Katholieke Universiteit Leuven Faculteit Bio-ingenieurswetenschappen Departement Biosystemen – Afdeling Plantenbiotechniek Laboratorium voor Tropische Plantenteelt
DISSERTATIONES DE AGRICULTURA
EFFECT OF THE NUTRITIONAL STATUS OF BANANA (MUSA spp.) ON LEAF DISEASE INFESTATION BY MYCOSPHAERELLA FIJIENSIS MORELET IN ECUADOR
Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Bio- ingenieurswetenschappen
Promotor: Prof. R. Swennen, K.U. Leuven Leden van de examencommissie: Prof. E. Decuypere, Chairman K.U. Leuven Prof. J. Coosemans, K.U. Leuven Prof. M. De Proft, K.U. Leuven Dr. R. Markham, Bioversity International Prof. R. Merckx, K.U. Leuven
door Ma. Isabel JIMENEZ F.
October 2008
ISBN 978-90-8826-074-2 Wettelijk depot D/2008/11.109/32
To Dr. Rodolpho Maribona, the founding father of CIBE To Jose Luis Jimenez, my dear brother ‘Que Dios los tenga en su gloria’
ACKNOWLEDGEMENTS I would like to express my special thanks to many people, who given their invaluable time, professional help and support, and boosted my moral when needed to finish my PhD degree. My very heartfelt thanks go in the first place to Professor Rony Swennen, head of the Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Katholieke Universiteit Leuven, for his strong support and scientific guidance. He devoted much time to me during this important period of my life. I feel privileged to have been his student and have had his direct supervision. I would like to express my gratitude to each member of the examination commission for their critical review. Their numerous and detailed comments contributed much to the quality of my thesis. I am grateful to the VLIR – ESPOL program and the PL480 grant, which sponsored my studies in Belgium and the research in Ecuador. My special thanks go also to the Ecuadorian banana farmers Simon Canarte and Jorge Encalada. They allowed me to conduct research on their farms. My gratitude goes to Mrs. Jorge Gonzalez, Fernando Torres, Danilo Egred, Xavier Romero, Hector Calle and Hernan Pozas, who gave me support during the different steps of this thesis. I also want to express my gratitude to the three Directors of CIBE. First to Dr. Rodolpho Maribona, who had started the cooperation with Prof. Rony Swennen and suddenly and sadly passed away. Dr. Helga Rodriguez took over and supported my research for about 2 years. Dr. Esther Lilia Peralta is the current Director of CIBE and I want to thank her for her moral and professional support during the last period of this thesis. I am indebted to my colleagues and friends: Omar Ruiz, who collaborated with the statistical analysis, Anita Armijos, Maria Jama and Mariuxi Quishpe, who have given their professional support but never forgot to share friendly words. Thanks to Sofia Korneva, Rufino Meza and Enrique Marquez for their assistance. ‘Gracias amigos y colegas’. I also appreciate the many efforts of each of my dear students, Belgians and Ecuadorians, who gave me the opportunity to participate in their professional formation. My special thanks to the staff of the Laboratory of Tropical Crop Improvement at KULeuven, for their hospitality and friendship during my stays in Belgium. Special thanks to Prof. Dirk De Waele, the doctoral students Sugantha, Lieselot, and Christine for commenting this document and Dr. Annemie Elsen. Thanks to my new friends I encountered in Europe: Cristal, Lyli, Mayo, Jo, Gaspard, Victoria, Any, Claudia, Veerle and her lovely parents. I will remember them for ever. This achievement would not have been possible without the support of my lovely parents, Juanita and Manuel. I cannot find the right words to express my gratitude. I can only say thanks for being as they are. ‘Gracias mis queridos viejitos’. I am grateful to my dear sister Martha and her husband, my brother Eduardo and his wife, to my brother-in-law Edwin, my parents-in-law Daysi and Jose and all my nephews, nieces, cousins and uncles for their interest in the progress of my work.
