Transcriptional Changes in Potato Sprouts upon Interaction with Rhizoctonia solani Indicate Pathogen-Induced Interference in the Defence Pathways of Potato.

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants' molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants' comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (-Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani. Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term "response to abiotic stimulus" was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.

Potato plant spheres and to a lesser extent the soil type influence the proportion and diversity of bacterial isolates with in vitro antagonistic activity towards Ralstonia solanacearum.

Ralstonia solanacearum biovar2-race3 (Rs r3b2) is an epidemic soil-borne bacterial phytopathogen causing brown rot disease in potato. In this study, we assessed how three soil types stored at the same field site influenced the proportion and diversity of bacterial isolates with in vitro antagonistic activity towards Rs in bulk soil and different potato plant spheres (rhizosphere, endorhiza and endocaulosphere; ecto- and endosphere of seed and yield tubers). In general, the plate counts observed for each sample type were not significantly different. A total of 96 colonies per sample type was picked and screened for in vitro antagonistic activity against Rs. Antagonists were obtained from all bulk soils and plant spheres with the highest proportion obtained from the endorhiza and endocaulosphere of potato plants. BOX-PCR fingerprints of antagonists showed that some were specific for particular plant spheres independent of the soil type, while others originated from different plant spheres of a particular soil type. The majority of antagonists belonged to Pseudomonas. A high proportion of antagonists produced siderophores, and interestingly antagonists from potato tubers frequently carried multiple antibiotic production genes. Our data showed an enrichment of bacteria with genes or traits potentially involved in biocontrol in the rhizosphere and in endophytic compartments. We report that the proportion and diversity of in vitro antagonists towards Rs isolated from bulk soil and different spheres of potato plants grown under field conditions in three different soil types was mainly shaped by the plant sphere and to a lesser extent by the soil type. Bacteria with antagonistic activity towards Ralstonia solanacearum were isolated from all plant spheres and bulk soils but their proportion was highest in endophytic compartments.

Necrotrophic lifestyle of Rhizoctonia solani AG3-PT during interaction with its host plant potato as revealed by transcriptome analysis.

The soil-borne pathogen Rhizoctonia solani infects a broad range of plants worldwide and is responsible for significant crop losses. Rhizoctonia solani AG3-PT attacks germinating potato sprouts underground while molecular responses during interaction are unknown. To gain insights into processes induced in the fungus especially at early stage of interaction, transcriptional activity was compared between growth of mycelium in liquid culture and the growing fungus in interaction with potato sprouts using RNA-sequencing. Genes coding for enzymes with diverse hydrolase activities were strongly differentially expressed, however with remarkably dissimilar time response. While at 3 dpi, expression of genes coding for peptidases was predominantly induced, strongest induction was found for genes encoding hydrolases acting on cell wall components at 8 dpi. Several genes with unknown function were also differentially expressed, thus assuming putative roles as effectors to support host colonization. In summary, the presented analysis characterizes the necrotrophic lifestyle of R. solani AG3-PT during early interaction with its host.

A non-invasive soil-based setup to study tomato root volatiles released by healthy and infected roots.

The role of root exudates in mediating plant-microbe interactions has been well documented. However, the function of volatile organic compounds (VOCs) emitted by plant roots has only recently begun to attract attention. This newly recognized relevance of belowground VOCs has so far mostly been tested using systems limited to a two-compartment Petri-dish design. Furthermore, many of the plant-microbe interaction studies have only investigated the effects of microbial VOCs on plant growth. Here, we go two steps further. First we investigated the volatile profile of healthy and pathogen (Fusarium oxysporum) infected tomato roots grown in soil. We then used a unique soil-based olfactometer-choice assay to compare the migration pattern of four beneficial bacteria (Bacillus spp.) towards the roots of the tomato plants. We demonstrate that the blend of root-emitted VOCs differs between healthy and diseased plants. Our results show that VOCs are involved in attracting bacteria to plant roots.

Draft genome sequence of the potato pathogen Rhizoctonia solani AG3-PT isolate Ben3.

The basidiomycetes fungus Rhizoctonia solani AG3 is responsible for black scurf disease on potato and occurs in each potato growing area world-wide. In this study, the draft genome sequence of the black scurf pathogen R. solani AG3-PT isolate Ben3 is presented. The genome sequence of R. solani AG3-PT isolate Ben3 consists of 1385 scaffolds. These scaffolds amount to a size of approx. 51 Mb. Considering coverage analyses of contigs, the size of the diploid genome was estimated to correspond to 116 Mb. Gene prediction by applying AUGUSTUS (3.2.1.) resulted in 12,567 identified genes. Based on automatic annotation using GenDBE, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the R. solani AG3-PT isolate Ben3 genome. Comparative analyses including the R. solani AG3 isolate Rhs1AP, also originating from potato, revealed first insights into core genes shared by both isolates and unique determinants of each isolate.

Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community.

A broad spectrum of fungal antagonists was evaluated as potential biocontrol agents (BCAs) against the soil-borne pathogen Rhizoctonia solani using a new combination of in vitro and in vivo assays. The in vitro characterisation of diverse parameters including the ability to parasitise mycelium and to inhibit the germination of Rhizoctonia sclerotia at different temperatures resulted in the selection of six potential fungal antagonists. These were genotypically characterised by their BOX-PCR fingerprints, and identified as Trichoderma reesei and T. viride by partial 18S rDNA sequencing. When potato sprouts were treated with Trichoderma, all isolates significantly reduced the incidence of Rhizoctonia symptoms. Evaluated under growth chamber conditions, the selected Trichoderma isolates either partly or completely controlled the dry mass loss of lettuce caused by R. solani. Furthermore, the antagonistic Trichoderma strains were active under field conditions. To analyse the effect of Trichoderma treatment on indigenous root-associated microbial communities, we performed a DNA-dependent SSCP (Single-Strand Conformation Polymorphism) analysis of 16S rDNA/ITS sequences. In this first assessment study for Trichoderma it was shown that the pathogen and the vegetation time had much more influence on the composition of the microbiota than the BCA treatment. After evaluation of all results, three Trichoderma strains originally isolated from Rhizoctonia sclerotia were selected as promising BCAs.

Effectiveness of 3 antagonistic bacterial isolates to control Rhizoctonia solani Kühn on lettuce and potato.

Rhizoctonia solani causes yield losses in numerous economically important European crops. To develop a biocontrol strategy, 3 potato-associated ecto- and endophytically living bacterial strains Pseudomonas fluorescens B1, Pseudomonas fluorescens B2, and Serratia plymuthica B4 were evaluated against R. solani in potato and in lettuce. The disease-suppression effect of the 3 biocontrol agents (BCAs) was tested in a growth chamber and in the field. In growth chamber experiments, all 3 BCAs completely or significantly limited the dry mass (DM) losses on lettuce and the disease severity (DS) caused by R. solani on potato sprouts. Strain B1 showed the highest suppression effect (52% on average) on potato. Under field conditions, the DS on both crops, which were bacterized, decreased significantly, and the biomass losses on lettuce decreased significantly as well. The greatest disease-suppression effect on potato was achieved by strain B1 (37%), followed by B2 (33%) and then B4 (31%), whereas the marketable tuber yield increased up to 12% (B1), 6% (B2), and 17% (B4) compared with the pathogen control at higher disease pressure. Furthermore, in all experiments, B1 proved to be the most effective BCA against R. solani. Therefore, this BCA could be a candidate for developing a commercial product against Rhizoctonia diseases. To our knowledge, this is the first report on the high potential of endophytes to be used as a biological control agent against R. solani under field conditions.

Strategy to select and assess antagonistic bacteria for biological control of Rhizoctonia solani Kühn.

A screening strategy was developed to assess the potential of plant-associated bacteria to control diseases caused by Rhizoctonia solani Kühn. About 434 already characterized antagonistic bacterial strains isolated from diverse plant species and microenvironments were evaluated for biocontrol and plant growth promotion by a hierarchical combination of assays. Analyzing in vitro antagonism towards different Rhizoctonia isolates resulted in a selection of 20 potential biocontrol agents. The strains were characterized by their antagonistic mechanisms in vitro as well as their production of the plant growth hormone indole-3-acetic acid. The plant growth promoting effect by antagonistic bacteria was determined using a microtiter plate assay on the basis of lettuce seedlings. Lettuce and sugar beet as host plant were included in the biocontrol experiments in which the antagonistic effect of 17 bacterial isolates could be confirmed in vivo. Sequencing of the 16S rDNA gene and (or) fatty acid methyl ester gas chromatography was used to identify the antagonistic isolates. Molecular fingerprints of isolates obtained by BOX-polymerase chain reaction were compared to avoid further investigation with genetically very similar strains and to obtain unique molecular fingerprints for quality control and patent licensing. According to our strategy, an assessment scheme was developed and four interesting biological control agents, Pseudomonas reactans B3, Pseudomonas fluorescens B1, Serratia plymuthica B4, and Serratia odorifera B6, were found. While S. plymuthica B4 was the best candidate to biologically control Rhizoctonia in lettuce, P. reactans B3 was the best candidate to suppress the pathogen in sugar beet.