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A study of molecular variation among Botrytis spp., the causal pathogen of scape and umbel blights of onion, as well as the biocontrol of the virulent pathogen using Bacillus subtilis PHYS7 under greenhouse conditions was carried out. Twenty-three isolates of Botrytis spp. were recovered from onion plants showing umbel blight symptoms. They were able to infect onion plants with varied severity. All Botrytis spp. produced cellulases; however, almost all of them produced pectinase. The highest activities of cellulases and pectinase were achieved by B. allii PHYOA1; however, B. cinerea PHYOC3 showed a high activity of cellulases but it failed to produce pectinase. The genetic variability among the pathogens was assessed by random amplified polymorphic DNA (RAPD) markers, using 5 random 10-mer primers: OPA03, OPA05, OPA06, OPI09, and OPW15. The results showed that 50 DNA bands ranging from 100 bp (OPA03) to 1600 bp (OPA05) were generated by the 5 primers that differentiated 9 isolates of Botrytis spp. The dual culture test showed that Bacillus subtilis PHYS77 and PHYS78 had a high antagonistic potentiality against the pathogen and involved in 60–62% reduction in its growth. Application of Ridomil Gold MZ and Bacillus subtilis PHYS77 on onion plants, 2 days after or before infection with the pathogen, significantly reduced the disease severity than the control. The study approved the molecular tool as a reliable and quick method to differentiate among the virulent and non-virulent strains of Botrytis spp. The application of B. subtilis PHYS77 as an effective biocontrol agent in the management of the onion blight disease can be recommended.

The present work focuses on the antifungal effect of volatile organic compounds (VOCs) produced by Bacillus amyloliquefaciens CPA-8 against Monilinia laxa, M. fructicola and Botrytis cinera, three postharvest fruit pathogens of sweet cherry fruit. VOCs were evaluated with a double petri dish assay against mycelial and colony growth of target pathogens. For this purpose, CPA-8 was grown on different media and cultured for 24 and 48 h at 30 °C before assays. Data showed that mycelial growth inhibition was higher when CPA-8 was grown on Tryptone Soya Agar (TSA) while no differences were generally observed when CPA-8 was cultured for either, 24 and 48 h. Moreover, no effects were observed on colony growth. The main volatile compounds emitted by CPA-8 were identified by solid-phase microextraction (SPME)-gas chromatography as 1,3 pentadiene, acetoin (3-hydroxy-2-butanone) and thiophene. Pure compounds were also tested in vitro on mycelial growth inhibition and their EC50 values against the three pathogens were estimated. Thiophene was the most effective VOC, showing more than 82% suppression of mycelial growth at the highest concentration (1.35 μL/mL headspace) and EC50 values ranging from 0.06 to 6.67 μL/mL headspace. Finally, the effectiveness of thiophene and CPA-8 VOCs was evaluated against artificially inoculated cherry fruits. Among the target pathogens, M. fructicola was clearly controlled by CPA-8 with less than 25% of rotten fruits compared to the control (65% disease incidence) and for all pathogens, less than 37.5% of CPA-8 treated decayed fruits produced spores (disease sporulation). Otherwise, pure thiophene showed no effect against any pathogen on disease incidence and disease sporulation. The results indicated that VOCs produced by B. amyloliquefaciens CPA-8 could develop an additive antifungal effect against postharvest fruit pathogens on stone fruit. This research was supported by the European project BIOCOMES FP7-612713 and by the Catalan government (Generalitat de Catalunya) for the PhD grant 2016-FI-B2 00143 (Amparo M. Gotor).

Metabolism and in vitro fungitoxicity of perinaphthenone against three economically important fungi of the citrus, Botryodiplodia spp., Botrytis spp. and Fusarium spp. were investigated. Perinaphthenone exhibited significant antifungal activity at 62.5 μM and above. Even, the inhibitory effect against Fusarium spp. was significantly enhanced by exposure to direct light. In addition, the metabolism of perinaphthenone by the three fungi was studied. Results show that perinaphthenone was transformed almost completely during the first 24 h; two major products, whose concentration increased progressively during the twelve days of the test, were isolated and identified as 2,3-dihydro-1H-phenalen-1-ol and 2,3-dihydro-phenalen-1-one. Although both metabolic products displayed a considerable fungistatic effect, their slightly lower activities in comparison to perinaphthenone indicate that the transformation was a detoxification process. Studies on the relationship between the effect of some substituents in the perinaphthenone core and the mycelial growth inhibition of Botryodiplodia spp. were also carried out. Results show that the α, β-unsaturated carbonyl system is an important structural requirement but not the only to be necessary for the strong antifungal activity of perinaphthenone. In general, the antifungal properties of perinaphthenone may be modulated through the incorporation of substituents in the naphthalene core or in the α, β-unsaturated carbonyl system. It is concluded that perinaphthenone could be used as an antifungal agent or as a structural template for the development of new fungicide compounds., Phenalenones; Botrytis spp.; Botryodiplodia spp.; Fusarium spp.; Antifungal activity; Metabolism; Reduction; Derivatives.

