments Fertilizer-N levels (mg/kg) 3.2 8 20 50 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 UPMB10 8.8 a 16.6 a 28.7 a 37.6 a Pseudostem Control 2.2 b 4.0 b 9.1 b 30.4 a 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 Leaf Control 9.0 b 14.0 b 37.5 b 104 a Sp7 11.4 a 19.2 a 49.0 a 105 a 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 Sp7 24.4 a 41.6 a 82.8 a 177 a UPMB10 24.3 a 43.1 a 86.8 a 183 a Means having same letter (s) in a column do not differ at 0.05 significant level by DMRT Table 1. Dry matter in root, pseudostem and leaves of banana plantlets inoculated with PGPR strains Sp7 and UPMB10 and supplied with various levels of fertilizer-N grown under hydroponics condition Dry matter (g/plant) Treatments Fertilizer-N levels (mg/kg) 3.2 8 20 50 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 UPMB10 2.7 a 4.5 a 8.4 a 12.0 a Root Control 0.96 b 1.57 c 2.52 b 2.0 c 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 Control 0.26 b 0.78 a 1.64 b 3.21 b Sp7 0.49 a 0.82 a 1.91 ab 3.69 b 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 Sp7 0.74 ab 1.14 a 2.67 a 4.22 a UPMB10 0.81 a 1.24 a 2.76 a 4.39 a Table 3. Distribution of % 15N atom excess in root, pseudostem and leaves of PGPR inoculated plants at various levels of fertilizer-N % Atom excess Treatments Fertilizer-N levels (mg/kg) 3.2 8 20 50 Root Control 5.85 a 7.55 a 8.48 a 9.02 a Sp7 3.71 b 5.81 b 7.38 b 8.49 b UPMB10 3.63 b 6.08 b 7.55 b 8.45 b 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 UPMB10 4.35 b 7.20 b 8.01 b 8.84 b 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 UPMB10 3.91 b 6.29 b 7.60 b 8.71 b Means having same letter (s) in a column of a parameter do not differ significantly at 0.05 significant levels by DMRT InfoMusa - Vol. 16 No. 1-2, June & December 200714 InfoMusa - Vol. 16 No. 1-2, June & December 2007 15 39.3 % Ndfa (Table 4). As expected, higher % Ndfa (37.0 to 39.3%) was recorded under a low fertilizer-N regime (3.2 mg/kg) by both strains Sp7 and UPMB10 and lowest (5.0- 5.3%) under higher fertilizer-N (50 mg/kg) condition. However, the total amount of N2 fixed (9.03 to 9.55 mg/plant) at the low level (3.2 mg/kg) was not significantly higher compared to N2 fixed at higher (50 mg/kg) fertilizer-N level (8.85 to 9.69 mg/plant) (Table 5). activity of banana leaves as observed in the previous studies. The higher dry matter production in the PGPR inoculated plants, however, resulted in greater N yield of these plants, which corresponded positively to the increased N2 fixation and nutrient uptake. The results of the current experiment indicated that bacterial inoculation in combination with minimal fertilizer-N (20 mg/kg) increased N yields through a highly synergistic effect. Similarly, Marschner (1995) concluded that contribution of N2 fixation to the total N per plant is increased by moderate level of soil or fertilizer-N but declines at high levels in legumes. The higher N yield of the inoculated plants was due to effective N2 fixation because the PGPR association with roots of banana plantlets successfully contributed 37-39% fixed-N to the host plant as estimated by 15N isotope dilution technique. The results of this experiment are in agreement with the findings of several investigators (Dobereiner and Baldani 1998, Amir et al. 2001) who reported that Azospirillum and rhizobacterial inoculation could contribute a substantial amount (25-50% in oil palm; 40% in non- legumes) of the total plant N requirement through BNF process. Malik et al. (1997) have also reported that N2 fixation ability by Azoracus in association with kallar grass contributed 26% of the plant-N content. The evidence of N2 fixation was further supported by nitrogenase activity of inoculated roots as estimated by acetylene reduction assay (ARA). Rhizobacteria could persist, multiply and had the nitrogenase activity at the end of experimental period. Both the PGPR strains used produced higher ARA values compared to the uninoculated controls. Higher ARA activity could be attributed to the presence of more bacterial cells on the root. The study on root colonization supported this finding that PGPR strains Sp7 and UPMB10 could effectively colonize roots of banana plantlets. The present study clearly shows that rhizobacterial inoculation combined with minimal fertilizer-N (50 mg/kg) could fix 8.85 to 9.69 mg N/plant (5.0-5.3% Ndfa). The total amount of N2 fixed was further increased (11.0 mg/plant; 12.5% Ndfa) by lowering the fertilizer-N (20 mg/kg). Similarly, Galal et al. (2000) found negative effect of using high fertilizer-N on BNF by Azospirillum in association with wheat roots 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 conditions N2-fixed (mg/plant) Fertilizer-N levels (mg/kg) Treatments 3.2 8 20 50 Control 0 b (x) 0 b (x) 0 b (x) 0 b (x) Sp7 9.03 a (x) 10.11 a (x) 10.26 a (x) 8.85 a (x) UPMB10 9.55 a (x) 9.53 a (x) 10.85 a (x) 9.69 a (x) Means having same letter (s) in a column do not differ significantly at 0.05 level by DMRT Table 4. Percentage of N derived from atmosphere (%Ndfa) in banana plantlets (total plant basis) inoculated with PGPR strains Sp7 and UPMB10 and supplied with various fertilizer-N levels grown under hydroponic conditions % Ndfa %Ndfa Fertilizer-N levels (mg/kg) Treatments 3.2 8 20 50 Control 0 b 0 b 0 b 0 b Sp7 37.0 a 24.3 a 12.4 a 5.0 a UPMB10 39.3 a 22.1 a 12.5 a 5.3 a Means having same letter (s) in a column do not differ significantly at 0.05 level by DMRT Discussion Bacterial inoculation combined with fertilizer-N application significantly increased dry matter production in root, pseudostem and leaves of banana plantlets and consequently the total dry matter yield grown under hydroponics condition for 45 days. In general, the higher dry matter production resulted in increased N yield in the inoculated plants. The N yield is the final product of fixed N2 in the nitrogen nutrition of crop plants. Plants inoculated with bacteria produced higher N yield when supplied with fertilizer-N from 3.2 to 20 mg/kg; but did not show any increment at 50 mg/kg. This difference is likely because of a lower contribution of BNF in the presence of higher inorganic-N concentration in the nutrient solution. Nitrogen distribution to different plant parts were also increased by the inoculation process. The leaves produced the highest amount of N due to a higher demand for photosynthesis and other physiological functions. PGPR inoculation stimulated the photosynthetic InfoMusa - Vol. 16 No. 1-2, June & December 200714 InfoMusa - Vol. 16 No. 1-2, June & December 2007 15 where grain utilized most of the fixed-N2. The results suggested that PGPR inoculation and fertilizer-N (20 mg/kg) application have a synergistic effect on N2 fixation in banana plantlets grown under hydroponic conditions in small pots (4.0 l) for 45 days. Conclusion Plant growth promoting rhizobacterial strain Sp7 and UPMB10 inoculation in combination with minimal fertilizer-N increased the N yield in banana plantlets. Inoculated plants together with the least fertilizer-N supply (3.2 mg/kg, 2.13% of the total N requirement) produced the highest Ndfa (37-39%) while those with higher inorganic-N (50 mg/kg, 33% of the total N requirement) showed the lowest Ndfa (5%). However, PGPR inoculation with 20 mg/kg fertilizer-N (13% of total plant N) produced a synergistic effect on N2 fixation with higher amounts of N2 fixed (11.0 mg/plant; 12.5 % Ndfa). The results suggest that PGPR strains Sp7 and UPMB10 are effective as biofertilizer for banana plantlets when applied together with 20 mg/kg (13% of total requirement) fertilizer-N supply. References Amir H.G., Z.H. Shamsuddin, M.S. Halimi, M. F.Ramlan & M. Marziah. 2001. Effects of Azospirillum inoculation on N2 fixation and growth of oil palm plantlets at nursery stage. Journal of Oil Palm Research 13(1):42-49. Boddey R.M., O.C. Oliveira, S. Urquiaga, V. M. Reis, F.L. Olovares, V.L.D. Baldani & J. Dobereiner. 1995. Biological nitrogen fixation associated with sugar cane and rice: contribution and prospects for improvements. Plant and Soil 174:195-209. Bremner J.M. 1996. Nitrogen-Total. pp. 1085-1121 in Methods of Soil Analysis (Part 3). Chemical Methods. (Spark D.L., Page A.L., Helmke P.A., Loeppert R.H., Sultanpour P.N., Tabatabi M.A., Johnsto C.T. and Sumner M.E., eds). American Society of Agronomy, Wisconsin. Brimecombe, J.M., F.A. De Leij & J.M. Lynch. 2001. The effect of root exudates on rhizosphere microbial population. Pp 95-140 in The Rhizosphere. (Pinton R. Varanini Z. and Nannipieri P., eds). Marcel Dekker Inc. New York, USA. Cocking E.C. 2000. Helping plants get more nitrogen from air. European Review 8(2):193-200. Cooper A. 1979. The ABC of NFT. Grower Books. London, 181pp. Dobereiner J. & V.L.D. Baldani. 1998. Biological nitrogen fixation by endophytic diazotrophs in non-legume crops in the tropics. Pp3-7 in Nitrogen Fixation with Non- legumes. (Malik K.A., Sajjad Mirza M.and Ladha J.K., eds). Kluwer Academic Publishers, London. El-Komy, H.M.A., T.M.M. Moharram & M.S.A. Safwat. 1998. Effects of Azospirillum inoculation on growth and N2 fixation of maize subjected to different levels of FYM using 15N-dilution method. Pp. 49-59 in Nitrogen Fixation with Non-legumes. (Malik K.A., Sajjad Mirza M.and Ladha J.K., eds). Kluwer Academic Publishers, London. Fried M. & Middelboe V. 1977. Measurement of amount of nitrogen fixed by a legume crop. Plant and Soil 47: 713-715. Galal Y.G.M., L.A. El Ghandour & S.S. Aly. 2000. Non- isotopic method for the quantification of biological nitrogen fixation and wheat production under field conditions. Biology and Fertility of Soils 32:47-51. Jensen E.S., A.J. Andersen & J.D. Thomsen. 1985. The influence of seed-borne N in 15N isotope dilution studies with legumes. Acta Agriculture of Scandinavian 35:438-443. Malik K.A., B. Rakhshanda, S. Mehnaz, G. Rasul, M.S. Mirza & S. Ali. 1997. Association of nitrogen-fixing plant-growth promoting rhizobacteria (PGPR) with kallar grass and rice. Plant and Soil 194:37-44. Malik K.A. & Y. Zafar. 1985. Quantification of root- associated nitrogen fixation as estimated by 15N isotopic dilution. Pp 161-171 in Nitrogen and the Environment (Malik K.A., Bhatti N.A. and Kausar F., eds). Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan. Marschner H. 1995. Functions of Mineral Nutrients: Macronutrients. Pp. 889 in Mineral Nutrition of Higher Plants. Academic Press, London. Okon Y., S.L. Albrecht & R.H. Burris. 1977. Methods for growing Spirillum lipoferum and for counting it in pure culture and different solution in association with plants. Applied and Environmental Microbiology 33:85-88. SAS. 1989. SAS/STAT. Guide to Personal Computers. Version 6.12. SAS Institute Inc., Cary, North Carolina. Shamsuddin Z.H. & A.W. Roslina. 1995. Isolation of Azospirillum from oil palm roots. 18th Microbiology Symposium, Kuching, Sarawak. Shrestha R.K. & J.K. Ladha. 1996. Genotypic variation in promotion of rice nitrogen fixation as determined by Nitrogen-15dilution. Soil Science Society America Journal 60(6):1815-1821. Warembourg F. R. 1993. Nitrogen fixation in soil and plant system. Pp 127-155 in Nitrogen Isotope Technique (Knowles R. and Blackburn T. H., eds). Academic Press, London. Md. Abdul Baset Mia is Associate Professor Department of Crop Botany, Bangabandhu Sheikh Mujibur Rahman Agricultural University Gazipur 1706, Bangladesh, e-mail: miabaset@yahoo.com, Zulkifli H. Shamsuddin and Zakaria Wahab work respectively at the Department of Land Management and Department of Crop Science, Faculty of Agriculture, and Marziah Mahmood at the Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. InfoMusa - Vol. 16 No. 1-2, June & December 200716 InfoMusa - Vol. 16 No. 1-2, June & December 2007 17 Fusarium wilt disease is a serious constraint in banana production worldwide (Stover 1962). Its causal agent, Fusarium oxysporum f. sp. cubense (Foc) is a genetically stable soil-borne fungus. The identification of wilt disease- producing strains is a big challenge scientists are facing because suitable methods for testing the virulence of the disease in greenhouse conditions are still not efficient. However, Sun and Su (1984) developed a method using tissue-cultured plantlets to study virulence, but the availability of such plantlets for all cultivars is a problem with this method. Hence, the aim of this research was to develop a suitable method to measure the virulence of Foc race 1 in banana. Materials and methods The experiment was carried out at the Department of Plant Pathology, Agricultural College and Research Institute, Madurai, India. Fusarium wilt (race 1) affected banana plants (cv Rasthali) were collected from banana growing areas of southern Tamil Nadu. Suckers showing brown discoloration were used for isolation of Foc. The suckers showing typical symptoms of the disease were washed in water and cut into small pieces using a sterilized scalpel and surface sterilized in 0.1 % mercuric chloride for 30 sec followed by washing in several changes of sterile distilled water. Sterilized potato dextrose agar (PDA) medium amended with 100 ppm of strephtomycin sulphate (to avoid bacterial contamination) was poured into sterile Petri plates at 15 ml per plate and 3 surface sterilized plant pieces were placed at equidistance in each plate. All these were carried out under aseptic condition. The plates were incubated at room temperatures (28± 2°C) for 7 days and observed for the fungal growth. The fungus was purified by a single spore isolation technique, transferring a single spore into PDA slant and incubated at 28°C for five days. The fungus was then multiplied in a sand medium (Ricker and Ricker 1936). Fine sand and maize flour were mixed at a ratio of 19:1 and sterilized at 1.4 kg/cm2 pressure for 2 hours. The sterilized sand medium was inoculated by transferring a disc (9mm) of the pathogenic culture grown on PDA plate into sterilized sand medium and shaken for 1 min in order to spread over the medium. All these were carried out under aseptic condition. They were incubated for a period of 3 weeks at room temperature (28 ± 2ºC). The fully grown inoculum of Foc was used for the virulence tests. Spore suspension was prepared by adding sterile distilled water to fungus grown in PDA slants at 10 ml per slant. The slant was gently shaken for 5 min and suspension was filtered through a muslin cloth. The filtered suspension was used as spore suspension for corm injection and sucker dipping in the study. Seven treatments were included in the study: T1 Sand inoculum of the isolate at 10% w/v of tank soil, T2 Sand inoculum of the isolate at 15% w/v of tank soil, T3 corm injection of the isolate at 3 ml/plant + Sand inoculum of the isolate at 10% w/v of tank soil, T4 sucker dipping for 30 min (106 colony forming unit of the isolate per ml - cfu/ml) + Sand inoculum of the isolate at 10% w/v of tank soil, T5 sucker dipping for 30 min (106 cfu/ml), T6 infected plant parts at 10% w/v of tank soil and T7 infected soil (105 cfu/g of soil) at 10% w/v of soil. Corm injection was done by injecting 3 ml of spore suspension (106 cfu/ml) of the isolate at the base of pseudostem of 3 month-old suckers of cv Rasthali using banana injector TNAU model. The suspension was injected into corm at an angle of 45°. Infected soil was collected from banana field where natural infection of the Fusarium wilt was observed as more than 80%. The soil was taken from rhizosphere regions of the infected plants. 