BIOCONTROL STRATEGIES AGAINST PLANT PATHOGENS

It is a known fact that the whole agriculture system is suffering from the diseases caused by plant pathogens, affecting negatively the crop yield production, food security, biodiversity, agricultural ecosystem and hence agricultural economy. In many countries, the containment strategies of plant pathogens are still depending on chemical pesticides that cause adverse effects in the long term. According to the implementations reinforced by European council 2009/128/EC, biocontrol strategies are considered as the most profound and integrated approach for sustainable disease management. Defining biocontrol in terms of plant pathology, it is the purposeful utilization of beneficial microbes, or its molecules, to suppress phytopathogens’ ability to colonize or induce symptoms in the host. In spite of their lesser shelf-life and unreliability as compared to conventional pesticides, their targeted biological interaction with the phytopathogens reduces the possibility of affecting non-target organisms, environment and the development of resistance in the pathogen. In this context, exploitation of bacterial endophytes has gained much attention during the past decades. Endophytic plant growth promoting bacteria (ePGPBs) mediate their biocontrol efficacy by targeting species through a multitude of direct or indirect biological interactions, often employing both modes of action, such as plant growth promotion, host’s resistance induction, allelochemicals secretion, and nutrients and niche competition. Another strategy that has gained popularity is the exogenous application of double stranded RNA (dsRNA), which is considered as the key trigger molecule of RNA interreference (RNAi), a post-transcriptional gene silencing mechanism, and has been shown to provide protection without the need for integration of dsRNA-expressing constructs as transgenes. In the present doctoral thesis, the above-mentioned biocontrol strategies were adapted, utilizing “ePGPBs as microbial inoculants” and “exogenously applied dsRNA as RNAi based natural product”, against several phytopathogens belonging to different families of viruses and fungi. Regarding ePGBPs as microbial inoculants, the objective of this study was to extend our understanding of five endophytic bacterial strains; Pantoea agglomerans (255-7), Pseudomonas syringae (260-02), Lysinibacillus fusiformis (S4C11), Paraburkholderia fungorum (R8), Paenibacillus pasadenensis (R16); that have shown a promising result in previous studies. In the present doctoral study, these strains were tested in planta to evaluate their role in providing plant growth promotion and broad-spectrum protection against two target pathosystems (viruses and fungi) that might have direct, indirect or simultaneous effects, proceeded with two following aims: (Aim 1) action against viruses: Cymbidium ringspot virus (CymRSV), Cucumber mosaic virus (CMV), Potato virus X (PVX), and Potato virus Y (PVY) on Nicotiana benthamiana plants, comparing their effects with those of three chitosan-based products, which are known to induce resistance in plants; and (Aim 2) action against fungal pathogens: Rhizoctonia solani, Pythium ultimum and Botrytis cinerea on Lactuca sativa plants, comparing their effects with Bacillus amyloliquefaciens strain (CC2) and Trichoderma spp. based product, under controlled conditions. To test the priming efficacy of ePGPBs against target viruses, several phenotypic parameters were observed along with the evaluation of three plant defense related genes (EDS1, PR2B and NPR1) on Nicotiana benthamiana plants. Interestingly, the symptoms reduction was successfully registered against CymRSV and CMV with increased heights of the plants. Some of the treatments were shown correlation between severity of symptoms and the virus concentration in the plants. Furthermore, the molecular interaction indicated the involvement of a salicylic acid (SA) mediated defense pathway as evidenced by the increased expression levels of EDS1 gene in strains R16 and 260-02. Whereas, strain S4C11 showed downregulation of PR2B gene, suggesting that SA-independent pathways could be involved. These findings opened queries regarding the duration of the protective effect, host-plant- pathogen interaction, and epidemiological implications of the use of similar biocontrol strains, that reduce the symptoms but not the concentration of virus in the host. To test the ePGPBs role against target fungi (pre- and post-harvest stage), several experiments were conducted including phenotypic paraments, gene expression analysis (PR1, PAL, ThlP3, ERF1 and ACCS1), microbiota analysis in bulk soil, rhizosphere, and root associated with Lactuca sativa in the presence or absence of the inoculants, and nutritional quality parameters at time of harvest and during shelf-life of Romaine lettuce. The results were accompanied in terms of symptoms reduction by strain R16 (P. ultimum, R. solani, B. cinerea) and strain 260-02 (R. solani, B. cinerea); % seed germination by strains R16, 260-02, 255-7, S4C11 in some healthy and R. solani infected lettuce varieties; inhibition of R. solani population in soil and rhizosphere soil by strains R16, 260-02 and 255-7. Furthermore, composition of the bacterial microbiota was radically different in the rhizosphere and the root endosphere among treatments, while the bulk soil formed a single cluster regardless of treatment, indicating that the use of these treatments did not have an ecological impact outside of the plant. Also, these strains were able to contribute to the maintenance of nutritional quality indexes of lettuce at harvest and during storage. All the obtained results indicated that these strains were involved directly (via antibiosis) and indirectly (via SA or ET/JA) in the observed reduction of symptoms. Particularly, strain R16 upregulated both PAL and ACCS1 gene in R. solani infected L. sativa (suggesting co-activation of SA- and JA/ ET mediated ISR resistance); strain 260-02 upregulated PAL gene in R. solani infected romaine lettuce and showed higher levels of ascorbic acid (AsA) production in B. cinerea infected romaine lettuce (suggesting the activation of SA- and AsA-mediated antioxidant resistance); and strain 255-7 triggered PAL and ThlP3 gene up-stream expression levels indicating SA mediated pathways in R. solani infected romaine lettuce. These findings affirmed the previous conclusions and added valuable pieces of information regarding the traits these ePGPB carried, most importantly, in individuating different mode of action of the different strains in different host plants with or without the presence of pathogen. Regarding the second approach, non-transgenic strategy was employed to induce resistance against Tomato Aspermy Virus (TAV) in N. benthamiana plants. DsRNA molecules for coat protein (CP) gene was produced by a two-step PCR assay followed by in vitro transcription and purification and was exogenously applied. The implementation of CP-derived dsRNA TAV was not successful in reducing observed symptoms (mosaics, blisters, crinkling, leaf distortion, and systemic vein clearing), regardless of treatments or days of post inoculation. Only a slight difference was found in plant heights indicating that the treatment managed to reduce stunted growth of the plant at dilution 01:10 (6 and 12 dpi). The reasons could involve inappropriate concentration of dsRNA inoculum. Therefore, future studies will be conducted to optimize in vitro dsRNA molecules production to obtain higher concentrations or more specific sequences, and more suitable viral genes. Both strategies have shown interesting outcomes and gave us the future direction which will help us in designing the adequate trials (in planta or semi-field) for the disease management and diseases control through the application of ePGPBs as a microbial inoculants and dsRNA-based product individually or in combination.