Long-term conservation tillage with reduced nitrogen fertilization intensity can improve winter wheat health via positive plant-microorganism feedback in the rhizosphere.

Microbiome-based solutions are regarded key for sustainable agroecosystems. However, it is unclear how agricultural practices affect the rhizosphere microbiome, plant-microorganism interactions and crop performance under field conditions. Therefore, we installed root observation windows in a winter wheat field cultivated either under long-term mouldboard plough (MP) or cultivator tillage (CT). Each tillage practice was also compared at two nitrogen (N) fertilization intensities, intensive (recommended N-supply with pesticides/growth regulators) or extensive (reduced N-supply, no fungicides/growth regulators). Shoot biomass, root exudates and rhizosphere metabolites, physiological stress indicators, and gene expression were analyzed together with the rhizosphere microbiome (bacterial/archaeal 16S rRNA gene, fungal ITS amplicon, and shotgun metagenome sequencing) shortly before flowering. Compared to MP, the rhizosphere of CT winter wheat contained more primary and secondary metabolites, especially benzoxazinoid derivatives. Potential copiotrophic and plant-beneficial taxa (e.g. Bacillus, Devosia, and Trichoderma) as well as functional genes (e.g. siderophore production, trehalose synthase, and ACC deaminase) were enriched in the CT rhizosphere, suggesting that tillage affected belowground plant-microorganism interactions. In addition, physiological stress markers were suppressed in CT winter wheat compared to MP. In summary, tillage practice was a major driver of crop performance, root deposits, and rhizosphere microbiome interactions, while the N-fertilization intensity was also relevant, but less important.

Does Constitutive Expression of Defense-Related Genes and Salicylic Acid Concentrations Correlate with Field Resistance of Potato to Black Scurf Disease?

Black scurf disease on potato caused by Rhizoctonia solani AG3 occurs worldwide and is difficult to control. The use of potato cultivars resistant to black scurf disease could be part of an integrated control strategy. Currently, the degree of resistance is based on symptom assessment in the field, but molecular measures could provide a more efficient screening method. We hypothesized that the degree of field resistance to black scurf disease in potato cultivars is associated with defense-related gene expression levels and salicylic acid (SA) concentration. Cultivars with a moderate and severe appearance of disease symptoms on tubers were selected and cultivated in the same field. In addition, experiments were conducted under controlled conditions in an axenic in vitro culture and in a sand culture to analyze the constitutive expression of defense-related genes and SA concentration. The more resistant cultivars did not show significantly higher constitutive expression levels of defense-related genes. Moreover, the level of free SA was increased in the more resistant cultivars only in the roots of the plantlets grown in the sand culture. These results indicate that neither expression levels of defense-related genes nor the amount of SA in potato plants can be used as reliable predictors of the field resistance of potato genotypes to black scurf disease.

Beneficial microbial consortium improves winter rye performance by modulating bacterial communities in the rhizosphere and enhancing plant nutrient acquisition.

The beneficial effect of microbial consortium application on plants is strongly affected by soil conditions, which are influenced by farming practices. The establishment of microbial inoculants in the rhizosphere is a prerequisite for successful plant-microorganism interactions. This study investigated whether a consortium of beneficial microorganisms establishes in the rhizosphere of a winter crop during the vegetation period, including the winter growing season. In addition, we aimed for a better understanding of its effect on plant performance under different farming practices. Winter rye plants grown in a long-time field trial under conventional or organic farming practices were inoculated after plant emergence in autumn with a microbial consortium containing Pseudomonas sp. (RU47), Bacillus atrophaeus (ABi03) and Trichoderma harzianum (OMG16). The density of the microbial inoculants in the rhizosphere and root-associated soil was quantified in autumn and the following spring. Furthermore, the influence of the consortium on plant performance and on the rhizosphere bacterial community assembly was investigated using a multidisciplinary approach. Selective plating showed a high colonization density of individual microorganisms of the consortium in the rhizosphere and root-associated soil of winter rye throughout its early growth cycle. 16S rRNA gene amplicon sequencing showed that the farming practice affected mainly the rhizosphere bacterial communities in autumn and spring. However, the microbial consortium inoculated altered also the bacterial community composition at each sampling time point, especially at the beginning of the new growing season in spring. Inoculation of winter rye with the microbial consortium significantly improved the plant nutrient status and performance especially under organic farming. In summary, the microbial consortium showed sufficient efficacy throughout vegetation dormancy when inoculated in autumn and contributed to better plant performance, indicating the potential of microbe-based solutions in organic farming where nutrient availability is limited.

