Biocontrol activities of yeasts or lactic acid bacteria isolated from Robusta coffee against Aspergillus carbonarius growth and ochratoxin A production in vitro.

Biocontrol Agents (BCAs) can be an eco-friendly alternative to fungicides to reduce the contamination with mycotoxigenic fungi on coffee. In the present study, different strains of bacteria and yeasts were isolated from Ivorian Robusta coffee. Their ability to reduce fungal growth and Ochratoxin A (OTA) production during their confrontation against Aspergillus carbonarius was screened on solid media. Some strains were able to reduce growth and OTA production by 85 % and 90 % and were molecularly identified as two yeasts, Rhodosporidiobolus ruineniae and Meyerozyma caribbica. Subsequent tests on liquid media with A. carbonarius or solely with OTA revealed adhesion of R. ruineniae to the mycelium of A. carbonarius through Scanning Electron Microscopy, and an OTA adsorption efficiency of 50 %. For M. caribbica potential degradation of OTA after 24 h incubation was observed. Both yeasts could be potential BCAs good candidates for Ivorian Robusta coffee protection against A. carbonarius and OTA contamination.

The Influence of Long-Term Storage on the Epiphytic Microbiome of Postharvest Apples and on Penicillium expansum Occurrence and Patulin Accumulation.

Patulin is a secondary metabolite primarily synthesized by the fungus Penicillium expansum, which is responsible for blue mold disease on apples. The latter are highly susceptible to fungal infection in the postharvest stages. Apples destined to produce compotes are processed throughout the year, which implies that long periods of storage are required under controlled atmospheres. P. expansum is capable of infecting apples throughout the whole process, and patulin can be detected in the end-product. In the present study, 455 apples (organically and conventionally grown), destined to produce compotes, of the variety "Golden Delicious" were sampled at multiple postharvest steps. The apple samples were analyzed for their patulin content and P. expansum was quantified using real-time PCR. The patulin results showed no significant differences between the two cultivation techniques; however, two critical control points were identified: the long-term storage and the deck storage of apples at ambient temperature before transport. Additionally, alterations in the epiphytic microbiota of both fungi and bacteria throughout various steps were investigated through the application of a metabarcoding approach. The alpha and beta diversity analysis highlighted the effect of long-term storage, causing an increase in the bacterial and fungal diversity on apples, and showed significant differences in the microbial communities during the different postharvest steps. The different network analyses demonstrated intra-species relationships. Multiple pairs of fungal and bacterial competitive relationships were observed. Positive interactions were also observed between P. expansum and multiple fungal and bacterial species. These network analyses provide a basis for further fungal and bacterial interaction analyses for fruit disease biocontrol.

Impacts of abiotic factors on the growth of three commercial biological control agents, on the growth and mycotoxinogenesis of Fusarium graminearum and on their interaction.

BACKGROUND: Evolving climatic conditions impact the behavior of microorganisms. The lack of efficiency of beneficial microorganisms against pathogens can be due to these evolving abiotic factors more favorable to the development and adaptation of pathogens. It is therefore of great interest to understand their impact (especially temperature increase and relative humidity (RH) variation) on pathogenic and non-pathogenic microorganisms. This work aimed to examine the possible effects of increasing temperature (20, 25, 30 and 33 °C) and RH (40%, 50%, 60% and 80%) on the growth and mycotoxin production (deoxynivalenol (DON) and zearalenone (ZEN)) of Fusarium graminearum, on the growth of three commercial biocontrol agents (BCAs; Mycostop®, Xedavir® and Polyversum®) and on the pathogen-BCA interaction. RESULTS: Results demonstrated that BCAs have contrasting impacts on the growth and mycotoxinogenesis of F. graminearum depending on abiotic factors. At 25 °C and regardless of RH, commercial BCAs limit DON production by F. graminearum, but at 30 °C and intermediate RH, Xedavir® is no longer effective. The ability of Xedavir® to control the production of ZEN production by F. graminearum is also affected by abiotic factors. However, increasing temperature has an opposite effect on its ability to control the accumulation of ZEN. Polyversum® oomycete is the BCA with the most resilient efficacy against F. graminearum toxinogenesis under the different abiotic factors. CONCLUSION: This work provides new knowledge of the effect of these abiotic parameters on the interaction between BCA and F. graminearum, especially on the production of mycotoxins. It paves the way for the development of efficient and resilient mycotoxin biocontrol strategies using beneficial microorganisms against F. graminearum, thus contributing to global food security. © 2023 Society of Chemical Industry.

