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 EC 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 EC 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.

Està subjecte a una llicència de Reconeixement-NoComercial-SenseObraDerivada 4.0 de Creative Commons Chapter V 175 Chapter V Biocontrol products based on Bacillus amyloliquefaciens CPA-8 using fluid-bed spray-drying process to control postharvest brown rot in stone fruit

Different treatments based on the biocontrol agent (BCA) Bacillus amyloliquefaciens CPA-8 to control brown rot under field conditions were evaluated as alternative to chemical applications. As part of a well-designed disease program that enables the integration of BCAs into cropping systems, testing of the sensitivity of Monilinia laxa and Monilinia fructicola at different doses of CPA-8 were conducted in stone fruit. CPA-8 dose of 10 7 CFU mL-1 reduced more than 60.0 and 75.5 % of brown rot incidence and severity, respectively. Once in the orchard, different degree of biocontrol activity was obtained depending on the inoculum pressure, which was mainly associated with meteorological conditions. Under drastic disease pressure, neither CPA-8 treatment nor the chemicals controlled the disease at harvest and only the chemical treatment reduced postharvest brown rot incidence. However, when Monilinia spp. incidence was close to the standard levels recorded in the area, treatments based on CPA-8 formulations proved to be efficacious. At harvest, BA3, BA4 treatments (CPA-8 optimised products) and PF+BA3 treatment (CPA-8 combined with Penicillium frequentans) reduced Monilinia spp. incidence compared to the control (54.7-64.1 %) although less than the chemicals (90.6 %). At postharvest, almost all CPA-8-based treatments (except PF+BA3) controlled the pathogen with BA4 treatment being as much effective as the chemicals (50.3 % of disease reduction). Finally, the population dynamics of CPA-8 on treated fruit surface remained after treatment application, at harvest and at postharvest shelf-life (>10 4 CFU cm-2). This study highlights the potential of B. amyloliquefaciens CPA-8 as alternative or complementary strategies to control Monilinia spp.

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA-8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA-8 that was sequenced and used to design SCAR (sequence-characterised amplified regions) primer pairs. The SCAR-4 marker amplified a semi-specific fragment of 665 bp not only for the strain CPA-8 but also for other 12 strains whose morphology was completely different from CPA-8. Another approach was developed to obtain a strain-specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA-8. Our results revealed that these two molecular markers, SCAR-4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA-8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA-8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.

The present work focuses on the assessment and comparison of three different formulation technologies and the effect of protectants on cell viability, storage stability, and antagonistic activity of the biocontrol agent Bacillus amyloliquefaciens CPA-8. Cultures were concentrated with different protective substances such as MgSO4, sucrose, and skimmed milk (SM) and subjected to liquid formulation, freeze-drying, and fluid-bed spray-drying. Results showed that CPA-8 freeze-dried cells without protectants or amended with SM suffered the highest losses in cell viability (0.41-0.48 log). Moreover, the cell viability of the tested freeze-dried products decreased after four months of storage at both tested temperatures (4 and 22 ºC). Otherwise, liquid and fluid-bed spray-dried products were stable for four months at 4 ºC and for twelve months at 22, 4 and-20 ºC, respectively, and no effect of the protectants was observed. The most suitable CPA-8 products were then tested against Monilinia laxa and Monilinia fructicola on artificially wounded nectarines and in all cases, the antagonistic activity was maintained similar to fresh cells. The efficacy results revealed that the formulation process did not affect the biocontrol potential of CPA-8. This work led us to conclude that effective formulations with final concentrations ranging from 1.93•10 9 to 2.98•10 9 CFU mL-1 and from 4.76•10 9 to 1.03•10 10 CFU g-1 were obtained for liquid and dried products, respectively. Additionally, the suitability of the fluid-bed spray-drying technology should be taken into account to develop a stable and effective CPA-8 product for practical applications to control brown rot in stone fruit.

Està subjecte a una llicència de Reconeixement-NoComercial-SenseObraDerivada 4.0 de Creative Commons Chapter VI 197 Chapter VI Environmental stress responses of the Bacillus amyloliquefaciens CPA-8-formulated products on nectarines and peaches

This work focuses on the biological understanding of the biocontrol agent Bacillus amyloliquefaciens CPA-8 in order to accomplish the characterization required in the registration process for the development of a microorganism-based product. The tolerance of CPA-8 to grow under different pH-temperature and water activity (a )-temperature conditions was widely demonstrated. Regarding the pH results, optimum growth at the evaluated conditions was observed at 37 °C and pH between 7 and 5. On the contrary, the slowest growth was recorded at 20 °C and pH 4.5. Moreover, the type of solute used to reduce a had a great influence on the minimum a at which the bacterium was able to grow. The lowest a values for CPA-8 growth in media modified with glycerol and glucose were 0.950 and 0.960, respectively. Besides, the lowest a for CPA-8 growth increased when the temperature decreased to 20 °C, at which CPA-8 was not able to grow at less than 0.990 a, regardless of the type of solute. Antibiotic susceptibility tests were carried out to determine which antibiotic could affect the behavior of the bacteria and revealed that CPA-8 was clearly resistant to hygromycin. Finally, a PCR amplification assay to detect the presence of enterotoxic genes from Bacillus cereus in CPA-8 was also performed. CPA-8 gave negative results for all the genes tested except for nheA gene, which is not enough for the toxicity expression, suggesting that fruit treated with this antagonist will not be a potential vehicle for foodborne illnesses.

Two effective biocontrol products (named as BA3 and BA4) based on Bacillus amyloliquefaciens CPA-8 have been reported as a potential alternative to chemical applications against brown rot caused by Monilinia spp. on stone fruit. To have practical use, this study aimed to describe the best packaging strategies (bags or flasks, atmosphere, and temperature of storage) to not only guarantee efficacy but also stability and ease of application of the products to be handled through the normal channels of distribution and storage. In terms of the viability neither the BA3 nor the BA4 product has been compromised after twelve months of storage. However, storage at 4 ºC affected the stability and visual aspect of both CPA-8 formulations, mainly associated not only to the increase of RH but also a w. Moreover, it should be pointed out that flasks did not conserve refrigerated BA3 samples in a suitable way, since RH and a w increased noticeably making their visual properties unsightly after 10 months of cold storage. At that time, the BA4 products were better preserved at 4 ºC when packaged in flasks. Finally, this study also demonstrated that the most suitable packaging conditions for long-term storability (stored at 22 ºC) did not show any negative effect in the biocontrol efficacy of CPA-8 in nectarines artificially infected with M. fructicola and provide suitable product delivery and field application. In conclusion, these results contribute to the final stage of development of these two CPA-8 products, practically ready for registration, thus contributing to the environmental-friendly management of postharvest diseases in stone fruit.