Monitoring of an Applied Beneficial Trichoderma Strain in Root-Associated Soil of Field-Grown Maize by MALDI-TOF MS.

The persistence of beneficial microorganisms in the rhizosphere or surrounding soil following their application is a prerequisite for the effective interaction with the plant or indigenous microbial communities in the respective habitats. The goal of the study was to analyze the establishment and persistence of the applied beneficial Trichoderma harzianum (OMG16) strain in the maize root-associated soil depending on agricultural practice (soil management practice, N-fertilizer intensity) in a field experiment. A rapid identification of the inoculated strain OMG16 is essential for its monitoring. We used a culture-based approach coupled to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis for the rapid identification of the inoculated Trichoderma strain as part of the beneficial microbe consortium (BMc). We isolated 428 fungal isolates from eight treatments of the field experiment. Forty eight percent of the isolated fungi equivalent to 205 fungal isolates were identified as Trichoderma, of which 87% (=179 isolates) were obtained from the fields inoculated with BMc. Gene sequence analysis showed a high similarity of the MALDI-TOF MS-identified Trichoderma, with that of the inoculated Trichoderma harzianum OMG16 confirming the re-isolation of the added beneficial fungus. This study highlighted the use of MALDI-TOF MS analysis as a quick, cost-effective detection and efficient monitoring tool for microbial-based bioinoculants in the field.

Influence of sewage sludge stabilization method on microbial community and the abundance of antibiotic resistance genes.

Municipal sewage sludge (MSS) and other biosolids are of high interest for agriculture. These nutrient-rich organic materials can potentially serve as organic fertilizers. Besides an increase of organic matter in soil, other positive effects were shown after their application. Especially the positive influence on circular economy increased the attention paid to management of MSS in recent years. Unfortunately, the use of sewage sludge has some drawbacks. Biosolids are frequently polluted with heavy metals, xenobiotic organic compounds and industrial chemicals, which may be hazardous for the environment and humans. Here, we investigated the influence of stabilization method and the size of wastewater treatment plant on the structure of microbial communities as well as the abundance of antibiotic resistance genes (ARG) and mobile genetic elements (MGE). All tested ARG and MGE were detectable in almost all of the samples. Interestingly, the presence of MGE as well as particular heavy metals correlated positively with the presence of several ARG. We conclude that the distribution of ARG and MGE in biosolids originated from municipal wastewater treatment plants, cannot be explained by the size of the facility or the applied stabilization method. Moreover, we postulate that the presence of pollutants and long-term impacts should be assessed prior to a possible use of sewage sludge as fertilizer.

What Drives the Assembly of Plant-associated Protist Microbiomes? Investigating the Effects of Crop Species, Soil Type and Bacterial Microbiomes.

In a field experiment we investigated the influence of the environmental filters soil type (i.e. three contrasting soils) and plant species (i.e. lettuce and potato) identity on rhizosphere community assembly of Cercozoa, a dominant group of mostly bacterivorous soil protists. Plant species (14%) and rhizosphere origin (vs bulk soil) with 13%, together explained four times more variation in cercozoan beta diversity than the three soil types (7% explained variation). Our results clearly confirm the existence of plant species-specific protist communities. Network analyses of bacteria-Cercozoa rhizosphere communities identified scale-free small world topologies, indicating mechanisms of self-organization. While the assembly of rhizosphere bacterial communities is bottom-up controlled through the resource supply from root (secondary) metabolites, our results support the hypothesis that the net effect may depend on the strength of top-down control by protist grazers. Since grazing of protists has a strong impact on the composition and functioning of bacteria communities, protists expand the repertoire of plant genes by functional traits, and should be considered as 'protist microbiomes' in analogy to 'bacterial microbiomes'.

Long-Term Fertilization Strategy Impacts Rhizoctonia solani-Microbe Interactions in Soil and Rhizosphere and Defense Responses in Lettuce.