Antifungal and antimycotoxic activities of 3 essential oils against 3 mycotoxinogenic fungi.

Fungal toxins can have various adverse health effects, including carcinogenic, teratogenic or hepatotoxic impacts. In addition, fungal alteration has also a negative impact on agricultural plant production. The use of chemical fungicides to control mycotoxin contamination is increasingly controversial and regulated. More environmentally friendly methods are therefore being explored. Essential oils, as compounds extracted from plants, are liquids whose specific aromatic compounds give each essential oil its own unique characteristics. Due to their rich chemical composition, essential oils (EOs) have many interesting properties, including antifungal activities. The objective of the present study was to analyze volatile chemical composition of EOs (Cymbopogon schoenanthus, Cymbopogon nardus and Eucalyptus camaldulensis) by GC/MS and to investigate their effects on the growth, sporulation and mycotoxin production of Aspergillus flavus, Aspergillus carbonarius and Fusarium verticillioides (aflatoxin B1, ochratoxin A and fumonisin B1, respectively). In addition, EOs influence on aflatoxin B1 (AFB1) and fumonisin B1 (FB1) biosynthesis pathways was explored using real-time qRT-PCR. The results obtained in vitro, by direct contact with the EOs and by diffusion of their volatile compounds, showed that the essential oils had inhibitory effects on the growth and the production of mycotoxins of the 3 fungal strains and modified the expression of some toxin synthesis genes. We conclude that the recorded effects were dependent on the combined effects of the EOs type, the fungal strains and the doses studied.

Biocontrol Agents Reduce Progression and Mycotoxin Production of Fusarium graminearum in Spikelets and Straws of Wheat.

The aim of this study was to evaluate the interactions between wheat plant (spikelets and straws), a strain of mycotoxigenic pathogen Fusarium graminearum and commercial biocontrol agents (BCAs). The ability of BCAs to colonize plant tissue and inhibit the pathogen or its toxin production was observed throughout two phases of the life cycle of pathogens in natural conditions (colonization and survival). All evaluated BCAs showed effective reduction capacities of pathogenic traits. During establishment and the expansion stage, BCAs provoked an external growth reduction of F. graminearum (77-93% over the whole kinetic studied) and mycotoxin production (98-100% over the whole kinetic studied). Internal growth of pathogen was assessed with digital droplet polymerase chain reaction (ddPCR) and showed a very strong reduction in the colonization of the internal tissues of the spikelet due to the presence of BCAs (98% on average). During the survival stage, BCAs prevented the formation of conservation perithecia of the pathogen on wheat straw (between 88 and 98% of perithecia number reduction) and showed contrasting actions on the ascospores they contain, or perithecia production (-95% on average) during survival form. The mechanisms involved in these different interactions between F. graminearum and BCAs on plant matrices at different stages of the pathogen's life cycle were based on a reduction of toxins, nutritional and/or spatial competition, or production of anti-microbial compounds.

Biocontrol Agents: Toolbox for the Screening of Weapons against Mycotoxigenic Fusarium.

The aim of this study was to develop a set of experiments to screen and decipher the mechanisms of biocontrol agents (BCAs), isolated from commercial formulation, against two major mycotoxigenic fungi in cereals, Fusarium graminearum and Fusarium verticillioides. These two phytopathogens produce mycotoxins harmful to human and animal health and are responsible for the massive use of pesticides, for the protection of cereals. It is therefore essential to better understand the mechanisms of action of alternative control strategies such as the use of BCAs in order to optimize their applications. The early and late stages of interaction between BCAs and pathogens were investigated from germination of spores to the effects on perithecia (survival form of pathogen). The analysis of antagonist activities of BCAs revealed different strategies of biocontrol where chronological, process combination and specialization aspects of interactions are discussed. Streptomyces griseoviridis main strategy is based on antibiosis with the secretion of several compounds with anti-fungal and anti-germination activity, but also a mixture of hydrolytic enzymes to attack pathogens, which compensates for an important deficit in terms of spatial colonization capacity. It has good abilities in terms of nutritional competition. Trichoderma asperellum is capable of activating a very wide range of defenses and attacks combining the synthesis of various antifungal compounds (metabolite, enzymes, VOCs), with different targets (spores, mycelium, mycotoxins), and direct action by mycoparasitism and mycophagy. Concerning Pythium oligandrum, its efficiency is mainly due to its strong capacity to colonize the environment, with a direct action via microbial predation, stimulation of its reproduction at the contact of pathogens and the reduction of perithecia formation.

