Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Antimicrobial activity of ClO2 gas against Salmonella Enteritidis on almonds.

Nuts, including almonds, are occasionally contaminated with Salmonella spp. In this study, we used chlorine dioxide (ClO2) gas to inactivate S. enterica subsp. Enterica serovar Enteritidis on almonds. Almonds inoculated with a single strain of S. Enteritidis (8.95 log cfu/mL) were exposed to ClO2 gas generated from 1.0 or 1.5 mL ClO2 solution in a sealed container at 50 or 60 °C (43% relative humidity) for up to 10 h. The concentration of ClO2 gas peaked at 354-510 and 750-786 ppm within 0.5 h upon deposition of 1.0 and 1.5 mL of aqueous ClO2, respectively, and gradually decreased thereafter. Population of S. Enteritidis on almonds treated at 50 °C decreased to 1.70-2.32 log cfu/sample within 1 h of exposure to ClO2 gas and decreased to below the detection limit (1.7 log cfu/sample) at all ClO2 concentrations after 8 h. At 60 °C, the microbial population fell below the detection limit within 1 h, regardless of the volume of ClO2 solution supplied. Microbial survival on almonds treated with ClO2 gas and stored at 12 or 25 °C was observed for up to 8 weeks and the organism was not recovered from the almonds treated for 10 h and stored at 12 °C for 2-8 weeks. The lightness (L value) and redness (a value) of almonds treated for 10 h were not changed by ClO2 gas treatment, but yellowness (b value) increased. Results showed that Salmonella on almonds was successfully inactivated by ClO2 gas treatment and the microbial survival did not occur during storage.

Potential probiotic and food protection role of wild yeasts isolated from pistachio fruits (Pistacia vera).

BACKGROUND: The biotechnological potential of yeasts from nuts such as pistachio, not only for health applications but also for industry use, has been scarcely studied. Interest in the probiotic capability of yeasts has increased in the past years as well as their utilization as food or feed preservatives. Their capabilities as biocontrol against problematic (spoilage or toxigenic) microorganisms or as antioxidants have been revalued. As a result, both abilities would be desirable to develop a new potential probiotic microorganism which could be added to food or feed to improve their properties. RESULTS: Molecular techniques allowed the identification of a total of seven different species and 15 strains. A screening of the probiotic potential of these strains was carried out. It was found that 65% of the strains resisted the gastrointestinal conditions as well as presented a generation time of < 22 h. Additionally, some strains showed better kinetic parameters than Saccharomyces boulardii (positive control). Complementary tests were done to determine their auto-aggregation capacity, cell surface hydrophobicity, behaviour in a sequential simulated digestion, biofilm formation capability and carbon source assimilation. Finally, 67% and 13% of the studied yeasts showed biocontrol and antioxidant activities, respectively. CONCLUSIONS: Diutina rugosa 14 followed by Diutina rugosa 8 were the best wild yeast from Pistacia vera as potential probiotic and in carbon source utilization. However, Hanseniaspora guilliermondii 6 and Aureobasidium proteae 5 could be used to improve food or feed product preservation because of their notable biocontrol and antioxidant capabilities. © 2020 Society of Chemical Industry.

Genetic diversity of aflatoxin-producing Aspergillus flavus isolated from selected groundnut growing agro-ecological zones of Uganda.