The long-term effects of agricultural management such as different fertilization strategies on soil microbiota and soil suppressiveness against plant pathogens are crucial. Therefore, the suppressiveness of soils differing in fertilization history was assessed using two Rhizoctonia solani isolates and their respective host plants (lettuce, sugar beet) in pot experiments. Further, the effects of fertilization history and the pathogen R. solani AG1-IB on the bulk soil, root-associated soil and rhizosphere microbiota of lettuce were analyzed based on amplicon sequencing of the 16S rRNA gene and ITS2 region. Organic fertilization history supported the spread of the soil-borne pathogens compared to long-term mineral fertilization. The fertilization strategy affected bacterial and fungal community composition in the root-associated soil and rhizosphere, respectively, but only the fungal community shifted in response to the inoculated pathogen. The potential plant-beneficial genus Talaromyces was enriched in the rhizosphere by organic fertilization and presence of the pathogen. Moreover, increased expression levels of defense-related genes in shoots of lettuce were observed in the soil with organic fertilization history, both in the absence and presence of the pathogen. This may reflect the enrichment of potential plant-beneficial microorganisms in the rhizosphere, but also pathogen infestation. However, enhanced defense responses resulted in retarded plant growth in the presence of R. solani (plant growth/defense tradeoff).

Role of Benzoic Acid and Lettucenin A in the Defense Response of Lettuce against Soil-Borne Pathogens.

Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO4) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.

Genome Analyses of the Less Aggressive Rhizoctonia solani AG1-IB Isolates 1/2/21 and O8/2 Compared to the Reference AG1-IB Isolate 7/3/14.

Rhizoctonia solani AG1-IB of the phylum Basidiomycota is known as phytopathogenic fungus affecting various economically important crops, such as bean, rice, soybean, figs, cabbage and lettuce. The isolates 1/2/21 and O8/2 of the anastomosis group AG1-IB originating from lettuce plants with bottom rot symptoms represent two less aggressive R. solani isolates, as confirmed in a pathogenicity test on lettuce. They were deeply sequenced on the Illumina MiSeq system applying the mate-pair and paired-end mode to establish their genome sequences. Assemblies of obtained sequences resulted in 2092 and 1492 scaffolds, respectively, for isolate 1/2/21 and O8/2, amounting to a size of approximately 43 Mb for each isolate. Gene prediction by applying AUGUSTUS (v. 3.2.1.) yielded 12,827 and 12,973 identified genes, respectively. Based on automatic functional annotation, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the AG1-IB genomes. The annotated genome sequences of the less aggressive AG1-IB isolates were compared with the isolate 7/3/14, which is highly aggressive on lettuce and other vegetable crops such as bean, cabbage and carrot. This analysis revealed the first insights into core genes of AG1-IB isolates and unique determinants of each genome that may explain the different aggressiveness levels of the strains.

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.

Distinct rhizomicrobiota assemblages and plant performance in lettuce grown in soils with different agricultural management histories.

A better understanding of factors shaping the rhizosphere microbiota is important for sustainable crop production. We hypothesized that the effect of agricultural management on the soil microbiota is reflected in the assemblage of the rhizosphere microbiota with implications for plant performance. We designed a growth chamber experiment growing the model plant lettuce under controlled conditions in soils of a long-term field experiment with contrasting histories of tillage (mouldboard plough vs cultivator tillage), fertilization intensity (intensive standard nitrogen (N) + pesticides/growth regulators vs extensive reduced N without fungicides/growth regulators), and last standing field crop (rapeseed vs winter wheat). High-throughput sequencing of bacterial and archaeal 16S rRNA genes and fungal ITS2 regions amplified from total community DNA showed that these factors shaped the soil and rhizosphere microbiota of lettuce, however, to different extents among the microbial domains. Pseudomonas and Olpidium were identified as major indicators for agricultural management in the rhizosphere of lettuce. Long-term extensive fertilization history of soils resulted in higher lettuce growth and increased expression of genes involved in plant stress responses compared to intensive fertilization. Our work adds to the increasing knowledge on how soil microbiota can be manipulated by agricultural management practices which could be harnessed for sustainable crop production.

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.

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.

Species-Specific Impact of Fusarium Infection on the Root and Shoot Characteristics of Asparagus.

Soil-borne pathogens can have considerable detrimental effects on asparagus (Asparagus officinalis) growth and production, notably caused by the Fusarium species F. oxysporum f.sp. asparagi, F. proliferatum and F. redolens. In this study, their species-specific impact regarding disease severity and root morphological traits was analysed. Additionally, various isolates were characterised based on in vitro physiological activities and on protein extracts using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The response of two asparagus cultivars to the different Fusarium species was evaluated by inoculating experiments. Differences in aggressiveness were observed between Fusarium species and their isolates on roots, while no clear disease symptoms became visible in ferns eight weeks after inoculation. F. redolens isolates Fred1 and Fred2 were the most aggressive strains followed by the moderate aggressive F. proliferatum and the less and almost non-aggressive F. oxysporum isolates, based on the severity of disease symptoms. Fungal DNA in stem bases and a significant induction of pathogenesis-related gene expression was detectable in both asparagus cultivars. A significant negative impact of the pathogens on the root characteristics total root length, volume, and surface area was detected for each isolate tested, with Fred1 causing the strongest effects. No significant differences between the tested asparagus cultivars were observed.

