Search

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium spp., which contaminates many crops, including pistachios. Pistachios contaminated with OTA may be subjected to border rejections resulting in significant economic losses to the United States agricultural revenues. The current study examined prevalence of OTA in California-grown pistachios and identified its causal agents. OTA was detected in 20% of samples from 2018 to 2021 ( n = 809), with 18% of samples exceeding the European Union regulatory limit of 5 μg/kg. Fungi potentially responsible for OTA contamination were isolated from leaves, nuts, and soil collected from 14 pistachio orchards across California. A total of 1,882 isolates of Aspergillus section Nigri and 85 isolates of section Circumdati were recovered. Within section Nigri , 216 (11.5%) isolates were identified as potential OTA producers using a boscalid-resistance assay. Phylogenetic analyses of partial gene sequences for β-tubulin and calmodulin genes resolved section Circumdati into four species: A. ochraceus (33%), A. melleus (28%), A. bridgeri (21%), and A. westerdijkiae (19%). A. westerdijkiae produced the highest levels of OTA in inoculated pistachios (47 μg/g), followed by A. ochraceus (9.6 μg/g) and A. melleus (3.3 μg/g). A. bridgeri did not produce OTA. OTA production by section Circumdati was optimal from 20 to 30°C. All 216 boscalid-resistant isolates from section Nigri were identified as A. tubingensis , and representative isolates ( n = 130) produced 3.8 μg/kg OTA in inoculated pistachios. This is the first detailed report on OTA contamination and causal fungi in California pistachios and will be helpful in devising effective management strategies., Competing Interests: The author(s) declare no conflict of interest.

Salmonella enterica subsp. enterica strain diversity in California pistachios is limited; some strains have persisted in the pistachio supply chain for ≥10 years. Representative isolates of six persistent strains and three sporadic strains isolated from California pistachios were selected to evaluate copper resistance, growth in pistachio hull slurry, biofilm formation, desiccation tolerance, and survival during subsequent storage. The presence of a copper homeostasis and silver-resistance island sequence in three of the persistent strains was associated with an increase in tolerance to CuSO 4 from 7.5 mM to 15 mM under anaerobic but not aerobic conditions; all isolates were resistant to ≥120 mM Cu-EDTA under both anerobic and aerobic conditions. When inoculated into pistachio hull slurry at 2.75 ± 0.04 log CFU/mL and incubated at 30 °C, the populations of Salmonella Enteritidis strain A (sporadic) increased to significantly lower levels than the other strains at 16, 20, 24, and 28 h but not at 40 and 48 h. Maximum populations of 8.70-8.85 log CFU/mL were observed for all strains at ≥40 h of incubation. All nine Salmonella strains produced weak to strong biofilms after 4 days at 25 °C; seven strains, including two sporadic strains, produced moderate biofilms, and Salmonella Liverpool strain A (persistent) produced a strong biofilm. The rdar+ and rdar- morphotypes were observed in both persistent and sporadic Salmonella strains. Population declines of 5.03 log were observed for Salmonella Enteritidis strain A within 18 h of drying on filter paper whereas reductions of 0.50-1.25 log were observed for the other eight Salmonella strains. Population reductions (3.98-5.12 log) of these eight strains were not significantly different after storage at 25 ± 1 °C and 35% relative humidity for 50 days. The phenotypic characteristics evaluated here do not independently account for the persistence of a small number of Salmonella strains associated with the California pistachio production chain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aflatoxins are carcinogens produced by the fungi Aspergillus flavus and A. parasiticus that contaminate pistachio crops. International markets reject pistachio when aflatoxins exceed permitted maximum levels. Releasing the atoxigenic strain AF36 of A. flavus is the leading aflatoxin pre-harvest control method. The product AF36 Prevail, sorghum grains coated with AF36 propagules, has been used in California since 2017. However, a high percentage of grains of the Prevail fail to sporulate in orchards. Here, the effect of soil moisture on the percentage of AF36 product grains sporulating (SG) and the quantity of spores per grain using a sporulation index (SI) was determined. Under controlled conditions, SG was higher than 85% when soil moisture was 13% or more, and SI increased with increasing soil moisture from 8.4 to 21%. The highest AF36 sporulation occurred near the micro-sprinklers when the grains were not impacted by the irrigation water drops. Arthropod predation was responsible for lost product grains, which was more pronounced in non-tilled soil than in tilled soil. Dispersal of the AF36 spores decreased markedly with the height and distance from the inoculum source, following a pattern of diffusion equations. However, AF36 spores easily reached canopies of pistachios located 10 m from the inoculum source. Our results indicate that AF36 Prevail should be applied close to the irrigation line in the moist soil area but avoiding the areas where excess irrigation causes water accumulation. The biocontrol of aflatoxins in California's pistachio production areas was optimized by improving the field realization of the biological control agent., Competing Interests: The author(s) declare no conflict of interest.

Pistachio nuts can become colonized by mycotoxigenic fungi, especially Aspergillus flavus , resulting in contamination with aflatoxins (AFs). We examined the effect of gaseous O 3 (50-200 ppm; 30 min; 6 L/min) on (a) in vitro germination, (b) mycelial growth, and (c) aflatoxin B1 (AFB 1 ) production on a milled pistachio nut-based medium at different water activity (a w ) levels and at 30 °C. This was complimented with in situ studies exposing raw pistachio nuts to 50-200 ppm of O 3 . Exposure of conidia to gaseous O 3 initially resulted in lower germination percentages at different a w levels. However, 12 h after treatment, conidial viability recovered with 100% germination after 24-48 h. Growth rates of mycelial colonies were slightly decreased with the increase of the O 3 dose, with significant inhibition only at 0.98 a w . The production of AFB 1 after O 3 treatment and storage for 10 days was stimulated in A. flavus colonies at 0.98 a w . Raw pistachio nuts inoculated with A. flavus conidia prior to O 3 exposure showed a significant decrease in population after 20 days of storage. However, AFB 1 contamination was stimulated in most O 3 treatments. The relationship between exposure concentration, time and prevailing a w levels on toxin control needs to be better understood for these nuts.

Pistachio ( Pistacia vera ) is an important crop in Italy, traditionally cultivated in Sicily (southern Italy) for several decades now. In recent years, new orchards have been planted in new areas of the island. Field surveys conducted in 2019 revealed the presence of symptomatic trees showing shoot dieback, cankers, fruit spots, and leaf lesions. Isolations from symptomatic samples consistently yielded fungal species in the Botryosphaeriaceae family. Identification of collected isolates was conducted using morphological and molecular analyses. Morphological characterization was based on conidia measurements of representative isolates and also effects of temperatures on mycelial growth was evaluated. DNA data derived from sequencing the ITS, tef1 -α, and tub2 gene regions were analyzed via phylogenetic analyses (maximum parsimony and maximum likelihood). Results of the analyses confirmed the identity of Botryosphaeria dothidea , Neofusicoccum hellenicum , and N. mediterraneum . Pathogenicity tests were conducted on detached twigs and in the fields both on shoots as well as on fruit clusters using the mycelial plug technique. The inoculation experiments revealed that among the Botryosphaeriaceae species identified in this study N . hellenicum (occasionally detected) and N . mediterraneum were the most aggressive based on lesion length on shoots and fruits. N . mediterraneum was the most widespread among the orchards while B. dothidea can be considered a minor pathogen involved in this complex disease of pistachio. Moreover, to our knowledge, this is the first report of N . hellenicum in Italy.

Pistachios have been implicated in two salmonellosis outbreaks and multiple recalls in the U.S. This study performed an in-depth retrospective data analysis of Salmonella associated with pistachios as well as a storage study to evaluate the survivability of Salmonella on inoculated inshell pistachios to further understand the genetics and microbiological dynamics of this commodity-pathogen pair. The retrospective data analysis on isolates associated with pistachios was performed utilizing short-read and long-read sequencing technologies. The sequence data were analyzed using two methods: the FDA's Center for Food Safety and Applied Nutrition Single Nucleotide Polymorphism (SNP) analysis and Whole Genome Multilocus Sequence Typing (wgMLST). The year-long storage study evaluated the survival of five strains of Salmonella on pistachios stored at 25 °C at 35% and 54% relative humidity (RH). Our results demonstrate: i) evidence of persistent Salmonella Senftenberg and Salmonella Montevideo strains in pistachio environments, some of which may be due to clonal resident strains and some of which may be due to preharvest contamination; ii) presence of the Copper Homeostasis and Silver Resistance Island (CHASRI) in Salmonella Senftenberg and Montevideo strains in the pistachio supply chain; and iii) the use of metagenomic analysis is a novel tool for determining the composition of serovar survival in a cocktail inoculated storage study., Competing Interests: The authors have declared that no competing interests exist.

