Hysterangium mats and associated bacteria under Eucalyptus gomphocephala in south-western Australia. By Nguyen Quang Dung

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1 Hysterangium mats and associated bacteria under Eucalyptus gomphocephala in south-western Australia By Nguyen Quang Dung BSc. Forestry (Vietnamese Forestry University) Thesis submitted in fulfilment of the requirements for the degree of Master of Philosophy in Biological Sciences School of Biological Sciences and Biotechnology Murdoch University Perth, Western Australia November 2012

2 Declaration I hereby declare that the work in this thesis is of my own research, except where reference is made, and has not previously been submitted for a degree at any educational institution. Contributions including professional advice and other help are detailed in the acknowledgements. Nguyen Quang Dung November 2012 i

3 ABSTRACT Eucalyptus gomphocephala (tuart) is an ecologically and culturally important woodland and forest tree native to south-western Australia. Unfortunately, tuart has been markedly declining in recent decades, and the mortality rate is as high as 90% in some populations. The cause of tuart decline is still poorly understood and we know nothing about how the decline changes populations of organisms such as ectomycorrhizal (ECM) fungi and beneficial bacteria. Therefore, this study investigated aspects of mycorrhizal fungal mats under healthy tuart, including effects on forest soil properties, and presence of beneficial bacteria. A common mat morphotype (grey, hydrophobic) was chosen for this research and the ECM fungus was identified as a Hysterangium sp. based on fungal morphology, ECM structure and ITS gene region analysis. The effect of the ECM mats on soil moisture, nutrients, microbial biomass (ninhydrin-reactive N) and microbial community (Biolog Ecoplate) was investigated. Potential PGPR with ability to solubilize phosphate (hydroxylapatite-ca 5 HO 13 P 3 ), produce IAA and use ACC acid as the sole carbon source were screened. Hysterangium mats significantly improved soil moisture, soil ph, nutrient level and microbial biomass compared to non-mat soils, however, there was no evidence of any difference in microbial diversity and activity between mat and non-mat soils. Twenty eight IAA producing bacterial isolates, 7 phosphate solubilizing isolates and 2 ACC deaminase producing isolates were screened from mat and non-mat soils. Interactions among beneficial bacteria, ECM fungi and eucalypt seedlings were then investigated. ECM synthesis was also conducted between Hysterangium MURU6276 and E. gomphocephala seedlings in vitro. The plant growth promoting ability of bacteria to eucalypt seedlings was assessed through a bioassay using petri dishes containing MMN medium overlaid with cellophane. Half plate petri dishes were used to screen for ii

4 mycorrhiza helper bacteria. ECM association between Hysterangium MURU6275 and E. gomphocephala seedlings was confirmed in vitro. Most bacterial isolates from under tuart had stimulating or inhibiting effects on the root growth of E. gomphocephala and/or E. grandis. The best 6 growth promoting isolates were T1M3, T1N2, T2N7, T3N4, T4N4 and T4N6. However, there was no evidence of the presence of mycorrhiza helper bacteria amongst the isolated bacteria. These findings provide the basis for further detailed research on biochemical processes and the diversity and functions of bacterial populations under Hysterangium mats associated with tuart. Areas for further study are suggested. iii

5 TABLE OF CONTENTS ABSTRACT... ACKNOWLEDGEMENTS ABBREVIATIONS CHAPTER 1 INTRODUCTION 1.1 Overview of the research program Background Thesis hypotheses and aims Thesis structure. 4 CHAPTER 2 LITERATURE REVIEW 2.1 Introduction Plant growth promoting rhizobacteria Mechanisms of PGPR action Research and applications of PGPR in forestry Mycorrhiza helper bacteria Mechanisms of MHB action Research and applications of MHB in forestry Mycorrhizal fungal mats Abundance of ECM mats Types of ECM mats Function of ECM fungal mats Soil microbial biomass Techniques for measuring soil microbial biomass Measuring soil microbial biomass by extracting ninhidryn reactive N Soil microbial activity Concluding remarks. 33 CHAPTER 3 HYSTERANGIUM-EUCALYPTUS GOMPHOCEPHALA MATS AND ASSOCIATED MICROORGANISMS 3.1 Introduction Materials and methods Site selection Soil and mat sampling Characterization of physiochemical and microbial properties 38 iv ii vi vii