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 Measuring Fusarium virulence InfoMusa - Vol. 16 No. 1-2, June & December 200716 InfoMusa - Vol. 16 No. 1-2, June & December 2007 17 The infected banana plants including pseudostem and corm were taken from the naturally infected field. They were then cut into small pieces and used in the study. Three month-old suckers of cv Rasthali were planted in tanks (90 x 60 x 45 cm). Each tank contains 10 suckers and was treated as a replicate, of which there were 4 per treatment- representing 40 suckers per treatment- arranged in randomized blocks. The tanks were kept in a screened house, regularly watered and constantly observed for disease symptoms. Three months after the initiation of the experiment, the incidence of wilt and vascular discoloration were assessed. The wilt incidence was assessed using the scale described by Ploetz et al. (1999): 1: Healthy, 2: Slight cholorosis and wilting with no petiole buckling, 3: Moderate chlorosis and wilting with some petiole buckling and or splitting of leaf bases, 4: Severe chlorosis, severe wilting, petiole buckling and dwarfing of the newly emerged leaf, 5: dead. The per cent wilt index was worked out using Mc Kinney’s (1923) formula: Total sum of numerical ratings x 100 Total number of plants observed x Maximum category value in the scale The plant was pulled out and vascular discoloration was identified by cutting of suckers horizontally. The percentage of the vascular discoloration was worked out by the scale described by Orjeda (1998): 1: Corm completely clean, no vascular discoloration, 2: Isolated points of discoloration in vascular tissue, 3: Discoloration of up to 1/3 of vascular tissue, 4: Discolouration of between 1/3 and 2/3 of vascular tissue, 5: Discoloration greater than 2/3 of vascular tissue, 6: Total discoloration of vascular tissue. The per cent vascular discoloration index was worked out using Mc Kinney’s (1923) formula described above. Results and discussion Five isolates of Foc were isolated from diseased suckers and used for this study. After three months, corm injection (T3) had induced the highest per cent wilt index, followed by sucker dipping (T4) (Table 1). The highest vascular discoloration incidence was also recorded in T3 and T4 (Table 2). Of the isolates, Foc 3 produced the highest wilt and vascular discoloration index in the plants. Earlier, Stover (1959) found that banana plants showed wilting after three months of pathogen inoculations. Hadi et al. (1987) observed that inoculation of banana roots with Foc induced the lesions after one week and mechanical punctures allowed the pathogen to colonize the cells in the cortex causing reddish brown lesions. Sivamani and Gnanamanickam (1998) reported that a 10% rice/sand mixture inoculum of Foc showed severe internal discoloration in corm after three to four weeks of pathogen inoculation. In our study, corm injection of the pathogen may have facilitated internal colonization, and soil application of the pathogen created 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 InfoMusa - Vol. 16 No. 1-2, June & December 200718 InfoMusa - Vol. 16 No. 1-2, June & December 2007 19 pathogen to colonize inside the cell system. Hence, corm injection of spore suspension of the isolate at 3 ml/plant + soil application of sand inoculum of the isolate at 10% w/v of tank soil should prove to be a rapid method for testing the virulence. References Hadi M.A.A., F. Fadel & A.I. Ghorab. 1987. Root rot of bananas and its control in Egypt. Pp. 161: 171 in Proceedings of the Conference of the Agricultural Development Research, Cairo, Egypt. Orjeda G. 1998. Evaluation of Musa germplasm for resistance to Sigatoka disease and Fusarium wilt. INIBAP Technical Guidelines 3. Inibap, Montpellier, France. p. 29. Mc Kinney H.H. 1923. A new system of grading plant diseases, Journal of Agricultural Research 26:195-218 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 Ploetz R.C., Haynes & A. Vazquez. 1999. Responses of new banana accessions in South Florida to Panama disease. Crop Protection 18(7):445-449. Ricker A.J. & R.S. Ricker. 1936. Introduction to research on plant diseases. Johns Swift Co. Mc., New York. 117pp. Sivamani E. & S.S. Gnanamanickam. 1988. Biological control of Fusarium oxysporum f. sp. cubense in banana by inoculation with Pseudomonas fluorescens. Plant Soil 107(1): 3-9. Stover R.H. 1959. A rapid and simple pathogenecity test for detecting virulent clones of Fusarium oxysporum f. sp. cubense using seedlings of Musa balbisiana. Nature 184:1591-1592. Stover R.H. 1962. Fusarial wilt (Panama disease) of bananas and Musa species. Common Wealth Mycological Institute, Kew, Surrey, UK, p 117. Sun E.J. & H.J. Su. 1984. Rapid method for determining differential virulence of Fusarium oxysporum f.sp. cubense using banana plantlets. Tropical Agriculture (Trinidad) 61(1):7-8. Bananas (Musa spp.) are grown in many districts of Kenya. They currently cover an area of 40 000 ha, producing 510 000 tonnes (FAOstat 2006). In the higher western regions of Kenya, the East African highland bananas (EAHB) (Musa AAA, ‘Matooke’ and ‘Mbidde’ cultivars) are most common, while in the central and eastern highlands and coastal areas, dessert cultivars are the most popular, especially ‘Cavendish’ and ‘Gros Michel’, which are an important component of diets and household incomes (INIBAP 1986). Banana production is faced by a number of constraints, although a complex of plant-parasitic nematodes, banana weevil, poor agronomic practices, Fusarium wilt, black Sigatoka disease and poor soil fertility combine to adversely affect yields in Kenya (Nguthi 1996, Inzaule et al. 2005). The plant-parasitic nematodes previously reported from Musa in Kenya include 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 T. Saravanan works at the Agricultural Research Station, Kovilpatti – 628 501, Tamil Nadu, India, e-mail: pathsaran75@rediffmail.com M. Muthusamy, E.G. Ebenezar and R. Bhaskaran work at the Department of Plant Pathology, Agricultural College and Research Institute, Madurai – 625 104, India InfoMusa - Vol. 16 No. 1-2, June & December 200718 InfoMusa - Vol. 16 No. 1-2, June & December 2007 19 Pratylenchus goodeyi, Radopholus similis and Helicotylenchus multicinctus (Gichure and Ondieki 1977, Inzaule et al. 2005). In neighbouring Uganda, severe production losses on EAHB, as a result of R. similis and H. multicinctus infection, have been documented (Speijer et al. 1994, Speijer et al. 1999, Gowen et al. 2005). Pratylenchus goodeyi is also common on Musa at altitudes above 1400 masl in Uganda, while R. similis tends to be more important below 1400 masl (Kashaija et al. 1994). H. multicinctus is highly prevalent across Uganda, but R. similis would appear to be responsible for the majority of the damage caused by nematodes to Musa. With interest in Musa production increasing in Kenya, surveys to establish nematode species profiles on Musa throughout the country were undertaken between 1990 and 1993. Details of the cropping systems and management practices relating to the farms visited during the current study have been presented previously, and included details of the distribution of Musa insect pests and preliminary information on nematode observations (Prasad and Seshu Reddy 2000). The current report, however, provides specific details concerning nematodes, including from the preliminary (pre-survey) studies undertaken within farms in Oyugis district, in addition to data from the nationwide survey assessed by Musa group. Materials and methods In 1990 observations were made on the nematode species profile on bananas in Oyugis district. The country-wide survey was conducted in 1993 across thirteen primary Musa growing districts (see Table 1). Oyugis district survey In Oyugis district, six farms, each with a minimum of ten mats/plant, aged between 2-3 years, of each of the two cultivars ‘Nakyetengu’ (Musa AAA, Matooke) and ‘Sukali Ndizi’ (Musa AB), were selected. On each farm, five mats per cultivar were sampled at the base of the most advanced plant in each mat. All the roots from an excavation of 20 cm x 20 cm x 20 cm, made using a spade, were bulked by cultivar for each farm and transported in a plastic bag in a cool-box to ICIPE’s Mbita Point Field Station for processing. Samples were stored overnight at 4°C in a fridge before being processed the following day. Roots were cut into ca. 1 cm segments, thoroughly mixed, and 5 x 5 g sub-samples were randomly removed for nematode extraction. Sub- samples were macerated for 2 s using a kitchen blender prior to extraction using a modified Baermann method for 48 h (Hooper et al. 2005). The recovered nematodes were identified and counted in 3 x 2 ml aliquots removed from a 25 ml suspension for each sub-sample. In the case of high nematode densities, the suspension was diluted to 50 ml or 75 ml. Mean nematode densities per farm per cultivar were determined and 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. InfoMusa - Vol. 16 No. 1-2, June & December 200720 InfoMusa - Vol. 16 No. 1-2, June & December 2007 21 order to assess within-site variability, three adjacent mats per cultivar were selected and sampled from a single farm, Silpa Odak, where root samples were collected and processed separately for each mat. Nationwide survey In the country-wide survey, one farm per banana-producing district was selected randomly for sampling where both EAHB and exotic cultivars were present. Three plants per Musa type were randomly selected on each farm and assessed for nematode incidence. Mats were sampled as in Oyugis district. Detached roots were bulked per cultivar per farm and transported to the laboratory at Mbita Point Field Station, where they were held at 4°C in the fridge until being processed within a few days. Roots of each sample were chopped finely (ca. 2-5 mm) and thoroughly mixed. Nematodes were extracted from a 10 g root sub-sample, removed from the whole sample and placed directly on a modified Baermann dish for ca. 48 h. Nematodes were identified and counted as above. Nematode data were subjected to ANOVA using Ln(x+1) transformed densities with the SAS statistical package. Data were presented as nematodes per 100 g root prior to transformation. Results Oyugis District Considerable variation in nematode densities between farms within Oyugis district was observed with P. goodeyi present at higher densities than other nematodes (Table 2). Radopholus similis was observed on three of the six farms on ‘Nakyetengu’, but at none on ‘Sukali Ndizi’, while Meloidogyne spp. (second stage juveniles) were observed on ‘Sukali Ndizi’ on all farms, but on only three farms on ‘Nakyetengu’. At the farm of Silpa Odak nematode root densities differed (P<0.05) between mats for the same cultivar, despite the fact that mats of similar age were sampled (Table 3). Although not subjected to analysis, the nematode species profile between cultivars appeared to vary considerably, especially for P. goodeyi and H. multicinctus. This apparent large variation therefore provided a basis for the separate sampling of exotic and EAHB cultivars in the country-wide survey. Nationwide survey The lesion nematode, P. goodeyi, was widespread on EAHB in most districts surveyed, except Busia near the Ugandan border and at Kwale at the coast (Table 1). In both the low altitude coastal districts of Kwale and Kilifi however, P. goodeyi was observed on exotic bananas. The presence of R. similis 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. 