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus , mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B 1 (AFB 1 ). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO 2 levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO 2 (400 vs. 1000 ppm), and water stress (0.98-0.93 water activity, a w ) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB 1 production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB 1 production was significantly stimulated ( p < 0.05), especially when exposed to 1000 ppm CO 2 at 0.98-0.95 a w and 35 °C, and sometimes in the 37 °C treatment group at 0.98 a w . The relative expression of the structural aflD gene involved in AFB 1 biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the a w level examined. For the regulatory aflR gene expression, there was a significant ( p < 0.05) increase in 1000 ppm CO 2 and 37 °C for both strains, especially at 0.95 a w . The in situ colonization of pistachio nuts resulted in a significant ( p < 0.05) stimulation of AFB 1 production at 35 °C and 1000 ppm CO 2 for both strains, especially at 0.98 a w . At 37 °C, AFB 1 production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO 2 . This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO 2 and the level of water stress on AFB 1 production.

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., (© 2020 Society of Chemical Industry.)

The objective of this survey was to estimate the prevalence, contamination level, and genetic diversity of Salmonella in selected raw, shelled tree nuts (Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts) at retail markets in the United States. A total of 3,374 samples of eight tree nuts were collected from different types of retail stores and markets nationwide between September 2015 and March 2017. These samples (375 g) were analyzed using a modified FDA's BAM Salmonella culture method. Of the 3,374 samples, 15 (0.44%) (95% confidence interval [CI] [0.25, 0.73]) were culturally confirmed as containing Salmonella; 17 isolates were obtained. Among these isolates, there were 11 serotypes. Salmonella was not detected in Brazil nuts (296), hazelnuts (487), pecans (510), pine nuts (500), and walnuts (498). Salmonella prevalence estimates in cashews (510), macadamia (278), and pistachios (295) were 0.20% (95% CI [<0.01, 1.09]), 2.52% (95% CI [1.02, 5.12]), and 2.37% (95% CI [0.96, 4.83]), respectively. The rates of Salmonella isolation from major/big-chain supermarkets (1381), small-chain supermarkets (328), discount/variety/drug stores (1329), and online (336) were 0.29% (95% CI [0.08, 0.74]), 0.30% (95% CI [0.01, 1.69]), 0.45% (95% CI [0.17, 0.98]), and 1.19% (95% CI [0.33, 3.02]), respectively. Salmonella prevalence in organic (530) and conventional (2,844) nuts was not different statistically (P = 0.0601). Of the enumerated samples (15), 80% had Salmonella levels ≤0.0092 most probable number (MPN)/g. The highest contamination level observed was 0.75 MPN/g. The prevalence and contamination levels of Salmonella in the tree nuts analyzed were generally comparable to previous reports. Pulsed-field gel electrophoresis, serotype, and sequencing data all demonstrated that Salmonella population in nuts is very diverse genetically. PRACTICAL APPLICATION: The prevalence, contamination level, and genetic diversity of Salmonella in eight types of tree nuts (3,374 samples collected nationwide) revealed in this survey could help the development of mitigation strategies to reduce public health risks associated with consumption of these nuts., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

Two decontamination methods were evaluated for inactivating a cocktail of Salmonella or Listeria monocytogenes inoculated onto model low moisture foods (LMFs; dried strawberry, dried apple, raisins, chocolate crumb, cornflakes, shell-on or deshelled pistachio nuts). One treatment was based on a peracetic acid-ethanol (PAA-ethanol) sanitizer combination with the other being an Advanced Oxidation Process (AOP) that simultaneously applied UV-C (254 nm), ozone and hydrogen peroxide. The low moisture food was spray inoculated then dried prior to treatment. With Salmonella it was found that a pre-incubation step in 1% w/v glycerol-tryptic soy broth for 1 h prior to plating, significantly increased recovery of the pathogen compared to TSB alone. However, no increased recovery of L. monocytogenes was observed using the TSB-glycerol pre-incubation step. No Salmonella was detected on cornflakes, chocolate crumb and strawberry using 1.25 parts per thousand (‰) PAA-ethanol. The inactivation of Salmonella on deshelled pistachio was significantly higher using 2.5‰ PAA-ethanol sanitizer compared to the AOP treatments tested. Only negligible reductions of Salmonella (<1 log cfu) were obtained with shell-on pistachio treated with PAA-ethanol sanitizer or AOP. Salmonella could be reduced on dried apple slices by >4 log CFU when 5.0‰ PAA-ethanol was applied. L. monocytogenes was more sensitive to PAA-ethanol compared to Salmonella and could be eliminated on all the LMFs apart from shell-on pistachio. An AOP treatment applied 10% v/v hydrogen peroxide, ozone and 54 mJ/cm 2 UV-C could significantly reduce Salmonella on dried apple slices compared to when the individual elements (hydrogen peroxide, ozone or UV-C) were applied. Salmonella was also eliminated by AOP on the other LMFs (apart from shell-on pistachio) although the same level of inactivation was achieved by spraying with 10% v/v hydrogen peroxide alone. L. monocytogenes was sensitive to hydrogen peroxide and AOP being eliminated from all the LMFs. Although this may suggest that hydrogen peroxide spray was equivalent to AOP treatment it was noted that no residual H 2 O 2 or changes in visual appearance was evident on samples treated with the latter process. The study has demonstrated that the two decontamination methods assessed can be applied to reduce Salmonella and L. monocytogenes on LMFs although efficacy is dependent on the pathogen and product type., Competing Interests: Declaration of competing interest The authors declare that the submitted work was conducted in the absence of any commercial or financial relationships that could be construed as a conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

In recent years, very many incidences of contamination with aflatoxin B 1 (AFB 1 ) in pistachio nuts have been reported as a major global problem for the crop. In Europe, legislation is in force and 12 μg/kg of AFB 1 is the maximum limit set for pistachios to be subjected to physical treatment before human consumption. The goal of the current study was to develop a mechanistic, weather-driven model to predict Aspergillus flavus growth and the AFB 1 contamination of pistachios on a daily basis from nut setting until harvest. The planned steps were to: (i) build a phenology model to predict the pistachio growth stages, (ii) develop a prototype model named AFLA-pistachio (model transfer from AFLA-maize), (iii) collect the meteorological and AFB 1 contamination data from pistachio orchards, (iv) run the model and elaborate a probability function to estimate the likelihood of overcoming the legal limit, and (v) manage a preliminary validation. The internal validation of AFLA-pistachio indicated that 75% of the predictions were correct. In the external validation with an independent three-year dataset, 95.6% of the samples were correctly predicted. According to the results, AFLA-pistachio seems to be a reliable tool to follow the dynamic of AFB 1 contamination risk throughout the pistachio growing season.

Aims: In this study, we aimed to explore the enzymatic diversity, the entomopathogenic and the antimicrobial potentialities of fungi associated with the pistachio bark beetle, Chaetoptelius vestitus., Methods and Results: A total of 40 isolates were screened for enzymatic diversity. Most of them, 92·5%, were able to produce at least two of the screened enzymes. Pathogenic assays performed on C. vestitus showed a high entomopathogenic activity of the isolates Ata&#95;io&#95;1 (A. tamarii), Fve&#95;io&#95;1 (F. verticillioides), Tpi&#95;io&#95;1 (T. pinophilus), Pal&#95;io&#95;1 (P. album), Pbi&#95;io&#95;2 (Penicillium bilaiae) and Pch&#95;io&#95;1 (P. chrysogenum), as based on mean mortality of C. vestitus. A screening of antimicrobial activity using well diffusion method showed that the isolates Tro&#95;io&#95;1 (T. pinophilus), Tat&#95;io&#95;1 (T. atroroseus) and Pch&#95;io&#95;1 (P. chrysogenum) presented the highest antibacterial activity. Furthermore, Mgu&#95;io&#95;1 (M. guilliermondii), Asc&#95;io&#95;1 (A. sclerotiorum), Ata&#95;io&#95;1 (A. tamarii), G. lavendula (Gla&#95;io&#95;1), Pva&#95;io&#95;1 (P. variotii), Pul&#95;io&#95;1 (P. ulaiense), Tat&#95;io&#95;1 (T. atroroseus) and Tro&#95;io&#95;1 (T. roseum) were active against at least two of the three tested fungal phytopathogens., Conclusions: Fungal isolates representing entomopathogenic activity and a broad spectrum of antimicrobial activities can be considered as promising resources for biological pistachio trees protection., Significance and Impact of the Study: Fungi associated with C. vestitus were investigated for detecting their potential biotechnological applications., (© 2020 The Society for Applied Microbiology.)