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I cannot express enough my gratitude to my beloved husband, friend and colleague Jose Manuel and my adorable angels Ma. Jose and Alexia. Their unconditional love, patience and understanding, especially during my long periods of abscence, have given me strength to finish this work. “Gracias mi gordo bello e hijitas mias”. In the end I want thank GOD for his daily guidance. Many thanks for each day that you have allowed me to experience this work between tears and joy. The human mind is not perfect and for sure I will have forgotten some people that contributed at different levels to this work. Therefore, many thanks to all of you. Ma. Isabel (MI)
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS.......................................................................................................................I TABLE OF CONTENTS....................................................................................................................... III LIST OF ABBREVIATIONS ................................................................................................................. V LIST OF FIGURES............................................................................................................................... VII LIST OF TABLES...................................................................................................................................XI SUMMARY............................................................................................................................................ XV CHAPTER 1 GENERAL INTRODUCTION, OBJECTIVES AND OUTLINE............................... 1 1.1. MUSA SPP. AND BLACK LEAF STREAK DISEASE (BLSD) ................................................................ 1 1.1.1. Global importance of banana ................................................................................................. 1 1.1.2. The Ecuadorian banana industry............................................................................................ 1 1.1.3. The banana crop and its production systems.......................................................................... 2 1.1.3.1. Morphology of the banana plant ......................................................................................................2 1.1.3.2. Banana production systems ..............................................................................................................4 1.1.3.2.1. Conventional system ................................................................................................................4 1.1.3.2.2. Organic system .........................................................................................................................4
1.1.4. Disease constraints.................................................................................................................. 5 1.1.4.1. BLSD symptoms and the life cycle of M. fijiensis Morelet.............................................................5 1.1.4.2. BLSD control methods .....................................................................................................................7
1.2. PLANT FUNGAL DISEASE CONTROL METHODS .................................................................................. 9 1.2.1. Conventional methods: chemical, physical and cultural........................................................ 9 1.2.1.1. Chemical methods ............................................................................................................................9 1.2.1.2. Physical methods ............................................................................................................................10 1.2.1.3. Cultural methods.............................................................................................................................10
1.2.2. Alternatives approaches to disease management ................................................................. 10 1.2.2.1. Mineral nutrition and plant disease ................................................................................................10 1.2.2.1.1. Macronutrients: Nitrogen, Phosphorus, Potassium, Calcium, Magnesium ..........................11 1.2.2.1.2. Micronutrients: Boron, Copper, Manganese and Zinc ..........................................................13 1.1.2.1.3. Silicon (Si)..............................................................................................................................15 1.2.2.2. Organic amendments and plant disease .........................................................................................16 1.2.2.2.1. Solid organic amendments .....................................................................................................16 1.2.2.2.2. Water based organic amendments or organic teas.................................................................18
1.3. RATIONALE, OBJECTIVES, HYPHOTHESES AND OUTLINE OF THE STUDY ........................................ 21 CHAPTER 2 BANANA PRODUCTION SYSTEMS, THEIR IMPLICATIONS FOR BLACK LEAF STREAK DISEASE, NUTRITIONAL STATUS AND FUNGUS CHARACTERIZATION .................................................................................................................................................................... 25 2.1. TWO BANANA PRODUCTION SYSTEMS: IMPLICATIONS ON BLSD AND THEIR NUTRITIONAL STATUS ............................................................................................................................................................... 25 2.1.1. Introduction ........................................................................................................................... 25 2.1.2. Materials and Methods.......................................................................................................... 25 2.1.3. Results and Discussion.......................................................................................................... 30 2.1.3.1. Yields, Incomes and Profits of the Organic and Conventional farms ...........................................30 2.1.3.2. BLSD development under in vitro conditions ...............................................................................32 2.1.3.3. BLSD development under greenhouse conditions.........................................................................34 2.1.3.4. BLSD and nutritional status under field conditions.......................................................................35 2.1.3.5. Nutrient composition of OT ...........................................................................................................40
