Epiphytic and endophytic microorganisms associated to different cultivar of tomato fruits in greenhouse environment and characterization of beneficial bacterial strains for the control of post-harvest tomato pathogens.

Several bacterial and fungal diseases affect greenhouse-grown tomato crops, causing severe annual yield losses worldwide. The need to reduce chemical compound applications has encouraged the search of alternative approach for the control of tomato diseases, including the use of biological control agents. The presence of total and beneficial microbial populations was investigated on the surface and in the pulp of seven cultivars of tomato fruit coming from eleven greenhouses in the Pachino district (south-east of Sicily), recognized by the European Community with the "Protected Geographical Indication" label. Principal component analysis (PCA) showed that epiphytic and endophytic microbial populations clustered into groups according to the areas of origin. Approximately 240 tomato fruit-associated bacterial isolates were selected and a high percentage of them showed antagonistic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, Xanthomonas euvesicatoria pv. perforans, Botrytis cinerea and Alternaria alternata. Analysis of the 16S rRNA gene sequences revealed a predominance of bacteria in Bacillus and Pseudomonas genera, followed by Citrobacter and Enterobacter. The presence of these genera differed according to the geographical areas of tomato samples, whereas their antagonistic capabilities varied according to the five tomato pathogens. The in vitro effectiveness of eight representative bacterial strains belonging to Pseudomonas, Bacillus and Enterobacter genera was confirmed in in vivo assays, carried out on tomato fruit artificially inoculated with A. alternata and B. cinerea. Altogether, these results revealed differences in population density of native microbiota of "Pomodoro di Pachino" fruits and allowed the selection of antagonistic bacterial strains that can be applied in field and in postharvest conditions as alternatives to chemical compounds.

Soil and Soilless Tomato Cultivation Promote Different Microbial Communities That Provide New Models for Future Crop Interventions.

The cultivation of soilless tomato in greenhouses has increased considerably, but little is known about the assembly of the root microbiome compared to plants grown in soil. To obtain such information, we constructed an assay in which we traced the bacterial and fungal communities by amplicon-based metagenomics during the cultivation chain from nursery to greenhouse. In the greenhouse, the plants were transplanted either into agricultural soil or into coconut fiber bags (soilless). At the phylum level, bacterial and fungal communities were primarily constituted in all microhabitats by Proteobacteria and Ascomycota, respectively. The results showed that the tomato rhizosphere microbiome was shaped by the substrate or soil in which the plants were grown. The microbiome was different particularly in terms of the bacterial communities. In agriculture, enrichment has been observed in putative biological control bacteria of the genera Pseudomonas and Bacillus and in potential phytopathogenic fungi. Overall, the study describes the different shaping of microbial communities in the two cultivation methods.

Plant Growth-Promoting Activity of Pseudomonas aeruginosa FG106 and Its Ability to Act as a Biocontrol Agent against Potato, Tomato and Taro Pathogens.

P. aeruginosa strain FG106 was isolated from the rhizosphere of tomato plants and identified through morphological analysis, 16S rRNA gene sequencing, and whole-genome sequencing. In vitro and in vivo experiments demonstrated that this strain could control several pathogens on tomato, potato, taro, and strawberry. Volatile and non-volatile metabolites produced by the strain are known to adversely affect the tested pathogens. FG106 showed clear antagonism against Alternaria alternata, Botrytis cinerea, Clavibacter michiganensis subsp. michiganensis, Phytophthora colocasiae, P. infestans, Rhizoctonia solani, and Xanthomonas euvesicatoria pv. perforans. FG106 produced proteases and lipases while also inducing high phosphate solubilization, producing siderophores, ammonia, indole acetic acid (IAA), and hydrogen cyanide (HCN) and forming biofilms that promote plant growth and facilitate biocontrol. Genome mining approaches showed that this strain harbors genes related to biocontrol and growth promotion. These results suggest that this bacterial strain provides good protection against pathogens of several agriculturally important plants via direct and indirect modes of action and could thus be a valuable bio-control agent.

Bioprospecting of Beneficial Bacteria Traits Associated With Tomato Root in Greenhouse Environment Reveals That Sampling Sites Impact More Than the Root Compartment.

Tomato is subject to several diseases that affect both field- and greenhouse-grown crops. To select cost-effective potential biocontrol agents, we used laboratory throughput screening to identify bacterial strains with versatile characteristics suitable for multipurpose uses. The natural diversity of tomato root-associated bacterial communities was bioprospected under a real-world environment represented by an intensive tomato cultivation area characterized by extraseasonal productions in the greenhouse. Approximately 400 tomato root-associated bacterial isolates, in majority Gram-negative bacteria, were isolated from three compartments: the soil close to the root surface (rhizosphere, R), the root surface (rhizoplane, RP), and the root interior (endorhizosphere, E). A total of 33% of the isolates produced siderophores and were able to solubilize phosphates and grow on NA with 8% NaCl. A total of 30% of the root-associated bacteria showed antagonistic activity against all the tomato pathogens tested, i.e., Clavibacter michiganesis pv. michiganensis, Pseudomonas syringae pv. tomato, Pseudomonas corrugata and Xanthomonas euvesicatoria pv. perforans, and Fusarium oxysporum f. sp. lycopersici. We found that the sampling site rather than the root compartment of isolation influenced bacterial composition in terms of analyzed phenotype. This was demonstrated through a diversity analysis including general characteristics and PGPR traits, as well as biocontrol activity in vitro. Analysis of 16S rRNA gene (rDNA) sequencing of 77 culturable endophytic bacteria that shared multiple beneficial activity revealed a predominance of bacteria in Bacillales, Enterobacteriales, and Pseudomonadales. Their in vitro antagonistic activity showed that Bacillus species were significantly more active than the isolates in the other taxonomic group. In planta activity against phytopathogenic bacteria of a subset of Bacillus and Pseudomonas isolates was also assessed.

The LuxR Regulators PcoR and RfiA Co-regulate Antimicrobial Peptide and Alginate Production in Pseudomonas corrugata.

Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR-RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.