Biological control of bacterial wilt disease caused by <em>Ralstonia solanacearum</em>: Insights into the ecology of antagonists and their complex interaction with the pathogen and the host plant rhizosphere microbiome

Abstract

Plant diseases are among the most limiting factors in agricultural production all over the world, particularly when the causal agent is characterized as quarantine pathogen. One of the most devastating diseases is bacterial wilt of potato and tomato caused by Ralstonia solanacearum (Rs) which is regarded as an epidemic soil-borne quarantine pathogen. The persistence and survival of Rs in soil for years and its ability to colonize a huge number of hosts and non-host crops poses a huge agricultural threat. In order to cope with such pathogen, investigations into the microbial diversity and suppressive potential in the soil and hizosphere of potential host plants are fundamental. In this context, we elucidated the effect of the soil types (DS, LL and AL) and the potato plant sphere (rhizosphere, endorhiza, endocaulosphere; tuber ecto- and endosphere) on the proportion and diversity of in vitro antagonists towards Rs. For this purpose, potato plants were grown under field conditions in three soil types that were stored at a unique experimental field site for more than ten years under the same weather conditions and cropping history. Screening equal numbers of dominant randomly picked colonies from each sphere for in vitro antagonistic activity towards Rs showed that in all spheres antagonists were detected among the dominant cultivable bacteria and that the highest proportion of antagonists was observed in the endophytic compartments. While several genotypes were detected only in one soil, other genotypes were isolated from two or three soils and in some cases with a preference to a particular soil. Based on these results, it is possible to conclude that both, plant sphere and soil types, affect the diversity and proportion of bacteria with in vitro antagonistic activity towards Rs. More antagonists with beneficial characteristics were isolated from plants grown in DS soils. However, to get more insights into the effect of the three soil types on the bacterial community associated with different plant spheres, denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from total community DNA (TC-DNA) was employed to compare the bacterial communities associated with the five spheres of potato plants grown in the three soil types. Our results showed that, based on the soil types, bacterial communities associated with potato plant spheres differed significantly only in the rhizosphere and in the soil attached to tuber while the endophytic bacterial communities were more variable and less influenced by the soil type. Additionally, bacterial community composition significantly differed between different potato spheres of the plants that were grown in the same soil type indicating niche-specific bacterial communities. Furthermore, our results indicated that the effect of the plant sphere was more pronounced than that of the soil type. Most of the antagonists obtained in our first study (Chapter 2) were affiliated with the genus Pseudomonas which is often enriched in the rhizosphere due to their good rhizosphere competence as well as their ability to rapidly respond to root exudates and to produce multiple compounds with plant beneficial activity. We explored the influence of the three soil types on the abundance of Pseudomonas populations and genes related to beneficial traits in different potato plant spheres. Pseudomonas community composition associated with potato plant spheres was analyzed by DGGE of Pseudomonas-specific 16S rRNA gene fragments amplified from TC-DNA and Pseudomonas-specific real-time PCR as influenced by the soil types and/or potato plant spheres. PCR-Southern blot hybridization was used to investigate the prevalence and distribution of Pseudomonas-specific genes encoding biological control and plant growth promoting related functions (phl, phz, prnD, hcnBC, pcbC, pqqE and gacA) in different plant spheres and soil types. The Pseudomonas community composition and abundance was significantly influenced by the soil type. The effect of soil types was only pronounced in the bulk soil and the soil tightly attached to the yield tubers. Both soil type and plant sphere had a very distinct effect on the distribution of Pseudomonas functional genes. Southern blot hybridization indicated the highest abundance of all tested genes was detected in the rhizosphere of potato plants grown in the LL soils, except for the 2,4-diacetyl phloroglucinol (phl). Our study indicated that Pseudomonas populations carrying the tested genes correlated with the type of soil and the enrichment of these genes, depended on the soil type-dependent rhizosphere effect. Finally, three greenhouse experiments with tomato plants were performed to evaluate the rhizocompetence of in vitro antagonists and their ability to control Rs. The population densities of Rs and antagonists were estimated by selective plating and in TC-DNA by means of real-time PCR and fliC gene PCR-Southern blot hybridization. Bacillus vallismortis (B63) and Pseudomonas brassicacearum (AL2YTEN-142)-inoculated plants showed a pronounced delay or no disease symptoms and significantly decreased in Rs population compared to the uninoculated plants. Amplicon sequencing of 16S rRNA gene fragments amplified from TC-DNA revealed pronounced treatment-dependent shifts in bacterial communities of the tomato rhizosphere. Most important, the strong reduction of Rs in the presence of the antagonists was confirmed and dynamic taxa in response to Rs or the inoculants were identified. Confocal laser scanning microscopy uncovered colonization patterns of the AL2YTEN-142. Gfp-positive cells were detected in lateral roots, root hairs and epidermal cells and within xylem vessels. Both inoculants hold great promise to control Rs under field conditions. In conclusion, this thesis provides deeper insights into the influence of different soil types and plant microenvironments on total bacterial and Pseudomonas communities regarding their composition and functional diversity, paying particular attention to the diversity and abundance of antagonists of Ralstonia solanacearum. A selection of antagonists was tested for in planta biological control of Rs, and the complex interactions between antagonists, pathogen and indigenous rhizosphere microbiome were investigated. In future studies, the biological control of Rs and the plant growth promotion will be tested on potatoes under field conditions in Egypt.

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