Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

Aims:  A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus. Methods and Results:  To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site‐directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application. Conclusions:  We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested. Significance and Impact of the Study:  We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.

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Bacterial apical necrosis of mango, elicited by Pseudomonas syringae pv. syringae, limits fruit production in southern Spain and Portugal. Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 59% were resistant to cupric sulfate. The survey of a mango orchard revealed an increase in frequencies of copper-resistant bacteria after repeated treatments with Bordeaux mixture. These data suggest that selection of copper-resistant strains could be a major reason for control failures following management with copper bactericides. Most copper-resistant isolates harbored plasmids, although the majority of them contained a 62-kb plasmid that also was present in copper-sensitive strains. The 62-kb plasmids were differentiated by restriction enzyme analysis and hybridization to copABCD DNA. The most frequently found copper-resistant plasmid type (62.1) was transferable by conjugation. Southern blot hybridizations showed that genetic determinants partially homologous to copABCD were present in all the copper-resistant strains examined, and usually were associated with plasmids; these determinants were not detected in copper-sensitive strains. The selective pressure exerted by copper bactericide sprays on the diversity of copper resistance determinants in bacterial populations of mango is discussed.

Pseudomonas syringae pv. syringae, which causes the bacterial apical necrosis of mango, produces the antimetabolite mangotoxin. We report here the cloning, sequencing, and identity analysis of a chromosomal region of 11.1 kb from strain P syringae pv. syringae UMAF0158, which is involved in mangotoxin biosynthesis. This chromosomal region contains six complete open reading frames (ORFs), including a large gene (ORF5) with a modular architecture characteristic of nonribosomal peptide synthetases (NRPS) named mgoA. A Tn5 mutant disrupted in mgoA was defective in mangotoxin production, revealing the involvement of the putative NRPS gene in the biosynthesis of mangotoxin. This derivative strain impaired in mangotoxin production also showed a reduction in virulence as measured by necrotic symptoms on tomato leaflets. Mangotoxin production and virulence were restored fully in the NRPS mutant by complementation with plasmid pCG2-6, which contains an 11,103-bp chromosomal region cloned from the wild-type strain P syringae pv. syringae UMAF0158 that includes the putative NPRS gene (mgoA). The results demonstrate that mgoA has a role in the virulence of P. syringae pv. syringae. The involvement of an NRPS in the production of an antimetabolite toxin from P. syringae inhibiting ornithine acetyltransferase activity is proposed.

a b s t r a c tBacillus amyloliquefaciens PPCB004 was selected as a potential antagonist to control Botrytis cinerea, Penicillium expansum and Rhizopus stolonifer on peach fruit. The HPLC data of PPCB004 indicated the lipopeptides iturin A, fengycin and surfactin as secondary metabolites. The GC/MS analysis of PPCB004 showed 3-hydroxy-2-butanone as the dominant compound (97.52% of relative peak area). Thyme (TO) and lemongrass (LO) oils showed over 50% and 25% inhibition of radial mycelial growth respectively with 8 ml oil per plate for all pathogens. Combination treatment with both oils failed to increase the percentage inhibition of radial mycelial growth of the pathogens. Combined application of PPCB004 with TO or LO was tested to assess the effectiveness in the control of these pathogens during postharvest storage. The biofilm formation of PPCB004 was significantly higher in LO than TO. LO (6 ml plate À1 ) and PPCB004 completely inhibited the mycelial growth of the pathogens. Fruit inoculation trials with PPCB004 þ LO in NatureFlexÔ modified atmosphere packaging (MAP), showed lower disease incidence and severity at 25 C for 5 d than treatments with PPCB004 þ MAP, PPCB004 þ TO þ MAP, LO þ MAP, TO þ MAP or stand-alone MAP. On naturally infected fruit, PPCB004 þ LO þ MAP and LO þ MAP treatments retained the total soluble solids/titratable acidity ratio and flesh firmness but failed to stimulate the levels of total phenolic content, phenylalanine ammonia-lyase, b-1,3-glucanase and chitinase activities. Combination of PPCB004 (spray treatment) þ LO (in pad delivery system) in NatureFlexÔ MAP showed absence of disease and off-flavour development, retained the overall appearance and increased the overall acceptance at market shelf conditions (20 C for 2 d) after cold storage at 4 C for 14 d.

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The Pseudomonas fluorescens complex of species includes plant-associated bacteria with potential biotechnological applications in agriculture and environmental protection. Many of these bacteria can promote plant growth by different means, including modification of plant hormonal balance and biocontrol. The P. fluorescens group is currently divided into eight major subgroups in which these properties and many other ecophysiological traits are phylogenetically distributed. Therefore, a rapid phylogroup assignment for a particular isolate could be useful to simplify the screening of putative inoculants. By using comparative genomics on 71 P. fluorescens genomes, we have identified nine markers which allow classification of any isolate into these eight subgroups, by a presence/absence PCR test. Nine primer pairs were developed for the amplification of these markers. The specificity and sensitivity of these primer pairs were assessed on 28 field isolates, environmental samples from soil and rhizosphere and tested by in silico PCR on 421 genomes. Phylogenomic analysis validated the results: the PCR-based system for classification of P. fluorescens isolates has a 98.34% of accuracy and it could be used as a rapid and simple assay to evaluate the potential of any P. fluorescens complex strain.