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DISSERTATIONES DE AGRICULTURA

EFFECT OF THE NUTRITIONAL STATUS OF BANANA (MUSA spp.) ON LEAF DISEASE INFESTATION BY

MYCOSPHAERELLA FIJIENSIS MORELET IN ECUADOR

October 2008

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

Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Bio- ingenieurswetenschappen door Ma. Isabel JIMENEZ F.

Katholieke Universiteit Leuven Faculteit Bio-ingenieurswetenschappen Departement Biosystemen – Afdeling Plantenbiotechniek Laboratorium voor Tropische Plantenteelt

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’

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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


3.3.2.1. 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


5.2.2.1. 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


5.3.2.1. 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 area under a curve ANOVA analysis of variance

BLSD Black leaf streak disease C conventional

oC degree Celsius CEC cation exchange capacity EC electrical conductivity

ED50 effective dose 50% GDP Gross domestic product

F fertilizer ha hectare

KOH potassium hidroxide L litre

LCV leaf critical values LM local microorganisms mm millimetre

dS/m decisiemens per metre cmol/kg cmols per kilogram

min minute ml millilitre MS Murashige and Skoog n number of observations

NF no fertilizer ns not significant O organic

OM organic matter OT organic tea PD potato dextrose

PDA potato dextrose agar mg/kg milligram per kilogram

psi pound per square inch PS potassium silicate r2 determination factor

ROS reactive oxygen species rpm revolution per minute SE standard error

SPAD soil plant analysis division ton tonnes yr year v/v volume – volume α 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<0.05.................................................................................................................................. 82 Figure 4.4 BLSD symptoms on banana plants (n = 10) established under greenhouse conditions and grown for 9 weeks. The plants received foliar applications of different micronutrients at three concentrations at 7 and 15 days intervals. Plants were inoculated with a conidia solution. Controls are fertilized and not fertilized plants ......................................... 87 Figure 5.1 Effect of Si sources and different concentrations on M. fijiensis Morelet colony growth after 7 and 15 days of cultivation on a solid in vitro medium (n = 25). ...................... 92 Figure 5.2 M. fijiensis Morelet mycelium growth under different concentrations of silicon (Si) and potassium hydroxide (KOH) in a liquid in vitro medium (n = 8). ............................. 93 Figure 5.3 Box plot of BLSD index on banana plants (n = 20) established under greenhouse conditions over a period of 8 weeks. These plants received weekly foliar application of different Si concentrations, ranging from 5-5000 mg/kg (from PS) with pH-adjusted (A) and pH-unadjusted (U) solution. These plants came from an in vitro assay where they had received the same Si concentrations. Reference treatments were plants receiving no fertilizer (NF) and fertilizer (F). ............................................................................................................. 99 Figure 5.4 Chlorophyll content (SPAD unit) of banana leaf with and without BLSD symptoms (n = 20) established under greenhouse conditions over a period of 8 weeks. Under

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greenhouse conditions, the plants received weekly foliar applications of different Si concentrations, ranging from 5-5000 mg/kg (from PS) with pH-adjusted and pH-unadjusted solution. Reference treatments were plants receiving no fertilizer and fertilizer. ................. 101 Figure 5.5 Si content (%) in the banana leaves after 8 weeks under greenhouse conditions. In these conditions, the plants received weekly foliar applications of different Si concentrations, ranging from 5-5000 mg/kg (from PS) with pH-adjusted and pH-unadjusted solution (n = 10). Reference treatments were plants receiving no fertilizer and fertilizer. ................................ 101 Figure 5.6 Foliar area (cm) of banana plants (n = 10) harvested after 8 weeks at greenhouse conditions. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. No fertilizer and fertilizer plants were evaluated as control treatments. ...... 102 Figure 5.7 Leaf thickness of banana plants (n = 10) recorded during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments. ............................................................................................................................. 105 Figure 5.8 Chlorophyll content of banana plants (n = 10) established during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments. ............................................................................................................................. 105 Figure 5.9 Si concentration in the banana corm (n = 10) growing during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments. ............................................................................................................................. 106 Figure 5.10 Si concentration in banana leaves (n = 10) growing during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments .............................................................................................................................. 106 Figure 5.11 Soil pH for banana plants (n = 10) growing during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments. .. 107 Figure 5.12 BLSD severity index of banana plants (n = 30) established under field conditions. The plants received during two intervals (7 and 14 days) and with two different modes of applications (foliar and root) different Si concentrations from PS. Reference treatments were plants receiving no fertilizer and fertilizer .................................................. 112

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LIST OF TABLES Table 1.1 Symptoms of BLSD (Meredith and Lawrence, 1969; Fouré, 1985)......................... 6 Table 2.1 BLSD symptom description for banana plants grown under greenhouse conditions.................................................................................................................................................. 28 Table 2.2 BLSD severity scoring system based on Gauhl’s modification of the Stover’s scale.................................................................................................................................................. 29 Table 2.3 Banana yields and incomes during 5 continuous years (each year being the total of 52 weekly values) from organic and conventional banana farms, located in the Guayas province, Taura village (7 km apart). ...................................................................................... 31 Table 2.4 BLSD symptom accumulation (area under the curve) in 6 leaves from banana plants in their vegetative stage sampled in an organic (O) and conventional (C) farm (n = 30).................................................................................................................................................. 34 Table 2.5 BLSD symptom accumulation (area under the curve) in 6 leaves from banana plants in their generative stage sampled in an organic (O) and conventional (C) farm (n = 30).................................................................................................................................................. 34 Table 2.6 BLSD symptom accumulation (area under the curve) in leaves 3 and 4 from banana plants evaluated in an organic (O) and conventional (C) farm (n = 80). ................................. 35 Table 2.7 Standing leaves and BLSD severity (%) in banana plants at different growth stages in an organic (O) and conventional (C) farm (n = 20 plants per growth stage)....................... 38 Table 2.8 Soil parameters and nutrients from organic (O) and conventional (C) farms. Analysis made by a subsidiary laboratory of BSI that operates in Ecuador............................ 39 Table 2.9 Nutrient concentration on dry matter basis in leaf 3 of banana plants from organic (O) and conventional (C) farms and the banana leaf critical values (LCV). Nutritien a......... 40 Table 2.10 Nutrient composition of OT prepared on the organic farm and used for foliar application on the banana crop in the organic farm. Analysis made by the Soil Service of Belgium. .................................................................................................................................. 41 Table 2.11 ANOVA (one way) of colony form-edge characteristics of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system. .................... 45 Table 2.12 ANOVA (one way) of texture characteristics of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system. ........................................... 45 Table 2.13 ANOVA (one way) of growth rate (mm/week) of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system.................................... 46 Table 2.14 ANOVA (one way) of amount of conidia (per ml) of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system.................................... 46 Table 3.1 In vitro inhibitory effects (0%: no inhibition; 100%: complete inhibition) on M. fijiensis Morelet colonies 7 days after inoculation grown on a solid medium amended with seven organic teas (OT) at 10 and 30 % v/v collected from one farm (Taura), (n = 35). Means with the same letter per OT and per concentration are not significantly different by Tukey test at P≤0.01.................................................................................................................................. 56 Table 3.2 Nutrient content of seven organic teas (OT) prepared by the farmer for foliar application. OT collected during 2004 in one organic farm in Guayas province. ................... 59 Table 3.3 Nutrient content of seven organic teas (OT) prepared by the farmer for root application. OT were collected during 2004 in one organic farm in Guayas province. .......... 60 Table 3.4 Nutrient content of four OT prepared in two different organic farms and in two periods of the year 2005. ......................................................................................................... 60 Table 3.5 Nutrient content of nine organic teas (OT) prepared in three locations and with three fermentation times. Coeficient of variance obtained per location (A) (Los Rios - R, El Oro - O and Guayas - G), and per fermentation time (B) (1, 2 and 4 months)........................ 62 Table 3.6 Proximity matrixes of the macronutrient (A) and micronutrient (B) content of nine OT prepared in three locations (Los Rios - R, El Oro - O and Guayas – G), and three fermentation time (1, 2 and 4 months). ................................................................................... 63 Table 3.7 Differences in BLSD symptoms (p-value by Mann-Whitney test) at 15 days intervals in three leaves of banana plantlets grown under greenhouse conditions (n = 20). The

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plantlets received organic teas (OT) from two origins (Taura and Balao). OT was applied once and three times per week and OT untreated plants were fertilized or not fertilized (control). OT was applied during 9 weeks before inoculation with M. fijiensis Morelet conidia (3x103conidia/ml). .................................................................................................................. 68 Table 3.8 Area under the curve (AUC) of plant height and number of leaves in greenhouse grown banana plants measured at weekly intervals for 9 weeks. Plants received organic teas from Balao at two frequencies and OT untreated plants were fertilized or not fertilized (control) (n = 20). Means in a column with the same letter are not significantly different by Duncan’s test at P≤0.05........................................................................................................... 69 Table 3.9 Area under the curve (AUC) of plant height and number of leaves in greenhouse grown banana plants receiving organic teas (OT) from Balao and Taura at two frequencies. Treatments were performed weekly for 9 weeks, (n = 20). Means in a column with the same letter are not significantly different by Duncan’s test at P≤0.05. ............................................ 69 Table 3.10 Area under the curve for BLSD severity index for the entire leaf canopy (A) and disease development in the youngest leaves 3 plus 4 (B) for banana plants growing under field conditions. The plants were treated weekly with organic teas (OT) prepared for foliar, root and foliar/root application or untreated (control). Data were collected weekly on non flowering mother plants for 23 weeks until flowering, and for 8 weeks on ratoon plants (n = 30). Means in the same column for the same variable with the same letter are not significantly different by Duncan’s test at P≤0.05. ...................................................................................... 72 Table 3.11 Area under the curve (AUC) for height, circumference at 1 m height and number of leaves of field grown banana plants in Taura treated weekly with organic teas (OT) prepared for foliar, root and foliar/root application versus control without OT. Data were collected weekly on vegetatively growing mother plants for 23 weeks until flowering, and for 8 weeks on ratoon plants (n = 30). Means, in the same column per cycle with the same letter are not significantly different by Duncan’s at P≤0.05............................................................. 74 Table 4.1 pH value of the culture medium (solid and liquid) after enrichment with different concentrations of a micronutrient. The pH value was measured before fungal inoculation.... 81 Table 4.2 Influence of different micronutrients and their concentration on M. fijiensis Morelet colony growth (% of inhibition) on a solid in vitro medium at two intervals. The experiments were carried out at least two times per micronutrient (n = 25). For inhibition percentages, colony diameter of treatments was compared with the control. .............................................. 82 Table 4.3 Plant height, total leaves and chlorophyll parameters of banana plants expressed as aea under the curve (AUC) over 9 weeks. . The plants were established under greenhouse conditions (n = 20) and received different micronutrient concentrations (1=low; 2=medium and 3=high) by foliar applications on a weekly basis. No fertilizer and fertilizer plants were used as control treatments. ...................................................................................................... 83 Table 4.4 Leaf thickness, fresh weight of banana plants and BLSD symptoms (n = 10) under greenhouse conditions. Micronutrients were applied weekly (9 weeks) at three concentrations (1=low; 2=medium and 3=high). Controls were plants receiving fertilizer and no fertilizer. After nutrient application, the plants were inoculated with a conidia solution. Data followed by the same letter per column are not significantly different (P≤0.05) according to Duncan’s test. .......................................................................................................................................... 84 Table 4.5 Parameters (n = 10) at plant harvesting, for banana plants established under greenhouse conditions, and which received different micronutrient concentrations (1=low; 2=medium and 3=high) by foliar applications at 7 and 15 days intervals. Fertilized and not fertilized plants acted as control. Data followed by the same small letter in each column are not significantly different (P≤0.05) according to Tukey’s test. Data in the same row for the same parameter and followed by the same capital letter are not significantly different (P≤0.05) according ANOVA (one-way) between both application intervals. ........................ 85 Table 4.6 Area under the curve (AUC) of leaf thickness and chlorophyll over 9 weeks from banana plants (n = 20), established under greenhouse conditions, which received different nutrient concentrations (1=low; 2=medium and 3=high) by foliar applications at 7 and 15 days intervals. Fertilized and not fertilized plants acted as control. Data followed by the same

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small letter in each column are not significantly different (P≤0.05) according to Duncan’s test. .......................................................................................................................................... 86 Table 5.1 Inhibition of M. fijiensis Morelet ascospore germination (%) on a solid in vitro medium with different concentrations of Si and K 48 hours after discharge (n = 50). Different small letters in the same row indicate significant differences at P<0.05 by Duncan’s test. Data followed by the same capital letter in each column are not significantly different (P≤0.05) according to Duncan’s test between both products. ................................................................ 93 Table 5.2 Area under the curve (AUC) over 8 weeks for plant height, number of roots and number of leaves of banana plantlets (n = 16) growing on MS solid medium (pH-adjusted and pH-unadjusted medium) amended with different Si concentrations from PS. ........................ 94 Table 5.3 Area under the curve (AUC) over 10 weeks for plant height, number of leaves, number and length of roots of banana plantlets (n = 16) growing on MS solid medium (pH-adjusted and pH-unadjusted medium) amended with different Si concentrations from PS and standard Si and different K concentrations coming from KOH. Means per column of each parameter with the same letter are not significantly different by Duncan’s test at P≤0.05. .... 95 Table 5.4 Area under the curve (AUC) over 8 weeks of plant height and number of roots from banana plants (n = 20) established under greenhouse conditions which received different Si concentrations during the in vitro period. Under greenhouse conditions, banana plants received foliar applications of different Si concentrations (from PS as a source) in a pH-adjusted and pH-unadjusted solution and both no fertilizer and fertilizer plants acted as control treatments. ................................................................................................................... 98 Table 5.5 Area under the curve (AUC) over 8 weeks of plant height, number of leaves and BLSD symptoms of banana plants (n = 20) under greenhouse conditions. These plants had not received any Si during their in vitro phase. The plants received weekly foliar application of different Si concentrations (from PS) with a pH-adjusted and pH-unadjusted solution. Reference treatments were plants receiving no fertilizer and fertilizer. Means per column of each parameter without a common letter are significantly different (P≤0.05) according to the Duncan’s comparison test...................................................................................................... 100 Table 5.6 Area under the curve (AUC) of plant height, number of leaves and foliar emission of banana plants (n = 20) under greenhouse conditions during 8 weeks. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. No fertilizer and fertilizer plants were evaluated as control treatments. Means per column of each parameter with the same letter are not significantly different by Duncan’s test at P≤0.05. .. 102 Table 5.7 Area under the curve (AUC) of BLSD on banana plants (n = 20) established under greenhouse conditions. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. Reference treatments were plants receiving no fertilizer and fertilizer. ................................................................................................................................ 103 Table 5.8 p-value (T-test) of differences in fresh (A) and dry (B) weight of the entire plant and some of its components (n = 10) which grew under greenhouse conditions during 8 weeks. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. Reference treatments were plants receiving no fertilizer and fertilizer. Data values equal or less than 0.05% are significantly different. .................................................. 104 Table 5.9 Area under the curve (AUC) over 8 weeks for plant height, number of leaves and BLSD symptoms of banana plants (n = 20) growing under greenhouse conditions. The plants received weekly root applications of different Si concentrations from PS and standard Si and different K concentrations coming from KOH. Reference treatments were plants receiving no fertilizer and fertilizer............................................................................................................ 108 Table 5.10 Fresh weight (g) of roots, corm and entire banana plant (n = 10) established under greenhouse conditions. The plants received weekly root applications of different Si concentrations from PS and Standard Si and different K concentrations coming from KOH. Not fertilized and fertilized plants were evaluated as control treatments.............................. 109 Table 5.11 ANOVA of area under a curve of chlorophyll content and leaf thickness of banana plants grown under greenhouse conditions. The plants received weekly root applications of different Si concentrations from potassium silicate (PS) and standard Si (Si) and different K

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concentrations coming from potassium hydroxide (KOH). Data followed by the same letter in each column are not significantly different (P≤0.05) according to Duncan’s test. ............... 110 Table 5.12 Area under the curve (AUC) of plant height and number of leaves over 8 months and days to flowering of banana plants established under field conditions (n = 30). The plants received during two intervals (7 and 14 days) and following two modes of application (foliar and root) different Si concentrations from PS. Reference treatments were plants receiving no fertilizer and fertilizer............................................................................................................ 111 Table 5.13 BLSD severity index and young leaves without symptoms (YLS) on second generation banana plants (sucker) established under field conditions during 3 months (n = 30). The plants received different Si concentrations from PS. Reference treatments were plants receiving no fertilizer and fertilizer. ...................................................................................... 112

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SUMMARY The major leaf disease on bananas is Sigatoka leaf spot caused by the

ascomycetous fungi Mycosphaerella spp. Two species of this genus are detrimental to this crop, i.e. Mycosphaerella musicola J.L. Mulder, the causal agent of yellow Sigatoka and Mycosphaerella fijiensis Morelet that causes the Back Leaf Streak Disease (BLSD). The latter disease arrived in Honduras in the early 1970s from where it spread rapidly through the Caribbean and Latin-America. It displaced yellow Sigatoka to some restricted geographical areas where the environment is cool. M. fijiensis Morelet is a heterothallic fungus with asexual and sexual stages, thereby producing conidia and ascospores.

Conidia and ascospores are spread by wind, water and planting material and infect young leaves as soon they emerge. First symptoms appear as yellow spots at the lower leaf side but after a few weeks evolve into black streaks which coalesce resulting in large dead patches on the entire leaf. If not treated, and under heavy infection pressure, banana plants have almost no green leaves left at harvest. Consequently plants grow slowly and produce small fruits which ripen prematurely. Yield losses can thus be up to 100%.

Over the past 40 years, BLSD has been controlled in commercial farms with fungicides. Routinely this happens once a week during the year but some farmers apply fungicides about 10-15 times per year with some advanced technologies and under less humid conditions. Systemic, protectant and contact fungicides, in water or in oil, are alternated to control this disease. However and because of the sexual stage and the fast cycling, the pathogen rapidly becomes resistant to fungicides. This results in heavier fungicide applications with more adverse consequences for the environment and human health while at the same time increasing substantially the cost of production.

For years, research has been going on to breed BLSD resistant banana plants but no hybrid has reached the market. Genetic engineering techniques offer now new prospects and fields tests are currently going on with bananas genetically modified at K.U.Leuven. Also the search for new fungicides continues. Yet, we believe that a complementary strategy which takes account of a balanced nutrition deserves attention as well. Indeed, it has been proven in several crops that poor fertilization or unbalanced nutrient applications increase disease problems. In other words, it seems that banana plants are fertilized to obtain high yields without taking care of the effect of current fertilizer practices on BLSD development. However for sustainable production there is a need to manage the farm in such a way that BLSD meets with an unfavourable environment. Therefore we investigated the effect of some nutrients on BLSD to design a better integrated pest management system for commercial banana production.

The research we designed dealt with the growth and development of the M. fijiensis Morelet pathogen, the banana plant and the disease. Experiments were therefore conducted in vitro, in the greenhouse and in the field. Two commercial farms were involved in the study; one growing bananas with only organic ingredients while in the other farm bananas received regular fungicide and conventional fertilizer applications. In the organic farm BLSD was under control.

In the in vitro study, M. fijiensis Morelet isolates from the organic and conventional farm were compared for traits such as morphology, aggressiveness, etc. in an attempt to explain why BLSD in the organic farm was not an important disease.

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In general, all studied parameters of both isolates were similar and the potential to generate the disease was comparable. In the organic banana farm, banana plants are treated regularly with organic teas (OT) that are produced locally and therefore the effect of these OT on the pathogen were investigated. Ascospore germination, colony and mycelium growth were evaluated after treatment with OT produced at different moments in the year. These OT inhibit the fungal growth but its effect is highly variable and depends on origin, time of sampling, concentration, etc. This is attributed to the on-farm OT production resulting in a highly variable composition.

Based on these results, the response of the fungus to some nutrients was investigated especially those which have had an effect on plant defence in other crops. B, Cu, Mn and Zn delayed the fungus development in their presence but after removal from the medium only Cu had a lasting inhibitory effect on the fungus. The inhibitory effect of the micronutrient was maximal at a high or low pH.

Special attention was given to Si as banana is a Si accumulator. M. fijiensis Morelet was inhibited by Si and inhibition increased with higher Si concentrations. However after Si removal from the medium, fungal growth recovered. In vitro banana plants grew better with Si and especially the roots benefited.

In vivo experiments were performed in the greenhouse and under field conditions where both the host and the disease were evaluated. Two banana production systems were compared; one being an organic farm whiles the other was a conventional farm. The farms were located 7 km one from each other, in the coastal region of Ecuador. The organic farm produced a lower yield than the conventional one but was more profitable because of lower costs and higher premium prices. It was shown that the organic farm is infected by the pathogen but that the disease does not cause much damage in contrast to the conventional farm. The nutritional status of both farms, both at the soil and leaf level was very similar. Hence the nutritional composition of organic teas (OT) were investigated and shown to be very variable. The preparation of the OT and other organic ingredients were described in detail.

In the greenhouse and under field conditions the effects of the organic products were further studied. In the greenhouse, plant growth and development was stimulated and BLSD reduced with these organic products but depended on frequent and continuous applications. In the field, organic products applied to the mother and the next generation (sucker) plants supported the results obtained in the greenhouse because the disease in the sucker was reduced. Conventional fertilizer however stimulated BLSD development.

Those micronutrients studied in vitro (B, Cu, Mn and Zn) were tested in greenhouse conditions too. Again these micronutrients reduced the disease symptoms and stimulated plant growth.

The effect of Si was also studied in the greenhouse and in the field. Although Si affects the pH of the solution applied, the pH itself has no effect on the results. Si applied to the root had a much greater effect on plant growth and disease reduction than when applied to the leaves. Best results were obtained with 500 – 1200 mg/kg Si and the results on BLSD under greenhouse conditions supported the results obtained under in vitro conditions. In the field, plant growth benefited from Si treatment but the effect on the disease was not clear presumably because the experiment did not last long enough to establish the effects in the following cycle.

CHAPTER 1: Introduction

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Chapter 1 GENERAL INTRODUCTION, OBJECTIVES AND OUTLINE

This thesis covers different aspects of direct and indirect effects caused by

some nutrients and liquid organic products on the leaf fungal pathogen Mycosphaerella fijiensis Morelet, its banana host and black leaf streak disease (commonly called Black Sigatoka) development on that host. Hence, this introductory chapter deals in section 1.1, with the importance of banana in the world as well as in Ecuador, with M. fijiensis Morelet, and black leaf streak disease. The second section (1.2) is focused on current methods used in the management of this plant disease and alternative strategies, studied in this thesis, to control plant diseases with special emphasis on fungal diseases. Finally, the third section (1.3) presents the rationale, objectives and the outline of this study.

1.1. Musa spp. and Black Leaf Streak Disease (BLSD)

1.1.1. Global importance of banana Bananas are grown in more than 120 countries throughout the tropics and sub-

tropics. The world production was around 100 million tonnes in 2006 (FAOSTAT, 2006). Small-scale farmers produce about 87% of all bananas grown worldwide, providing a staple food for millions of people. They are a source of energy, certain minerals and vitamins A, C and B6 (Sharrock, 1996). Bananas become more and more important because they also provide income and play a role in poverty alleviation (Frison and Sharrock, 1999). Bananas will grow in a range of environments and produce fruit throughout the year, which means the crop is important in food supplies when other crops are not available. They are particularly fitted to intercropping systems and to mixed farming with livestock (Bekunda and Woomer, 1996). As a perennial crop, bananas protect the soil against rain and wind and their biomass is used as mulch to improve the soil fertility and quality. Especially in Latin America and the Caribbean, bananas provide foreign exchange and the last FAO statistics (2005) showed that more than 14% of total exported banana come from those areas. The value of banana exports is greatly comparable to that of other fruits, such as apples and oranges, as well as vegetables such as tomatoes and potatoes. In many countries, bananas are more than just a food crop. They provide an important source of fibre; they can also be fermented to produce alcohol (Chandler, 1995; Thompson, 1995; Sharrock, 1996).

In many countries where the population increases, bananas provide a vital food source, so productivity increases are essential, but diseases hinder this goal.

1.1.2. The Ecuadorian banana industry The banana industry has a great economic and social importance in Ecuador.

The evolution of the sector can be analyzed from the following data: during the 1980s, the average contribution of the banana export sector to total exports was 9.38%, and bananas accounted for 38.6% of all agricultural exports. Thus bananas generate a significant amount of hard foreign currency. However, during the 1990s, banana exports represented 21.1% of total exports and 64.7% of all agricultural exports. In 2006, the Ecuadorian banana industry earned eleven hundred million


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dollars and around fifty eight million dollars in taxes. These figures signify 3.84% of total gross domectic product (GDP) and 50% of the agricultural GDP of the country. During 2007, the exports of bananas contributed 55.0% of total revenues from traditional exports and 23.2% of non-oil exports. These data clearly show the economical importance of the banana crop for Ecuador (UNEP, 2007).

In social terms, the banana sector has become one of the most important production activities in Ecuador. Banana production is labour intensive, thus generating a wide range of employment. By 1998, around 98,000 workers were directly involved in banana plantations. Furthermore, by 2007, more than 500,000 families, which results in more than 2.5 million people, depended on the banana industry. The banana crop covers more than 180,000 hectares and bananas are exported by several companies (AEBE, 2006). During the 1990s, the Noboa Company exported 43% of total fruit, Reybanpac 12%, Dole 19% and 26% was exported by other companies. In 2006, 55% of Ecuadorian banana exports came from other companies illustrating the dynamics of the business (AEBE, 2006).

1.1.3. The banana crop and its production systems

1.1.3.1. Morphology of the banana plant

The banana plant is a giant herb, which consists of three main parts: the corm bearing the roots and the suckers (lateral shoots, ratoon), the pseudostem with the leaves and finally, the inflorescence.

The corm, bulb or rhizome, is the real stem and is located below the soil surface. Roots emerge from the corm in groups of 3 or 4 during the first 6 months after planting. Their ramification and length depend on the variety and growing conditions. The apical meristem called the “growing tip” is located in the central top part of the corm. The growing tip remains there during the vegetative stage. The corm bears the leaves and each internode carries a bud, which can develop into a sucker. Suckers are the lateral shoots from the main plant (also called mother plant) and have strong vascular connections with it. The number of suckers depends on the variety and growing conditions. Usually the biggest sucker is selected during the vegetative growth of the mother plant to succeed it after harvest. This selected sucker, is called ratoon. After flowering, a mother plant bears an inflorescence which develops into a fruit bunch that can be harvested 9 to 14 months after planting depending on the variety and production system (Swennen and Rosales, 1994; Price, 1995).

The pseudostem (Figure 1.1) is non-woody and formed by encircling and overlapping leaf sheaths. The length of the pseudostem depends on the number of emerged leaves and its height is measured from the soil to the “Y” shape formed by the youngest two leaf petioles. Indeed leaves emerge on top of the pseudostem. Initially they are enrolled (called “cigar leaf”) but once emerged they fully open. They consist of two lamina halves separated by the midrib which is an extension of the petiole and leaf sheath. Veins run in parallel and perpendicular to the midrib. As there are no secondary veins, wind damage is quite common. The leaf canopy consists of nearly 15 leaves, all of a different age, and emerged at a 7-15 day interval. The pseudostem and petiole color, waxiness and leaf orientation depend on the variety (Swennen and Rosales, 1994; Price, 1995).

The banana growth cycle consists of two phases: the vegetative stage, when leaves are produced and which ends when the inflorescence appears (called shooting); and the generative stage that starts with the emission of the inflorescence, followed by fruit filling and bunch harvesting (Figure 1.1).


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The low multiplication rate of the banana plant and phytosanitary problems arising from the use of infected planting material from the field, stimulated the use of tissue cultured or in vitro developed planting material. This starts with the initiation of cultures from sterilized shoot tips obtained from selected banana plants followed by proliferation and then regeneration (Teisson and Côte, 1997; Heslop-Harrison and Schwarzacher, 2007). Such small plants are then acclimatized in the greenhouse or screenhouse before field planting. This period is called hardening and consists of two phases. During phase 1, the small plants are maintained for 4 weeks in plastic pots containing 98 holes of approximately 0.09 L volume. Afterwards, the plants are transplanted for the second phase into individual plastic containers of 0.9L. During 4 to 6 weeks, plants are then further maintained until transplanting to the field.

Figure 1.1 Morphology of a banana plant of the variety Valery (Cavendish AAA group) (taken by CIBE – ESPOL).


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1.1.3.2. Banana production systems

1.1.3.2.1. Conventional system

Global chemical fertilizer consumption increased tenfold between 1950 and 2000. Its over-consumption or inappropriate use causes environmental problems including loss of biodiversity and underground water contamination but also health problems such as birth defects, nerve damage and cancer (Rekha and Prasad, 2006; Ghorbani et al., 2008a).

The conventional production system of banana is based on the use of large amounts of synthetic fertilizers and pesticides. The banana crop earmarked for export is produced in monocultures, resulting in a continuous loss of organic material and impoverishment of soils. Around 978 kg urea/ha/year and 1,250 kg KCl/ha/year is applied. Fertilizers are also more and more applied on leaves (Lopez and Espinoza, 1995). BLSD is managed by a combination of cultural practices and chemical methods. This disease was controlled by a Bordeaux mixture (suspension of Cu sulphate, hydrated lime and water) in the mid 1930s. In the 1950s, petroleum oil and dithiocarbamate fungicides superseded the Bordeaux mixture. The first systemic fungicide used was Benomyl, followed by propiconazole (triazoles). Currently used fungicides are mancozeb, chlorothalonil (protectants), benzimidazoles, triazoles, morpholine and strobilurins (systemic fungicides). However the chemical control of BLSD must be seen as part of an integrated disease management strategy, which should involve also good sanitation practices, adequate drainage systems, optimal plant densities and nutrients for plant growth (Jones, 2000). In general, the conventional production system in all production zones is especially aimed at eliminating or reducing the pathogen by regular applications of fungicides.

1.1.3.2.2. Organic system

In general, organic agriculture is a holistic production system which emphasizes the use of agronomic, mechanical and biological methods to enhance the whole farm ecosystem (FAO/WHO, Codex Alimentarius, 1999).

