Molecular Interactions of Endophytic Actinobacteria in Wheat and Arabidopsis

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the TRFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonisation were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in waterrepellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonisation within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species. Conn, V.M., and Franco, C.M.M. (2004). Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism. Applied and Environmental Microbiology 70, 6407-6413. Abstract: The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction ____________________________________________Appendix Six 253 fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonisation of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonisation by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonisation levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonisation level could be confirmed even though the indigenous endophyte population was not adversely affected. A.6.2 Conference Presentations 13 International Symposium on Biology of Actinomycetes 2003, 1-5 December, Melbourne, Australia Poster Title: Analysis of the Endophytic Actinobacteria Population in the Roots of Wheat (Triticum aestivum) in response to different field soils with and without the addition of microbial inoculants by Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Partial Sequencing of 16S rDNA. Abstract Introduction: Endophytes are microorganisms that live within healthy plant tissue causing no apparent disease symptoms. A number of the biologically active endophytes have been isolated and belong to the actinobacteria phylum (Coombs, 2002). Actinobacteria are high G+C, gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and anti-fungal compounds. It is well known that soil type, host plant, cropping practices, growth stage and other environmental factors are capable of affecting the microbial population present in the rhizosphere, however, the effect of soil type and microbial inocula on the endophyte population is not known (Smalla et al., 2001). Endophytic populations have been isolated and characterised primarily by cultivation-based methods, but as not all microorganisms can be grown in vitro, molecular techniques need to be used for microbial community analysis. Aim: The aim of this study was to analyse how the endophytic actinobacteriaIntroduction: Endophytes are microorganisms that live within healthy plant tissue causing no apparent disease symptoms. A number of the biologically active endophytes have been isolated and belong to the actinobacteria phylum (Coombs, 2002). Actinobacteria are high G+C, gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and anti-fungal compounds. It is well known that soil type, host plant, cropping practices, growth stage and other environmental factors are capable of affecting the microbial population present in the rhizosphere, however, the effect of soil type and microbial inocula on the endophyte population is not known (Smalla et al., 2001). Endophytic populations have been isolated and characterised primarily by cultivation-based methods, but as not all microorganisms can be grown in vitro, molecular techniques need to be used for microbial community analysis. Aim: The aim of this study was to analyse how the endophytic actinobacteria ____________________________________________Appendix Six 254 population in wheat roots change in response to cultivation in different field soils with and without microbial inocula using the terminal restriction fragment length polymorphism (T-RFLP) technique. Methods: Cultivation of wheat (cv. Krichauff) in three field soils, a soil with NutriLife 4/20 microbial inoculum, and in a field soil with added actinobacteria endophyte inoculants. The endophytic bacterial DNA was extracted from wheat roots and the endophytic actinobacteria population determined by T-RFLP analysis of 16S rRNA. Results: The endophytic actinobacteria population was significantly affected by the soil type. The highest diversity and level of endophytic colonisation was found in wheat grown in a dark loam (Swedes Flat) and the lowest in a non-wetting sand (Western Flat). Over 40 different endophytic actinobacteria genera were identified in three field soils, of these 10 genera were common in all soils. The addition of NutriLife 4/20 reduced the endophytic actinobacteria diversity from 40 genera to 21, and the endophytic colonisation by approximately half. In contrast the addition of a single endophyte inoculum by seed application has a very small effect on endophytic colonisation and diversity. Of 28 genera identified in the roots there was a change of colonisation levels in only six genera. Conclusions: The endophytic actinobacteria population of wheat is affected by the soil type. Soils that support a higher, indigenous microbial population result in higher endophytic actinobacteria diversity and level of colonisation. The addition of a mixed microbial inoculum to the soil reduces the natural endophytic actinobacteria population. It would appear microbes present in the NutriLife 4/20 inoculum out compete the indigenous actinobacteria microflora, preventing their access to the seed and ultimately endophytic colonisation. This is in contrast to the addition of a single actinobacteria endophyte to the wheat plant, where the population remained relatively stable. International Joint Workshop on PR-Proteins and Induced Resistance Denmark, Helsingor, May 5-9, 2004 Poster Title: Induction of Key Genes in the Systemic Acquired Resistance snd Jasmonic Acid/Ethylene Pathways of Arabidopsis Thaliana by Endophytic Actinobacteria. Conn, Vanessa; Franco, Christopher and Walker, Mandy* Department of Medical Biotechnology, Flinders University, Flinders Medical Centre, Adelaide, Australia, 5043 *CSIRO Plant Industry, PO Box 350, Glen Osmond, Adelaide, Australia, 5064 ____________________________________________Appendix Six 255 Abstract: Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Arabidopsis thaliana was used as a model system to investigate the mechanism of resistance. Real Time RT-PCR was used to determine if key genes in the Systemic Acquired Resistance (PR-1 and PR-5) and Jasmonic Acid/Ethylene (Pdf1.2 and HEL) pathways were induced. Coating the A. thaliana (Col-0) seeds with the endophytic actinobacteria, Streptomyces sp. (EN27 and EN28), Micromonospora sp. (EN43) and Nocardioides albus (EN46) induced a low level of PR-1, Pdf1.2 and HEL gene expression. The level of gene induction was significantly lower compared with plants infected with a pathogen. Infection with the bacterial pathogen Erwinia carotovora triggers the JA/Ethylene pathway and all endophytic actinobacteria tested were able to induce Pdf1.2 gene expression above the level detected in the wild-type infected plant, with EN43 and EN46 inducing the highest Pdf1.2 expression (6.5-fold increase over the wild-type infected plant). In comparison, challenge with the fungal pathogen Fusarium oxypsorum strongly induced the SAR pathway, with EN28 inducing PR-1 and PR-5 genes 86 and 21-fold over the wild-type infected plant, respectively. The endophytic actinobacteria appear to be able to prime both the SAR and JA/Ethylene pathways, upregulating genes in either pathway depending on the infecting pathogen. Application of the culture filtrate of the endophytic actinobacteria EN43 induced PR-1 and PR-5 in FL026 media, and Pdf1.2 and HEL in FL031 media suggesting that a secreted metabolite(s) or a cell factor(s) produced under different culture conditions are capable of priming the plant for pathogen infection. These results indicate the endophytic actinobacteria are capable of priming the plant when the spores are coated onto the seed before sowing, triggering induced systemic resistance. Inoculation with individual, or combinations of endophytic actinobacteria may provide a greater level of disease resistance in crops when used as biocontrol agents.