Ecology and diversity of indigenous Trichoderma species in vegetable cropping systems A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by Emmanuel BOURGUIGNON National Centre for Advanced Bio-Protection Technologies, Lincoln University, Canterbury, New Zealand 2nd of July 2008 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy (Ph.D.) Ecology and diversity of indigenous Trichoderma species in vegetable cropping systems Emmanuel Bourguignon Email: [email protected] The overall aim of this research was to improve the understanding of the ecology and diversity of Trichoderma species within the soil and rhizosphere of onion (Allium cepa L.) and potato (Solanum tuberosum L.) under intensive management in New Zealand. The indigenous Trichoderma population was measured in a field trial at Pukekohe over a three year period under six different crop rotation treatments. The treatments included two continuous onion and potato rotations (intensive), two onion/potato mixed rotation (conventional), and two green manure rotations (sustainable). Results showed that Trichoderma populations were stable in both the rhizosphere and bulk soil (1.5 x 102 to 8.5 x 103 CFU g-1 ODS). The planting and incorporation of an oat (Avena sativa L.) green manure in the sustainable rotations positively increased Trichoderma colony forming unit (CFU) numbers in the rhizosphere soil from 3.4 x 102 to 2.5 x 103 g-1 ODS. A Trichoderma species identification method was developed based on colony morphology. Representative isolates were verified using restriction fragment length polymorphism (RFLP) and DNA sequencing. The method allowed for rapid and reliable identification of isolated Trichoderma species. Five species were identified in the Pukekohe soil: T. asperellum, T. atroviride, T. hamatum, T. harzianum and T. koningii. Results showed identical species diversity between the rhizosphere, rhizoplane and bulk soil. The species did not strongly compete between each other for the rhizosphere ecological niche and differences in species proportions seemed to be caused by environmental factors rather than the rotation treatments. i The incorporation of oat green manure in pots did not significantly promote the indigenous Trichoderma population size and diversity in the rhizosphere of onion plants up to 4 months old. The identified species were the same as in the field trial. The incorporation of onion scale residues was shown to result in low Trichoderma and high Penicillium CFU numbers and a reduction in plant size. Additionally, the presence of high levels (6.0 x 105 CFU g-1 ODS) of Penicillium CFU was negatively correlated with the presence of Trichoderma CFU. The effect of oat incorporation on Trichoderma saprophytic growth was also investigated in a soil sandwich assay and revealed no significant differences. A series of experiments indicated that onion extract obtained from dry onion scale residues had no antifungal activity against either Trichoderma or Penicillium and instead tended to promote their hyphal growth and sporulation. It also showed that competition between Penicillium and Trichoderma isolates was limited despite the ability of Penicillium to produce a wide range of inhibitory substances. Four indigenous Trichoderma species (T. atroviride, T. hamatum, T. harzianum and T. koningii) were shown to be rhizosphere competent in a split tube experiment over a 6 week period. The results of this experiment revealed that, the Trichoderma species clearly displayed differences in their ability to colonise the rhizosphere of young onion seedlings. Species such as T. koningii had the greatest rhizosphere colonising ability regardless of soil depth while T. harzianum displayed the weakest ability. Results also indicated that when inoculated as a mixture the four species competed with one another to colonise the rhizosphere. Overall, this research indicated that the studied crop rotation treatments and the use of oat as a green manure did not strongly promote indigenous Trichoderma populations. Species diversity was constant throughout the research with T. hamatum and T. koningii being the most frequently isolated species. Keywords: Trichoderma, rhizosphere, ecology, indigenous, population, crop rotation, Allium cepa L., Solanum tuberosum L., Avena sativa L., green manure, amendments, species diversity, identification, RFLP, Penicillium, rhizosphere competence. ii Contents ABSTRACT I CONTENTS III LIST OF TABLES VII LIST OF FIGURES X ABBREVIATIONS AND SYMBOLS XIV ACKNOWLEDGEMENTS XVI CHAPTER ONE: GENERAL