New Isolated Metschnikowia pulcherrima Strains from Apples for Postharvest Biocontrol of Penicillium expansum and Patulin Accumulation.

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application.

Aspergillus flavus Growth Inhibition and Aflatoxin B1 Decontamination by Streptomyces Isolates and Their Metabolites.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus, mainly during grain storage. As pre-harvest methods are insufficient to avoid mycotoxin presence during storage, diverse curative techniques are being investigated for the inhibition of fungal growth and aflatoxin detoxification. Streptomyces spp. represent an alternative as they are a promising source of detoxifying enzymes. Fifty-nine Streptomyces isolates and a Streptomyces griseoviridis strain from the commercial product Mycostop®, evaluated against Penicillium verrucosum and ochratoxin A during previous work, were screened for their ability to inhibit Aspergillus flavus growth and decrease the aflatoxin amount. The activities of bacterial cells and cell-free extracts (CFEs) from liquid cultures were also evaluated. Fifty-eight isolates were able to inhibit fungal growth during dual culture assays, with a maximal reduction going down to 13% of the control. Aflatoxin-specific production was decreased by all isolates to at least 54% of the control. CFEs were less effective in decreasing fungal growth (down to 40% and 55% for unheated and heated CFEs, respectively) and aflatoxin-specific production, with a few CFEs causing an overproduction of mycotoxins. Nearly all Streptomyces isolates were able to degrade AFB1 when growing in solid and liquid media. A total degradation of AFB1 was achieved by Mycostop® on solid medium, as well as an almost complete degradation by IX20 in liquid medium (6% of the control). CFE maximal degradation went down to 37% of the control for isolate IX09. The search for degradation by-products indicated the presence of a few unknown molecules. The evaluation of residual toxicity of the tested isolates by the SOS chromotest indicated a detoxification of at least 68% of AFB1's genotoxicity.

Microbiome Status of Cider-Apples, from Orchard to Processing, with a Special Focus on Penicillium expansum Occurrence and Patulin Contamination.

Patulin is a secondary metabolite produced primarily by the fungus Penicillium expansum, responsible for the blue mold disease on apples. It is found in apple products including apple cider when apple juice is added after fermentation. In the present study, two hundred and twenty-five cider-apples of the variety "Bedan", cultivated in Brittany in France, were sampled from the orchard during harvesting until the storage step, right before processing. The patulin analysis on these samples reported a low contamination at the orchard and a significantly higher-level of contamination in the cider-apples starting from the transporting bin. The percentage of positive samples increased from 6% to 47% after 12 h in the harvesting bin before transporting and reached 95% after 24 h of transporting, decreasing then to 69% at the end of the storage. Penicillium expansum was quantified on the surface of apples using real-time PCR and was observed to be mostly consistent between the harvest and post-harvest steps. It was detected on average, on the surface of 85% of all sampled apples with a mean value around 2.35 × 106Penicillium expansum DNA/g of apple. Moreover, the changes in the fungal and bacterial epiphytic microbiota in the different steps were studied using a metabarcoding approach. The alpha and beta diversity analysis revealed the presence of unique and more diverse bacterial and fungal communities on the surface of apples picked from the orchard compared to the rest of the sampling steps. Potential indigenous biological control agents were identified on the surface of sampled apples. Future perspective includes developing actions of prevention and control of the contamination by Penicillium expansum during the harvest and along the various critical post-harvest stages before transformation in a sustainable development concern.