BACKGROUND: Groundnut pre- and post-harvest contamination is commonly caused by fungi from the Genus Aspergillus. Aspergillus flavus is the most important of these fungi. It belongs to section Flavi; a group consisting of aflatoxigenic (A. flavus, A. parasiticus and A. nomius) and non-aflatoxigenic (A. oryzae, A. sojae and A. tamarii) fungi. Aflatoxins are food-borne toxic secondary metabolites of Aspergillus species associated with severe hepatic carcinoma and children stuntedness. Despite the well-known public health significance of aflatoxicosis, there is a paucity of information about the prevalence, genetic diversity and population structure of A. flavus in different groundnut growing agro-ecological zones of Uganda. This cross-sectional study was therefore conducted to fill this knowledge gap. RESULTS: The overall pre- and post-harvest groundnut contamination rates with A. flavus were 30.0 and 39.2% respectively. Pre- and post-harvest groundnut contamination rates with A. flavus across AEZs were; 2.5 and 50.0%; (West Nile), 55.0 and 35.0% (Lake Kyoga Basin) and 32.5 and 32.5% (Lake Victoria Basin) respectively. There was no significant difference (χ2 = 2, p = 0.157) in overall pre- and post-harvest groundnut contamination rates with A. flavus and similarly no significant difference (χ2 = 6, p = 0.199) was observed in the pre- and post-harvest contamination of groundnut with A. flavus across the three AEZs. The LKB had the highest incidence of aflatoxin-producing Aspergillus isolates while WN had no single Aspergillus isolate with aflatoxin-producing potential. Aspergillus isolates from the pre-harvest groundnut samples had insignificantly higher incidence of aflatoxin production (χ2 = 2.667, p = 0.264) than those from the post-harvest groundnut samples. Overall, A. flavus isolates exhibited moderate level (92%, p = 0.02) of genetic diversity across the three AEZs and low level (8%, p = 0.05) of genetic diversity within the individual AEZs. There was a weak positive correlation (r = 0.1241, p = 0.045) between genetic distance and geographic distance among A. flavus populations in the LKB, suggesting that genetic differentiation in the LKB population might be associated to geographic distance. A very weak positive correlation existed between genetic variation and geographic location in the entire study area (r = 0.01, p = 0.471), LVB farming system (r = 0.0141, p = 0.412) and WN farming system (r = 0.02, p = 0.478). Hierarchical clustering using the unweighted pair group method with arithmetic means (UPGMA) revealed two main clusters of genetically similar A. flavus isolates. CONCLUSIONS: These findings provide evidence that genetic differentiation in A. flavus populations is independent of geographic distance. This information can be valuable in the development of a suitable biocontrol management strategy of aflatoxin-producing A. flavus.

Source Attribution of Salmonella in Macadamia Nuts to Animal and Environmental Reservoirs in Queensland, Australia.

Salmonella enterica is a common contaminant of macadamia nut kernels in the subtropical state of Queensland (QLD), Australia. We hypothesized that nonhuman sources in the plantation environment contaminate macadamia nuts. We applied a modified Hald source attribution model to attribute Salmonella serovars and phage types detected on macadamia nuts from 1998 to 2017 to specific animal and environmental sources. Potential sources were represented by Salmonella types isolated from avian, companion animal, biosolids-soil-compost, equine, porcine, poultry, reptile, ruminant, and wildlife samples by the QLD Health reference laboratory. Two attribution models were applied: model 1 merged data across 1998-2017, whereas model 2 pooled data into 5-year time intervals. Model 1 attributed 47% (credible interval, CrI: 33.6-60.8) of all Salmonella detections on macadamia nuts to biosolids-soil-compost. Wildlife and companion animals were found to be the second and third most important contamination sources, respectively. Results from model 2 showed that the importance of the different sources varied between the different time periods; for example, Salmonella contamination from biosolids-soil-compost varied from 4.4% (CrI: 0.2-11.7) in 1998-2002 to 19.3% (CrI: 4.6-39.4) in 2003-2007, and the proportion attributed to poultry varied from 4.8% (CrI: 1-11) in 2008-2012 to 24% (CrI: 11.3-40.7) in 2013-2017. Findings suggest that macadamia nuts were contaminated by direct transmission from animals with access to the plantations (e.g., wildlife and companion animals) or from indirect transmission from animal reservoirs through biosolids-soil-compost. The findings from this study can be used to guide environmental and wildlife sampling and analysis to further investigate routes of Salmonella contamination of macadamia nuts and propose control options to reduce potential risk of human salmonellosis.

Microbiological, Physicochemical, and Immunological Analysis of a Commercial Cashew Nut-Based Yogurt.