Background: Pistachio fruits suffer from postharvest decay, caused by Aspergillus flavus. This results in aflatoxin B1 (AFB 1 ) accumulation in kernels, which is hazardous for human health due to its carcinogenic activity. In this study, the mechanism used by exogenous β-aminobutyric acid (BABA) treatment for attenuating Aspergillus decay, minimizing aflatoxin B 1 (AFB 1 ) accumulation, and maintaining nutritional quality in fresh-in-hull pistachio kernels, infected by A. flavus during storage at 25 °C for 18 days, was investigated., Result: Results of an in vivo assay showed that the spore germination and germ tube elongation of A. flavus was repressed by BABA treatment at 7.5 mM. Aspergillus decay accompanied by AFB 1 accumulation was also minimized in fresh-in-hull pistachio kernels treated with BABA at 7.5 mM and infected by A. flavus. Fresh-in-hull pistachio kernels, infected by A. flavus, treated with BABA at 7.5 mM, also exhibited higher phenol and flavonoid accumulation and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity accompanied by higher phenylalanine ammonia lyase (PAL) enzyme activity., Conclusion: Promoting phenylpropanoid pathway activity with higher PAL enzyme activity in fresh-in-hull pistachio kernels treated with BABA may not only reduce Aspergillus decay in kernels by cell wall fortification but also may be favorable for maintaining the kernels' nutritional quality through its effects on ROS scavenging capacity. As oxidative stress, represented by ROS accumulation, is responsible for A. flavus growth and AFB1 accumulation, higher phenol and flavonoid accumulation in fresh-in-hull pistachio kernels treated with BABA may be beneficial for attenuating Aspergillus decay and minimizing AFB1 accumulation. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

A survey was conducted during 2015 and 2016 in pistachio orchards throughout the San Joaquin Valley of California to investigate the occurrence of canker diseases and identify the pathogens involved. Cankers and dieback symptoms were observed mainly in orchards aged >15 years. Symptoms of canker diseases included brown to dark brown discoloration of vascular tissues, wood necrosis, and branch dieback. In total, 58 fungal isolates were obtained from cankers and identified based on multilocus phylogenetic analyses (internal transcribed spacer, glyceraldehyde 3-phosphate dehydrogenase, β-tubulin, calmodulin, actin 1, and translation elongation factor 1α) representing 11 fungal species: Colletotrichum karstii , Cytospora californica , Cytospora joaquinensis , Cytospora parapistaciae , Cytospora pistaciae , Diaporthe ambigua , Didymella glomerata , Diplodia mutila , Neofusicoccum mediterraneum , Phaeoacremonium canadense , and Schizophyllum commune . Pathogenicity tests conducted in the main pistachio cultivars Kerman, Golden Hills, and Lost Hills using the mycelium-plug method indicated that all fungal species were pathogenic to Pistacia vera . All species tested caused cankers in pistachio branches, although virulence among species varied from high to moderate. Overall, N. mediterraneum and Cytospora spp. were the most widespread and virulent species associated with canker diseases of pistachio in California.

California produces 99.1% of pistachios grown in the United States, and diseases affecting pistachio rootstocks represent a constant challenge to the industry. Field surveys of fungi associated with pistachio rootstocks with symptoms of crown rot and stem canker in three central California counties followed by phylogenetic analyses of translation elongation factor 1-α and second largest subunit of RNA polymerase II gene fragments identified three Fusarium species ( Fusarium equiseti , Fusarium oxysporum , and Fusarium proliferatum ) and two Neocosmospora species ( Neocosmospora falciformis and Neocosmospora solani ). F. oxysporum and N. falciformis were the fungal species most frequently recovered from symptomatic pistachio trees. Inoculations of detached twigs of cultivar Kerman pistachio Pioneer Gold I and clonal University of California, Berkeley I (UCBI) rootstocks showed that all five species could colonize pistachio wood and cause vascular discolorations. Pathogenicity tests in potted pistachio trees completed Koch's postulates and confirmed that F. oxysporum , F. proliferatum , N. falciformis , and N. solani were capable of producing rot and discoloration in stems of clonal UCBI rootstocks, the most widely planted pistachio rootstock in California. To our knowledge, this study is the first to present insights into the biodiversity and biology of Fusarium and Neocosmospora species associated with pistachio trees in California.

Aim: This study investigated the biodiversity of fungi associated with the pistachio bark beetle, Chaetoptelius vestitus, in Tunisia. We evaluated the phytopathogenic activities and tested antagonistic potentialities with respect to phytopathogens., Methods and Results: A total of 41 fungal isolates were randomly isolated from C. vestitus adults and galleries. We identified 28 species belonging to 13 genera using ITS sequences of the ribosomal RNA operons. Pathogenicity assays performed using the excised shoot method revealed that isolates Aal&#95;io&#95;1 (Alternaria alternata), Feq&#95;io&#95;1 (Fusarium equiseti), Fgra&#95;io&#95;1 (Fusarium graminearum), Fve&#95;io&#95;1 (Fusarium verticilloides), Tro&#95;io&#95;1 (Trichothecium roseum) and Nqu&#95;io&#95;1 (Nothophoma quercina) displayed a high pathogenic activity on pistachio stems. Estimation of the antagonistic potentialities of isolated fungi against several phytopathogenic isolates as tested using a dual-culture method showed that isolates Tpi&#95;io&#95;1 (Talaromyces pinophilus), Pbi&#95;io&#95;2 (Penicillium bilaiae), Asc&#95;io&#95;1 (Aspergillus sclerotiorum) and Gla&#95;io&#95;1 (Geosmithia lavendula) displayed a broad range of antagonistic activities., Conclusion: Fungi associated with C. vestitus had a variable range of pathogenic activity on pistachio stem. Phytopathogenic fungi were antagonized by different fungal isolates which could be promising in pistachio protection against phytopathogenic fungi., Significance and Impact of the Study: This study is the first that investigated the diversity of fungi associated with C. vestitus and evaluated both their phytopathogenic activity and antagonistic potential against fungal phytopathogens., (© 2019 The Society for Applied Microbiology.)