Organic systems promote integrated, balanced and stable production systems, with maximal use of resources from the farm, including wastes, thereby reducing dependency on external inputs. An organic system is aimed at the re-establishment of a natural ecological equilibrium, based on a high soil biodiversity, and chemical and physical soil qualities. It respects the natural resources, but entails additional production and certification costs (IFOAM, 1998; FAO, 1999).

Banana varieties are cultivated for export and demand high yield. In ecological and natural systems, banana plants are planted in new plantations, and usually replaced by other plants after 5-10 years. Organic material from prunings and harvest residues are used to maintain a good quality/active life soil. Mulch is uniformly scattered on the soil surface. Animal manure is used as a complementary fertilizer, but not as a main nutrient source (CORPEI, 2001). In organic banana systems, the management of phytosanitary problems needs to rely on an environment favourable for the host, but adverse to the pathogens (Holderness et al., 2000)).

The certification process for organic banana farms is imperative and necessary to check that the product fulfils certain minimal ecological production standards. Such standards can be elaborated by private firms or associations as well as certification bodies or by a public administration. It is however important that the applied local


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practice is internationally recognized. More than ten international as well as national certification agencies are active in Ecuador. Nowadays, Ecuador has more than 10,000 hectares producing banana with organic certification (FAO, 2003; SICA, 2007).

1.1.4. Disease constraints Bananas are affected by a range of diseases and pests caused by fungi,

bacteria, viruses and nematodes (Gowen, 1995; Moore et al., 1995; Sarah et al., 1996; Bridge et al., 1997; Mourichon et al., 1997; De Waele and Davide, 1998; Jones, 2000; De Beer et al., 2001) which damage the foliage, roots, corm, pseudostem and fruits. Leaf spot disease is the most important problem in banana production worldwide because the varieties used are extremely susceptible to the causal agent of the disease. Ecuador is not an exception with its banana monocultures.

1.1.4.1. BLSD symptoms and the life cycle of M. fijiensis Morelet

BLSD is considered a major economical constraint for banana production because both plant growth and yield are affected. The disease reduces the photosynthetic area extensively. Yield losses range between 30% to 100% when control measures are inadequate or fail respectively (Stover, 1983; Mobambo et al., 1993). In addition, it causes premature fruit ripening in the field and during transportation or storage (Mourichon et al., 1997; Carlier et al., 2000b).

Mycosphaerella fijiensis Morelet, the causal agent of BLSD is a heterothallic ascomycete with asexual (conidia) and sexual (ascospores) reproduction stages (Carlier 2000a). The fungus produces conidia and ascospores when the leaf surface is wetted either by rain or dew. The conidia are spread by rain and irrigation from plant to plant. Ascospores are however spread by wind over longer distances and are considered the most important means of spreading the disease in plantations (Stover, 1980 and 1983; Mourichon and Zapater, 1990; Gauhl, 1994). Conidia production is higher during the dry season (Fouré and Moureau, 1992) while ascospores are more abundant during the rainy season (Meredith et al., 1973). The production, maturation and release of ascospores are related to increases in rainfall and relative humidity (98 to 100%) (Carlier et al., 1994; Fullerton and Olsen, 1991, 1995). Once on a leaf, spore germination occurs rapidly and germ tubes of both ascospores and conidia penetrate through the stomata after 48-72h when the ambient temperature is above 20ºC (Fouré and Moureau, 1992). Irrespective whether the infection occurs by ascospores or conidia, the same stages of symptoms are produced and the disease development is the same (Figure 1.2). Ascospores play an important role in the M. fijiensis Morelet population structure as they are sexual recombinants. Indeed, a large genetic variability has been observed between ascospores coming from different geographical locations, from different plants and varieties, between different lesions on the same plant and even within a single lesion. Hence this allows for the rapid adaptation to fungicides and host varieties (Carlier et al., 1994; Fullerton and Olsen, 1995; Muller et al., 1997; Hayden et al., 2003).

The disease development is divided into six symptomal stages (Table 1.1).


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Table 1.1 Symptoms of BLSD (Meredith and Lawrence, 1969; Fouré, 1985).

Disease symptoms Description Stage 1: initial speck stage Symptoms occur initially as feint, minute (up

to 0.25 mm in diameter), reddish brown specks on the lower surface of the leaf. They are often more abundant near the margin, particularly towards the tip.

Stage 2: first streak stage

The specks elongate, becoming slightly wider, and form a narrow, reddish brown streak up to 20 x 2 mm, with the long axis parallel to the leaf venation. The streaks are more clearly visible on the lower surface of the leaf than the upper surface and its distribution on the leaf is variable. Streaks can be numerous and coalesce to form larger streaks.

Stage 3: second streak stage The streaks change colour from reddish brown to dark brown or almost black, sometimes with a purplish tinge, becoming clearly visible on the upper surface of the leaf. When they are numerous and more or less evenly distributed, the entire leaf blackens.

Stage 4: first spot stage The streak broadens and becomes somewhat fusiform or elliptical and develops a light brown, water-soaked border around the spot.

Stage 5: second spot stage The dark brown or black central area of the spot becomes slightly depressed and the water-soaked border becomes more pronounced due to darkening. At this stage, a slight yellowing of the leaf tissue immediately surrounding the water-soaked border may occur.

Stage 6: third or mature spot stage

The centre of the spot dries out, becoming light grey or buff-coloured, and further depressed. The spot is surrounded by a narrow, well-defined, dark brown or black border. Between this border and the normal green colour of the leaf, there is often a bright yellow transitional zone. After the leaf has collapsed and withered, spots remain clearly visible with the light-coloured centres and dark borders.


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Figure 1.2 Conidia and ascospores (40x) of M. fijiensis Morelet and disease symptoms after spore germination at field conditions (photo taken CIBE - ESPOL).

The period between infection, the appearance of the first symptoms and

subsequent necrotic spots until the production of disseminating organs, depends on the variety, climatic conditions and the severity of the infection (Fullerton, 1994; Jacome and Schuh, 1992). The high rainfall and humidity of the tropical regions where bananas are grown are especially favourable for the development of this disease (Figure 1.3).

In Ecuador, BLSD was first reported in 1987 in Cavendish plantations near the northern coastal province of Esmeraldas (Fernandez, 1993). From there, the disease spread rapidly throughout the country and affected all banana plantation areas. Nowadays, as the banana crop is planted in more than 10 provinces of the country, BLSD occurs everywhere (Jimenez, 2000).

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).

1.1.4.2. BLSD control methods

In conventional plantations of the Cavendish subgroup, BLSD is managed by a combination of cultural practices and chemical methods. Chemical control is the basis of BLSD management strategies (Jones, 2000; Marin et al., 2003). Bordeaux

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mixture, first used in the mid-1930s, has been replaced by several succeeding generations of protectant and systemic fungicides, all with a different mode of action. Benzimidazoles impede the binding of β-tubulin proteins as they form microtubes, and strobilurins inhibit electron transport between cytochrome b and cytochrome c1 in the mitochondrial respiration chain of the pathogen (Clough et al., 1994; Beresford et al., 1999; Bartlett et al., 2002). These groups of products have a high risk of inducing resistance (Brent, 1995). Triazoles inhibit the pathogen ergosterol biosynthesis pathway via the inhibition of 14-sterol demethylase, whereas morpholines affect the synthesis of C14 reductase, ∆7 and ∆8 isomerase of the ergosterol biosynthesis. These products are considered to be multi-site inhibitors and of medium resistance risk (Brent and Hollomon, 1998; Ruiz et al., 1999).

Aircraft are used for aerial applications. In small farms, motorized backpack sprayers or tractors are used. Since systemic fungicides tend to build up resistance or tolerance by M. fijiensis Morelet, these fungicides are combined or alternated with broad-spectrum, protectant fungicides, such as the dithiocarbamates and chlorothalonil. With the exception of chlorothalonil, these fungicides are usually mixed with petroleum-based oil sprays. The oils themselves are fungistatic and retard the development of the pathogen in the infected leaf. When they are mixed in water emulsions with fungicides, the resulting “cocktails” provide superior disease control (Ploetz, 2004; Marin et al., 2003).

Disease forecasting systems rely on fungicides to arrest infections during the early stages of development. Based on measurements of temperature and evaporation, in conjunction with the assessment of early infection stages, the application of fungicides is planned. Weekly evaluations of the first 3 unfolded leaves a ‘stage of evolution of the disease’ is deduced, and used to programme fungicide applications (Marín and Romero, 1992).

Cultural practices are focused on inoculum reduction, such as excision (and removal) of necrotic leaves, reduction of relative humidity inside the crop by efficient draining systems to remove water excess and reduction of the relative humidity (Wielemaker, 1990; Calvo and Bolaños, 1998; Carlier et al., 2000a).

The delivery of BLSD resistant bananas and plantains is considered to be the best and most sustainable alternative. However cross breeding has to overcome numerous obstacles especially high sterility (no seed set) and long generation times. Members of the Cavendish subgroup are completely sterile as the fruit is produced parthenocarpically (Simmonds and Shepherd, 1955; Robinson, 1999).

This explains why banana and plantain breeding resulted only recently in the delivery of some BLSD resistant plants after more than 70 years intensive efforts (Rowe, 1984; Shepherd, 1987; Bakry et al., 1990; Shepherd, 1990; Swennen and Vuylsteke, 1990; Bakry and Horry, 1992; Swennen and Vuylsteke, 1993; Rowe and Rosales, 1993; Ortiz and Vuylsteke, 1994a; Ortiz and Vuylsteke, 1994b; Ortiz et al., 1995; Rowe and Rosales, 1996; Ortiz, 1997; Vuylsteke, 2000). Hence genetic engineering offers many prospects because this technique allows the delivery of varieties identical to the preferred ones with additional resistance to BLSD. Transformation techniques for banana have been developed (Sagi et al., 1995a and 1995b; May et al., 1995), genes for BLSD resistance inserted and field tests are ongoing. However this strategy is handicapped by the fact that for each desired variety, transgenic plants need to be developed, tested and accepted.

Research is ongoing to control BLSD by inducing the systemic resistance response in banana plants (Holderness et al., 2000). Several products have been tested


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but limitations are great because the line between phytotoxicity and effective control is indeed very fine.

The cultivation of bananas under certain organic conditions indicates that BLSD can be reduced to a certain extent. In this system, all pesticides are prohibited and disease management relies on prevention rather than control. Key practices are the application of biological products and organic solutions, proper soil management stimulating high biological activity, efficient drainage, balanced nutrition, removal of infected leaves, use of Cu salts and mineral or vegetable derived oils, etc. (Holderness et al., 2000).

1.2. Plant fungal disease control methods This section is divided in two parts. First, information about the conventional

control methods for plant diseases is given focused on the three methods used in banana production. Then extensive information serves for the second section dealing with the two strategies studied in this thesis.

1.2.1. Conventional methods: chemical, physical and cultural Plant diseases are governed by the direct interaction of the host, pathogen and

environment. Other components such as the workforce in the plantations that perform the phytosanitary activities and move infected plant material, and time, related to the biological cycle of the pathogen, are also important for disease management. Consequently, the manipulation of one or several of these factors influences the plant disease level. By integrating available techniques, diseases can be kept under a threshold where there is minimal economic and environmental damage. In addition this slows down or avoids pathogens becoming resistant and maintains biodiversity (Browning et al., 1977; Agrios, 2005).

1.2.1.1. Chemical methods

In general, chemical methods use synthetic pesticides that affect in one or more aspects the pathogen. The last generation of fungicides are so effective that only small amounts are needed to kill the pathogen. However, this does not reduce the risk to humans, animals and the environment. Usually, several fungicides control a broad spectrum of plant fungi whereas others are specific to some group of pathogens. This forms also the basis for fungicide rotation which is essential to avoid pathogen resistance. There are two groups of fungicides: protectant and systemic. The protectant fungicides cover the plant surface where they avoid the entrance of pathogens. To be effective, repeated applications on the crop are needed. Systemic fungicides enter and spread through the plant and are then everywhere toxic to the pathogen. Resistant, insensitive or tolerant are terms to describe a pathogen’s behaviour towards the fungicides. Two categories of resistance are discerned: discrete resistance and multi-step resistance. The first involves a rapid change from sensitive to resistant but the latter appears slowly or less suddenly and is linked to the circumstances of fungicide application. In the end both kinds of resistance cause serious problems for disease management (Brent, 1995).

Fungicides are classified in classes with low, moderate and high risks of generating resistance. Pathogens are also grouped in three classes with low, medium and high risks of developing resistance. For example Mycosphaerella fijiensis Morelet and Phytophthora infestans are high risk pathogens; Mycosphaerella musicola


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J.L.Mulder and Monilia spp. are pathogens considered with medium risk; Pythium and Rhizoctonia spp. have a low risk to develop resistance towards fungicides. To avoid the build up of resistance, several strategies can be implemented such as product rotation, follow the recommended dosage and avoid fungicide strategies which try to completely eliminate the pathogen (Delp and Dekker, 1985; Brent and Hollomon, 1998; Brent, 1995).

In response to public concern and to increase sustainability and disease control, there is a need to reduce the use of pesticides. This should involve the training of farmers, research on disease control measures other than pesticides, better understanding of threshold levels of infection that cause economic losses and development of disease forecasting systems to allow the optimal use of chemicals.

1.2.1.2. Physical methods

Physical control methods are complementary in the management of plant diseases. They can be passive or active in eliminating pathogens. In general, physical methods are non-polluting but some cause other problems. For instance, tillage can increase soil erosion, open-field burning destroys organic matter and increase air pollution, and others are not economical such as solar-heating practices using a plastic covering the soil. Definitely the future application of these practices depends on more research (Cook, 1988; Katan, 2000; Vincent et al., 2003; Yoneyama, 2004; Agrios, 2005).

1.2.1.3. Cultural methods

Cultural practices are important in the management of plant diseases and complement other methods. Their goal is to prevent disease establisment or reduce the pathogen population or inoculum. Deleafing of infected leaf tissue is an example of a cultural practice used to manage black leaf streak disease. Cultural practices are not always economical (Ellett et al., 1996; Carlier et al., 2000a; Katan, 2000 and 2004).

1.2.2. Alternatives approaches to disease management For good disease management, different control methods need to be used

simultaneously to reduce economic and environmental problems. Some strategies related with individual nutrients influence disease development and fermented solid and/or liquid organic products apparently are involved in the reducction of plant diseases.

Hence in the next two sections an overview is presented on the positive effects and the possibilities that both strategies offer on disease management. First, nutrients are discussed which are used in Ecuador for sustainable banana production. In addition, these nutrients are discussed with respect to fungal diseases in other crops as there is not much such information for the banana crop. In the second part, the presented information deals with organic amendments affecting foliar and soil diseases.

1.2.2.1. Mineral nutrition and plant disease

Nutrients are classified as macronutrients and micronutrients depending on whether they are needed in large or small quantities. There are sixteen nutrients needed by a plant; carbon, hydrogen and oxygen comes from the air but the other nutrients come from the soil. N, P and K are needed in highest amounts followed by


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Ca, Mg and S. The micronutrients (Fe, Mn, Zn, Cu, B, Mo and Cl) are essential but the plant requires them in minute amounts (Fritz, 1985). Other elements like Ni, Se, Co or Si are beneficial to some specific crops.

Plants are deficient when some of these essential nutrients are not or under-supplied while oversupply results in toxicity problems (Stevens et al., 2002). The location of nutrient deficiency symptoms depends on the mobility of the nutrient. Therefore some macronutrients (N, P, K, Mg) become deficient in the older leaves while other elements (Ca, Zn, B, Mn) become deficient in new leaves when supply is insufficient (Mengel, 2002). Different soil conditions influence availability such as pH, texture, moisture, temperature, as well as mineral solubility, and soil microbial activity.

The influence of mineral nutrition on plant disease development is well studied, especially for the macronutrients (Huber, 1980; Datnoff, 1994; Walters and Bingham, 2007). However, many aspects are not well understood when micronutrients are involved. Nutrients are involved in different processes like enzyme activation, and metabolic regulation but also in structural components (Daurob and Snyder, 2007; Moreno et al., 2003). Clearly plants with balanced fertilization are less susceptible to diseases while plants with unbalanced fertilization become more susceptible to diseases (Henn, 2004). According to Huber (1980) "Plant nutrition has a big effect on the plant's susceptibility to disease" and "Not much micronutrient is needed to mobilize a plant's disease resistance, but it is critical”. Moreover, micronutrients are needed for the health of the consumers especially in developing countries where their lack is linked to the so-called “hidden hunger” (IFIA, 2007).

We now briefly highlight the role of different nutrients with special focus on micronutrients as these are part of the research focus of this thesis.

1.2.2.1.1. Macronutrients: Nitrogen, Phosphorus, Potassium, Calcium, Magnesium

Nitrogen (N) is essential for the formation of proteins, enzymes, energy transfer and chlorophyll (Tucker, 1999). In excess or undersupply it results in more disease development but if applied optimally it will contribute to resistance (Huber and Watson, 1974). The effects on the infection of Uromyces rumicis in Rumex obtusifolius leaves were linked to N sources and concentration (Hatcher and Ayres, 1998). In wheat varieties infected by Pyrenospora tritici-repentis (tan spot disease) different disease expression levels were also correlated with different concentrations of N (Huber and Lee, 1987). This was similar in two genotypes of wheat with different resistance expression to yellow rust (Puccinia striiformis) (Danial and Parlevliet, 1995). In potatoes, the lowest doses of N reduced the intensity of late blight and blackleg. The highest population of Alternaria alternata, Botrytis cinerea, Colletotrichum coccodes and Fusarium colonizing the potato “Rybitwa” was found with the lowest amount of N applied (Cwalina-Ambroziak, 2004). Sheath blight Rhizoctonia solani in rice increased with excessive N fertilization (Slaton et al., 2003). Rice in rotation with soybean in areas where high N rates were used, increased sheath blight incidence in susceptible rice cultivars (Belmar et al., 1987). In another trial it was shown that disease increased with increased N applications in four slow-blasting rice genotypes (Mukherjee et al., 2005). The form of N affected the incidence of some diseases like Fusarium Wilt (Fusarium oxysporum f.sp. lycopersici) in tomatoes where ammonium fertilization is not recommended (Gleason and Edmunds, 2006). In turfgrasses the excessive use of N increased leaf spot and crown rot, and high amounts of N in St. Augustinegrass increased gray leaf spot, caused by the


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fungus Pyricularia grisea. In contrast, rust disease caused by Puccinia, increased under low N (Hagan, 2005). High amounts of N in tea plants were related to anthracnose in Japan (Chen and Chen, 1999).

Potassium (K) affects plant diseases because it plays important regulatory roles. It is required in the carbohydrate and lipid metabolism and is essential in the transport of amino acids and major steps of protein synthesis (Thompson, 2008). Deficiencies in K are associated with thin cell walls, shorter roots and sugar accumulation. The incidence of leaf spot in cotton caused by Cercospora, Stemphylium and Alternaria were related to low K levels in the soil (PPI, 1998). In greenhouse conditions, carnation infected with Gibberella zeae, demonstrated a higher disease severity with high N and low K levels (Stack et al., 1986). In China, where tea plants grow in K-deficient soils, leaf anthracnose and brown blight in tea seedlings could be overcome with K dressings (Ruan et al., 2000). In Arabidopsis thaliana infected with Phytophthora palmivora K application induced the plant defence response (Daniel and Guest, 2006). Field experiments during 12 years, showed the positive influence of K chloride on the corn stalk rot caused by Fusarium graminearum. Even, the change in the pathogen populations in the soil was associated to the disease reduction and the KCl application over this long time (Xiaoyan et al., 2007). The incidence of stalk rot in sorghum and the stem cancer in soybean were influenced by K fertilization (PPI, 1998). In rice, the incidence of several diseases such as sheath blight (Rhizoctonia solani), blast (Pyricularia oryzae), stem rot (Sclerotium oryzae), black sheath rot (Gaeumannomyces graminis var. graminis), scab (Fusarium proliferatum) were managed by a balanced K fertilization. However, K fertilization needs to be used as a tool to improve plant nutrition and not for disease prevention (Williams and Goldan, 2001).

Phosphorus (P) is important in the formation of cell membranes and organelles; it is a structural component of DNA and RNA. As adenosine triphosphate (ATP), P is part of the major energy source in the cell and acts in different processes of photosynthesis. It has a role in the formation of oil, sugar and starch. It improves the root development and has a role in plant defence. In combination with K, this element reduced leaf rust in wheat both in the field and in the greenhouse and in cucumber it contributed to 94% protection against powdery mildew (PPI, 1999). Wheat infected with Gaeumannomyces graminis var. tritici (take-all) showed highest disease pressure in the absence of P and decreasing root damage with increasing P applications (Brennan, 1988). In cowpea, yield was improved and brown blotch disease, caused by Colletotrichum capsici, reduced with applications of 90 and 120 kg/ha P during three consecutives years (Owolade et al., 2006). The soil-born disease caused by Phytophthora spp. was controlled after P applications (Guest and Grant, 1991; Jackson et al., 2000). Soybean fertilization with P and K reduced the pod and stem blight; in wheat leaf rust was reduced by applications of P (PPI, 1999). In commercial fields of rice (1000 ha) the incidence of brown leaf spot (caused by Cochliobolus miyabeanus) was inversely linked to the P level in the plants (Phelps and Shand, 1995).

Calcium (Ca) is an essential macronutrient for plant vigour and strength. It has a structural role in plants and is essential in the formation of cell wall and membranes. It acts as an intracellular messenger in plant responses to development and the environment (White and Broadley, 2003). Its deficiency provokes physiological disorders like the blossom-end rot in tomato, pepper, eggplant, and watermelon (Roberts and Kucharek, 2006). Ca is also involved in cross tolerance (Bowler and Fluhr, 2000). In strawberries, anthracnose was reduced with P and Ca


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dressings (Nam et al., 2006). Different Ca salts in vitro reduced the peach cancer fungus, Leucostoma persoonii between 73% and 85% (Biggs et al., 1994). In rice, Ca deficiency increases brown spot (caused by Helminthosporium oryzae) (Datnoff et al., 1991).

Magnesium (Mg) is essential for chlorophyll and enzyme activation for photosynthesis (Shaul, 2002). Its role in plant resistance remains unclear and there are some reports linking it to plant diseases caused by Plasmodiophora brassicae in crucifers (Myers and Campbell, 1985). In wheat, Mg deficiency predisposes it to the take-all disease caused by Gaeumannomyces graminis (Huber, 1989).

1.2.2.1.2. Micronutrients: Boron, Copper, Manganese and Zinc

Micronutrients also influence resistance or tolerance mechanisms to pathogens in plants (Krauss, 1999).

Boron (B) is a structural component because it promotes the stability and rigidity of the cell wall. B is involved in the lignin synthesis and cell wall cross-linking of pectin polymers (Römheld and Marschner, 1991; Blevins and Lukaszewski 1994; Brown et al., 2002; García-Hernández and Cassab-López, 2005) and the integrity of the plasma membrane (Roldan et al., 1992; Ferrol et al., 1993; Brown et al., 2002; Dordas and Brown, 2005). Because of its role in lignification and as a structural component it seems that it is involved in forming a barrier against pathogen invasion, though a profound understanding of the physiological and biochemical mechanisms in plant defence are still lacking (Stangoulis and Graham, 2007).

B reduces diseases caused by Plasmodiophora brassicae in crucifers; Fusarium solani in bean; Verticillium albo-atrum in tomato; Gaeumannomyces graminis in cotton (Graham and Webb, 1991; Dixon, 1996) and Blumeria graminis in wheat (Marschner, 1995). Pathogenic fungi supposedly do not need B, but excess of B may be toxic. This is supported by the use of borate as a stump treatment for controlling annosus butt rot of Sitka spruce in Britain (Pratt, 2000). The perennial canker of grapevine is caused by Eutypa tata. Boric acid was shown to be a good inhibitor of ascospore germination under in vitro conditions and a potential alternative for benlate. Similar results were found under field conditions (Rolshausen and Gubler, 2005).

Copper (Cu) is involved in chlorophyll synthesis. This metal is a component of plastocyanin, peroxidases, multi Cu-protein and several oxidases (Sandmann and Boger, 1980). Cu is involved in the lignin biosynthesis of the cell walls (Marschner, 1995; Lin et al., 2005). Excess Cu causes inhibition of plant growth and problems in cellular processes like electron transport (Yruela, 2005). Many common diseases and disorders are associated with Cu. It is recognized as a biocide and therefore a component of many pesticides, but resulting in soil residual problems (Graham and Webb, 1991; Krauss, 1999; Van Zwieten et al., 2004; Fishel, 2005). Physiological processes influencing disease resistance or susceptibility are not well understood but Cu is a regulator of various enzyme systems linked to plant defence and the production of antimicrobial compounds (Graham and Grahan, 1991; Lebeda et al., 2001). Harker et al. (1990) reported that CuCl2 was an abiotic elicitor in the induction of chalcone synthetase, an enzyme necessary for the biosynthesis of different flavonoids, involved in plant disease resistance. Cu amine oxidase was essential in the signaling molecules of H2O2 (hydrogen peroxide) production in wound tissue of chickpea cv Sultano infected by Ascochyta rabiei (Rea et al., 2002). Leaf rust (Puccinia triticina sp. tritica), tan spot (Pyrenophora tritica-repentis), and Fusarium


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head blight (Fusarium graminearum) incidence and disease severity in wheat were reduced with Cu treatments (Franzen et al., 2008).

Manganese (Mn) is involved in enzyme activation, in chlorophyll formation, redox processes, and RNA and DNA synthesis. It is involved as a co-factor in many reactions (Porro, 2002). Mn controls lignin and suberin biosynthesis which are important biochemical barriers to fungal invasion (Marschner, 1995; Ehara et al., 2000) such as in wheat resistance against powdery mildew and take-all disease (Graham and Webb, 1991; Huber and Wilhelm, 1988; Krauss, 1999).

Wheat plants in Mn deficient soils were more susceptible to Gaeumannomyces graminis and Mn sulphate reversed that situation (Graham and Rovira, 1984; Brennan, 1992). Soil applications of Mn also reduced common scab in potato (Keinath and Loria, 1996), Fusarium spp. infection in cotton and Sclerotinia sclerotiorum in squash (Graham and Webb, 1991; Agrios, 2005). Infection of cowpea by Rhizoctonia solani and R. bataticola was reduced after applications of Mn sulphate. This was associated with increased polyphenol oxidase, peroxidase and total phenols (Kalim et al., 2003). Take-all patch in creeping bentgrass could be controlled by Mn (Heckman et al., 2003).

Zinc (Zn) is essential for carbohydrate formation and an enzyme activator involved in protein, hormone, RNA and DNA synthesis and growth regulation (Marschner, 1995; Graham and McDonald, 2001). Zn-deficiency caused the activation/inhibition of Cu/Zn-SOD acid phosphatase and ribonuclease (Pandey et al., 2002). Zn protects the cell membrane against oxidative damage through the detoxification of superoxide radicals which favours pathogenesis (Cakmak, 2000; Mengel and Kirkby, 2000). Numerous reports confirmed the role of Zn in relation to plant diseases. Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani in tomato were reduced by increasing Zn concentrations in the soil (Siddiqui et al., 2002). Zn sulphate was effective in reducing potato late blight caused by Phytophthora infestans and Phytophthora root rot of avocado (Whiley et al., 1991; Baider and Cohen, 2003). Wheat plants receiving Zn via soil applications exhibited a reduced infection by Fusarium graminearum and root-rot diseases caused by Gaeumannomyces graminis (Graham and Webb, 1991). Soil amendments with Zn reduced the severity of maize smut, caused by Ustilago maydis, by over 10% and, in combination with N fertilizers, induced severity by over 20% (Kostandi et al., 1997). In upland rice, the occurrence of panicle blast (Pyricularia grisea) was inversely related to the Zn content in the plant (Filippi and Prabhu, 1998). The production of mycotoxins by Aspergillus species was strongly affected by Zn (Cuero et al., 2003). Zn is also associated with citrus blight (Tucker et al., 1984; Derrick and Timmer, 2000; Wutscher et al., 1997). High Zn concentrations in the soil affect the growth and metabolism of beneficial microorganisms, with consequences on plant health and biological control (Knight et al., 1997; Van Elsas et al., 2002; Moffet et al., 2003). In Medicago truncatula Zn did not directly inhibit Rhizoctonia solani but stimulated root development partly offsetting fungal damage (Streeter et al., 2001). However, in the same crop and in rotation with pasture, Zn reduced two other diseases: root rot disease and common leaf spot disease caused by Phytophthora megasperma f. sp. medicaginis, and Pseudopeziza medicaginis respectively (Grewal, 2001). In wheat high concentrations of Zn decreased Rhizoctonia root rot (Thongbai et al., 1993). Zn applications and Pseudomonas fluorescens together contributed to controlling Fusarium crown in wheat and tomatoes. A similar positive influence of Zn content in the soil and Pseudomonas fluorescens 2-79 improved in wheat the biocontrol against


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Gaeumannomyces graminis var. tritici (Thongbai et al., 1993; Duffy and Defago, 1997; Ownley et al., 2003).

1.1.2.1.3. Silicon (Si)

Plant nutritionists and physiologists focus on the 13 elements which are normally supplied via the soil as discussed above because deficiency leads to impaired growth, an incomplete life cycle and lower yield (Savant et al., 1997b). Si is not included in the shortlist of macro- or micronutrients because it does not fit with the definition of essentiality, i.e. (1) mineral element when absent, results in an incomplete life cycle; (2) the function of the element is not replaceable by another mineral element and (3) the element is directly involved in plant metabolism or is required for a distinct metabolic step such as an enzyme reaction (Marschner, 1995). Si only proved to be essential for Chrysophyceae and Equisitaceae and is not considered an essential element for any other plant family. This may explain why Si is routinely omitted in any fertilizer scheme (Epstein, 1999). However, soluble Si has enhanced the growth and development of several plant species including rice, sugarcane, and most other cereals and several dicotyledons such as cucumber and watermelon. It is a vital component of the epidermal cell wall. Plants containing Si can fight off diseases and resist insects, drought, heat and stress. But the exact and complete mechanisms associated with Si in the plant remain unclear. Si is linked to barrier formation against the entrance of pathogens either by direct effects or by reinforcement of the cell wall. Physiological effects link Si with the activation of the induced systemic resistance processes (Fauteux et al., 2005, Rodrigues and Datnoff, 2005; Park et al., 2006b; Ma et al., 2006; Ma and Yamaji, 2006).

