Effects of endophytic actinobacteria on lucerne growth and the development of its N -fixation 2 symbiosis with rhizobia A thesis submitted for the award Doctor of Philosophy at Flinders University of South Australia Hoang Xuyen Le Department of Medical Biotechnology School of Medicine, Faculty of Medicine, Nursing and Health Sciences Flinders University of South Australia 2015 Declarations I certify that this thesis does not contain material which has been accepted for award of any degree of diploma, and to the best of my knowledge and belief it does not contain any material previously published or written by another person except where due reference is made in the text of this thesis or in the notes. Hoang Xuyen Le i Contents Declarations .............................................................................................................................. i List of tables ............................................................................................................................ ix List of figures .......................................................................................................................... xi Acknowledgment .................................................................................................................. xiii Abbreviations .........................................................................................................................xiv Abstract .................................................................................................................................. xv Chapter 1 ................................................................................................................................ 1 Introduction and Literature review ..................................................................................... 1 1.1 Global importance of legumes and nitrogen fixation ......................................................... 2 1.1.1 General information on legumes ................................................................................. 2 1.1.2 Lucerne globally and in Australia .............................................................................. 3 1.2 Importance of soil micro-flora to legume production ........................................................ 4 1.2.1 Rhizobia and nitrogen fixation .................................................................................... 4 1.2.1.1 Rhizobium spp. ..................................................................................................... 4 1.2.1.2 The nodulation process ........................................................................................ 5 1.2.1.3 Lucerne symbiosis .............................................................................................. 12 1.2.2 Effects of other soil microorganisms on plant growth and nitrogen fixation by legumes .............................................................................................................................. 12 1.2.2.1 Mycorrhiza spp. ................................................................................................. 12 1.2.2.2 Trichoderma spp. ............................................................................................... 14 1.2.2.3 Pseudomonas spp. .............................................................................................. 14 1.2.3 Endophytic actinobacteria ........................................................................................ 15 1.2.3.1 Definition of endophyte ...................................................................................... 15 1.2.3.2 General characteristics of actinobacteria and endophytic actinobacteria ........ 16 1.2.3.3 Occurrence and diversity of endophytic actinobacteria in plants ..................... 19 1.2.3.4 Evidence of actinobacterial effects on agricultural plants ................................ 21 1.2.3.5 Potential mechanisms of action ......................................................................... 24 1.3 Summary, potential for legume improvement and critical knowledge gaps. ................... 28 1.4. Research plan (Objectives) ............................................................................................. 29 1.4.1 Aims of research ....................................................................................................... 29 1.4.2 Hypothesis ................................................................................................................. 29 1.4.3 Research plan ............................................................................................................ 29 Chapter 2 .............................................................................................................................. 31 ii General materials and methods .......................................................................................... 31 2.1 Legume seeds, rhizobia and actinobacteria ...................................................................... 32 2.2 Treatment of seeds and application of inoculants ............................................................ 33 2.2.1 Surface sterilisation of seeds..................................................................................... 33 2.2.2 Isolation, growth and application of actinobacteria ................................................. 33 2.2.3 Growth of Rhizobium ................................................................................................ 34 2.2.4 Plant growth media, nutrition, sowing and water supply ......................................... 34 2.2.5 Harvest of plants ....................................................................................................... 35 2.3 Data collection and analysis ............................................................................................. 35 2.4 Identification of endophytic actinobacteria by 16S rRNA gene amplification and sequencing.............................................................................................................................. 35 2.4.1 DNA extraction ......................................................................................................... 36 2.4.2 Quantify DNA concentration .................................................................................... 37 2.4.3 PCR 16S rRNA .......................................................................................................... 37 2.4.4 Clean up and sequencing the PCR products ............................................................. 38 Chapter 3 .............................................................................................................................. 39 Isolation and characterisation of actinobacterial endophytes from pasture legumes ... 39 3.1. Introduction ..................................................................................................................... 40 3.2 Materials and methods ..................................................................................................... 42 3.2.1 Source of legumes ..................................................................................................... 