Pineapple Mycobiome Related to Fruitlet Core Rot Occurrence and the Influence of Fungal Species Dispersion Patterns.

Fruitlet Core Rot (FCR) is a fungal disease that negatively impacts the quality of pineapple, in particular the 'Queen Victoria' cultivar. The main FCR causal agent has been identified as Fusariumananatum. This study focused on the correlation between FCR disease occurrence, fungal diversity, and environmental factors. FCR incidence and fungal species repartition patterns were spatially contextualized with specific surrounding parameters of the experimental plots. The mycobiome composition of healthy and diseased fruitlets was compared in order to search for potential fungal markers. A total of 240 pineapple fruits were sampled, and 344 fungal isolates were identified as belonging to 49 species among 17 genera. FCR symptom distribution revealed a significant gradient that correlated to that of the most abundant fungal species. The association of wind direction and the position of proximal cultivated crops sharing pathogens constituted an elevated risk of FCR incidence. Five highly represented species were assayed by Koch's postulates, and their pathogenicity was confirmed. These novel pathogens belonging to Fusariumfujikuroi and Talaromycespurpureogenus species complexes were identified, unravelling the complexity of the FCR pathosystem and the difficulty of apprehending the pathogenesis over the last several decades. This study revealed that FCR is an airborne disease characterized by a multi-partite pathosystem.

Mass spectrometry-based detection and risk assessment of mycotoxin contamination of 'kankankan' used for roasted meat consumption in Abidjan, Côte d'Ivoire.

'Kankankan' is a popular spice powder used to season roasted meat in Côte d'Ivoire. However, produced in a traditional way, the conditions of production and storage of kankankan favour the proliferation of mycotoxin-producing fungal strains. The aim of this study was to carry out an inventory of mycotoxin contamination of this spice powder and to assess risk exposure to consumers. In total, 75 samples of kankankan were collected from wholesalers (6), sellers of kankankan in the markets (35) and sellers of roasted meat (34) across three municipalities of Abidjan, the economic capital of Côte d'Ivoire. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to detect and quantify nine different mycotoxins. Dietary exposure was calculated by using estimated daily intake (EDI), whereas risk characterisation was assessed using the margin of exposure (MOE) approach. Aflatoxins and fumonisins were found in 99% of samples assessed, while contamination with beauvericin was proportionally lowest (28%). At all the three types of actors within the food production chain (wholesalers, kankankan sellers and roasted meat sellers) the mean concentrations of aflatoxin B1 (AFB1) in samples exceeded the European standard for spice mixtures, with concentrations reaching up to 502 µg/kg. The estimated daily intakes of aflatoxins observed in the different populations were above the recommended level of 0.017 ng kg-1 b.w. day-1. The MOES values for adolescents and adults were 8.10 and 12.78, respectively, well below the safe margin of 10,000. The co-occurrence of mycotoxins in kankankan samples together with high aflatoxin exposure to consumers represent a potential risk to public health, calling for immediate risk management and education of kankankan producers and consumers.

Minimizing Ochratoxin A Contamination through the Use of Actinobacteria and Their Active Molecules.

Ochratoxin A (OTA) is a secondary metabolite produced by fungal pathogens such as Penicilliumverrucosum, which develops in food commodities during storage such as cereals, grapes, and coffee. It represents public health concerns due to its genotoxicity, carcinogenicity, and teratogenicity. The objective of this study was to evaluate the ability of actinobacteria and their metabolites to degrade OTA and/or to decrease its production. Sixty strains of actinobacteria were tested for their ability to prevent OTA formation by in vitro dual culture assays or with cell free extracts (CFEs). In dual culture, 17 strains strongly inhibited fungal growth, although it was generally associated with an increase in OTA specific production. Seventeen strains inhibited OTA specific production up to 4% of the control. Eleven actinobacteria CFEs reduced OTA specific production up to 62% of the control, while no substantial growth inhibition was observed except for two strains up to 72% of the control. Thirty-three strains were able to degrade OTA almost completely in liquid medium whereas only five were able to decrease it on solid medium, and two of them reduced OTA to an undetectable amount. Our results suggest that OTA decrease could be related to different strategies of degradation/metabolization by actinobacteria, through enzyme activities and secretion of secondary metabolites interfering with the OTA biosynthetic pathway. CFEs appeared to be ineffective at degrading OTA, raising interesting questions about the detoxification mechanisms. Common degradation by-products (e.g., OTα or L-ß-phenylalanine) were searched by HPLC-MS/MS, however, none of them were found, which implies a different mechanism of detoxification and/or a subsequent degradation into unknown products.

