Isolation, Characterisation and Identification of Plant Growth Promoting Bacteria exhibiting activity against Fusarium pseudograminearum A thesis submitted in fulfilment of the requirements for the degree of Master of Science (Applied Biology & Biotechnology) by NARESH TALARI School of Science College of Science, Engineering and Health RMIT University DECLARATION I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed. Naresh Talari June 2017 2 ABSTRACT The main constraints to Australian chickpea and wheat production include several factors such as drought, biotic and abiotic stresses such as crown rot, salinity and cold; which totally contribute to losses of 10-70%. It has been found that there are practices that are helpful in controlling these stresses, such as the tolerant varieties, pesticides and crop rotation, transgenic crops and conventional breeding techniques, but these methods are not completely successful. It can be thus said that new methods need to be developed in order to minimise the biotic as well as abiotic stresses in chickpea.
Plant growth promoting bacteria (PGPB) potentially represent one such novel approach and are the focus of this research. The generally perceived mechanisms of PGPB which result in reduced plant stress include competition (with a plant pathogen) for an ecological niche, secretion of inhibitory bioactive compounds, and secondary metabolic induction of systemic resistance in the plant host to a range of soil born-pathogens and abiotic stresses. The current study focuses on the potential use of PGPB to enhance the tolerance of chickpea and wheat to crown rot caused by Fusarium pseudograminearum. Two strains, NM-12 and NM-33, identified as Bacillus subtilis and Stenotrophomonas rhizophila were isolated from the rhizosphere soils in Victoria, Australia (Perry Bridge).
The beneficial bacterium, Bacillus subtilis and Stenotrophomonas rhizophila were analysed for their direct plant growth promoting effects. Direct antagonistic effect on Fusarium pseudograminearum was demonstrated by a dual culture assay and culture filtrate assays together with estimation of spores and fungal biomass dry weight in vitro. 3 To identify the mechanisms underlying the inhibition of the fungus by the two isolates, the bacterial exudates were assessed for the presence of a range of potential antifungal products, including lytic enzymes, hormones, antibiotics and other secondary metabolites. Strain NM-12 was shown to produce indole acetic acid (IAA) at different concentrations even at 6% salt concentration.
In comparison, no IAA production was observed by strain NM-33. Further, siderophore production was moderate under control conditions but significantly increased at high salt concerntration (6%). In contrast, β- glucanase production was observed under normal as well as high salt concentrations. Interestingly, NM- 12 which exhibited enhanced ability to suppress the fungal pathogen was found to possess genes encoding cyanide production and 1-Aminocyclopropane-1- carboxylate (ACC) deaminase both of which are indirectly responsible for plant growth promotion.
In conclusion, the two bacterial isolates, Bacillus and Stenotrophomonas were found to be capable of promoting growth and improving the survivability of chickpea and wheat plants exposed to crown rot. These findings could be potentially extended to other crops to improve crop productivity under biotic stress. 4 Dedicated to my Mother 5 ACKNOWLEDGEMENTS I would like to express sincere gratitude and appreciation to my supervisors Dr Nitin Mantri and Prof Andy Ball. Their patience, motivation and immense knowledge guided me to successful completion of this project.
They helped improve my experimental design, analysis, scientific thinking and academic writing to a great extent. I would like to thank the financial support from Royal Melbourne Institute of Technology (RMIT University) that covered my tuition fees and funding for the project. I would like to express my gratitude to the staff and students working with me in the laboratory. Dr Lisa Dias provided generous help on all aspects of my research.
I would like to express my thanks to Dr Esmaeil Shahsavari for helping with the operations of laboratory equipment and in demonstrating basic laboratory techniques. Also, I would like to express my gratitude to Dipesh Parekh for his generous help in terms of research advice, thesis editing and submission. Finally, I would like to thank all my family members for their concern and support.1 Background and Aims .2 Research Focus and hypotheses of the thesis. 17 2 Chapter 2: Review of Literature .2 Importance of Wheat and Chickpea in Australia .3 Crown Rot Disease and its Pathogen .4 Current Management of Crown Rot .5 Economic Loss across the Globe and to Australia .6 Plant Growth Promoting Bacteria (PGPB) .3 Modulating the levels of phytohormones .1 Production of Antibiotics and Lytic Enzymes .2 Production of Siderophores.
34 3 Chapter 3: Isolation, screening, selection and identification of plant growth promoting bacteria .2 Materials and Methods .1 Chemicals and Raw materials .4 Preparation of different media to obtain maximum recovery of PGPB during isolation .5 Rapid isolation and in vitro screening of effective bacteria .6 Selection of antagonistic bacteria by dual culture assay .7 Identification of selected bacteria .8 Culture filtrate assay .9 Antifungal activity in broth.10 Antagonistic activity by fungal biomass dry weight .3 Results and Discussion .2 Dual culture assay for identification of antagonistic bacteria .3 16S rRNA sequencing to identify the two antagonistic bacterial strains 53 Strains .4 Culture filtrate assay on agar plates .5 Antifungal activity assessed using by cell count in broth using a haemocytometer .6 Antagonistic activity by fungal biomass dry weight. 63 4 Chapter 4: Characterization of plant growth promoting bacteria in terms of secondary metabolite production .2 Materials and methods .1 Indole acetic acid (IAA) assay .4 Volatile components assay.5 Identification of ACC deaminase and HCN producing genes .3 Results and Discussion .4 Production of volatile compounds. 86 5 Chapter 5: General Discussion and Conclusions. 98 9 Table of Figures Figure 2.1: Symptoms of Fusarium Crown rot in Chickpea and Wheat (Boucher et al.2: Overview of the mechanisms of biocontrol (Beauregard et al.1: Rapid isolation of plant growth promoting bacteria plates (A) and B) Bacterial and fungal colonies.
