Interactions of fungal pathogens and antagonistic bacteria in the rhizosphere of Brassica napus
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The rhizosphere is an active interface where plants and microorganisms (pathogenic, beneficial and neutral) establish a complex and varied molecular dialogue, however knowledge of the functional mechanisms mediating interactions is still limited. Plants invest a significant proportion of their photosynthetically fixed carbon in maintaining the rhizosphere microbiome via root exudation and in return beneficial microbes provide profitable functions to the plant. The potential of naturally occurring soil microorganisms to control phytopathogens and to promote plant growth is well documented, but the functional mechanisms governing the reciprocal signaling between microbial communities and plants are not well understood. The aim of the studies described in this thesis was to gain insight into the functional basis of interactions between the fungal root pathogen Rhizoctonia solani and root associated antagonistic bacteria of the genus Serratia in the rhizosphere of Brassica napus.
Transcriptomic responses of the oilseed rape pathogen R. solani, to the plant-associated and pathogen- antagonistic bacteria Serratia proteamaculans S4 and S. plymuthica AS13, were studied using RNA-sequencing. The results demonstrate a major shift in the fungal gene expression with simultaneous alterations in primary metabolism, activation of defense and attack mechanisms and distortions in hyphal morphology.
Stable isotope probing coupled with high throughput sequencing allowed the description of the composition of bacterial and fungal communities in the rhizosphere soil and the roots of B. napus and the identification of active taxa capable of assimilating recently fixed plant carbon. Our results support the idea of active selection of microbial communities from the more diverse rhizosphere environment by the roots. Furthermore, the data confirm the potential of some active genera (Streptomyces, Rhizobium, Clonostachys and Fusarium) to be used as microbial inoculants for improved productivity and health of oilseed rape.
Patterns of gene expression in B. napus exposed to factorial combinations of R. solani and S. proteamaculans S4 were examined in-vitro using RNA-sequencing. Plants inoculated with R. solani only were almost dead at 240h post-inoculation and massive transcriptional reprogramming was observed, whereas the presence of S4 modulated the transcriptional responses and resulted in healthy plants. With R. solani present, we observed an interplay between stress and defense involving salicylic acid, jasmonic acid, ethylene and abscisic acid as common regulators. Induced systemic resistance when S4 present potentially depends on jasmonic acid, auxin and salicylic acid. Downregulation of stress-related and upregulation of defense-related genes were associated with transcriptional responses suggesting floral induction and plant development.Keywords:
Bulk soil
Serratia
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Abstract Aims Plants host communities of fungal and bacterial endophytes, establishing a complex network of multipartite interactions, but the mechanisms whereby they interact are poorly understood. Some fungi, such as the beneficial mycorrhiza-like fungus Serendipita (= Piriformospora ) indica , can be helped by bacteria for establishment, survival and colonization. Although this fungus harbors a Rhizobium as an endofungal bacterium, we hypothesized that other bacteria might also establish associations with the fungus and combining S. indica with bacteria might enhance plant growth and health. Methods The interactions among S. indica and four endophytic Proteobacteria belonging to Methylobacterium , Tardiphaga , Rhodanobacter and Trinickia spp. were characterized in vitro and for their effect on tomato growth and biocontrol of Fusarium oxysporum and Rhizoctonia solani . Possible mechanisms behind these interactions were described based on genome and microscopic analyses, using fungal and bacterial strains tagged with fluorescent markers. Results All bacteria stimulated S. indica growth in vitro. Moreover, several of the bacteria stimulated growth of tomato plants, but co-inoculations with S. indica and bacteria did not perform better than single inoculations. Contrarily, combinations of S. indica and bacteria significantly reduced disease progression of fungal pathogens. These microbes seem to cooperate in the process of root colonization for instance by increasing fungal sporulation and hyphae expansion, showing multipartite interaction between microbes and plants. Interestingly, the strain of Trinickia internally colonizes spores of S. indica as an endofungal bacterium during in vitro-co-culturing, suggesting further that the fungus might acquire formerly unrecognized genera of bacteria and genome analysis of the bacteria revealed many genes potentially involved in fungal and plant growth stimulation, biocontrol and root colonization, highlighting putative mechanisms of plant-fungal-bacterial interaction. Conclusions Our study represents an important step towards unraveling the complex interactions among plants, S. indica , endophytic bacteria and fungal pathogens, and indicates that adding bacteria to fungal inoculum could have a remarkable impact on the plant- S. indica symbiosis.