Nut-based milks and yogurts are gaining popularity, but may not offer the same benefits as dairy yogurts to consumers. Cashew nuts often cause severe allergic reactions, and cashew nut allergens are stable to several types of processing. To compare its characteristics to dairy yogurt and characterize the effects of fermentation on the Ana o 1-3 cashew nut allergens, a commercial yogurt made from cashew nuts (Cashewgurt) was evaluated for microbiological, physiochemical, and immunological properties. Average counts for lactobacilli and Streptococcus thermophilus were greater than 10 million colony forming units per milliliter, indicating the capacity to provide a health benefit. Cashewgurt pH and viscosity values were comparable to cow milk yogurts, and it was off white in color. SDS-PAGE analysis indicated a clear reduction in Ana o 1 and 2, and immuno-assay with polyclonal anti-cashew IgG antibody and cashew-allergic IgE indicated an overall reduction in allergen content. In contrast, SDS-PAGE, mass spectrometry, immunoblot, and ELISA all revealed that Ana o 3 was relatively unaffected by the fermentation process. In conclusion, Ana o 1 and Ana o 2 are sensitive to degradation, while Ana o 3 survives lactic acid bacterial fermentation during yogurt production. The analysis presented here indicates that cashew nut yogurt is not suitable for those with cashew nut allergy.

Ecology and Epidemiology of Diseases of Nut Crops and Olives Caused by Botryosphaeriaceae Fungi in California and Spain.

In recent decades, the cultivated area and production of nuts and olives have increased, driven by an increasing consumer interest in healthier food. Diseases of almond, pistachio, olive, and walnut crops caused by species belonging to the Botryosphaeriaceae family have caused concern worldwide. Although considerable progress has been made in elucidating the etiology of these diseases, scientific knowledge of other aspects of these diseases is more limited. In this article, we present an overview of the most important diseases caused by Botryosphaeriaceae fungi affecting almond, pistachio, olive, and walnut crops by focusing on ecology and epidemiology, primarily in California and Spain.

Understanding the Process of Latent Infection of Canker-Causing Pathogens in Stone Fruit and Nut Crops in California.

The Botryosphaeriaceae family is considered a fungal family that includes pathogens causing latent infection of woody plants, and a number of species were identified as causal pathogens of canker and shoot blight diseases. To better understand the process of latent infection of major canker-causing pathogens in woody tissues in different tree crops important in California, shoot and bud samples were randomly collected from four tree crops: almond, dried plum, pistachio, and walnut. The previously developed DNA primers and quantitative real-time PCR (qPCR) assay systems were applied to detect six canker-causing pathogen groups, including Botryosphaeria dothidea, and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis. The concepts of molecular severity (MS) and latent infection index (LII) were introduced and applied to quantify the latent infection levels for these samples. Variation in incidence of latent infection among pathogen groups was observed, whereas the incidences were relatively low among species of Phomopsis and Diplodia. High incidences of Cytospora spp. were observed in two dried plum (prune) orchards. Most orchards showed high incidences of B. dothidea and Lasiodiplodia spp. and moderate incidences of Neofusicoccum spp. Variations in MS were observed among samples of the studied orchards, ranging from 4 to 8. The overall results of LII demonstrated that species of Diplodia and Phomopsis were less important in population development of canker-causing pathogens at the latent phase. Lasiodiplodia spp. were the most aggressive and had been well developed in populations among the studied tree crops. Cytospora spp. became predominant in two of the three dried plum orchards, whereas B. dothidea and Neofusicoccum spp. showed trends of increase in incidence across various tree crops. This study also demonstrated the usefulness of this sensitive qPCR approach in providing evidence of the latent phase of major canker-causing pathogens of stone fruit and nut crops at an early stage of latent infection in woody plant tissues.

Ozone gas as a storage treatment to control Gnomoniopsis castanea, preserving chestnut quality.

BACKGROUND: Chestnuts are gluten-free, low-fat, cholesterol-free products. Postharvest decay reduces chestnut shelf life and can cause severe economic losses. In this study we investigated the effect of ozone (O3 ) gaseous treatment on chestnut rot caused by Gnomoniopsis castanea and the quality parameters of chestnuts. RESULTS: The results showed that ozone treatment (150 ppb during the day, and 300 ppb during the night) reduced the decay of chestnuts and had a fungistatic effect on isolates of G. castanea. The exposure of chestnuts to ozone did not alter weight losses, sugar content and titratable acidity. The concentration of total phenolics decreased during the storage period, both for treated and untreated nuts. However, after 150 days of treatment the polyphenol content of the chestnuts exposed to ozone was significantly higher than in control nuts. CONCLUSIONS: Our results suggested that ozone is an appropriate and economical tool to maximize the quality of chestnut shelf life, enabling it to be stored for long periods. © 2019 Society of Chemical Industry.

