Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae, and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum, and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.

High-efficiency fungal pathogen intervention for seed protection: new utility of long-chain alkyl gallates as heat-sensitizing agents.

Control of food-contaminating fungi, especially pathogens that produce mycotoxins, is problematic since effective method for intervening fungal infection on food crops is often limited. Generally Regarded As Safe (GRAS) chemicals, such as natural compounds or their structural derivatives, can be developed as antimicrobial agents for sustainable food/crop production. This study identified that long-chain alkyl gallates, i.e., octyl-, nonyl-, and decyl gallates (OG (octyl 3,4,5-trihydroxybenzoic acid), NG, DG), can function as heat-sensitizing agents that effectively prevent fungal contamination. Out of twenty-eight candidate compounds and six conventional antifungal agents examined, the heat-sensitizing capacity was unique to the long-chain alkyl gallates, where OG exhibited the highest activity, followed by DG and NG. Since OG is a GRAS compound classified by the United States Food and Drug Administration (FDA), further in vitro antifungal studies were performed using OG. When OG and mild heat (57.5°C) were co-administered for 90 seconds, the treatment achieved > 99.999% fungal death (> 5 log reduction). Application of either treatment alone was significantly less effective at reducing fungal survival. Of note, co-application of OG (3 mM) and mild heat (50°C) for 20 minutes completely prevented the survival of aflatoxigenic Aspergillus flavus contaminating crop seeds (Brassica rapa Pekinensis), while seed germination rate was unaffected. Heat-sensitization was also determined in selected bacterial strains (Escherichia coli, Agrobacterium tumefaciens). Altogether, OG is an effective heat-sensitizing agent for control of microbial pathogens. OG-mediated heat sensitization will improve the efficacy of antimicrobial practices, achieving safe, rapid, and cost-effective pathogen control in agriculture/food industry settings.

Valorizing Tree-Nutshell Particles as Delivery Vehicles for a Natural Herbicide.

The United States is a principal producer of tree nuts (almonds, pistachios, and walnuts), resulting in the generation of excess of tree-nutshell by-products each year, with few market outlets. A nutshell is an essential, lignocellulosic layer that protects a kernel (seed) from the environment during cultivation. The objective of this study was to develop nutshell by-products as herbicide delivery systems, which would not only enable sustainable weed control in fields but also increases nutshell value and reduce the cost of waste disposal. We recently identified a natural salicylaldehyde (SA) that emits volatiles with both herbicidal and antifungal properties. In this study, walnut shell particles saturated with 0.8 to 1.6 M SA were developed as delivery vehicles for SA to soil, which allowed for the controlled release of an SA fumigant for weed control. The pre- and post-emergent herbicidal efficacy of SA was investigated using model monocot (Lolium arundinaceum (Schreb.) Darbysh; turfgrass) and dicot (Brassica rapa var. pekinensis; Chinese cabbage) plants. We compared (1) the effects of different types of solvents for dissolving SA (dimethyl sulfoxide (DMSO) and ethanol (60%, v/v)), and (2) the effect of covering soil with plastic layers (i.e., soil pasteurization) or not covering soil during SA fumigation using nutshells. Results: In the pre-emergent herbicidal testing with the soil covered, the dicot plants exhibited levels of higher susceptibility to SA in DMSO emitted from nutshells when compared to the monocot plants. The seed germination frequencies in the dicots were 15% and 1% with 0.8 and 1.6 M SA, respectively, while those in the monocots were 32% and 18%, respectively, under the same test conditions. In the post-emergent herbicidal testing with the soil covered, the growth of both the monocot and dicot plants was completely prevented after 5 to 7 days of SA fumigation, resulting in the deaths of entire plants. It was noteworthy that in the post-emergent herbicidal testing, SA dissolved in ethanol (60%, v/v) completely disrupted the growth of the monocot and dicot plants as early as 3 days after SA emission from the nutshells, even without the soil being covered. Tree-nutshell particles could serve as effective SA delivery vehicles with controlled release capabilities for SA. The SA exhibited pre- and post-emergent herbicidal activities against the monocot and dicot plants at most growth stages. SA (0.8 and 1.6 M) dissolved in ethanol (60%, v/v) might exert a synergism for higher herbicidal activity after emission from nutshells. Since tree nuts capture/store a substantial amount of carbon over their life-cycles, the new and sustainable utility of using nutshells not only reduces carbon emissions but also valorizes tree-nut by-products, thus benefitting the tree-nut industry.

