Identifying the Active Microbiome Associated with Roots and Rhizosphere Soil of Oilseed Rape.

RNA stable isotope probing and high-throughput sequencing were used to characterize the active microbiomes of bacteria and fungi colonizing the roots and rhizosphere soil of oilseed rape to identify taxa assimilating plant-derived carbon following 13CO2 labeling. Root- and rhizosphere soil-associated communities of both bacteria and fungi differed from each other, and there were highly significant differences between their DNA- and RNA-based community profiles. Verrucomicrobia, Proteobacteria, Planctomycetes, Acidobacteria, Gemmatimonadetes, Actinobacteria, and Chloroflexi were the most active bacterial phyla in the rhizosphere soil. Bacteroidetes were more active in roots. The most abundant bacterial genera were well represented in both the 13C- and 12C-RNA fractions, while the fungal taxa were more differentiated. Streptomyces, Rhizobium, and Flavobacterium were dominant in roots, whereas Rhodoplanes and Sphingomonas (Kaistobacter) were dominant in rhizosphere soil. "Candidatus Nitrososphaera" was enriched in 13C in rhizosphere soil. Olpidium and Dendryphion were abundant in the 12C-RNA fraction of roots; Clonostachys was abundant in both roots and rhizosphere soil and heavily 13C enriched. Cryptococcus was dominant in rhizosphere soil and less abundant, but was 13C enriched in roots. The patterns of colonization and C acquisition revealed in this study assist in identifying microbial taxa that may be superior competitors for plant-derived carbon in the rhizosphere of Brassica napusIMPORTANCE This microbiome study characterizes the active bacteria and fungi colonizing the roots and rhizosphere soil of Brassica napus using high-throughput sequencing and RNA-stable isotope probing. It identifies taxa assimilating plant-derived carbon following 13CO2 labeling and compares these with other less active groups not incorporating a plant assimilate. Brassica napus is an economically and globally important oilseed crop, cultivated for edible oil, biofuel production, and phytoextraction of heavy metals; however, it is susceptible to several diseases. The identification of the fungal and bacterial species successfully competing for plant-derived carbon, enabling them to colonize the roots and rhizosphere soil of this plant, should enable the identification of microorganisms that can be evaluated in more detailed functional studies and ultimately be used to improve plant health and productivity in sustainable agriculture.

Transcriptomic changes in the plant pathogenic fungus Rhizoctonia solani AG-3 in response to the antagonistic bacteria Serratia proteamaculans and Serratia plymuthica.

BACKGROUND: Improved understanding of bacterial-fungal interactions in the rhizosphere should assist in the successful application of bacteria as biological control agents against fungal pathogens of plants, providing alternatives to chemicals in sustainable agriculture. Rhizoctonia solani is an important soil-associated fungal pathogen and its chemical treatment is not feasible or economic. The genomes of the plant-associated bacteria Serratia proteamaculans S4 and Serratia plymuthica AS13 have been sequenced, revealing genetic traits that may explain their diverse plant growth promoting activities and antagonistic interactions with R. solani. To understand the functional response of this pathogen to different bacteria and to elucidate whether the molecular mechanisms that the fungus exploits involve general stress or more specific responses, we performed a global transcriptome profiling of R. solani Rhs1AP anastomosis group 3 (AG-3) during interaction with the S4 and AS13 species of Serratia using RNA-seq. RESULTS: Approximately 104,504 million clean 75-100 bp paired-end reads were obtained from three libraries, each in triplicate (AG3-Control, AG3-S4 and AG3-AS13). Transcriptome analysis revealed that approximately 10% of the fungal transcriptome was differentially expressed during challenge with Serratia. The numbers of S4- and AS13-specific differentially expressed genes (DEG) were 866 and 292 respectively, while there were 1035 common DEGs in the two treatment groups. Four hundred and sixty and 242 genes respectively had values of log2 fold-change > 3 and for further analyses this cut-off value was used. Functional classification of DEGs based on Gene Ontology enrichment analysis and on KEGG pathway annotations revealed a general shift in fungal gene expression in which genes related to xenobiotic degradation, toxin and antioxidant production, energy, carbohydrate and lipid metabolism and hyphal rearrangements were subjected to transcriptional regulation. CONCLUSIONS: This RNA-seq profiling generated a novel dataset describing the functional response of the phytopathogen R. solani AG3 to the plant-associated Serratia bacteria S4 and AS13. Most genes were regulated in the same way in the presence of both bacterial isolates, but there were also some strain-specific responses. The findings in this study will be beneficial for further research on biological control and in depth exploration of bacterial-fungal interactions in the rhizosphere.

Transcriptional responses of the bacterial antagonist Serratia plymuthica to the fungal phytopathogen Rhizoctonia solani.