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 InfoMusa - Vol. 16 No. 1-2, June & December 200720 InfoMusa - Vol. 16 No. 1-2, June & December 2007 21 was limited to Oyugis and Homa Bay districts only. In Kwale and Kilifi H. multicinctus and in Kilifi Meloidogyne spp., were recorded only from exotic banana cultivars and not EAHB. In Muranga district P. coffeae was recovered extensively from both EAHB and exotic banana and was also recorded on exotic bananas in Nyeri and Vihiga districts. The nematodes Rotylenchus clavicaudatus, Scutellonema spp., Cricomema spp., Xiphinema spp, Hemicycliophora spp. and an undescribed species of Trophurus were also observed infrequently. Discussion The most widespread nematode observed attacking EAHB and exotic bananas in Kenya in 1993 was P. goodeyi. It was also found at the highest densities. H. multicinctus was also prevalent, while R. similis, which is prevalent in neighbouring Uganda and Tanzania, was observed at relatively low densities and infrequently. The relatively high density at which some of the nematode species was observed would likely correspond to levels suggesting they are causing production losses (Gowen et al. 2005). However, while there are good data establishing the adverse effect of R. similis on Musa, the relation between P. goodeyi, Meloidogyne spp., H. multicinctus and Musa is less clear, although information relating to their damage potential is gradually increasing (Walker et al. 1984, Barekye et al. 1999, Bridge 2000, Ssango et al. 2004, Moens et al. 2005). There also appears to be a strong Musa genotype influence when considering the association of these nematodes (Pinochet and Rowe 1978, Hartman J., IITA, unpublished), although the highly pathogenic nature of P. coffeae on Musa is becoming increasingly apparent (Speijer et al. 2000, Sundararaju 2001, Brentu et al. 2004). Of particular interest from the current study therefore, are the relatively high densities of P. coffeae in Muranga, and its occurrence in Nyeri and Vihiga districts. Previously P.coffeae was not observed on Musa in Kenya (Gichure and Ondieki 1977), but appears to be increasingly observed in East Africa, albeit erratically. In North West Tanzania, for example, it was not recorded in 1984 (Walker et al. 1984), but was soon after (Sikora et al. 1989), while it was not detected in Zanzibar in 1992 (Maas 1992), but occurred frequently more recently (Rajab et al. 1999). Given the damage potential observed from studies on plantain in West Africa (Speijer et al. 2001, Brentu et al. 2004), P. coffeae appearance on Musa in the region is a cause for concern, even though ‘P. coffeae’ appears to be composed of a number of closely related species which may differ in their pathogenic potential and host specificity (e.g. Duncan et al. 1999). Currently the effect of the nematode on EAHB and dessert banana is not known. Of additional interest is the observation of P. goodeyi at the low altitude sites on the Kenyan coast. Pratylenchus goodeyi is usually found at higher altitudes (above 1400 masl in mainland Africa) where conditions are cooler (which it is less able to survive) than at lower altitudes (Bridge et al. 1995). Its occurrence therefore on the relatively hot coastal areas of Kenya is unusual. With the exception of its occurrence in the Canary Islands (Price 2000) where it is a problem on commercial dessert bananas, this would appear to be a unique observation on mainland Africa, and perhaps an indication of a heat-tolerant strain. It is not known whether the P. coffeae in Muranga district is native or was introduced through imported material. Its presence is of potential concern, however, while information on its current distribution and pathogenicity would be useful to the Kenyan Musa industry. Similarly, the presence of P. goodeyi at the coast may possibly be a result of introduction, and may indicate that sampling in the current study may have taken place within a relatively short period after its introduction on planting material, before the nematode died out. It would therefore be similarly useful to establish the current situation and, if necessary, take avoiding action. The infrequent occurrence of other nematode species is not viewed as a cause for any concern, although other species, such as Rotylenchus spp. are recognized as serious pests in some Musa production areas (De Waele et al. 2000). The results of the current study reinforce the need for the assessment of introduced Musa material against key pests such as P. coffeae as well as R. similis. It also reinforces the need to restrict the movement of (potentially) infected material, and / or to improve the promotion and dissemination of information on the use of Musa sanitation practices and use of healthy planting material. The current study was undertaken some years ago, InfoMusa - Vol. 16 No. 1-2, June & December 200722 InfoMusa - Vol. 16 No. 1-2, June & December 2007 23 and its publication has unfortunately been delayed due to unforeseen circumstances. It remains however highly relevant, providing an outline of the plant-parasitic nematode distribution on Musa in Kenya upon which to guide future activities and studies to benefit the industry. In our study, the high level of variation in nematode species’ profiles between sites and cultivars creates difficulty in assessing their importance to Musa in Kenya. However, with the relatively high densities observed and the wide variation of nematode species, including some which are associated with severe production loss to bananas in other countries, considerable losses due to nematodes are to be expected. Screening of popular cultivars or those to be released in Kenya against such nematodes is recommended. Acknowledgments Dedicated to the memory of Paul Speijer. The authors wish to thank the Federal Ministry of Economic Cooperation (BMZ), Germany for its financial support to conduct this research. References Barekye A., I.M. Kashaija, E. Adipala & W.K. Tushemereirwe. 1999. Pathogenicity of Radopholus similis and Helicotylenchus multicinctus on bananas in Uganda. Pp. 319–326 in Mobilizing IPM for sustainable banana production in Africa, Proceedings of a workshop on banana IPM, Nelspruit, South Africa 23-28 November 1998 (E.A. Frison, C.S.Gold, E.B. Karamura and R.A. Sikora, eds). INIBAP, Montpelier, France. Brentu C.F., P.R. Speijer, K.R. Green, B.M.S. Hemeng, D. De Waele & D.L. Coyne. 2004. Micro-plot evaluation of the yield reduction potential of Pratylenchus coffeae, Helicotylenchus multicinctus and Meloidogyne javanica on plantain cv. Apantu-pa (Musa spp., AAB-group) in Ghana. Nematology 6:455-462. Bridge J. 2000. Nematodes of bananas and plantains in Africa: research trends and management strategies relating to the small-scale farmer. In Proceedings of the first international conference on banana and plantain for Africa. Acta Horticulturae 540: (Craenen, K., R. Ortiz, E.B. Karamura & D.R. Vuylsteke, eds). Acta Horticulturae 540:391-408. Bridge J., N.S. Price & P. Kofi. 1995. Plant parasitic nematodes of plantain and other crops in Cameroon, West Africa. Fundamental and Applied Nematology 18: 251-260. De Waele D., S.R. Gowen, M. Tessera & A.J. Quimio. 2000. Other Nematodes. Pp. 314–315 in Diseases of bananas, abacá and enset (D.R. Jones, ed.). CAB International, Wallingford, UK. Duncan L.W., R.N. Inserra, W.K. Thomas, D. Dunn, I. Mustika, M.L. Frisee, L.M. Mendes, K. Morris & D.T. Kaplan. 1999. Molecular and morphological analysis of isolates of Pratylenchus coffeae and closely related species. Nematropica 29:61-80. FAOstat. 2006. Food and Agriculture Organization of the United Nations, Rome. Gichure E. & J.J. Ondieki. 1977. A survey of banana nematodes in Kenya. Zeitshchrift fur Pflanzenkrankeiten und Pflanzenschutz 84:724-728. Gowen S.C., P. Quénéhervé & R. Fogain. 2005. Nematode parasites of bananas and plantains. Pp. 611-643 in Plant parasitic nematodes in subtropical and tropical agriculture, 2nd ed. (M. Luc, R.A. Sikora and J. Bridge, eds). CAB International, Wallingford, U.K. Hooper D.J., J. Hallman & S. Subbotin. 2005. Methods for extraction, processing and detection of plant and soil nematodes. Pp. 53-86 in Plant parasitic nematodes in subtropical and tropical agriculture, 2nd ed. (M. Luc, R.A. Sikora and J. Bridge, eds). CAB International, Wallingford, U.K. INIBAP. 1986. Banana research in Eastern Africa. Proposal for a regional research and development network. INIBAP/86/3/001 rev. INIBAP, Montpellier, France. 106pp. Inzaule S.S.S., F. Kimani, S. Mwatuni & M. Makokha. 2005. Status of banana pests and diseases in Western Kenya. African Crop Science Proceedings 7:309-312. Kashaija I.N., P.R. Speijer, C.S. Gold & S.R. Gowen. 1994. Occurrence, distribution and abundance of plant parasitic nematodes on bananas in Uganda. Preliminary results of a diagnostic survey. African Journal of Crop Science 2:99-104. Maas P.W. 1992. Observations on plant parasitic nematodes in bananas and some other crops in Zanzibar. 12-26 July, 1992. IPO-DLO Report No. 92-14, DLO. Research Institute for Plant Protection, Wageningen, The Netherlands. Moens T., M. Araya, R. Swennen & D. De Waele. 2005. Screening of Musa cultivars for resistance to Helicotylenchus multicinctus, Meloidogyne incognita, Pratylenchus coffeae and Radopholus similis. Australasian Plant Pathology 34:299-309. Nguthi F. 1996. Banana production and research in Kenya. MusAfrica 12. Pinochet J. & P.R. Rowe. 1978. Reaction of two banana cultivars to three different nematodes. Plant Disease Reporter 152:727-729. Prasad J.S. & K.V. Seshu Reddy. 2000. Pest problems of banana in Kenya: a survey report. Pest Management in Horticultural Ecosystems 6:50-54. Price N.S. 2000. The Biogeography of the Banana Nematodes Radopholus similis and Pratylenchus goodeyi in Proceedings of the First International Conference on Banana and Plantain for Africa, Kampala, Uganda, 14 -18 October 1996. (K. Craenen, R. Ortiz, E.B. Karamura & D.R. Vuylsteke, eds). Acta Horticulturae 540:431-440. Rajab K.A., S.S. Salim & P.R. Speijer. 1999. Plant- parasitic nematodes associated with Musa in Zanzibar. African Plant Protection 5:105-110. Sikora R.A., N.D. Bafokazara, A.S.S. Mbwana, G.W. Oloo, B. Uruno & K.V. Seshu Reddy. 1989. Interrelationships between banana weevil, root lesion nematode and agronomic practices and their importance for banana decline in Tanzania. FAO Plant Protection Bulletin 37(4): 151-157. Speijer P.R., C.S. Gold, E.B. Karamura & I.N. Kashaija. 1994. Assessment of nematode damage in East African Highland banana systems. Pp. 191-203 in Banana nematodes and weevil borers in Asia and the Pacific: Proceedings of a conference/workshop on nematodes and weevil borer affecting banana in Asia and the Pacific, Kuala Lumpur, Malaysia, April 18-22, 1994, Los Baños, Laguna, Philippines, (R.V. Valmayor, R.G. Davide, J.M. Stanton, N.L. Treverrow and V.N. Roa, eds). INIBAP/ ASPNET, Los Baños, Philippines. K.V. Seshu Reddy – formerly researcher at the International Centre of Insect Physiology and Ecology (ICIPE) - P.O. Box 30772, Nairobi, Kenya, his current address is 202 K.K. Heights, Masab Tank, Hyderabad-500 028, India. J.S. Prasad - formerly researcher at ICIPE, can now be contacted at the Directorate of Rice Research, Rajendranagar, Hyderabad-500 030, India. R.A. Sikora is a professor at the University of Bonn, Nussalee 9, 5300 Bonn, Germany. D.L. Coyne (corresponding author, e-mail: d.doyne@cgiar.org) works at the International Institute of Tropical Agriculture (IITA) – P.O. Box 7878, Kampala, Uganda - correspondence address: c/o Lambourn & Co., Carolyn House, 26 Dingwall Road, Croydon CR9 3EE . Paul Speijer also worked at IITA-Uganda. He died on 30 January 2000. InfoMusa - Vol. 16 No. 1-2, June & December 200722 InfoMusa - Vol. 16 No. 1-2, June & December 2007 23 Speijer P.R., C. Kajumba & F. Ssango. 1999. East African highland banana production as influenced by nematodes and crop management in Uganda. International Journal of Pest Management 45:41-49. Speijer P.R., M.O. Rotimi & D. De Waele. 2001. Plant parasitic nematodes associated with plantain (Musa spp., AAB-group) in Southern Nigeria and their relative importance compared to other biotic constraints. Nematology 3:423-436. Speijer P.R., F. Ssango & D. Vuylsteke. 