Complete Genome Sequences of Two Bacillus velezensis Strains Isolated from California Raisin Vineyard Soils.

Bacillus velezensis strains JP3042 and JP3144 were isolated from California raisin vineyard soils and were selected for further study of in vitro antifungal activity. Here, we present the complete genome sequences of these strains to aid in the understanding of their antifungal activity and diversity within the species.

Method for high-throughput antifungal activity screening of bacterial strain libraries.

Our laboratory developed a high-throughput screen for antifungal phenotypes in which bacterial libraries in 96-well format are transferred to agar pour plates inoculated with spores of the target fungus. This method is an improvement over bacterial/fungal coculture plate methods that measure antifungal activity as inhibition of radial fungal growth.

Fate of Aflatoxins during Almond Oil Processing.

ABSTRACT: Almonds rejected as inedible are often used for production of almond oil. However, low-quality almonds are frequently contaminated with aflatoxins, and little is known regarding transfer of aflatoxins to almond oil during processing. In this study, oil was produced from reject almonds by hexane extraction. Of 19 almond samples that were naturally contaminated with aflatoxins, 17 oil samples contained measurable amounts of aflatoxins, and aflatoxin content of contaminated oil was correlated with aflatoxin content of the nuts. However, oil aflatoxin levels were not correlated with the oxidation level of the oil as measured by percent free fatty acids and peroxide value. Adsorbents used in oil refining were tested for their ability to remove aflatoxins from contaminated oil. Fuller's earth and bentonite were the most effective, removing 96 and 86% of total aflatoxins from contaminated oil samples, respectively. Treatment with diatomaceous earth, in contrast, had no effect on aflatoxin levels in oil. These results show that oil refining steps using mineral clay adsorbents may also function to remove aflatoxins from contaminated oil.

Effect of Blanching on Aflatoxin Contamination and Cross-Contamination of Almonds.

ABSTRACT: Blanching of almonds was examined for reducing the aflatoxin content of contaminated nuts. Almonds with intact pellicles were spiked with aflatoxin B1 (AFB1) and blanched at 85°C. Following blanching, almond kernels and pellicles contained 20 and 19% of the spiked AFB1, respectively. The blanching water contained an additional 41% of the spiked AFB1. In a separate study, postblanching water was spiked with AFB1 and used for subsequent blanching of uncontaminated almonds. The resulting blanched kernels acquired 3.3% of the AFB1 from the spiked water, demonstrating a low level of cross-contamination from reused contaminated blanching water. The effect of the blanching temperature on partitioning of AFB1 from almonds to blanching water was significant at a 20-ppb spiking level, but not at 100 ppb. AFB1 levels that were unaccounted for in the mass balance of blanching components were presumed to be lost due to binding to water-solubilized almond components and were independent of pH and blanching time. Blanching reduced total aflatoxins in naturally contaminated almonds by 13 to 76%, depending on almond quality, as well as blanching time and temperature. These results indicate that the association between almond components and aflatoxin generated through mold contamination is more complex than in spiking experiments.

Comparison of Aspergillus Section Nigri Species Populations in Conventional and Organic Raisin Vineyards.

Species belonging to Aspergillus section Nigri are widespread in the vineyard environment, both in soil and on plant surfaces. We used plate counts and droplet digital PCR (ddPCR) methods to compare populations of the four most prevalent species (A. carbonarius, A. niger, A. welwitschiae, and A. tubingensis) over two consecutive years in conventional and organic vineyards, to determine whether management affects the potential distribution of mycotoxigenic Aspergillus species. In 2016, plate counts showed that soil populations of total filamentous fungi and of Aspergillus section Nigri species were not significantly different between conventional and organic vineyards. In 2017, while total fungal populations in soil were not significantly different, Aspergillus section Nigri populations were significantly higher in organic vineyard soil. In both years, there were no significant differences in total fungal populations and in Aspergillus section Nigri populations on fruit surfaces collected from conventional and organic vineyards. Likewise, ddPCR methods did not show significant differences in percent distribution of Aspergillus species in soil and fruit between conventional and organic vineyards. These results suggest that intervention strategies for preharvest control of potential mycotoxigenic fungi are likely to be equally compatible with either vineyard management strategy.

Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic Aspergillus flavus mixtures in soil.

Aflatoxin B1 is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28 °C for 2 weeks and 1 month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.

A risk assessment of dietary Ochratoxin a in the United States.