The feasibility of using gaseous ozone, alone or in combination with other treatments, to decontaminate in-shell almonds and pistachios, prepared under different pathogen-inoculation conditions, was explored. Nuts were inoculated with either Salmonella enterica serovar Enteritidis or a potential Salmonella surrogate, Enterococcus faecium OSY 31284. The effect of inoculation method (with or without vacuum application), and of drying inoculated nuts (up to three days) on treatment efficacy was investigated. Inoculated nuts were subjected to gaseous ozone alone (almonds, pistachios) or ozone in combination with heating in brine solution (pistachios). Ozone treatment included application of vacuum (10 in Hg), followed by vessel pressurization to 12.5 psig with ozone‑oxygen mixture (160 g ozone/m 3 gas mixture) and holding for 30 min. Heating was conducted in a brine solution (5% NaCl) at 70 °C, for 10 min. Ozone-based treatments were significantly more effective (P < 0.05) on almonds than pistachios, with maximum S. Enteritidis reduction of 2.9 vs. 0.8 log CFU/g, regardless of inoculation method or the drying time. Treatment of inoculated pistachios with heated brine and gaseous ozone reduced S. Enteritidis population by 5.0 to 7.0 log CFU/g and was not significantly more effective than treatment with heated brine alone (reduction of 4.8 to 7.1 log CFU/g). Application of vacuum during inoculation increased bacterial population on nut kernels by approximately 1.2 log CFU/g, but the increase in inoculum population had no effect on inactivation of either species of inoculated bacteria. Decontamination treatments were less effective against both bacteria by up to 2 log CFU/g when drying time of inoculated nuts increased. E. faecium was significantly more resistant to heat and ozone treatment (P < 0.05) than was S. Enteritidis on pistachios, but not on almonds. Results of this study show that laboratory methodology affects observed treatment effectiveness. Considering its high resistance to the heat-ozone combination, E. faecium may not be a suitable surrogate for S. Enteritidis during processing of pistachios by this treatment. Efficacy of ozone gas to decontaminate S. Enteritidis-inoculated nuts depends heavily on the type of nut. Although reductions of S. Enteritidis populations on in-shell pistachios are low, treatment of in-shell almonds resulted in greater reductions, indicating the promise of this technology to enhance the safety of specific nut products., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In California, aflatoxin contamination of almond, fig, and pistachio has become a serious problem in recent years due to long periods of drought and probably other climatic changes. The atoxigenic biocontrol product Aspergillus flavus AF36 has been registered for use to limit aflatoxin contamination of pistachio since 2012 and for use in almond and fig since 2017. New biocontrol technologies employ multiple atoxigenic genotypes because those provide greater benefits than using a single genotype. Almond, fig, and pistachio industries would benefit from a multi-strain biocontrol technology for use in these three crops. Several A. flavus vegetative compatibility groups (VCGs) associated with almond, fig, and pistachio composed exclusively of atoxigenic isolates, including the VCG to which AF36 belongs to, YV36, were previously characterized in California. Here, we report additional VCGs associated with either two or all three crops. Representative isolates of 12 atoxigenic VCGs significantly ( P < 0.001) reduced (>80%) aflatoxin accumulation in almond and pistachio when challenged with highly toxigenic isolates of A. flavus and A. parasiticus under laboratory conditions. Isolates of the evaluated VCGs, including AF36, constitute valuable endemic, well-adapted, and efficient germplasm to design a multi-crop, multi-strain biocontrol strategy for use in tree crops in California. Availability of such a strategy would favor long-term atoxigenic A. flavus communities across the affected areas of California, and this would result in securing domestic and export markets for the nut crop and fig farmer industries and, most importantly, health benefits to consumers.

During harvest, pistachios are hulled, separated in water into floater and sinker streams (in large part on the basis of nut density), and then dried before storage. Higher prevalence and levels of Salmonella were previously observed in floater pistachios, but contributing factors are unclear. To examine the behavior of pathogens on hulled pistachios during simulated drying delays, floater and sinker pistachios collected from commercial processors were inoculated at 1 or 3 log CFU/g with cocktails of Salmonella and in some cases Escherichia coli O157:H7 or Listeria monocytogenes and incubated for up to 30 h at 37°C and 90% relative humidity. Populations were measured by plating onto tryptic soy agar and appropriate selective agars. In most cases, no significant growth ( P > 0.05) of Salmonella was observed in the first 3 h after inoculation in hulled floaters and sinkers. Growth of Salmonella was greater on floater pistachios than on corresponding sinkers and on floater pistachios with ≥25% hull adhering to the shell surface than on corresponding floaters with <25% adhering hull. Maximum Salmonella populations (2 to 7 log CFU/g) were ∼2-log higher on floaters than on corresponding sinkers. The growth of E. coli O157:H7 and Salmonella on hulled pistachios was similar, but a longer lag time (approximately 11 h) and significantly lower maximum populations (4 versus 5 to 6 log CFU/g; P < 0.05) were predicted for L. monocytogenes . Significant growth of pathogens on hulled pistachios is possible when delays between hulling and drying are longer than 3 h, and pathogen growth is enhanced in the presence of adhering hull material.

This study was conducted to investigate the effect of salt content during radio-frequency (RF) heating on rate of temperature increase, dielectric properties (DPs), and reduction of pathogens in pistachios. Also, the effect of RF heating on pistachio quality of varying salt content was determined. Pistachios of different salt content (0, 100, and 330 mg sodium/serving) were inoculated with Salmonella enterica and treated in a 27.12-MHz RF heater. The RF heating rate increased when salt content was in the range of 0-100 mg sodium/serving, but there were no significant (P > 0.05) differences in the rate of temperature rise after salt content reached to 100 mg sodium/serving. Both dielectric constant and dielectric loss factor of pistachios increased with rising salt content. Along with increased salt content, RF treatment time required to reduce this pathogen by 4 log CFU/g decreased first and then remained the same above an upper limit of salt content corresponding to the peak heating rate. RF treatment did not significantly (P > 0.05) cause changes in the color and level of lipid oxidation of pistachios. The results of the current study imply that RF heating may be a potential intervention for inactivating foodborne pathogens in pistachios and that the effect of pasteurization is influenced by dielectric loss factor relative to salt content.

Salmonella has been isolated from dried pistachios in both postharvest and retail surveys. The source of Salmonella in pistachios is unknown, but introduction is possible at points during production, harvest, and postharvest activities. To examine the behavior of Salmonella on pistachios during simulated postharvest conditions, early-, mid-, and late-season inhull pistachios were collected from two commercial processors over five different harvests. Pistachios were inoculated with cocktails of nalidixic acid- or rifampin-resistant Salmonella at 0.64 to 1.59 log CFU/g (low) or 2.73 to 3.27 or 4.29 to 4.31 log CFU/g (high) and were incubated for up to 30 h under commercially relevant conditions (23, 35, or 37°C and 50 or 90% relative humidity [RH]). Populations of Salmonella were measured by plating onto tryptic soy agar and CHROMagar Salmonella with added nalidixic acid or rifampin. Individual growth curves at the same temperature and RH differed significantly among different lots of pistachios. Except for a single late-season lot in which no significant growth was observed, Salmonella multiplied under all storage conditions. In the first 3 h after inoculation, insignificant (most cases) to small (0.41 to 0.67 log CFU/g) but significant ( P < 0.05) mean increases in Salmonella populations were measured; the mean predicted time to achieve maximum populations (5 to 8 log CFU/g) was 16 ± 4 h. In paired samples, longer lag phases, lower growth rates, and lower maximum increases were observed with inoculated inhull pistachios incubated at 23°C and 50% RH compared with 35 or 37°C and 90% RH. Similar growth curves were observed at the low and high inoculum levels; throughout the 30 h of incubation, Salmonella populations were consistently ∼1 to 2 log CFU/g lower on pistachios inoculated at the low inoculum level. Managing the time between harvesting and hulling will reduce the potential for growth of Salmonella on pistachios during postharvest handling.

This study evaluated the efficacy of gamma irradiation to inactivate food-borne pathogens on pistachios (Pistacia vera L.). Pistachios inoculated with Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes were subjected to gamma irradiation in the range of 0, 0·5, 1, 3 and 5 kGy, and colour change was evaluated after treatment and during storage at room temperature. Pathogen populations decreased with increasing treatment doses. A gamma irradiation dose of 5 kGy decreased the three pathogens on pistachios to under the detection limit (1·0 log CFU per g) without effecting colour change. During storage following treatment, pathogens were reduced due to the postirradiation effect. D-values of pathogens on pistachios showed that L. monocytogenes was more resistant to gamma irradiation than was E. coli O157:H7 or S. Typhimurium. During gamma irradiation treatment, L, a and b values of pistachios did not significantly change but these values changed during storage. These results show that gamma irradiation has potential as a nonthermal process for inactivating food-borne pathogens in pistachios without inducing colour changes. SIGNIFICANCE AND IMPACT OF THE STUDY: Generally, nuts have lower water activity which precludes the growth of food-borne pathogens. But, food-borne outbreaks due to pathogen-contaminated nuts have been reported in the last few decades. Pistachios are one of the most popular nuts and have many health beneficial effects. However, many pasteurization interventions have been used to reduce pathogens on pistachios, but most of them are not effective. This study confirms the effectiveness of gamma irradiation on pasteurization of pistachios. This may be helpful in nut processing industries to ensure the microbial safety of nuts., (© 2018 The Society for Applied Microbiology.)