Higher plants vary in their capacity to accumulate Si. Plant Si accumulators belong to the family of wetland grasses such as paddy rice which contain between 4.6- 7% Si on a dry matter basis. Other plants like sugarcane, cereals and turfgrasses contain between 0.5 to 1.5% Si on a dry matter basis. Recent studies demonstrated that the banana (Musa spp.) is a Si accumulator. In Cameroon, the banana variety Grande Naine (Musa acuminata AAA Cavendish group) contained 0.13% Si in the petiole and 0.49% in the lamina. These same authors report that the Si content of in vitro banana plantlets grown in hydroponics increased as follows within a plant: roots < pseudostem < petiole and midrib < young lamina < old leaf (Opfergelt et al., 2005; Henriet et al., 2006; Henriet, 2008). Si accumulation is an active process regulated by specific transport proteins (Datnoff and Rodrigues 2005; Tamai and Ma, 2003; Hull 2004). Si amendments proved to be effective in controlling both soilborne and foliar fungal diseases in cucumber, rice, sugarcane, turf and several other plant species (Datnoff et al., 2001). Several studies in rice demonstrated that Si could be as effective as fungicides against rice blast and improved resistance in genotypes which are partially resistant (Seebold et al., 2000 and 2004). Si appears to be as effective as a fungicide in controlling grey leaf spot in St. Augustinegrass (Brecht et al., 2003) and powdery mildew in Kentucky bluegrass (Kanto et al., 2004; Hamel and Heckman, 2000). Si also protected tomato against Fusarium oxysporum sp. lycopersici (Carvalho, 2006) and cucumber against Sphaerotheca fuliginea (Schuerger and Hammer, 2003). It is suggested that Si activates the plant defence mechanism (Cherif et al., 1992a, 1992b and 1994).


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1.2.2.2. Organic amendments and plant disease

1.2.2.2.1. Solid organic amendments

Organic soil amendments, including animal and green manures and wastes from processed animal products have been used for centuries as soil complements. During the chemical era, the impact of such amendments on plant diseases was ignored, but in the past, their potential as disease control measures was recognized (Hawke and Lazarovits, 1994; Lazarovits et al., 2001; Raviv, 2008). Solid amendments are obtained with the assistance of microorganisms which transform organic wastes (from animal, plant and human) into useful products. Around the world, different composting processes are used, which are based on aerobic and anaerobic decomposition steps. High and low temperatures are critical (Misra et al., 2003) during the process. At the end of the decomposition process, the compost is sometimes enriched with nutrient sources like rock phosphate (Zayed and Abdel-Motaal 2005).

Solid organic amendments improve the soil by increasing water infiltration, water holding capacity, nutrient retention and release, increase the microfauna population and reduce erosion. These results are obtained after longer periods of application of high quality compost (Brady and Weil, 1999; Ghorbani et al., 2008a). Solid organic amendments added to the soil are decomposed by anaerobic and aerobic processes and increase the soil organic matter content. In compost decomposition has already taken place before application. This has implications for soil and nutrient management, as well as plant health and pest management (Trankner, 1992; Kuepper and Sullivan, 2004). In Puerto Rico addition of compost increased the quantity and the quality of organic matter and in bean fields soil physical characteristics depended on the type and rate of organic amendments (Rivero et al., 2003; Darby et al., 2006). Mediterranean fields are lower in organic matter, but the addition of quality compost improved the organic carbon content and the soil structure in comparison with untreated fields (Sparvoli et al., 2008). In general, the positive impact of organic amendments on the soil is clear but the exact relationship between physical, chemical and microbiological aspects needs more clarification.

There is much evidence that organic amendments suppress soilborne diseases by the reduction of pathogens. This has been reported for Phytophthora and Pythium in different crops (Drinkwater et al., 1995; Lewis et al., 1992; Lumsden et al., 1983; Trankner and Brinton 1997; Vandesteene et al., 2003). In tomato, organic amendments from different origins, caused 80% less disease incidence (Ghorbani et al., 2008b). Compost reduced anthracnose incidence (Abbasi et al., 2002). Development of Botrytis cinerea was reduced by compost in cucumber (Segarra and Casanova, 2007). In alfalfa, clover tiredness was treated with compost (Logsdon, 1995; De Ceuster and Hoitink, 1999). In barley and wheat powdery mildew, caused by Drysiphe graminis, was 95% suppressed when a 1:1 soil:compost mix was used (Trankner, 1992). Rhizoctonia sp. in bean was reduced by 80% in areas with the highest compost rates, and by 40% when intermediate rates were applied (Hudson, 1994). Soil:compost mix (1:1) in cucumber reduced the Sphaerotheca sp. and powdery mildew by 20%; and with a 1:3 mix the infection decreased by 40% (Trankner, 1992). Disease suppressive effects of compost were obtained in field trials on snap beans and southern peas (black-eyed peas). Ashy stem blight in beans was severe in areas without compost applications and was almost eliminated where compost was applied. Leaf wilting and leaf death were pronounced where compost


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was not applied (Ozores-Hampton et al., 1994). Similarly, compost reduced diseases in turfgrass resulting in less use of fungicides (Boulter et al., 2000). Root rot diseases in snap bean and sweet corn in the field and severity of damping-off of cucumber in greenhouse was reduced by 29, 67 and 30% respectively after addition of organic amendments (Darby et al., 2006).

Composts in soils can induce a systemic resistance response in several crops but the exact mechanism is not well understood and not all organic amendments are able to provoke these responses (Krause et al., 2003).

Induced resistance, direct parasitism, nutrient competition, and direct inhibition through antibiotics and the biocontrol agent’s variability depend on the raw material used for the solid organic amendments and its decomposition processes (Hoitink and Boehm, 1999; Abbasi et al., 2002). Zhang et al. (1998) demonstrated that beta-1,3-glucanase activity was induced significantly when cucumber infected with C. orbiculare was grown in a compost mix. Similar results were obtained for transgenic Arabidopsis plants grown in a compost mix. Induced systemic resistance response was established in the pathosystem Arabidopsis thaliana–Verticillium dahliae with five composts and in eggplant, and Verticillium wilt was suppressed with compost (Paplomatas et al., 2005).

Other organic amendments may increase a plant’s induced resistance. For example, mycorrhizal plants are more resistant to soilborne pathogens, by the accumulation of components like ROS (reactive oxygen species) molecules, activation of phenylpropanoid metabolism, and accumulation of specific isoforms of hydrolytic enzymes such as chitinases and glucanases. Their protection is linked to improved nutrition and activated plant defence mechanisms. Pozo and Azcon (2007) presented a recent overview on arbuscular mycorrhiza and plant defence mechanisms against biotic stresses. Also Trichoderma spp. contribute to systemic and localized resistance against fungi (Marra et al., 2006) as was demonstrated for Botrytis cinerea and Rhizoctonia solani in bean. This demonstrates that the diversity of microorganisms in the solid amendments is important (Jenkins, 2005) which will influence and activate the disease defence system of plants before pathogen invasion (Hoitink et al., 1997; Goldstein, 1998; Sullivan, 2001). For example, expression of disease resistance genes was demonstrated in tomatoes after organic amendment application and tested on the foliar pathogen Septoria lycopersici (Kavroulakis et al., 2005).

Mature compost is more suppressive than immature compost (Nelson et al., 2004). Beneficial microorganisms such as Trichoderma hamatum and T. harzianum in immature composts were not able to suppress Rhizoctonia. The suppression of Pythium in the soil is directly related to the microbiological activity, microbial biomass and organic amendments. Indeed, Pythium and R. solani are pathogens which depend on a reduced soil biodiversity (Cook and Baker, 1983). In fact raw organic materials favour colonization by pathogens but during decomposition temperature increases kill plant pathogens (Jenkins, 2005). After decomposition, the temperature decreases and biological activity increases again but with microorganisms that are suppressive to plant diseases (Ryckeboer et al., 2001; Fuchs and Larbi, 2005). The decomposition of organic ingredients depend on ambient temperature, aeration, moisture, C:N ratio of material, etc. (Boulter et al., 2000). Compost has been used effectively in the nursery industry, in high-value crops, and in potting soil mixtures for controlling root rot diseases (Stone et al., 2001) and microbial products have been found to be effective in a greenhouses where sterile potting mixtures are used (Hoitink and Boehm, 1999).


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Compost also provides nutrients, especially micronutrients and changes soil conditions resulting in improved soil fertility (Bollen, 1993; Engeli et al., 1993; Fuchs and Larbi, 2005). The activity of a compost treatment for disease control depends also on the nature of the raw materials from which the compost was prepared, the maturity of the compost, and the composting process (Hoitink, 1986; Chung and Hoitink, 1990). Compost with high quality should contain disease-suppressive organisms and mycorrhizal inoculum related to the microbial biodiversity (Sances and Ingham, 1997; Sullivan 2001).

Composts do not only reduce soilborne diseases but also foliar pathogens such as Blumeria (Erysiphe) graminis f.sp. hordei, the causal agent of barley powdery mildew. Disease reduction varied considerably from compost batch to batch (Fuchs and Larbi, 2005). There are far less studies on foliar disease suppression by organic amendments. Soil and rhizosphere microorganisms could induce resistance to foliar diseases in plants grown in containers (Liu et al., 1995; Meera et al., 1995) and in the field (Wei et al., 1996). Composted pine bark container mix suppressed Pythium root rot and foliar Anthracnose on cucumber, whereas a dark peat container mix failed to suppress both diseases (Zhang, 1996, 1998). Studies using a container system showed that organic matter-mediated suppression of soilborne diseases was fairly common, but less than 10% of composts tested in container mixes suppressed foliar diseases (Krause et al., 2003). Foliar disease suppression may be generated less frequently than root rot suppression in container mixes because foliar disease suppression requires the presence of specific organisms that are relatively infrequent colonizers of organic wastes. Established organic systems typically exhibit reduced root disease incidence relative to conventional farming systems, but there is no clear trend for foliar disease incidence in comparative farming systems (van Bruggen, 1995). The suppressiveness of foliar diseases by soil amendments was studied for Pseudomonas brown spot in snap beans, and the results showed a significant relationship between N-availability and disease expression (Rotenberg et al., 2005).

1.2.2.2.2. Water based organic amendments or organic teas

For centuries, farmers have mixed and soaked plant wastes, manures and composts in water, and used the rich decanted brew as a liquid fertilizer, or "organic tea". There are a great number of observations that indicate that organic teas probably have some sort of benefit for growing plants. These observations are supported by scientific research, which shows that organic slurries not only have dissolved nutrients, but provide humic acids, vital enzymes and beneficial microorganisms, all of which contribute to a more vigorous plant growth (Hadar et al., 1992; Peavy, 1993; Bess, 2000; Scheuerell and Mahaffee, 2002; Kelley, 2004).

Organic tea systems were described as “anaerobic” or “aerobic”, depending on the degree of aeration during preparation. However, several authors state that this classification is ambiguous because in the end, all properly designed organic tea systems should be completely aerobic. The real distinction is the degree of aeration given to the system during the extraction period over an extended period (Merill and McKeon, 1999).

The terms “active, passive, and aerated, non aerated” are widespread (Merill and McKeon, 1999; Jackson, 2001; Scheuerell, 2003; Kelley et al., 2004). Active or aerated tea devices extend the extraction time so that more nutrients can be drawn from the organic raw materials. There are several good references on the use and production of compost teas from a variety of feedstock (Andrews and Harris, 1992;


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Elad and Shtienberg, 1994). Passive, non aerated or anaerobic extraction systems are terms used when a feedstock is simply left to soak in water. After a few days, the system will become anaerobic and produces various organic acids such as butyric, proprionic and acetic acids and reduced forms of N (NH4) and S (H2S). There are some reports that these types of products from anaerobic systems can be harmful to plant roots when they are fresh or when used in some specific crops (Merill and McKeon, 1999).

In general, the quality of organic teas ("slurry", "leachate” or "extract") depends on the quality of the raw materials processed. Since organic materials are very variable, their effects tend to be variable as well. The variability of the manure varies with the kind, age, diet and origin of the animal, while the quality of green materials depends on kind, age, growing conditions, and nutritional status of the plant.

Fresh manure teas have soluble salts, especially macronutrients (N, P, K, Ca, Mg and S) and micronutrients (Fe, Zn, Mn and Cu). Aged feedstock and immature composts can contain some nutrients that are not fixed in the microbial biomass but, they also can provide sugars, amino acids, humic and fulvic acids and some micronutrients (Fe, Zn, Mn and Cu) (Merill and McKeon, 1999; Ingham, 2005).

The organic teas from manures are a good source of N, P, K and trace elements. Only a few manures are really suitable for the making of organic teas. These include horse, cow and goat manures. Horse manure yields more nutrients, but cow manures provide more humic acids. Organic tea from fresh composts (less than 6 months old) contains fewer nutrients than manures, but they can provide humic acids and some chelated micronutrients. Organic tea from aged and suppressive composts has beneficial microorganisms, especially if they were well aerated and if the fermentation was for several days (Merill and McKeon, 1999; Granatstein, 1999; Scheuerell and Mahaffee, 2004). In others words suppressive teas, from manure or compost origin, can be considered as a concentrated liquid inoculum of beneficial microorganisms (Bess, 1999). Ingham (2005) reported that an organic tea from compost needs to contain at least the following amounts of organisms per litre: active bacteria - 2 to 10 mg; total bacteria -150 to 300mg; active fungi - 2 to 10 mg; total fungi - 150 to 300 mg; protozoa - 1,000 individuals; beneficial nematodes - 5 to 30.

Reports mention that an organic tea can operate in two ways: chemically and biologically. The former will supply different components for plant growth and defence, and provide nutrients for growth and multiplication of beneficial microorganisms as well (Merrill and Mackeon, 1999). Biologically, organic tea provides beneficial organisms that cover the different plant parts. By root or foliar applications these microorganims occupy infection and non-infection sites, consume substances released by the plant and produce compounds that inhibit the growth of plant pathogens. The microorganisms from the organic tea applied to the soil can help in the retention and availability of nutrients for the plants. Moreover they can detoxify some compounds in the soil and water thereby improving the growth conditions for the plant (Ingham and Alms, 2003).

Organic tea can be applied directly to the soil or to the plant foliage. Soil applications can be made by drenching into the rooting zone and by irrigation but filtration is needed to avoid the obstruction of the system. Foliar applications need to cover both sides of the leaves (Grobe, 2003a). However the combinations of soil and foliar applications can offer the best results.

The ability of organic tea to suppress many plant diseases was already recognized by Hunt et al. (1973). The other pioneer Heinrich Weltzien (1989, 1991) showed that the organic tea (“water extracts of compost”) used as a foliar spray


20

inhibited Phytophthora on tomatoes and potatoes and that its suppressive effect was linked to microorganisms.

Seed-treated peas with compost extract germinated significantly better than untreated seeds in soil artificially inoculated with Pythium ultimum (Trankner, 1992). Organic teas from manure obtained at different periods were applied against Botrytis cinerea. All extracts inhibited conidial germination and lesion development in vitro. Under glasshouse conditions however, these extracts did not have any effect but high amounts of microbial populations were found (McQuilken and Whipps, 1994). The apple disease Venturia inaequalis was treated weekly by an anaerobic organic tea originating from a mushroom source. A significant reduction of the affected leaf area was obtained and more bacteria were detected in the treated leaves. Under in vitro conditions similar results were obtained (Yohalem et al., 1996). Also more bacteria were found after foliar applications against the following diseases: gray mold on strawberry and tomato, powdery mildew and downy mildew on grapes, potato late blight, and apple scab. In Germany, studies with compost tea from horse and cattle manure were effective in the control of gray mold on strawberry and powdery mildew on grape (Granatstein, 1999). Soilborne disease on potatoes as common scab and Verticillium wilt were reduced and the total microorganism population increased by liquid organic products (swine manure). The toxic compounds identified were volatile fatty acids (Lazarovits et al., 2001, Tenuta et al., 2002). In vineyards, powdery mildew was largely controlled by weekly applications of compost tea. The treatments did not improve the soil biology (Ingham and Alms, 2003). Organic tea from manures was tested in Tunisia on Fusarium roseum var. sambucinum, F. oxysporum, F. solani var. coeruleum, Phytophthora erythroseptica and Rhizoctonia solani. Results showed that the organic tea controlled all pathogens including Fusarium solani in potato tubers during storage (Znaïdi et al., 2002). Organic strawberry in Finland were treated by organic tea composed of seaweed, garlic, compost extracts, Trichoderma spp., and Gliocladium catenulatum for grey mould (Botrytis cinerea). The biological treatments had no effect on the incidence of grey mould and shelf life of the berries (Prokkola et al., 2003). Vermicompost and vermicompost tea was studied in the greenhouse and field for the control of Septoria leaf spot of tomato (Septoria lycopersici) and early blight caused by Alternaria solani. The organic tea was able to control the pathogen under both conditions (Gangaiah, 2004).

Compost leachates prepared with plant waste were used in the treatment of onion seeds inoculated with Aspergillus niger (black mold disease). The leachates reduced the disease incidence and stimulated bacteria populations having an antagonistic activity (Özer and Koycu, 2006). The use of different compost extracts resulted in different degrees of blight control (Phytophthora infestans), but the suppression of blight lesion growth was not improved after adding antagonistic microorganisms to the organic tea (Ghorbani et al., 2005). Late blight (Phytophthora infestans) severity in potato was reduced by 27% after foliar applications with compost tea (Al-Mughrabi 2007). Also in tomato foliar treated organic teas were beneficial (Zaller, 2006).

Numerous studies in Germany reported that the use of organic tea from 3 days old cattle and horse manure extracts showed excellent results. The effects did not appear to be systemic but high levels of microorganisms on the leaf surface showed antagonistic effects on Uncinula necator, the causal agent of powdery mildew in grape (Tränkner and Brinton, 1997). As a consequence of the leaf spraying with tea, beneficial microorganisms can colonize the leaf surfaces and help in the protection of the plants against some leaf and soilborne diseases (Scheuerell, 2003). Research


21

showed that some chemicals such as siderophores, pseudobactins and pseudomycins produced by Pseudomonas spp. had a powerful suppressive effect on several microorganisms (Kloepper et al., 1980) such as Fusarium oxysporum f. sp. lycopersici, F. culmorum, Cladosporium cucumerinum, Colletotrichum lagenarium and Phytophthora infestans (Fakhouri et al., 2001). Proteins of plant or microbial origin effectively suppressed certain pathogenic fungi. In some cases cyanids and antibiotics interact with the host plant and create resistance to diseases (Kai et al., 1990; Weltzien, 1989, 1991).

The principles of suppressiveness caused by microorganisms in organic teas can be summarized as follow: (i) growth restriction of pathogens; (ii) inhibition of pathogen spore germination; (iii) competition for nutrients; (iv) production of chemical inhibitors; (v) direct parasitism on pathogens and/or (vi) stimulation of the plant defence system (Brinton, 1995; Scheuerell and Mahaffee, 2004; ROU, 2006).

Extracts from 30 composts were studied on apple scab (Venturia inaequalis) and downy mildew (Plasmopara viticola). The incubation time had no effect on the protection but all treatments reduced the severity caused by both pathogens. Sterilization by either autoclaving or filtration had no effect. Thus these authors concluded that the microbial activity was not the main effect of these extracts and that the active ingredient must be a water soluble, heat stable metabolite produced during composting (Larbi et al., 2006). Hence in conclusion, pathogen control with organic amendments is linked to a better nutrient supply to the plant, changed gene expression in the plant, mechanical barrier development in the plant, beneficial microorganisms and the supply of inhibitory molecules.

1.3. Rationale, objectives, hyphotheses and outline of the study

Bananas are the main crop cultivated under large monoculture systems in Ecuador. Because of the current banana production volume, Ecuador is the leading export country of the world with a market share of around 28%. The banana industry employs about half a million people.

Every year, new threats appear in the banana industry, but BLSD control remains undoubtedly the biggest challenge. In all areas where bananas are grown, especially where conventional production systems prevail, the climatic conditions are optimal for disease development. There, synthetic fungicides are used, which account for 25 to 30% of total production costs. Ecuador invests around 90-100 million dollars a year in controlling this disease in 180,000 hectares of banana. The continuous use of synthetic fungicides not only affects the ecosystems and human health, but also reduces over time the effectiveness of the fungicides.

Hence it is important to design efficient strategies to control or at least reduce BLSD, which is the main aim of this study. We postulate therefore that BLSD can only be controlled in a sustainable way, if strategies are designed and evaluated which simultaneously focus on the pathogen, the banana plant and disease development.

Specific hypotheses were: 1. Banana can be grown commercially in an organic way in the presence

of M. fijiensis Morelet (Chap. 2). 2. Isolates of M. fijiensis Morelet originating from an organic field

behave as isolates from a conventionally sprayed field (Chap. 2). 3. The nutritional status of bananas in an organic plantation is different

from those in a conventional plantation (Chap. 2).


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4. The organic products used in the organic banana production system have direct and indirect effects on the fungus, the host and disease and some specific nutrients are responsible for it (Chap. 3).

5. The micronutrients Zn, B, Cu and Mn contribute in controlling M. fijiensis Morelet and can improve banana growth and development (Chap. 4).

6. The nutrient Si contributes in controlling M. fijiensis Morelet and can improve banana growth and development (Chap. 5).

Therefore research was conducted on the fungal pathogen, the banana plant and disease development. The research was performed under field conditions, but to speed it up experiments were also performed under in vitro and nursery conditions. The results of the thesis are presented as follow (Figure 1.4): First the banana production system under organic and conventional management conditions was studied and the disease development and the nutritional status in both fields compared (chapter 2, section 2.1). The fungal isolates from both systems were characterized (chapter 2, section 2.2). Then the liquid amendments (called organic teas) from the organic farms were evaluated and tested on the pathogen, disease and the banana plant (chapter 3, sections 3.2 and 3.3). As these organic teas are rich in many nutrients, the effect of several micronutrients (Zn, B, Mn and Cu) was evaluated with respect to their direct or indirect effect on the pathogen, the host and the disease (chapter 4). Reports demonstrated the importance of Si in several pathosystems and its effects was evaluated on M. fijiensis Morelet, the banana host and their relationship (chapter 5, sections 5.2 and 5.3). The obtained results form the basis for the formulation of more efficient and alternative strategies to control BLSD (chapter 6) which should be both more environmentally friendly and efficient than the current practices in the conventional and organic banana farms. Hence these suggestions should lead to a more sustainable and economically viable banana production system.


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Figure 1.4 Research outline of the study.

CHAPTER 2: Banana production systems

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Chapter 2 BANANA PRODUCTION SYSTEMS, THEIR IMPLICATIONS FOR BLACK LEAF STREAK DISEASE, NUTRITIONAL STATUS AND FUNGUS CHARACTERIZATION1

2.1. Two banana production systems: implications on BLSD and their nutritional status

2.1.1. Introduction Nearly 0.6% of Ecuador‘s land area is used for banana cultivation.

Commercial banana production takes place chiefly on the alluvial plains of the coastal lowlands in the provinces of Los Rios, Guayas and El Oro. About 10 to 12% of all economically active people in the country obtain some benefit from banana production. In 2008 there are 5,871 producers of banana and plantain. Unlike banana production in other exporting countries, production in Ecuador is not in the hands of multinational companies cultivating large areas. Instead, 80% of the total export production comes from growers maintaining areas smaller than 30 ha (SICA, 2007).

Because of environmental concerns regarding heavy pesticide use and soil degradation, many banana growers have shifted towards organic production. In such systems nutrients come from large amounts of organic matter, animal manure, salts sources and rock phosphate.

Since BLSD is such an aggressive airborne fungus, especially in large banana plantations consisting of Cavendish varieties only (Mourichon et al., 1997), it is puzzling to note that more and more farmers in Ecuador adopt a fungicide-free production system. Thus the main aim is now to describe the specific agronomic practices of the organic production system and to compare the disease and nutritional status between an organic and conventional farm. The goal is to understand why BLSD is not devastating the farm with the organic banana production system.

2.1.2. Materials and Methods Geographical situation and climatic characteristics Two neighbouring farms (7 km apart) were selected: one farm receiving only

inputs of organic origin and products approved for organic agriculture, henceforth called the organic farm (O), and a farm receiving conventional fertilizers and fungicides, hereafter called the conventional farm (C). Both farms were located in the

1 Some results were published in: Jiménez, M., Van der Veken, L., Neirynck, H., Rodríguez, H., Ruiz, O., and Swennen, R., 2007.

Organic banana production in Ecuador: its implications on black Sigatoka development and plant - soil nutritional status. Renewable Agriculture Food System 22(4): 1-10.


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Guayas province (southern coast of Ecuador), near the village of Taura (2°18’00”S, 79°42’00”W). The close proximity was essential for conducting a comparative study on BLSD because weather conditions (such as temperature, amount of rainfall and sunshine) influence fungal development (Gauhl, 1994).

Based on 10-year averages, weather data for this area were as follows: maximum temperature 30.6oC; minimum temperature 20.4oC; annual rainfall 842 mm; and relative humidity 80.6% (FAOCLIM, 2001). The rainy season lasts from January until May, and the dry season from June until December.

General facts about the organic and conventional farm The organic farm covered 196 ha cultivated with the Cavendish cultivars

(varieties) (Daniells et al., 2001) Williams (40%), Valery (40%), Grand Naine (10%) and Taura (10%), a local Cavendish selection. Plant density varied between 1450 and 1500 plants/ha. The plantation was at least 10 years old and had initially been treated with synthetic fertilizers and pesticides. Eight years ago, these practices ceased. The organic farm no longer uses pesticides against BLSD. Presently, BLSD infected leaves are pruned and nutrients are applied via solid (compost) and liquid amendments such as organic teas. Annual yields varied from 1100 to 1500 boxes/ha/year depending on the different plots within the farm.

The conventional farm consisted of 87.5ha of the cultivar Williams. Plant density was 1450 plants/ha. This plantation was about 15 years old and annual yields were 2100 boxes/ha/year. BLSD was controlled by both protectant (carbamates, chlorothalonil) and systemic (triazols, benzimidazols, morpholins and strobirulins) fungicides with seven or more fungicide cycles per year. Weeds were controlled with glyphosate every 6 to 8 weeks in the rainy season and every 10 weeks in the dry season. The banana fertilization consisted of 400kg/ha N mostly from Urea, 600kg/ha K, 80kg/ha Mg, 2kg/ha Zn and 1kg/ha B spread over 13 application cycles within a year.

The production data per week during five years were obtained from each farm and related with income and cost data.

General facts relating to organic tea (OT) Capture of local microorganisms (LM) In the preparation of the OT, a microorganism solution was used as raw

material. The solution was prepared as follows: the growth medium for the microorganisms consisted of boiled rice (150g) and 400 ml of a mixture of sugarcane molasses (40L), fish mill (27kg), NaCl (680g) and water (80L). This mixture was boiled, put into small plastic containers covered by a mosquito net and placed on the soil close to trees bordering the banana field.

After 21 days, unidentified microorganisms were harvested. Simultaneously another sugarcane molasses-fish mill-NaCl-water mixture was made and transferred to a 500L tank. Fifty to seventy pots with microorganisms were transferred to a big tank and hermetically sealed. Microorganisms were multiplied by transferring 50L of the raw material to another tank (500L), where 20L of sugarcane molasses were added to 430L of water. The tank was then hermetically sealed for 7 days, after which it was ready for use in the preparation of organic teas. It is important to mention that this microorganism solution was prepared during the dry season to avoid water accumulation in the containers.


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Organic tea preparation OT was a fermented solution prepared from raw materials used in the organic

farm. The production of OT was based principally on fresh cow dung (40kg), sugarcane molasses (4L), soybeans and bean leaves (10kg), burned rice husks (10kg) and LM (8L from the diluted LM tank). A 200L tank was filled with water, and the mixture was left to ferment under anaerobic conditions.

Once the fermentation process was finished, and prior to foliar application, each tank of 200L was enriched with the following micronutrients: MgSO4 (0.9 - 1.3kg), H3BO3 (0.9 - 1.3kg), ZnSO4 (0.9 - 1.3kg), CuSO4 (0.9 - 1.3kg) and MnSO4 (0.9 - 1.3kg). In the case of OT for soil application, K and P were added after fermentation and with the following quantity per 200L tank: 3.2 - 6.4kg of K2SO4 and 3.2 - 6.4kg of rock phosphate. After 60 days of anaerobic fermentation, the OT was used for foliar or/and soil applications. The OT was sprayed on the leaves with a motorized backpack sprayer at a solution of 3L pure OT diluted in 17L water per ha per week. The soil OT was applied weekly through the irrigation system at 100L per ha.

For approximately one year six samples were collected from the organic banana plantation to analyze the nutrient composition of the OT used as a main alternative in the management of BLSD. These samples were sent to Belgium and their nutritional composition analyzed by the Soil Service of Belgium.

Experiment one: BLSD in vitro development Thirty randomly selected plants in their vegetative and generative growth

stage were selected from the organic and conventional farm. Pieces from the middle leaf part were collected in the field and carried to the laboratory. The evolution of the disease development was measured in the fully extended leaves 1 to 6, leaf 1 being the youngest leaf (Brun, 1963). In the laboratory, an incubation medium was prepared with bacto-agar Difco TM (4g/L) added to distilled water and sterilized for 25min at 121°C and 15psi. In order to avoid the growing of other fungi in the medium, Benzimidazole® at 50mg/kg was added to the Petri dish with a sterile syringe (Millex®-GP 0.22µm) under sterile conditions. Thirty leaf discs (5cm2) per number of leaves and per farm were placed with the upper leaf surface on the medium and were incubated under continuous light (4000 Lux ± 200) at 26°C for 30 days approximately (Mourichon et al., 1987). For each leaf disc, symptom development was recorded following the Table 1.1.

Experiment two: BLSD development under greenhouse conditions The experiment was repeated twice. Each time, 80 micropropagated

Cavendish plantlets (Williams variety) were established in a greenhouse at 28oC and 96% relative humidity using a randomized design. Eight weeks after pot planting, 40 plants were inoculated with in vitro conidia of M. fijiensis Morelet (3x103 conidia/ml) per isolate from the organic and conventional farm. M. fijiensis Morelet colonies and conidia were produced according to the protocols mentioned in Chapter 2, section 2.2.

The disease in the greenhouse was measured according to Alvarado et al. (2003), which is a modification of the scale presented by Fullerton and Olsen (1995), (Table 2.1). The observed symptoms on the leaves are shown in Figure 2.1.