Rhizobacteria with biocontrol ability exploit a range of mechanisms to compete successfully with other microorganisms and to ensure their growth and survival in the rhizosphere, ultimately promoting plant growth. The rhizobacterium Serratia plymuthica AS13 is able to promote oilseed rape growth and improve seedling survival in the presence of the fungal pathogen, Rhizoctonia solani AG 2-1; however, our understanding of the mechanisms underlying the antagonism of Serratia is limited. To elucidate possible mechanisms, genome-wide gene expression profiling of S. plymuthica AS13 was carried out in the presence or absence of R. solani. We used RNA sequencing methodology to obtain a comprehensive overview of Serratia gene expression in response to R. solani. The differential gene expression profiles of S. plymuthica AS13 revealed significantly increased expression of genes related to the biosynthesis of the antibiotic pyrrolnitrin (prnABCD), protease production and transporters. The results presented here provide evidence that antibiosis is a major functional mechanism underlying the antagonistic behaviour of S. plymuthica AS13.

Influence of soil type, cultivar and Verticillium dahliae on the structure of the root and rhizosphere soil fungal microbiome of strawberry.

Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar.

Non-contiguous finished genome sequence of plant-growth promoting Serratia proteamaculans S4.

Serratia proteamaculans S4 (previously Serratia sp. S4), isolated from the rhizosphere of wild Equisetum sp., has the ability to stimulate plant growth and to suppress the growth of several soil-borne fungal pathogens of economically important crops. Here we present the non-contiguous, finished genome sequence of S. proteamaculans S4, which consists of a 5,324,944 bp circular chromosome and a 129,797 bp circular plasmid. The chromosome contains 5,008 predicted genes while the plasmid comprises 134 predicted genes. In total, 4,993 genes are assigned as protein-coding genes. The genome consists of 22 rRNA genes, 82 tRNA genes and 58 pseudogenes. This genome is a part of the project "Genomics of four rapeseed plant growth-promoting bacteria with antagonistic effect on plant pathogens" awarded through the 2010 DOE-JGI's Community Sequencing Program.

Complete genome sequence of Serratia plymuthica strain AS12.

A plant-associated member of the family Enterobacteriaceae, Serratia plymuthica strain AS12 was isolated from rapeseed roots. It is of scientific interest because it promotes plant growth and inhibits plant pathogens. The genome of S. plymuthica AS12 comprises a 5,443,009 bp long circular chromosome, which consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome was sequenced within the 2010 DOE-JGI Community Sequencing Program (CSP2010) as part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens".

Complete genome sequence of the rapeseed plant-growth promoting Serratia plymuthica strain AS9.

Serratia plymuthica are plant-associated, plant beneficial species belonging to the family Enterobacteriaceae. The members of the genus Serratia are ubiquitous in nature and their life style varies from endophytic to free-living. S. plymuthica AS9 is of special interest for its ability to inhibit fungal pathogens of rapeseed and to promote plant growth. The genome of S. plymuthica AS9 comprises a 5,442,880 bp long circular chromosome that consists of 4,952 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome is part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens" awarded through the 2010 DOE-JGI Community Sequencing Program (CSP2010).

Complete genome sequence of the plant-associated Serratia plymuthica strain AS13.

Serratia plymuthica AS13 is a plant-associated Gammaproteobacteria, isolated from rapeseed roots. It is of special interest because of its ability to inhibit fungal pathogens of rapeseed and to promote plant growth. The complete genome of S. plymuthica AS13 consists of a 5,442,549 bp circular chromosome. The chromosome contains 4,951 protein-coding genes, 87 tRNA genes and 7 rRNA operons. This genome was sequenced as part of the project entitled "Genomics of four rapeseed plant growth promoting bacteria with antagonistic effect on plant pathogens" within the 2010 DOE-JGI Community Sequencing Program (CSP2010).

Interactions among Glomus irregulare, arbuscular mycorrhizal spore-associated bacteria, and plant pathogens under in vitro conditions.

