Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.     

Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer

The antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer were investigated. The MIC (minimal inhibitory concentration) of fengycin without commercial surfactin added was 0.4 mg/ml while the MIC of fengycin with commercial surfactin added was 2.0 mg/ml. Fengycin acted on cell membrane and cellular organs and inhibited DNA synthesis. The antifungal effect of fengycin was reduced after commercial surfactin was added. All these results suggest that the fungal cell membrane may be the primary target of fengycin action and commercial surfactin may reduce the antifungal activity of fengycin.     

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.     

Putative new plant viruses associated with Plasmopara viticola-infected grapevine samples

In this study, we analysed a total of 16 libraries from over 150 grapevine leaf and grape samples infected with Plasmopara viticola (downy mildew of grapevine) to characterise the virome associated to this oomycete. Samples were collected in five distinct regions in Italy and in four different regions in Spain, representative of different pedoclimatic conditions and different grapevine cultivars during 2018 growing season. Due to the metagenomics nature of the samples (containing at least both downy mildew hyphae and spores, and grapevine cells residues), we were able to assemble several plant viruses and a few possible novel plant virus genomes with our in silico analysis. We detected several plant virus variants already reported in grapevine, and a putative new ilarvirus previously unreported in grapevine. Furthermore, we characterised three new phenui-like viruses (in the order Bunyavirales), one of which shares some commonalities with plant coguviruses. Finally, we report a new strict association of three viral segments (one flavi-like and two virga-like) that we propose to be a new virus taxon named jivivirus.     

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.     

Two imide substances from a soil-isolated Streptomyces atratus strain provide effective biocontrol activity against grapevine downy mildew

Grapevine downy mildew (Plasmopara viticola) is a devastating disease of grapevine. In this study, 151 actinomycete isolates were obtained and tested for antagonistic activity against P. viticola. The assay suggested that 28 isolates displayed antagonistic effects to varying degrees. The greatest reduction in disease severity was observed with isolate PY-1, which reduced disease severity by 92.13% in the detached leaf assay, and by 83% in a field assay. It was identified as Streptomyces atratus using the 16S rDNA sequence analysis. To elucidate the antagonistic mechanism of PY-1 against P. viticola, scanning electron microscopy showed that major damage to the pathogens sporangia and sporangiophores was observed after treatment for PY-1. Furthermore, PY-1 showed antagonistic activity against other pathogens, including: Botrytis cinerea, Colletotrichum orbiculare, Fusarium oxysporum, Phytophthora capsici and Phytophthora infestans. Two imide compounds were purified from the fermentation liquid using silica gel chromatography and high-performance liquid chromatography and identified as 5-acetoxycycloheximide and cycloheximide using nuclear magnetic resonance. Both compounds showed significant antagonistic activity against P. viticola, determining a reduction in disease severity by 65% and 84%, respectively. In conclusion, 5-acetoxycycloheximide and cycloheximide were identified for the first time in a new S. atratus strain able to effectively control grapevine downy mildew.     

Levels of 4-hydroxystilbene-oxidizing isoperoxidases related to constitutive disease resistance in in vitro-cultured grapevine

A zymographic screening of peroxidases (EC 1.11.1.7) capable of oxidizing 4-hydroxystilbene was carried out by means of the peroxidase-catalyzed oxidative coupling of 4-hydroxystilbene and 4-aminoantipyrine. This screening reveals that only a few isoperoxidases are active in oxidizing 4-hydroxystilbene to viniferin-type compounds in in vitro cultures of grapevine. Unlike total peroxidase activity measured with 4-methoxy--naphthol, the levels of total peroxidase activity measured using 4-hydroxystilbene are related to disease resistance against downy mildew in axillary bud cultures of Vitis vinifera and (Vitis vinifera x Vitis rupestris) x Vitis riparia. From this observation, and using the above zymographic assay, it was found that a 4-hydroxystilbene-oxidizing isoperoxidase was overexpressed in both leaves and stems of the hybrid in relation to the increase in disease resistance of this species. These results suggest that constitutive 4-hydroxystilbene-oxidizing isoperoxidases may participate through their role in viniferin synthesis in the constitutive resistance mechanism that grapevines show against downy mildew.Abbreviations 4-AAP 4-aminoantipyrine - HRP horseradish peroxidase - 4-HS hydroxystilbene - HSPrx 4-hydroxystilbene-oxidizing peroxidase - 4-MN 4-methoxy--naphthol     