Alternaria late blight caused by Alternaria alternata is a major disease affecting pistachios grown in California and to some degree those grown in Arizona. Alternaria alternata is prone to quick fungicide resistance selection when single-mode of action fungicides are used. For the specific detection of five possible amino acid alterations associated with Alternaria alternata resistance to succinate dehydrogenase inhibitors used in California and Arizona pistachio orchards, we have designed five primer sets to be used as an allele-specific PCR assay (AS-PCR). Within a couple of hours from DNA extraction, the new specific-primers amplify the different PCR product sizes, according to the chosen target, allowing their differentiation on gel agarose. In our study, the AS-PCR assay consistently detected the mutations H277L and H277R at the AaSdhB gene, the mutations H134R and S135R at the AaSdhC gene, and the mutation D123E at the AaSdhD gene from A. alternata isolates collected from pistachio orchards in California and Arizona., Significance and Impact of the Study: This study provides a rapid and cost-effective way to identify five different genotypes associated with Alternaria alternata resistance to succinate dehydrogenase inhibitors fungicides, used to control Alternaria late blight in pistachio. With the allele-specific method developed here, users will be able to identify genotypes with nucleotide substitutions which lead to a reduced sensitivity or resistance selection using a one-step PCR assay, without the use of restriction enzymes which elevates the reaction costs and the chance for errors. In addition, this study formally includes the identification and sequence accession for SdhB-H277L and SdhC-S135R amino acid substitution in A. alternata sampled from California and Arizona pistachio orchards., (© 2018 The Society for Applied Microbiology.)

Some thermal processes, such as pistachio roasting, are not yet well characterized with respect to the impact of product and process variables on Salmonella lethality. This study aimed to quantify the effects of process temperature, humidity, and initial product water activity (a w ), on Salmonella lethality for in-shell pistachios. In-shell pistachios were inoculated with Salmonella Enteritidis PT 30 (∼8.5 log CFU/g), equilibrated (0.45 or 0.65 a w ), and heated without soaking ("dry") or after a pure-water or 27% NaCl brining pretreatment ("presoaked"). Inoculated pistachio samples (15 g) were heated in a laboratory-scale, moist-air convection oven at 104.4 or 118.3°C, humidities of ∼3, 15, or 30%, v/v (∼24.4, 54.4, or 69.4°C dew point), and air speed of 1.3 m/s. Salmonella survivors were quantified at six times during each treatment, targeting total reductions of ∼3 to 5 log. Survivor data were analyzed using analysis of variance to identify main effects (time, temperature, humidity, and initial a w ) and two-term interactions with time. As expected, lethality increased ( P < 0.05) with temperature and humidity. For example, the time to achieve a 4-log reduction decreased 50 to 80% when humidity increased from ∼3 to 30%. When the dry and presoaked treatments were analyzed separately, initial product a w (0.45 versus 0.65 a w or 0.75 versus 0.95 a w ) did not affect lethality ( P > 0.05). However, when comparing dry against presoaked treatments, the time to achieve a 4-log reduction decreased 55 to 85% ( P < 0.05) for presoaked pistachios subjected to the same temperature-humidity treatment. Salt had no effect ( P > 0.05) on lethality outcomes. These results, relative to initial a w , process humidity, brining, and salt effects on process lethality, are critically important and must be considered in the design and validation of thermal processes for Salmonella reduction in pistachio processing.

One of the most important fungal agents of pistachio dieback disease belongs to the ascomycete genus Paecilomyces that has been identified as P. variotii. In 2012-2014, 700 plant samples from pistachio trees and 27 other plant species with dieback symptoms were collected from 10 provinces of Iran. Of the 567 pistachio samples, 277 Paecilomyces strains were obtained and from the 133 samples of other plants (except pistachio and including Pistacia mutica, Punica granatum, Prunus amygdalus, Caesalpinia gilliesii, Nerium oleander, Tamarix aphylla, Tamarix hispida and Haloxylon sp.), 23 fungal isolates were recovered and five isolates were obtained from the air of infected pistachio orchards. Based on morphology, all 305 isolates were identified as P. variotii. Physiological studies revealed that 299 isolates belong to P. formosus. Three isolates were assigned to P. variotii, while three isolates could not be assigned to any of the known species. Of the 305 isolates, 62 were selected for phylogenetic analysis based on DNA variation (ITS, β-tubulin and calmodulin). This analysis showed that all of our isolates form a clade with P. formosus. P. formosus consists of the three former species P. formosa, P. lecythidis and P. maximus. This study shows that our isolates form a strongly supported clade with strains of P. lecythidis. So, the causal agent of dieback disease of pistachio and other examined trees is P. formosus which is closely related to the former species P. lecythidis and has some differences with the former species P. formosa and P. maximus. Based on phylogenetic studies P. formosus thus seems to be a species complex that could be divided into three separate species., Competing Interests: The authors have declared that no competing interests exist.

Interactions between pathogenic and nonpathogenic fungal species in the tree canopy are complex and can determine if disease will manifest in the plant and in other organisms such as honey bees. Seasonal dynamics of fungi were studied in an almond orchard in California where experimental release of the atoxigenic biopesticide Aspergillus flavus AF36 to displace toxigenic Aspergillus strains has been conducted for five years. The presence of the vegetative compatibility group (VCG) YV36, to which AF36 belongs, in the blossoms, and the honey bees that attend these blossoms, was assessed. In blossoms, A. flavus frequencies ranged from 0 to 4.5%, depending on the year of study. Frequencies of honey bees carrying A. flavus ranged from 6.5 to 10%. Only one A. flavus isolate recovered from a blossom in 2016 belonged to YV36, while members of the VCG were not detected contaminating honey bees. Exposure of pollinator honey bees to AF36 was detected to be very low. The density of several Aspergillus species was found to increase during almond hull split and throughout the final stages of maturation; this also occurred in pistachio orchards during the maturation period. Additionally, we found that AF36 effectively limited almond aflatoxin contamination in laboratory assays. This study provides knowledge and understanding of the seasonal dynamics of Aspergillus fungi and will help design aflatoxin management strategies for almond. The evidence of the low levels of VCG YV36 encountered on almond blossoms and bees during pollination and AF36's effectiveness in limiting aflatoxin contamination in almond provided additional support for the registration of AF36 with USEPA to use in almond in California., Competing Interests: Having funding support from the Almond Board of California does not alter our adherence to PLOS ONE policies on sharing data and materials.

We developed a quantitative risk assessment model to assess the risk of human nontyphoidal salmonellosis from consumption of pistachios in the United States and to evaluate the impact of Salmonella treatments (1- to 5-log reductions). The exposure model estimating prevalence and contamination levels of Salmonella at consumption included steps in pistachio processing such as transport from grower to huller, removal of the hull through wet abrasion, separation of pistachio floaters (immature, smaller nuts) and sinkers (mature, larger nuts) in a flotation tank, drying, storage, and partitioning. The risks of illness per serving and per year were evaluated by including a Salmonella dose-response model and U.S. consumption data. The spread of Salmonella through float tank water, delay in drying resulting in growth, increased Salmonella levels through pest infestation during storage (pre- and posttreatment), and a simulation of the 2016 U.S. salmonellosis outbreak linked to consumption of pistachios were the modeled atypical situations. The baseline model predicted one case of salmonellosis per 2 million servings (95% CI: one case per 5 million to 800,000 servings) for sinker pistachios and one case per 200,000 servings (95% CI: one case per 400,000 to 40,000 servings) for floater pistachios when no Salmonella treatment was applied and pistachios were consumed as a core product (>80% pistachio) uncooked at home. Assuming 90% of the pistachio supply is sinkers and 10% is floaters, the model estimated 419 salmonellosis cases per year (95% CI: 200 to 1,083 cases) when no Salmonella treatment was applied. A mean risk of illness of less than one case per year was estimated when a minimum 4-log reduction treatment was applied to the U.S. pistachio supply, similar to the results of the Salmonella risk assessment for almonds. This analysis revealed that the predicted risk of illness per serving is higher for all atypical situations modeled compared with the baseline, and delay in drying had the greatest impact on consumer risk.

To identify predominant isolates for potential use as biocontrol agents, Aspergillus flavus isolates collected from soils of almond, pistachio and fig orchard in the Central Valley of California were tested for their membership to 16 atoxigenic vegetative compatibility groups (VCGs), including YV36, the VCG to which AF36, an atoxigenic isolate commercialized in the United States as biopesticide, belongs. A surprisingly large proportion of isolates belonged to YV36 (13.3%, 7.2% and 6.6% of the total almond, pistachio and fig populations, respectively), while the percentage of isolates belonging to the other 15 VCGs ranged from 0% to 2.3%. In order to gain a better insight into the structure and diversity of atoxigenic A. flavus populations and to further identify predominant isolates, seventeen SSR markers were then used to genetically characterize AF36, the 15 type-isolates of the VCGs and 342 atoxigenic isolates of the almond population. There was considerable genetic diversity among isolates with a lack of differentiation among micro-geographical regions or years. Since isolates sharing identical SSR profiles from distinct orchards were rare, we separated them into groups of at least 3 closely-related isolates from distinct orchards that shared identical alleles for at least 15 out of the 17 loci. This led to the identification of 15 groups comprising up to 24 closely-related isolates. The group which contained the largest number of isolates were members of YV36 while five groups were also found to be members of our studied atoxigenic VCGs. These results suggest that these 15 groups, and AF36 in particular, are well adapted to various environmental conditions in California and to tree crops and, as such, are good candidates for use as biocontrol agents., (Published by Elsevier B.V.)