2.2. CHARACTERIZATION OF M. FIJIENSIS MORELET ISOLATES FROM ORGANIC AND CONVENTIONAL BANANA PRODUCTION SYSTEMS............................................................................................................ 41 2.2.1. Introduction ........................................................................................................................... 41 2.2.2. Material and Methods ........................................................................................................... 42 2.2.3. Results.................................................................................................................................... 45 2.2.3.1. Characterization of colonies and conidia production.....................................................................45 2.2.3.2. Isolate aggressiveness.....................................................................................................................48
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2.2.4. Discussion.............................................................................................................................. 49 2.3. CONCLUSIONS ................................................................................................................................ 50 CHAPTER 3 ORGANIC TEAS AND THEIR EFFECTS ON M. FIJIENSIS MORELET, THE BLACK LEAF STREAK DISEASE AND THE BANANA HOST ................................................... 51 3.1. INTRODUCTION ............................................................................................................................... 51 3.2. EFFECTS OF OT ON M. FIJIENSIS MORELET.................................................................................... 52 3.2.1. Materials and Methods.......................................................................................................... 52 3.2.2. Results.................................................................................................................................... 56 3.2.2.1. In vitro evaluation of OT ................................................................................................................56 3.2.2.2. OT nutrient content.........................................................................................................................59
3.2.3. Discussion.............................................................................................................................. 63 3.3. EFFECTS OF OT ON BLSD UNDER GREENHOUSE AND FIELD CONDITIONS ..................................... 64 3.3.1. Materials and Methods.......................................................................................................... 64 3.3.2. Results.................................................................................................................................... 66 3.3.2.1. OT assessment on banana growth and BLSD under greenhouse conditions ................................66 3.3.2.2. OT assessment on banana growth and BLSD under field conditions ...........................................72
3.3.3. Discussion.............................................................................................................................. 74 3.4. CONCLUSIONS ................................................................................................................................ 75 CHAPTER 4 IMPACT OF PH ON M. FIJIENSIS MORELET AND INFLUENCE OF SOME MICRONUTRIENTS ON THE PATHOGEN, THE DISEASE AND THE HOST........................ 77 4.1. INTRODUCTION ............................................................................................................................... 77 4.2. MATERIALS AND METHODS ............................................................................................................ 78 4.3. RESULTS ......................................................................................................................................... 80 4.3.1. pH influence on M. fijiensis Morelet growth.....................................................................................80 4.3.2. Effect of the micronutrients B, Cu, Mn and Zn on M. fijiensis Morelet under in vitro conditions ..81 4.3.3. Effect of the micronutrients B, Cu, Mn and Zn on M. fijiensis Morelet under greenhouse conditions ......................................................................................................................................................................83
4.4. DISCUSSION .................................................................................................................................... 88 4.5. CONCLUSIONS ................................................................................................................................ 88 CHAPTER 5 THE EFFECT OF SILICON ON M. FIJIENSIS MORELET, ON THE BANANA PLANT AND ON BLACK LEAF STREAK DISEASE UNDER IN VITRO AND IN VIVO CONDITIONS ......................................................................................................................................... 89 5.1. INTRODUCTION ............................................................................................................................... 89 5.2. EFFECTS OF SI ON M. FIJIENSIS MORELET AND THE BANANA PLANT UNDER IN VITRO CONDITIONS ............................................................................................................................................................... 90 5.2.1. Materials and Methods.......................................................................................................... 90 5.2.2. Results.................................................................................................................................... 92 5.2.2.1. Si effect on M. fijiensis Morelet under in vitro conditions ............................................................92 5.2.2.2. Si effect on banana plantlets under in vitro conditions..................................................................93