42 3.2.2 Isolation of endophytic actinobacteria ...................................................................... 42 3.2.2.1 Surface-sterilization procedure .......................................................................... 42 3.2.2.2 Media for isolation ............................................................................................. 43 3.2.2.3 Purification of actinobacterial endophytes ........................................................ 43 3.2.3 Characterisation of endophytic actinobacteria ........................................................ 43 3.2.3.1 Morphology (frequency and distribution) .......................................................... 43 3.3 Results and discussion ..................................................................................................... 44 3.3.1 Isolation of endophytic actinobacteria from legumes ............................................... 44 3.3.2 Morphological characterisation of actinobacteria ................................................... 47 3.4 Conclusion ....................................................................................................................... 49 Chapter 4 .............................................................................................................................. 50 Effects of actinobacteria on lucerne germination, seedling growth, and tolerance of soil- borne disease......................................................................................................................... 50 4.1 Introduction ...................................................................................................................... 51 4.2 Materials and methods ..................................................................................................... 54 iii 4.2.1 Effects of actinobacteria on lucerne germination ..................................................... 54 4.2.1.1 Lucerne germination on agar ............................................................................ 54 4.2.1.2 Lucerne growing in a sandy loam ...................................................................... 54 4.2.2 Inhibition of the growth of pathogenic fungi on agar ............................................... 55 4.2.3 Effect of actinobacteria on the development of root damage symptoms caused by Rhizoctonia solani .............................................................................................................. 56 4.2.3.1 Preliminary screening in Falcon tubes .............................................................. 56 4.2.3.2 Pot experiment in glasshouse ............................................................................. 58 4.2.4 Detection of plant growth promotion properties ...................................................... 59 4.2.4.1 Indole acetic acid (IAA) production ................................................................... 59 4.2.4.2 Phosphate solubilisation .................................................................................... 59 4.2.4.3 Siderophore production ..................................................................................... 60 4.3 Results and discussion ..................................................................................................... 61 4.3.1 Effects of actinobacteria on germination .................................................................. 61 4.3.2 Inhibition of pathogen mycelial growth on agar....................................................... 62 4.3.3 Effect of actinobacteria on root damage from R. solani ........................................... 64 4.3.3.1 Preliminary screening in Falcon tubes .............................................................. 64 4.3.3.2 Pot assay ............................................................................................................ 65 4.4 Conclusions ...................................................................................................................... 69 Chapter 5 .............................................................................................................................. 71 Effects of selected actinobacteria on the nodulation and growth of lucerne and its rhizobial partner (Sinorhizobium meliloti strain RRI 128) .............................................. 71 5.1 Introduction ...................................................................................................................... 72 5.2 Materials and methods ..................................................................................................... 73 5.2.1 Actinobacteria, rhizobia and lucerne ........................................................................ 73 5.2.2 Interaction of twelve endophytic actinobacteria with symbiosis of lucerne and rhizobia .............................................................................................................................. 75 5.2.2.1 In sandy loam ..................................................................................................... 75 5.2.2.2 Assessment of promising actinobacteria in sand and vermiculite media ........... 76 5.2.2.2.1 Microscopy ...................................................................................................... 76 5.2.2.2.2 Elemental composition and biochemical analyses .......................................... 77 5.2.3 Interaction of five actinobacteria on the growth of three rhizobial strains .............. 78 5.2.4 Effects of actinobacteria LuP30 and LuP47B on the growth of lucerne nodulating rhizobia strain RRI 128 ...................................................................................................... 79 5.2.4.1 Growth in Yeast Mannitol Broth ........................................................................ 79 iv 5.2.4.2 In sand and vermiculite ...................................................................................... 79 5.3 Results and discussion ..................................................................................................... 80 5.3.1 Effects of actinobacteria on lucerne growth and symbiosis ...................................... 80 5.3.1.1 Preliminary screening in a sandy loam ............................................................. 80 5.3.1.2 Validation of best actinobacteria in sand and vermiculite (low N) media ......... 81 5.3.2 Effect of actinobacteria on rhizobial growth ............................................................ 88 5.3.3 Effects of actinobacteria on the growth of the rhizobial strain RRI 128 ................... 89 5.4 Conclusions ...................................................................................................................... 91 Chapter 6 .............................................................................................................................. 93 6.1 Introduction ...................................................................................................................... 94 6.2 Materials and methods ..................................................................................................... 