The study reports efficacy of Illicium verum essential oil (IvEO) against food borne moudls and its nanoencapsulation for enhancing antifungal and antiaflatoxigenic potency. Chemical characterization of the IvEO showed anethole (89.12%) as major compound followed by estragole (4.859%). The IvEO showed broad fungitoxic spectrum against common food borne moulds. It's minimum inhibitory concentration (MIC) and minimum aflatoxin B 1 inhibitory concentration (MAIC) against aflatoxigenic strain Aspergillus flavus LHP-PV-1 were 0.7, and 0.5 μL/mL respectively. Morphological observations of treatment sets by SEM and TEM along with decrease in ergosterol content and enhanced leakage of Ca 2+ , K + and Mg 2+ ions denoted fungal cell membrane as site of action. The IvEO showed promising free radical scavenging activity and favourable safety profile with high LD 50 value on mice. The IvEO also exhibited considerable protection of Pistacia vera from fungal contamination and complete protection from aflatoxin B 1 contamination in storage containers. Nanoencapsulated IvEO in gel form and lyophilized form exhibited enhanced efficacy as fungal inhibitor and aflatoxin suppressor. The chemically characterised IvEO may be recommended as plant based preservative having favourable safety and its nanocapsules may be of industrial significance as shelf life enhancer of food items. This is the first report on in situ antiaflatoxigenic efficacy and nanoencapsulation of IvEO., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus , and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses.

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.

The risk of salmonellosis from consumption of pistachios produced and consumed in the U.S. was assessed through quantitative microbial risk assessment. Data on Salmonella prevalence and concentration on pistachios, nut crop volume, storage times and temperatures during processing and handling, and reductions during storage or from roasting were derived from laboratory experiments, published literature, and industry expert opinion. Uncertainty was analyzed via what-if scenarios for Salmonella prevalence, concentration, storage reduction, treatment variability, portion of crop treated, and increased consumption. The estimated U.S. incidence of salmonellosis when 100% of pistachios were exposed to a 4 ± 0 log reduction treatment averaged 1.4 cases per billion servings, or <1 case/year, without considering Salmonella decline during storage. Including Salmonella decline during storage reduced the salmonellosis estimates approximately 10-fold. The predicted arithmetic mean number of cases associated with individual 500,000-kg storage silos, contaminated at the highest observed levels, ranged from 5 to 530 when the product was consumed untreated, but was reduced to below 1 case per silo when a 4 ± 0 log reduction treatment was applied. Assuming a uniform 4-log reduction treatment is applied to 100% of the crop and there is no decline of Salmonella during storage, the assessment indicates the following: 10-fold increases in either Salmonella prevalence or concentration, 2-fold increases in both prevalence and concentration, or consumption of >0.05% of untreated product volume yield an arithmetic mean risk of >1 case/year., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pistachio (Pistacia vera) is an important nut for its economic, nutritional and health aspects but it can be contaminated by aflatoxigenic fungi in the field and during storage. Biological control could be considered as an alternative to chemical treatment. In this study, we evaluated the antifungal and anti-mycotoxigenic capability of two Bacillus spp. both in vitro and on pistachio kernels. In in vitro conditions, both strains were able to reduce the mycelial growth and they were able to degrade the four aflatoxins during the first three days after inoculation. AFG 1 and AFG 2 were rapidly degraded within two days of incubation with the bacterial strains. No aflatoxin was found in the bacterial cell walls, permitting exclusion of mycotoxin adsorption and hypothesis of an in vitro biodegradation as a mode of action. The cultivar of pistachio most susceptible to fungal colonization was 'Ahmad-Aghaei', selected among four main Iranian cultivars. A. parasiticus was able to grow and produce aflatoxins on pistachios, but at longer inoculation periods, a natural decrease of aflatoxins was registered. Both strains were able to reduce the fungal incidence and number of spores on pistachio with a stronger effect during the first 5dpi. The effect on aflatoxin content in vivo was less pronounced than in vitro, with a maximum effect at 8dpi. At longer times, there was a contrasting effect due to the lower activity of Bacillus spp. in stationary phase and higher growth of Aspergillus species. This consideration could explain the lack of aflatoxin reduction at 12dpi. Both bacterial strains showed good antifungal activity and aflatoxin reduction in in vitro conditions and on pistachio kernels. Altogether, these results indicate that Bacillus species could be considered as potential biocontrol agents to combat toxigenic fungal growth and subsequent aflatoxin contamination of nuts and agricultural crops in practice., (Copyright © 2017. Published by Elsevier B.V.)

Starch suspensions were crosslinked with trisodium citrate for either 0 or 17h, gelled and then freeze-dried to corresponding aerogels. The aerogel from the 17h-crosslinked suspension was loaded with the antifungal compound, trans-2-hexenal, and coated with the surfactant, sorbitan monooleate. Aerogel hardness was increased by the citrate-mediated crosslinking, whereas its adhesiveness decreased. Starch gelation decreased the crystallinity index (CrI) from 59% to ≈23%; however, the pre-gelation crosslinking resulted in a higher CrI value (i.e. ≈38%) for the aerogel. The voids at the internal microstructure of the 17h-crosslinked aerogel were more uniform and coating with surfactant closed the surface openings. The latter accordingly resulted in a more sustained release of the volatile, trans-2-hexenal, from the crosslinked starch aerogel and led to slower lethality of Aspergillus parasiticus cells inoculated on pistachio nuts compared with the non-coated condition., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pistachio nuts have been associated with outbreaks of foodborne disease and the industry has been impacted by numerous product recalls due to contamination with Salmonella enterica. The current hot air drying of pistachios has low energy efficiency and drying rates, and also does not guarantee the microbial safety of products. In the study described herein, dehulled and water-sorted pistachios with a moisture content (MC) of 38.14% (wet basis) were dried in a sequential infrared and hot air (SIRHA) drier to <9% MC. The decontamination efficacy was assessed by inoculating pistachios with Enterococcus faecium, a surrogate of Salmonella enterica used for quality control in the almond industry. Drying with IR alone saved 105min (34.4%) of drying time compared with hot air drying. SIRHA drying of pistachios for 2h with infrared (IR) heat followed by tempering at a product temperature of 70°C for 2h and then by hot air drying shortened the drying time by 40min (9.1%) compared with drying by hot air only. This SIRHA method also reduced the E. faecium cell population by 6.1-logCFU/g kernel and 5.41-logCFU/g shell of pistachios. The free fatty acid contents of SIRHA dried pistachios were on par with that of hot air dried samples. Despite significant differences in peroxide values (PV) of pistachio kernels dried with the SIRHA method compared with hot air drying at 70°C, the PV were within the permissible limit of 5Meq/kg for edible oils. Our findings demonstrate the efficacy of SIRHA drying in achieving simultaneous drying and decontamination of pistachios., (Published by Elsevier B.V.)

Bioaugmentation-assisted phytoremediation implies the administration of selected plant growth promoting bacteria, which significantly improve plant growth and sequestration of heavy metals. In this work, 184 bacterial strains associated with roots of Pistacia lentiscus were isolated from plants spontaneously growing in the abandoned Sardinian mining areas (SW Sardinia, Italy) and phylogenetically characterised. Twenty-one bacterial isolates were assayed for properties relevant for plant growth promotion and metal tolerance. Five different strains, belonging to the genera Novosphingobium, Variovorax, Streptomyces, Amycolatopsis, Pseudomonas, were selected based on their properties for the greenhouse phytoremediation tests. Among the tested inocula, the strain Variovorax sp. RA128A, able to produce ACC deaminase and siderophore, was able to significantly enhance germination and increase length and weight of shoots and roots. Irrespective of the applied treatment, mastic shrub was able to accumulate Cd, Pb and Zn especially in roots.

