Enhanced shelf-life of the formulated biocontrol agent Bacillus amyloliquefaciens CPA-8 combining diverse packaging strategies and storage conditions

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 °C affected the stability and visual aspect of both CPA-8 formulations, mainly associated not only to the increase of RH but also aw. Moreover, it should be pointed out that flasks did not conserve refrigerated BA3 samples in a suitable way, since RH and aw increased noticeably making their visual properties unsightly after 10 months of cold storage. At that time, the BA4 products were better preserved at 4 °C when packaged in flasks. Finally, this study also demonstrated that the most suitable packaging conditions for long-term storability (stored at 22 °C) 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.info:eu-repo/semantics/acceptedVersio

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)

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

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 EC50values 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 EC50values 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

Biological control of brown rot in stone fruit using Bacillus amyloliquefaciens CPA-8 under field conditions

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 under laboratory conditions. CPA-8 dose of 107 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 (Penicillium frequentans combined with CPA- 8) reduced Monilinia spp. incidence compared to the control (54.7–64.1%) and similar to the chemicals. 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 (>104 CFU cm−2). This study highlights the potential of B. amyloliquefaciens CPA-8 as alternative or complementary strategies to control Monilinia spp. © 2017 Elsevier Lt