Salmonella source attribution in a subtropical state of Australia: capturing environmental reservoirs of infection.

Salmonellosis is a leading cause of hospitalisation due to gastroenteritis in Australia. A previous source attribution analysis for a temperate state in Australia attributed most infections to chicken meat or eggs. Queensland is in northern Australia and includes subtropical and tropical climate zones. We analysed Queensland notifications for salmonellosis and conducted source attribution to compare reservoir sources with those in southern Australia. In contrast to temperate Australia, most infections were due to non-Typhimurium serotypes, with particularly high incidence in children under 5 years and strong seasonality, peaking in summer. We attributed 65.3% (95% credible interval (CrI) 60.6-73.2) of cases to either chicken meat or eggs and 15.5% (95% CrI 7.0-19.5) to nuts. The subtypes with the strongest associations with nuts were Salmonella Aberdeen, S. Birkenhead, S. Hvittingfoss, S. Potsdam and S. Waycross. All five subtypes had high rates of illness in children under 5 years (ranging from 4/100 000 to 23/100 000), suggesting that nuts may be serving as a proxy for environmental transmission in the model. Australia's climatic range allows us to conduct source attribution in different climate zones with similar food consumption patterns. This attribution provides evidence for environment-mediated transmission of salmonellosis in sub-tropical regions.

Characterization of Aspergillus section Flavi isolated from fresh chestnuts and along the chestnut flour process.

An extensive sampling of Aspergillus section Flavi considered to be the main agent responsible for aflatoxin contamination, was carried out in the field and along the processing phases of chestnut flour production in 2015. Fifty-eight isolates were characterized by means of biological, molecular and chemical assays. The highest incidence of Aspergillus section Flavi was found in the field. The identification of the isolates was based on ß-tubulin and calmodulin gene sequences. A. flavus was found to be the dominant species, and this was followed by A. oryzae var effusus, A. tamarii, A. parasiticus and A. toxicarius. Nineteen percent of the strains produced aflatoxins in vitro and forty percent in vivo. The pathogenicity assay on chestnut showed 56 virulent strains out of 58. The molecular, morphological, chemical and biological analyses of A. flavus strains showed an intraspecific variability. These results confirm that a polyphasic approach is necessary to discriminate the species inside the Aspergillus section Flavi. The present research is the first monitoring and characterization of aflatoxigenic fungi from fresh chestnut and the chestnut flour process, and it highlights the risk of a potential contamination along the whole chestnut production chain.

Several species of Penicillium isolated from chestnut flour processing are pathogenic on fresh chestnuts and produce mycotoxins.

A collection of 124 isolates of Penicillium spp. was created by monitoring fresh chestnuts, dried chestnuts, chestnut granulates, chestnut flour and indoor chestnut mills. Sequencing of the ITS region, ß-tubulin and calmodulin, macro-morphology and secondary metabolite production made it possible to determine 20 species of Penicillium. P. bialowiezense was dominant in the fresh chestnuts, while P. crustosum was more frequent in the other sources. A pathogenicity test on chestnut showed that around 70% of the isolates were virulent. P. corylophilum and P. yezoense were not pathogenic, while the other 18 species had at least one virulent isolate. P. expansum and P. crustosum were the most virulent. The isolates were characterized to establish their ability to produce 14 toxic metabolites in vivo: 59% were able to produce at least one mycotoxin. P. expansum was able to produce patulin, chaetoglobosin A and roquefortine, while P. bialowiezense produced C. Mycophenolic acid. Cyclopenins and viridicatins were produced by most of the P. crustosum, P. polonicum, P. solitum and P. discolour isolates. Some of the P. crustosum isolates were also able to produce roquefortine C or penitrem A. Information about the occurrence of Penicillium spp. and their mycotoxins will help producers to set up management procedures that can help to control the fungal growth and the mycotoxin production of chestnuts.

Dynamics of Fungicide Sensitivity in Venturia effusa and Fungicide Efficacy under Field Conditions.