The objective of the present study was to assess the differences in modeled growth/AFB1 production probability and kinetic growth parameters for Aspergillus flavus inoculated as single spores or in a concentrated inoculation point (~500 spores). The experiment was carried out at 25°C and at two water activities (0.85 and 0.87) on pistachio extract agar (3%). Binary data obtained from growth and AFB1 studies were modeled using linear logistic regression analysis. The radial growth curve for each colony was fitted to a linear model for the estimation of the lag phase for growth and the mycelial growth rate. In general, radial growth rate and lag phase for growth were not normally distributed and both of them were affected by the inoculation type, with the lag phase for growth being more affected. Changing from the multiple spore to the single spore inoculation led to a delay of approximately 3-5days on the lag phase and higher growth rates for the multiple spore experiment were found. The same trend was observed on the probability models, with lower predicted probabilities when colonies came up from single spores, for both growth and AFB1 production probabilities. Comparing both types of models, it was concluded that a clear overestimation of the lag phase for growth occurred using the linear model, but only in the multiple spore experiment. Multiple spore inoculum gave very similar estimated time to reach some set probabilities (t10, t50 and t100) for growth or AFB1 production due to the abruptness of the logistic curve developed. The observed differences suggest that inoculum concentration greatly affects the outcome of the predictive models, the estimated times to growth/AFB1 production being much earlier for the concentrated inoculum than for a single spore colony (up to 9days). Thus the number of spores used to generate data in predictive mycology experiments should be carefully controlled in order to predict as accurately as possible the fungal behavior in a foodstuff., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Fungal-contaminated tissues are known to produce volatile profiles that are different from uncontaminated tissues. Fungi require certain water activity levels before growth can occur. For nonxerophilic fungi, a water activity of 0.85 is typical for growth, and for extreme xerophilic fungi, the water activity can be as low as 0.64. Recent investigations with stored pistachios (kernels in shell, no hull tissue) at varying relative humidities showed differences among the collected volatile profiles at the tested humidities (ambient, 63, 75, and 84%). Water activities of the kernel and shell were also measured. Results showed significant changes in volatile profiles as a function of water activity of the corresponding pistachio tissue with measured water activity levels at or below that of what is considered extreme xerophilic activities. Because fungal growth, including mycotoxigenic fungi, is dependent upon water activity, the detected volatile profiles could be used for early detection of fungal presence. Multivariate analysis of the volatile data demonstrated significant differences among the volatile profiles at the tested relative humidity levels, and several volatiles were identified as biomarkers of increased humidity and likely fungal development.

Three bacterial strains (TE1, TD3 and FB2) were isolated from date palm (degla), pistachio and barley. The presence of nitrate reductase (narG) and nitrite reductase (nirS and nirK) genes in the selected strains was detected by PCR technique. Molecular identification based on 16S rDNA sequencing method was applied to identify positive strains. In addition, the D-optimal mixture experimental design was used to optimize the optimal formulation of probiotic bacteria for denitrification process. Strains harboring denitrification genes were identified as: TE1, Agrococcus sp LN828197; TD3, Cronobacter sakazakii LN828198 and FB2, Pedicoccus pentosaceus LN828199. PCR results revealed that all strains carried the nirS gene. However only C. sakazakii LN828198 and Agrococcus sp LN828197 harbored the nirK and the narG genes respectively. Moreover, the studied bacteria were able to form biofilm on abiotic surfaces with different degree. Process optimization showed that the most significant reduction of nitrate was 100% with 14.98% of COD consumption and 5.57 mg/l nitrite accumulation. Meanwhile, the response values were optimized and showed that the most optimal combination was 78.79% of C. sakazakii LN828198 (curve value), 21.21% of P. pentosaceus LN828199 (curve value) and absence (0%) of Agrococcus sp LN828197 (curve value)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study was undertaken to evaluate the effectiveness of superheated steam (SHS) on the inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, Salmonella Enteritidis phage type (PT) 30 and Listeria monocytogenes on almonds and in-shell pistachios and to determine the effect of superheated steam heating on quality by measuring color and texture changes. Almonds and in-shell pistachios inoculated with four foodborne pathogens were treated with saturated steam (SS) at 100 °C and SHS at 125, 150, 175, and 200 °C for various times. Exposure of almonds and pistachios to SHS for 15 or 30s at 200 °C achieved >5l og reductions among all tested pathogens without causing significant changes in color values or texture parameters (P>0.05). For both almonds and pistachios, acid and peroxide values (PV) following SS and SHS treatment for up to 15s and 30s, respectively, were within the acceptable range (PV<1.0 meq/kg). These results show that thermal application of 200 °C SHS treatment for 15s and 30s did not affect the quality of almonds and pistachios, respectively. Therefore, SHS treatment is a very promising alternative technology for the tree nuts industry by improving inactivation of foodborne pathogens on almonds and pistachios while simultaneously reducing processing time., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Salmonella is commonly found in a variety of food products and is a major cause of bacterial foodborne illness throughout the world. In this study, we investigated the prevalence and diversity of Salmonella in eight different food types: sheep ground meat, cow ground meat, chicken meat, cow offal, traditional Sanliurfa cheese, unripened feta cheese, pistachios, and isot (a spice blend of dried red peppers specific to Sanliurfa), traditionally and commonly consumed in Turkey. Among 192 food samples, Salmonella was detected in 59 samples, with the highest prevalence in raw poultry parts (58%) and offal (58%) samples, while Salmonella was not detected in pistachios and dried red pepper. Resultant Salmonella isolates were characterized by serotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Ten different serotypes represented 10 MLST sequence types (STs) with 1 novel ST and 17 PFGE types. Antimicrobial resistance profiling revealed that 30.5% of the isolates were resistant to two or more antimicrobials. Salmonella enterica subsp. enterica serotype Telaviv, which is rare throughout the world, was the second most common serotype isolated from food samples in this study, suggesting that this serotype might be one of the subtypes that is endemic to Turkey.

Human exposure to aflatoxins in foods is of great concern. The aim of this work was to use predictive mycology as a strategy to mitigate the aflatoxin burden in pistachio nuts postharvest. The probability of growth and aflatoxin B1 (AFB1) production of aflatoxigenic Aspergillus flavus, isolated from pistachio nuts, under static and non-isothermal conditions was studied. Four theoretical temperature scenarios, including temperature levels observed in pistachio nuts during shipping and storage, were used. Two types of inoculum were included: a cocktail of 25 A. flavus isolates and a single isolate inoculum. Initial water activity was adjusted to 0.87. Logistic models, with temperature and time as explanatory variables, were fitted to the probability of growth and AFB1 production under a constant temperature. Subsequently, they were used to predict probabilities under non-isothermal scenarios, with levels of concordance from 90 to 100% in most of the cases. Furthermore, the presence of AFB1 in pistachio nuts could be correctly predicted in 70-81 % of the cases from a growth model developed in pistachio nuts, and in 67-81% of the cases from an AFB1 model developed in pistachio agar. The information obtained in the present work could be used by producers and processors to predict the time for AFB1 production by A. flavus on pistachio nuts during transport and storage., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Various species of phytopathogenic Botryosphaeriaceae were identified previously from pistachio trees worldwide. Disease symptoms caused by pathogens in Botryosphaeriaceae on pistachio include panicle and shoot blight, leaf defoliation, fruit discoloration and decay. In this study species of Botryosphaeriaceae were collected from blighted pistachio shoots in Arizona, USA, and Greece. The aims of this study were to identify these Botryosphaeriaceae isolates and to test their pathogenicity to pistachio. The fungi were identified based on comparisons of DNA sequence data of the nuclear rDNA internal transcribed spacer region (ITS), a partial translation elongation factor 1-alpha gene (TEF1), a partial β-tubulin gene (TUB2) and morphological characteristics. Results indicated that some isolates collected from pistachio represent two previously undescribed species, which we described here as Lasiodiplodia americana sp. nov. from the United States and Neofusicoccum hellenicum sp. nov. from Greece. Field inoculations of L. americana and N. hellenicum on branches of four pistachio cultivars showed that both L. americana and N. hellenicum are pathogenic on pistachio. The four pistachio cultivars differed in their susceptibility to the Botryosphaeriaceae species. Results of this study suggested that the two new species of Botryosphaeriaceae need to be monitored carefully to determine the distribution of these pathogens and the possible spread to other areas., (© 2015 by The Mycological Society of America.)