2000. Evaluation of host plant response to nematodes in Musa germplasm in Uganda in Proceedings of the First International Conference on Banana and Plantain for Africa, Kampala, Uganda, 14-18 October 1996 (Craenen K., R. Ortiz, E.B. Karamura and D.R. Vuylsteke, eds). Acta Horticulturae 540:225-232. Ssango F., P.R. Speijer, D.L. Coyne & D. De Waele, 2004. Path Analysis: a novel approach to determine the contribution of nematode damage to East African Highland banana (Musa spp., AAA) yield loss under two crop management practices in Uganda. Field Crops Research 90:177-187. Sundararaju P. 2001. Research on nematodes at the National Research Centre for banana in India. INFOMUSA 10:16-18. Walker P.Y., M.J. Hebblethwaite & J. Bridge, 1984. Project for Banana Pest Control and Improvement in Tanzania. EEC Report for the Government of Tanzania. London. TDRI. 129pp. Fungi promote growth of bananas 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 A rbuscular mycorrhizal fungi (AMF) are endophytic and biotrophic mutualistic symbionts that colonize most crops’ roots. Mycorrhization increases the capacity to absorb certain mineral nutrients and is particularly efficient in the assimilation of phosphorus. The mycorrhizal infection causes physical, biochemical and physiological changes in the colonized roots that lead to improvements in the general condition of the plant and help to alleviate biotic and/or abiotic stresses (Barea et al. 2004). The benefits from inoculating micropropagated banana plants with AMF during the first stages of growth have already been clearly proved (Yano- Melo et al. 1999). The beneficial effects of early mycorrhization of this crop have even been detected one year after inoculation in plants that have been in the field for nine months (Jaizme-Vega et al. 2002). Since AMF are obligate symbionts, it is impossible to multiply and reproduce them in the absence of live roots, so this has been a serious limitation to the production of inocula on a large scale. Nevertheless, over the last few years, several substrata and methods have been described for the production of inocula for horticulture, fruitgrowing and forestry (von Alten et al. 2002). Today, the optimization and commercialization of arbuscular mycorrhizal inoculants on a large scale represents the biggest difficulty facing the necessary transfer of this biotechnology from the scientific field to the agricultural sector. The increasing number of small and medium-sized businesses producing inoculants bears witness to the high demand of this kind of product. Its publicity is based on conceiving AMF as a guarantee of healthy and beneficial products of natural origin (von Alten et al. 2002). Aspects such as the content of nutrients of the inoculant, its medium, pH and recommended doses are part of the information that must be provided to consumers (von Alten et al. 2002). However, the density of propagules (quantification of infective units) or the concentration of spores constitute quality criteria for these products. However, the quality control of AMF-based products is so far carried out only by the companies themselves, there being no public regulations on such checks (von Alten et al. 2002). This experiment was undertaken with the purpose of comparing the effectiveness of three AMF-based products. At the first stage, the optimum dose of each product was determined on the basis of growth- related and economic criteria (amount of product consumed). Once the optimum dose for each formulation was established, a comparative study was done to determine the effectiveness of the products. InfoMusa - Vol. 16 No. 1-2, June & December 200724 InfoMusa - Vol. 16 No. 1-2, June & December 2007 25 Materials and methods The experiment was carried out in the Plant protection laboratory of the Instituto Canario de Investigaciones Agrarias in La Laguna, Tenerife, Canary Islands during 2005. The material used was micropropagated Musa acuminata Colla (AAA) cv. Petite Naine (Dwarf Cavendish) and cv. Gruesa (a commercial cultivar widely used in Canary Islands). The plants, which were 7±1 cm tall and had 2-3 fully grown leaves, were received on an agar medium in a glass bottle under aseptic conditions. They were carefully taken out of the bottles to wash the roots and eliminate the remains of the agar. The roots were cut, leaving 0.5-1 cm long ends. Then, the plants were transplanted to black polyethylene multi-pot trays, each of which contained 15 cells of 250 ml. The cells were filled with a steam-sterilized substrate consisting of a mix of equal parts of soil, volcanic ash and peat (TKS1 Instant, Floragard, Germany). The AMF products were applied at the time of transplanting. The Table 1 below shows the characteristics of the products and the doses. The inoculation was carried out by adding the amount of product volume determined for each dose right underneath the plants. The decision on the doses to apply was made after consulting the manufacturers. The intermediate dose of each formulation was that recommended by the growers. This was the starting dose, which was then increased or decreased, depending on the experimental conditions, with special attention being paid to the volume of substrate. Each treatment (dose and type of inoculum) was replicated 10 times. There was also a control treatment (no application), also consisting of 10 replications. The multi-pot trays were put into the greenhouse under black mesh and plastic, and at controlled temperature (25ºC) and humidity (90%). The plants were irrigated with distilled water (50-75 ml/plant, depending on their needs). The stage of acclimatization ended eight weeks after the trial setup. At this time, the plants were moved into individual 2-liter pots of black polyethylene with a steam sterilized medium consisting of equal parts of soil, volcanic ash and peat (TKS1Instant). Each pot was fertilized with 0.5 g of Osmocote 17-10-10, a slow-release mineral fertilizer. The test lasted for another eight weeks after transplanting into the individual-pot stage, after which time the plants were taken out of the pots and analysed to determine the percentage of mycorrhizal colonization of their roots (Koske and Gemma 1989) and the following variables associated with growth response: fresh weight of the roots and aerial parts (g), pseudostem and root length (cm) and leaf area, which was measured with a Li-3100 area meter (LI-COR, USA). Since the inoculants and doses were evaluated in the same test, the data were grouped in two different ways: a) comparison between different doses of the same inoculant and selection of the optimum dose of each; b) comparison between the three inoculants. The data were statistically analysed through ANOVA and the averages were compared through Tukey’s multiple range test (P≤0.050). All this was performed with Systat 7.0 software (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’. InfoMusa - Vol. 16 No. 1-2, June & December 200724 InfoMusa - Vol. 16 No. 1-2, June & December 2007 25 Results Selection of the optimum dose for each product Effects on plant growth Each inoculant behaved in a different way depending on the dose used, there being no evidence of a direct relationship between dose and effect. Although all the products and doses tested resulted in a better performance than that of the control plants, such improvement was not always significant for each and every one of the growth variables measured. The positive effect of Endorize Mix was significant for dose II (Table 2). The highest values of the five variables measured were recorded with this dose. Doses I and III did not cause a significant a priori increase in growth. This fact, considered together with the amount of product used, led us to select dose II as the optimum one. In the case of Terra Vital, and evaluating all the variables combined, it was the lowest dose (dose I = 12ml) that had the maximum effect on the plants (Table 3). The biomass production of the plants inoculated with dose I outperformed that of those plants that received 25 and 40 ml. However, the differences between doses were not significant in statistical terms, in spite of which dose I was selected as the optimum one, since the highest values were recorded with it for most of the variables studied. Furthermore, economic criteria were considered in the selection of such dose. The study of TRI TON revealed that there were no significant differences between doses for 4 out of the 5 variables of growth (Table 4). Despite that, dose I (10 ml) seems to be the most effective and economically attractive. Mycorrhizal colonization The mycorrhizal colonization of the root turned out to be another indicator of the differing behaviours of the products (Figure 1). In the case of Endorize Mix, the percentage of colonization increase did not surpass 35%, there being a direct correlation between dose and colonization. Terra Vital showed the most variable results: whereas 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). InfoMusa - Vol. 16 No. 1-2, June & December 200726 InfoMusa - Vol. 16 No. 1-2, June & December 2007 27 of up to 63%, doses I and II generated increases of 29 and 23%, respectively. In this case, the data turned out clearly divided into two statistical groups. As for TRI TON, it showed a similar behaviour to that of Endorize, with a direct relation between root colonization and doses, as well as significant differences between levels I and III (23 and 48%, respectively). The combined results concerning mycorrhizal colonization and plant growth made it possible to confirm the selection of the optimum dose for each product. The doses selected for the next stage of the study were 10 ml for Endorize Mix and TRI TON and 12 ml for Terra Vital. Comparative assessment of the three products The data obtained show that Endorize Mix had a significant advantage over the other two products for most of the growth variables 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, that is, higher than the control group by 30%. As for TRI TON, it only increased the aerial biomass by 15%. The growth estimated in terms of plant height repeated this trend, with some variations (Figure 2). Here, Terra Vital proved to be the most effective, with a significant increase in height of 18% in comparison to the control plants. Endorize Mix increased the plant height by 15%. Unlike TRI TON, these two treatments were statistically grouped together. Regarding impact on the leaf area, this variable showed similar results (Figure 3). Here, Endorize Mix was the most effective, with 1352.15 cm2, 39% more than the control plants (974.08 cm2), whereas Terra Vital produced an increase of 27%, with 1241.98 cm2. Although TRI TON generated an increase in comparison to the control plants, the increase was not significant. The root colonization by AMF at this second stage of the test is represented in Figure 4. Despite the difference in absolute values between products, the percentage increases in mycorrhizal colonization were not significantly different in statistical terms. Figure 1. Percentage of mycorrhizal colonization for each product and dose at the end of the experiment. EM I: Endorize Mix (5 mL) EM II: Endorize Mix (10 mL) EM III: Endorize Mix (15 mL) TV I: Terra Vital (12 mL) TV II: Terra Vital (25 mL) TV III: Terra Vital (40 mL) T I: TRITON (10 mL) T II: TRITON (15 mL) T III: TRITON (20 mL) % 100 80 60 40 20 0 EM I EM II EM III TV I TV II TV III T I T II T III g 150 120 90 60 30 0 Control Endorize Mix Terra Vita I TRITON Fresh weight of aerial part Plant height cm 25 20 15 10 5 0 Figure 2. Effect of the products on plant growth: fresh weight of aerial part and length of pseudostem. cm2 1600 1200 800 400 0 Control Endorize Mix Terra Vita I TRITON Figure 3. Effect of the products on plant growth: leaf area. % 30 20 10 0 EM II TV I T I Figure 4. Percentage of mycorrhizal colonization for the three products at the optimum dose selected. Discussion Although references on the use of commercial mycorrhizal inoculants for nursery production of plants of agronomic InfoMusa - Vol. 16 No. 1-2, June & December 200726 InfoMusa - Vol. 16 No. 1-2, June & December 2007 27 interest are not abundant, they generally describe positive results in terms of increases in growth and biomass (Lovato et al. 1992, Niemira et al. 1995, Busch and Lelley 1997, Bourrain et al. 1999, Corkidi et al. 2005) and/or the rates of survival during the acclimatization of micropropagated material (Bourrain et al. 1999). Furthermore, field testing of ecological production of strawberry showed an increase in the harvest after the application of diverse mycorrhizal inoculants, even though the yield within the categories of commercially produced fruit was not affected (Bull et al. 2005). Some of these findings indicate – as does this test – the variability of the responses, which depend on both the product and the cultivar (Bull et al. 2005, Corkidi et al. 2005). Therefore, most authors propose conducting preliminary tests that make it possible to select the most appropriate inoculant for each specific situation, this way ensuring a successful application (Busch and Lelley 1997, Bull et al. 2005, Corkidi et al. 2005). However, the lack of correlation between infectivity and effectiveness of the fungi inoculated – as found in this experiment - was also detected in some of these studies (Bull et al. 2005). The results contributed by this experiment allow us to confirm without any room for doubt – even with the variability of response recorded – the positive effect of the products tested on the banana cultivar Petite Naine and the infective capacity of the fungi that these products contain. The species of mycorrhiza present in each product and their physico- chemical support substrates influence this behavioural variability. The good results of this test support the possibility of using AMF commercial products on a large scale during the post vitro acclimatization of banana in nurseries, as indicated for other crops by other authors (Bull et al. 2005, Corkidi et al. 2005). This assertion is also supported by the small amount of inoculant needed to increase growth and lower the average time of stay in the nursery, which directly influences the commercial profitability of the inoculants analysed. By publicising the guarantee of health and natural origin benefits for plants, the commercial brands have serious possibilities of occupying a market in need of such products and still lacking exhaustive regulations by the public authorities (von Alten et al. 2002). The results of this experiment demonstrate the benefits from the inoculation of banana during the post vitro stage. The use of commercial mycorrhizal products could help to simplify and optimize the utilization of this biotechnology in nurseries, with favourable effects on the quality of the vegetal material. However, an initial assessment of the products should be made in order to adjust the doses and types of inoculants to the specific conditions of each situation. References Barea J.M., R. Azcon & C. Azcon-Aguilar. 2004. Mycorrhizal fungi and plant growth promoting rhizobacteria. Pp. 351-372 in Plant Surface Microbiology (A. Varma, L. Abbott, D. Werner, R. Hampp, eds). Springer-Verlag, Germany. Bourrain L., J.C. Navatel, C.B. Blal & J. Parat. 1999. Les mycorrhizes: des champignons symbiotiques en aide aux pépinièristes. Infos Paris 154: 36-39. Bull C.T., J. Muramoto, S.T. Koike, J. Leap, C. Shennan & P. Goldman. 2005. Strawberry cultivars and mycorrhizal inoculants evaluated in California organic production fields. Crop Management doi:10.1094/CM- 2005-0527-02-RS. Busch E. & J.L. Lelley. 