Venturia effusa, which causes pecan scab, has developed resistance to fungicides that were once effective. Over 2 years, laboratory-based sensitivity of fentin hydroxide (TPTH) and tebuconazole in V. effusa and their efficacy under field conditions were compared. Leaf and nut scab were assessed on pecan trees receiving 10 applications of TPTH, tebuconazole, azoxystrobin, azoxystrobin plus tebuconazole, TPTH plus tebuconazole, or no fungicide (NTC) per year. Sensitivity of V. effusa on leaflets collected from treated and nontreated trees was assessed in June and September, respectively. The mean relative germination (RGe) on TPTH at 30 µg/ml was 10.9 and 40.9% in 2016 and 4.2 and 0.6% in 2017. Mean relative growth (RGr) on tebuconazole at 1 µg/ml was 45.5 and 34.6% in 2016 and 69.3 and 56.3% in 2017. In both years, leaf and nut scab were significantly lower on trees treated with azoxystrobin, azoxystrobin + tebuconazole, or TPTH + tebuconazole when compared with NTC and tebuconazole-treated trees. Compared with the NTC, tebuconazole did not significantly reduce leaf scab in 2017 or nut scab in either year, indicating that an RGr value between 34.6 and 69.3% is likely to result in a control failure on tebuconazole-treated trees. Although better activity was expected, TPTH reduced scab with RGe values between 0.6 and 40.9%. These results are valuable for developing fungicide sensitivity thresholds to better predict fungicide performance.

Development of qPCR systems to quantify shoot infections by canker-causing pathogens in stone fruits and nut crops.

AIMS: To develop real-time PCR assays for quantification of shoot infection levels of canker disease of stone fruits and nut crops caused by six fungal pathogen groups. METHODS AND RESULTS: This study focused on six major canker-causing fungal pathogen groups: Phomopsis sp., Botryosphaeria dothidea, Lasiodiplodia sp., Cytospora sp., Neofusicoccum sp. and Diplodia sp., occurring in stone fruits and nut crops in California. DNA primers were designed to specifically target each of the six pathogen groups after the specificity tests using canker-causing and non-canker-causing pathogens and by using DNA sequences of other species from GenBank using blast. The quantitative real-time PCR (qPCR) systems were developed and used to quantify the infection levels of inoculated dried plum shoots. CONCLUSIONS: For Neofusicoccum sp. and Phomopsis sp., which were used in inoculation of walnut shoots, the values of the molecular severity ranged from 5·60 to 6·94 during the 16 days of latent infection period. The qPCR assays were more efficient, accurate and precise to quantify latent infections caused by canker-causing pathogens as compared to the traditional plating methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the potential of using the developed qPCR systems for epidemiological studies on canker diseases of woody plants.

Biodiversity of mycobiota throughout the Brazil nut supply chain: From rainforest to consumer.

A total of 172 Brazil nut samples (114 in shell and 58 shelled) from the Amazon rainforest region and São Paulo state, Brazil was collected at different stages of the Brazil nut production chain: rainforest, street markets, processing plants and supermarkets. The mycobiota of the Brazil nut samples were evaluated and also compared in relation to water activity. A huge diversity of Aspergillus and Penicillium species were found, besides Eurotium spp., Zygomycetes and dematiaceous fungi. A polyphasic approach using morphological and physiological characteristics, as well as molecular and extrolite profiles, were studied to distinguish species among the more important toxigenic ones in Aspergillus section Flavi and A. section Nigri. Several metabolites and toxins were found in these two sections. Ochratoxin A (OTA) was found in 3% of A. niger and 100% of A. carbonarius. Production of aflatoxins B and G were found in all isolates of A. arachidicola, A. bombycis, A. nomius, A. pseudocaelatus and A. pseudonomius, while aflatoxin B was found in 38% of A. flavus and all isolates of A. pseudotamarii. Cyclopiazonic acid (CPA) was found in A. bertholletius (94%), A. tamarii (100%), A. caelatus (54%) and A. flavus (41%). Tenuazonic acid, a toxin commonly found in Alternaria species was produced by A. bertholletius (47%), A. caelatus (77%), A. nomius (55%), A. pseudonomius (75%), A. arachidicola (50%) and A. bombycis (100%). This work shows the changes of Brazil nut mycobiota and the potential of mycotoxin production from rainforest to consumer, considering the different environments which exist until the nuts are consumed.