Control of Downy Mildew, Plasmopara viticola (de Bary) Berlese et de Toni, on Grapevine Leaves through Water Extracts from Composted Organic Wastes

Watery extracts of composted manure-straw-soil mixtures induced increased resistance of grapevine leaves against downy mildew, Plasmopara viticola, if applied by dipping or spraying. The extracts had no direct fungicidal or fungitoxic effects.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

Optimization of Inactivation of Endospores of <Emphasis Type="Italic">Bacillus cereus</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sterilization of surfactin and fengycin to Bacillus cereus was observed, and the optimization of the inactivation of surfactin and fengycin to spores of B. cereus by a response surface methodology was studied. Results showed that surfactin and fengycin had high sterilization to B. cereus, whose minimal inhibitory concentration was 31.25 μM and 62.5 μM respectively. The optimization result indicated that spores of B. cereus could be inactivated by two orders of magnitude when the temperature was 20.41°C, the action time was 21.13 h, and the concentration (surfactin/fengycin molar ratio 1:1) was 54.20 μM.     

Detection and identification of antibiotic biosynthesis genes in Bacillus subtilis strains

Two Bacillus subtilis strains L10 and G1, previously isolated from fermented pickles, were tested for antibacterial activity against Vibrio harveyi using a well-diffusion agar assay. Antibiotic biosynthesis genes were then detected in both bacterial strains and included the following genes: bacA, bacB and bacD genes for bacilysin production, ppsE/fenB gene for plipastatin/fengycin production, albF and albA genes for subtilosin production, and srfAB and sfP genes for surfactin production. Based on these results, two B. subtilis strains could be considered as potential biological control agents in aquaculture.     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Optimization of Sterilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sensitivity of Escherichia coli to surfactin and fengycin was observed, and the optimization of the antimicrobial activity of surfactin and fengycin to E. coli in milk by a response surface methodology was studied. Results showed that E. coli had high sensitivity to these antibiotics, whose minimal inhibitory concentrations were 15.625 μg·mL⁻¹ and 31.25 μg·mL⁻¹, respectively. The optimization result indicated that E. coli could be sterilized by 5 orders of magnitude when the temperature was 5.5°C, the action time was 15.8 h, and the concentration (surfactin/fengycin weight ratio 1:1) was 14.63 μg·mL⁻¹.     

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety

Background  

Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola.     

Identification and biochemical characteristics of lipopeptides from Bacillus mojavensis A21

This study reports the potential of a marine bacterium, Bacillus mojavensis A21, to produce lipopeptide biosurfactants. The crude lipopeptide mixture was found to be very effective in reducing surface tension to 31 mN m⁻¹. PCR experiments using degenerate primers revealed the presence of nonribosomal peptide synthetases genes implied in the biosyntheses of fengycin and surfactin. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) performed on whole cells of B. mojavensis A21 confirmed the presence of lipopeptides identified as members of surfactin and fengycin families. Further, a detailed analysis performed by MALDI-TOF-TOF revealed the presence of pumilacidin compounds. The crude lipopeptide mixture was tested for its inhibitory activity against Gram-positive and Gram-negative bacteria, and fungal strains. It was found to display significant antimicrobial activity. Strain A21 lipopeptide mixture was insensitive to proteolytic enzymes, stable between pH 3.0 and 11.0, and resistant to high temperature. Production of lipopeptides is a characteristic of several Bacillus species, but to our knowledge this is the first report involving identification of pumilacidin, surfactin and fengycin isoforms in a B. mojavensis strain.     