1997. Use of endomycorrhizal fungi for plant cultivation on buildings. Angewandte Botanik 71: 50-53. Corkidi, L., E.B. Allen, D. Merhaut, M.F. Allen, J. Downer, J. Bohn & M. Evans. 2005. Effectiveness of commercial mycorrhizal inoculants on the growth of Liquidambar styraciflua in plant nursery conditions. Journal of Environmental Horticulture 23 (2): 72-76 Jaizme-Vega M.C., M. Esquivel Delamo, P. Tenoury Domínguez & A.S. Rodríguez-Romero. 2002. Effects of mycorrhization on the development of two cultivars of micropropagated banana. INFOMUSA 11(1): 25-28. Koske R.E. & J.H. Gemma. 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycological Research 92: 486-505. Lovato P., J.P. Guillemin & S. Gianinazzi. 1992. Applications of commercial arbuscular endomycorrhizal fungal inoculants to the establishment of micropropagated grapevine rootstock and pineapple plants. Agronomie 12 (10): 873-880. Niemira B.A., G.R. Safir, V. Hammerschmidt & G.W. Bird. 1995. Production of prenuclear minitubers of potato with peat based arbuscular mycorrhizal fungal inoculum. Agronomy Journal 87 (5): 942-946. Von Alten H., B. Blal, J.C. Dodd, F. Feldmann & M. Vosatka. 2002. Quality control of arbuscular mycorrhizal fungi inoculum in Europe. Pp. 281- 296 in Mycorrhizal Technology in Agriculture: from Genes to Bioproducts (S. Gianinazzi, H. Schüepp, J.M. Barea & K. Haselwandter, eds). Birkhäuser Verlag, Switzerland. Yano-Melo A.M., J.S. Jr. Orivaldo, J.M. Lima-Filho, N.F. Melo & L.C. Maia. 1999. Effect of arbuscular mycorrhizal fungi on the acclimatization of micropropagated banana plantlets. Mycorrhiza 9 (2): 119-123 Ana Sue Rodríguez- Romero and María del Carmen Jaizme-Vega* work at the Dpto. de Protección Vegetal, Instituto Canario de Investigaciones Agrarias, Apdo. 60, 38200 La Laguna, Tenerife, Spain *E-mail: mcjaizme@icia.es InfoMusa - Vol. 16 No. 1-2, June & December 200728 InfoMusa - Vol. 16 No. 1-2, June & December 2007 29 “Strengthening the capacity of the regional National Agricultural Research Systems (NARS) to sustainably manage the outbreak of banana Xanthomonas wilt in East and Central Africa” - a brief project report. The banana industry in East and Central Africa is a major contributor to regional food and income security. Since 2001, a new wave of Xanthomonas wilt epidemics in bananas has become a serious threat to regional food security. Entire crop holdings have been wiped out in areas where highly susceptible banana cultivars were dominating farming systems. The Banana Research Network for Eastern and Southern Africa (BARNESA) appealed for resources to search for a solution to the problem. A Ugandan impact assessment study estimated that up to US$ 4 billion could be lost by 2010 if no measures were taken to arrest the epidemic (Karamura 2006).Subsequently, the Inter- national Network for the Improvement of Banana and Plantains (INIBAP) (now Bioversity International), in collaboration with the Food and Agriculture Organization (FAO) convened a regional stakeholders’ meeting in February, 2005 and developed a strategy that envisioned multi-disciplinary, multi-sectoral approaches in a coherent regional response. This took account of the countries/regions that were Xanthomonas wilt-free but threatened; the frontline areas where the disease had just arrived; and the endemic areas where the disease was already established. It also sought to raise the awareness of all stakeholders along the production-consumption chains and to provide them with knowledge and skills for the diagnosis and management of the disease. Importantly, it was realised that phenologically wilt-resistant banana cultivars could still be infected via contaminated pruning tools. Hence, awareness-raising and associated training were seen as an integral component of the intervention strategy to control the epidemic and restore productivity in the banana-based farming systems. The spread of the disease can be significantly arrested by increasing farmers’ and other stakeholders’ knowledge and skills for diagnosis, spread mechanisms and control measures (Molina 2006). Low cost management technologies developed by research institutions needed to be transferred to the affected farmers. There was a need to involve more stakeholders along the production-consumption chains to help manage the disease in a coordinated regional strategy (Karamura et al. 2006). The aims of the regional strategy were boosted when the Catholic Relief Services (CRS) and the International Institute of Tropical Agriculture (IITA) secured funds from USAID to execute the Crop Crisis Control Project (C3P). This is an 18-month initiative to facilitate and intensify a coordinated fight against Xanthomonas wilt of bananas and Cassava Mosaic Disease in six countries of Central and East Africa: Burundi; Democratic Republic of Congo (DRC); Kenya; Rwanda; Tanzania and Uganda (See figure 1). The CRS sub-contracted Bioversity to strengthen the capacity of regional NARS to sustainably manage banana Xanthomonas wilt in the countries participating in the C3P. Bioversity, IITA and the NARS used the regional strategy developed in the framework of the BARNESA and the Association for Strengthening Agricultural Research in East and Central Africa (ASARECA) to develop, test and disseminate Xanthomonas wilt diagnostic and management tools to stakeholders along the banana production- consumption chains. The specific objectives were: • To equip stakeholders with skills know- ledge/tools for sustainable management of Xanthomonas wilt at the farm level; • To raise public awareness of the disease threat and appropriate control measures • To develop, evaluate, and disseminate information materials to stakeholders Focus on building knowledge for BXW control An effective strategy for combating outbreaks of banana Xanthomonas wilt Compiled by E. Karamura and V. Johnson This article is a synthesis of the results of the Crop Crisis Control Project (C3P) funded by USAID and executed by the Catholic Relief Services together with the International Institute of Tropical Agriculture. CRS subcontracted Bioversity to strengthen the capacity of the regional NARS to sustainably manage the outbreak of Banana Xanthomonas wilt in the C3P participating countries. Central Africa Republic Angola Dem. Republic of Congo Congo Gabon Zambia Cameroon Uganda Burundi Tanzania Kenya Malawi Mozambique Rwanda Project area InfoMusa - Vol. 16 No. 1-2, June & December 200728 InfoMusa - Vol. 16 No. 1-2, June & December 2007 29 • To mainstream information on the ecology and management of Xanthomonas wilt of bananas in field demonstrations; • To establish an early warning/surveillance system to facilitate timely response/actions against the Xanthomonas wilt epidemic; • To strengthen farmer capacity to introduce and manage clean banana planting material of farmer-selected varieties; • To evaluate on-farm the effectiveness of the Xanthomonas wilt of bananas Diagnostic and Management tools; • To facilitate the drawing up of national frameworks (Nation Action Plans) for the control and management of Xanthomonas wilt of bananas and other banana pests and diseases. • To strengthen NARS linkages with regional (CIALCA, BARNESA) and international platforms (Bioversity International, IITA, ARIs). Trainers were trained at regional and country levels and community level trainings were back-stopped. The sub- project also deployed novel approaches for communicating its messages to target groups aimed at mass mobilization against the disease. Diagnostic and management tools and sensitization materials were developed, tested and disseminated. National level training produced trainers for community-based trainers who then trained farmers in disease recognition and management. The sub-project also developed com- munication and public awareness tools, and organized appropriate exchange visits for farming communities and/or extension teams to gain field experiences and exploit synergies between NARS. Finally, sub-project effectiveness and impact were evaluated. The IITA and country project managers (CPMs) collaborated in this capacity building initiative The ‘onion-peel‘ training concept adopted a level of technical content appropriate to each successive training tier, with the regional trainings having the most technical content. At regional and national level, Training of Trainers (ToT) participants were given electronic and hard copies of all the tools used in the training, so they could produce or modify training materials as need arose. The farmer-trainings were more visual demonstrations of symptoms and management techniques than theoretical disease-management presentations. Research and extension team leading national level campaigns against Xanthomonas wilt, were given specific training aimed at developing responses appropriate to the stage of the epidemic (disease-free, frontline and endemic regions) relative to crop phenology. Training accommodated for the needs and roles of farmers, extension providers and researchers and covered the use and management of ‘clean’ planting materials. Each country team had different frame- works for training, developed according to disease pressure and existing levels of knowledge. In general, national level training of research, extension and policy managers involved four components: 1. An overview of pest and diseases in banana-based cropping systems, of their impact on the banana industry and of the concomitant potential for improvement in regional livelihoods. 2. A description of Xanthomonas wilt in bananas, its diagnosis, mechanisms of spread and its control, and a review of strategies to raise public awareness of the disease. Disease management strategies Figure 1. C3P project area InfoMusa - Vol. 16 No. 1-2, June & December 200730 InfoMusa - Vol. 16 No. 1-2, June & December 2007 31 targeted the eradication of disease pockets and longer-term disease management. 3. Planning skills for sustainable integrated pest and disease control strategies, including developing national action plans and establishing farm demonstrations and other farmer-learning approaches. Field visits included demonstrations of symp- toms and control measures, and a participatory critique. 4. Visits to research centres working against wilt, and to private sector laboratories involved in the production and distribution of banana planting materials. This strengthened linkages and generated information and technology updates. At the end of each tier training, trainees developed and presented a training programme for their respective target areas and subsequently published their training schedules to enable IITA and Bioversity to backstop the country level training. The back-stopping support included providing teaching tools, field demonstration of symptoms and control measures. It also provided opportunity for cross-fertilization of ideas and experiences from other countries with similar activities. At the end of the workshop, the back-stopping scientist facilitated a workshop review so that trainees were equipped to address the needs of the next level. At the start, different NARS had different levels of capacity to manage Xanthomonas wilt. To address capacity-gaps, the project facilitated sharing cross-border information and technologies via research/extension/ farmer teams exchange visits. Appropriate approaches were designed for the different contexts: the Rwandese/Congolese teams visited Uganda targeting farmer-empowering approaches; the Ugandan teams visited Tanzania to learn about stakeholder mobi- lization and to target policy makers; the Kenyans visited Uganda to learn skills for raising public awareness. Communities including researchers, extension, local councils, universities and 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. Figure 2. BXW awareness-raising poster 100 50 0 % w ith B XW Recommended BXW control measure * not statistically significantly different Yes No Yes No Yes No De-budding Disinfecting tools Removing infected mats 66.7 94.4 38.9 82.8 69.4* 85.0* InfoMusa - Vol. 16 No. 1-2, June & December 200730 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 equivalent geographical zones (12 districts in all). • From each district, 2 X 25 km2 were randomly selected (24 sites in all) • From each site, 4 farms were randomly selected.