6 3.2.4 Characterization of fungal mat and mat-forming fungi Characterization of bacteria Results Characterization of fungal mat Soil properties Characterization of bacteria Discussion ECM mat and fungi Soil properties Plant growth promoting properties of bacteria 68 CHAPTER 4.. INTERACTION AMONG TUART-ASSOCIATED RHIZOBACTERIA, ECTOMYCORRHIZAL FUNGI AND EUCALYPTUS SEEDLINGS 4.1 Introduction Materials and methods Effect of bacteria on eucalypt seedling growth In vitro ECM synthesis between Hysterangium MURU6276 and tuart Effect of bacteria on the root growth of ECM mycelium Data analysis Results Effect of bacteria on the growth of eucalypt seedlings ECM synthesis in vitro Effect of bacteria on fungal mycelium Discussion Effect of bacteria on the growth of eucalypt seedlings ECM synthesis Effect of bacteria on ECM mycelia growth 90 CHAPTER 5.. GENERAL DISCUSSION 5.1 Properties of fungal mats and associated organisms Future research ECM fungi Fungal mats Bacterial associated with fungal mats. 99 EPPENDIX. 101 REFERENCES v

7 ACKNOWLEDGMENTS I would like to thank the Agricultural Science Technology Project, Ministry of Agriculture and Rural Development, Vietnam for providing me with a scholarship without which this Master of Philosophy would not have been possible. I am grateful to Associate Professor Pham Quang Thu and Associate Professor Nguyen Hoang Nghia, Forest Science Institute of Vietnam, for their support in doing research in Australia and advice on research topics. I wish to express my immense gratitude to my principal supervisor Professor Bernie Dell for his conscientious supervision throughout. This MPhil would not have been possible without his financial support. The good advice and assistance in soil microbial skills of my second supervisor, Dr Lambert Brau, have been invaluable. Much support was received by staff and fellow research students at Murdoch University, for which I am indebted. I would like to thank Murdoch technical staff Ms Rebecca Swift for her helpful advice and supply of plant growth promoting rhizobacteria, Ms Diane White and Dr William Dunstan for their help in fungal identification by DNA analysis, Dr Katinka Ruthof for providing tuart seed, and Mr Gordon Thomson for his invaluable input into preparation of the histological specimens. Furthermore, my deep thanks to fellow students, Mr Dang Thanh Tan for his help when I first arrived at Murdoch University, Ms Lily Ishaq for her useful discussion throughout, and Mr Harry Eslick for his help in measuring soil microbial biomass and helpful statistical discussion. I would like to thank my parents for their support. Lastly, I would particularly like to thank my beloved wife, Hoa Hong who sacrificed a lot for my research and our lovely daughter, Thao Nhi during my MPhil. vi

8 LIST OF ABBREVIATIONS ACC AHL AM AMR ANOVA ARDRA ATP BLAST CFE CFI CLPP CMD CNN DF DGGE DNA DTPA ECM et al. Exc. FAME FISH G+C GPB IAA IAM ICP IPyA IR ISR ITS LPS 1-aminocyclopropane-1-carboxylic acid N-acryl-L-hormonerine lactone arbuscular mycorrhiza average metabolic response analysis of variance amplified ribosomal deoxyribonucleic acid restriction analysis adenosine triphosphate Basic Local Aligement Search Tool chloroform fumigation extraction chloroform fumigation incubation community-level physiological profiling community metabolic diversity competition for nutrients and niches Dworkin and Foster denaturing gradient gel electrophoresis deoxyribonucleic acid diethylene-triamine-penta-acetic acid ectomycorrhiza et alia exchangable fatty acid methyl ester fluorescent in situ hybridization guanine + cytosine glucose peptone broth indole-3-acetic acid indoleacetamine inductively coupled plasma indolepyruvic acid infrared reflectance induced systemic resistance internal transcribed spacer lipopolysaccharidses vii

9 L-TRP MEA MHB ml MMN medium MPVK medium MW NA NB NRC PCA PCR PDA PGP PGPR Phl PLFA PLFA PPQ rdna rrna SA SD SE SIR SMB SPSS SSCP TGGE T-RFLP L-tryptophan malt extract agar mycorrhiza helper bacteria milliliter Modified Melin Norkrans medium Modified Pikovskaya medium microwave irradiation nutrient agar nutrient broth ninhydrin reactive compounds principal component analysis polymerase chain reaction potato dextrose agar plant growth promoting plant growth promoting rhizobacteria 2,4-diacetyl phloroglucinol phospholipid fatty acids phospholipid fatty acid analysis cofactor pyrrolquinoline quinine ribosomal deoxyribonucleic acid ribosomal ribonucleic acid salicylic acid standard deviation standard error of the mean substrate induced respiration soil microbial biomass Statistical Package for the Social Sciences single strand conformation polymorphism temperature gradient gel electrophoresis terminal restriction fragment length polymorphism viii