Diversity and Toxigenicity of Fungi that Cause Pineapple Fruitlet Core Rot.

The identity of the fungi responsible for fruitlet core rot (FCR) disease in pineapple has been the subject of investigation for some time. This study describes the diversity and toxigenic potential of fungal species causing FCR in La Reunion, an island in the Indian Ocean. One-hundred-and-fifty fungal isolates were obtained from infected and healthy fruitlets on Reunion Island and exclusively correspond to two genera of fungi: Fusarium and Talaromyces. The genus Fusarium made up 79% of the isolates, including 108 F. ananatum, 10 F. oxysporum, and one F. proliferatum. The genus Talaromyces accounted for 21% of the isolated fungi, which were all Talaromyces stollii. As the isolated fungal strains are potentially mycotoxigenic, identification and quantification of mycotoxins were carried out on naturally or artificially infected diseased fruits and under in vitro cultures of potential toxigenic isolates. Fumonisins B1 and B2 (FB1-FB2) and beauvericin (BEA) were found in infected fruitlets of pineapple and in the culture media of Fusarium species. Regarding the induction of mycotoxin in vitro, F.proliferatum produced 182 mg kg⁻1 of FB1 and F. oxysporum produced 192 mg kg⁻1 of BEA. These results provide a better understanding of the causal agents of FCR and their potential risk to pineapple consumers.

Commercial Biocontrol Agents Reveal Contrasting Comportments Against Two Mycotoxigenic Fungi in Cereals: Fusarium Graminearum and Fusarium Verticillioides.

The aim of this study was to investigate the impact of commercialized biological control agents (BCAs) against two major mycotoxigenic fungi in cereals, Fusarium graminearum and Fusarium verticillioides, which are trichothecene and fumonisin producers, respectively. With these objectives in mind, three commercial BCAs were selected with contrasting uses and microorganism types (T. asperellum, S. griseoviridis, P. oligandrum) and a culture medium was identified to develop an optimized dual culture bioassay method. Their comportment was examined in dual culture bioassay in vitro with both fusaria to determine growth and mycotoxin production kinetics. Antagonist activity and variable levels or patterns of mycotoxinogenesis inhibition were observed depending on the microorganism type of BCA or on the culture conditions (e.g., different nutritional sources), suggesting that contrasting biocontrol mechanisms are involved. S. griseoviridis leads to a growth inhibition zone where the pathogen mycelium structure is altered, suggesting the diffusion of antimicrobial compounds. In contrast, T. asperellum and P. oligandrum are able to grow faster than the pathogen. T. asperellum showed the capacity to degrade pathogenic mycelia, involving chitinolytic activities. In dual culture bioassay with F. graminearum, this BCA reduced the growth and mycotoxin concentration by 48% and 72%, respectively, and by 78% and 72% in dual culture bioassay against F. verticillioides. P. oligandrum progressed over the pathogen colony, suggesting a close type of interaction such as mycoparasitism, as confirmed by microscopic observation. In dual culture bioassay with F. graminearum, P. oligandrum reduced the growth and mycotoxin concentration by 79% and 93%, respectively. In the dual culture bioassay with F. verticillioides, P. oligandrum reduced the growth and mycotoxin concentration by 49% and 56%, respectively. In vitro dual culture bioassay with different culture media as well as the nutritional phenotyping of different microorganisms made it possible to explore the path of nutritional competition in order to explain part of the observed inhibition by BCAs.

Study of in vitro interaction between Fusarium verticillioides and Streptomyces sp. using metabolomics.