96 6.2.1 Actinobacteria, Rhizobium meliloti and lucerne seeds ............................................. 96 6.2.2 Growth of actinobacteria and rhizobia ..................................................................... 96 6.2.3 Plant growth media, nutrition, sowing and water supply ......................................... 96 6.2.4 Effects of actinobacteria on the growth and symbiosis of lucerne ............................ 96 6.2.5 General parameters measured and statistical analyses ............................................ 99 6.3 Results and discussion ................................................................................................... 100 6.3.1 Effect of actinobacteria on lucerne growth in absence of rhizobia ........................ 100 6.3.2 Effect of actinobacteria on lucerne growth when applied with rhizobia in presence of different levels of nitrogen ........................................................................................... 103 6.3.3 15N experiment ......................................................................................................... 110 6.3.4 Early effects of actinobacteria on nodulation of lucerne plants ............................. 113 6.4 Conclusion ..................................................................................................................... 117 Chapter 7 ............................................................................................................................ 119 Efficacy of LuP30 and LuP47B on other symbiotic associations (lucerne, sub-clover and serradella) and wheat ................................................................................................. 119 7.1 Introduction .................................................................................................................... 120 7.2 Materials and methods ................................................................................................... 121 7.2.1 Effects of LuP30 and LuP47B on plant growth of lucerne in two different soil extracts ............................................................................................................................. 121 7.2.1.1 MPN of two soil extracts .................................................................................. 121 7.2.1.2 Experimental design ......................................................................................... 122 7.2.2 Effects of LuP30 and LuP47B on nodulation of lucerne at different concentrations of rhizobia ............................................................................................................................ 123 7.2.3 Effects of actinobacteria on growth and symbiosis of sub-clover and serradella .. 124 v 7.2.3.1 Actinobacteria, rhizobia and seeds .................................................................. 124 7.2.3.2 Interaction of LuP30 and LuP47B on the growth of two different species of rhizobia ........................................................................................................................ 124 7.2.3.3 Experimental design ......................................................................................... 125 7.2.4 Nitrogen fixing ability, plant growth promotion and nitrogen use efficiency in non- legume, wheat .................................................................................................................. 126 7.3 Results and discussion ................................................................................................... 126 7.3.1 Different soil extracts containing rhizobia ............................................................. 126 7.3.2 Effects of actinobacteria on the growth and nodulation of lucerne plants inoculated with different concentrations of rhizobia ......................................................................... 130 7.3.3 Effects of LuP30 and LuP47B on clover and serradella ......................................... 131 7.3.3.1 Interaction tests between actinobacteria and the rhizobia .............................. 131 7.3.3.2 Response of sub-clover growth symbiosis to inoculation with by LuP30 and LuP47B ........................................................................................................................ 134 7.3.3.3 Response of serradella to inoculation with LuP30 and LuP47B ..................... 136 7.3.4 Effects of LuP30 and LuP47B on the growth of wheat ........................................... 137 7.4 Conclusion ..................................................................................................................... 139 Chapter 8 ............................................................................................................................ 140 Identification and characterisation of the Streptomyces spp. LuP30 and LuP47B ...... 140 8.1 Introduction .................................................................................................................... 141 8.2 Materials and methods ................................................................................................... 142 8.2.1 Morphological studies ............................................................................................ 142 8.2.1.1 Culture morphological characteristics ............................................................ 142 8.2.1.2 Scanning electron microscopy (SEM) of cellular and spore morphological characteristics .............................................................................................................. 142 8.2.2 Physiological and biochemical characteristics ...................................................... 143 8.2.2.1 Growth at various temperatures, pH, sodium chloride and utilisation of carbohydrates .............................................................................................................. 143 8.2.2.2 Hydrolysis of Gelatine ..................................................................................... 144 8.2.2.3 Hydrolysis of Esculin ....................................................................................... 144 8.2.2.4 Hydrolysis of Starch ......................................................................................... 144 8.2.2.5 Decomposition of Urea .................................................................................... 145 8.2.2.6 Decomposition of Casein ................................................................................. 145 8.2.2.7 Decomposition of adenine, xanthine and L-tyrosine: ...................................... 146 8.2.2.8 Catalase production ......................................................................................... 146 8.2.2.9 Use of organic acids ........................................................................................ 146 vi 8.2.3 Chemotaxonomy studies .......................................................................................... 147 8.2.3.1 DAP cell wall analysis ..................................................................................... 147 8.2.3.2 Sugar cell wall analysis ................................................................................... 