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Table 2.1 BLSD symptom description for banana plants grown under greenhouse conditions.

Scale Description

0 Leaf symptoms absent 1 Reddish flecks on lower leaf surface. No symptoms on the

upper surface 2 Regular or irregular circular spots on the lower leaf surface.

No symptoms on the upper surface 3 Regular or diffused light brown circular spots on the upper

leaf surface 4 Black or brown circular spots, possibly with a yellow halo or

chlorosis of adjacent tissues, on the upper leaf surface. Areas of green tissue sometimes present

5 Black spots with dry centre of grey colour and the leaf completely necrotic

Figure 2.1 Evolution of BLSD symptoms under greenhouse conditions (photo CIBE - ESPOL). Numbers correspond to the scale in Table 2.1.


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Experiment three: BLSD and nutritional status under field conditions During 2003 and 2004, 80 banana plants of 2 m height were randomly chosen

per season (rainy and dry) in the organic and conventional farm. For each plant, leaf number 3 and 4 (counted down from the top of the plant) were evaluated weekly during 10 weeks for BLSD symptom development (Table 1.1). The disease severity was evaluated on 20 plants per stage (vegetative -2m height-, flowering and harvest) with Gauhl’s modification of Stover’s severity scoring system (Carlier et al., 2002). This is based on the proportion of leaf area covered by disease symptoms (Table 2.2 and Figure 2.2).

Table 2.2 BLSD severity scoring system based on Gauhl’s modification of the Stover’s scale.

Scale Description

0 no symptoms 1 not more than 1% (stages 1, 2, or 3) 2 less than 5% of the leaf affected 3 from 6 to 15% of the leaf affected 4 from 16 to 33% of the leaf affected 5 from 34 to 50% of the leaf affected 6 more than 50% of the leaf affected

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.


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At the experimental sites the nutritional status was monitored during the years 2003 and 2004. Leaf samples (Martin-Prével 1992) consisting of 15cm wide strips were collected from the central part of leaf number 3 (counted down from the top of the plant) in 30 randomly chosen plants. The leaf nutrient content was compared with the leaf critical values (LCV) (Lahav and Turner, 1992). After removal of the mulch on the soil surface, soil samples at 0-30cm depth were collected from fifteen randomly selected locations between the banana plants. This was done twice a year, in each farm. This sampling depth was sufficient because the majority of banana roots develop in the topsoil (Blomme et al., 2001).

After collection, the samples (soil and leaves) were sent to a subsidiary laboratory of BSI (British Standard Institution) that operates in Ecuador. Elements as K, Ca, Mg, Cu, Mn were determined by Atomic Absorption Spectrophotometry. Other elements as B and S were determined by UV – VIS Spectrophotometry and the analytical methods were based on the Official Methods of Analysis of AOAC International (Jones, 2001). Soil samples were processed by Mehlich extraction, whereas foliar samples were treated by wet-acid digestion. N was analyzed by Kjeldahl digestion for plant tissue (Jones, 2001).

Statistical analysis The variables were classified into two types: discrete variables for the disease

symptoms, and continuous variables such as nutrient content or yield per farm and the data of area under a curve (annex 2) of index disease evolution during evaluation time. Univariate descriptive statistics were applied for the estimation of central tendency parameters such as averages, and dispersion parameters such as standard deviation and coefficient of variance. Inferential statistics - Analysis of Variance (ANOVA) and the Central Limit Theorem - were applied to compare the leaf nutrient content from the experimental sites versus. The statistical differences were established by T-test and Mann Whitney-test. All data were analysed by running SPSS version 11 and MINITAB 13 for Windows.

2.1.3. Results and Discussion

2.1.3.1. Yields, Incomes and Profits of the Organic and Conventional farms Yield expressed in ton per ha or boxes per ha was significantly higher in the

conventional farm than in the organic farm. Hence on an annual basis the conventional farm was more productive than the organic farm (Table 2.3). However, as organic fruit fetches much higher prices, the lower yield in the organic farm was more than compensated. Therefore, on average a 16% higher net profit was achieved in the organic farm compared to the conventional farm during 2001-2005.

Table 2.3 Banana yields and incomes during 5 continuous years (each year being the total of 52 weekly values) from organic and conventional banana farms, located in the Guayas province, Taura village (7 km apart).

Parameters 2001 2002 2003 2004 2005 Mean 2001 2002 2003 2004 2005 MeanBoxes/ha/year 1,507 1,084 1,379 1,273 1,118 1,272 1,773 2,009 2,181 2,269 2,543 2,155Ton/ha/year 29 21 26 24 21 24 34 38 41 43 48 41Mean annual price per box (USA dollars)

6* 6.5* 7* 7.5* 7.5* 6.9 2.9** 3** 3.2** 3.25* 3.25* 3.1

Income/ha/year(USA dollars) 9,039 7,047 9,653 9,548 8,386 8,735 5,141 6,027 6,978 7,376 8,264 6,757

Profit/ha/year (USA dollars) 3,789 1,797 4,403 4,298 3,136 3,485 -159 727 1,678 2,076 2,964 1,457

Production cost/ha5 yr. average 5,250* 5,300*

*Personal comunication from farmers **Arias et al., 2003

Organic Farm Conventional Farm


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2.1.3.2. BLSD development under in vitro conditions Leaf discs of banana leaves 1 to 6 were collected from plants in the vegetative and

generative growing stage in the organic and conventional fields. BLSD symptoms under in vitro conditions were more pronounced in leaf discs from the organic farm than in leaf discs from the conventional farm, both in plants in the vegetative stage and in the generative stage (Figure 2.3). The pattern of BLSD symptom evolution was similar for all leaves, in both farms and at both plant stages. However, differences in symptom evolution in the organic and conventional farm were more pronounced in the generative plants and with increasing leaf age.

While in the generative plant growth stage BLSD in leaf 5 and 6 reached about stage 5 in the organic farm after 28 days incubation, it reached only stage 3 in the conventional farm (Figure 2.3 B). In the vegetative growth stage BLSD was also more pronounced in the organic farm than in the conventional farm but differences were smaller (score 2 and 1 for leaf 5 after 28 days incubation for the organic and conventional farm respectively) (Figure 2.3 A).

The symptom evolution in the vegetative growth stage did not show differences in the leaf numbers 1, 3 and 6 (Table 2.4) but was significantly larger in leaf numbers 2, 4 and 5 in the organic farm whereas the symptom stages in all leaves in the generative plants were again larger in the organic farms (Table 2.5).

These results correspond to the symptom development under field conditions (Stover, 1980; Gauhl, 1994). Ineed, the lower infection level in the conventional farm was attributed to fungicide residues. Also the increase in BLSD development stages was linked to increasing leaf age and plant stage. This might be caused by the lower nutrient status of the leaves (Lahav, 1974; Turner and Barkus, 1980; Vorm and van Diest, 1982; Hedge and Srinivas, 1989) as aging leaves export nutrients to younger leaves and the developing bunch. The translocation of nutrients from the older to the younger plant tissue could also contribute to higher disease development (Restrepo, 2000).


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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).


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Table 2.4 BLSD symptom accumulation (area under the curve) in 6 leaves from banana plants in their vegetative stage sampled in an organic (O) and conventional (C) farm (n = 30).

Leaf number

T-test(p-value )

1 2.8 1.4 1.4 0.8 0.39072 10.6 2.1 2.4 1.1 0.0009*3 17.4 3.0 10.5 2.0 0.06354 34.3 3.4 14.2 2.7 0.0001*5 38.3 3.7 16.1 2.2 0.0001*6 41.9 3.3 35.4 2.9 0.1455

* Significant differences at α=0.05

OMean ± SE

CMean ± SE

Table 2.5 BLSD symptom accumulation (area under the curve) in 6 leaves from banana plants in their generative stage sampled in an organic (O) and conventional (C) farm (n = 30).

Leaf number

T-test(p-value )

1 74.3 3.6 29.0 1.5 0.0001*2 90.3 4.4 33.4 3.4 0.0001*3 103.2 7.3 43.5 3.9 0.0001*4 114.7 7.9 50.5 3.5 0.0001*5 136.8 5.8 69.0 2.6 0.0001*6 140.5 5.4 70.5 4.0 0.0001*

* Significant differences at α=0.05

OMean ± SE

CMean ± SE

2.1.3.3. BLSD development under greenhouse conditions In order to eliminate environmental effects and the possible effects of different M.

fijiensis Morelet isolates, disease symptoms were monitored on micropropagated plants grown under greenhouse conditions. Disease symptoms were similar to those observed in the field on mature plants for conidia originating from both farms (Gauhl, 1994). The disease development generated by isolates from the organic and conventional farm was similar regardless of the origin of the conidial suspension during each evaluation time (Figure 2.4).


35

AA

B B

C

B B

C

0

1

2

3

4

5

15 30 45 60 15 30 45 60

C O

Days after inoculation per fungal isolate

BLS

D

sym

ptom

s

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).

2.1.3.4. BLSD and nutritional status under field conditions In both growing seasons (rainy and dry season), BLSD symptoms were more

pronounced in the organic farm than in the conventional farm (Figure 2.5 and 2.6), and the accumulation of the disease symptoms in leaf 3 and leaf 4 confirmed significant differences between organic and conventional farms in relation to the season (Table 2.6). These results confirm that the speed of disease development is faster in the rainy season when leaves are wet and thus cause more problems in banana fields because of frequent re-infestations; also there is again confirmation that BLSD is more pronounced in older than younger leaves. However, the differences of disease expression between farms are attributed to the fact that BLSD was kept under control in the conventional farm by frequent fungicide applications. In the organic farm however, the fungus was not controlled by fungicides, but leaves received OT on a regular basis.

Table 2.6 BLSD symptom accumulation (area under the curve) in leaves 3 and 4 from banana plants evaluated in an organic (O) and conventional (C) farm (n = 80).

O CANOVA (p-value) O C

ANOVA (p-value)

Leaf 3 172.8 121.9 0.0001* 149.6 91.2 0.0001*SE 0.8 0.4 0.5 3.6

Leaf 4 194.4 111.3 0.0001* 173.1 124.6 0.0001*SE 1.2 0.4 0.5 5.3

*Significant differences at α=0.05

Rainy season Dry season


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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).


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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).

Disease severity was always lower in the conventional farm during any plant

growth stage, indicating good chemical control (Table 2.7). This resulted in a significantly higher number of standing leaves in the conventional farm at any growing stage except flowering.

Despite the better BLSD control in the conventional farm, the disease seemed to have been adequately controlled in the organic farm. Indeed, at harvesting 8.5 functional leaves were observed, while 6-7 functional leaves are considered adequate (Zapata et al., 1999). Moreover there is the general belief, that in the absence of fungicide applications against BLSD, banana plants have virtually no leaves at harvest. Hence, BLSD in the organic farm was properly controlled, avoiding serious yield losses.


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For bananas little is known about optimal nutrient requirements in relation to disease resistance. Unpublished data from Mendez et al., mentioned by Romero (1998), reveal that N and K imbalances favour BLSD development. Results in Uganda showed that the intensity of BLSD is linked to organic matter and Ca and Mg content in the topsoil (Holderness et al., 2000). It has been demonstrated that deficiencies in Ca and P predisposes a plant to fungal and bacterial infection (Gunifer et al., 1980). BLSD severity can be reduced if soil fertility and organic matter is high, such as in Nigerian backyards that receive ample amounts of household refuse (Mobambo et al., 1994).

Table 2.7 Standing leaves and BLSD severity (%) in banana plants at different growth stages in an organic (O) and conventional (C) farm (n = 20 plants per growth stage).

Vegetative Flowering HarvestingParameters

Number of standing leaves per plant 11.9 ± 0.14 12.5 ± 1.19 8.5 ± 0.16Leaf area with BLSD severity (%) 2.9 ± 0.27 3.3 ± 0.33 6.3 ± 0.44

Number of standing leaves per plant 13.03 ± 0.14 13.4 ± 0.17 9.7 ± 0.17Leaf area with BLSD severity (%) 0 ± 0.02 0 ± 0.02 1.2 ± 0.16

0.0001* 0.00017* 0.559 ns0.0001* 0.0001* 0.0001*


Plant growth stage

Mean ± SEO

C

ns = not significant at α=0.05

Number of standing leaves per plant (O vs C)Leaf area with BLSD severity (%) (O vs C)

ANOVA (p-value )

Since both farms received nutrients in different ways, the soil nutrient status

was evaluated. Both the organic and conventional farms had the same nutrient content (Table 2.8) except for Zn, which was significantly higher in the conventional farm.

Also, macro and micronutrient content in leaf 3 of banana plants from organic and conventional farms were similar, and all nutrients were in ample supply as recommended for banana production (Lahav and Turner, 1983; Alvarez et al., 2001). Moreover, the leaf nutrient content from organic and conventional farms was compared with each other and found not to be significantly different (Table 2.9).

The leaf critical values (LCV) are used to guide farmers for their sustainable banana production. With respect to this value, both farms presented a much higher level of K, Mg, Cu, Fe and B than needed. In contrast both farms have a lower level of Ca, Zn and Mn. In addition the organic farm had a very high level of P but which was not different from the LCV of P in the conventional farm (Table 2.9).


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Table 2.8 Soil parameters and nutrients from organic (O) and conventional (C) farms. Analysis made by a subsidiary laboratory of BSI that operates in Ecuador

O CT-test

(p -value)pH 6.70 ± 0.10 6.35 ± 0.05 0.12

OM (% ) 1.89 ± 0.19 1.61 ± 0.21 0.44EC

(dS/m) 0.74 ± 0.19 0.84 ± 0.17 0.75

CEC (cmol/kg soil) 21.38 ± 1.23 22.88 ± 1.48 0.52

(mg/kg)NH4 6.00 ± 2.00 5.50 ± 2.50 0.89

P 15.50 ± 2.50 12.50 ± 0.50 0.44(cmol/kg soil)

K 0.41± 0.07 0.62 ± 0.09 0.24Ca 14.08 ± 0.72 13.79 ± 1.26 0.86Mg 6.65 ± 0.30 8.23 ± 0.02 0.12Na 0.10 ± 0.005 0.11 ± 0.01 0.35

(mg/kg)Cu 8.40 ± 0.70 9.2 ± 1.10 0.61Fe 52.85 ± 12.75 52.85 ± 13.95 1.00Zn 4.55 ± 0.25 7.50 ± 0.30 0.019*B 0.60 ± 0.24 0.67 ± 0.28 0.86

Mn 3.90 ± 0.70 4.50 ± 1.70 0.79S 45.95 ± 15.25 21.40 ± 2.20 0.35

Macronutrients

Micronutrients

Mean ± SE

* Significant difference at α=0.05


40

Table 2.9 Nutrient concentration on dry matter basis in leaf 3 of banana plants from organic (O) and conventional (C) farms and the banana leaf critical values (LCV). Nutritien analysis made by a subsidiary laboratory of BSI that operates in Ecuador.

O CContrast means

(O vs. C)(p-values )

Banana leaf critical values

(LCV)

(% ) (% )N 2.51 ± 0.21 2.54 ± 0.24 0.934 ns 2.6P 0.27 ± 0.06 0.19 ± 0.00 0.455 ns 0.17K 4.47 ± 0.09 4.96 ± 0.44 0.458 ns 3.6Ca 0.65 ± 0.07 0.69 ± 0.12 0.781 ns 0.9Mg 0.35 ± 0.04 0.39 ± 0.02 0.445 ns 0.29S 0.23 ± 0.06 0.16 ± 0.04 0.448 ns 0.2

(mg/kg) (mg/kg)Cu 10.05 ± 1.05 14.20 ± 4.40 0.514 ns 8.5Fe 123.15 ± 4.95 145.15 ± 56.65 0.764 ns 72Zn 20.65 ± 3.75 16.45 ± 2.75 0.469 ns 27B 28.83 ± 5.04 25.88 ± 0.75 0.662 ns 21

Mn 141.75 ± 25.65 106.45 ± 7.55 0.388 ns 300ns = not significant (α=0.05)

Mean ± SE

Macronutrients

Micronutrients

2.1.3.5. Nutrient composition of OT The nutrient composition of the OT was monitored over a period of 10 months

(Table 2.10). The high coefficient of variation (from 77.4 up to 241.5%) indicated that the nutrient composition of the OT varied substantially during the year. This was not surprising since the OT was made from locally available materials that also varied during the year (NOSB, 2004; Restrepo, 2000). Moreover, the quantities of the different materials used for the fermentation process were not well controlled. Table 2.10 shows that macronutrients like K, Na, and Ca and micronutrients such as Si, Fe and Mn were present in the largest quantities. Strikingly, the nutrient analysis shows very low levels of Cu, despite that Cu is applied during the preparation of the OT. Sometimes levels of Zn and B are also very low despite that these nutrients are added. This also indicates that OT production is variable and that the mixing of different ingredients might be inadequate or that some ingredients do not dissolve.


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Table 2.10 Nutrient composition of OT prepared on the organic farm and used for foliar application on the banana crop in the organic farm. Analysis made by the Soil Service of Belgium.

Mean Sdev Cv%03/2003 06/2003 09/2003 10/2003 11/2003 01/2004

K 3955.0 601.5 68.6 5808.8 7258.3 4252.8 3657.5 2837.5 77.6P 1339.0 10.7 10.7 87.5 808.3 3693.2 991.6 1427.9 144.0

Ca 2634.0 49.4 34.3 3004.8 3165.8 2297.7 1864.3 1443.4 77.4Na 2623.0 89.5 46.2 4944.9 6873.3 3754.3 3055.2 2708.3 88.6Mg 912.0 35.9 19.8 1550.7 1685.0 1022.4 871.0 717.1 82.3

N (total) 5927.0 1030.2 412.9 637.0 261.0 1023.0 1548.5 2167.7 140.0

Si 93.5 0.9 0.5 494.1 607.5 180.2 229.5 260.2 113.4Fe 293.3 2.5 2.6 253.7 226.7 239.3 169.7 131.4 77.4Cu 1.5 0.0 0.0 1.5 1.2 0.6 0.8 0.7 85.0Mn 38.2 1.6 11.9 15.3 6134.2 5.9 1034.5 2498.3 241.5Zn 8.0 0.1 0.2 11.0 17.8 2.9 6.7 7.0 104.5B 0.6 0.1 0.1 4.7 10.4 3.7 3.3 4.0 123.3

ElementsOT samples

Macronutrients (mg/100 g dry material)

Micronutrients (mg/100 g dry material)

2.2. Characterization of M. fijiensis Morelet isolates from organic and conventional banana production systems

2.2.1. Introduction

The conversion from a conventional to an organic production system implicates many changes in the management of the crop, not the least the arrest of the application of fungicides against BLSD. However, in Ecuador, some of these organic farms exhibit surprisingly a low level of BLSD with plants having 8 to 9 leaves at harvest. This requires an in-depth study of what is going on in those organic farms. Hence it is necessary to compare the morphological and aggressiveness characteristics of M. fijiensis Morelet isolates from an organic banana production system with those from a conventional banana production system. Some authors already reported that M. fijiensis Morelet colony growth, conidia production and aggressiveness of isolates can vary (Williams, 1990; Porras and Perez, 1997; Jacome and Schuh, 1993a and 1993b; Rosa and Menendez, 2001, Hanada et al., 2002; Etebu et al., 2002; Etebu, et al., 2005). Likewise the colour of colonies can vary (Manzo-Sanchez et al., 2001).

The objectives were to examine the (i) morphological characteristics; (ii) conidia production capability and (iii) aggressiveness of fungal isolates obtained from a conventional and an organic production site where BLSD is well controlled in a yet unknown way.


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2.2.2. Material and Methods Fungal material: M. fijiensis Morelet ascospores, colonies and conidia M. fijiensis Morelet isolates from banana leaves infected with BLSD were

isolated from farms using an organic and conventional banana production system. Colonies and conidia were obtained under laboratory conditions from single ascospores discharged from infected banana leaves. The infected banana leaf samples were examined under a stereo-microscope to select leaf pieces with abundant pseudothecia in lesions of stage 5 and 6 of BLSD. The selected material was sliced in pieces from ± 1cm² and stapled to filter paper, the adaxial side towards the paper. To stimulate ascospore discharge, mature pseudothecia were impregnated with water. For that purpose, filter papers with attached leaf pieces were incubated for 48hrs in a plastic box with moist cotton. After the incubation period, filter papers were secured to the lids of plastic Petri dishes of 100mm diameter, containing 10 ml of water agar medium (14g Difco® agar per litre of distilled water). Discharge was allowed to take place for 1 hour. Then, the Petri dishes with the ascospores were incubated for 48 hours at 26°C, under dark conditions. Afterwards, single ascospores with germinated tubes were picked up and transferred to PDA (Potato Dextrose Agar Difco®, 39g/L) medium, pH 6. Single ascospores were grown at 26oC under dark conditions for 7 days.

Three different media were used to produce in vitro conidia from monosporic colonies (from isolates from an organic and conventional production system). Potato dextrose agar (4%) Difco® was used for colony growth for 7 days at 26oC and in complete darkness. Then, colonies were transferred to a Mycophyl medium, pH 6 (200g soybean boiled in 1L of distilled water, 16g agar -Difco®- and 20g dextrose Merck®) for 20 days under the above mentioned conditions. Conidiophores were induced by transferring small pieces of mycelium to V8 sporulation medium (200ml of vegetable juice -V8®-, 16g -Difco®- agar, 1.5g CaCO3 -Mallinckrodt®- and 800ml distilled water) at pH 6. Conidia were produced after 7 days at 26oC and with continuous light (4,000 Lux ± 200). Conidia were collected by washing the colonies using 10ml of a solution of sterilized distilled water with 0.005% of Tween-20-Mallinckrodt®. The conidia solution was maintained in Eppendorf tubes of 15 ml at 16oC until use.

Morphological characterisation of colonies Twenty single ascospores per banana production system were selected and,

once transferred to the PDA media, the colony diameter was measured every 3 days for 2 months. At the same time, the morphological characteristics were qualified, i.e. colony colour, colony form of the edge, shape and texture (Figure 2.7). Nine colour combinations among which black, grey, white and pink were evaluated. The colony form (edge) was classified as regular or irregular; the shape was registered as with or without elevation and the colony texture was evaluated as either smooth or creasy. During evaluation the colonies remained in the incubator at 26oC and in the dark.


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Evaluation of conidia production Per banana production system, the production of conidia was evaluated.

Following the above mentioned protocol, each colony was subcultured on PDA, mycophyl and V8 medium until the production of fungal conidia. The Petri dishes used were completely randomized with five Petri dishes per isolate. Then the conidia production per colony was counted by using a Neubauer counting chamber under a light microscope.

Isolate aggressiveness The aggressiveness of the isolates from both production systems was

evaluated under in vitro conditions. For this purpose banana leaves from micropropagated "Williams" banana plants (AAA Cavendish group) were used. Leaf pieces (5cm2) were excised and placed with their adaxial side towards the medium and inoculated with fungal conidia. They were incubated in a benzimidazole-rich medium, pH 6.5 and the Petri dishes of 100mm diameter were sealed. Factors assessed were: material of the Petri dish (plastic and glass); duration of light incubation (12 and 24 hours) and inoculum concentrations (1000, 5000 and 10000 conidia per ml). A factorial design was applied with three observations per factor, 36 per production system, and the disease symptom development was evaluated. Lesions on epidermal cells caused by the growth of fungal hyphae were checked once a week as the leaves remained green.

Data analysis Analysis of Variance (ANOVA) and the Central Limit Theorem were applied

to analyse the morphological data. All data, including morphological as well as conidia production, were used to compare similarities between isolates from the organic and conventional production system by the Clustering Method, Single Linkage and Euclidean Distance Type. The statistical differences were obtained by T-test at 5% of significance and 95% confidence interval. All data were analysed by running MINITAB 13 for Windows.


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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).


45

2.2.3. Results

2.2.3.1. Characterization of colonies and conidia production The experiment started with twenty monoascospore colonies per production

system (organic and conventional) but three colonies from the organic production system were lost. The morphological characteristics of the isolates from both production systems did not show any differences in colony form (edge) (Table 2.11) nor in their colony shape (with and without elevations). However, the colony texture was significantly different between isolates from both production systems (Table 2.12).

Table 2.11 ANOVA (one way) of colony form-edge characteristics of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system.

Analysis of Variance Source DF SS MS F P Prod.Sys. 1 0.004 0.004 0.01 0.904 Error 35 9.185 0.262 Total 36 9.189 Individual 95% CIs for Mean Prod.Sys. n Mean StDev ---------+---------+---------+------- O 17 1.4706 0.5145 (----------------*----------------) C 20 1.4500 0.5104 (---------------*--------------) ---------+---------+---------+-------

1.35 1.50 1.65

Table 2.12 ANOVA (one way) of texture characteristics of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system.

Analysis of Variance Source DF SS MS F P Prod.Sys. 1 7.64 7.64 7.20 0.011 Error 35 37.12 1.06 Total 36 44.76 Individual 95% CIs for Mean Prod.Sys. n Mean StDev ---+---------+---------+---------+--- O 17 2.412 1.176 (-------*--------) C 20 1.500 0.889 (-------*-------) ---+---------+---------+---------+---

1.20 1.80 2.40 3.00


46

Based on the ANOVA analysis, the growth rate of the isolate from the organic production system was faster than the isolate from the conventional farm (<0.05%) but the confidence interval analysis (95%) did not show a difference (Table 2.13). In contrast, the amount of conidia was significantly less with the isolates from the organic farm (Table 2.14).

Table 2.13 ANOVA (one way) of growth rate (mm/week) of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system.

Analysis of Variance Source DF SS MS F P Prod.Sys. 1 0.001332 0.001332 5.91 0.020 Error 35 0.007884 0.000225 Total 36 0.009217

Individual 95% CIs for Mean Prod.Sys. n Mean StDev --+---------+---------+---------+---- O 17 0.37734 0.01385 (---------*--------) C 20 0.36530 0.01592 (--------*-------) --+---------+---------+---------+----

0.3600 0.3680 0.3760 0.3840

Table 2.14 ANOVA (one way) of amount of conidia (per ml) of M. fijiensis Morelet colonies from an organic (O) and conventional (C) banana production system.

Analysis of Variance Source DF S MS F P Prod.Sys. 1 1.596E+09 1.596E+09 10.11 0.004 Error 28 4.422E+09 157915992 Total 29 6.018E+09 Individual 95% CIs for Mean Prod.Sys. n Mean StDev --+---------+---------+---------+---- O 10 31105 9874 (---------*---------) C 20 46579 13658 (------*------) --+---------+---------+---------+----

24000 32000 40000 48000


47

In general, isolates from both production systems were very similar (>98%) based on the evaluation of all morphological data, colony colour and colony growth rate (Figure 2.8).

However, it is interesting to highlight the grouping of isolates from the organic (3 groups) and from the conventional (2 groups) production system (Figure 2.8). The texture of the colony seemed to be a determining feature for the conventional colony grouping because all colonies grouped in C1 had a creasy texture while all colonies of C2 had a smooth texture. For the organic colonies, the combination of colony shape (with and without elevation) and texture could explain the grouping because the O2 group clustered all colonies without elevations and creasy texture whereas the O3 group clustered all colonies with elevations and the O1 group clustered colonies with and without elevations and with a smooth and creasy texture. All colonies had initially a black colour that evolved into a pink colour after two months.

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.


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2.2.3.2. Isolate aggressiveness In general, similar patterns were established for BLSD symptom development

under in vitro conditions on leaf discs inoculated by conidia produced from organic and conventional isolates. Also the concentration of inoculum was not a key factor. However, the disease evolved much faster on leaf pieces incubated for 24 hours than after 12 hours (Figure 2.9). Also the disease evolved much faster in plastic than glass Petri dishes (Figure 2.9) which might be attributed to a higher light reflectance in plastic Petri dishes than in glass Petri dishes (data not shown, courtesy Prof. M. De Proft, Division of Crop Biotechnics, K.U. Leuven University).

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.

The interaction between the different factors was also evaluated (Figure 2.10),

with parallel lines indicating no interaction and crossing lines showing an interaction. Thus a strong interaction existed between the material of the Petri dish and the inoculum concentration on disease symptom development. This indicates that both environmental as well as fungal characteristics influence the outcome of the disease.


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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.

2.2.4. Discussion Isolates from the organic and conventional banana farm were compared.

Morphologically they were similar but isolates from the organic farm produced less conidia. However taking all parameters into account, we conclude that all isolates are quite similar. Most importantly the results prove that M. fijiensis Morelet isolates from the organic production system do indeed cause BLSD. This underscores that the organic farm has BLSD and that its fungal isolates have the ability to provoke the disease in the studied conditions.

The composition of the medium and light illumination has been reported to influence the production of conidia under in vitro conditions (Jacome and Schuh, 1993b; Rosa and Menendez, 2001, Hanada et al., 2002). We complement these findings by illustrating that the material of the Petri dish had a marked effect. Indeed plastic Petri dishes result in a higher conidia production most probably because of a higher relative humidity and/or other characteristics from this Petri dish material such as light reflection. As such and under these conditions we can use lower amounts of inoculum to run the experiment especially when we combine this with a 24 hour incubation period.


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2.3. Conclusions Cavendish banana plantations are reported to depend on synthetic fungicides

to remain productive where BLSD occurs. However, the presented results indicate that bananas can be grown commercially without fungicides in Ecuador, at least in the studied area (Taura village, Guayas province, southern coast of Ecuador) and under conditions of the evaluated organic banana field (196 ha). The studied organic farm recorded a yield of 23 ton/ha/year whereas the conventional farm produced 42 ton/ha/year. The higher value received per exported box from the organic bananas compensates for the lower yield. Our research also demonstrates that the M. fijiensis isolates from both farms (organic and conventional) exhibit similar morphological characteristics and the same aggressiveness under controlled conditions as well as in the field. Hence more research is required to find out why the banana plants in the organic field do not succumb to BLSD.

A first step was taken by investigating the nutrient composition of the soil and banana plants grown under both growing conditions. Indeed this is needed, as it has been reported that nutrients have an effect on fungal resistance and since in both production conditions nutrient supplies were completely different. In the conventional field conventional fertilizers were applied while in the organic field locally made organic brews were applied with a very variable composition. However, banana plants in both farms (the organic and the conventional farm) had a similar leaf nutrient content which was sufficient according to the reported leaf critical values for this crop.

