Bacillus lipopeptides: versatile weapons for plant disease biocontrol

In the context of biocontrol of plant diseases, the three families of Bacillus lipopeptides - surfactins, iturins and fengycins were at first mostly studied for their antagonistic activity for a wide range of potential phytopathogens, including bacteria, fungi and oomycetes. Recent investigations have shed light on the fact that these lipopeptides can also influence the ecological fitness of the producing strain in terms of root colonization (and thereby persistence in the rhizosphere) and also have a key role in the beneficial interaction of Bacillus species with plants by stimulating host defence mechanisms. The different structural traits and physico-chemical properties of these effective surface- and membrane-active amphiphilic biomolecules explain their involvement in most of the mechanisms developed by bacteria for the biocontrol of different plant pathogens.     

Characterization of fungal antagonistic bacilli isolated from aerial roots of banyan (Ficus benghalensis) using intact‐cell MALDI‐TOF mass spectrometry (ICMS)

Aims

To characterize fungal antagonistic bacilli isolated from aerial roots of banyan tree and identify the metabolites responsible for their antifungal activity.

Methods and Results

Seven gram positive, endospore‐forming, rod‐shaped endophytic bacterial strains exhibiting a broad‐spectrum antifungal activity were isolated from the surface‐sterilized aerial roots of banyan tree. The isolates designated as K1, A2, A4 and A12 were identified as Bacillus subtilis, whereas isolates A11 and A13 were identified as Bacillus amyloliquefaciens using Biolog Microbial Identification System. The antifungal lipopeptides, surfactins, iturins and fengycins with masses varying in the range from m/z 900 to m/z 1550 could be detected using intact‐cell MALDI‐TOF mass spectrometry (ICMS). On the basis of mass spectral and carbon source utilization profile, all seven endophytes could be distinguished from each other. Furthermore, ICMS analysis revealed higher extent of heterogeneity among iturins and fengycins produced by B. subtilis K1, correlating well with its higher antifungal activity in comparison with other isolates.

Conclusion

Seven fungal antagonistic bacilli were isolated from aerial roots of banyan tree, exhibiting broad spectrum of antifungal activity, among which B. subtilis K1 isolate was found to be most potent. The ICMS analysis revealed that all these isolates produced cyclic lipopeptides belonging to surfactin, iturin and fengycin families and exhibited varying degree of heterogeneity.

Significance and Impact of the study

The endophytes are considered as a potential source of novel bioactive metabolites, and this study describes the potent fungal antagonistic bacilli from aerial roots of banyan tree. The isolates described in this study have a prospective application as biocontrol agents. Also ICMS analysis described in this study for characterization of antifungal metabolites produced by banyan endophytic bacilli may be used as a high throughput tool for screening of microbes producing novel cyclic lipopeptides.     

Cyclic Lipopeptide Biosynthetic Genes and Products,and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria

The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (98.4%) produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the production of antimicrobial cLPs.     

Characterization of Novel Lipopeptides Produced by Bacillus tequilensis P15 Using Liquid Chromatography Coupled Electron Spray Ionization Tandem Mass Spectrometry (LC–ESI–MS/MS)

Microbes are known to interact and communicate with their neighbouring cells by releasing diverse types of low molecular weight diffusible metabolites. This paper describes the identification of iturins, fengycins and surfactins secreted by Bacillus tequilensis P15 isolated from sea coast of Jakhao, Kutch, India, using liquid chromatography coupled electron spray ionization tandem mass spectrometry. In methanol soluble fraction of acid precipitate harvested from cell free supernatant of B. tequilensis P15, 5 variants of iturins, 6 of fengycins and 39 surfactins could be identified. In particular, new surfactins with Ile/Leu at position 5 and Asp at position 6 in the peptide chain were discovered, which have not been previously reported. A novel class of novel surfactin consisting of Glu/methyl ester of Asp at position 5 in peptide chain was also identified. In addition, several linear forms of surfactins were also identified in the methanol soluble extracellular fraction of B. tequilensis P15. This is the first report on co-production of all the three classes of cyclic lipopeptides by a marine isolate B. tequilensis.     

Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants

Multiple strains of Bacillus spp. were demonstrated to stimulate plant defence responses. However, very little is known about the nature of molecular determinants secreted by these Gram-positive bacteria that are responsible for the elicitation of the induced systemic resistance (ISR) phenomenon. This study shows that the lipopeptides surfactins and fengycins may be involved in this elicitation process. In bean, pure fengycins and surfactins provided a significant ISR-mediated protective effect on bean plants, similar to the one induced by living cells of the producing strain S499. Moreover, experiments conducted on bean and tomato plants showed that overexpression of both surfactin and fengycin biosynthetic genes in the naturally poor producer Bacillus subtilis strain 168 was associated with a significant increase in the potential of the derivatives to induce resistance. In tomato cells, key enzymes of the lipoxygenase pathway appeared to be activated in resistant plants following induction by lipopeptide overproducers. To our knowledge, such lipopeptides constitute a novel class of compounds from non-pathogenic bacteria that can be perceived by plant cells as signals to initiate defence mechanisms.     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis

Of the 13 strains of Bacillus subtilis tested for the coproduction of the lipopeptide surfactin and the antifungal lipopeptides of the iturin family, only 1 produced both lipopeptides with a high yield. The cultures were made in a synthetic medium. Several L-amino acids and various carbon sources were good substrates for the lipopeptide production. The maximum yield of surfactin was about 110 mg/liter and that of iturin A about 39 mg/liter/absorbance unit for the best strain, B. subtilis S 499.     

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.     

Cyclic lipopeptide profile of the plant-beneficial endophytic bacterium Bacillus subtilis HC8

In a previous study (Malfanova et al. in Microbial Biotech 4:523–532, 2011), we described the isolation and partial characterization of the biocontrol endophytic bacterium B. subtilis HC8. Using thin-layer chromatography, we have detected several bioactive antifungal compounds in the methanolic extract from the acid-precipitated supernatant of HC8. In the present study, we have further analyzed this methanolic extract using liquid chromatography-mass spectrometry. Based on the comparison of retention times and molecular masses with those of known antifungal compounds, we identified three families of lipopeptide antibiotics. These include four iturins A having fatty acyl chain lengths of C14 to C17, eight fengycins A (from C14 to C18 and from C15 to C17 containing a double bond in the acyl chain), four fengycins B (C15 to C18), and five surfactins (C12 to C16). Evaluation of the antifungal activity of the isolated lipopeptides showed that fengycins are the most active ones. To our knowledge, this is the first report of an endophytic Bacillus subtilis producing all three major families of lipopeptide antibiotics containing a very heterogeneous mixture of homologues. The questions remain open which of these lipopeptides (1) are being produced during interaction with the plant and (2) are contributing to the biocontrol activity of HC8.     

Biological control of postharvest diseases of apples,peaches and nectarines by Bacillus subtilis S16 isolated from halophytes rhizosphere

Penicillium expansum, Botrytis cinerea and Colletotrichum acutatum are the most common postharvest pathogens of apples, peaches and nectarines. In this study, 96 bacteria were isolated from halophytes rhizosphere and assayed for biocontrol activity under in vitro conditions. Among the 96 isolates tested, isolate S16 effectively inhibited the growth of P. expansum, B. cinerea and C. acutatum. The isolate S16 has reduced 78.33±1.53 to 82.98±2.13% of disease severity in apples, peaches and nectarines. Matrix-assisted laser desorption ionisation-time of flight mass spectrometry of the antifungal compounds revealed three lipopeptide complexes, namely surfactins, iturins and fengycins. Lipopeptides and hydrolytic enzymes produced by the isolate S16 play an important role in the antifungal activity. Polymerase chain reaction analysis using ituD, srfAD, fenD and fenE gene-specific primers showed that the isolate S16 carry sequences similar to ituD, srfAD, fenD and fenE genes. Based on the 16S rDNA sequencing, the effective isolate S16 was identified as Bacillus subtilis.     

