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.

A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.

Diseases of fruit trees caused by pathovars of Pseudomonas syringae van Hall are of major concern in fruitproducing areas worldwide, are exceedingly difficult to control, and result in significant economic losses. The pathogen has the ability to kill both young and older trees. Systemic infection and death of young trees is a perennial problem in nurseries, and canker development leading to the girdling and death of scaffold limbs and entire trees is a common event that can lead to the rapid demise of older orchards. For example, bacterial canker of plum caused by P. syringae pv. syringae causes annual tree mortality rates as high as 30% in Germany (43), and bacterial canker of hazelnut has resulted in the mortality of large numbers of trees in Italy and other European countries (77). Yield losses due to lesions on fruit are typically more sporadic in occurrence and variety dependent, but significant yearly losses have been reported in some instances. Finally, the pathogen's trait of ice nucleation activity also could exacerbate the importance of frost injury as a predisposing factor for infection. Frost damage in commercial orchards is a sporadic problem that facilitates colonization and the initiation of pathogenesis for these opportunistic pathogens.P. syringae and its close relatives cause diseases of monocots, herbaceous dicots, and woody dicots, and these pathogens utilize an impressive array of virulence factors such as effectors, toxins, and phytohormones to incite disease symptoms. Aspects of the systematics, ecology, and genetics of P. syringae have been reviewed and discussed by many authors (cited literature examples: 6,27,44,47,52). Extensive recent research efforts have focused on Pseudomonas diseases of herbaceous plants, and our understanding of P. syringae-host interactions has been facilitated by genome sequencing, comparative genomics, and functional studies (cited literature examples: 21,31,96).Our understanding of and ability to manage P. syringae diseases of fruit trees is relevant to our understanding of P. syringae diseases of herbaceous hosts, and vice versa. The exciting new insights into host-pathogen interactions from the model systems can give us clues on how to pro-ceed with the less genetically pliable fruit tree systems. Studies of the fruit tree systems, and comparisons with the model systems, can provide insights into the interactions of P. syringae strains with woody hosts and address relevant questions, such as: What is the role of type III secretion in infection of woody tissues? What virulence traits enable the colonization of woody tissue by P. syringae? Are there differences in the roles of toxins in diseases of woody hosts and herbaceous hosts? How did some pathogens evolve the ability to induce galls in woody hosts? Can resistance mechanisms characterized in the model-system herbaceous hosts be exploited in fruit trees?The objectives of this article are (i) to provide an overview of several Pseudomonas diseases of fruit trees, (ii) to discuss the current and emerging unde...

Aim:  This study was undertaken to isolate Bacillus subtilis strains with biological activity against soil‐borne phytopathogenic fungi from the avocado rhizoplane. Methods and Results:  A collection of 905 bacterial isolates obtained from the rhizoplane of healthy avocado trees, contains 277 gram‐positive isolates. From these gram‐positive isolates, four strains, PCL1605, PCL1608, PCL1610 and PCL1612, identified as B. subtilis, were selected on the basis of their antifungal activity against diverse soil‐borne phytopathogenic fungi. Analysis of the antifungal compounds involved in their antagonistic activity showed that these strains produced hydrolytic enzymes such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin, and/or iturin A. In biocontrol trials using the pathosystems tomato/Fusarium oxysporum f.sp. radicis‐lycopersici and avocado/Rosellinia necatrix, two B. subtilis strains, PCL1608 and PCL1612, both producing iturin A, exhibited the highest biocontrol and colonization capabilities. Conclusions:  Diverse antagonistic B. subtilis strains isolated from healthy avocado rhizoplanes have shown promising biocontrol abilities, which are closely linked with the production of antifungal lipopeptides and good colonization aptitudes. Significance and Impact of the Study:  This is one of the few reports dealing with isolation and characterization of B. subtilis strains with biocontrol activity against the common soil‐borne phytopathogenic fungi F. oxysporum f.sp. radicis‐lycopersici and R. necatrix.

The antibacterial potential of four strains of Bacillus subtilis, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, previously selected on the basis of their antifungal activity and efficacy against cucurbit powdery mildew, was examined. Among these strains, UMAF6614 and UMAF6639 showed the highest antibacterial activity in vitro, especially against Xanthomonas campestris pv. cucurbitae and Pectobacterium carotovorum subsp. carotovorum. These strains produced the three families of lipopeptide antibiotics known in Bacillus spp.: surfactins, iturins, and fengycins. Using thin-layer chromatography analysis and direct bioautography, the antibacterial activity could be associated with iturin lipopeptides. This result was confirmed by mutagenesis analysis using lipopeptide-defective mutants. The antibacterial activity was practically abolished in iturin-deficient mutants, whereas the fengycin mutants retained certain inhibitory capabilities. Analyses by fluorescence and transmission electron microscopy revealed the cytotoxic effect of these compounds at the bacterial plasma membrane level. Finally, biological control assays on detached melon leaves demonstrated the ability of UMAF6614 and UMAF6639 to suppress bacterial leaf spot and soft rot; accordingly, the biocontrol activity was practically abolished in mutants deficient in iturin biosynthesis. Taken together, our results highlight the potential of these B. subtilis strains as biocontrol agents against fungal and bacterial diseases of cucurbits and the versatility of iturins as antifungal and antibacterial compounds.

Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis . The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.

Summary Plant‐beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant‐growth promotion and/or disease‐suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine‐1‐carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil‐dwelling plant pathogens and play a role in the ecological competence of phenazine‐producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant‐beneficial phenazine‐producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein‐coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant‐beneficial phenazine‐producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant‐growth promotion and rhizosphere competence.

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.