Antonio de Vicente

The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca

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.

Plant protection and growth stimulation by microorganisms: biotechnological applications of Bacilli in agriculture

The increasing demand for a steady, healthy food supply requires an efficient control of the major pests and plant diseases. Current management practices are based largely on the application of synthetic pesticides. The excessive use of agrochemicals has caused serious environmental and health problems. Therefore, there is a growing demand for new and safer methods to replace or at least supplement the existing control strategies. Biological control, that is, the use of natural antagonists to combat pests or plant diseases has emerged as a promising alternative to chemical pesticides. The Bacilli offer a number of advantages for their application in agricultural biotechnology. Several Bacillus-based products have been marketed as microbial pesticides, fungicides or fertilisers. Bacillus-based biopesticides are widely used in conventional agriculture, by contrast, implementation of Bacillus-based biofungicides and biofertilizers is still a pending issue.

Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings

Seedlings of Pinus pinaster Alton accumulated chlorophyll (Chl) when grown in complete darkness. Contents of Chl a and Chl b increased during germination, reaching similar levels in light- and dark-grown plants. Glutamine-synthetase (GS; EC 6.3.1.2) activity was detected in the embryo and its level increased markedly in cotyledons of dark-germinated seedlings. Similar levels of GS activity were observed when the seeds were germinated in the presence of white light. Only one GS form, which eluted at about 0.1 M KCl, was found by ion-exchange chromatography. A predominant GS polypeptide of 43 kDa was detected in cotyledons, and its steady-state level increased with development in a lightindependent fashion. In roots and needles, a related GS polypeptide of 43 kDa was the unique species detectable by western blot analysis. Immunoblots of soluble proteins from isolated chloroplasts showed low abundance of GS protein, indicating that glutamine synthesis in pine cotyledons occurs mainly in the cytosol. Nitrogen-feeding experiments carried out with detached shoots indicated that neither NO3−nor NH4+regulate GS levels and the polypeptide pattern. Our results indicate that environmental factors, such as light and nitrogen supply, have a limited role in GS accumulation during pine development.

Coliphages as an indicator of faecal pollution in water. Its relationship with indicator and pathogenic microorganisms

Abstract The study was designed to test the proposal that Escherichia coli specific bacteriophages might serve as universal faecal pollution indicators in water. A highly specific, sensitive and rapid technique for the detection and quantification of these virus particles was developed. The numerical relationship between E. coli and its parasitic phages was investigated in three different aqueous ecosystems such as sea water in the vicinity of sewage outfalls, river water contaminated by domestic and industrial sewage discharges, and estuarine waters, and found to be very close. In addition, the results obtained indicate that the coliphages are good indicators of the presence of the pathogenic microorganisms studied. In nearly all the water samples tested, the results suggest that coliphages are better indicators of faecal pollution than the classical indicator systems currently employed.

The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits

UNLABELLED Numerous vegetable crops are susceptible to powdery mildew, but cucurbits are arguably the group most severely affected. Podosphaera fusca (synonym Podosphaera xanthii) is the main causal agent of cucurbit powdery mildew and one of the most important limiting factors for cucurbit production worldwide. Although great efforts have been invested in disease control, by contrast, many basic aspects of the biology of P. fusca remain unknown. TAXONOMY Podosphaera fusca (Fr.) Braun & Shishkoff. Kingdom Fungi; Phylum Ascomycota; Subdivision Pezizomycotina; Class Leotiomycetes; Order Erysiphales; Family Erysiphaceae; genus Podosphaera; species fusca. IDENTIFICATION Superficial persistent mycelium. Conidia in chains, hyaline, ellipsoid to ovoid or doliform, about 24-40 x 15-22 microm, with cylindrical or cone-shaped fibrosin bodies, which often germinate from a lateral face and produce a broad, clavate germ tube and cylindrical foot-cells. Unbranched erect conidiophores. Cleistothecia globose, mostly 70-100 microm in diameter, dark brown/black. One ascus per cleistothecium with eight ascospores. HOST RANGE Angiosperm species that include several families, such as Asteracea, Cucurbitaceae, Lamiaceae, Scrophulariaceae, Solanaceae and Verbenaceae. DISEASE SYMPTOMS White colonies develop on leaf surfaces, petioles and stems. Under favourable environmental conditions, the colonies coalesce and the host tissue becomes chlorotic and usually senesces early. CONTROL Chemical control and the use of resistant cultivars. Resistance has been documented in populations of P. fusca to some of the chemicals registered for control.