Effects of Wound Size, Amount of Sap, and Number of Blighted Nuts on Infection of Pistachio Organs by Neofusicoccum mediterraneum.

Laboratory and field studies were conducted to determine the effects of wounding of nut exocarp, susceptibility period after wounding, and sap nut on infection of pistachio nut by Neofusicoccum mediterraneum, the main causal agent of panicle and shoot blight of pistachio. Under controlled conditions and in the field, detached nuts were inoculated with a conidial suspension 30 min before or after wounding. In addition, a 30-µl drop of pistachio sap was placed on the surface of noninjured nuts 30 min before or after they were wounded and then inoculated. Wounding increased the disease severity under both controlled and field conditions. The addition of sap increased the susceptibility of nuts under controlled conditions but not in the field, possibly due to dried sap blocking the pathogen infection. When nuts of Kerman, Kalehghouchi, and Golden Hills pistachio were wounded and inoculated at different time periods after wounding; the nuts of the three cultivars were highly susceptible to pathogen infection during at least the first 24 h after wounding. Under field conditions, there was not a clear effect of increasing the number of inoculated nuts per panicle or the inoculation position (basal or apical) in killing (blight) of the panicle. Conversely, inoculations conducted with mycelial plugs resulted in higher disease, increased the proportion of dead panicles, and resulted in faster symptom expression than inoculations conducted with a conidial suspension. To determine the temporal infection pattern, leaves and panicles were regularly collected from different orchards from 2004 to 2007 and the pathogen was isolated on medium. Important differences in latent infection were detected between years and orchards, with nut and rachis being, in general, the tissues most susceptible to infection. Results of this study help in better understanding the dynamic of infection and colonization of pistachio by N. mediterraneum.

Antibacterial and cytotoxic cytochalasins from the endophytic fungus Phomopsis sp. harbored in Garcinia kola (Heckel) nut.

BACKGROUND: The continuous emergence of multidrug-resistant (MDR) bacteria drastically reduced the efficacy of our antibiotic armory and consequently, increased the frequency of therapeutic failure. The search for bioactive constituents from endophytic fungi against MDR bacteria became a necessity for alternative and promising strategies, and for the development of novel therapeutic solutions. We report here the isolation and structure elucidation of antibacterial and cytotoxic compounds from Phomopsis sp., an endophytic fungus associated with Garcinia kola nuts. METHODS: The fungus Phomopsis sp. was isolated from the nut of Garcinia kola. The crude extract was prepared from mycelium of Phomopsis sp. by maceration in ethyl acetate and sequentially fractionated by column chromatography. The structures of isolated compounds were elucidated on the basis of spectral studies and comparison with published data. The isolated compounds were evaluated for their antibacterial and anticancer properties by broth microdilution and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods respectively. The samples were also tested spectrophotometrically for their hemolytic properties against human red blood cells. RESULTS: The fractionation of the crude extract afforded three known cytochalasins including 18-metoxycytochalasin J (1), cytochalasins H (2) and J (3) together with alternariol (4). The cytochalasin compounds showed different degrees of antibacterial activities against the tested bacterial pathogens. Shigella flexneri was the most sensitive microorganism while Vibrio cholerae SG24 and Vibrio cholerae PC2 were the most resistant. Ampicillin did not show any antibacterial activity against Vibrio cholerae NB2, Vibrio cholerae PC2 and Shigella flexneri at concentrations up to 512 µg/mL, but interestingly, these multi-drug resistant bacterial strains were sensitive to the cytochalasin metabolites. These compounds also showed significant cytotoxic properties against human cancer cells (LC50 = 3.66-35.69 µg/mL) with low toxicity to normal non-cancer cells. CONCLUSION: The three cytochalasin compounds isolated from the Phomopsis sp. crude extract could be a clinically useful alternative for the treatment of cervical cancer and severe infections caused by MDR Shigella and Vibrio cholerae.

Quantification and identification of microorganisms found on shell and kernel of fresh edible chestnuts in Michigan.