INTRODUCTION 1 1.1 RESEARCH BACKGROUND 1 1.2. LITERATURE REVIEW 5 1.2.1 Trichoderma 5 1.2.1.1 The genus Trichoderma 5 1.2.2 The ecology of Trichoderma species 6 1.2.2.1 Habitats 6 1.2.2.2 Environmental parameters 7 1.2.2.3 Substrate diversity 8 1.2.3 Metabolism 9 1.2.3.1 Enzyme production for carbon and nitrogen metabolism 9 1.2.3.2 Antibiotic production 11 1.2.3.3 Other secondary metabolite production 13 1.2.4 Biological control capacity of Trichoderma species 13 1.2.4.1 Mycoparasitism 13 1.2.4.2 Antibiosis 14 1.2.4.3 Competition 15 1.2.4.4 Plant growth promotion and induced resistance 15 1.2.5 Factors affecting antagonistic activity 16 1.2.5.1 Abiotic factors 16 1.2.5.2 Biotic factors 18 1.2.5.3 Rhizosphere competence 18 1.3. TECHNIQUES REQUIRED TO STUDY TRICHODERMA ECOLOGY IN SOIL 19 1.3.1 Isolation techniques 19 1.3.2 Identification techniques 20 1.3.2.1 Morphology based techniques 20 1.3.2.2 Molecular based techniques 21 1.3.3 Quantification techniques 22 1.3.3.1 Non-molecular based techniques 22 1.3.3.2 Molecular based techniques 24 1.3.4 Species diversity assessment 24 1.3.4.1 Biochemical based techniques 24 1.3.4.2 Molecular based techniques 25 1.3.5 Species distribution assessment 26 1.3.5.1 Microscopic techniques 26 1.3.6 Species functionality assessment 27 1.3.6.1 Biochemical assays 27 1.3.6.2 Molecular techniques 28 1.4. SUMMARY 29 1.5 RESEARCH AIM AND OBJECTIVES 29 1.5.1 Aim 29 iii 1.5.2 Objectives 30 1.5.3. Thesis format 30 CHAPTER TWO: EFFECT OF CROP ROTATION ON DIVERSITY AND ABUNDANCE OF INDIGENOUS TRICHODERMA IN THE RHIZOSPHERE OF ONION AND POTATO 31 2.1. INTRODUCTION 31 2.2. MATERIALS AND METHODS 33 2.2.1. Field trial 33 2.2.1.1. Location 33 2.2.1.2. Soil type and crops 33 2.2.1.3. Rotation treatments 34 2.2.1.4. Field trial layout 35 2.2.1.5. Assessments 36 2.2.2. Soil sampling, and isolation of Trichoderma species 37 2.2.2.1. Rhizosphere soil sampling 37 2.2.2.2. Rhizosphere soil dilution 38 2.2.2.3. Rhizoplane soil sampling 39 2.2.2.4. Rhizoplane soil dilution 39 2.2.2.6. Trichoderma colony purification and isolation 40 2.2.2.7. Single spore isolation 40 2.2.3. Trichoderma species identification using morphological characteristics 42 2.2.3.1. Morphology on TSM-LU plates 42 2.2.3.2. Morphology on ½ PDA+c plates 43 2.2.4. Trichoderma species identification using molecular tools 44 2.2.4.1. DNA extraction 44 2.2.4.2. DNA amplification 44 2.2.4.3. Restriction Fragment Length Polymorphism (RFLP) identification 45 2.2.4.4. DNA sequencing 46 2.2.5. Data analysis 47 2.2.5.1. Trichoderma CFU numbers 47 2.2.5.2. Trichoderma species presence frequency 48 2.3. RESULTS 49 2.3.1. Trichoderma CFU analysis in rhizosphere, rhizoplane and bulk soil 49 2.3.2. Trichoderma species identification 56 2.3.2.1. Trichoderma species morphology groups on TSM-LU agar 56 2.3.2.2. Trichoderma species morphology on ½ PDA+c 59 2.3.2.3. Trichoderma species diversity 67 2.3.2.4. Trichoderma species presence frequency 69 2.4. DISCUSSION 73 2.4.1. Trichoderma CFU numbers in the rhizosphere, rhizoplane and bulk soil 73 2.5.2. Trichoderma species identification 78 2.4.3. Trichoderma species diversity and proportion 79 2.4.4. Species presence frequency 82 2.5. SUMMARY 84 CHAPTER THREE: EFFECT OF GREEN MANURE AND ORGANIC MATTER AMENDMENTS ON THE ABUNDANCE, DIVERSITY AND SAPROPHYTIC GROWTH OF INDIGENOUS TRICHODERMA 85 SECTION 1: EFFECT OF GREEN MANURE AMENDMENTS, IN TWO POT EXPERIMENTS, ON THE ABUNDANCE AND DIVERSITY OF INDIGENOUS TRICHODERMA IN THE RHIZOSPHERE OF ONION PLANTS. 85 3.1. INTRODUCTION 85 iv 3.2. MATERIALS AND METHODS 87 3.2.1. Experiment one: Green oat shoots amendment 87 3.2.1.1. Rationale 87 3.2.1.2. Preparation of plant material 88 3.2.1.3. Pukekohe soil mix 88 3.2.1.4. Treatments and green manure amendments 89 3.2.1.5. Experimental design 90 3.2.1.6. Assessments 91 3.2.1.7. Rhizosphere soil sampling and soil dilution 91 3.2.1.8. Trichoderma species identification 92 3.2.1.9. Onion plant dry weight 93 3.2.2. Experiment two: Green oat crop amendment 93 3.2.2.1. Rationale 93 3.2.2.2. Soil Sampling 93 3.2.2.3. Treatments 94 3.2.2.4. Experimental set up and assessments 94 3.2.2.5. Rhizosphere soil sampling and soil dilution 95 3.2.2.6. Trichoderma species identification 95 3.2.2.7. Onion plant harvest 96 3.2.3. Data analysis 96 3.3. RESULTS 97 3.3.1. Experiment one: Green oat shoots amendments 97 3.3.1.1. Trichoderma CFU numbers 97 3.3.1.2. Penicillium CFU numbers 101 3.3.1.3. Trichoderma species diversity 104 3.3.1.4. Trichoderma species proportion and presence frequency 104 3.3.1.5.