芽孢杆菌三种抗菌素基因的杂交检测

以三对特异引物PCR合成表面活性素(surfactin)、伊枯草菌素(iturin)和抗菌蛋白TasA的地高辛标记探针,采用斑点杂交技术对24个菌株的相关基因加以检测,并用竞争酶联免疫吸附法(enzyme-linked immu-nosorbent assay,ELISA)分析阳性菌株中表面活性素基因的表达情况。结果表明:以阳性质粒为模板,获得1 200bp、1 479 bp和790 bp的3个特异性探针,其检测灵敏度分别为2 ng、20 ng和0.1 ng;从24个菌株中检测到10个菌株含有表面活性素合成相关基因,10个菌株含有伊枯草菌素合成相关基因和4个菌株中存在tasA基因;ELISA结果表明:从10个阳性菌株的KMB发酵液中均检测到表面活性素,其中菌株EN1和菌株SB1的产量较高,分别达32.9 mg/L和41.0 mg/L。     

Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens

Some isolates of the Bacillus subtilis/amyloliquefaciens species are known for their plant protective activity against fungal phytopathogens. It is notably due to their genetic potential to form an impressive array of antibiotics including non-ribosomal lipopeptides (LPs). In the work presented here, we wanted to gain further insights into the relative role of these LPs in the global antifungal activity of B. subtilis/amyloliquefaciens. To that end, a comparative study was conducted involving multiple strains that were tested against four different phytopathogens. We combined various approaches to further exemplify that secretion of those LPs is a crucial trait in direct pathogen ward off and this can actually be generalized to all members of these species. Our data illustrate that for each LP family, the fungitoxic activity varies in function of the target species and that the production of iturins and fengycins is modulated by the presence of pathogens. Our data on the relative involvement of these LPs in the biocontrol activity and modulation of their production are discussed in the context of natural conditions in the rhizosphere.     

Co-producing lipopeptides and poly-gamma-glutamic acid by solid-state fermentation of Bacillus subtilis using soybean and sweet potato residues and its biocontrol and fertilizer synergistic effects

A Bacillus subtilis strain B6-1, previously isolated from the rhizosphere of vegetable, selectively produced antibiotics or poly-gamma-glutamic acid (gamma-PGA) in two kinds of liquid media and their co-productions were obtained when using soybean and sweet potato residues in solid-state fermentation. The antibiotics were purified and identified as fengycins. After these residue cultures were introduced, cucumber wilts were effectively suppressed. The introduction also significantly increased the dry weights of roots and shoots of cucumber seedlings, and the roots to shoots ratio, especially at lower nutrition, which indicated the fertilizer synergistic effects. So the product of soybean and sweet potato residues, cultivated with B6-1 co-producing lipopeptides and gamma-PGA, can be expected to be used as both biocontrol agents and fertilizer synergists.     

Interaction of antimicrobial cyclic lipopeptides from Bacillus subtilis influences their effect on spore germination and membrane permeability in fungal plant pathogens

Bacillus subtilis cyclic lipopeptides are known to have various antimicrobial effects including different types of interactions with the cell membranes of plant pathogenic fungi. The various spectra of activities of the three main lipopeptide families (fengycins, iturins, and surfactins) seem to be linked to their respective mechanisms of action on the fungal biomembrane. Few studies have shown the combined effect of more than one family of lipopeptides on fungal plant pathogens. In an effort to understand the effect of producing multiple lipopeptide families, sensitivity and membrane permeability of spores from four fungal plant pathogens (Alternaria solani, Fusarium sambucinum, Rhizopus stolonifer, and Verticillium dahliae) were assayed in response to lipopeptides, both individually and as combined treatments. Results showed that inhibition of spores was highly variable depending on the tested fungus-lipopeptide treatment. Results also showed that inhibition of the spores was closely associated with SYTOX stain absorption suggesting effects of efficient treatments on membrane permeability. Combined lipopeptide treatments revealed additive, synergistic or sometimes mutual inhibition of beneficial effects.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge

An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants. This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B. subtilis C-1. This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B. subtilis strains. These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry. The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization. Iturin compounds which contain unusual fatty acid components were detected.     

New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction

Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.     

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.     

Enhanced Production of Surfactin from Bacillus subtilis by Continuous Product Removal and Metal Cation Additions

The lipopeptide, surfactin, is produced by Bacillus subtilis. A study has been made on large-scale production of this surfactant. A good yield was obtained from a glucose substrate fermentation by continuously removing the product by foam fractionation. The surfactin could be easily recovered from the collapsed foam by acid precipitation. The yield was also improved by the addition of either iron or manganese salts. Hydrocarbon addition to the medium, which normally increases biosurfactant production, completely inhibited surfactin production by B. subtilis.