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Discovery of a Paenibacillus isolate for biocontrol of black rot in brassicas A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by Hoda Ghazalibiglar Lincoln University 2014 DECLARATION This dissertation/thesis (please circle one) is submitted in partial fulfilment of the requirements for the Lincoln University Degree of ________________________________________ The regulations for the degree are set out in the Lincoln University Calendar and are elaborated in a practice manual known as House Rules for the Study of Doctor of Philosophy or Masters Degrees at Lincoln University. 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Candidate ________________________________________ Date ______________________ Supervisor ________________________________________ Date ______________________ Or: 2 Parts of this dissertation/thesis (please circle one) have been submitted and/or accepted for publication in advance of submission of the dissertation/thesis (please circle one) for examination. In this case, please set out on a separate page information on: • which sections have been submitted, which have been accepted and which have appeared; • which journals they have been submitted to; • who are the co-authors. Candidate ________________________________________ Date ______________________ Supervisor ________________________________________ Date ______________________ Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy. Abstract Discovery of a Paenibacillus isolate for biocontrol of black rot in brassicas by Hoda Ghazalibiglar Black rot, one of the most devastating diseases of brassicas worldwide and a major problem for New Zealand’s seed industry, is caused by the seed borne bacterium, Xanthomonas campestris pv. campestris (Xcc). The pathogen can spread rapidly from plant to plant during the growing season and high losses in both yield and quality can subsequently occur in vegetable and forage brassicas. Currently, there is no effective control method. Biological control may offer an option for control of this seed borne disease. Potential microbial biocontrol agents were isolated from commercial cabbage and rape seed lots as part of the “Smart Seeds for Export” research programme at the Bio-protection Research Centre and one of the promising microbes was the bacterium Paenibacillus. This research therefore addresses the use of Paenibacillus applied as a seed treatment for biological control of black rot on cabbage. Based on dual culture assays, 24 isolates of Paenibacillus were categorized for their interactions with Xcc. Eight of these isolates with different bioactivity in suppression of Xcc in vitro were then screened for their capacity to reduce black rot symptoms on cabbage in pot trial assays. From these results one Paenibacillus isolate (P16), at the concentration of 5 × 109 CFU/ml was selected as a potential biocontrol agent. To investigate if the disease control was provided via plant growth promotion, the P16 isolate was co-applied with Xcc as a seed treatment. In the presence of Xcc, P16- treated seedlings had significantly (P ˂ 0.05) greater growth parameters including root length, leaf area, and root and shoot dry weight compared to the control. However, there was no significant difference in plant growth parameters between P16-treated seedlings and the control in the absence of the pathogen. This suggests that the P16 isolate enabled plants to survive and grow normally by reducing Xcc infection. A real-time PCR method was developed to facilitate studies on the population dynamics of the P16 isolate on the seed, developing seedling and in the surrounding soil environment. For this purpose a P16-specific Primer set was designed based on the gyrB region with the highest discriminatory area. The specificity, sensitivity and reliability of the real-time PCR to detect and iii quantify P16 were confirmed. Endophytic activity and rhizosphere competency of the P16 isolate were assessed. P16 was recovered from cabbage seedlings grown from P16-treated seeds (1.5 × 107 CFU/seed) and also their rhizosphere and bulk soil using the developed real-time PCR assay. Standard curves were conducted for soil and plant samples individually, and the detection limits of 1 × 103 CFU/g of dried soil or plant were determined for both substrates. P16 was not recorded in plant samples, indicating either that the BCA is not endophytic or its density in the plant was below the detection limit. In rhizosphere soil, P16 density had decreased from 9.9 × 105 to 1.1 × 103 CFU/g by 11 days after sowing (DAS), and thereafter it was below the limit of detection. A P16 population in the bulk soil was only detected up to 6 DAS. Overall, the P16 isolate is most probably not endophytic and is rhizosphere competent only during early cabbage seedling growth. Induced systemic resistance as a mode of action of the P16 isolate in suppression of black rot in cabbage was studied. The P16 isolate was applied as a seed treatment and 2- and 4-week old plants were challenged with Xcc. A significant (P ˂ 0.05) reduction in disease severity of P16-treated seedling was observed compared to the non-treated control when Xcc was injected into 4-week old seedlings. As the biocontrol agent and the pathogen were spatially separated, induced systemic resistance would appear to be the mode of action. However, a study of seven defense-related genes showed no differential gene expression in P16- protected seedlings in response to Xcc challenge. P16 appeared to prime plants in response to wounding. This study has provided a starting point for further research at the molecular level to better understand the apparent systemic resistance induced by P16 in cabbage in response to Xcc infection. Keywords: Biocontrol agent, Paenibacillus, black rot, brassicas, plant growth promotion, rhizosphere competence, endophytic activity, induced systemic resistance, real-time PCR. iv Acknowledgements I would like to take this opportunity to thank all those who have contributed in any way to the completion of this research. I wish to express my gratitude and respect to my main supervisor, Prof. John Hampton for giving me the opportunity to work on this project. John, I definitely loved your supervisory style; trusting in me and leaving enough space for me to explore problems encountered in the research, at the same time offering invaluable guidance and advice. Special thanks to you for being a person open to ideas and for encouraging and helping me to shape my interests. This study would not have been as smooth and pressure free without your support, patience and enthusiasm. My sincere thanks also go to my co-supervisor, Dr. Andrew Holyoake who helped me to understand the molecular techniques and find the answers to my endless questions in a different way. Special thanks to him for allowing me to grow as a research scientist. His encouragement, criticism and support have been greatly appreciated. I would like to thank my advisor, Dr. Eline van Zijll de Jong for all her precious advice, continuous support and friendship which kept me motivated throughout my study. She was always open to discussion and her attitude to research inspired me to work tirelessly through all the ups and downs of the study. I could not have imagined having a better advisor at both an academic and personal level, for which I am extremely grateful.