Red circle shows the small zones of clearance within the plate, C) Isolation plate without fungal spores, D) Established pure bacterial strains, E) Agar slants for culture maintenance) .2: Dual culture assay of bacterial isolates and Fusarium pseudograminearum on potato dextrose agar plates .3: Suppression of fungal growth by bacterial isolates NM-12 and NM-33. radial growth of fungus in presence and absence of the bacterial isolates, B. Percent inhibition of fungus after 7 days incubation at 28oC) .4: PCR gel image of 16s rRNA amplification .5: Phylogenetic tree obtained from Clustal W.6: Effect of bacterial cell culture filtrates on the growth of Fusarium pseudograminearum in PDA (A. NM-12 cell filtrate with fungus & control (fungus only), B.
NM-33 cell filtrate with fungus & control (fungus only).7: Influence of the culture filtrate from bacterial isolates NM-12 and NM- 33 on fungal growth (A. Radial growth of fungus after seven days in presence and absence of cell filtrates, B. Percent inhibition of fungus after 7 days incubation at 28oC) .8: The antifungal activity of the two bacterial isolates NM-12 and NM-33 and a mixture of both strains during co-culture in liquid medium for 24 h. fungal spores (A) and bacterial cells (B) were enumerated as number per mL.9: Determination of the antifungal activity of the two bacterial isolates, NM-12 and NM-33, and a mixture of both strains by co-culture with fungus in liquid medium for 24 h.
fungal growth is expressed as g/ml dry weight.1: Oxidation of IAA .2: Candidate strain plate inverted on the fungal plate and double taped with paraffin .3: IAA production by NM-12 and NM-33 isolates under normal growth conditions.4: Influence of salt on IAA production (µg/mL) by strain NM-12 .5: The calibrated graph according to the standard OD values: .6: Siderophore production (%) by selected isolates, NM-12 and NM-13 under normal and stressed (saline) conditions .7: Glucose production under normal and saline conditions as seen by the development of red colour in NM-12 AND NM-33 tubes compared to the control (DNS) .8: A comparison of β- glucanase production (units) by the two bacterial isolates, NM-12 (blue) and NM-33 (red) under normal and saline conditions .9: Standard graph with concentration on X-axis and OD values on Y-axis .10: Percentage inhibition of Fusarium pseudograminearum growth on PDA plates by volatile compounds from bacterial isolates NM-12 and NM-33 .11: PCR gel image showing amplification of both HCN and ACC deaminase producing gene s in NM-12. 89 11 List of Tables Table 3.1: Composition of Soil extract agar .2: Composition of Tryptone soy agar .3: Composition of Nutrient agar .4: Primers used for PCR amplification of bacterial and fungal genes .5: Composition of Potato Dextrose agar (PDA) .6: Composition of Glucose Yeast (GY) extract .7: 16S rDNA sequences (5’- 3’) of bacteria isolated from Australian soils54 Table 4.1: Composition of Starch-casein broth (SCB) .2: Preparation of standards for IAA estimation .3: Composition of King’s B broth .4: Composition of Tryptone soy agar .5: Preparation of glucose standards .6: Composition of Bennet agar .7: Composition of Potato dextrose Agar .8 Absorbance values of cultures producing IAA at 530nm .9 Standard OD values .10: OD values of the two strains at 530nm .11: Standard OD values .1 BACKGROUND AND AIMS Fusarium crown rot (FCR) is a severe chronic cereal disease that infects the crown, basal stems and root tissues. It has recently become a common disease among cereals grown in Australia and worldwide. This is because the moist conditions at the beginning of the season enable the fungus to grow from infected stubble to an adjacent seedling (Hogg et al.
FCR is found in all the semi-arid regions that exist around the world (Chakraborty et al. It is caused by several Fusarium sp. Several studies reported that F. pseudograminearum is the common fungus that is generally related to the crown rot as observed in New South Wales and Queensland, Australia (Akinsanmi et al.
All the wheat and chickpea cultivating regions in Australia are affected by this disease and it is estimated that annual losses due to crown rot are $80 million and $30-$60 million for wheat and chickpea, respectively (Verrell, 2016). Studies show that 35% of wheat crop yield loss in the Pacific Northwest of USA is due to crown rot (Smiley et al. Apart from yield associated loss, FCR infected plants may produce mycotoxins in the grains that are detrimental to human health (http://www. It is therefore crucial to control FCR pathogen in the field.
Fungi, viruses, nematodes and bacterial are the most commonly observed causes of diseases in agricultural plants. Some species of fungi are known to cause important plant diseases and increased loss of agricultural crops. Plant pathogens need to be controlled to maintain the average level of yield both, quantitatively and qualitatively. Farmers often rely heavily on using chemical fungicides to control 13 these plant diseases.
However, the environmental problems surrounding the widespread use of the chemicals including synthetic fungicides have led to public concern towards the use of synthetic pesticides in agriculture. Extensive use of chemical pesticides and fungicides has become a major environmental threat; for example, the use of fertilizers, pesticides and fungicides is one of the main drivers of species extinction, leading not only to a reduced global biodiversity but also to significant changes in ecosystem dynamics (Aktar et al. Despite these disadvantages, the use of agrochemicals continues to be an invaluable and powerful method to control plant disease. However, due to the negative impacts of the application of these chemicals, there is a research drive to develop sustainable, microbial-based biocontrol agents as an alternative or supplement to agrochemicals.