1 SYMBIOTIC AGRICULTURE: INCREASING KNOWLEDGE ON THE MODE OF ACTION OF BENEFICIAL MICROORGANISMS A thesis Submitted to the University of Modena and Reggio Emilia For the Degree of Doctor of Philosophy In the faculty of AGRI-FOOD SCIENCES, TECHNOLOGIES AND BIO- TECHNOLOGIES (Plant Pathology) Vurukonda Sai Shivakrishnaprasad Reggio Emilia, Italy, 2016-2019 2 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revision, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. 3 ABSTRACT Bacteria that are beneficial to plants are considered to be plant growth- promoting bacteria (PGPB) and can facilitate plant growth by a number of direct and indirect mechanisms. Non-pathogenic, soil microbes that occupy the rhizosphere can influence plant growth and induce changes in the plant’s physiological, chemical, metabolic, molecular activities, influencing plant-microbe interactions with abiotic and biotic stressors. Plants colonized by these microbes express unique plant phenotypes that show increased root and shoot mass, enhanced nutrient uptake, and stress mitigation. Additionally, the microbes may fix nitrogen and phosphate or produce siderophores for plant use. Among the plant-associated microbes, plant growth-promoting rhizobacteria (PGPR) are the most commonly used as inoculants for biofertilization. Plant growth-promoting rhizobacteria are non-pathogenic, free- living soil and root-inhabiting bacteria that colonize seeds, root tissue (endophytic/epiphytic), or the production of root exudates. In addition to these adaptations, PGPRs may utilize other mechanisms to facilitate plant growth including IAA synthesis, siderophore production, phosphate solubilization activity, ammonia production, and antifungal and antibacterial compounds production. Plant growth-promoting bacterial endophytes employ similar plant growth promotion mechanisms to those used by rhizospheric PGPB. In fact, bacterial endophytes are PGPBs that go one step further and colonize the inside of the plant tissues and provide more efficient and prompted protection to their hosts compared to those that bind exclusively to the plant’s rhizosphere. Therefore, it is likely that endophytic plant growth-promoting bacteria will be superior to similar non- endophytic bacterial strains in promoting plant growth under a wide range of environmental conditions. In the present study, Chapter I describes a set of beneficial plant growth promoting strains were evaluated for their in vitro plant growth promoting traits and 4 antagonistic activity on various phytopathogenic bacteria and fungi. We identified plant growth-promoting endophytes within the bacterial groups as part of the core bacterial consortium. Among all the strains tested, two prospective isolates Streptomyces sp. strain SA51 and Pseudomonas sp. strain PT65 used in the present study were extensively characterized to evaluate their in vitro plant growth promoting (PGP) traits and their biocontrol activity. Here, we characterized both the strains SA51 and PT65 for their colonization ability, plant growth promotion and protection against tomato spot disease caused by Xanthomonas vesicatoria on tomato (Solanum lycopersicum) as model plant. In this study, direct inhibitory action against X. vesicatoria by the bacterized tomato plants showed significantly good plant growth, as compared to unbacterized controls. Protection against X. vesicatoria by the bacterized tomato plants was confirmed in the greenhouse: disease was reduced by approximately 96%. Additionally, plants bacterized by strain SA51 showed significant plant growth, particularly in aerial parts as compared to non-bacterized controls. Finally, benefit was seen in inoculated healthy plants in terms of a significant increase in dry weight and length of roots and shoots, as compared to the uninoculated controls. A GFP mutant of strain SA51 was produced to study its endophytic colonisation in tomato plants: results confirmed that SA51 was able to efficiently colonise tomato endophytically, from the roots to the leaves. Field experiments confirmed the ability of strain SA51 to act as plant growth promoting agent: such promoting activity was also reflected into an increase of fruit production by approximately 7%. Furthermore, we performed whole genome sequence (WGS) analysis for the strain SA51, which provided in detailed properties of the strain metabolic profile using the Kyoto Encyclopedia of Genes and Genomes (KEGG), thus providing evidence for the presence of genes involved in the pathway for indole alkaloid biosynthesis and in iron transport and metabolism, together with genes coding for proteins acting in the regulation of iron homeostasis. At the same time, based on RAST