Clearly the results at this stage do not explain why BLSD in the organic field is controlled to a certain extent by a specific nutrient. This calls for a more systematic approach of different nutrients under more controlled experimental conditions which is the subject of the following chapters.

CHAPTER 3: Organic teas

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Chapter 3 ORGANIC TEAS AND THEIR EFFECTS ON M. FIJIENSIS MORELET, THE BLACK LEAF STREAK DISEASE AND THE BANANA HOST2

3.1. Introduction

There are several reports showing that different kinds of organic products control plant fungi like Verticillium, Rhizoctonia, Fusarium and Phytophthora both in vitro and in the field (Khalifa, 1965; Conn and Lazarovits, 1999; Wickland et al., 2001; Tenuta et al., 2002). A potential alternative to fungicides are organic teas (OT) applied either to the soil or the plant foliage or both. These OT are obtained from various organic sources such as harvest residues, different manures, compost, vermicompost, etc. (Merrill and McKeon, 1999; Romero, 2000) after aerobic or anaerobic preparation (Ingham, 2005). Once applied to the soil, they move into the root zone and affect the rhizosphere (Hoitink and Fahy, 1986). Their nutrients are taken up by the plant as well as by soil microorganisms (Ingham, 2005). The type and nutrient composition of OT depends on the age and kind of ingredients. The microorganisms from the OT include mycoparasites, hyperparasitic fungi, epiphytic microorganisms as well as specific bacteria such as Pseudomonas, Azotobacter, and certain fungi like Trichoderma and Gliocladium, which then become part of the soil and rhizosphere microbial ecology (Hubbard et al., 1983; Meshram, 1984).

OT from compost intended for foliar application should be made with care to avoid leaf damage by salts and pathogens (Bess, 2000; Wickland et al., 2001). They provide microorganisms and nutrients to the leaf surface and reduce plant diseases caused by, for example, Botrytis (Elad and Shtienberg, 1994), Phytophthora (Hoitink et al., 1977) Plasmopora, and Venturia (Merrill and McKeon, 1999). In potato fields, OT from liquid swine manure reduced the incidence of wilt caused by Verticillium dahliae Kleb, common scab caused by Streptomyces scabies (Thaxter) Lambert & Loria, and damage by parasitic nematodes, for up to 3 years after a single application. This organic product was shown to kill microsclerotia of Verticillium dahliae in acid soils (Conn and Lazarovits, 1999). In another study, several acids from liquid swine manure OT were tested and formic and n-caproic acids shown to be most toxic against microsclerotia of Verticillium dahliae at approximately 4 mM (Tenuta et al., 2002). Effective control of the pathogens was achieved especially when the OT was composed of 50% cattle manure + 20% sheep manure + 20% poultry manure + 10% crushed wheat straw. In potato this product reduced dry rot caused by Fusarium solani during storage (Khalifa, 1965; Wickland et al., 2001).

In recent years, BLSD control in banana without fungicides has been investigated. OT with Effective Microorganisms from some commercial products called efficient microorganism (EM®) were shown to block the coalescence of BLSD lesions, thereby reducing significantly the severity of BLSD in banana leaves (Tabora

2 Some results were submitted as follows: Jiménez, M., Quito, D., Maura, F., Rodríguez, H., Ruiz, O., and Swennen, R., The effects of organic

teas on Mycosphaerella fijiensis (Morelet) and their potential as a tool for black Sigatoka disease management and banana growth enhancement. European Journal Plant Pathology (2008).


52

et al., 1997). In the laboratory, M. fijiensis Morelet ascospores treated with EM solutions became deformed and their germination reduced (Bayro, 1998). OT from compost and vermicompost were demonstrated to delay BLSD epidemics in the greenhouse by 18 days after foliar applications on the plantain var. Curare (Larco et al., 2004). Other OT made from liquid harvest residues in combination with fungicides, reduced the number of fungicide sprays needed for BLSD control by at least 40% (Arango, 2002).

In Ecuador, more than 10,000 ha of organic bananas are produced for export and the majority receives liquid organic tea for disease control, resulting in a yield average of 24 ton/ha/year (Table 2.1). As BLSD is one of the main reasons causing yield loss (Mourichon et al., 1987; Ramsey et al., 1990; Carlier et al., 2000a and Ploetz, 2004) and these organic banana fields are commercially viable, it is indicative that some of the management practices do indeed reduce BLSD development.

This prompted us to investigate the effects of OT. The following questions were addressed: have the OT direct effects on M. fijiensis Morelet and do OT affect growth of banana plants as well?

3.2. Effects of OT on M. fijiensis Morelet

3.2.1. Materials and Methods

Fungal isolation Isolation of ascospores and colonies, and conidia produced was according to

the protocols in Chapter 2, section 2.2. Organic Teas (OT) Experiments were done with OT prepared by the banana producers: three

different batches were evaluated. The raw materials used for the preparation of these products were as follows: 20kg fresh cattle manure, 4L sugarcane molasses, 4L microorganism solution (from a product known as EM® and commercially available. It is claimed to be a mixture of microorganisms consisting mainly of lactic acid bacteria, cyanogenic bacteria, and yeasts), and 150L water. Their mixture was fermented on-farm anaerobically in closed plastic tanks. The tanks were placed for 2 months under a shed at ambient temperature.

In the first in vitro experiments, we collected all OT from the farm in Taura and screened two groups of OT: (i) one prepared for foliar application and enriched with sulphate salts (1.3kg ZnSO4, 0.9kg CuSO4 and 0.9kg MnSO4) and (ii) the other prepared for root application and amended with 3.2kg K2SO4 and 6.4kg rock phosphate. For the second experiment, we sampled OT immediately after fermentation in June and September (both in the dry season, Figure 1.3), from two organic banana farms (in Taura and Balao in the Guayas province) that routinely produce OT for banana production. The organic tea from Taura was enriched with salts (see above) whereas in the OT from Balao no extra nutrients were added. Once collected from the farms, both OT were kept in plastic bottles at room temperature. In


53

these experiments, the ascospore germination was tested with the OT that showed the best results in the second experiment.

Others experiments were done with OT prepared by us (Figure 3.1) according to the process performed by the farmers. However, we measured weekly the pH with a portable pH meter (Mettler Toledo® SG2) and the electrical conductivity (dS/m) with a portable conductimeter (Mettler Toledo SG7FK2) during the OT preparation. For the OT preparation other vairables were considered such as: (i) preparation time: 1, 2 and 4 months in a shed at ambient temperature and (ii) three different farms in three locations: Los Rios, Guayas and El Oro. These factors were selected to know whether the preparation time and/or the location of preparation affect the OT nutritional content and the direct effect of the OT on the fungus. The materials and the methodology used for these OT were as mentioned above.

In the field all OT were filtrated with a home made filter to separate the liquid from the solid rest of the raw materials. The pH of the OT fluctuated then between 4.5 and 5.5 as measured with pH indicator strips (Whatman® with a color scale between 4.5 -10).

Immediately after collection, the OT samples were sent to a subsidiary laboratory of BSI (British Standards Institution), located in Guayaquil, Ecuador, for nutritional content analysis. The elements K, Ca, Mg, Cu, Mn were determined by Atomic Absorption Spectrophotometry; B and S were determined by UV – VIS Spectrophotometry and the analytical methods were based on the Official Methods of Analysis of AOAC International (AOAC, 2008).

In vitro evaluation of OT against M. fijiensis Morelet A first batch of seven OT (foliar and root) originating from the Taura farm

were screened under in vitro conditions. The OT were prepared by the farmer for foliar and root application but their mixture (foliar:root was 1:1) was made in the lab. Each OT and their mixture were tested separately at two concentrations (10 and 30% v/v) for their inhibitory effects on M. fijiensis Morelet colony growth. The design was completely randomized with 10 observations per treatment and the experiment was repeated twice. Each OT was centrifuged at 4000rpm 20mn and the solid residues removed. Then the OT were filter sterilised using a Millipore filtration system (Millex® 0.45µm), connected to a vacuum pump. The solution was then passed through a hydrophilic Durapore® membrane filter (0.22µm Millipore filter). The different OT concentrations were consequently mixed with a cooled down sterilized PDA medium (39g/L). For each concentration, seven plastic Petri dishes (15 x 100mm of diameter) were filled with 10ml medium-OT mixture. The M. fijiensis Morelet conidia solution (3x103conidia/ml) was inoculated onto the Petri dishes and incubated at 26oC in the dark. Seven days after inoculation, 5 separate colonies were randomly selected per plate and their diameter measured through a light microscope (Leica), using a micrometric scale. Only the growth of these selected colonies was again measured with a ruler fifteen days after inoculation.


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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).


55

With the second batch of OT (from two organic farms and at two different sampling dates), three OT concentrations (10, 30 and 70% v/v) were compared with an untreated control; two sterilization methods (autoclaved and filtration), and two media (solid and liquid) were evaluated for colony and mycelium growth. The design was completely randomized with 25 observations and 8 observations respectively, per treatment and the experiments were repeated twice. Before autoclaving, OT were mixed with the PDA medium (pH 6) and then autoclaved for 20 min (121°C and 15psi) and left to cool before inoculation. For the filter sterilization, OT was added to the sterilized medium through a hydrophilic Durapore® membrane filter (0.22µm Millipore filter). Fungus inoculation, incubation and colony evaluation was done as described above. The mycelial growth took place in a liquid PD-V8 medium (200g potatoes boiled in 1000ml distilled water, 20g dextrose Merck®, 16ml vegetable juice V8®) at pH 6. Eight Erlenmeyers with fungal mycelium were placed on a rotatory shaker (140rpm) at room temperature (26oC ± 2) under permanent light. Dry weight data of mycelium was collected 15 days after inoculation.

In the third in vitro experiment, the effect on ascospores of one OT from Balao was tested using five concentrations (0.5, 1, 3, 5 and 10% v/v) and compared with a medium without OT. The design was completely randomized and repeated twice. Five plastic Petri dishes of 15 x 100mm diameter were filled (10ml) with Difco®Agar mixed with each OT concentration. Then M. fijiensis Morelet ascospores were discharged from banana leaves directly onto the medium. Fifty single ascospores were chosen randomly and their germinative tubes measured under an inverse microscope (Zeis® Axiont Vert 25, 40x) with a micrometric scale. Ascospore germination was evaluated after incubation in an incubator (Lab Line Instruments Inc®) at 26oC for 48 hours under dark conditions.

In the fourth experiment, nine OT (from 3 times and 3 places) were tested. Colony growth as well as mycelium development were measured in three concentrations per product (10, 30 and 70% v/v) sterilized by autoclaving (as mentioned above), and compared with an untreated control. The design was completely randomized with 25 observations per treatment.

The percentage of inhibition was determined by measuring colony diameter, mycelium weight and length of germinative tube of ascospores of different OT treatments and compared to treatments without OT. Inhibition was calculated as follows: % inhibition = (mean fungal measurement of control treatment – fungal measurement of an OT treatment) x 100/ mean fungal measurement of the control treatment.

Statistical analyses Univariate descriptive statistics were applied for the estimation of central

tendency and dispersal parameters. Inferential statistics - Analysis of Variance (ANOVA) and Central Limit Theorem - were applied to analyze the quantitative variables such as percentage of inhibition; the homogeneous subgroups were obtained at 1 and 5% significance, by Duncan and Tukey test. A proximity matrix with the nutrient content of nine OT was obtained using Euclidean Distance. Data were analysed by running SPSS version 11 and MINITAB 13 for Windows.


56

3.2.2. Results 3.2.2.1. In vitro evaluation of OT

The first batch of OT demonstrated that the extent of inhibition depended on the sampled product, and thus the moment of sampling, and varied between less than 10% inhibition to complete inhibition (Table 3.1). In general, seven days after inoculation, the foliar OT were more inhibitory than the root OT. The combination of foliar OT with root OT was in general better than the root OT alone. Similar response patterns were obtained 15 days after inoculation. Moreover, there were no significant differences at 1% between both concentrations (10 and 30% v/v) at both evaluation times. There was no correlation between the content of a single nutrient in the OT and the OT inhibitory effect.

Table 3.1 In vitro inhibitory effects (0%: no inhibition; 100%: complete inhibition) on M. fijiensis Morelet colonies 7 days after inoculation grown on a solid medium amended with seven organic teas (OT) at 10 and 30 % v/v collected from one farm (Taura), (n = 35). Means with the same letter per OT and per concentration are not significantly different by Tukey test at P≤0.01.

# of OT

1 100 a 100 a 72 b 100 a 100 a 100 a2 37 c 100 a 27 d 76 b 11 d 70 b3 100 a 100 a 100 a 100 a 100 a 100 a4 100 a 100 a 41 c 100 a 100 a 100 a5 51 b 79 b 9 e 47 c 43 b 72 b6 100 a 100 a 44 c 100 a 100 a 100 a7 6 d 95 a 7 f 8 d 24 c 57 c

30

Foliar/Root

10 30 3010

Root Foliar

10Concentration (%)

When comparing two OT from the two farms (Taura and Balao) the 30% and

70% v/v concentrations always completely inhibited fungal growth. At 10% in the Taura farm, colony inhibition was less effective than with OT from Balao and less effective in June than in the September sampling (Figure 3.2). The method of sterilisation had no effect on growth inhibition, suggesting that the active ingredient (s) is heat-resistant. The results after 7 days gave the same pattern as those after 15 days. Similar results were obtained for mycelial growth (Figure 3.3).


57

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).

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).


58

Ascospore germination depended on the OT concentration, i.e. the higher OT concentration gave the higher inhibition of germination (R2=0.81) (P=0.000) starting with 5% inhibition at 0.5% v/v up to complete inhibition at 5% v/v (Figure 3.4).

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).

All nine OT, coming from three places (Los Rios, El Oro and Guayas) and

three fermentation times (1, 2 and 4 months), completely inhibited the fungus development (colony and mycelium) at all tested concentrations (Figure 3.5).

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 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).


59

3.2.2.2. OT nutrient content In the search of the direct effect of OT on M. fijiensis Morelet structures under

in vitro conditions, the nutrient content of different OT were analyzed. No relationship was found between a nutrient and fungus inhibition. In addition a tremendous variation in nutrient composition was ascertained between all OT whether prepared for foliar or root application. Coefficients of variation ranged between 11.4 and 175.6% (Tables 3.2 and 3.3).

Some inconsistencies were noted. First the P content was quite similar in the foliar and in the root OT although rock phosphate was added to the root OT. This points to a not-standardized methodology and/or problems with solubilisation of this and possibly other ingredients as well causing OT variability. Second, Zn, Cu and Mn were added during the preparation of the foliar OT at a fixed rate, yet the content of these nutrients varied extensively.

Plants treated with OT from the second OT batch exhibited phytotoxicity symptoms. Presumably this was caused by the very high concentration of some micronutrients, unlike in the first batch. Again a very high variability in composition was ascertained (CV of 5.7 – 141.1%), while there was no pattern linked to location nor time of sampling (Table 3.4).

Table 3.2 Nutrient content of seven organic teas (OT) prepared by the farmer for foliar application. OT collected during 2004 in one organic farm in Guayas province.

1 2 3 4 5 6 7 Mean CV (%)

N 1.59 0.90 0.16 0.92 0.12 0.09 0.16 0.16 103.60P 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.05 11.39K 0.25 0.79 0.53 0.56 0.53 0.71 0.33 0.33 36.17Ca 0.10 0.13 0.12 0.14 0.07 0.06 0.06 0.06 35.02Mg 0.04 0.06 0.08 0.06 0.05 0.07 0.06 0.06 21.52

Zn 695.3 182.6 279.5 112.8 266.4 82.6 107.6 246.69 86.16Cu 9.4 41.1 73.8 36.7 90.2 69.1 10.5 47.26 66.69Mn 396.2 387.1 18.18 175.5 151.2 338.2 2.52 209.84 79.49Si 20.5 81.0 79.0 77.5 58.5 45.0 59.5 60.14 36.53

Macronutrients (%)

Micronutrients (mg/kg)

Foliar OT


60

Table 3.3 Nutrient content of seven organic teas (OT) prepared by the farmer for root application. OT were collected during 2004 in one organic farm in Guayas province.

1 2 3 4 5 6 7 Mean CV (%)

N 0.14 0.09 0.12 0.12 0.15 0.19 0.16 0.14 23.34P 0.05 0.04 0.04 0.04 0.04 0.04 0.05 0.04 11.39K 0.60 0.44 0.56 0.35 0.39 0.82 0.66 0.55 30.40Ca 0.11 0.11 0.14 0.09 0.09 0.21 0.12 0.12 33.45Mg 0.12 0.07 0.06 0.05 0.06 0.10 0.13 0.08 38.05

Zn 807.8 154 112.8 75.5 10.4 497.6 625.5 326.23 96.01Cu 1125.0 71.1 36.7 72.2 2.2 47.8 251.7 229.53 175.55Mn 27.9 103.3 175.5 324.3 17.2 124.5 6.6 111.34 101.41Si 108.0 37.5 77.5 40.7 45.0 90.0 73.0 67.34 40.19

Root OT

Macronutrients (%)


Table 3.4 Nutrient content of four OT prepared in two different organic farms and in two periods of the year 2005.

June September Mean CV (%) June September Mean CV (%)

N 0.17 0.36 0.27 50.70 0.36 0.39 0.38 5.66P 0.04 0.07 0.06 38.57 0.07 0.06 0.07 10.88K 0.36 1.05 0.71 69.21 0.07 0.11 0.09 31.43Ca 0.09 0.07 0.08 17.68 0.40 0.51 0.28 120.85Mg 0.08 0.19 0.14 57.62 0.02 0.04 0.03 47.14

Zn 1344.0 243.5 793.7 98.04 12.7 50.9 31.8 85.04Cu ND ND --- --- ND 0.2 0.2 ---Mn 2505.0 2.6 1253.8 141.13 4.5 4.2 4.3 6.19Si 89.0 120.0 104.5 20.98 95.0 105.0 100.0 7.07

ND = not determined

Macronutrients (%)


Taura OT Balao OT

Given this tremendous variability in nutrient content of OT, one is left to

wonder how OT, presumed to control BLSD, can be standardized. Simultaneously this means that one has to explore what other variables need to be taken into account when evaluating OT. Therefore OT were prepared in three locations and with three different fermentation times. For the third batch of OT, nine products were analyzed.

During preparation the pH (Figure 3.6) and conductivity (Figure 3.7) was measured. The coefficient of variation fluctuated between 0.5-8.7% and 0.2-17.5% respectively. The pH after 1 month of fermentation was always the highest while there was no clear pattern in conductivity with time. The results on the fungus confirmed again the potential of the OT as explained above. The nutrient content is displayed in Table 3.5 (A and B). Again variation in nutrient composition was very high whether comparing OT between different localities or OT between different time periods. Hence no link could be made between a specific nutrient composition and the inhibitory effect of the OT.


61

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).

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).

0

1

2

3

4

5

6

R1 R2 R4 O1 O2 O4 G1 G2 G4

Treatments

pH v

alue

0

2

4

6

8

10

12

14

R1 R2 R4 O1 O2 O4 G1 G2 G4

Treatments

Conductivity (mS/cm)

Table 3.5 Nutrient content of nine organic teas (OT) prepared in three locations and with three fermentation times. Coeficient of variance obtained per location (A) (Los Rios - R, El Oro - O and Guayas - G), and per fermentation time (B) (1, 2 and 4 months).

A

R1 R2 R4 Mean CV(%) O1 O2 O4 Mean CV(%) G1 G2 G4 Mean CV(%)

N 0.38 0.36 0.14 0.29 46.45 0.10 0.29 0.16 0.18 54.26 0.24 0.19 0.28 0.24 18.09P 0.03 0.01 0.01 0.02 58.24 0.02 0.01 0.01 0.01 55.97 0.01 0.01 0.01 0.01 20.00K 1.38 0.07 0.16 0.54 136.06 0.13 1.57 0.34 0.68 113.82 0.71 2.10 1.08 1.30 55.08Ca 0.09 0.05 0.06 0.07 35.60 0.07 0.05 0.05 0.06 18.80 0.04 0.06 0.06 0.05 24.88Mg 0.02 0.01 0.02 0.02 28.29 0.02 0.01 0.01 0.01 23.41 0.01 0.01 0.02 0.01 11.64

Zn 8.63 13.13 3.25 8.33 59.33 3.00 2.28 5.80 3.69 50.42 3.55 5.00 2.78 3.78 29.92Cu 2.88 2.28 2.05 2.40 17.77 2.13 2.00 0.45 1.53 61.19 0.90 0.78 0.38 0.68 40.13Mn ND* -- -- -- -- ND* -- -- -- -- ND* -- -- -- --Si 0.91 0.11 0.69 0.57 72.47 0.05 0.06 2.01 0.71 160.02 3.11 3.60 1.97 2.89 29.02

OT

Macronutrients (%)


B

R1 O1 G1 Mean CV(%) R2 O2 G2 Mean CV(%) R4 O4 G4 Mean CV(%)

N 0.38 0.10 0.24 0.24 59.57 0.36 0.29 0.19 0.28 30.29 0.14 0.16 0.28 0.19 39.44P 0.03 0.02 0.01 0.02 38.31 0.01 0.01 0.01 0.01 7.67 0.01 0.01 0.01 0.01 9.64K 1.38 0.13 0.71 0.74 83.84 0.07 1.57 2.10 1.25 84.37 0.16 0.34 1.08 0.53 92.50Ca 0.09 0.07 0.04 0.07 41.90 0.05 0.05 0.06 0.05 11.32 0.06 0.05 0.06 0.06 8.07Mg 0.02 0.02 0.01 0.02 18.51 0.01 0.01 0.01 0.01 12.37 0.02 0.01 0.02 0.01 20.63

Zn 8.63 3.00 3.55 5.06 61.31 13.13 2.28 5.00 6.80 83.01 3.25 5.80 2.78 3.94 41.27Cu 2.88 2.13 0.90 1.97 50.69 2.28 2.00 0.78 1.68 47.44 2.05 0.45 0.38 0.96 98.73Mn ND* -- -- -- -- ND* -- -- -- -- ND* -- -- -- --Si 0.91 0.05 3.11 1.36 116.42 0.11 0.06 3.60 1.26 161.67 0.69 2.01 1.97 1.55 48.31

OT

Macronutrients (%)


*ND: not determined


63

Proximity matrixes (Table 3.6 A and B) constructed with the content of macro and micronutrients of the nine OT confirmed that there are few similarities between the OT and that no pattern could be linked neither to the zones nor fermentation times.

Table 3.6 Proximity matrixes of the macronutrient (A) and micronutrient (B) content of nine OT prepared in three locations (Los Rios - R, El Oro - O and Guayas – G), and three fermentation time (1, 2 and 4 months).

3.2.3. Discussion

In general, the organic tea (OT) was shown to have inhibitory properties in vitro on M. fijiensis Morelet. This is in line with results on BLSD control in other locations and with other OT reports (Tabora et al., 1997; Arango, 2002; Larco et al., 2004).

While the OT inhibitory effect in vitro was fast (within 7 days) and complete, the effect of the OT depended on its preparation, as foliar OT was superior over root OT. Second, its effect was concentration-dependent as a 30% v/v seemed to give a much stronger inhibition than a 10% v/v. Thirdly, its effect at 10% v/v depended on the moment and location of preparation. Like other authors (Merrill and McKeon, 1999; Restrepo, 1998), we showed that the nutrient composition was very variable, but in addition we could not link the nutrient composition nor fermentation times with

A Proximity Matrix Euclidean Distance

Zone/months Los Ríos/4 El Oro/4 Guayas/4 Los Ríos/2 El Oro/2 Guayas/2 Los Ríos/1 El Oro/1 Guayas/1Los Ríos/4 0El Oro/4 2.444 0Guayas/4 3.231 2.507 0

Los Ríos/2 1.370* 2.660 3.344 0El Oro/2 3.309 2.698 1.703 2.726 0Guayas/2 1.832 2.613 2.395 1.355* 1.914 0

Los Ríos/1 5.290 4.059 3.289 4.601 2.405 3.745 0El Oro/1 3.717 2.411 3.356 3.158 2.832 3.211 3.335 0Guayas/1 2.029 2.718 3.096 0.958* 2.227 1.589* 4.225 2.840 0

* no significant differences

Zone/months Los Ríos/4 El Oro/4 Guayas/4 Los Ríos/2 El Oro/2 Guayas/2 Los Ríos/1 El Oro/1 Guayas/1Los Ríos/4 0El Oro/4 1.917 0Guayas/4 2.346 1.455 0

Los Ríos/2 0.618* 2.226 2.594 0El Oro/2 1.023* 2.676 2.594 0.886* 0Guayas/2 3.952 3.297 2.178 4.342 4.02 0

Los Ríos/1 1.794 2.269 1.895 2.289 1.951 2.436 0El Oro/1 1.005* 2.435 2.278 0.883* 0.350* 3.792 1.857 0Guayas/1 2.712 1.538 2.543 3.216 3.613 3.529 2.589 3.469 0

B Proximity Matrix Euclidean Distance

* no significant differences


64

the inhibitory properties of the OT. This implies that some OT components that were not measured might contribute to the inhibition of the fungus. Since autoclaved sterilized OT maintained their inhibitory effect, one or several heat stable inhibitory components are implied. This is important because it increases the potential for use of this type of products.

3.3. Effects of OT on BLSD under greenhouse and field conditions


Fungal isolation M. fijiensis Morelet conidia were obtained under laboratory conditions from

single ascospores discharged from banana leaves infected with BLSD following the protocols mentioned in Chapter 2, section 2.2.

Organic Teas (OT) The experiments in the greenhouse and field were executed using the OT

produced in the farms (Chapter 3, section 3.2). OT assessment on banana growth and BLSD under greenhouse conditions Greenhouse experiments were carried out with micropropagated banana

plantlets of the ‘Williams’ variety (Cavendish, Musa AAA group). Plantlets of 3cm size were planted in black 0.09L polyethylene plastic containers filled with a substrate composed of sand, rice husks and coffee residues (1:1:2). Plants of approximately 15 cm height were transplanted to individual 0.9L black plastic containers filled with the same substrate. The experiment was established as a completely randomized design with 20 plants per treatment. Two OT (from Taura and Balao), and three OT concentrations (10%, 30% and 70% v/v) and two spraying time regimes per week were tested (once or three times). Per plant 25ml OT solution were sprayed with a garden hose. Non-OT treated control plants were either fertilized (1.5g/L of urea/application) or not fertilized but received only water during the daily irrigation.

OT applications lasted 9 weeks and stopped just before inoculation with the M. fijiensis Morelet conidia solution. Leaves 2, 3 and 4 (counting down from the top of the plant), were sprayed with a concentration of 3x103 conidia/ml using an aerograph Badger® type 100. During the course of this experiment, the used OT were tested in vitro, as was their antifungal activity against M. fijiensis Morelet colony development. Disease development was recorded every two weeks, as presented in Table 2.1 (Chapter 2, section 2.1). The plant height (measured from the soil surface up to the angle between the two youngest leaves) and number of leaves was also measured.


65

OT assessment on banana growth and BLSD under field conditions The field trial was conducted during 2005-2006 at Taura in an organic banana

plantation which was naturally infected with BLSD and established on a clay loam soil (Inceptisol) with a very gentle slope (<1%). It was located in the province of Guayas (2º15`36.4``S, 79º42`32.6``W) at 10.3m above sea level. Annual rainfall was 830 mm and concentrated from November until May. In a plot of 30 x 30m containing 135 plants, thirty banana plants (variety Williams) of around 1-1.50m height were evaluated during the experimental time. The experiment was conducted in a randomized block design with 4 treatments and 3 replications and 30 observations. The treatments were: (i) weekly foliar spraying of OT prepared for foliar application (10L ha-1); (ii) weekly soil spraying of OT prepared for root application at 100L ha-1; (iii) a combination of the previous two treatments; (iv) no OT applications (control). Applications lasted for 8 months and were made with a manual pump CP3 at 20 psi. Two disease parameters were recorded. The first was to obtain a global view of the status of BLSD by assessing the disease severity using Gauhl’s modification of Stover’s severity scoring system (Carlier et al., 2000a; Vawdrey and Grice, 2005). The whole plant was evaluated according to the total leaf area affected by BLSD. A disease severity index was calculated for each plant in each replicate according to the formula:

(Σnb) x 100

Severity index = (N-1)T

Where: n = number of leaves in each grade; b = grade; N = number of grades

used in the scale (Table 2.2); T = total number of leaves scored The proportion of the leaf area covered by disease symptoms is displayed in

the Table 2.2 (Chapter 2, section 2.1). The second system was based on weekly observations of disease symptoms of

the youngest leaves of the plant. The developmental stages of Sigatoka diseases was recorded and quantified and the most advanced stage was established by a coefficient according to the following scale:

Stage Grade Coefficient1 - 20

+ 402 - 60

+ 803 - 100

+ 1204 - 140

+ 1605 - 180

+ 2006 - 220

+ 240


66

The grade of the dominant symptom was determined as a minus (-) when up to 50 necrotic lesions per leaf are observed and a plus (+) when there are more than 50 lesions. Moreover, this variable was calculated using the total symptoms in the leaves and the foliar emission period (Corrales and Marin, 1992; Fouré and Moureau, 1992; Gomez and Castaño, 2001; Marin et al., 2003). Plant height, circumference at 1 m, and number of leaves were measured as well.

Statistical analyses Quantitative variables such as area under a curve (annex 2) for the disease and

plant parameters were analyzed by univariate descriptive statistics applied for the estimation of central tendency and dispersal parameters. The disease infection in plants from greenhouse experiments was analyzed by a non-parametric Kruskal-Wallis and Mann-Whitney test. Data were analysed by running SPSS version 11 and MINITAB 13 for Windows.

3.3.2. Results 3.3.2.1. OT assessment on banana growth and BLSD under greenhouse conditions

The OT used in the greenhouse experiments consistently inhibited colony growth in vitro. Under greenhouse conditions, and in the absence of OT, BLSD developed very quickly, with faster symptom development in older leaves (Figure 3.8). In general, the different OT concentrations had the same effect. The Balao OT significantly inhibited BLSD symptom development with one and three weekly applications relative to the controls (not fertilized and fertilizer).

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).


68

In contrast, the Taura OT had no effect when sprayed weekly (P=0.269) while it became significant (P=0.044) when applied three times per week. However, on a per leaf basis, the Balao OT inhibitory effect was clearer both at weekly and three-times weekly application in leaves 3 and 4 but absent in the younger leaf two (Table 3.7).