Salmonella persistence in soil depends on reciprocal interactions with indigenous microorganisms.

Fresh fruits and vegetables have numerous benefits to human health. Unfortunately, their consumption is increasingly associated with food-borne diseases, Salmonella enterica being their most frequent cause in Europe. Agricultural soils were postulated as reservoir of human pathogens, contributing to the contamination of crops during the growing period. Since the competition with the indigenous soil microbiota for colonization sites plays a major role in the success of invading species, we hypothesized that reduced diversity will enhance the chance of Salmonella to successfully establish in agricultural environments. We demonstrated that the abundance of Salmonella drastically decreased in soil with highly diverse indigenous prokaryotic community, while in soil with reduced prokaryotic diversity, Salmonella persisted for a long period. Furthermore, in communities with low diversity, Salmonella had an impact on the abundance of other taxa. The high physiological plasticity allows Salmonella to use agricultural soils as alternative habitat which might provide a route of animal/human infections. In addition, adjusted transcriptional profile with amino acid biosynthesis and the glyoxylate cycle most prominently regulated, suggests an adaptation to the soil environment. Our results underline the importance of the maintenance of diverse soil microbiome as a part of strategy aiming at reduced risk of food-borne salmonellosis outbreaks.

Impact of Long-Term Organic and Mineral Fertilization on Rhizosphere Metabolites, Root-Microbial Interactions and Plant Health of Lettuce.

Fertilization management can affect plant performance and soil microbiota, involving still poorly understood rhizosphere interactions. We hypothesized that fertilization practice exerts specific effects on rhizodeposition with consequences for recruitment of rhizosphere microbiota and plant performance. To address this hypothesis, we conducted a minirhizotron experiment using lettuce as model plant and field soils with contrasting properties from two long-term field experiments (HUB-LTE: loamy sand, DOK-LTE: silty loam) with organic and mineral fertilization history. Increased relative abundance of plant-beneficial arbuscular mycorrhizal fungi and fungal pathotrophs were characteristic of the rhizospheres in the organically managed soils (HU-org; BIODYN2). Accordingly, defense-related genes were systemically expressed in shoot tissues of the respective plants. As a site-specific effect, high relative occurrence of the fungal lettuce pathogen Olpidium sp. (76-90%) was recorded in the rhizosphere, both under long-term organic and mineral fertilization at the DOK-LTE site, likely supporting Olpidium infection due to a lower water drainage potential compared to the sandy HUB-LTE soils. However, plant growth depressions and Olpidium infection were exclusively recorded in the BIODYN2 soil with organic fertilization history. This was associated with a drastic (87-97%) reduction in rhizosphere abundance of potentially plant-beneficial microbiota (Pseudomonadaceae, Mortierella elongata) and reduced concentrations of the antifungal root exudate benzoate, known to be increased in presence of Pseudomonas spp. In contrast, high relative abundance of Pseudomonadaceae (Gammaproteobacteria) in the rhizosphere of plants grown in soils with long-term mineral fertilization (61-74%) coincided with high rhizosphere concentrations of chemotactic dicarboxylates (succinate, malate) and a high C (sugar)/N (amino acid) ratio, known to support the growth of Gammaproteobacteria. This was related with generally lower systemic expression of plant defense genes as compared with organic fertilization history. Our results suggest a complex network of belowground interactions among root exudates, site-specific factors and rhizosphere microbiota, modulating the impact of fertilization management with consequences for plant health and performance.

Salmonella adapts to plants and their environment during colonization of tomatoes.

Humans and animals are considered typical hosts for Salmonella, however, also plants can be colonized. Tomatoes were linked to salmonellosis outbreaks already on several occasions. The aim of this study was, therefore, to establish a comprehensive view on the interaction between Salmonella enterica and tomatoes, and to test the hypothesis that colonization of plants is an interactive process. We assessed the persistence of Salmonella in agricultural soil, the colonization pattern in and on tomatoes, as well as the reciprocal responses of tomatoes to different Salmonella strains and Salmonella to root exudates and tomato-related media. This study revealed that Salmonella can persist in the soil and inside the tomato plant. Additionally, we show that Salmonella strains have particular colonization pattern, although the persistence inside the plant differs between the tested strains. Furthermore, the transcriptome response of tomato showed an up-regulation of several defense-related genes. Salmonella transcriptome analysis in response to the plant-based media showed differentially regulated genes related to amino acid and fatty acid synthesis and stress response, while the response to root exudates revealed regulation of the glyoxylate cycle. Our results indicate that both organisms actively engage in the interaction and that Salmonella adapts to the plant environment.