5.3. EFFECTS OF SI ON THE BANANA PLANT AND BLSD UNDER GREENHOUSE AND FIELD CONDITIONS ............................................................................................................................................................... 96 5.3.1. Materials and Methods.......................................................................................................... 96 5.3.2. Results.................................................................................................................................... 97 5.3.2.1. The response of banana plants to Si amendments under greenhouse conditions ..........................97 5.3.2.2. Banana response to Si applications under field conditions..........................................................110
5.4. DISCUSSION .................................................................................................................................. 113 5.5. CONCLUSIONS .............................................................................................................................. 113 CHAPTER 6 CONCLUSIONS AND PERSPECTIVES .................................................................. 115 IN VITRO STUDIES ................................................................................................................................ 116 IN VIVO STUDIES .................................................................................................................................. 116 PERSPECTIVES ..................................................................................................................................... 117 REFERENCES ...................................................................................................................................... 119 ANNEXES .............................................................................................................................................. 135 ANNEX 1: LIST OF PRESENTATIONS, PUBLICATIONS AND MANUSCRIPTS .......................................... 135 ANNEX 2: AREA UNDER A CURVE (AUC) ......................................................................................... 137
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LIST OF ABBREVIATIONS AUC ANOVA BLSD C o C CEC EC ED50 GDP F ha KOH L LCV LM mm dS/m cmol/kg min ml MS n NF ns O OM OT PD PDA mg/kg psi PS r2 ROS rpm SE SPAD ton yr v/v α
area under a curve analysis of variance Black leaf streak disease conventional degree Celsius cation exchange capacity electrical conductivity effective dose 50% Gross domestic product fertilizer hectare potassium hidroxide litre leaf critical values local microorganisms millimetre
decisiemens per metre cmols per kilogram minute millilitre Murashige and Skoog number of observations no fertilizer not significant organic organic matter organic tea potato dextrose potato dextrose agar milligram per kilogram pound per square inch potassium silicate determination factor
reactive oxygen species revolution per minute standard error soil plant analysis division tonnes year volume – volume alfa
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LIST OF FIGURES Figure 1.2 Conidia and ascospores (40x) of M. fijiensis Morelet and disease symptoms after spore germination at field conditions (photo taken CIBE - ESPOL). ....................................... 7 Figure 1.3 Rainfall and BLSD severity, during two periods: average of 2000 to 2006 and during 2007 in an Ecuadorian banana zone (Data from DOLE Cia., with permission). ........... 7 Figure 1.4 Research outline of the study. ............................................................................... 23 Figure 2.1 Evolution of BLSD symptoms under greenhouse conditions (photo CIBE ESPOL). Numbers correspond to the scale in Table 2.1. ........................................................ 28 Figure 2.2 Schematic representation of Gauhl’s modification of the Stover’s severity scale used to evaluate the status of BLSD in the banana plant......................................................... 29 Figure 2.3 BLSD stages recorded during 28 days on banana leaf discs cultured under in vitro conditions from vegetative (A) and generative (B) plants. Leaves 1 to 6 were sampled from an organic (O) and a conventional (C) farm (n = 30)................................................................... 33 Figure 2.4 BLSD symptom evaluations over 60 days on micropropagated banana plants grown in the greenhouse and inoculated with M. fijiensis Morelet. The conidial solution came from isolates from an organic (O) and conventional (C) farm. Columns with the same letter and for the same time (days after inoculation) are not significantly different by Mann Whitney-test (n = 40)............................................................................................................... 35 Figure 2.5 BLSD symptoms evolution in leaf 3 and leaf 4 in an organic (O) and conventional (C) farm during the rainy season 2004 (n = 80). ..................................................................... 36 Figure 2.6 BLSD symptoms evolution in leaf 3 and leaf 4 in an organic (O) and conventional (C) farm during the dry season 2004 (n = 80). ........................................................................ 37 Figure 2.7 Morphological characteristics of M. fijiensis Morelet colonies grown in a Petri dish on solid medium for 30 days (approximately 4.5x magnified) (photo CIBE – ESPOL). 44 Figure 2.8 Dendogram using Clustering Method, Single Linkage and Euclidean Distance Type, based on the combination of colour, morphological characteristics and growth rate of all selected M. fijiensis Morelet colonies from an organic (O) and a conventional (C) production system.................................................................................................................... 