Endophyte
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Summary Plant‐associated microbial communities are crucial for plant growth and play an important role in disease suppression. Community composition and function change upon pathogen attack, yet to date, we do not know whether these changes are a side effect of the infection or actively driven by the plant. Here, we used a split‐root approach to test whether barley plants recruit bacteria carrying antifungal traits upon infestation with F usarium graminearum . Split‐root systems allow disentangling local infection effects, such as root damage, from systemic, plant‐driven effects on microbiome functionality. We assessed the recruitment of fluorescent pseudomonads, a taxon correlated with disease suppression, and of two well‐described antifungal genes ( phl D coding for 2,4‐ DAPG and hcn AB coding for HCN ). We show an enrichment of fluorescent pseudomonads, phl D and hcn AB , upon pathogen infection. This effect was only measurable in the uninfected root compartment. We link these effects to an increased chemotaxis of pseudomonads towards exudates of infected plants. Synthesis . We conclude that barley plants selectively recruited bacteria carrying antifungal traits upon pathogen attack and that the pathogen application locally interfered with this process. By disentangling these two effects, we set the base for enhancing strategies unravelling how pathogens and plant hosts jointly shape microbiome functionality.
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Trichoderma
Jasmonic acid
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The plant root microbiota, consisting of diverse microbial communities within and around the plant roots, can significantly influence plant development and stress tolerance. The goal of this thesis was to analyse the microbe-microbe and plantmicrobe interactions in the root. For this purpose, Serendipita indica, a mutualistic fungal root endophyte, able to improve plant performance and disease resistance in host plants, was studied. Strategies of the fungus to establish colonisation within the plant were studied by investigating the effect of putative S. indica effector proteins on the plant auxin signalling pathway. Single S. indica effectors were found to alter the expression of auxin-related genes, with consequent effects on plant growth, indicating a potential competitive advantage of S. indica gained through effectors against other microbes. Furthermore, by analyzing the effect of two beneficial fungal endophytes, S. indica and Colletotrichum tofieldiae on the rhizobiome composition of Arabidopsis thaliana, inoculated individually on plant roots, I aimed to reveal plant- and/or microbe-derived patterns that are involved in shaping the root microbiome. Using a gnotobiotic system and a synthetic bacterial community, I demonstrated the persistence of the bacterial community in the plant root, as well as the transition of C. tofieldiae to a potentially pathogenic lifestyle hereby affecting plants’ survival. To further test these patterns in a natural environment, a bacterial taxon commonly utilized in agriculture, Rhizobia, was used as a mutualist root symbiont of the legume Medicago truncatula. Rhizobia strains were tested for their ability to stimulate plant growth and nutrient supply as well as their competitiveness in representative UK soil types and their different natural microbiomes. Sinorhizobium meliloti 1022 was found to enhance plant growth and nitrogen acquisition in all soil types, altering the bacterial community structure and composition of the root endosphere, while the fungal community structure and composition was driven only by the soil characteristics. Overall, this work contributes towards elucidating the stability of beneficial plant-microbe interactions in natural communities and in different environments. The long-term aim of such study is to integrate molecular and environmental factors to generate customized beneficial microbiomes that can be applied to sustainably support crop production.
Medicago truncatula
Endophyte
Root hair
Colonisation
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Summary An emerging experimental framework suggests that plants under biotic stress may actively seek help from soil microbes, but empirical evidence underlying such a ‘cry for help’ strategy is limited. We used integrated microbial community profiling, pathogen and plant transcriptive gene quantification and culture‐based methods to systematically investigate a three‐way interaction between the wheat plant, wheat‐associated microbiomes and Fusarium pseudograminearum ( Fp ). A clear enrichment of a dominant bacterium, Stenotrophomonas rhizophila (SR80), was observed in both the rhizosphere and root endosphere of Fp‐ infected wheat. SR80 reached 3.7 × 10 7 cells g −1 in the rhizosphere and accounted for up to 11.4% of the microbes in the root endosphere. Its abundance had a positive linear correlation with the pathogen load at base stems and expression of multiple defence genes in top leaves. Upon re‐introduction in soils, SR80 enhanced plant growth, both the below‐ground and above‐ground, and induced strong disease resistance by boosting plant defence in the above‐ground plant parts, but only when the pathogen was present. Together, the bacterium SR80 seems to have acted as an early warning system for plant defence. This work provides novel evidence for the potential protection of plants against pathogens by an enriched beneficial microbe via modulation of the plant immune system.