The Streptomyces sp. strain AV05 isolated from an organic amendment was found to impact both growth and fumonisin production of Fusarium verticillioides during in vitro direct confrontation. In order to investigate the interactions between the Streptomyces sp. strain AV05 and F. verticillioides, a metabolomic approach was used. The study of the endometabolomes of the microorganisms was carried out in two different conditions: the microorganisms were cultivated alone or in confrontation. The aim of this study was to examine the modifications of the endometabolome of F. verticillioides in confrontation with the Streptomyces strain. The metabolites involved in these modifications were identified using 2D NMR. Many metabolites were found to be overproduced in confrontation assays with the Streptomyces strain, notably 16 proteinogenic amino acids, inosine, and uridine. This suggested that fungal metabolic pathways such as protein synthesis have been affected due to interaction. Thus, metabolomic studies, as well as proteomics or transcriptomics, are useful for deciphering the mechanisms of interactions between biological control agents and mycotoxigenic fungi. This comprehension is one of the key elements of the improvement of the selection and use of antagonistic agents.

An Imaging Approach to Identify Mechanisms of Resistance to Pineapple Fruitlet Core Rot.

Fruitlet core rot is one of the major postharvest disease of pineapple (Ananas comosus var. comosus). In the past, control strategies were designed to eliminate symptoms without addressing their causes or mechanisms, thus achieving only moderate success. In this study, (i) we focused on the anatomy of the fruitlets in the resistant "MD-2" and susceptible "Queen" pineapple cultivars; (ii) we identified the key role of the carpel margin in the infection process; (iii) we identified the key role of the sinuous layer of thick-walled cells in the inhibition of Fusarium ananatum colonization; and (iv) we linked the anatomy of the fruitlets with the phenolic content of cell walls. The fruitlet anatomy of the two cultivars was studied using X-ray, fluorescence, and multiphoton microscopy. Sepals and bracts were not perfectly fused with each other, allowing the pathogen to penetrate the fruit even after flowering. In fact, the fungi were found in the blossom cups of both cultivars but only became pathogenic in the flesh of the "Queen" pineapple fruit under natural conditions. The outer layer of the "MD-2" cavity was continuous with thick cell walls composed of ferulic and coumaric acids. The cell walls of the "Queen" blossom cup were less lignified at the extremities, and the outer layer was interspersed with cracks. The carpel margins were fused broadly in the "MD-2" pineapple, in contrast to the "Queen" pineapple. This blemish allows the fungus to penetrate deeper into the susceptible cultivar. In pineapple fruitlets, the hyphae of F. ananatum mainly progressed directly between cell walls into the parenchyma but never reached the vascular region. A layer of thick-walled cells, in the case of the resistant cultivar, stopped the colonization, which were probably the infralocular septal nectaries. Anatomical and histochemical observations coupled with spectral analysis of the hypodermis suggested the role of lignin deposition in the resistance to F. ananatum. The major phenolics bound to the cell walls were coumaric and ferulic acids and were found in higher amounts in the resistant cultivar postinoculation. The combination of fruitlet anatomy and lignification plays a role in the mechanism of host resistance to fruitlet core rot.

Differentiation and quantification of the ochratoxin A producers Aspergillus ochraceus and Aspergillus westerdijkiae using PCR-DGGE.

Ochratoxin A (OTA) is a nephrotoxic, teratogenic, immunotoxic, and carcinogenic mycotoxin which is produced in tropical zones mainly by Aspergillus carbonarius, A. niger, A. ochraceus, and A. westerdijkiae. A. ochraceus and A. westerdijkiae species are phenotypically and genomically very close but A. westerdijkiae produce OTA at a very higher level than A. ochraceus. These species have been differentiated recently. The DNA primer pairs which were drawn so far are not specific and a genomic region of the same size is amplified for both species or they are too specific, and in this case, the DNA of a single species is amplified. To help preventing OTA contamination of foodstuffs, the PCR-DGGE (Denaturing Gradient Gel Electrophoresis) method was used to discriminate between A. ochraceus and A. westerdijkiae DNA fragments of the same size but with different sequences and thus faster access to a diagnosis of the toxigenic potential of the fungal microflora. The proposed methodology was able to differentiate A. westerdijkiae from A. ochraceus with only one primer pairs in a single run. A calibration based on initial DNA content was obtained from image analysis of the DGGE gels and a method of quantification of the two strains was proposed.