47 Figure 2.9 BLSD symptoms evaluation over 45 days on leaf pieces inoculated with three concentrations of conidia per ml: 1 (1000), 2 (5000) and 3 (10000), produced from organic (O) and conventional (C) isolates, using plastic (p) and glass (g) Petri dishes and incubated for 12 and 24 hours (n = 36). Treatments with the same letters are not significantly different by T-test................................................................................................................................... 48 Figure 2.10 Relationship between all factors involved in the evaluation of the aggressiveness of isolates. Production systems: organic (O) and conventional (C); hours of light: 12 and 24; Petri dish materials: plastic (p) and glass (g); inoculum concentrations: 1000, 5000 and 10000 M. fijiensis Morelet conidia per ml.......................................................................................... 49 Figure 3.1 Raw materials and different steps in the preparation and fermentation of the organic tea in an organic banana farm in Ecuador (photo CIBE-ESPOL). ............................. 54 Figure 3.2 Diameter of M. fijiensis Morelet colonies grown under in vitro conditions on agar medium amended with organic teas collected in Taura and Balao banana farms in June and September 2004. Organic teas were at 0, 10, 30 and 70% v/v concentration and sterilized by autoclaving or filtration. Evaluation was made 7 and 15 days after inoculation (n = 25). ...... 57 Figure 3.3 Weight of M. fijiensis Morelet mycelium grown in in vitro liquid medium amended with organic teas collected in Taura and Balao banana farms in June and September 2004. Organic teas were at 0, 10, 30 and 70% v/v concentration and sterilized by autoclaving or filtration. Evaluation was made 15 days after inoculation (n = 8). ..................................... 57 Figure 3.4 Ascospore inhibition (%) of M. fijiensis Morelet in in vitro solid medium amended with organic teas (OT). OT were at 0.5, 1, 3, 5 and 10% v/v concentration. Evaluation was made 48 hours after discharge of ascospores (n = 50)............................................................. 58 Figure 3.5 Weight of M. fijiensis Morelet mycelium grown in in vitro liquid medium amended with organic teas (OT) collected in three zones (Los Rios (R), El Oro (O) and
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Guayas (G)) and after three fermentation times (1, 2 and 4 months). OT were evaluated at 0, 10, 30 and 70% v/v concentration and sterilized by autoclaving. Evaluation was made 15 days after inoculation (n = 8)........................................................................................................... 58 Figure 3.6 pH of nine organic teas produced in three different zones (Los Rios (R), El Oro (O) and Guayas (G)), and during three fermentation times (1, 2 and 4 months)..................... 61 Figure 3.7 Conductivity of nine organic teas produced in three different zones (Los Rios (R), El Oro (O) and Guayas (G)), and during three fermentation times (1, 2 and 4 months). ........ 61 Figure 3.8 BLSD symptoms over 60 days in leaves 2, 3 and 4 of banana plantlets. OT from Taura and Balao were applied for 9 weeks before inoculation with a M. fijiensis Morelet conidia solution (3x103conidia/ml). The disease development was evaluated four times after inoculation at 15 days intervals. The products were applied once or three times weekly at 10, 30 and 70% and compared with treatments with and without fertilizer under greenhouse conditions (n = 20)................................................................................................................... 67 Figure 3.9 Effect of OT sampled at Taura and Balao farms on height of banana plantlets grown under greenhouse conditions. The products were applied once or three times weekly at 10, 30 and 70% and compared with treatments with and without fertilizer under greenhouse conditions (n = 20)................................................................................................................... 70 Figure 3.10 Damage caused on micropropagated banana plants (Williams variety, Cavendish group) by applications of OT from Taura at 70% v/v. From the group of plants that received one application weekly only few plants survived and the group that received three applications weekly all plants died. The green groups of plants around corresponded to the others treatments with Balao OT that didn’t cause toxic effects at any factors studied (concentration and application time) and the lower concentrations of OT from Taura (10, 30%) at both application time.................................................................................................. 71 Figure 3.11 BLSD severity index in banana plants under commercial growing conditions treated weekly with organic tea (OT) prepared for foliar, root and foliar/root application versus treatment without OT (no product). Data were collected on vegetatively growing mother plants until flowering. Once mother plants started flowering, data were collected on ratoon plants (n = 30). ............................................................................................................. 73 Figure 4.1 M. fijiensis Morelet colony growth on a solid in vitro medium in the 4 to 12 pH range. Colony diameter was measured 7 and 15 days after inoculation. The bars are standard errors (n = 25).......................................................................................................................... 80 Figure 4.2 Radial distribution of mycelium weight of M. fijiensis Morelet grown in in vitro liquid medium in the 4 to 12 pH range. Mycelium weight was measured 15 days after inoculation (n = 8). .................................................................................................................. 81 Figure 4.3 Regeneration of M. fijiensis Morelet mycelium on a nutrient-free solid in vitro medium after earlier exposure to different micronutrients on a solid in vitro medium. Observations took place after 7 days. Bars with different letters indicate significant difference at P