This study evaluated the efficacy of aqueous (aQUAT) and isopropyl alcohol-based quaternary ammonium (ipQUAT) sanitizers for reducing Salmonella spp., Escherichia coli O157:H7, or Listeria monocytogenes populations on peanut and pistachio shell pieces. Inoculated nutshells were mixed with QUAT sanitizers, water, or 70% ethanol and enumerated immediately or after incubation at 30 °C for 48 h. None of the treatments had any immediate effect on Salmonella or E. coli O157:H7 populations on the peanut or pistachio shells. L. monocytogenes populations declined immediately on the peanut and pistachio shells treated with aQUAT or ipQUAT. After incubation, Salmonella and E. coli O157:H7 populations increased significantly on the water- or aQUAT-treated peanut and pistachio shells. L. monocytogenes populations also increased significantly on the water- or aQUAT-treated peanut shells, but levels did not change on the water-treated pistachio shells and levels were just above the limit of detection on the aQUAT-treated pistachio shells. After treatment with ipQUAT and 48-h incubation, Salmonella and E. coli O157:H7 populations decreased to or below the limit of detection on both shell types; L. monocytogenes populations remained at or below the limit of detection on both shell types., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium and is a potential contaminant of a wide variety of food products. To determine the incidence of OTA contamination in dried fruits and tree nuts, retail packaged and bulk raisins, dates, figs, prunes, almonds, pistachios, and walnuts were collected from small and large supermarkets in seven areas of the United States between 2012 and 2014. Of the 665 samples analyzed, OTA was detected in 48 raisin samples, 4 fig samples, 4 pistachio samples, and 1 date sample. OTA contamination levels ranged from 0.28 to 15.34 ng/g in dried fruits and 1.87 to 890 ng/g in pistachios; two raisin samples and one pistachio sample exceeded the European Union regulatory limit of 10 ng/g. PCR detection of potential OTA-producing Aspergillus species revealed the presence of A. niger, A. welwitschiae, and A. carbonarius in 20, 7, and 7 of the 57 OTA-contaminated samples, respectively. However, OTA-producing A. carbonarius was isolated from only one raisin sample, and no other OTA-producing Aspergillus species were found. These results suggest that raisins are more frequently contaminated with low levels of OTA than are other dried fruits and nuts and that Aspergillus species are the likely source of that contamination.

The effects of two exposure times per day (6 and 16 h) of UV-A or UV-B radiation, combined with dark and dark plus light incubation periods during 7-21 d on fungal growth and mycotoxins production of Aspergillus species were studied. Aspergillus carbonarius and Aspergillus parasiticus were inoculated on grape and pistachio media under diurnal and nocturnal temperatures choosing light photoperiod according to harvest conditions of these crops in Spain. Ultraviolet irradiation had a significant effect on A. carbonarius and A. parasiticus colony size (diameter, biomass dry weight, and colony density) and mycotoxin accumulation, although intraspecies differences were observed. Inhibition of A. carbonarius fungal growth decreased when exposure time was reduced from 16 h to 6 h, but this was not always true for ochratoxin A (OTA) production. OTA reduction was higher under UV-A than UV-B radiation and the reduction increased along time conversely to the aflatoxins (AFs). Aflatoxin B1 (AFB1) was the main toxin produced by A. parasiticus except in the UV-B light irradiated colonies which showed a higher percentage of AFG than AFB. Morphological changes were observed in colonies grown under UV-B light., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Background: One of the most important saprophytic infections in fresh pistachio fruits after harvesting is Aspergillus flavus colonization, which significantly reduces fruit quality. Salicylic acid plays a crucial role in plant tissues and has a suppression effect on some fungi., Results: The inhibitory effect of salicylic acid on the growth of A. flavus was assessed in vitro and in vivo. For this purpose, seven concentrations (0, 1, 3, 5, 7, 9 and 11 mmol L(-1)) of salicylic acid were used in both experiments. Also, aflatoxin B1 contents of the samples were analysed using immunoaffinity chromatography. The results obtained from in vitro experiments showed that salicylic acid significantly reduced Aspergillus growth at all concentrations, and at 9 mmol L(-1) growth was completely suppressed. In vivo evaluation showed relatively high levels of inhibition, though the intact treated fruits as compared with the injured treated fruits demonstrated higher inhibitory effects., Conclusion: Regarding the inhibitory effects of salicylic acid on the control of A. flavus contamination, its application on pistachio fruits after harvesting could be a promising approach to control the fungus infection and reduce aflatoxin production in treated fruits., (© 2013 Society of Chemical Industry.)

Aim: The aim of this study was to investigate the effect of plasma-enhanced chemical vapour deposition (PECVD) treatment on selected bacteria and spores and to contribute to the understanding of the synergistic effect of UV-directed plasma., Methods and Results: The experiments were conducted on pure cultures of Aspergillus brasiliensis and Escherichia coli and on naturally contaminated pistachios that were exposed to pure oxygen-, pure argon- and to a mixture of oxygen-argon-generated plasma for different treatment times and at different micro-organism concentrations. Optical emission spectroscopy (OES) measurements were performed to observe the active species in the plasma. After exposure, the effectiveness of decontamination was assessed through microbiological techniques by calculating the growth reduction on a logarithmic scale. A treatment time of 30 min resulted in a 3·5 log reduction of A. brasiliensis using pure oxygen or argon, while treatment times of 5 min, 1 min and 15 s resulted in a 5·4 log reduction using a mixture of argon and oxygen (10 : 1 v/v). Treatment times of 1 min and 30 s resulted in a 4 log reduction of E. coli with oxygen and argon, respectively, which led to a complete elimination of the micro-organisms. Two-log reductions of fungi were achieved for pistachios after a treatment time of 1 min., Conclusions: These results suggest that this newly designed plasma reactor offers good potential applications for the reduction in micro-organisms on heat-sensitive materials, such as foods. The plasma that was generated with Ar/O2 was more effective than that which was generated with pure oxygen and pure argon., Significance and Impact of the Study: An improvement in the knowledge about PECVD mechanisms was acquired from the chemical and biological points of view, and the suitability of the method for treating dry food surfaces was demonstrated., (© 2014 The Society for Applied Microbiology.)

Aflatoxins, carcinogenic toxins produced by Aspergillus fungi, contaminate maize, peanuts, and tree nuts in many regions of the world. Pistachios are the main source of human dietary aflatoxins from tree nuts worldwide. Over 120 countries have regulations for maximum allowable aflatoxin levels in food commodities. We developed social network models to analyze the association between nations' aflatoxin regulations and global trade patterns of pistachios from 1996-2010. The main pistachio producing countries are Iran and the United States (US), which together contribute to nearly 75% of the total global pistachio market. Over this time period, during which many nations developed or changed their aflatoxin regulations in pistachios, global pistachio trade patterns changed; with the US increasingly exporting to countries with stricter aflatoxin standards. The US pistachio crop has had consistently lower levels of aflatoxin than the Iranian crop over this same time period. As similar trading patterns have also been documented in maize, public health may be affected if countries without aflatoxin regulations, or with more relaxed regulations, continually import crops with higher aflatoxin contamination. Unlike the previous studies on maize, this analysis includes a dynamic element, examining how trade patterns change over time with introduction or adjustment of aflatoxin regulations.

The spiroketal (E)-conophthorin has recently been reported as a semiochemical of the navel orangeworm moth, a major insect pest of California pistachios and almonds. Conophthorin and the isomeric spiroketal chalcogran are most commonly known as semiochemicals of several scolytid beetles. Conophthorin is both an insect- and plant-produced semiochemical widely recognized as a nonhost plant volatile from the bark of several angiosperm species. Chalcogran is the principal aggregation pheromone component of the six-spined spruce bark beetle. Recent research has shown conophthorin is produced by almonds undergoing hull-split, and both spiroketals are produced by mechanically damaged almonds. To better understand the origin of these spiroketals, the volatile emissions of orchard fungal spores on fatty acids common to both pistachios and almonds were evaluated. The volatile emission for the first 13 days of spores placed on a fatty acid was monitored. The spores investigated were Aspergillus flavus (atoxigenic), A. flavus (toxigenic), Aspergillus niger, Aspergillus parasiticus, Penicillium glabrum, and Rhizopus stolonifer. The fatty acids used as growth media were palmitic, oleic, linoleic, and linolenic. Spores on linoleic acid produced both spiroketals, those on linolenic acid produced only chalcogran, and those on palmitic and oleic acid did not produce either spiroketal. This is the first report of the spiroketals conophthorin and chalcogran from a fungal source.