Arbuscular mycorrhizal (AM) fungi interact with bacteria (AM fungi-associated bacteria, AMB) in the mycorrhizosphere. We previously identified a set of AMB that enhance AM fungal colonization, plant growth, and inhibit pathogens. Here, we used transformed carrot root cultures in a two-compartment plate system for further in vitro studies on interactions taking place among Glomus irregulare (syn.Glomus intraradices), AMB, and plant pathogens. We found that exudates of G. irregulare stimulated growth of all ten AMB isolates tested in multi-well plates. AMB growth stimulation was observed also during co-cultivation of three of these AMB with G. irregulare in the hyphal compartment. In addition, co-cultivation stimulated growth of G. irregulare hyphae and spore production, as well as G. irregulare root colonization. GC/MS analysis in a preliminary screening of metabolites revealed differences in concentrations of several identified but also unidentified compounds in G. irregulare hyphal exudates. Exudates in presence of three different AMB isolates co-cultivated with G. irregulare contained several additional compounds that differed in amount compared with G. irregulare alone. The results indicate that G. irregulare exudates contain carbohydrates, amino acids, and unidentified compounds that could serve as a substrate to stimulate AMB growth. With regard to effects on plant pathogens, growth inhibition of Rhizoctonia solani, Verticillium dahliae, and Pectobacterium carotovorum ssp. carotovorum was evident in the presence of the AMB isolates tested together with the G. irregulare exudates. These in vitro studies suggest that G. irregulare and AMB stimulate growth of each other and that they together seem to provide an additive effect against growth of both fungal and bacterial pathogens.

Soil, but not cultivar, shapes the structure of arbuscular mycorrhizal fungal assemblages associated with strawberry.

Arbuscular mycorrhizal fungi are widespread plant symbionts occurring in most agricultural crops, where they can play key roles in the growth and health of their plant hosts. Plant benefits can depend on the identity of the associated arbuscular mycorrhizal fungi (AMF), but little is known about the identity of the fungal partners in most agricultural systems. In this study, we describe the AMF assemblages associated with four cultivars of strawberry in an outdoor experiment using two field soils with different origin and management history. Assemblages were characterised by clone library sequencing of 18S rRNA gene fragments. Soil dramatically influenced the degree of mycorrhizal colonisation and AMF assemblage structure in the roots. No differences were observed between cultivars. Fungi belonging to the genus Acaulospora dominated the AMF assemblages in one soil, but they were not detected in the other. These results suggest that physicochemical soil characteristics and management can play a role in determining the identity and structure of microbial communities associated with particular hosts in agricultural systems.

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores.

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.

Rhizoplane colonisation of peas by Rhizobium leguminosarum bv. viceae and a deleterious Pseudomonas putida.

Pseudomonas putida strain A313, a deleterious rhizosphere bacterium, reduced pea nitrogen content when inoculated alone or in combination with Rhizobium leguminosarum bv. viceae on plants in the presence of soil under greenhouse conditions. When plants were grown gnotobiotically in liquid media, mixed inocula of A313 and rhizobia gave a higher proportion of small evenly distributed nodules when compared with a single rhizobial inoculation. In addition, the rhizobial root establishment was reduced by A313 irrespective of inoculum density, indicating that A313 has the capacity to interact with the early rhizobial infection process. When pea seedlings were simultaneously inoculated with A313 and rhizobia, A313 colonised the root hairs to the same extent as the rhizobia, according to analysis by immunofluorescence microscopy. This suggests that the root hair colonisation trait of P. putida interferes with the onset of the symbiotic process.

A study on microbial diversity in different cultivars of Brassica napus in relation to its wilt pathogen, Verticillium longisporum.

Oilseed rape (Brassica napus) is one of the major oilseed crops in the world but is vulnerable to attack by many pathogens and insect pests. In addition to the host plant genotype, micro-organisms present in the rhizosphere and within plant tissues affect the susceptibility to plant pathogens. While rapid progress has been achieved concerning the concept of plant resistance genes, information on the role of the microbial community in plant protection is less apparent. We have studied the endophytic bacterial populations present in different tissues of oilseed rape and also analysed several cultivars (Express, Libraska, Maluka and Westar), which differ in their susceptibility to the wilt pathogen Verticillium longisporum. The population diversity was studied using agar plating assay, fatty acid methyl ester analysis and functional characterisation of isolated strains. Our work shows that already in the seeds there exists diversity in populations as well as in the total microbial load between two of the four tested cultivars. About 50% of the strains isolated from cultivars Express and Libraska showed moderate to strong direct inhibition of V. longisporum. The diversity of the endophytic flora isolated from oilseed rape and its implications in crop protection are discussed.

Plant-affecting streptomycin-sensitive micro-organisms in barley monoculture soils.

Soils from barley monoculture and from crop-rotation plots of three long-term field experiments were tested for their effects on barley growth in the glasshouse, using plant growth tubes. Test plants of young barley developed significantly shorter and thinner root axes when grown in soil from monoculture plots than when grown in soil from crop rotation plots, but the difference in shoot growth was only small. Addition of small doses of streptomycin sulphate neutralized the retarded root growth in soil from monoculture plots, whereas the fungicide metalaxyl induced no such effect. The difference in root growth of barley test plants could be transmitted into a soil-free root environment by inoculation with the microflora from roots grown in soil. These findings indicate that some component(s) of rhizosphere bacteria is the causal agent of the early monoculture effect measured.