Histological and Ultrastructural Studies on the Curative Effects of Mandipropamid on Plasmopara viticola

The mandelic acid amide, mandipropamid, which belongs to the carboxylic acid amide (CAA) fungicides, is active against Plasmopara viticola, the causal agent of grapevine downy mildew. The fungicide primarily inhibits the germination of encysted zoospores, thus preventing the pathogen’s penetration into the host tissues, but it also shows curative effects. In this study, the infection structures of P. viticola in both leaves and berries were investigated to detect the histological and ultrastructural alterations induced by mandipropamid when applied after inoculation. Compared to the untreated samples characterized by a diffuse colonization of the tissues and by a normal ultrastructure of the pathogen, the application of mandipropamid 24 h after inoculation with P. viticola reduced pathogen colonization in leaves and berries. In addition, detachment of the plasmalemma from the hyphal and haustorial walls was observed 72 h after inoculation. In the berries, an abnormal proliferation of the pathogen plasma membrane was observed. Collapsed hyphae and haustoria in treated leaves were surrounded by callose or encapsulated in an amorphous material inside the host cell 72 h after inoculation, while a similar effect was observed in later stages (7 days) in berries. The results confirm that mandipropamid, which acts at the interface between the pathogen plasmalemma and cell wall, has curative activity against P. viticola, appearing more rapidly in leaves than in berries.     

Exploiting medicinal plants and phylloplane microflora for the management of grapevine downy mildew

Abstract

Grapevine downy mildew is the most devastating disease throughout the world causing huge monetary losses. Twenty medicinal plant extracts and six phylloplane microfloras were evaluated for their efficacy against sporangial germination of grapevine downy mildew pathogen Plasmopara viticola in vitro. The results revealed that the Neem Seed Kernel Extract (NSKE) at 5% significantly inhibited the sporangial germination (75.36%) of P. viticola. Among the phylloplane microflora Pseudomonas fluorescens was highly effective in reducing the sporangial germination (64.26%). Post inoculation spraying of NSKE (5%) and P. fluorescens (0.2%) effectively inhibited the disease development in the greenhouse. Three sprays with NSKE (5%) and phylloplane P. fluorescens (0.2%): first spray after initial appearance of disease and the second and third at 10 day intervals were found to be promising in reducing disease incidence in the field.     

An ethoxylated surfactant enhances the penetration of the sulfated laminarin through leaf cuticle and stomata,leading to increased induced resistance against grapevine downy mildew

Some β‐1,3‐glucans and particularly sulfated laminarin (PS3) are known as resistance inducers (RIs) in grapevine against the downy mildew. However, their efficacy in vineyard is still often too low, which might be caused by a limited penetration through the leaf cuticle following spray application. We used ¹⁴C‐sucrose uptake experiments with grapevine leaves in order to select a surfactant as saccharide penetration enhancer. Our results showed that although sucrose foliar uptake was low, it was strongly enhanced by Dehscofix CO125 (DE), a highly ethoxylated surfactant. Fluorescent saccharides were then produced and laser scanning microscopy was used to analyze their foliar diffusion pattern in Arabidopsis thaliana and grapevine. Interestingly, sucrose and PS3 were seemingly able to penetrate the leaf cuticle only when formulated with DE. Diffusion could preferentially occur via stomata, anticlinal cell walls and trichomes. In grapevine, PS3 penetration rate was much higher on the stomateous abaxial surface of the leaf than on the adaxial surface. Finally, using DE allowed a higher level of downy mildew control by PS3, which corroborated diffusion observations. Our results have practical consequences for the improvement of treatments with saccharidic inducers on grape. That is, formulation of such RIs plays a critical role for their cuticular diffusion and consequently their efficacy. Also, spray application should preferentially target the abaxial surface of the leaves in order to maximize their penetration.