Screening for candidate bacterial biocontrol agents against soilborne fungal plant pathogens

Over the years, many bacterial isolates have been evaluated as potential biocontrol agents against soilborne fungal phytopathogens. However, few of them were ultimately successful after evaluation in field trials. One of the major reasons for this failure is the lack of appropriate screening procedures to select the most suitable microorganisms for disease control in diverse soil environments. For this reason, the study of bacterial screening has a future that is characterised by many technical and conceptual challenges. In this review, we summarise and discuss the convenience of use of the main screening methods currently applied to select bacterial candidates for biocontrol of fungal and oomycete soilborne phytopathogens. Also, a comparative case study of the application of different screening methods applied to an experimental pathosystem is shown, revealing the success of bacterial candidates selected by different strategies for biocontrol of the phytopathogenic fungus Rosellinia necatrix in avocado plants. Screening for antagonism against this fungal pathogen, one of the more straightforward methods used for the selection of bacterial biocontrol agents, was proven to be a valid strategy for this experimental system.

The antagonistic strain Bacillus subtilis UMAF6639 also confers protection to melon plants against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses

Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF6639. First, we examined the signalling pathways elicited by UMAF6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF6639 as potential determinants for ISR activation. Our results demonstrated that UMAF6639 confers protection against cucurbit powdery mildew by activation of jasmonate‐ and salicylic acid‐dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent.

Surfactin triggers biofilm formation of Bacillus subtilis in melon phylloplane and contributes to the biocontrol activity.

The biocontrol activity of many Bacillus species has been traditionally related to the direct antagonism of pathogens. In previous works, we reported that B. subtilis strain UMAF6614 was an efficient biocontrol agent that produced bacillomycin, fengycin and surfactin lipopeptides. Bacillomycins and fengycins were shown to have antagonistic activity towards fungal and bacterial pathogens of cucurbits; however, the functionality of surfactin remained unclear. In this study, the role of surfactin in the biocontrol activity of this strain was investigated. We observed that a deficiency in surfactin production led to a partial reduction of disease suppression by this biocontrol agent, which coincided with a defect in biofilm formation and the colonization of the melon phylloplane. These effects were due to a dramatic reduction in the production of exopolysaccharide and the TasA protein, which are the two major components of the extracellular matrix. We propose that the biocontrol activity of this strain is the result of the coordinated action of the three families of lipopeptides. B. subtilis UMAF6614 produces surfactin to trigger biofilm formation on melon phylloplane, which ensures the long-term persistence and the adequate secretion of suppressive lipopeptides, bacillomycins and fengycins, which efficiently target pathogens.

Biological control of peach brown rot (Monilinia spp.) by Bacillus subtilis CPA-8 is based on production of fengycin-like lipopeptides

Bacillus subtilis CPA-8, a strain with demonstrated ability to control Monilinia spp. in peaches, was studied to elucidate its mechanisms of antifungal activity. Growth inhibition assays using cell-free supernatants and butanolic extracts showed strong antifungal activities against Monilinia laxa and Monilinia fructicola. By comparison with the reference B. subtilis strains UMAF6614 and UMAF6639, fengycin, iturin and surfactin lipopeptides were identified by thin layer chromatography in butanolic extracts from cell-free supernatants, indicating that antibiosis could be a major factor involved in the biological control ability of CPA-8. TLC-bioautography analysis confirmed the presence of fengycin, iturin and surfactin lipopeptides but strong antifungal activity could be associated only with fengycin lipopeptides. These results were definitively supported by mutagenesis analysis targeted to suppress fengycin biosynthesis by disruption of the B. subtilis fenB gene. By TLC-bioautography analysis it was possible to identify transformants from CPA-8 with reduced or suppressed antifungal activity, and this phenotype was associated with the lack of fengycin bands. Fruit trials confirmed that fengycin-defective mutants and their cell-free supernatants lost their ability to control peach brown rot disease in comparison with CPA-8 wild type strain or Serenade Max®, a commercial formulation based on B. subtilis. Furthermore, population dynamics studies determined that CPA-8 fengycin-deficient mutants survived in wounds in peach fruit equally well as the CPA-8 wild type. Taken together our data indicate that fengycin-like lipopeptides play a major role in the biological control potential of B. subtilis CPA-8 against peach brown rot.

The Iturin-like Lipopeptides Are Essential Components in the Biological Control Arsenal of Bacillus subtilis Against Bacterial Diseases of Cucurbits

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.