Bacillus subtilis MBI600 (Bs MBI600) is a recently commercialized plant-growth-promoting rhizobacterium (PGPR). In this study, we investigated the effects of Bs MBI600 on the growth of tomato and its biocontrol efficacy against three main soilborne tomato pathogens (Rhizoctonia solani, Pythium ultimum, and Fusarium oxysporum f.sp. radicis-lycopersici-Forl). Furthermore, the root colonization ability of the Bs MBI600 strain on tomato roots was analyzed in vivo with a yellow fluorescence protein (yfp)-labeled strain, revealing strong colonization ability, which was affected by the root growth substrate. The application of Bs MBI600 on tomato plants resulted in significant increases in shoot and root lengths. Transcriptional activation of two auxin-related genes (SiPin6 and SiLax4) was observed. Single applications of Bs MBI600 on inoculated tomato plants with pathogens revealed satisfactory control efficacy compared to chemical treatment. Transcriptomic analysis of defense-related genes used as markers of the salicylic acid (SA) signaling pathway (PR-1A and GLUA) or jasmonic acid/ethylene (JA/ET) signaling pathway (CHI3, LOXD, and PAL) showed increased transcription patterns in tomato plants treated with Bs MBI600 or Forl. These results indicate the biochemical and molecular mechanisms that are activated after the application of Bs MBI600 on tomato plants and suggest that induction of systemic resistance (ISR) occurred.

Background: Grapevine trunk diseases (GTDs) is a disease complex caused by wood pathogenic fungi belonging to genera like Phaeomoniella, Phaeoacremonium, Fomitiporia, Eutypa and members of the family Botryosphaeriaceae. However, the co-occurrence of these fungi in symptomatic and asymptomatic vines at equivalent abundances has questioned their role in GTDs. Hence, we still lack a good understanding of the fungi involved in GTDs, their interactions and the factors controlling their assemblage in vines. We determined the fungal and bacterial microbiome in wood tissues of asymptomatic and symptomatic vines of three main Greek cultivars (Agiorgitiko, Xinomavro, Vidiano), each cultivated in geographically distinct viticultural zones, using amplicon sequencing. Results: We noted that cultivar/biogeography (lumped factor) was the strongest determinant of the wood fungal microbiome (p < 0.001, 22.7%), while GTD symptoms condition had a weaker but still significant effect (p < 0.001, 3.5%), being prominent only in the cultivar Xinomavro. Several fungal Amplicon Sequence Variants (ASVs), reported as GTD-associated pathogens like Kalmusia variispora, Fomitiporia spp., and Phaemoniella chlamydosporα (most dominant in our study), were positively correlated with symptomatic vines in a cultivar/viticultural zone dependent manner. Random Forest analysis pointed to P. chlamydosporα, K. variispora, A. alternata and Cladosporium sp., as highly accurate predictors of symptomatic vines (0% error rate). The wood bacterial microbiome showed similar patterns, with biogeography/cultivar being the main determinant (p < 0.001, 25.5%) of its composition, followed by the GTD status of vines (p < 0.001, 5.2%). Differential abundance analysis revealed a universal positive correlation (p < 0.001) of Bacillus and Streptomyces ASVs with asymptomatic vines. Network analysis identified a significant negative co-occurrence network between these bacterial genera and Phaemoniella, Phaeoacrominum and Seimatosporium. These results point to a plant beneficial interaction between Bacillus/Streptomyces and GTD pathogens. Conclusions: Our study (a) provides evidence that GTD symptomatic plants support a wood fungal microbiome, showing cultivar and biogeography-dependent patterns, that could be used as a proxy to distinguish between healthy and diseased vines, (b) points to strong interactions between the bacterial and fungal wood microbiome in asymptomatic vines that should be further pursued in the quest for discovery of novel biocontrol agents.