Table 3.7 Differences in BLSD symptoms (p-value by Mann-Whitney test) at 15 days intervals in three leaves of banana plantlets grown under greenhouse conditions (n = 20). The plantlets received organic teas (OT) from two origins (Taura and Balao). OT was applied once and three times per week and OT untreated plants were fertilized or not fertilized (control). OT was applied during 9 weeks before inoculation with M. fijiensis Morelet conidia (3x103conidia/ml).

2 3 4 2 3 4

30 0.47 0.44 0.57 0.81 0.68 0.5945 0.74 0.52 0.78 0.72 0.00* 0.00*60 0.51 0.70 1.00 0.04 0.02 0.00*

30 0.15 0.33 0.59 0.29 0.00* 0.00*45 0.19 0.71 0.06 0.27 0.00* 0.00*60 0.12 0.05 0.40 0.41 0.15 0.04

30 0.01 0.00* 0.00* 0.22 0.497 0.8145 0.01 0.12 0.85 0.88 0.00* 0.00*60 0.05 0.32 0.64 0.96 0.00* 0.00*

30 0.41 0.00* 0.00* 0.04 0.00* 0.0145 0.13 0.02 0.05 0.25 0.00* 0.0160 0.19 0.32 0.21 0.41 0.04 0.04


OT vs. fertilized

Leaf number

OT vs. fertilized

Three applications/weekly

OT vs. not fertilized

OT vs. not fertilized

One application/weekly

OT originTaura Balao

Days after inoculation


69

Fertilizer significantly stimulated plant growth (height) and plant development (leaf number) (Table 3.8). Balao OT applied at 3 applications per week resulted in even higher plants while one weekly application had no effect (Table 3.8 and Figure 3.9). In general, the Balao OT was superior over the Taura OT for both parameters and for both frequencies of application (Table 3.9). In addition, the Taura OT at a high concentration (70%) caused irreversible damage to all plantlets when it was applied three times weekly but was less toxic when it was applied once per week (Figure 3.10).

Table 3.8 Area under the curve (AUC) of plant height and number of leaves in greenhouse grown banana plants measured at weekly intervals for 9 weeks. Plants received organic teas from Balao at two frequencies and OT untreated plants were fertilized or not fertilized (control) (n = 20). Means in a column with the same letter are not significantly different by Duncan’s test at P≤0.05.

Treatments Plant height Number ofleaves

1 weekly OT application 387.80 b 379.12 b3 weekly OT application 564.26 a 420.44 aFertilizer 538.85 a 425.78 aNot Fertilized 315.35 b 341.84 b

AUC

Table 3.9 Area under the curve (AUC) of plant height and number of leaves in greenhouse grown banana plants receiving organic teas (OT) from Balao and Taura at two frequencies. Treatments were performed weekly for 9 weeks, (n = 20). Means in a column with the same letter are not significantly different by Duncan’s test at P≤0.05.

Treatments

1 weekly OT applicationTaura OT 299.03 b 289.80 bBalao OT 387.80 a 379.12 a

3 weekly OT applicationTaura OT 391.45 b 258.63 bBalao OT 564.26 a 420.44 a

AUC

Plant height Number ofleaves

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).

Figure 3.10 Damage caused on micropropagated banana plants (Williams variety, Cavendish group) by applications of OT from Taura at 70% v/v (red encircled). 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 correspond to the other treatments with Balao OT that did not cause toxic effects at any concentration and application time. Green groups of plants also correspond to the lower concentrations of OT from Taura (10, 30%) at both application times.


72

3.3.2.2. OT assessment on banana growth and BLSD under field conditions BLSD severity in mother plants grown under field conditions was only significantly

reduced by OT applied to the roots while foliar OT reduced BLSD insignificantly. However in the ratoon crop both the root and foliar OT significantly reduced BLSD (Table 3.10). The disease development in the youngest leaves (3 and 4) was significantly reduced by the root applied OT in the mother plant (Table 3.10) while all three OT application systems reduced the disease development in the ratoon (Table 3.10, Figure 3.11). Moreover, when the reduction of disease severity between mother and ratoon plants was analysed (paired T-test) significant differences were found for foliar, foliar/root and control treatments with a p-value of 0.0121, 0.0127 and 0.022 respectively.

Table 3.10 Area under the curve for BLSD severity index for the entire leaf canopy (A) and disease development in the youngest leaves 3 plus 4 (B) for banana plants growing under field conditions. The plants were treated weekly with organic teas (OT) prepared for foliar, root and foliar/root application or untreated (control). Data were collected weekly on non flowering mother plants for 23 weeks until flowering, and for 8 weeks on ratoon plants (n = 30). Means in the same column for the same variable with the same letter are not significantly different by Duncan’s test at P≤0.05.

AFoliar 78.07 ab 35.21 abRoot 58.73 a 32.51 a

Foliar/Root 105.01 b 53.36 bControl

(no product) 99.99 b 96.35 c

BFoliar 2820 ab 638 aRoot 2048 a 672 a

Foliar/Root 3536 b 839 aControl

(no product) 8097 c 1440 b

RatoonMotherTreatments Cycle

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).


74

In the mother crop, all three OT stimulated plant growth (height) but this was only significant for all three OT in the ratoon (Table 3.11). Root OT and foliar/root OT gave also the biggest plants in the ratoon. There was no effect on the number of leaves neither in the mother plants nor ratoon (Table 3.11).

Table 3.11 Area under the curve (AUC) for height, circumference at 1 m height and number of leaves of field grown banana plants in Taura treated weekly with organic teas (OT) prepared for foliar, root and foliar/root application versus control without OT. Data were collected weekly on vegetatively growing mother plants for 23 weeks until flowering, and for 8 weeks on ratoon plants (n = 30). Means, in the same column per cycle with the same letter are not significantly different by Duncan’s at P≤0.05.

Treatments

Mother

Foliar 19641.77 a 4088.64 a 2435.94 a

Root 19182.10 ab 3923.15 b 2431.73 a

Foliar/Root 19620.13 a 4064.73 a 2414.76 a

Control (no product)

18722.55 b 3795.87 b 2405.84 a

Ratoon

Foliar 3944.38 b 834.52 ab 628.25 a

Root 3975.65 b 866.02 a 633.15 a

Foliar/Root 4132.68 a 868.23 a 624.05 a

Control (no product)

3794.23 c 801.15 b 618.80 a

Circumferenceat 1 m height

Number ofleaves

AUC

Plant height

3.3.3. Discussion

All applied OT inhibited M. fijiensis Morelet growth in vitro. Yet, in the greenhouse BLSD development could not be stopped when the same OT was applied to the banana plants. However, there was a striking difference between the in vitro and greenhouse experiments. In the in vitro experiments OT was all the time in contact with the fungus, while in the greenhouse, banana plants stopped receiving OT (after 8 weeks OT treatment) just before fungal inoculation. Hence it seems imperative that the OT need to be applied continuously during the infection of M. fijiensis Morelet, for the OT to become effective.

This is supported by field research where regularly treated OT becomes more effective (with time) in the ratoon crop than in the mother crop. Therefore research needs to be conducted to find out whether good control of BLSD in the field with the OT is the result of a direct effect of the OT on the pathogen or whether it is due to the fact that leaves treated with


75

OT are protected by an OT layer reducing the fungus-leaf contact. Also we cannot exclude whether our OT supported microbiological life on the banana leaf or contained certain microorganisms with possible effects against M. fijiensis Morelet. Finally we can also presume that a nutritional effect is involved as OT are rich in nutrients (Bess, 2000) and banana plants grew better with OT applications, especially after root or foliar/root applications but also after foliar applications.

The OT preparation process needs to be studied and adjusted because the nutrient variability is simply too high. Sometimes the concentration of some nutrients might then even lead to toxicity effects as we experienced when treating young plants.

3.4. Conclusions

The results of this chapter demonstrated that there is great potential to control M. fijiensis Morelet, the causal agent of BLSD with home-made OT. However, since the inhibitory action is location- time- and concentration-specific, there is a need to identify the exact effect of the OT on the fungus and the plant. Only an in-depth understanding of the mode of action of the OT can lead to the standardization of the OT preparation, which is now very variable, cumbersome and time-consuming.

OT with a constant inhibitory effect on M. fijiensis Morelet will lay the foundation for the cultivation of organically grown bananas on a larger scale. Its use in banana conventional systems could also reduce the number of fungicide applications; reduce fungal resistance problems and consequently some environmental problems too. Therefore, OT has the potential to become part of an integrated disease management system and could make the banana industry more sustainable especially for smallholders.

The chemical characterization of OT showed that these products contain considerable concentrations of nutrients that banana demand for good growth. According to literature, some of these nutrients seem to have the potential to control disease development. Hence, in what will follow, we address the effects of some micronutrients on the fungal pathogen, the plant host and the BLSD.

CHAPTER 4: Micronutrients

77

Chapter 4 IMPACT OF pH ON M. FIJIENSIS MORELET AND INFLUENCE OF SOME MICRONUTRIENTS ON THE PATHOGEN, THE DISEASE AND THE HOST

4.1. Introduction

The pH of a tissue is recognized to directly influence fungus development. This factor was shown to act as a key signal for growth, differentiation and virulence in plant fungal pathogens (Caddick et al., 1986). Indeed, it affects the activity of several enzymes that are essential for survival and food assimilation. Several microorganisms are known to survive over large pH ranges. They secrete different sets of enzymes at different ambient pH values. Botrytis cinerea is such a plant pathogenic fungus that secretes large quantities of toxins and host-degrading enzymes and it acts at a pH ranging from 2 to 7 (Manteau et al., 2003). For example, at a pH above 5 the virulence of Botrytis is enhanced because this microorganism secretes oxalic acid that increases its virulence (Manteau et al., 2003). Gene expression was first shown to be pH-dependent in Aspergillus nidulans (Caddick et al., 1986). This microorganism can secrete acid phosphatases under acidic conditions and alkaline phosphatases in an alkaline environment. It can survive in the very broad pH range of 2.5 to 9.0, and expresses either of two sets of genes according to the ambient pH (Denison, 2000). Many other fungi change their gene expression according to the extracellular pH (Penalvan and Arst, 2002). Oxysporus sp., Schizophyllum commune and Ganoderma sp. fungi are sensitive to pH changes of less than 0.5 units (Haddadin et al., 2002).

On the other hand, the role of nutrients, used in crop fertilization, has been well investigated in terms of their effects on susceptibility, tolerance, and resistance of plants against pathogens (Huber 1990). However, their direct effect on the pathogen has been inadequately studied. For example, B in combination with a fungicide increased a plant’s tolerance to Botrytis spp., Cercospora spp., or Fusarium spp., as it increased the biosynthesis and oxidation of phenolic compounds relating to changes in the cell wall lignifications (Rodrigues et al., 2004; Ruiz et al., 1998). Bordeaux mixture, one of the first Cu fungicides, applied on banana leaves, reduced leaf spotting by 86% (Klein, 1961; St Leger et al., 1998; Russell, 2003). Reuveni et al. (1997) demonstrated a level of protection against powdery mildew induced by B, Mn, and Cu, applied to the upper surface of the first leaf of cucumber plants. Mn deficient wheat plants became infected by Gaeumannomyces graminis. Indeed, Mn nutrition influences the host-pathogen balance (Graham and Rovira, 1984). Four wheat genotypes (Triticum aestivum L.) were tested in soils that received different doses of Zn and Mn. Wheat genotypes that were more efficient in micronutrient uptake were also less affected by G. graminis (Reigart and Roberts, 1999). These results suggest that bacterial microflora may play a role in the expression of Mn and Zn efficiency and tolerance to take-all in some wheat genotypes (Penalvan and Arst, 2002). Foliar applications of B, Mn and Zn on tan spot disease in winter durum wheat, caused by Drechslera tritici-repentis, reduced significantly the number of lesions per leaf (Simoglou et al., 2006). Experiments with Zn (10 mM) have been reported to inhibit the yeast to mycelium transition in Sporothrix schenckii (Rengel et al., 1996). Other in vitro studies with Fusarium oxysporum f. sp. radicis-lycopersici, showed that Zn-EDTA at concentrations below 10 µg/ml eliminated the production of fusaric acid, a pathogenicity factor, and reduced total biomass. The same authors reported similar results with Cu-EDTA on the same pathogen (Duffy and Défago, 1997). Experiments under controlled conditions demonstrated that Zn concentrations in shoots of Medicago truncatula


78

were inversely related to the severity of Rhizoctonia solani in the root (Streeter, 2001). The development of Panama disease on banana caused by Fusarium oxysporum f. cubensis, is supposedly related to nutritional imbalances, especially Zn, Ca and Mg (Borges, 1983).

Fungal pathogens induce changes in the photosynthetic process of a host plant (Chou et al., 2000; Meyer et al., 2001; Soukupova et al., 2003; Chaerle et al., 2004). Photosynthesis becomes severely impaired both in symptomatic and asymptomatic leaf areas after fungal infection (Scholes and Rolfe, 1996; Chou et al., 2000; Lohaus et al., 2000). As such, lesions caused by fungal rust could be detected by an increased chlorophyll fluorescent emission in Phaseolus (Scholes, 1992; Peterson and Aylor, 1995). Similarly for wheat infected by Cochliobolus sativus chlorophyll measurements were useful (Rosyara et al., 2007). Chlorophyll contents were determined before and after inoculation with Fusarium subglutinans in mango and the results showed no changes after infection (Donald et al., 2002). A poor correlation between leaf chlorophyll content and the reflectance indices was also found after Mycosphaerella leaf infection in commercial plantations of young Eucalyptus (Pietrzykowski et al., 2006).

This chapter presents data on the effect of pH and micronutrients such as B, Cu, Mn, and Zn under in vitro conditions on M. fijiensis Morelet and under greenhouse conditions on BLSD and the banana plant.

4.2. Materials and methods

Fungal material: M. fijiensis Morelet ascospores, colony and mycelium M. fijiensis Morelet ascospores were acquired directly from banana leaves infected by

BLSD (stage 6) in the field. Plants were sampled in the Guayas province (southern coast of Ecuador) in an organic farm (Taura). This was done on dry days to avoid ascospore release during transport. Samples were transported in paper bags to prevent drying and to keep ascospores on the leaves. The isolation of ascospores and production of colonies and conidia were according to the protocols mentioned in Chapter 2, section 2.2.

The fungus mycelium was produced using PD-V8 liquid medium (200ml of vegetable juice -V8®-, 16g Difco®-agar, 1.5g CaCO3 -Mallinckrodt®- and 800ml distilled water) and inoculated with M. fijiensis Morelet conidia solution at 3x103 conidia/ml. Mycelium growth took place in Erlenmeyers on a rotatory shaker (140rpm) at room temperature (26oC ± 2) under light conditions.

Micronutrients: B, Cu, Mn, and Zn B (5%), Cu (4%), Mn (5.6%) and Zn (6.8%) were obtained from liquid amino acid

chelate (Metalosate®). pH influence on M. fijiensis Morelet structures The effect of the medium pH was assessed under in vitro conditions against M.

fijiensis Morelet ascospore germination, colony and mycelium growth. Three experiments were carried out using a completely randomized design. In the case of the colony 25 observations per treatment were evaluated; the mycelium growth was measured in eight observations per treatment and fifty single ascospores were chosen randomly and their germinative tubes measured. The pH was measured with a Thermo Orion® 210 and depending on the treatment, regulated either with citric acid (1M) or sodium hydroxide (1M)


79

to obtain a 4-12 pH range. Colony and mycelium growth was determined by measuring colony diameter after 7 and 15 days and mycelium weight 15 days after inoculation. Experiments were inoculated with a M. fijiensis Morelet conidia solution with the above mentioned concentrations. Colony growth was at 26oC under dark conditions while mycelium growth was on a rotatory shaker (140rpm) at room temperature (26oC ± 2) under light conditions.

Effects of micronutrients: B, Cu, Mn and Zn on M. fijiensis Morelet under in vitro

conditions The 4 selected plant micronutrients were checked for their effect on M. fijiensis

Morelet at the following concentrations: 50, 500, 1200, 2500, and 5000 mg/kg. The same protocols for media preparation, fungus inoculation, incubation and evaluation time were used as aforementioned. Nutrients were added by filtering through a micropore filter (0.22µm, Millex) and the pH value was measured using pH indicator strips Whatman® with a color scale between 4.5 -10. Subsequently the fungus was inoculated. The following components of fungal development were evaluated: ascospore germination, colony development and mycelium growth. All experiments were performed in a complete randomized design. In the case of the colony 25 observations per treatment were evaluated; the mycelium growth was measured in eight observations per treatment and fifty single ascospores were chosen randomly and their germinative tubes measured.Inhibition was calculated with the formula: % inhibition = (mean fungal measurement of control treatment – fungal measurement of each OT treatment) x 100/ mean fungal measurement of control treatment. The effective dosage 50% (ED50) was estimated by a linear regression model between the logarithm of the five concentrations and its inhibition percentage caused on the pathogen. Twenty four different experiments were assessed in a complete randomized design to evaluate the effect of each nutrient element on each fungal structure.

Effects of micronutrients: B, Cu, Mn and Zn under greenhouse conditions For the greenhouse experiments micropropagated plantlets were used. The

micronutrients selected were assessed at three concentrations, classified as low, medium and high: B (0.75, 1.25, 1.75 ml/L); Cu (0.5, 1, 1.5 ml/L); Mn (0.5, 1, 1.5 ml/L); Zn (1.5, 2, 2.5ml/L) sprayed on the leaves. In the first experiment, the plants received the treatments once per week during 9 weeks. The second experiment was performed with 1 application every 7 and 15 days during 9 weeks. The applications of micronutrients were stopped once the fungus was inoculated. Plants with N fertilization (1.5g/L of urea/application) and only water irrigation were included as controls in each experiment. In both experiments, 20 banana plantlets per treatment were transplanted in black polyethylene plastic containers of 0.9L filled with a substrate composed of sand, rice husks and peat moss (1:1:2). Irrigation was manual. Plant parameters were measured on a weekly basis and the disease development, after pathogen inoculation, was evaluated every 15 days.

Both experiments were inoculated with a M. fijiensis Morelet conidia solution on leaves 2, 3 and 4 (counting down from the top of the plant). The inoculation was sprayed with an aerograph (Badger® 100). The disease development was measured according to Alvarado et al. (2003), which is a modification of the scale presented by Fullerton and Olsen (1995) (Table 2.1, chapter 2, section 2.1). Plant growth and its response were measured by total leaf number, plant height, chlorophyll content, leaf thickness and plant biomass weight. The chlorophyll content was measured by a chlorophyll meter (Konica-Minolta®, SPAD 502). This apparatus measured in a fast and non-destructive way the differences between light


80

absorption at 650nm and 940nm in SPAD (Soil Plant Analysis Division) units. The leaf thickness was measured by a digital micrometer (Mitutoyo® Micron, HM 430).

Statistical analysis Descriptive statistics were applied for the estimation of central tendency and dispersal

parameters. Analysis of Variance (ANOVA) and the Central Limit Theorem were applied to analyze the percentage of inhibition of the ascospore germination tubes. The fungal colony inhibition was analyzed by a T-test. The analysis of mycelium weight was made by the non-parametric Kruskal-Wallis and Mann-Whitney test. With the data about the development of the plant over a time interval, the area under the curve value was calculated (annex 2). The homogeneous subgroups for the greenhouse data were obtained at 5% significance, using the Duncan, Tukey and T-test. The latter was applied when the data number was small and followed a normal distribution pattern. All data were analysed by running SPSS version 11 and MINITAB 13 for Windows.

4.3. Results 4.3.1. pH influence on M. fijiensis Morelet growth

Maximum colony growth was attained at pH 7 (Figure 4.1). Colony growth was lower at an acid pH (5 and 6) and an alkaline pH (8 and 9). A further increase/decrease of pH in the medium caused a further reduction in colony growth. These differences were significant (α=0.005) at 15 days against 7 days (Figure 4.1). The same pattern was observed for mycelium weight (Figure 4.2). Meanwhile, ascospore germination was significantly inhibited at pH 6 (P=0.000). Growth recovered once the pH became neutral again.

0.0

1.5

3.0

4.5

4 5 6 7 8 9 10 11 12

pH

M. fijiensis Morelet colony diameter

(mm)

Seven days

Fifteen days

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).


81

Weight of M. fijiensis Morelet mycelium (mg)

-0.20.30.8

1.31.82.3

2.84

5

6

7

89

10

11

12

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).

4.3.2. Effect of the micronutrients B, Cu, Mn and Zn on M. fijiensis Morelet under in vitro conditions

All micronutrients changed the pH of the medium with increasing concentrations (Table 4.1). B increased the pH and Cu, Mn and Zn reduced the pH with increasing concentrations. Cu had the most pronounced effects on pH. With increasing concentrations of Cu the pH changed from 5 to 2.5. M. fijiensis Morelet colony growth was likewise affected by increasing concentrations of micronutrients. Cu and Zn gave complete inhibition of colony growth at 500mg/kg after 7 and 15 days. However Mn and B gave complete inhibition at 1200 mg/kg after 7 days but their effects decreased after 15 days, indicating a temporary effect on the pathogen (Table 4.2).

Table 4.1 pH value of the culture medium (solid and liquid) after enrichment with different concentrations of a micronutrient. The pH value was measured before fungal inoculation.

50 500 1200 2500 5000

B 7 7 8 8 8.5Cu 5 4 3.5 3 2.5Mn 5 4.5 4 4 4Zn 6 6 5 4.5 4.5

Concentration (mg/kg)Nutrient

pH


82

Table 4.2 Influence of different micronutrients and their concentration on M. fijiensis Morelet colony growth (% of inhibition) on a solid in vitro medium at two intervals. The experiments were carried out at least two times per micronutrient (n = 25). For inhibition percentages, colony diameter of treatments was compared with the control.

Nutrient50 500 1200 2500 5000

B 2.4 c 28.9 b 100 a 100 a 100 aCu 4.6 b 100 a 100 a 100 a 100 aMn 4.5 c 82.5 b 100 a 100 a 100 aZn 32.7 b 100 a 100 a 100 a 100 a

B 2.7 c 4.5 bc 11.8 b 100 a 100 aCu 1.3 b 100 a 100 a 100 a 100 aMn 5.8 d 28.9 c 61.2 b 100 a 100 aZn 8.6 b 100 a 100 a 100 a 100 a

7 days

15 days

Concentrations (mg/kg)

Inhibition of M. fijiensis Morelet colony growth (%)

The ED50 value ranged between 50 to 600 mg/kg for all nutrients studied: B = 440

mg/kg; Cu = 168 mg/kg; Mn = 574 mg/kg and Zn = 149 mg/kg. Mycelium weight and ascospore germination were significantly inhibited (α=0.05) by increasing micronutrient concentrations just like for colony growth. The lasting effect of inhibition on fungal growth was also evaluated. Mycelium growth was fastest and complete again after 7 days when Zn was removed from the medium, while Cu had the longest inhibitory after-effect (Figure 4.3).

d

c

b

a

0

20

40

60

80

100

120

B Cu Mn Zn

Nutrient

Regeneration of M. fijiensis

mycelium(%)

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<0.05.


83

4.3.3. Effect of the micronutrients B, Cu, Mn and Zn on M. fijiensis Morelet under greenhouse conditions

All micronutrients significantly increased plant height and the number of leaves in comparison to the fertilized and unfertilized control plants (Table 4.3). B/2 (medium concentration) gave the best growth and development. Fertilizer increased the chlorophyll content like all nutrient treatments with B, Mn and Zn being superior over Cu (Table 4.3). There was no clear picture emerging when considering the concentration of a micronutrient.

Table 4.3 Plant height, total leaves and chlorophyll parameters of banana plants expressed as aea under the curve (AUC) over 9 weeks. . The plants were established under greenhouse conditions (n = 20) and received different micronutrient concentrations (1=low; 2=medium and 3=high) by foliar applications on a weekly basis. No fertilizer and fertilizer plants were used as control treatments.

B/1 417.83 efg 326.73 ab 1997.45 bcdB/2 520.48 a 357.65 a 2209.13 aB/3 448.25 cdef 277.89 c 2139.79 ab

Cu/1 398.60 fg 333.86 ab 1704.20 eCu/2 404.63 fg 325.48 ab 1819.88 deCu/3 470.10 abcd 335.84 ab 1940.82 cdMn/1 490.20 abc 370.47 a 2237.58 aMn/2 434.13 defg 298.02 bc 2079.86 abcMn/3 463.73 bcde 325.90 ab 2204.39 aZn/1 513.80 ab 333.85 ab 2275.47 aZn/2 505.60 ab 337.83 ab 2284.02 aZn/3 486.05 abc 344.49 ab 2132.94 abc

Fertilizer 396.35 g 236.29 d 2208.03 aNo fertilizer 211.30 h 208.10 d 1280.76 f

Nutrient/concentration

Parameters (AUC)

Total leaves ChorophyllPlant height

Means per column of each parameter without a common letter are significantly different (P≤0.05) according to Duncan’s comparison test.

Fertilizer reduced leaf thickness but micronutrients tended to increase leaf thickness

(Table 4.4). With Cu at the medium and high concentration the leaf thickness became significantly thicker. Plant fresh weight was also increased with fertilizer and any micronutrient tested which did not differ amongst the different nutrients.


84

Table 4.4 Leaf thickness, fresh weight of banana plants and BLSD symptoms (n = 10) under greenhouse conditions. Micronutrients were applied weekly (9 weeks) at three concentrations (1=low; 2=medium and 3=high). Controls were plants receiving fertilizer and no fertilizer. After nutrient application, the plants were inoculated with a conidia solution. Data followed by the same letter per column are not significantly different (P≤0.05) according to Duncan’s test.

B/1 0.26 bcd 77.00 ab 3.8 abB/2 0.26 bcd 87.23 ab 3.6 abB/3 0.25 cd 80.70 ab 3.6 ab

Cu/1 0.28 abcd 60.02 b 3.0 abCu/2 0.31 a 66.96 ab 3.2 abCu/3 0.29 ab 74.32 ab 2.6 aMn/1 0.27 abcd 89.70 ab 4.0 abMn/2 0.28 abcd 69.61 ab 3.4 abMn/3 0.26 bcd 79.29 ab 4.2 abZn/1 0.27 bcd 91.49 ab 4.2 abZn/2 0.25 cd 77.32 ab 3.2 abZn/3 0.27 abcd 93.07 a 3.0 ab

Fertilizer 0.24 d 78.37 ab 4.8 bNo fertilizer 0.28 abc 8.17 c 3.8 ab

BLSDsymptoms

Nutrient/concentration Leaf thickness

(mm)Plant fresh weight (g)

Parameters

Regarding BLSD development, there were no differences between any micronutrient concentration and controls except that Cu treated plants (at the highest concentration) resulted in the lowest BLSD development and the fertilized controls in the highest disease development (Table 4.4).

In a second experiment micronutrients were applied on a weekly (as the first

experiment) and two-weekly interval during 9 weeks. At the end of the experiment, the interval had no effect on any plant parameter except that a 7 day interval application with Zn resulted in taller plants. The total number of leaves was not different between any micronutrient treatments at any concentration and not different from the two controls (Table 4.5). However micronutrients stimulated plant growth for the 15 day treatment in comparison to the unfertilized control. Similarly micronutrients resulted in higher fresh weight in most treatments.

Table 4.5 Parameters (n = 10) at plant harvesting, for banana plants established under greenhouse conditions, and which received different micronutrient concentrations (1=low; 2=medium and 3=high) by foliar applications at 7 and 15 days intervals. Fertilized and not fertilized plants acted as control. Data followed by the same small letter in each column are not significantly different (P≤0.05) according to Tukey’s test. Data in the same row for the same parameter and followed by the same capital letter are not significantly different (P≤0.05) according ANOVA (one-way) between both application intervals.

B/1 11.20 a A 11.13 aA 12.75 abcB 15.33 abA 44.97 bcdB 92.19 abAB/2 9.65 a A 11.32 aA 14.00 abcA 16.10 abA 67.21 abcdA 63.38 abAB/3 10.47 a A 9.88 aA 14.00 abcA 16.20 abA 44.13 bcdA 61.70 abA

Cu/1 11.00 a A 11.10 aA 16.50 abcA 13.75 abB 86.54 abcA 72.62 abACu/2 11.31 a A 11.15 aA 16.88 abcA 16.25 abA 80.24 abcA 77.94 abACu/3 11.07 a A 10.88 aA 16.83 abcA 13.50 abB 82.29 abcA 67.43 abAMn/1 7.76 a A 10.10 aA 12.20 bcB 17.00 abA 48.24 bcdA 66.50 abAMn/2 10.68 a A 9.80 aA 18.60 abA 13.63 abB 98.28 abA 46.65 bcBMn/3 10.75 a A 9.85 aA 15.63 abcB 18.67 aA 74.44 abcdB 112.79 aAZn/1 10.68 a A 11.28 aA 17.80 abA 14.30 abB 87.07 abcA 69.02 abAZn/2 10.80 a A 9.48 aA 18.88 abA 12.70 bcB 107.79 aB 46.21 bcBZn/3 11.32 a A 9.85 aA 19.20 aA 14.25 abB 93.77 abB 54.11 abB

Fertilizer 10.20 a A 10.27 aA 10.50 cB 13.17 bcA 49.56 bcdA 52.01 bcANo fertilizer 7.90 a A 7.96 aA 8.03 cA 8.10 cA 21.54 dA 22.54 cA


Total Leaves (#)

Plant height (cm)

Plant fresh weight(g)

7 15 7 15 7 15


86

Leaf thickness during plant development was not affected by fertilizer application as compared with the unfertilized treatment (Table 4.6). Most micronutrient treatments increased leaf thickness if applied at 15 days intervals. This was the case for B at the medium and high concentration, Cu, Mn and Zn at the high concentration. Fertilizer and micronutrients increased the chlorophyll content both at the 7 and 15 days interval treatments. There were no differences between nutrients and concentrations. In general BLSD was significantly reduced by each micronutrient and concentration when applied at a 7 day interval. However at 15 days interval applications, disease control was less effective (Figure 4.4).

Table 4.6 Area under the curve (AUC) of leaf thickness and chlorophyll over 9 weeks from banana plants (n = 20), established under greenhouse conditions, which received different nutrient concentrations (1=low; 2=medium and 3=high) by foliar applications at 7 and 15 days intervals. Fertilized and not fertilized plants acted as control. Data followed by the same small letter in each column are not significantly different (P≤0.05) according to Duncan’s test.