(96 farms in all) All the sites selected had been exposed to Xanthomonas wilt sensitization, training 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 SPSS and means separated using a chi- square test. The training reached 51 400 people (7 times the original target size). This was partly because bananas and this threatening disease are important to regional livelihoods, and partly due to the C3P mobilization. Conversely, it was also observed that in a few sites farmers did not readily adopt recommendations that included high short- term losses via plant destruction. Training evaluation showed that farm competencies gained were very high (60- 100%) for disease symptoms and control measures, but that farmers could not explain the various disease spread mechanisms. Similarly in all countries, farmers were effectively applying two of the first-line-of- defence recommendations (de-budding and destruction of infected material) but sterilization of infected tools remained a problem. This was attributed to the constraints of flaming in fire where building fires is restricted or the expense and scarity of bacterial products. In conclusion, strengthening the capacity of stakeholders, especially at the grass-root level, significantly reduced Xanthomonas wilt disease incidence on farm by 4-6 times. Those applying the recommendations had significantly higher levels of disease-free bananas than those that were not (see figure 3). For effective control, all management options (cultural-biological and institutional mechanisms) need to be applied at the same time. De-budding, destruction of infected materials and the disinfection of Figure 3. Effect of C3P - enhanced awareness on BXW control levels Competence levels to recognise and manage the disease Numbers of: Baseline data of disease incidence & distribution levels Farmer’s ability to recognise Xanthomonas wilt symptoms Farmers’ knowledge on disease Table 1. Data collection in C3P project. Data type Data characteristics Capacity to distinguish between Xanthomonas wilt (XW) and Fusarium wilt • development (how XW enters and develops within plants) • spread (plant to plant, mat to mat, location to location) • 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. • male-bud • bunch • within plant (sectioned banana fingers & pseudo- stems) • XW task forces formed at different levels • participants trained • organizations participating in the fight against XW • When sites were established • 4-6 months later (on disease incidence on farm) InfoMusa - Vol. 16 No. 1-2, June & December 200732 InfoMusa - Vol. 16 No. 1-2, June & December 2007 33 tools, as well as mobilization of stakeholders and monitoring and evaluating the implementation of control options were seen as the first lines of defence within the overall management strategy against the Xanthomonas wilt of bananas (and Enset). As a result of C3P project activities, the rate of reported disease outbreaks in all countries has gone down by about 20-40%, except in the northern part of the western rift valley, which brings together DR Congo, Uganda, and Rwanda. From here it is threatening to expand southwards into Burundi and western Tanzanian region of Kigoma. More resources will be required to keep the disease out of northern Tanzania, central Kenya, and the Indian Ocean islands and coastal regions. In order to raise awareness in those disease -free but threatened banana-growing agro- ecologies, the sub-project has prepared both electronic and hard-copy information tools, key among which are: BXW diagnostic and management manual that outlines the approaches that were tested and used in the subproject to control the disease, available both as hard copies and on the web (http://platforms.inibap.org/ xanthomonaswilt/). Pest risk assessment (PRA) that was published with the collaboration of Central Science Laboratory (UK) and which provides all available information on the disease and assesses the risks to the banana industry in the region. The document is available as hard copies and on the web. Management tools including posters, pamphlets, brochures and audiovisual material were produced and disseminated. These were translated into French and subsequently to local languages by the NARS participating in the project Two monitoring and review meetings were conducted in Kigali, Rwanda (July 2007) to evaluate management and diagnostic tools, and in Bujumbura, Burundi (December 2007) to discuss sub-projects outputs). The following recommendations arose: • to develop the best approaches for managing the disease more research should be conducted on: • host-vector-pathogen interactions; • developing user-friendly methods for disinfecting tools at farm level; • the role of other pests and diseases in the transmission of the wilt • pathogen longevity in soil and on decaying plant debris under field conditions. • To create a regional Xanthomonas wilt coordination center where further developing mechanisms for regional surveillance and information exchange would be facilitated. This would provide for data management as a basis for consolidating national surveillance methods and the formulation of regional strategies against the disease. References Karamura E., Kayobyo G., Blomme G., Benin S., Eden- Green S.J. and Markham R. 2006. Impacts of XW epidemic on the livelihoods of rural communities in Uganda. Proceedings of the 4th International Bacterial Wilt Symposium. Central Science Laboratory, UK. Molina A. 2006. Managing bacterial wilt/fruit rot disease of banana in Southeast Asia. Pp 26-31 in Developing a regional strategy to address the outbreak of banana Xanthomonas wilt in East and Central Africa. INIBAP, Montpellier, France. Eldad Karamura is the Regional coordinator of the banana and plantain research group of Bioversity International for East and central Africa in Kampala, Uganda and Vincent Johnson works as scientific editor at Bioversity in Montpellier. Thesis An Improved Agrobacterium-mediated transformation method for banana and plantain (Musa spp.) Geofrey Arinaitwe PhD thesis submitted in February 2008* to the Katholieke Universiteit Leuven, Leuven, Belgium. Banana (commonly known as Matooke) provides food for over eight million Ugandans. More than half of Uganda’s population is employed in its production process. Banana production is continuously declining in Uganda due to pests and diseases. A leaf defoliating disease called black Sigatoka (BSD), is most destructive and reduces banana yield by 50% in susceptible cultivars. Resistance breeding for BSD, using conventional methods, is complicated by the crop’s biology and genetics. InfoMusa - Vol. 16 No. 1-2, June & December 200732 InfoMusa - Vol. 16 No. 1-2, June & December 2007 33 Through the use of modern biotechnological tools, genes with potential to control BSD have been isolated. Cell and tissue culture techniques are also available. In the current study we compared an existing Agrobacterium-mediated transfor- mation method of banana with the particle bombardment-mediated transformation method of banana. Then we improved the Agrobacterium-mediated transformation method by changing infection length, age and volume of the embryogenic suspension. The effects of polyamine spermidine on regenerability of transformed embryogenic cell clones was demonstrated. Transformation and integration of rice chitinase genes Cht-2 and Cht-3 in banana are presented. Chitinases are enzymes that hydrolyze chitin, a major component of most fungi. We also evaluated the possibility of introducing more than one gene and developed a fast method to detect multiple genes. Effects of the use of single versus two different selectable marker genes are presented. This study is on-going as chitinase activity is analysed in the generated transgenic lines and 26 lines are field tested in Uganda. ThesisCharacterization and isolation of T-DNA tagged banana promoters active during in vitro regeneration and low temperature stress Efrén Germán Santos Ordóñez PhD thesis submitted in April 2008* to the Katholieke Universiteit Leuven, Leuven, Belgium A genome-wide T-DNA tagging strategy was pursued for the characterization and isolation of novel banana promoters. Banana embryogenic cell suspensions were transformed via Agrobacterium tumefaciens containing a promoterless, codon-optimized luciferase (luc+) gene next to the right T-DNA border. Approximately 89,000 transgenic banana cell colonies were screened for luminescence two to three months after transformation using a sophisticated camera system in a light-tight box. Screening occurred under controlled temperature conditions in which luminescence was monitored in real- time at 26°C followed by a gradual decrease to different low temperatures (LT) including 18°C, 16°C, 12°C and 8°C. The frequency of cell colonies showing luminescence at 26°C and the different LT treatments ranged from 0.17% to 2.69%. Transgenic cell colonies responsive to 8°C (i.e. showing an enhanced or repressed luciferase expression pattern) were regenerated to plantlets and luminescence was monitored at different in vitro developmental stages. With increasing differentiation the number of lines with LT up-regulated LUC expression decreased. Banana plants showing luminescence were analyzed for the T-DNA copy number by Southern hybridization. T-DNA inserts averaged 3.6 (range from 1 to 5) in 10 independent lines tested. DNA sequences flanking the right and left T-DNA borders were isolated via TAIL-PCR and inverse PCR. Continuity of sequence between the corresponding right and left border flanking sequences was revealed by linking PCR using flanking sequence specific primers. RT-PCR analysis was performed in lines with multiple inserts to identify and confirm the sequence that activated luciferase expression. Four candidate promoter sequences which were transcriptionally fused to the luc+ were cloned upstream of a uidAINT reporter gene and back-transformed to banana, which confirmed promoter activity for two sequences in in vitro cultures. Promoter activity in mature banana plants remains to be investigated. To summarize, a high-throughput T-DNA tagging system has enabled us to characterize and isolate novel banana promoters with potential for a number of downstream applications including banana improvement via genetic modification. * Note from the editor: The delayed printing of this last issue of INFOMUSA provided us the opportunity to add the abstracts of two theses submitted in 2008 that may be of great interest for our readers. InfoMusa - Vol. 16 No. 1-2, June & December 200734 InfoMusa - Vol. 16 No. 1-2, June & December 2007 35 PhD thesis submitted in February 2007 to the Katholieke Universiteit Leuven, Leuven, Belgium. The Laboratory of Tropical Crop Improvement (KULeuven, Belgium) hosts, under the authority of Bioversity International, the global in vitro collection of banana varieties (Musa and Ensete spp.). The aim of this international genebank is to conserve all banana and plantain genetic resources safely and to supply the germplasm to any bona fide users. However, in vitro preserved germplasm needs regular sub-culturing, creating the possibility of losses due to contamination and human error. Moreover, an important problem associated with in vitro culture is the occurrence of somaclonal variation. KULeuven was one of the pioneers to explore the possibilities of storing plant material in liquid nitrogen (at -196°C). However, for successful storage at -196°C, cells need to survive first a severe dehydration process prior to freezing and then to regenerate after thawing. In banana, meristems are the most suitable structures to be cryopreserved. Although meristems have the potential to regenerate, they still need to survive the dehydration associated with cryopreservation. In general, dehydration tolerance is achieved by a sucrose- mediated osmotic stress acclimation. However, more than half of the collection consists of varieties that have a non- existent or low cryopreservation survival rate. Hence, there is a need to understand the mechanisms behind acclimation during the pre-culture of meristems on sucrose and to obtain insight into the genotype-specific diversity. Understanding gene function and gene expression profiling can be approached via several techniques. A concise overview of the existing technologies is given with the emphasis on proteomics and proteomic technologies. Protein separation via two-dimensional gel electrophoresis and protein identification via tandem mass spectrometry (MS/MS) is the most informative approach for a poorly characterized organism like banana. Proteins are relatively well conserved, making the identification of non-model gene products in comparison to well-known orthologous proteins quite efficient. Intact proteins (separated via 2DE) are essential for a good and reliable identification. Protein sample preparation is a critical step and is absolutely essential for obtaining good results. Most plant tissues are not a ready source for protein extraction and need specific precautions. The cell wall and the vacuole make up the majority of the cell mass, with the cytosol representing only 1 to 2 % of the total cell volume. Subsequently, plant tissues have a relatively low protein content compared to bacterial or animal tissues. Banana is a good representative of a recalcitrant plant species since it contains numerous interfering compounds. Banana contains extremely high levels of oxidative enzymes (polyphenol oxidase), phenolic compounds (simple phenols (dopa), flavonoids, condensed tannins, lignin), carbohydrates, terpenes, suberin, and waxes. The majority of the plant protocols introduce a precipitation step to concentrate the proteins and to separate them from the interfering compounds. A phenol extraction protocol was optimized for small amounts of banana plant tissue (down to 50 mg fresh weight (FW)) and different sample preparation methods were evaluated. Only TCA/acetone precipitation and phenol extraction methanol/ammonium acetate precipitation are useful as standard methods for recalcitrant plant tissue. The optimized phenol extraction was most efficient in removing interfering substances and resulted in the highest quality gels. This Optimized proteomic methods to unravel biochemical processes in banana meristems during in vitro osmotic stress acclimation S. Carpentier Thesis InfoMusa - Vol. 16 No. 1-2, June & December 200734 InfoMusa - Vol. 16 No. 1-2, June & December 2007 35 method proved also to be useful for other plant species like apple, pear and potato. Proteins can be identified by comparing the proteins of interest to orthologous proteins of species that are well characterised. In one approach, peptide mass fingerprints (PMF) and peptide sequence information were combined to search and to correlate the un-interpreted spectra with the theoretical product ion spectra from different databases in an automated way. However, some orthologous genes retain a low percentage of identity and the chance of finding significant and conserved peptides decreases, so a PMF-MS/MS approach might fail. A method that extracts