Stenotrophomonas
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Competitive behaviours of plant growth promoting rhizobacteria (PGPR) are integral to their ability to colonize and persist on plant roots and outcompete phytopathogenic fungi, oomycetes and bacteria. PGPR engage in a range of antagonistic behaviours that have been studied in detail, such as the production and secretion of compounds inhibitory to other microbes. In contrast, their defensive activities that enable them to tolerate exposure to inhibitory compounds produced by their neighbours are less well understood. In this study, the genes involved in the Pseudomonas protegens Pf-5 response to metabolites from eight diverse rhizosphere competitor organisms, Fusarium oxysporum, Rhizoctonia solani, Gaeumannomyces graminis var. tritici, Pythium spinosum, Bacillus subtilis QST713, Pseudomonas sp. Q2-87, Streptomyces griseus and Streptomyces bikiniensis subspecies bikiniensi, were examined. Proximity induced excreted metabolite responses were confirmed for Pf-5 with all partner organisms through HPLC before culturing a dense Pf-5 transposon mutant library adjacent to each of these microbes. This was followed by transposon-directed insertion site sequencing (TraDIS), which identified genes that influence Pf-5 fitness during these competitive interactions. A set of 148 genes was identified that were associated with increased fitness during competition, including cell surface modification, electron transport, nucleotide metabolism, as well as regulatory genes. In addition, 51 genes were identified for which loss of function resulted in fitness gains during competition. These included genes involved in flagella biosynthesis and cell division. Considerable overlap was observed in the set of genes observed to provide a fitness benefit during competition with all eight test organisms, indicating commonalities in the competitive response to phylogenetically diverse micro-organisms and providing new insight into competitive processes likely to take place in the rhizosphere.
Oomycete
Transposon mutagenesis
Pseudomonas chlororaphis
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Summary Emerging experimental framework suggests that plants under biotic stress may actively seek help from soil microbes, but empirical evidence underlying such a ‘cry for help’ strategy is limited. We used integrated microbial community profiling, pathogen and plant transcriptive gene quantification and culture-based methods to systematically investigate a three-way interaction between the wheat plant, wheat-associated microbiomes and Fusarium pseudograminearum ( Fp ). A clear enrichment of a dominant bacterium, Stenotrophomonas rhizophila (SR80), was observed in both the rhizosphere and root endosphere of Fp- infected wheat. SR80 reached 3.7×10 7 cells g -1 in the rhizosphere and accounted for up to 11.4% of the microbes in the root endosphere. Its abundance had a positive linear correlation with the pathogen load at base stems and expression of multiple defense genes in top leaves. Upon re-introduction in soils, SR80 enhanced plant growth, both the below- and above-ground, and induced strong disease resistance by priming plant defense in the aboveground plant parts, but only when the pathogen was present Together, the bacterium SR80 seems to have acted as an early warning system for plant defense. This work provides novel evidence for the potential protection of plants against pathogens by an enriched beneficial microbe via modulation of the plant immune system.
Stenotrophomonas
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Here we discuss the advantages of the majority of this versatile and diverse group of microorganisms for plant health and growth as demonstrated by their contribution to disease-suppressive soils, their antifungal and antibacterial activities, their ability to produce volatile compounds and their capacity to enhance plant biomass. Although much is still to be discovered about the colonization strategies and molecular interactions between plant roots and these microorganisms, they are destined to become important players in the field of plant growth-promoting rhizobacteria for agriculture.
Plant disease
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Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting "soldier" rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.
Exudate
Defence mechanisms
Secondary metabolism
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Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.
Pythium ultimum
Pseudomonas fluorescens
Pythium
Human pathogen
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