Antifungal effect of volatile organic compounds produced by Bacillus amyloliquefaciens CPA-8 against fruit pathogen decays of cherry

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)

New advances in the control of brown rot in stone fruit using the biocontrol agent Bacillus amyloliquefaciens CPA-8

La podridura marró causada pel fong Monilinia spp. és responsable d'importants pèrdues en la postcollita de la fruita de pinyol. Entre les estratègies de control respectuoses amb el medi ambient, cal destacar l'ús d'agents de biocontrol (ABCs). Aquesta tesi té com a objectiu completar el desenvolupament de l'ABC B. amyloliquefaciens CPA-8 per així obtenir un producte eficaç que proporcioni una estratègia comercialment viable. Els principals resultats es classifiquen en (i) caracterització de CPA-8 (disseny de marcadors moleculars, respostes ecofisiològiques, sensibilitat a antibiòtics, producció d’enterotoxines); (ii) desenvolupament de productes basats en CPA-8 (millora del medi de creixement, selecció de la tecnologia de formulació, optimització de l'assecat per llit fluïditzat-atomització); i (iii) definició de l'estratègia de control de CPA-8 (activitat en diferents hostes, definició de llindars tècnics i eficàcia en assajos de camp). La integració dels productes basats en CPA-8 en els sistemes de cultiu habituals contribueix en el maneig de les malalties de postcollita en fruita d'os en el marc d'una agricultura sostenible i / o ecològica.\\\\La podredumbre marrón causada por el hongo Monilinia spp. es responsable de importantes pérdidas en la poscosecha de la fruta de hueso. Entre las estrategias de control respetuosas con el medio ambiente, cabe destacar el uso de agentes de biocontrol (ABCs). Esta tesis tiene como objetivo completar el desarrollo del ABC B. amyloliquefaciens CPA-8 para así obtener un producto eficaz que proporcione una estrategia comercialmente viable. Los resultado se clasifican en (i) caracterización de CPA-8 (diseño de marcadores moleculares, respuestas ecofisiológicas, sensibilidad a antibióticos, producción de enterotoxinas); (ii) desarrollo de productos basados en CPA-8 (mejora del medio de crecimiento, selección de la tecnología de formulación, optimización del secado por lecho fluido-atomización); y (iii) definición de la estrategia de control de CPA-8 (actividad en diferentes huéspedes, definición de umbrales técnicos y eficacia en ensayos de campo). La integración de los productos basados en CPA-8 en los sistemas de cultivo habituales contribuye en el manejo de las enfermedades de poscosecha en fruta de hueso en el marco de una agricultura sostenible y/o ecológica.Brown rot caused by the fungus Monilinia spp. is responsible for substantial postharvest losses of stone fruit. Among the environment-friendly strategies of control, the application of biological control agents (BCAs), has been strongly considered. Therefore, this thesis aimed to complete de development of the BCA Bacillus amyloliquefaciens CPA-8 to obtain efficacious CPA-8-based products that provide a plausible commercial strategy. The main findings are classified into (i) CPA-8 characterisation (molecular markers design, ecophisiological responses, sensibility to antibiotics, production of enterotoxins); (ii) CPA-8-based products development (improvement of the growth medium, selection of the formulation approach, fluid-bed spray-drying optimisation); and (iii) definition of the CPA-8 control strategy (activity in a wide range of hosts, definition of some technical thresholds and field efficacy at harvest and postharvest time). The integration of the CPA-8-based products here developed into the usual cropping systems can contribute to the management of post-harvest diseases in stone fruit in the framework of a sustainable and/or organic agriculture