Evaluating the response to Fusarium ananatum inoculation and antifungal activity of phenolic acids in pineapple.

Fusarium ananatum causes fruitlet core rot (FCR) in pineapple (Ananas comosus var. comosus) when the fruit reaches maturity. Hidden symptoms make it difficult to assess the disease, regardless of its stage, and basic questions concerning the involvement of the phenolic compounds in response to infection remain unknown. A direct inoculation method of F. ananatum in pineapple fruitlets was developed to monitor the growth of black spots and the changes in phenolic acids and ascorbic acid concentration under controlled conditions. After inoculation, infection began with a flesh discolouration at the inoculation point and then spread in a darker shade to form a black spot. Coumaroyl-isocitric and caffeoyl-isocitric acids levels respectively showed a 150- and 200-fold increase in infected fruitlet when compared to healthy fruitlet. These hydroxycinnamic acids increased minimally in the adjacent fruitlet and remained stable in the other parts of the fruit. By contrast, sinapic acid and hydroxybenzoic acid isomers (HBA) decreased after F. ananatum inoculation in the infected fruitlet, whereas they remained stable in the adjacent and healthy fruitlets. Ascorbic acid decreased to zero in the infected fruitlet. The antifungal activity of phenolic compounds and ascorbic acid was evaluated against the mycelial growth of F. ananatum. p-Coumaric acid exhibited a total inhibition of the mycelial growth at 1000 µg g-1. Ferulic acid inhibited 64 % of mycelial growth at a concentration of 1000 µg g-1. Caffeoylquinic acid, sinapic acid, and ascorbic acid also showed significant antifungal activity, but to a lesser extent. Finally, coinoculation of the hydroxycinnamic acids with the pathogen restrains its development in the fruit. This is the first study to highlight the involvement of phenolic compounds in the pineapple FCR disease.

Effect of different light wavelengths on the growth and ochratoxin A production in Aspergillus carbonarius and Aspergillus westerdijkiae.

The effects of light at different wavelengths and photoperiod on growth and ochratoxin A production of Aspergillus carbonarius and Aspergillus westerdijkiae were studied: far-red (740 nm), red (625 nm), blue (445 nm), and UV-A (366 nm). Fungal growth was not significantly affected by photoperiod or light wavelength; the only exception was A. westerdijkiae which showed reduced growth under UV-A light (366 nm). Short-wavelength blue light (445 nm) and UV-A light caused a reduction in ochratoxin A production of both fungal species. However, long-wavelength red light (625 nm) and far-red light (740 nm) reduced ochratoxin A production only in A. westerdijkiae but not in A. carbonarius. It is believed that this difference in reactivity to light is due to differences in the melanin content of the two fungal species: A. carbonarius is a black fungus with higher melanin content than A. westerdijkiae, a yellow fungus. Other possible explanations for the reduction of ochratoxin A production by light were also discussed.

Aeration effects on metabolic events during sporulation of Bacillus thuringiensis.

The metabolism of Bacillus thuringiensis during its sporulation process was investigated under different concentrations of oxygen. At the beginning of sporulation, the aeration conditions were regulated to obtain different oxygen transfer rates (OTR) in four separate fermentations, representing interrupted, limited, non-limited, and saturated oxygenation, respectively. A higher OTR resulted in a higher pH, up to about 9 in the case of saturated oxygenation, while the interrupted oxygenation resulted in a significantly acidic culture. In contrast, the absence of oxygen resulted in rapid sporangia lysis and caused acidification of the medium, indicating a distinctly different sporangia composition and different metabolism. The bacterium also showed different CO2 production rates during sporulation, although a maximum point was observed in every case.With a higher OTR, the maximal value was observed after a longer time and at a lower value (40, 26, and 13 mmol/L/h for limited, non-limited, and saturated cases, respectively). Despite the exhaustion of glucose prior to the sporulation phase, the interrupted oxygenation resulted in acetate, lactate, and citrate in the medium with a maximum concentration of 4.8, 1.3, and 5.0 g/L, respectively. Notwithstanding, while the metabolic events differed visibly in the absence of oxygen, once sporulation was triggered, it was completed, even in the case of an interrupted oxygen supply.