protein-identifying information directly from spectra without being dependent on existing databases is extremely useful. However, deriving sequences from MS/ MS spectra is not straightforward. MALDI TOF/TOF ion spectra contain in general incomplete ion series typically rich in various ion types. Attempts to simplify the de novo identification rely mainly on the simplification of the fragmentation pattern. The addition of a sulfonic acid group to the N-termini of tryptic peptides facilitates de novo peptide sequencing. The strong acidic group at the N-terminus is deprotonated and therefore neutralizes the positive charge of the b-ion by its negative charge. This results in simple spectra displaying mainly y-ions series. In this chapter, an innovative approach in which the extracted tryptic peptides are N-terminal sulfonated without any further sample purification is reported and successfully applied to banana. It was demonstrated that this approach results in a high and reliable identification rate and that it is adequate to identify different isoforms resulting from allelic variations, or from different gene loci. Banana is a unique model for the study of meristems thanks to the development of the shoot meristem tip culture technique. For the first time, a large scale study of a meristematic proteome is reported. The results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration tolerant variety (ABB) with a dehydration sensitive variety (AAAh), it was possible to distinguish several genotype specific proteins (isoforms) and to associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g. phosphoglycerate kinase, phosphoglucomutase, UGPase) and proteins that are associated with stress adaptation (e.g. OSR40-like protein, ASR). This work proves that proteome analysis is successful to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop. To get a better insight into the acclimation process and to correlate acclimation with cryopreservation, both a dynamic study (screening over time) and a comparison between varieties were performed, with the focus on the validation of candidate proteins. Although the sucrose pre-treatment and the concomitant high intracellular sucrose concentration are required for a low mortality rate after cryopreservation, it also induces negative stress effects. Sucrose pre-culture is correlated to high relative abundance of phosphoglycerate kinase, UGPase and OSR40 and a low relative abundance of SuSy, lectin and chitinase and abscisic stress, ripening protein-like protein (ASR). We hypothesise that a low mortality rate after cyopreservation can be achieved by a low relative abundance of SuSy and high relative abundance of phosphoglycerate kinase, OSR 40, UGPase, ASR and storage proteins such as lectin and chitinase. A screening of candidate proteins links several proteins and specific isoforms to the tolerant B genome, which opens perspectives to apply proteomics for taxonomical characterization and classification. InfoMusa - Vol. 16 No. 1-2, June & December 200736 InfoMusa - Vol. 16 No. 1-2, June & December 2007 37 Study of fungal endophytes as resistance inductors for black Sigatoka (Mycosphaerella fijiensis) control in plantain M. Barrios Murillo Thesis MSc thesis submitted in 2006 to the Tropical Agricultural Research and Higher Education Center - CATIE, Turrialba, Costa Rica Black Sigatoka is a foliar disease caused by Mycosphaerella fijiensis fungus. It attacks banana and plantain crops around the world, causing enormous production losses and requiring large agrochemical investments for its control. It is very important to develop alternative control methods to reduce economic and environmental costs. The Market access and agricultural production: The case of banana production in Uganda F. Bagamba Thesis PhD thesis submitted in 2007 to Wageningen University, The Netherlands. The study analyses smallholder household response to production constraints (crop pests and diseases, soil constraints) and the development of product markets and off-farm employment opportunities. It was carried out in the central region, Masaka and southwest, which have different production constraints and opportunities. Results for the cost-benefit analysis show that banana is the most profitable of all the crops grown in terms of gross margin. However, imperfections in labour and food markets cause farmers in the central region to allocate more land and labour to the less profitable annual crops (sweet potatoes, maize and cassava) which are more satisfactory in terms of household food requirements. High food prices and limited off-farm opportunities induce farmers to rely on their own farm production for their household food needs. Results from the technical efficiency analysis show that banana farmers in Uganda are technically inefficient, and output can be increased by 30% in the southwest and 58% in the central region. Improved roads, formal education, access to credit and extension work are some of the factors that improve productivity and technical efficiency. Results confirm that infestation by the banana weevil and Sigatoka contribute to the low banana production in the central region. Farm size is positively related to farm productivity. However, production is more efficient on smaller plots (decreasing returns to scale). The low productivity on small farms leads to question the sustainability of smallholder agriculture, given the imperfections in labour and food markets and limited access to purchased inputs. Findings from labour supply analysis show that farmers are responsive to economic incentives. Access to off-farm opportunities takes away the most productive labour from farm production. Education and road access tend to increase the time allocated to off- farm activities while farm size is negatively correlated to hours worked on off-farm activities. The study reveals that policies that promote income diversification into off- farm activities can contribute to sustained development in the rural sector. In particular, policies that reduce transaction costs are likely to improve productivity and efficiency in both the off-farm sector and farm sector. Investment in road infrastructure, education and financial institutions that are suited to smallholder production needs could help in alleviating the bottlenecks in the labour, food and financial markets, and improve resource allocation between the farm and non-farm sectors. InfoMusa - Vol. 16 No. 1-2, June & December 200736 InfoMusa - Vol. 16 No. 1-2, June & December 2007 37 objective of this study was to evaluate colonization, growth promotion and black Sigatoka control in plantain tissue culture plantlets, inoculated with four Fungal Endophytes (FE) isolate under controlled greenhouse conditions. Results showed the highest FE colonization percentage in the roots, followed by the corm, pseudostem and leaf. Trichoderma atroviride isolates lost their colonization power over time, while Fusarium oxysporum strains gradually increased theirs. Significant differences were found for growth promotion of leaf area (p<0.0001) and pseudostem diameter (p<0.0093); combined FE strain treatments had a positive effect, attributed to synergic action by the combined strains. Leaf analysis (Ca-Mg-K relationship), showed a slight unbalance in the treatments evaluated (except the control), probably due to FE soil nutrient uptake. In relation to black Sigatoka control, a series of comparative measurements using Fouré (1985) and Romero (2005) scales, T4 (Fusarium oxysporum isolate) was the only treatment showing a significant delay (five days) in the development of disease symptoms compared to the control. There are already literature reports on resistance induction using FE inoculated into roots to control foliar diseases. Although the results obtained from this study do not demonstrate the expected response with enough certainty, it is recommended to continue working in this direction, since this is the first research effort with FE conducted in plantain. ThesisEvaluation of endophytic fungi and botanical extracts for black Sigatoka (Mycosphaerella fijiensis Morelet) control in banana G. Osorio M.Sc thesis submitted in 2006 to the Tropical Agricultural Research and Higher Education Center - CATIE, Turrialba, Costa Rica Black Sigatoka (Mycosphaerella fijiensis Morelet) causes important losses to banana production. The use of fungicides has been the most efficient mean to control this foliar disease. The development of innovative strategies to diminish conventional chemical control dependency is a permanent challenge for a sustainable and environmentally respectful agriculture. The objective of this research was to evaluate mutualist endophytic fungi and botanical extracts to control black Sigatoka in the field. Banana (cv. Grand naine) vitroplants and corms inoculated separately with Trichoderma atroviride strains (E1 E2) were utilized. These materials were planted in bags and acclimatized for three months before their establishment in the field, under a completely randomized block design in split plots. Foliar applications of botanical extracts, Momordica charantia (B1) and Senna reticulata (B2) started six months after planting in the field. Black Sigatoka severity and incidence were evaluated using Fouré (1985) and Gauhl (1989) scales and following Marin and Romero (1998) methodology. During the vegetative period, the following variables were recorded: Evolution State (ES), Foliar Emission Rythm (FER), Total Leaves (TL), Youngest Diseased Leaf (YDL) and Infection Index (IND). At flowering, only one reading was done for TLF, YDLF and INDF, along with the following phenological variables: Period from Planting to Flowering (PPF), Plant Height at Flowering (PHF) and Pseudostem Circumference at Flowering (PCF). Parallel to this, phytochemical analysis of B1 and B2 were conducted to determine main secondary metabolites. An ANOVA and a Duncan test were run using the SAS system. The Area under the progress curve of the disease (ABCPE, Spanish acronym) expressed in the ES and IND was calculated. Results at root level reveal statistically significant differences (p<0.05) for ES and IND variables, outstanding E2 after Q, with low values in comparison to remaining treatments. Nevertheless, InfoMusa - Vol. 16 No. 1-2, June & December 200738 InfoMusa - Vol. 16 No. 1-2, June & December 2007 39 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 considered as one of the most serious virus disease affecting bananas worldwide (Thomas et al. 1994). Banana bunchy top virus (BBTV) is disseminated through infected planting materials. Within the field, the virus is transmitted by a banana aphid, Pentalonia nigronervosa. Symptoms of the disease include dark green spots along the leaf veins, midrib and petiole, upright leaves with wavy margins, stunted growth, more erect leaves giving the plant a rosetted or ‘bunchy top’ appearance (Jones 2000). The disease has been reported and 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, BBTV symptoms on cv. Williams at Vila Ulongwe government banana nursery block. there was no evidence of positive effects on the other analyzed variables. At foliar level, there were statistically significant differences (p<0.05) favoring B2 with a less ABCPE for ES and less IND infection percentage. However, the chemical treatment (chlorothalonil) was the most effective control. Comparing the planting material type (“seeds”), vitroplants showed the best behavior regarding severity, TL, YDL and APF. Significant differences (p<0.05) were observed for some interactions, standing out TL and YDL variables in the tri-factorial relationship: seeds with roots treatment and the chemical application at foliar level. In this case, seed with nematicide and fungicide interaction showed the best behavior for disease protection. Phytochemical analysis indicated a variety of secondary metabolites (polyphenols, coumarins, quinones, saponins, triterpenes, flavonoids, among other), some of which have been reported in the literature for their antifungal or resistance induction activity. This research allowed to select B2 as “highly promising” to be proposed as a candidate for banana black Sigatoka integrated management due to its protectant action. However, it is not known which molecule exerts the disease control effect. If an opportunity to continue with this research exists, it is recommended to isolate the active compound. Regarding endophytic fungi, it is necessary to conduct more studies to optimize their efficiency with subsequent reinforcement application, extending and inducing molecules liberation, which are intermediate signs of a possible systemic resistance. InfoMusa - Vol. 16 No. 1-2, June & December 200738 InfoMusa - Vol. 16 No. 1-2, June & December 2007 39 W.T. Gondwe and B.M.L. Mwenebanda work at IITA/SARRNET, Chitedze Agricultural Research Station, P.O. Box 30258, Lilongwe 3, Malawi, E. Natha at IITA/ SARRNET, c/o Total Land Care, Vila Ulongwe, Angonia, Mozambique and P. Mufale at IITA/SARRNET, c/o Msekera Agricultural Research Station, Chipata, Zambia. both ‘Bluggoe’ and ‘Pisang awak’ which surrounded the Cavendish banana farm were not affected. Cultivars of the Cavendish subgroup have been reported to be highly susceptible to BBTV (Thomas and Iskra- Caruana 2000). In BBTV hot spots in Malawi, Cavendish bananas, other AAA bananas and some plantains (AAB) were first wiped out before ABB cultivars. There was a slow symptoms appearance in the ABB cvs such as ‘Bluggoe’ and ‘Pisang awak’ (Mwenebanda 1998, pers. observation). In Angola, the disease was spotted on False horn plantains (AAB) and Cavendish bananas (Pillay et al. 2005). Tushemereirwe and Bagabe (1998) reported that no study to establish yield losses due to BBTV has been reported in Africa. However, it was reported that severely infected plants of highly susceptible cultivars fail to produce bunches resulting into 100% yield loss. Geering (2003) reported that there was partial resistance to BBTV in various banana genotypes. The same reporter cited ‘Gros Michel’ as having a low susceptibility to BBTV with delayed symptom expression. He also stated that ‘Highgate’, a member of the ‘Gros Michel’ banana subgroup, is used by the breeding programme of the Fundación Hondureña de Investigación Agrícola (FHIA) as a potential source of partial resistance to BBTV. This is the first reported case of BBTV in both Mozambique and Zambia. References Geering Andrew. 