148 8.3.3.3 Menaquinone analysis...................................................................................... 149 8.3.3.4 Fatty acid methyl ester (FAME) analysis......................................................... 150 8.2.4 Genomic studies ...................................................................................................... 151 8.2.4.1 Phylogenetic and genomic studies ................................................................... 151 8.2.4.2 DNA-DNA hybridisation .................................................................................. 151 8.2.5 In vitro N free living ability..................................................................................... 154 8.3 Results and discussion ................................................................................................... 154 8.3.1 Streptomyces strain LuP30 ..................................................................................... 154 8.3.1.1 Morphological characteristics ......................................................................... 154 8.3.1.3 Physiological and biochemical characteristics ............................................... 157 8.3.1.4 Chemotaxonomy studies ................................................................................... 160 8.3.1.5 Genomic characteristics .................................................................................. 160 8.3.2 Streptomyces strain LuP47B ................................................................................... 162 8.3.2.1 Morphological characteristics ......................................................................... 162 8.3.2.2 Physiological and biochemical characteristics ............................................... 165 8.3.2.3 Chemotaxonomy studies ................................................................................... 167 8.3.2.4 Genomic characteristics .................................................................................. 168 8.3.3 Growth of actinobacteria on nitrogen free media................................................... 170 8.4 Conclusion ..................................................................................................................... 170 Chapter 9 ............................................................................................................................ 172 Major findings and future directions ............................................................................... 172 9.1 Major findings ................................................................................................................ 173 The hypothesis is supported. The results showed that six endophytic actinobacteria act as biocontrol agents against a fungal pathogen, R. solani, and two of the six actinobacteria, LuP30 and LuP47B, also increased the plant growth and nitrogen fixation of lucerne with its rhizobial partner. ......................................................................................................... 173 9.1.1 Endophytic actinobacteria from legumes: genus, temperature, isolation media .... 173 9.1.2 Effects of endophytic actinobacteria on the plant growth and symbiotic function of lucerne ............................................................................................................................. 173 9.1.3 Antifungal and bio-control properties of actinobacterial endophytes .................... 175 9.1.4 Novel species ........................................................................................................... 175 9.2 Future directions ............................................................................................................ 176 vii 9.2.1. Understanding the roles of LuP30 and LuP47B involved in nodulation and nitrogen fixation processes ............................................................................................................. 176 9.2.2 Mechanism of LuP30 and LuP47B in biocontrol activity ....................................... 176 9.2.3 Efficacy of LuP30 and LuP47B on other symbioses and field trials ....................... 177 9.3 Publications (Conferences and Journals) ....................................................................... 177 Appendices .......................................................................................................................... 180 References ........................................................................................................................... 202 viii List of tables Title Page Table 1.1 Production and area harvested of legumes in the world from 1999 2 to 2009 Table 1.2 Australian lucerne hay production 2002 4 Table 1.3 Examples of endophytic actinobacterial genera described within 20 the past 10 years Table 2.1 Details of microbes used in the experiments 32 Table 3.1 Number of endopytic actinobacteria isolated from roots and 45 nodules of four legumes using different media and incubation temperatures Table 3.2 Number of Streptomyces and non-Streptomyces isolated from 48 roots and nodules of four different legumes in South Australia. Table 4.1 Effect of different isolate sources on germination of seeds and 62 early growth of lucerne Table 4.2 Number of endophytic actinobacteria showing antifungal activity 63 Table 4.3 Effect of selected actinobacteria isolates on pathogen mycelial 64 growth, IAA, phosphate solubilisation and siderophores Table 4.4 Number of endophytic actinobacteria showing biocontrol activity 65 Table 4.5 Effect of actinobacterial isolates applied to seed on the shoot and 66 root weight of lucerne seedlings growing in soil infected with Rhizoctonia solani AG8 Table 4.6 The 16S rRNA gene sequence similarity of the six actinobacteria 68 with their closest type cultures Table 5.1 Summary of some general properties of thirteen cultures used in 74 the first expermiment Table 5.2 Effect of endophytic actinobacteria on the growth (shoot and root 80 length and weight) and nodulation (number and weight) of lucerne plants inoculated with RRI 128 and harvested 7 weeks after planting on sany loam. Table 5.3 Effect of endophytic actinobacteria on the growth (shoot and root 82 length and weight) and nodulation (number and weight) of lucerne plants inoculated with RRI 128 and harvested 7 days after planting on sand and vermiculite. Table 5.4 Effects of endophytic actinobacterial isolates EN23, LuP30 and 86 LuP47B on the concentration (mg/kg) and total accumulation of nutrients (ug) in lucerne shoots harvested 7 weeks after planting Table 5.5 Effect of endophytic actinobacteria on the growth of three strains 88 of lucerne- rhizobia spread onto agar plates at two concentrations CFU/ml Table 6.1 Effect of endophytic actinobacteria (Streptomyces spp. EN23, 101 LuP30 and LuP47B alone or in combination with S. meliloti strain RRI 128) and soil N on lucerne shoot and root weight at 7 weeks after inoculation. Table 6.2 Effect of inoculation with Streptomyces spp. (alone or in 102 combination with rhizobia) on the concentration (mg/kg) and total accumulation of nutrients (mg or ug) in lucerne shoots at 4 and 7 weeks after inoculation. ix