B/1 16.94 b 18.83 cde 2933.73 b 2749.65 aB/2 35.98 a 21.50 a 3109.33 b 3169.89 aB/3 23.33 ab 21.55 a 2692.47 bc 3017.19 aCu/1 18.06 b 18.21 de 3110.67 b 2725.56 aCu/2 17.86 b 19.69 bcd 3262.47 b 2928.63 aCu/3 19.03 ab 21.45 a 3156.66 b 3129.52 aMn/1 18.39 b 19.30 bcd 3311.07 b 3247.69 aMn/2 22.52 ab 19.57 bcd 3236.62 b 2934.66 aMn/3 16.99 b 20.49 abc 3020.33 b 2871.40 aZn/1 17.12 b 18.12 de 3238.65 b 3107.79 aZn/2 23.99 ab 19.33 bcd 4011.55 a 2800.10 aZn/3 19.15 ab 20.74 ab 3316.02 b 2936.29 a

Fertilizer 17.89 b 17.89 de 3100.54 b 2819.84 aNo fertilizer 17.03 b 17.03 e 2073.61 c 2074.62 b

15

Parameters (AUC)

Leaf thickness Chorophyll

7 15 7

00.20.40.60.8

11.21.41.61.8

2

low

med

ium

high

lo

w

med

ium

high

low

med

ium

high

lo

wm

ediu

m

high

F

NF

low

med

ium

high

lo

w

med

ium

high

low

med

ium

high

lo

wm

ediu

m

high

B Cu Mn Zn Controls B Cu Mn Zn

7 15Treatments

BLSD symptoms

Figure 4.4 BLSD symptoms on banana plants (n = 10) established under greenhouse conditions and grown for 9 weeks. The plants received foliar applications of different micronutrients at three concentrations at 7 and 15 days intervals. Plants were inoculated with a conidia solution. Controls are fertilized and not fertilized plants.


88

4.4. Discussion

There is a need for new fungicides or alternative methods of fungal control. This is especially relevant for banana, where large amounts of fungicides are applied continuously and fungal resistance is an increasing problem (Brent, 1995; Brent and Hollomon, 1998; Beresford et al., 1999; Ruiz et al., 1999; Bartlett et al., 2002). We proved that the micronutrients B, Cu, Mn and Zn had a direct inhibitory effect on fungal growth and development under in vitro conditions. Cu and Zn resulted in significant inhibition of M. fijiensis Morelet. Once the contact is halted between the nutrient and this pathogen, the inhibitory effect ceased, except for Cu were there was a large after-effect, confirming the usefulness of Cu containing fungicides for Sigatoka control in the past. Similarly, the pH of the in vitro media adversely affects M. fijiensis Morelet; in particular, a high or low pH has a high but incomplete inhibitory effect on colony growth. This effect is temporary because the inhibition ceased when the pH was brought back to a neutral pH. This suggests that this pathogen manifests a wide adaptability to a wide pH range, as reported for some other fungi (Caddick et al., 1986; Denison, 2000; Manteau et al., 2003).

The micronutrients and pH effects also demonstrate that regular applications are needed if inhibition of M. fijiensis Morelet needs to be maintained in the field. Under greenhouse conditions, foliar applied micronutrients at different concentrations show in general that plants grow and develop as good as the fertilized control plants. However, the BLSD was significantly reduced with any of the micronutrients if weekly applied. This reinforces the point that the micronutrients need to be in constant contact with the pathogen if we want a good disease control.

4.5. Conclusions

The present chapter provides evidence on the inhibitory effect of pH and some micronutrients against M. fijiensis Morelet under specific conditions. The micronutrients Cu, Zn, Mn and B reduce BLSD in greenhouse plants if applied weekly but not at two-weekly intervals. Similarly, in in vitro conditions the inhibitory effect against the pathogen is only working when these micronutrients are present, but once taken away only Cu has an inhibitory after-effect. This supports the further use of these elements in organic teas which are weekly applied in the organically grown banana fields (chapter 2.1) and where BLSD is well controlled. Our results also show that the regular application prevails over the concentration of the micronutrient when it comes to the inhibition of the growth of M. fijiensis Morelet. In addition, we note that the applied micronutrients stimulate plant growth to some extent. These results also explain why the autoclaving of organic teas did not show a reduced effect on BLSD (chapter 3.1). The results also show that conventionally cultivated banana fields, i.e. conventional cultivation, would benefit if they would receive less N as this nutrient stimulates the BLSD. In addition, our results suggest that aerial applications of fungicides would become more efficient against M. fijiensis Morelet, if the fungicide solution would be kept at a pH 7 and contains the tested micronutrients.

The current research however provides no hint whether any of the micronutrients tested (Cu, Zn, Mn and B) affects the plant’s response against the fungal disease. Hence research is needed to explore whether the defence mechanisms of the banana plant were improved. This has indeed been proved for Si, which can upregulate a plant’s resistance.Therefore, the effects of this nutrient are presented in the following chapter.

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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

5.1. Introduction

Si is considered to be useful for a wide range of crops which are under biotic and/or abiotic stress (Jones and Handreck, 1967; Elawad and Green, 1979; Datnoff et al., 1991, 1992; Savant et al., 1997a; Seebold et al., 2000; Korndörfer and Lepsch, 2001). Si has direct effects on Sphaerotheca fuliginea as it reduces the area of individual colonies, the germination percentage of conidia on inoculated leaves, and the number of haustoria per colony (Menzies et al., 1991a; 1991b). Si and plant growth and health has been studied to some extent, especially in Gramineae (Datnoff et al., 1991; Datnoff et al., 1992). Plants absorb Si as monosilicic or orthosilicic acid (H2SiO4) by diffusion and/or uptake driven by transpiration-induced root absorption (Elawad and Green, 1979; Epstein, 1999, 2001; Ma et al., 2002; Epstein and Bloom, 2005). Leaves, stems, and culms of rice grown in the presence of Si, showed an erect growth which improved the distribution of light within the canopy (Elawad and Green, 1979; Epstein, 1999; Ma and Takahashi, 1990; Savant et al., 1997a; Ma et al., 2002). Moreover, in cucumber leaf senescence was retarded, leaves looked greener and the canopy was larger with added Si (Adatia and Besford, 1986). The growth stimulation is due to protection that Si provides to plants against detrimental effects (Seebold et al., 2001 and 2004). In addition, Si contributes to cell mechanical properties, including rigidity and elasticity (Perry and Keeling-Tucker, 2000; Taiz and Zeiger, 2002), facilitates roots to grow through hard substrates (Epstein, 1994) and prevents banana seed coats to shrink during germination (Graven et al., 1996). Importantly, this mineral increases resistance to a range of diseases and pests (Ma et al. 2002). Extracts from horsetail (Equisetum arvense L.) containing 15% Si on a dry weight basis, have been used as protection against diseases like damping off and powdery mildew (Bélanger et al., 1995). Further, pea seedlings treated with Si and inoculated with Mycosphaerella pinoides developed fewer lesions (Dann and Muir, 2002). Moreover, the inhibitory effects of Si against Powdery mildew and Pythium infer that soluble Si activates defence mechanisms in cucumber by showing enhanced activity of chitinases, peroxidases, polyphenol oxidases and phenolic compounds (Samuels et al., 1991; Chérif et al., 1994; Fawe et al., 1998, 2001). However, these beneficial effects were lost when the Si supply was interrupted even though Si had been irreversibly accumulated (Samuels et al., 1991; Seebold et al., 2001, 2004). Si application in rice reduces leaf blast and sheath blight, thereby contributing to a yield increase (Rodrigues et al., 2001, 2003, 2004). A recent study demonstrated that the banana (Musa spp.) is a Si accumulator which is taken up actively under hydroponic conditions (Henriet et al., 2006; Henriet, 2008). Earlier it was shown that banana can induce silicate dissolution and thus increase the Si availability around roots (Hinsinger et al., 2001; Rufyikiri et al., 2004).

On the other hand, chlorophyll content and photosynthesis activity are influenced by many factors such as plant development, and biotic and abiotic stress (Mazumdar, 2007). In rice infected with the blast pathogen (Pyricularia grisea) it was demonstrated that added Si changes the chlorophyll content (Ranganathan et al., 2006). Likewise in avocado, inoculated with Phytophthora cinnamomi and receiving soluble Si, the chlorophyll content increased (Bekker et al., 2005). In tomato Si increased also the chlorophyll content (Al-Aghabary et al.,

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2005; Romero-Aranda et al., 2006). The chlorophyll content can also be a tool to direct the right amount of fertilizer which in wheat stands for a SPAD units (Soil Plant Analysis Division) between 35 and 40 (Vidal et al., 1999). In banana however it is not known whether the chlorophyll content can be linked to a fungal disease and Si treatments.

Here we investigate the contribution of Si to plant health and growth in banana. This chapter comprises two parts: in a first part we measure the effects of Si on M. fijiensis Morelet and on banana plants under in vitro conditions and in a second part we investigate the response of the plant to Si applied via the soil or the leaf under greenhouse and field conditions.

5.2. Effects of Si on M. fijiensis Morelet and the banana plant under in vitro conditions


M. fijiensis Morelet fungal material Isolation and obtention of fungal structures was according to the protocols mentioned

in Chapter 2, section 2.2. Plant material Micropropagated banana plantlets of the variety Williams (AAA, Cavendish group)

were used in the in vitro experiments. Plants were transplanted in phase 1 with approximately 6cm height and plants transplanted in phase 2 with around 15cm height.

Si sources For the fungal bioassays, two water-soluble sources were used: (i) Potassium Silicate

(PS) (PC Corp.) with 20.8% SiO2 and 8.3% K2O, pH 11; (ii) Monosilicic Acid (MA) (TerraTech Corp®) with 22% Si(OH)4, pH 14. For the effect on micropropagated banana plants, PS was used. Si, henceforth called Standard Silicon, was also used as a pure reagent (0.1% Si, Atomic Spectroscopy Standard, PerkinElmer®). Potassium Hydroxide (KOH) (85% KOH, Merck®) was used to evaluate the effect of K application.

Si effects on M. fijiensis Morelet Si was tested for its direct effect on M. fijiensis Morelet at the following

concentrations: 50, 500, 1200, 2500, and 5000 mg/kg. This nutrient was added via a micropore filter (0.22µm, Millex) and the pH value was measured using pH indicator strips of Merck®. Subsequently the fungus was inoculated. The fungal development was monitored as follows: ascospore germination, colony and mycelium growth. The same protocols for media preparation, fungus inoculation, incubation and evaluation time per fungus structure were used at similar experiments (chapter 4, section 4.2). All experiments were performed in a complete randomized design. For the colony, 25 observations per treatment were evaluated; the mycelium growth was established from eight observations per treatment and fifty single ascospores were chosen randomly and their germinative tubes measured. The percentage of inhibition of the different M. fijiensis Morelet structures was determined using the

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measurement of the concerned parameter (colony diameter, mycelium weight and length of germinative tube of ascospore) from treatments with nutrient concentrations and treatments without nutrient concentration. Inhibition percentage was calculated as follows: % inhibition = (mean fungal measurement of control treatment – fungal measurement of each OT treatment) x 100/ mean fungal measurement of control treatment. The effective dose for 50% inhibition (ED50) was estimated by a linear regression model between the five concentrations and the inhibition percentages caused on the pathogen.

Si effect on banana plantlets under in vitro conditions The layout of two experiments was a completely randomized design, using 20 plants

per treatment. Murashige and Skoog (MS) medium at pH 6.12-6.15 was the basis for the regeneration of the plants. The experiments started with a corm bearing a meristem, after the entire root system, pseudostem and leaves were removed from an in vitro plant. In a first experiment two factors were investigated: Si concentrations (0, 5, 50, 500, 5000 mg/kg supplied by PS) and pH of the culture medium, adjusted and unadjusted. Indeed, when PS was supplied to the medium, the pH increased. With citric acid (1M) the pH of the medium was brought back to 6.12-6.15 and called adjusted medium. If this was not done, we speak of an unadjusted medium. The plantlets were placed in a growth chamber under natural light conditions (±2200 Lux) and a temperature of 28°C for 8 weeks. Every week the plant height, number of leaves and number of roots were measured.

In the second experiment, six Si concentrations applied as PS were evaluated: 0, 50, 500, 1200, 2500, 5000 mg/kg and again the pH was adjusted and unadjusted following the procedure mentioned above. In this experiment, the same concentrations and the same pH conditions of Si and K as with PS, were applied with Standard Si (Si) and potassium hydroxide (KOH) respectively. For both experiments, once solutions were prepared, they were autoclaved (Market Forge®) for 20min at 121ºC and 15psi, and then immediately used for culturing the plantlets. The plantlets were kept in the same conditions as for the first experiment for 10 weeks and every fifteen days, plant height, number of leaves and number and lenght of roots were measured.

Statistical analysis The data distribution was analyzed by Kolmogorov-Smirnov (K-S). Number of

observations less than 30 was tested by Kruskal-Wallis and Mann-Whitney tests. Analysis of Variance (ANOVA) and the Central Limit Theorem were applied to analyze the percentage of inhibition of the ascospore germinating tubes. The fungal colony inhibition was analyzed by a T-test. The analysis of mycelium weight was made by the non-parametric Kruskal-Wallis and Mann-Whitney test. With the data about the development of the plant over a time interval, the area under the curve value (AUC) was calculated (annex 2). This parameter was then analyzed for normality and following the results parametric and none parametric tests were applied. The homogeneous subgroups were obtained at 5% significance, using the Duncan and T-test. The latter was applied when the data number was small and follows a normal distribution pattern. All data were analysed by running SPSS version 11 and MINITAB 13 for Windows.

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5.2.2. Results

5.2.2.1. Si effect on M. fijiensis Morelet under in vitro conditions The pH increased with the Si concentration since with 50 mg/kg and 5000 mg/kg it

became 7.5 and 10.5 respectively. The potassium silicate (PS) and monosilicic acid (MA) also had an inhibitory effect on M. fijiensis Morelet (colonies, mycelium and ascospores). Both products followed a similar pattern at both evaluation times (Figure 5.1). Significant differences (α=0.05) were established in relation to concentrations and evaluation times (7 and 15 days) on fungal colonies, with the exception of the high concentrations (2500 and 5000 mg/kg) of MA. The mycelium weight was significantly reduced (α=0.05) by increasing concentrations of both Si sources (R2 = 0.94 for PS and R2 = 0.88 for MA). In a medium without Si but after Si treatment, the mycelium recovered partially (60%) except for the highest concentrations. The ascospore germination was also significantly affected. The ED50 values recorded for both Si sources were 100 mg/kg for PS and 40 mg/kg for MA.

Because one Si source was formulated as PS, the effect of K was evaluated. Both Si and KOH reduced ascospore germination with increasing concentrations (Table 5.1). As from 500 mg/kg the inhibitory effect of KOH was stronger than of Si. Nevertheless, only the highest concentration of KOH caused complete inhibition.

0

20

40

60

80

100

120

7Potassium Silicate

15 7Monosilicic Acid

15

Evaluation time, Si sources

M. fijiensisMoreletcolony

inhibition(%)

50 500 1200 2500 5000

Figure 5.1 Effect of Si sources and different concentrations on M. fijiensis Morelet colony growth after 7 and 15 days of cultivation on a solid in vitro medium (n = 25).

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Table 5.1 Inhibition of M. fijiensis Morelet ascospore germination (%) on a solid in vitro medium with different concentrations of Si and K 48 hours after discharge (n = 50). Different small letters in the same row indicate significant differences at P<0.05 by Duncan’s test. Data followed by the same capital letter in each column are not significantly different (P≤0.05) according to Duncan’s test between both products.

Treatments 50 500 1200 2500 5000

Silicon (Si) 20.9 aA 28.5 bB 53.1 cB 63.3 dA 89.7 eB

Potassium hydroxide

(KOH)29.5 aA 48.5 bA 64.2 cA 71 cA 100 dA

Concentration (mg/kg)Inhibition of M. fijiensis Morelet ascospore germination (%)

Regarding mycelium inhibition, KOH had a higher inhibitory effect and resulted in complete inhibition at 2500 mg/kg while for Si the inhibitory effect increased only up to 500 mg/kg (Figure 5.2).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 50 500 1200 2500 5000

Concentrations (mg/kg)

M. fijiensis Moreletmycelium weight

(mg)

Silicon Potassium hydroxide

Figure 5.2 M. fijiensis Morelet mycelium growth under different concentrations of silicon (Si) and potassium hydroxide (KOH) in a liquid in vitro medium (n = 8).

5.2.2.2. Si effect on banana plantlets under in vitro conditions Increasing concentrations of Si up to 500 mg/kg in the pH adjusted in vitro medium,

had no effect on banana growth and development. However at 5000 mg/kg number of roots and leaves were reduced (Table 5.2). In the unadjusted medium, banana plants at 5 and 50 mg/kg grew and developed as the control plants (without Si) but at 500 mg/kg and higher concentrations, growth and development were reduced. Hence, for correct interpretation of Si effects, one need to correct the pH, otherwise the results are confounded.

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Table 5.2 Area under the curve (AUC) over 8 weeks for plant height, number of roots and number of leaves of banana plantlets (n = 16) growing on MS solid medium (pH-adjusted and pH-unadjusted medium) amended with different Si concentrations from PS.

Treatments

MS mediumwithout Si 86.89 ab 197.00 ab 142.70 a

Si (mg/kg) Adjusted pH

5 90.55 ab 167.38 ab 135.20 ab50 116.55 a 213.65 a 158.39 a

500 112.39 a 209.63 a 149.58 a5000 90.15 ab 36.28 de 95.23 bc

Si (mg/kg) Unadjusted pH5 89.40 ab 153.38 ab 135.31 ab

50 96.72 ab 121.00 bc 127.45 ab500 52.32 c 82.04 cd 82.79 bc

5000 0.00 d 0.00 e 0.00 c

Plant height

AUCNumber of

rootsNumber of

leaves

Means per column for plant height and number of roots with the same letter are not

significantly different by Duncan’s test at P≤0.05. In a second experiment two sources of Si were evaluated as well as any possible

confounding effect from K added together with Si. With PS, any Si concentration resulted in smaller plants but leaf and root parameters were not affected if the pH was adjusted. When the pH was not adjusted, plant size was further reduced while all root and leaf parameters were reduced as from 1200 mg/kg. A similar picture emerged for the KOH treatments. With Standard Si (Si) treatment the adjusting of the pH did not change the results (Table 5.3). In general any type of Si and K application never improved plant growth and development but gave either the same results as the control or worse results. In general at the concentrations of 50 to 500 mg/kg of Si or K or in combination, root number was significantly stimulated (Table 5.3).

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Table 5.3 Area under the curve (AUC) over 10 weeks for plant height, number of leaves, number and length of roots of banana plantlets (n = 16) growing on MS solid medium (pH-adjusted and pH-unadjusted medium) amended with different Si concentrations from PS and standard Si and different K concentrations coming from KOH. Means per column of each parameter with the same letter are not significantly different by Duncan’s test at P≤0.05.

Treatments

MS medium without Si 1648.6 abcd 151.4 a 245.6 bcde 1444.2 abc

Si (mg/kg)

50 1219.5 efghji 161.3 a 314.3 abcd 1512.8 abc500 1245.0 cdefghij 168.0 a 295.5 abcd 1977.8 a1200 1002.8 ghijkl 133.5 ab 373.5 ab 1403.3 abcd2500 1146.0 fghijk 171.0 a 331.5 abc 1620.0 abc5000 1202.3 efghijk 184.5 a 330.0 abc 1617.0 abc

50 1218.8 efghji 155.3 a 339.0 abc 1585.5 abc500 1057.5 ghijkl 158.3 a 383.3 ab 1493.3 abc1200 1131.0 fghijkl 97.5 bc 125.3 ef 597.8 efghi2500 947.7 jkl 77.7 c 131.6 ef 688.6 defghi5000 0.0 m 0.0 d 0.0 f 0.0 i

Si (mg/kg)

50 1454.1 abcdefg 165.9 a 378.8 ab 1676.3 abc500 1581.6 abcde 157.5 a 312.2 abcd 1455.0 abc1200 1425.0 abcdefgh 148.1 a 315.9 abcd 1394.1 abcd2500 731.3 l 90.0 bc 175.3 de 558.8 fghi5000 795.9 lk 92.8 bc 199.7 cde 548.4 ghi

50 1391.3 bcdefghi 162.2 a 349.7 ab 1303.1 abcd500 1250.6 cdefghij 171.6 a 365.6 ab 1455.9 abc1200 1424.1 abcdefgh 185.6 a 400.3 a 1323.8 abcd2500 1231.9 defghij 160.3 a 308.4 abcd 1051.9 bcdefg5000 95.6 m 19.7 d 36.6 f 83.4 i

K (mg/kg)

50 1423.1 abcdefgh 159.4 a 432.2 a 1791.6 ab500 1515.9 abcdef 160.3 a 295.3 abcd 1272.2 abcd1200 1726.9 ab 174.4 a 430.3 a 1364.1 abcd2500 1664.1 abc 164.1 a 328.1 abc 1377.2 abcd5000 1838.4 a 155.6 a 113.4 ef 288.8 hi

50 1540.3 abcdef 156.6 a 389.1 ab 1673.4 abc500 1307.8 cdefghij 150.9 a 424.7 a 1561.9 abc1200 972.2 ijkl 82.5 c 186.6 de 950.6 cdefgh2500 0.0 m 18.8 d 0.0 f 0.0 i5000 0.0 m 0.0 d 0.0 f 0.0 i

AUC

Unadjusted pH

Adjusted pHPotassium Hydroxide (KOH)

Unadjusted pH

Adjusted pHStandard Silicon (Si)

Potassium silicate (PS)Adjusted pH

Unadjusted pH

Plant height Number of leaves Number of roots Length of roots

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5.3. Effects of Si on the banana plant and BLSD under greenhouse and field conditions

5.3.1. Materials and Methods Plant material, pathogen isolation and mineral sources Micropropagated banana plantlets of the variety Williams (AAA, Cavendish group)

were used in greenhouse and field experiments. In the greenhouse, plants with approximately six complete unfolded leaves were inoculated with a conidia solution of M. fijiensis obtained under laboratory conditions following the protocols mentioned in Chapter 2.2. Mineral sources of Si were potassium silicate (PS) with 20.8% SiO2 and 8.3% K2O, pH 11. Standard silicon (Si) (0.1% Si, Atomic Spectroscopy Standard, PerkinElmer®) and potassium hydroxide (85% KOH, Merck®) were used as a pure reagent to evaluate the effect of Si and K.

Response of banana plants to Si amendments under greenhouse conditions A first experiment used the plants of the first in vitro experiment and can thus be

considered as a continuation of that experiment. This means that after 8 weeks in vitro the plantlets were transplanted in the greenhouse and each treatment (0, 5, 50, 500, 5000 mg/kg Si from PS and pH adjusted and unadjusted) continued but was applied via the leaves. In the second experiment, the plants in the greenhouse received the same treatments as in experiment one but had no Si history during the in vitro phase. In the third experiment Si (from PS) concentrations were increased (50, 500, 1200, 2500, 5000 mg/kg) and applied via the root system and the foliage. The pH of the solution was unadjusted. In a fourth experiment Si (from PS) concentrations were 500, 1200 and 2500 mg/kg and applied via the root system. To better investigate the effect of Si and K in PS, two other sources were added, i.e. standard silicon solution (Si) and potassium hydroxide (KOH). All greenhouse experiments had plants with N fertilization (1.5g/L of urea/application) and only water irrigation included as controls. In all experiments 20 banana plantlets per treatment were planted in black polyethylene plastic containers of 0.09L filled with a substrate composed of sand, rice husks and peat moss (1:1:2). Si applications were applied on a weekly basis and each plant received 25 ml water or a Si and or K solution. Irrigation was manually. Plant growth parameters were measured on a weekly basis and disease development, after pathogen inoculation, was measured each fifteen days.

The fungus inoculation procedure was as in other similar experiments and disease development evaluation was recorded as presented in Table 2.1 (Chapter 2, section 2.1). Plant growth and response was measured by total leaf number, plant height, chlorophyll content, leaf thickness and plant biomass weight. The chlorophyll content and leaf thickness was measured as explained in chapter 4. The soil pH was measured in a soil/water ratio 1:1 as stated in the Soil Survey Laboratory Method Manual and determined by a portable pH meter (Mettler Toledo®). Chemical analysis of Si content was executed by the Western Hemisphere Analytical Laboratory using Atomic Absorption Spectrophotometry and the analytical methods were based on the Official Methods of Analysis of AOAC International.

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Si effect on banana plants under field conditions The experimental site was characterized by a silty loam soil (Inceptisol) with a gentle

slope (<1.2%). It was located at the central part of Ecuador, Los Rios province (00º59`59.8``S, 79º20`43.6``W) at 276.3 m above sea level. The field trial was conducted from September 2006 to May 2007 during which precipitation was 1168.3mm and the temperature varied between 32.5oC and 19.9oC. Si was applied as PS and at a concentration of 500, 1200 and 2500 mg/kg via the root system or the foliage at 7 and 14 days application cycles. The root application was made with a backpack sprayer bearing a modified hose to inject the solution into the root zone. Foliar applications were executed with a motor backpack sprayer carrying an electrostatic nozzle. The experiment was a completely randomized block design with three repetitions. Each plot of 30 x 30m was composed of 10 plants. Cultural practices were as in commercial plantations. Plant growth (plant height and number of leaves) parameters were collected every 15 days. The BLSD index represents the disease status of the entire leaf canopy according to Gauhl’s modification of the Stover’s scale (Gauhl, 1994), based on the percentage of the leaf covered by disease symptoms. A second phytosanitary parameter was based on disease symptoms in leaves 2-4 according to Fouré (Corrales and Marin, 1992; Fouré and Moureau, 1992). Arbitrary coefficients were assigned to calculate the disease evolution (Marin et al., 2003). Both scales are explained in Chapter 3, section 3.3.

Statistical analyses In all experiments data distribution was analyzed with the Kolmogorov-Smirnov (K-S)

test. With the data about the development of plant material over a time interval, the area under the curve value (AUC) was calculated (annexe 2). This parameter was then analyzed for normality and following the results parametric and none parametric tests were applied. The Central Limit Theorem was applied with the field data followed by analysis of variance (ANOVA) and homogeneous subgroups were carried out by post hoc test.

5.3.2. Results

5.3.2.1. The response of banana plants to Si amendments under greenhouse conditions Plantlets with an in vitro history of Si treatment, developed very well in the

greenhouse during 8 weeks whether the pH was adjusted or not. Consequently a pH adjustment of a Si solution prior to leaf treatment was not critical (Table 5.4). Up to the highest level of applied Si (and K because PS was used), treated plants grew better than the non-fertilized plants and similar to the conventionally fertilized plants. Root development of Si treated plants was the same as for the fertilized plants except at the highest concentration. However conventionally fertilized plants were significantly more BLSD susceptible than Si treated and unfertilized plants (Figure 5.3). Hence N seemed to increase susceptibility while PS could stimulate plant growth without increasing BLSD susceptibility.

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Table 5.4 Area under the curve (AUC) over 8 weeks of plant height and number of roots from banana plants (n = 20) established under greenhouse conditions which received different Si concentrations during the in vitro period. Under greenhouse conditions, banana plants received foliar applications of different Si concentrations (from PS as a source) in a pH-adjusted and pH-unadjusted solution and both no fertilizer and fertilizer plants acted as control treatments.

Treatments

No Fertilizer 426.13 c 393.87 cFertilizer 573.05 ab 431.00 ab

Si (mg/kg)5 594.74 a 437.09 a50 605.45 a 414.78 ab

500 601.42 a 415.04 ab5000 538.18 b 389.67 c

Si (mg/kg)5 604.76 a 438.38 a50 614.08 a 430.33 ab

500 599.78 a 437.85 a5000 -- --

Unadjusted pH

Adjusted pH

AUCNumber of rootsPlant height

Means per column of each parameter without a common letter are significantly

different (P≤0.05) according to the Duncan’s comparison test.

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Figure 5.3 Box plot of BLSD index on banana plants (n = 20) established under greenhouse conditions over a period of 8 weeks. These plants received weekly foliar application of different Si concentrations, ranging from 5-5000 mg/kg (from PS) with pH-adjusted (A) and pH-unadjusted (U) solution. These plants came from an in vitro assay where they had received the same Si concentrations. Reference treatments were plants receiving no fertilizer (NF) and fertilizer (F).

Effect of Si treatments on plant growth and disease development became more

pronounced when plants received foliar Si once they were planted in the greenhouse (Table 5.5). Si treated plants grew similarly with an adjusted and unadjusted pH but were better than non-fertilized plants but not always as good as fertilized plants. Leaf development of Si treated plants was superior over non-fertilized plants and similar or slightly inferior to the fertilized plants except at 5000 mg/kg when development was much lower. Again, all Si treated plants were significantly less affected by BLSD than conventionally fertilized plants, and were comparable with non-fertilized plants.

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Table 5.5 Area under the curve (AUC) over 8 weeks of plant height, number of leaves and BLSD symptoms of banana plants (n = 20) under greenhouse conditions. These plants had not received any Si during their in vitro phase. The plants received weekly foliar application of different Si concentrations (from PS) with a pH-adjusted and pH-unadjusted solution. Reference treatments were plants receiving no fertilizer and fertilizer. Means per column of each parameter without a common letter are significantly different (P≤0.05) according to the Duncan’s comparison test.

Treatments

No Fertilizer 537.22 d 512.97 cd 34.20 bcdFertilizer 1220.53 a 573.38 a 50.48 e

Si (mg/kg)5 993.16 b 535.57 bc 38.65 d50 1026.66 b 539.21 bc 38.34 d500 1038.00 b 549.33 ab 31.35 abc

5000 870.37 c 490.77 de 28.78 ab

Si (mg/kg)5 1053.69 b 545.30 ab 33.98 bcd50 1022.82 b 536.97 bc 35.86 cd500 1072.10 b 559.72 ab 36.10 cd

5000 851.76 c 470.40 e 26.96 a

Adjusted pH

Unadjusted pH

AUC

BLSD symptoms

Number of leavesPlant height

At the end of the experiment, the chlorophyll content was measured on leaves

with/without BLSD symptoms. The unfertilized plants had significantly the lowest SPAD value and BLSD increased the SPAD value (Figure 5.4). At the same time, the Si content in the leaves was analyzed. The results demonstrated that the pH of the Si solution did not affect the Si content. All Si treated plants had a significant higher level of Si than the fertilized and unfertilized plants and 50 and 500mg/kg Si treated plants had the highest Si levels (0.1 %) which were double the amount in the fertilized and unfertilized plants (Figure 5.5).