2003. Banana bunchy top virus - screening for virus resistance. 3rd meeting of the PROMUSA Virology working group. PROMUSA Newsletter10:8. Jones D. 2000. Diseases of banana, abacá and enset. CAB International, Wallingford, Oxon, UK. Pillay, M., Blomme, G., Rodrigues, E. & A.L. Ferreira. 2005. Presence of banana bunchy top virus in Angola. InfoMusa 14(1):44–45 Thomas J.E & M-L. Iskra-Caruana. 2000. Diseases caused by Viruses. Pp 241-253 in Diseases of banana, abacá and enset (D.R. Jones, ed.). CAB international, Wallingford, Oxon, UK. Thomas J.E, M-L. Iskra-Caruana & D.R. Jones. 1994. Banana Bunchy Top Disease. Musa Disease Fact Sheet No. 4. International Network for the Improvement of Banana and Plantain, Montpellier, France. Tushemereirwe W.K. & M. Bagabe. 1998. Review of disease distribution and pest status in Africa Pp. 139- 147 in Mobilizing IPM for Sustainable banana production in Africa. Proceedings of a workshop on banana IPM held in Nelspruit, South Africa- 23 – 28 November 1998. International Network for the Improvement of Banana and Plantain, Montpellier, France. MusaNewsINIFAT 02: a new cooking-banana hybrid is born in Cuba A. Rodríguez Nodals, A. Rodríguez Manzano and A. Rodríguez Manzano In recent years, geneticists have produced more disease-resistant banana and plantain hybrids that have contributed to worldwide food security. This was favoured by the introduction of clones from the Fundación Hondureña de Investigación Agrícola (FHIA). In Cuba, important results have also been achieved, such as the clone Burro CEMSA (ABB), highly resistant to Fusarium wilt and to yellow Sigatoka and, to some extent, to black leaf streak (black Sigatoka). For a further breeding programme, several clones were selected as parental material (Table 1), and among them, Burro CEMSA ( ) and Pelipita (ABB) ( ) produced 508 seeds and (after discarding abnormals) in turn 84 seedlings for evaluation. The promising clone INIFAT 02 was successfully produced from this cross, despite the triploid condition of both parents, due to their residual fertility and to the Musa phenomenon of unreduced meiosis – or reduced though irregular meiosis InfoMusa - Vol. 16 No. 1-2, June & December 200740 InfoMusa - Vol. 16 No. 1-2, June & December 2007 41 INIFAT 02 was found to contain two genomes of Musa balbisiana Colla and one of Musa acuminata Colla. The following are some of INIFAT 02’s main agronomically valuable characteristics: • Shorter cycle than those of its parents under similar conditions. This clone was harvested 270 days after planting, whereas Burro CEMSA and Pelipita were harvested at 300 and 395 days, respectively. • Tall plants (3-5 m): height depends on such factors as climate, soil and management. This clone is intermediate between its parents under equal conditions. On a typical red ferralitic soil, the average height was 3.05 m during the first harvest cycle, whereas Burro CEMSA and Pelipita reached, on average, 3.50 and 2.90 m, respectively. • Assessments eight months after planting showed that the average INIFAT 02 tiller-number was five per plant, whereas Burro CEMSA and Pelipita produced, on average, seven and three suckers per plant, respectively. • The bunches consist of 5-9 hands. Figure 1 shows a clone grown in an urban patio. • Finger-size was intermediate between those of both parents (Figure 2). In the first hand, central finger average length was 16 cm, whereas Burro CEMSA and Pelipita averaged 13 and 20 cm, respectively. From a genetic point of view it is interesting that the above characteristics show a type of “intermediate” inheritability, except for the harvest precocity. • Like its parents, INIFAT 02 is highly resistant to black leaf streak. It has shown no wilting caused by Fusarium, which was Table 1. Names and genomic groups of the parents. Parents Paka (AA) Highgate (AAA) SH-3362 (AA) Burro CEMSA (ABB) Pelipita (ABB) Congo (AAA) Pelipita (ABB) Gros Michel (AAA) At the beginning of flowering, male flowers were selected from those clones where good levels of fertility had been reported (Simmonds 1966, Rowe and Rosales 1992, Román et al. 1990). The flowers were rubbed against the stigma of the female flowers which opened in the morning. This process was continually repeated as more flowers opened. Eleven types of crosses were performed whose pulp was extracted from ripe Figure 1. Bunch of ‘INIFAT 02’ cultivated in urban patios in La Habana. bunches. Hybrids displaying undesirable characteristics were eliminated (e.g. albino plants, broader-than-long or very thick leaves, or dwarfism (expressed as resetting). Bunches were selected with notable agronomic value (e.g. appropriate finger length, high bunch weight, short or medium cycle, and tolerance/resistance to black leaf streak). The selected clones’ genomic groups were determined using Simmonds and Shepherd’s principle (1955), quoted by Simmonds (1966). Physical characteristics and yield com- ponents were described with international descriptors for the genus Musa. The triploid genetic constitution was determined according to Simmonds’ morphogenetic principle. Triploidy, common to most bananas and 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 green) and long growth cycle. InfoMusa - Vol. 16 No. 1-2, June & December 200740 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 A total of 84 seedlings were obtained by the crossbreeding of Burro CEMSA x Pelipita; 28 of them were transplanted for field testing, and 5 promising clones were selected. INIFAT 02 was the clone that featured the best agronomic characteristics. INIFAT 02 is an ABB triploid and a hybrid between the subgroups Bluggoe and Pelipita, both of which are ABB. INIFAT 02 features better culinary properties than Pelipita, and is more precocious and gives a similar yield. For the first time a successful 3x/3x crossbreeding has been reported, which was possible thanks to the female fertility of the selected parent, to the low though sufficient fertility of Pelipita pollen and to the genetic phenomena of meiosis. 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). It is recommended that further ecological- zone studies of this clone be carried out to assist its launching as a new commercial hybrid. With regard to most of the significant agronomic characteristics, it stands out for combining the virtues of both parents and is more precocious at harvest than either of them. Research should be done on the behaviour of INIFAT 02 in those municipalities of Cuba most affected by drought. References Román M., M. J. Manzano & A. Rodríguez Nodals. 1990. Determinación de la fertilidad masculina en clones de plátano. Ciencia y Técnica en la Agricultura, Serie Viandas, Hortalizas y Granos, La Habana, Cuba. Rowe P. & F. Rosales. 1992. Genetic improvement of bananas, plantains and cooking bananas in FHIA, Honduras. Pp.243-266 in Breeding bananas and plantains: proceedings of an International Symposium on Genetic Improvement of Bananas for their Resistance to Diseases and Pests (J. Ganry, ed.). Cirad-Flhor, Montpellier, France. Simmonds N.W. 1966. Bananas. 2nd ed. Longmans, London. Simmonds N.W. & K. Shepherd. 1955. The taxonomy and origins of the cultivated bananas. J. Linnean Soc. of London, Botany 55:302-312 Tomekpé K., P. Rowe, H. Tezenas du Montcel & D. Vuylsteke. 1995. Plantain and Popoulou/Maia Maoli breeding: Current approaches and future opportunities. Workshop INIBAP/MARDI, Serdang, Malaysia. Figure 2. Size and format of the fingers of ‘INIFAT 02’. For more information, contact Adolfo Rodríguez Nodals, Arlene Rodríguez Manzano or Adolfo Rodríguez Manzano, Instituto de Investigaciones Fundamentales en Agricultura Tropical (INIFAT), Calle 1, esq 2. Santiago de las Vegas, Ciudad de La Habana, Cuba. E-mail: adolforn@inifat.co.cu ; arlenerm@inifat.co.cu InfoMusa - Vol. 16 No. 1-2, June & December 200742 InfoMusa - Vol. 16 No. 1-2, June & December 2007 43 For the past several years, Bioversity has been working with its partners around the world to achieve a consensus on how best to conserve the diversity of bananas. It was a tribute to the effectiveness of this concerted effort that the Global Strategy for Conservation Musa was one of the first crop-specific conservation strategies to be approved and published by the Global Crop Diversity Trust. The conservation effort received further encouragement when, at the end of 2007, banana was among the first crops to receive a long-term grant, under a partnership agreement between the Trust and Bioversity. Now there are yet more developments that will provide additional impetus to the conservation effort. As this issue goes to press, many key banana collections around the world are beginning to receive grants from the Trust, via Bioversity, to regenerate accessions that are at risk from disease or other hazards, and to provide a ‘safety back-up’ of unique accessions in vitro – on site, if the facilities are available, and at the International Transit Centre in Belgium. The Trust is also providing additional support for cryopreservation work, previously supported by the World Bank and Gatsby Charitable Foundation, which seeks to assure the long-term security of Musa diversity by placing a copy of the international collection (and a ‘black-box’, additional back-up) in liquid nitrogen. This time around, the National Bureau of Plant Genetic Resources in India will share the workload with the Katholieke Universiteit Leuven in Belgium. The international in vitro collection, alth- ough it has existed and has been growing steadily for over twenty years, has previously 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. For more information, visit the website dedicated to the conservation and use of the genetic diversity of bananas at www.musa- net.org. Progress in conserving banana diversity MusaNews Banana wild relative Musa acuminata burmannicoides, popularly known as Calcutta 4, used as a source of disease resistance in many breeding programmes, survives now only in ex situ collections, such as this one at Solok in Indonesia On March 7, 2007, the banana community lost an important African agronomist, Dr Antoine Bakelana Ba-Kifumfutu. Born in the Democratic Republic of Congo in 1947, Dr Bakelana started to study agronomy in his country and then in the USA, obtaining a PhD from Ohio State University in 1992. From 1994, he headed the Fruits and Banana programme of the Institut national pour l’étude et la recherche agronomique (INERA) in M’Vuazi and became Director In memory of Dr Bakelana Congo loses banana champion InfoMusa - Vol. 16 No. 1-2, June & December 200742 InfoMusa - Vol. 16 No. 1-2, June & December 2007 43 Le bananier et sa culture A. Lassoudière Books 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 since 1967, has worked as a researcher within the banana production chain, but also as an expert with banana producers and professional bodies in this sector. In addition to his long stays in Côte d’Ivoire, Cameroon and the French West Indies, he has made numerous expert missions in all the world. If you are interested to buy one or more copies, please contact : Éditions Quæ, c/o Inra, RD 10, 78026 Versailles Cedex, France Tel. + 33 1 30 83 34 06 Fax + 33 1 30 83 34 49 email : serviceclients@quae.fr www.quae.com Dr Bakelana (on the left) is happy to see the future harvest from a plot of the CFC project. of the INERA M’Vuazi research center in 1998. Dr Bakelana dedicated most of his life to bananas, and was an active member of the African banana research networks MUSACO 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 Bioversity. Those who knew and worked with Dr Bakelana will remember him for his good humour, even under difficult conditions, for his dedication to banana research, and his ability to get along with his fellow banana scientists. InfoMusa - Vol. 16 No. 1-2, June & December 200744 As explained in the editorial, we have been very busy establishing a new range of on- line resources. We urge you to visit these resources using the links indicated below. The new technologies used to develop these web resources allow all those interested to easily contribute and enrich them through constructive debates and knowledge sharing. A bunch of web resources Using these technologies, we hope to help to bring new dynamism to the banana research community and facilitate knowledge sharing. All these websites can be accessed through our institutional web site (http://bananas.biov ersityinternational.org/) with a single click or through their own address. 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. Musa web ressources InfoMusa - Vol. 16 No. 1-2, June & December 200744 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 Publications ht tp :// ba na na s. bi ov er si ty in te rn at io na l.o rg 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:l.er-rachiq@cgiar.org" Bioversity-France Parc Scientifique Agropolis II 34397 Montpellier Cedex 5 - FRANCE e-mail: bioversity-france@cgiar.org Fax: (33) 467 61 03 34