Genetic Potential of the Biocontrol Agent Pseudomonas brassicacearum (Formerly P. trivialis) 3Re2-7 Unraveled by Genome Sequencing and Mining, Comparative Genomics and Transcriptomics.

The genus Pseudomonas comprises many known plant-associated microbes with plant growth promotion and disease suppression properties. Genome-based studies allow the prediction of the underlying mechanisms using genome mining tools and the analysis of the genes unique for a strain by implementing comparative genomics. Here, we provide the genome sequence of the strain Pseudomonas brassicacearum 3Re2-7, formerly known as P. trivialis and P. reactans, elucidate its revised taxonomic classification, experimentally verify the gene predictions by transcriptome sequencing, describe its genetic biocontrol potential and contextualize it to other known Pseudomonas biocontrol agents. The P. brassicacearum 3Re2-7 genome comprises a circular chromosome with a size of 6,738,544 bp and a GC-content of 60.83%. 6267 genes were annotated, of which 6113 were shown to be transcribed in rich medium and/or in the presence of Rhizoctonia solani. Genome mining identified genes related to biocontrol traits such as secondary metabolite and siderophore biosynthesis, plant growth promotion, inorganic phosphate solubilization, biosynthesis of lipo- and exopolysaccharides, exoproteases, volatiles and detoxification. Core genome analysis revealed, that the 3Re2-7 genome exhibits a high collinearity with the representative genome for the species, P. brassicacearum subsp. brassicacearum NFM421. Comparative genomics allowed the identification of 105 specific genes and revealed gene clusters that might encode specialized biocontrol mechanisms of strain 3Re2-7. Moreover, we captured the transcriptome of P. brassicacearum 3Re2-7, confirming the transcription of the predicted biocontrol-related genes. The gene clusters coding for 2,4-diacetylphloroglucinol (phlABCDEFGH) and hydrogen cyanide (hcnABC) were shown to be highly transcribed. Further genes predicted to encode putative alginate production enzymes, a pyrroloquinoline quinone precursor peptide PqqA and a matrixin family metalloprotease were also found to be highly transcribed. With this study, we provide a basis to further characterize the mechanisms for biocontrol in Pseudomonas species, towards a sustainable and safe application of P. brassicacearum biocontrol agents.

Salmonella Establishment in Agricultural Soil and Colonization of Crop Plants Depend on Soil Type and Plant Species.

Human pathogenic bacteria, such as Salmonella enterica, are able to colonize crop plants. So far, not much is known about biotic and abiotic factors influencing this colonization in field soil. This understanding, however, is imperative for the provision of safe fresh produce to the consumer. In this study, we investigated the effects of soil type, organic fertilization, plant species and the way of Salmonella entry into the plant production system, on the survival of S. enterica in soil as well as the colonization of plants. The selected S. enterica serovar Typhimurium strain 14028s, S. Typhimurium strain LT2 and S. Senftenberg were able to persist in soil for several weeks. Salmonella's persistence in soil was prolonged in loamy, if compared to sandy soil, and when applied together with organic fertilizer. The leaves of lettuce and corn salad were colonized by S. enterica providing evidence for internalization from the soil via the root. Colonization rates were affected by soil type, plant species and S. enterica strain. Overall, S. enterica was detected in leaves of 0.5-0.9% of the plants, while lettuce was more frequently colonized than corn salad. Plants grown in sandy soil were more often colonized than plants grown in loamy soil. After spray inoculation, S. enterica could be detected on and in leaves for several weeks by cultivation-depending methods, confirmed by confocal microscopy using GFP-labeled S. Typhimurium 14028s. On the one hand, transcriptome data from S. Typhimurium 14028s assessed in response to lettuce medium or lettuce root exudates showed an upregulation of genes associated with biofilm formation and virulence. On the other hand, lettuce inoculated with S. Typhimurium 14028s showed a strong upregulation of genes associated with plant immune response and genes related to stress response. In summary, these results showed that organic fertilizers can increase the persistence of Salmonella in soil and that soil type and plant species play a crucial role in the interactions between human pathogens and crop plants. This understanding is therefore a starting point for new strategies to provide safe food for the consumer.