BACKGROUND: Chestnut is a relatively new cultivated crop for Michigan, and postharvest loss due to decay has been problematic as production has increased each year. In 2007, more than 25% of chestnuts were lost to postharvest decay, equivalent to approximately 5300 kg of fresh product. To determine the organisms responsible for decay, a microbiological survey was performed in 2006 and 2007 to identify microorganisms involved in postharvest shell (external surface) mold and internal kernel (edible portion) decay of chestnuts. RESULTS: Filamentous fungi including Penicillium expansum, Penicillium griseofulvum, Penicillium chrysogenum, Coniophora puteana, Acrospeira mirabilis, Botryosphaeria ribis, Sclerotinia sclerotiorum, Botryotinia fuckeliana (anamorph Botrytis cinerea) and Gibberella sp. (anamorph Fusarium sp.) were the predominant microorganisms that negatively impacted fresh chestnuts. Populations of microorganisms varied between farms, harvesting methods and chestnut parts. CONCLUSION: Chestnuts harvested from the orchard floor were significantly (P < 0.05) more contaminated than chestnuts harvested directly from the tree, by more than 2 log colony-forming units (CFU) g(-1) . In addition, a significant difference (P < 0.05) in the microbial population was seen between chestnuts submitted by different growers, with average count ranges of fungi, mesophilic aerobic bacteria (MAB) and yeasts equal to 4.75, 4.59 and 4.75 log CFU g(-1) respectively. © 2016 Society of Chemical Industry.

Monitoring of aflatoxin G1, B1, G2, and B2 occurrence in some samples of walnut.

This research was conducted to monitor the aflatoxigenic fungi and aflatoxin contamination of walnut in the Hamedan province. For this purpose, 40 samples were analyzed. Aspergillus, Alternaria, Rhizopus, Cladosporium, Fusarium, yeast, and some different bacteria were isolated from walnuts. Aspergillus is the most frequent genus. Aspergillus flavus was predominantly isolated. HPLC was used for evaluation of aflatoxin contamination of walnut samples. Aflatoxins G1 (AFG1), B1 (AFB1), G2 (AFG2), and B2 (AFB2) were produced by 20 isolates. AFG1 and AFB1 were being predominant at concentration ranges of 1.7-18.2 and 0-8.2 ngg-1, respectively. Highest levels were found in one sample that was highly contaminated with Aspergillus flavus/Aspergillus parasiticus. Methyl beta cyclodextrin also was performed for detection of aflatoxigenic Aspergillus isolates. The results showed that only 31.6% (p < 0.05) of A. flavus and A. parasiticus isolates were able to produce aflatoxin. A significant difference was shown between shielded and unshielded walnut in aflatoxin contamination. The content of aflatoxin in most of the walnut samples did not reach to maximum tolerable limit for aflatoxin B1 in EU standard (p > 0.05). Thus, systematic and continues monitoring of walnuts is recommended.

Combining Lactic Acid Spray with Near-Infrared Radiation Heating To Inactivate Salmonella enterica Serovar Enteritidis on Almond and Pine Nut Kernels.

The aim of this study was to investigate the efficacy of near-infrared radiation (NIR) heating combined with lactic acid (LA) sprays for inactivating Salmonella enterica serovar Enteritidis on almond and pine nut kernels and to elucidate the mechanisms of the lethal effect of the NIR-LA combined treatment. Also, the effect of the combination treatment on product quality was determined. Separately prepared S. Enteritidis phage type (PT) 30 and non-PT 30 S. Enteritidis cocktails were inoculated onto almond and pine nut kernels, respectively, followed by treatments with NIR or 2% LA spray alone, NIR with distilled water spray (NIR-DW), and NIR with 2% LA spray (NIR-LA). Although surface temperatures of nuts treated with NIR were higher than those subjected to NIR-DW or NIR-LA treatment, more S. Enteritidis survived after NIR treatment alone. The effectiveness of NIR-DW and NIR-LA was similar, but significantly more sublethally injured cells were recovered from NIR-DW-treated samples. We confirmed that the enhanced bactericidal effect of the NIR-LA combination may not be attributable to cell membrane damage per se. NIR heat treatment might allow S. Enteritidis cells to become permeable to applied LA solution. The NIR-LA treatment (5 min) did not significantly (P > 0.05) cause changes in the lipid peroxidation parameters, total phenolic contents, color values, moisture contents, and sensory attributes of nut kernels. Given the results of the present study, NIR-LA treatment may be a potential intervention for controlling food-borne pathogens on nut kernel products.