annotations, we provided evidence for the presence of genes and operons related to 5 metal transporters and antibiotic biosynthesis, suggesting that SA51 could be involved in the biological control of plant pathogens and/or in the reshaping of the soil microbiota. In chapter II, we have tested a set of microbes as microbial consortium (a set of prospective microbes) for biocontrol, biochemical and differential gene expression as compare to control (un-inoculated) in grapevine plants. Since plant growth promotion is a multigenic process under the influence of many factors, an understanding of these processes and the functions regulated may have profound implications. The gene expression changes, represented by different time points hours post inoculation (hpi) have been studied to gain insight into various genes responsible for pathogen related (PR) proteins, lytic enzymes, growth hormones and to maintain cell wall integrity assisted plant growth promotion of grapevine leaves. It was observed that the microbial consortium profusely induced the upregulation of grapevine genes involved in maintenance of biocontrol and plant growth promotion activity. Almost all the genes were downregulated initially after 0 hpi and 2 hpi, but later from 4 hpi genes like ACC, CHS, PAL & PER were significantly upregulated. Particularly, PR11 and PR12 genes were significantly upregulated after 4 hpi. In case of biochemical aspects microbial treatments has increased the quality in terms of pigmentation and stability towards oxidation. Microbial consortium also tested for the biocontrol activity of two diseases like Flavescence Dorée and Esca. In case of Flavescence Dorée, results were not satisfactory and no difference in disease progression and quantity was observed between treated and untreated plots. This can be explained with the lack of vector control. Whereas, in case of Esca, experiments showed a remarkable effect of sprays with the microbial consortium in slowering disease progression. Since grapevine is a multiannual crop – a vineyard may last over 30 years – a continuous disease slowering may have a positive impact in grapevine longevity and productivity 6 In summary, we were able to confirm the ability of single beneficial microbes and a microbial consortium to act as promoting factor for plant growth and health; this study was done in vitro and in planta. 7 ACKNOWLEDGEMENTS I would like to acknowledge many people who helped me throughout the process of completion of my thesis in many ways. Without their help, it would have not been possible to successfully accomplish this degree. First and foremost, I would like to thank my supervisor Prof. Emilio Stefani who has given me the opportunity to work in his laboratories by accepting me as graduate student. I deeply appreciate his tireless and dedicated guidance throughout my research period. His motivation will remain a great energy source for me in the future as well. Many people with excellent technical expertise made our project possible from mere idea to workable and reproducible piece of work. For this, my greatest gratitude goes to Dr. Davide Giovanardi, for taking care of my experimental procedures and co- tutoring me for some time, Prof. Stefano Cassanelli, who helped me in designing qPCR experiments and analysing results, to Prof. Mauro Mandrioli, for the help with bioinformatics and timely guidance, and to my colleague Dr. Noel Mekam who helped me a lot in field studies and Irem Altin for her help in lab as a wonderful lab mate for being good friend. I am grateful to my professors Dr. Sk.Z. Ali and Dr. V. Sandhya Rao (Agri Biotech Foundation, India) who has motivated me towards research and to pursue Ph.D., without their support and guidance this degree would not be possible. I would also like to thank our Ph.D. School faculty and my other colleagues, my special thanks to Prof. Alessandro Ulrici, Prof. Andrea Pulvarenti, Prof. Luisa Antonella Volpelli, Prof. Maria Gullo and Dr. Luciana De Vero. I would also thank CCS AOSTA Srl. company for providing funds for the Ph.D. programme and their great support. 8 I deeply appreciate and thank my external examiners, Dr. Jeffery B. Jones (University of Florida, USA) and Dr. Subramaniam Gopalakrishnan (ICRISAT, INDIA) for taking their precious time in reading and reviewing my thesis. I would also like to thank my friends Nihar Sahu, Dr. Divya Dhanasekar, Dr. Mounika Nagabhairava, Bindu Prathyusha, Dr. Shrey Bodhankar, Manjari Shrivastava and Bindu Sunkar for being part of my journey to achieve doctoral degree. Of course, without thanking my family this journey would not be possible. 9 LIST OF CONTENTS Authors Declaration 2 Abstract 3-6 Acknowledgements 7-8 List