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0

10

20

30

40

50

60

70

5 50 500 5000 5 50 500 F NF

Adjusted pH Unadjusted pH Controls

Treatments

Chlorophyll content (SPAD value)

Leaf with BLSD Leaf without BLSD

Figure 5.4 Chlorophyll content (SPAD unit) of banana leaf with and without BLSD symptoms (n = 20) established under greenhouse conditions over a period of 8 weeks. Under greenhouse conditions, the plants received weekly foliar applications of different Si concentrations, ranging from 5-5000 mg/kg (from PS) with pH-adjusted and pH-unadjusted solution. Reference treatments were plants receiving no fertilizer and fertilizer.

Figure 5.5 Si content (%) in the banana leaves after 8 weeks under greenhouse conditions. In these conditions, the plants received weekly foliar applications of different Si concentrations, ranging from 5-5000 mg/kg (from PS) with pH-adjusted and pH-unadjusted solution (n = 10). Reference treatments were plants receiving no fertilizer and fertilizer.

In a third experiment executed under the same conditions as the previous experiment,

all Si treatments resulted in taller plants with more leaves and faster foliar emission than non-fertilized and sometimes even fertilized plants (Table 5.6). In general, root treated plants were

cc

abaab

bb

aab

b

0.00

0.10

0.20

5 50 500 5000 5 50 500 5000 F NF

Adjusted pH Unadjusted pH Controls

Treatments

Silicon in the leaf (%)

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better than the fertilized plants except at 5000 mg/kg (Table 5.6) and the foliar area was much larger than in the controls and the foliar applied plants, except at 5000mg/kg (Figure 5.6).

Table 5.6 Area under the curve (AUC) of plant height, number of leaves and foliar emission of banana plants (n = 20) under greenhouse conditions during 8 weeks. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. No fertilizer and fertilizer plants were evaluated as control treatments. Means per column of each parameter with the same letter are not significantly different by Duncan’s test at P≤0.05.

Treatments

No Fertilizer 763.88 e 508.52 f 30.39 eFertilizer 846.37 cd 554.68 e 45.41 d

Si (mg/kg) Foliar Root Foliar Root Foliar Root50 816.87 de 1042.81 a 566.98 de 655.55 a 55.13 bcd 66.22 a

500 881.47 bcd 1021.04 a 556.33 e 604.40 bc 54.71 cd 62.30 abc1200 891.71 bc 1059.82 a 561.33 e 625.78 b 54.37 cd 60.86 abc2500 879.58 bcd 996.90 a 572.20 de 618.50 bc 57.75 abc 56.68 abc5000 915.80 b 840.08 cd 564.60 de 593.95 cd 51.98 cd 65.19 ab

AUCPlant height Number of leaves Foliar emission

Figure 5.6 Foliar area (cm) of banana plants (n = 10) harvested after 8 weeks at greenhouse conditions. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. No fertilizer and fertilizer plants were evaluated as control treatments.

0

50

100

150

200

250

No

ferti

lizer

Ferti

lizer 50 500

1200

2500

5000 50 500

1200

2500

5000

Foliar applic. Root applic.

Treatments

Foliar area (cm)

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103

Again conventional fertilization increased BLSD symptoms while Si reduced it. Si treated via the root system seemed to be much more effective in controlling BLSD than via the leaves (Table 5.7).

Table 5.7 Area under the curve (AUC) of BLSD on banana plants (n = 20) established under greenhouse conditions. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. Reference treatments were plants receiving no fertilizer and fertilizer.

TreatmentsNo Fertilizer 16.44 bc

Fertilizer 34.31 a

Si (mg/kg)50 12.92 bc 17.33 bc500 17.90 bc 8.00 cd

1200 16.06 bc 4.84 d2500 21.51 b 10.40 cd5000 22.29 b 2.91 d

BLSD

Foliar Root

Data followed by the same letter in each column are not significantly different

(P≤0.05) according to Duncan’s test. Before pathogen inoculation, half of the plants were harvested and the fresh and dry

weight recorded of the plant with leaves, roots and corm. The Si treatments resulted in significantly (heavier) plants in comparison to the non-fertilized treatment, irrespective how it was applied (foliar or root). In contrast and in comparison with the fertilized treatment, again the root applied Si had the most pronounced (i.e. higher) effect on plant growth, except for the highest concentration (Table 5.8).

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Table 5.8 p-value (T-test) of differences in fresh (A) and dry (B) weight of the entire plant and some of its components (n = 10) which grew under greenhouse conditions during 8 weeks. The plants received weekly foliar and root applications of Si (from PS) at different concentrations. Reference treatments were plants receiving no fertilizer and fertilizer. Data values equal or less than 0.05% are significantly different.

Si (mg/kg) Foliar Root Foliar Root Foliar Root50 0.001 0.000 0.005 0.000 0.002 0.000

500 0.002 0.000 0.001 0.000 0.001 0.0001200 0.001 0.000 0.000 0.000 0.000 0.0002500 0.000 0.000 0.000 0.002 0.006 0.0005000 0.006 0.000 0.003 0.001 0.017 0.000


500 0.173 0.000 0.040 0.002 0.157 0.0011200 0.021 0.005 0.021 0.006 0.049 0.0002500 0.133 0.012 0.099 0.280 0.445 0.0035000 0.628 0.127 0.337 0.846 0.588 0.115


500 0.002 0.000 0.010 0.005 0.002 0.0021200 0.002 0.001 0.027 0.003 0.014 0.0022500 0.008 0.000 0.002 0.001 0.021 0.0015000 0.020 0.000 0.002 0.004 0.043 0.004


500 0.986 0.002 0.043 0.014 0.030 0.0041200 0.298 0.003 0.058 0.009 0.056 0.0042500 0.960 0.004 0.014 0.005 0.166 0.0055000 0.793 0.160 0.017 0.023 0.230 0.082

Silicon treatments compared with fertilized plantsPlant Root Corm

A Silicon treatments compared with not fertilized plantsPlant Root Corm

B Silicon treatments compared with not fertilized plantsPlant Root Corm

Silicon treatments compared with fertilized plantsPlant Root Corm

CHAPTER 5: Silicon

105

Also the leaf thickness (Figure 5.7) was positively affected by Si applications especially if applied via the root system. The chlorophyll content (Figures 5.8) showed that fertilized plants had significantly higher levels over non-fertilized plants but the same levels as Si treated plants via the root system. There was however no effect of Si concentration.

Figure 5.7 Leaf thickness of banana plants (n = 10) recorded during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments.

Figure 5.8 Chlorophyll content of banana plants (n = 10) established during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments.

0

10

20

30

40

50

60

50 500 1200 2500 5000 50 500 1200 2500 5000 F NF

Foliar Root Controls

Treatments

Chlorophyll content (SPAD unit)

0.220.230.240.250.260.270.280.29

0.3

50 500 1200 2500 5000 50 500 1200 2500 5000 F NF


Treatments

Leaf thickness (mm)

CHAPTER 5: Silicon

106

The Si content in the corm (Figure 5.9) and in the leaves (Figure 5.10) gave a similar pattern between the treatments; all Si treated plants had higher levels (irrespective of the mode of application) than fertilized and unfertilized plants. In the corm Si content reached levels up to 1.4% while in the leaves it reached up to 2.5%. The Si in the fertilized and unfertilized plants most probably came from the Si of decaying rice husks used as part of the substrate.

Figure 5.9 Si concentration in the banana corm (n = 10) growing during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments.

Figure 5.10 Si concentration in banana leaves (n = 10) growing during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments.

0.00.51.01.52.0

2.53.03.54.0

50 500 1200 2500 5000 50 500 1200 2500 5000 F NF


Treatments

Silicon in the corm

(%)

0.00.51.01.52.02.53.03.54.0

50 500 1200 2500 5000 50 500 1200 2500 5000 F NF


Treatments

Silicon in the leaf

(%)

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107

The soil pH at low Si concentrations was similar to the fertilized plants but with

increasing Si concentrations the pH tended also to increase (Figure 5.11).

Figure 5.11 Soil pH for banana plants (n = 10) growing during 8 weeks under greenhouse conditions. The plants received weekly foliar and root applications at different concentrations of Si (from PS). Not fertilized and fertilized plants were evaluated as control treatments.

In a fourth experiment and under the same conditions, Si was applied via the root

system at three concentrations (500, 1200 and 2500 mg/kg) thereby using two Si sources and a K source, to get insight in a possible confounding effect caused by K. In general, plant height with Si and K was always superior to the non-fertilizer treatment and equal to the fertilizer treatment, and only superior to the fertilized plants at 500 and 1200 mg/kg Si when using Standard Silicon. The same pattern emerged for the number of leaves with only 1200 mg/kg Si coming from Standard Silicon being superior to the fertilized plants (Table 5.9).

Again conventional fertilizer increased the BLSD (in comparison to the not fertilized plants) while Si applied as Standard Silicon significantly decreased it (in comparison to the conventionally fertilized plants). However K applied as PS seems to stimulate BLSD development as all treatments gave the same BLSD response as with the fertilized plants (Table 5.9). When K was applied as KOH, BLSD was the same as with the fertilized plants at the highest concentration (2500 mg/kg of K). Fresh weight (Table 5.10) of the root, corm and plant (including pseudostem, leaves, roots and corm) was significantly increased by Si and K in comparison to the non-fertilized plants and the same or higher than in fertilized plants.

0123456789

50 500 1200 2500 5000 50 500 1200 2500 5000 F NF


Treatments

pH o

f soi

l

CHAPTER 5: Silicon

108

Table 5.9 Area under the curve (AUC) over 8 weeks for plant height, number of leaves and BLSD symptoms of banana plants (n = 20) growing under greenhouse conditions. The plants received weekly root applications of different Si concentrations from PS and standard Si and different K concentrations coming from KOH. Reference treatments were plants receiving no fertilizer and fertilizer.

Treatments

No Fertilizer 399.15 d 305.05 d 3.15 abcFertilizer 531.85 cd 363.06 bc 5.10 d

Si (mg/kg) Potassium Silicate (PS)500 623.13 abc 399.50 ab 4.53 cd

1200 585.30 abc 358.32 bc 4.45 cd2500 607.75 abc 353.39 c 4.73 d

Si (mg/kg) Standard Silicon (Si)500 655.50 a 380.78 abc 2.83 ab

1200 635.03 ab 419.37 a 3.28 abc2500 552.40 bc 387.87 abc 2.93 ab

K (mg/kg) Potassium Hydroxide (KOH)500 551.90 bc 403.61 ab 3.20 abc

1200 519.25 cd 393.06 abc 2.05 a2500 536.30 cd 365.64 bc 3.90 bcd

AUCBLSD

symptomsNumber of

leavesPlant height


(P≤0.05) according to Duncan’s test.

CHAPTER 5: Silicon

109

Table 5.10 Fresh weight (g) of roots, corm and entire banana plant (n = 10) established under greenhouse conditions. The plants received weekly root applications of different Si concentrations from PS and Standard Si and different K concentrations coming from KOH. Not fertilized and fertilized plants were evaluated as control treatments.

No fertilizer 2.29 c 2.01 c 15.11 cFertilizer 18.37 b 10.97 ab 96.60 ab

Si (mg/kg)500 19.85 b 10.51 ab 93.33 ab

1200 21.00 ab 9.29 b 88.17 b2500 26.44 a 9.07 b 102.09 ab

Si (mg/kg)500 19.98 b 10.81 ab 106.06 ab

1200 19.14 b 9.97 ab 101.25 ab2500 18.59 b 9.57 b 91.01 ab

K (mg/kg)500 24.37 ab 10.98 ab 119.05 a

1200 21.60 ab 9.26 b 96.44 ab2500 26.52 a 12.18 a 98.44 ab

Fresh weight (g)

Potassium Hydroxide (KOH)

Standard Silicon (Si)

Potassium silicate (PS)

Corm PlantRootTreatments


(P≤0.05) according to Duncan’s test. The chlorophyll content was significantly higher in plants receiving Si from both

sources than in plants receiving only K. The lowest concentration (500 mg/kg) of any of these nutrient sources gave also the lowest chlorophyll content (Table 5.11). The leaf thickness did not show significant differences between different Si and K sources and their concentration had no effect (Table 5.11).

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110

Table 5.11 ANOVA of area under a curve of chlorophyll content and leaf thickness of banana plants grown under greenhouse conditions. The plants received weekly root applications of different Si concentrations from potassium silicate (PS) and standard Si (Si) and different K concentrations coming from potassium hydroxide (KOH). Data followed by the same letter in each column are not significantly different (P≤0.05) according to Duncan’s test.

Product PS 2239.59 a 10.92 aSi 2237.16 a 11.02 a

KOH 2156.35 b 10.72 a

Concentrationmg/kg

500 2120.01 b 10.79 a1200 2220.15 a 10.78 a2500 2279.34 a 10.93 a

Leaf thicknessChlorophyllAUC

5.3.2.2. Banana response to Si applications under field conditions Si applied (as PS) via the foliage and root system significantly increased the plant

growth and leaf number related to both control treatments (with and without fertilizer). However, there were no clear differences between different Si concentrations and intervals of application except that root application gave taller plants in the 14 day intervals. Non-fertilized plants flowered later than fertilized plants (Table 5.12) while in general Si treated plants flowered at the same time or earlier than the fertilized plants. Root application at 14 days intervals resulted in the shortest cycle.

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111

Table 5.12 Area under the curve (AUC) of plant height and number of leaves over 8 months and days to flowering of banana plants established under field conditions (n = 30). The plants received during two intervals (7 and 14 days) and following two modes of application (foliar and root) different Si concentrations from PS. Reference treatments were plants receiving no fertilizer and fertilizer.

Treatments

No Fertilizer 10995.52 e 2340.48 d 232.38 eFertilizer 13956.25 d 2351.02 d 194.75 d

Si (mg/kg)

500 17712.15 ab 2665.66 b 190.27 bcd1200 18247.10 a 2649.88 bc 185.37 abc2500 17397.37 ab 2686.62 b 189.10 bcd

500 17571.30 ab 2587.35 c 183.50 ab1200 17477.00 ab 2733.30 a 190.03 bcd2500 18138.77 a 2685.61 b 186.53 abc

Si (mg/kg)

500 16817.75 bc 2632.64 bc 190.27 bcd1200 17035.52 ab 2622.61 bc 192.60 cd2500 16644.73 bc 2690.08 b 190.73 bcd

500 17938.25 a 2652.24 bc 190.27 bcd1200 17870.75 a 2627.70 bc 187.00 abc2500 17869.63 a 2600.97 c 181.87 a

AUC

Foliar application7 days

14 days

Root application7 days

14 days

Plant height Number of leaves

Flowering days


(P≤0.05) according to Duncan’s test The disease expression parameter was similar between all treatments (Figure 5.12).

However the beneficial effect of Si applications on the disease expression on the next generation (sucker) was very clear with more leaves being free of the disease than fertilized and not fertilized plants (Table 5.13).

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Figure 5.12 BLSD severity index of banana plants (n = 30) established under field conditions. The plants received during two intervals (7 and 14 days) and with two different modes of applications (foliar and root) different Si concentrations from PS. Reference treatments were plants receiving no fertilizer and fertilizer.

Table 5.13 BLSD severity index and young leaves without symptoms (YLS) on second generation banana plants (sucker) established under field conditions during 3 months (n = 30). The plants received different Si concentrations from PS. Reference treatments were plants receiving no fertilizer and fertilizer.

No Fertilizer 789.35 ab 367.25 bFertilizer 847.43 b 369.75 b

Si (mg/kg)500 831.01 b 414.25 a

1200 717.48 a 412.25 a2500 746.91 ab 401.75 ab

YLSBLSD severityAUC

Treatments

Means per column of each parameter with the same letter are not significantly

different by Duncan’s test at P≤0.05.

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113

5.4. Discussion

The beneficial effects of Si in many crops are attributed to a reduction in biotic and abiotic stress (Korndörfer and Lepsch, 2001) and many reports exist that Si reduces fungal diseases in plants. Banana is a Si accumulator (Henriet et al., 2006) and suffers much from BLSD, caused by the leaf pathogen Mycosphaerella fijiensis Morelet. Hence a study of the potential effect of Si on BLSD is warranted.

Bananas are massively multiplied in vitro and then planted in the nursery prior to field planting. Hence we investigated the effect of Si on plant growth and development in 3 growing conditions (in vitro, nursery and the field) and BLSD under nursery and field conditions. Si was applied as PS and since this changes drastically the pH, the pH was adjusted and not adjusted. Under in vitro conditions, a pH adjustment was favourable on plant growth but under nursery conditions became unnecessary. As PS contains both Si and K, other treatments were included as Standard Si and KOH. Fertilized and non-fertilized plants acted as a control.

In general, conventionally fertilized plants developed much better than non-fertilized plants, but were more affected by BLSD indicating that nitrogen fertilizer increased fungal infection. The content of Si treated plants was up to 2.5% in the leaves and 1.5% in the corm. Si treated plants showed less BLSD while K might seem to have increased BLSD. The Si treatments under field conditions resulted in more leaves being free of BLSD symptoms. Also plant growth and more distinctly leaf development and leaf area was much improved by Si especially when applied via the root system. The chlorophyll content and the leaf thickness were positively affected by the root applications of Si.

5.5. Conclusions

This chapter demonstrated that Si had direct effects on both actors of the pathosystem: banana and M. fijiensis Morelet. In in vitro conditions, the inhibitory effect of Si on the pathogen increased with increasing concentrations. In vitro Si treated banana plants became BLSD susceptible in the nursery most probably because the Si treatment had stopped once the infection was performed. Hence, it seems that for Si to be effective, continuous application is necessary. This is supported by the field data of 8 months cultivated bananas, were Si treated banana plants showed more leaves being free of BLSD.

Bananas plants are one of the crops with the highest demands for fertilizer. For example, N levels can reach up to 300kg per plant per year. Also they are sprayed heavily with fungicides and in some places at almost weekly intervals. Our study shows that bananas should also receive Si fertilizer and that N dosages should be diminished as part of an integrated pest management strategy to reduce infection by M. fijiensis Morelet. Hence different Si sources warrant to be investigated because they contain other nutrients with possible effect on M. fijiensis Morelet. The effect of K on BLSD needs a more in depth study.

CHAPTER 6: Conclusions and perspectives

115

Chapter 6 CONCLUSIONS AND PERSPECTIVES Bananas are the fourth most important crop in the tropics with an annual production of

100 million tonnes. For more than 400 million people it is a staple food crop while for another 600 million they are an important extra food or income. 13% of the bananas are exported especially from Latin America where dessert bananas are cultivated in large monocultures. However, the bulk of the bananas are grown by smallholders in small fields.

Most bananas in the world are affected by black leaf streak (BLSD) which replaced the less virulent yellow Sigatoka as the dominant leaf spot. Cultivars belonging to the Cavendish subgroup that produce bananas for export suffer from this leaf spot disease as the plantains, and the highland cooking and beer bananas. This leaf spot disease reduces the yield up to 100% but in commercial plantations where bananas are produced for export, the disease is controlled with heavy applications of fungicides. Given the fact that the pathogen, Mycosphaerella fijiensis Morelet has become resistant to certain fungicides and that large amounts of fungicides are costly and hazardous to humans and the environment, research is needed to develop integrated disease management strategies applicable to all types of farms.

Most banana farms focus all their resources to secure high yields with high amounts of conventional fertilizers and control diseases with high amounts of pesticides. Nutrition and pests/diseases are managed separately as if nutrition has no positive or negative effect on pests/diseases. Not surprisingly and after years of monocultures under such conditions, soils deteriorate, pathogens become resistant and yields start to decline. To reverse this trend, there is a need to gain a better understanding between banana plant nutrition and pest/disease pressures within the context of the entire farming system so that fields can be managed in a more sustainable way.

Organically grown banana plantations pretend to have a more sustainable approach. They claim that production is viable and that the soil is not depleted, that disease pressure is low and that the environment does not suffer from their practices. As BLSD is present in almost any banana growing area in the world, there is a need a to verify whether organically grown bananas for export have less or no BLSD, and if so to understand how this disease is controlled. An in-depth understanding is indeed needed if one wants to extend the areas under organic banana farming conditions because currently organic banana farming is quite artisanal and rely on bulky amounts of organic products. In addition, many practices are questionable as their value has not been demonstrated. Thus we investigated this production system and compared it with the classical production system which uses conventional fertilizer and fungicides. The goal was to gather information which can lead to a reduced BLSD infection in any production system, thus whether under commercial or backyard conditions. This is indeed needed as one cannot expect that the solution should come only from breeding and/or genetic engineering or the application of fungicides.

The thesis starts first with an overview on the link between nutrition and disease development in several crops other than banana. Then an overview is given on the use of organic products and their effect on diseases which forms the basis of the study of an organic banana production system.

The research was conducted at three levels: in vitro, greenhouse and field conditions which in fact correspond to a complete control of the environment up to an incomplete control but also reflects an artificial situation up to the real conditions. Attention was paid to the pathogen (M. fijiensis Morelet), the host (banana plant) and the effect of the pathogen on the host, i.e. the disease (BLSD). Chapter 2 characterizes an organic production farm in Ecuador cultivated with export bananas. Its management is described in detail and compared with a conventional production system. Data are provided which show that BLSD is present but well


116

controlled. The fungal isolate from the organic farm was shown to behave as the isolate from the conventional farm. As both farms have the same soil and leaf nutrient composition, attention was than given to the specific preparation and use of organic inputs. In the organic farm liquid organic products are applied (organic teas-OT). Therefore and in chapter 3, the effect of these OT which varied very much in composition, were tested on the pathogen and the disease. As these OT are rich in micronutrients, which the conventional banana farm does not receive, the individual effect of Zn, B, Cu, Mn and Si on the pathogen and the banana plant were investigated (chapter 4 and 5).

In vitro studies The fungus and the host were evaluated under in vitro conditions. First isolates were

obtained from the organic and conventional farm. Then their morphology and growth were studied which revealed no important differences. The fungal aggressiveness was the same for both isolates (chapter 2, section 2.2) but the amount of conidia produced by the organic isolate was less than with the conventional isolate. Yet, the potential to generate the disease was similar between both isolates. Consequently, the pathogen characteristics in the organic banana field are not the reason why in that field banana plants can be grown without fungicide applications.

In chapter 3, section 3.2, several organic teas produced after fermentation of organic wastes was evaluated against M. fijiensis Morelet. Results were variable but several OT inhibited fungal growth under in vitro conditions even after autoclaving. As this could point to the effect of (a) specific nutrient(s) the nutrient content of these OT were analyzed. Macro and micronutrient composition were found to be highly variable and no direct link could be made between the inhibitory effect and a specific nutrient composition. Hence several micronutrients were separately investigated.

B, Cu, Mn and Zn displayed a direct effect on the fungus under in vitro conditions (chapter 4). Once these nutrients were eliminated from the medium, there was no inhibition on fungal growth anymore except for Cu that has a lasting after-effect. The tested micronutrients also modify the pH of the growth medium and hence the nutrient effect needed to be separated from the pH effect. In general, an acid or alkaline pH reduced the fungal growth while there was no effect at a neutral pH.

The applied OT also contained Si and since banana is a Si accumulator and Si was shown to inhibit some fungi in other crops, this nutrient was also investigated (chapter 5, section 5.2). Higher amounts of Si resulted in a higher pH and this was linked to more fungal inhibition. However, the Si ED50 for ascospore inhibition was less than for the other micronutrients assessed. Si also stimulated plant growth at a rate of 50-500 mg/kg.

In vivo studies In vivo studies were conducted in the greenhouse and the field to assess both the

banana plant growth and the BLSD. In chapter 2, section 2.1, it was shown that the organic farm is indeed infected by BLSD but that plants have still a high number of green leaves at harvest. The nutritional status (soils and leaves) of the organic and conventional farms was similar.

In chapter 3, section 3.3 it was demonstrated that the OT positively affects plant development and growth in the greenhouse and the field. Also the disease development was low especially if OT are applied weekly whether to the soil or the leaves. However it takes time to see the effect and this explains why the OT effect is much clearer in the ratoon crop than in the mother crop.

The micronutrients (B, Cu, Mn, and Zn) improved plant growth but their effect on BLSD were clearer with 7 days interval applications than with 15 days interval applications


117

(chapter 4). These results were in line with the in vitro conditions where the fungus stopped growing in the presence of the same micronutrients. Similarly Si was tested (chapter 5, section 5.3). In the greenhouse plant growth was stimulated especially if applied via the root system and this depended on the concentrations used. Also BLSD symptom expression was reduced by Si. In general, the positive contributions of Si on plant growth and reduced disease expression were confirmed in the field experiment as well. In conclusion we can state that nutrients affect both plant growth and development, and BLSD development. Under the experimental conditions described N increases BLSD while several micronutrients show a potential to reduce it.

Perspectives The organic banana farming system studied, showed that commercial farming is

possible without the use of fungicides in semi-humid conditions although Mycosphaerella fijiensis Morelet was present and did cause some damage. The research pointed out that the used OT were a key factor in this system as they contributed several nutrients some of which with a potential to reduce the growth of the pathogen and the BLSD. More research is needed to unequivocally demonstrate the effect of the tested micronutrients and identify the optimal amount needed of each nutrient. Such experiments should always evaluate the effect of the specific micronutrient over a long time and under field conditions. In addition micronutrients need to be applied regularly as it seemed that at least some micronutrients had a direct effect on the pathogen. Once these field experiments have provided clear results, one needs to improve the OT preparation as now the ingredients are bulky and very variable. In a next step one needs to test the effect of the micronutrients and improved OT on BLSD development under humid conditions. One cannot exclude that some micronutrients also had an indirect effect and thus improved the defence system of the plant. This need more detailed research and should also involve the study of gene expression and proteomics research.

As for the short term, it would be useful to incorporate in the aerial applied fungicides the micronutrients we have studied (Zn, B, Cu, Mn and Si) alone and in combination, as these will contribute to fungal inhibition and plant growth. However the optimal dosage needs to be elaborated. Consequently we believe that our results can contribute to both organic and conventional banana farming. Also the amount of N applied needs to be revisited as it does not only contribute to improved plant growth but also to a higher BLSD.

During current OT preparation one allows infection with local microorganisms. Hence also research is needed to verify whether some microorganisms contribute to the direct and indirect inhibition of M. fijiensis Morelet growth.

References

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ANNEXES

ANNEX 1: List of presentations, publications and manuscripts Jiménez M., Bermeo J., Neirynck, H., Van der Veken L., Jama M., Maribona R., and

Swennen R. Characterization of two Mycosphaerella fijiensis isolates from an organic and inorganic banana plantation. 1st International Congress on Musa. Harnessing research to improve livelihoods. Penang, Malaysia, 8 July 2004.

Jiménez M., Márquez E., Jama M., Cañarte S., Encalada J., Rodríguez H., Ruiz, O., Ruiz

J., and Swennen R. 2006. Black Sigatoka management in an organic banana system in Ecuador. International Congress: Black Sigatoka management in banana and plantain in Latin America and the Caribbean. San Jose, Costa Rica, 23 March 2006.

Jiménez M., Bermeo J., Márquez E., Cañarte S., Encalada J., Rodríguez H., Ruiz, O.,

Ruiz J., and Swennen R. 2006. Contribution to the knowledge of banana nutrition and the development of Mycosphaerella fijiensis, causal agent of black Sigatoka. International Congress: Black Sigatoka management in banana and plantain in Latin America and the Caribbean. San Jose, Costa Rica, 23 March 2006.

Jiménez M., Van der Veken L., Neirynck H., Rodríguez H., Ruiz O., and Swennen R.,

2007. Organic banana production in Ecuador: its implications on black Sigatoka development and plant - soil nutritional status. Renewable Agriculture Food System 22 (4): 1-10.

Jimenez M., Van der Veken L., Rodríguez H., Ruiz O. and Swennen R. Controlling black

leaf streak disease with soil supplements in an organic banana plantation in Ecuador. In: Jones D.R. and Van den Bergh I. (eds.). Proceedings of the ISHS-ProMusa symposium on Recent advances in banana crop protection for sustainable production and improved livelihoods. Acta Horticulturae. (Accepted).

Jiménez, M., Quito, D., Maura, F., Rodríguez, H., Ruiz, O., and Swennen, R. The effects

of organic teas on Mycosphaerella fijiensis (Morelet) and their potential as a tool for black Sigatoka disease management and banana growth enhancement. Submitted to European Journal Plant Pathology (2008).

Jiménez, M., Espinoza, L., Verguts, V., Ruiz, O., and Swennen, R. The inhibitory effect

of pH and some micronutrients on the development of Mycosphaerella fijiensis Morelet, under in vitro conditions (in preparation).

Jiménez, M., Heasing, M., Rodriguez, H., Peralta, E., Ruiz, O., and Swennen, R. The

effect of silicon on banana plant development in vitro and in soil, and its potential benefits for black Sigatoka management (in preparation).

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ANNEX 2: Area under a curve (AUC)

The area under a curve is a mathematical value calculated for parameters measured over time such as plant growth (height, diameter, number of leaves, number of roots, etc.) and disease expression. This value represents the sum of the measured parameter over a certain time interval.

The AUC per parameter and per plant was calculated by integration as described by Campbell and Madden (1990).

The trapezoidal model is the simplest and common calculation value to obtain the AUC.

Where: Ʃ is the sum of the areas of all individual trapezoidal integration from i to

n – 1; n is the number of evaluations, X is the value of the disease scale (symptoms or severity index) or the plant growth data (height, diameter, number of roots or leaves, etc.) and the ti+1 – ti is the time interval between two consecutives evaluations.

Figure A1. Linear plot of plant height versus time showing the data points used

for AUC calculation. The AUC is calculated per segment (Figure A1). The area of each segment can be

calculated by multiplying the average of the plant height (for example) with the segment width (time). For the segment Ph2 to Ph3:

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The area under a curve is thus the sum of an entire series of segments.

References: 1. Campbell and Madden, 1990 2. De Jesus et al., 2004 3. Kesmala et al., 2004 4. Kesmala et al., 2006 5. Meles et al., 2004

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