Bacillusspp. MBI 600 is a gram-positive bacterium and is characterized as a PGPR strain involved in plant growth promotion and control of various plant pathogens which has recently been introduced into the agricultural practice. In this study we performed a Next Generation Sequencing analysis, to analyze the full genome of this microorganism and to characterize it taxonomically. Results showed that MBI 600 strain was phylogenetically close to otherBacillusspp. strains used as biocontrol agents and identified asB. subtilis. GOG analysis showed clusters contributed to secondary metabolites production such as fengycin and surfactin. In addition, various genes which annotated according to other plant-associated strains, showed that play a main role in nutrient availability from soil. The root colonization ability of MBI 600 strain was analyzedin vivowith a yellow fluorescence protein (yfp) tag. Confocal laser scanning microscopy of cucumber roots treated withyfp-tagged MBI 600 cells, revealed that the strain exhibits a strong colonization ability of cucumber roots, although it is affected significantly by the growth substrate of the roots.In vitroandin plantaexperiments with MBI 600 strain andF. oxysporumf.sp.radicis cucumerinumandP. aphanidernatum, showed a high control ability against these soilborne pathogens. Overall, our study demonstrates the effectiveness of MBI 600 in plant growth promotion and antagonism against different pathogens, highlighting the use of this microorganism as a biocontrol agent.

Bacillus subtilis MBI600 is a commercialized plant growth-promoting bacterial species used as a biocontrol agent in many crops, controlling various plant pathogens via direct or indirect mechanisms. In the present study, a detailed transcriptomic analysis of cucumber roots upon response to the Bs MBI600 strain is provided. Differentially expressed genes (DEGs) analysis showed altered gene expression in more than 1000 genes at 24 and 48 h post-application of Bs MBI600. Bs MBI600 induces genes involved in ISR and SAR signaling. In addition, genes involved in phytohormone production and nutrient availability showed an upregulation pattern, justifying the plant growth promotion. Biocontrol ability of Bs MBI600 seems also to be related to the activation of defense-related genes, such as peroxidase, endo-1,3(4)-beta-glucanase, PR-4, and thaumatin-like. Moreover, KEGG enriched results showed that differentially expressed genes were classified into biocontrol-related pathways. To further investigate the plant’s response to the presence of PGPR, a profile of polar metabolites of cucumber treated with Bs MBI600 was performed and compared to that of untreated plants. The results of the current study gave insights into the mechanisms deployed by this biocontrol agent to promote plant resistance, helping to understand the molecular interactions in this system.

Olive leaf spot (OLS) caused by Fusicladiumoleagineum is mainly controlled using copper fungicides. However, the replacement of copper-based products with eco-friendly alternatives is a priority. The use of plant resistance-inducers (PRIs) or biological control agents (BCAs) could contribute in this direction. In this study we investigated the potential use of three PRIs (laminarin, acibenzolar-S-methyl, harpin) and a BCA (Bacillus amyloliquefaciens FZB24) for the management of OLS. The tested products provided control efficacy higher than 68%. In most cases, dual applications provided higher (p < 0.05) control efficacies compared to that achieved by single applications. The highest control efficacy of 100% was achieved by laminarin. Expression analysis of the selected genes by RT-qPCR revealed different kinetics of induction. In laminarin-treated plants, for most of the tested genes a higher induction rate (p < 0.05) was observed at 3 days post application. Pal, Lox, Cuao and Mpol were the genes with the higher inductions in laminarin-treated and artificially inoculated plants. The results of this study are expected to contribute towards a better understanding of PRIs in olive culture and the optimization of OLS control, while they provide evidence for potential contributions in the reduction of copper accumulation in the environment.

Lettuce is the most commonly cultivated leafy vegetable in Greece, available in the market throughout the year. In this study, an emerging foliar disease observed in commercial farms has been associated to the pathogen Fusarium equiseti, a member of the Fusarium incarnatum-equiseti species complex (FIESC). Thirty F. equiseti isolates obtained from symptomatic lettuce plants were identified on the basis of morphology and evaluated for their pathogenicity. The isolates were further characterized using amplification and sequence analysis of the internal transcribed region (ITS-rDNA), and of the translation elongation factor 1-alpha (TEF1-a), calmodulin (CAM), beta-tubulin (Bt), and small subunit (SSU) genes. Moreover, a novel RT-qPCR assay was developed, designing a primer pair and a probe based on the TEF1-a sequences. This assay showed high specificity, amplifying F. equiseti DNA samples, while no amplification product was observed from samples of other common soilborne fungi. The generated RT-qPCR assay could be a useful tool for the detection and quantification of F. equiseti in soil samples deriving from fields cultivated with lettuce and other leafy vegetables, hosts of this specific pathogen.