Phytopathogens cause undeniably serious damage in agriculture by harming fruit cultivations and lowering harvest yields, which as a consequence substantially reduces food production efficiency. Fungi of the Botrytis, Colletotrichum and Verticillium genera are a main concern in berry production. However, no rapid detection method for detecting all of these pathogens simultaneously has been developed to date. Therefore, in this study, a multiplex real-time PCR assay for this purpose was established. Universal fungal primers for the D2 region of the large subunit ribosomal DNA and three multiplexable fluorogenic probes specific for the chosen fungi were designed and deployed. The triplex approach for the molecular detection of these fungi, which was developed in this study, allows for the rapid and effective detection of crucial berry pathogens, which contributes to a more rapid implementation of protective measures in plantations and a significant reduction in losses caused by fungal diseases.

The effect of biofumigation, through slow-release diffusors, of thyme and savory essential oils (EO), was evaluated on the control of postharvest diseases and quality of peaches and nectarines. EO fumigation was effective in controlling postharvest rots. Naturally contaminated peaches and nectarines were exposed to EO vapors for 28 days at 0 °C in sealed storage cabinets and then exposed at 20 °C for five days during shelf-life in normal atmosphere, simulating retail conditions. Under low disease pressure, most treatments significantly reduced fruit rot incidence during shelf-life, while, under high disease pressure, only vapors of thyme essential oil at the highest concentration tested (10% v/v in the diffusor) significantly reduced the rots. The application of thyme or savory EO favored a reduction of brown rot incidence, caused by Monilinia fructicola, but increased gray mold, caused by Botrytis cinerea. In vitro tests confirmed that M. fructicola was more sensitive to EO vapors than B. cinerea. Essential oil volatile components were characterized in storage cabinets during postharvest. The antifungal components of the essential oils increased during storage, but they were a low fraction of the volatile organic compounds in storage chambers. EO vapors did not influence the overall quality of the fruit, but showed a positive effect in reducing weight loss and in maintaining ascorbic acid and carotenoid content. The application of thyme and savory essential oil vapors represents a promising tool for reducing postharvest losses and preserving the quality of peaches and nectarines.

Antifungal compounds such as ergosterol biosynthesis inhibitors are widely used to control crop diseases. Among them, one of the most recent, the hydroxyanilide fenhexamid, is efficient principally against Botrytis cinerea, the major causal agent of grey mould. Fenhexamid is a new type of ergosterol biosynthesis inhibitor affecting the sterol C4 demethylation processes due to its specific interaction with one of the four proteins of the enzymatic complex, the 3-ketoreductase. Our regular monitoring conducted on French vineyards allowed the identification of the first isolates of B. cinerea with acquired resistance. Two types of resistant isolates named HydR3- and HydR3+ were distinguished by their resistance level. This acquired resistance is due to point mutations in the erg27 gene leading to target modifications. These modifications induce a reduced in affinity of fenhexamid towards its target, the 3-ketoreductase. Because of their high resistant level, the HydR3+ strains have to be considered relative to the risk of resistance phenomenom occurrence in vineyards. Fitness studies conducted in vitro on isogenic mutants showed altered "overwintering" capacities of HydR3+ mutants suggesting that they probably do not impact fenhexamid's field efficacy. While B. cinerea's acquired resistance could be explained only by target modifications, as in most cases of fungicide resistance, the situation is different for the related species Botrytis pseudocinerea naturally resistant to fenhexamid. We show that erg27 polymorphism only slightly contributes to resistance whereas fenhexamid detoxification by a cytochrome P450 named cyp68.4 is the major mechanism responsible for the resistance. This is the first case of a functional validation of fungicide detoxification involved in resistance.

Antifungal compounds such as ergosterol biosynthesis inhibitors are widely used to control crop diseases. Among them, one of the most recent, the hydroxyanilide fenhexamid, is efficient principally against Botrytis cinerea, the major causal agent of grey mould. Fenhexamid is a new type of ergosterol biosynthesis inhibitor affecting the sterol C4 demethylation processes due to its specific interaction with one of the four proteins of the enzymatic complex, the 3-ketoreductase. Our regular monitoring conducted on French vineyards allowed the identification of the first isolates of B. cinerea with acquired resistance. Two types of resistant isolates named HydR3- and HydR3+ were distinguished by their resistance level. This acquired resistance is due to point mutations in the erg27 gene leading to target modifications. These modifications induce a reduced in affinity of fenhexamid towards its target, the 3-ketoreductase. Because of their high resistant level, the HydR3+ strains have to be considered relative to the risk of resistance phenomenom occurrence in vineyards. Fitness studies conducted in vitro on isogenic mutants showed altered ”overwintering” capacities of HydR3+ mutants suggesting that they probably do not impact fenhexamid’s field efficacy. While B. cinerea’s acquired resistance could be explained only by target modifications, as in most cases of fungicide resistance, the situation is different for the related species Botrytis pseudocinerea naturally resistant to fenhexamid. We show that erg27 polymorphism only slightly contributes to resistance whereas fenhexamid detoxification by a cytochrome P450 named cyp68.4 is the major mechanism responsible for the resistance. This is the first case of a functional validation of fungicide detoxification involved in resistance.

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Soilborne diseases (e.g. Rhizoctonia, Pythium, Botrytis spp., Pratylenchus penetrans) threaten flower bulb production in the Netherlands. Since measures against these diseases do not result in sufficient control or result in a high input of pesticides Applied Plant Research investigates the development of integrated management strategies to control these diseases. Integrated management combines preventive and control measures. Different aspects and results of several preventive and control measures are discussed. The contributions of soil suppressiveness, biofumigation crops, precrops, biological control, pesticides of natural origin in integrated disease management are considered