Ochratoxin A (OTA) is a mycotoxin (fungal toxin) found in multiple foodstuffs. Because OTA has been shown to cause kidney disease in multiple animal models, several governmental bodies around the world have set maximum allowable levels of OTA in different foods and beverages. In this study, we conducted the first exposure and risk assessment study of OTA for the United States' population. A variety of commodities from grocery stores across the US were sampled for OTA over a 2-year period. OTA exposure was calculated from the OTA concentrations in foodstuffs and consumption data for different age ranges. We calculated the margin of safety (MOS) for individual age groups across all commodities of interest. Most food and beverage samples were found to have non-detectable OTA; however, some samples of dried fruits, breakfast cereals, infant cereals, and cocoa had detectable OTA. The lifetime MOS in the US population within the upper 95% of consumers of all possible commodities was >1, indicating negligible risk. In the US, OTA exposure is highest in infants and young children who consume large amounts of oat-based cereals. Even without OTA standards in the US, exposures would not be associated with significant risk of adverse effects.

Population Dynamics of Aspergillus Section Nigri Species on Vineyard Samples of Grapes and Raisins.

Several species of Aspergillus section Nigri, including potential mycotoxin producers, are common residents of grape vineyards, but the relative population size of individual species throughout the growing season is difficult to determine using traditional isolation and identification methods. Using a quantitative droplet digital PCR (ddPCR) method in combination with dilution plating, total Aspergillus section Nigri populations and relative proportions of A. niger, A. welwitschiae, A. carbonarius, and A. tubingensis were measured from vineyard samples without the need for identifying individual fungal isolates. Grapes were sampled from two raisin vineyards (vineyards A and B) at berry set, veraison, harvest, and raisin stages in two consecutive years. Plate counts showed that the total population of Aspergillus section Nigri present on the fruit increased from berry set to raisin and became a larger component of the total recovered fungal population in both vineyards in both years. Results from ddPCR analysis showed that the relative proportion of A. carbonarius among the four species assayed increased later in the season (harvest and raisin) in comparison to earlier in the season (berry set and veraison). Total fungal and Aspergillus section Nigri plate counts were not significantly different between vineyards in either year. However, vineyard A generally showed higher proportions of A. carbonarius in harvest and raisin samples than vineyard B. This coincided with higher incidence and levels of ochratoxin A in vineyard A harvest and raisin fruit than in vineyard B fruit. This work demonstrates that this ddPCR method is a useful tool for culture-independent monitoring of populations of mycotoxigenic Aspergillus species during grape and raisin production.

Effect of oxidant stressors and phenolic antioxidants on the ochratoxigenic fungus Aspergillus carbonarius.

BACKGROUND: There are few studies dealing with the relationship between oxidative stress and ochratoxin A (OTA) biosynthesis. In this work, we analyzed the effect of the oxidant stressor menadione and the antioxidants 3,5-di-tert-butyl-4-hydroxytoluene (BHT), catechin, resveratrol and a polyphenolic extract on growth, generation of reactive oxygen species (ROS), OTA production and gene expression of antioxidant enzymes of Aspergillus carbonarius. RESULTS: Exposure to menadione concentrations higher than 20 µmol L(-1) led to increases in ROS and OTA levels and a decrease in growth rate. Exposure to 2.5-10 mmol L(-1) BHT also led to higher ROS and OTA levels, although growth rate was only affected above 5 mmol L(-1). Naturally occurring concentrations of catechin, resveratrol and polyphenolic extract barely affected growth rate, but they produced widely different effects on OTA production level depending on the antioxidant concentration used. In general, gene expression of antioxidant enzymes superoxide dismutase (SOD) and peroxiredoxin (PRX) was downregulated after exposure to oxidant and antioxidant concentrations that enhanced OTA production level. CONCLUSION: Aspergillus carbonarius responds to oxidative stress, increasing OTA production. Nevertheless, the use of naturally occurring concentrations of antioxidant phenolic compounds to reduce oxidative stress is not a valid approach by itself for OTA contamination control in grapes.

Occurrence of ochratoxin a contamination and detection of ochratoxigenic Aspergillus species in retail samples of dried fruits and nuts.

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.

Spread of Aspergillus flavus by Navel Orangeworm (Amyelois transitella) on Almond.

Navel orangeworm (NOW) damage to almond is correlated with increased incidence of aflatoxin contamination caused by Aspergillus flavus. However, no reports demonstrate a causative relationship between NOW feeding and A. flavus infection. To demonstrate the potential of NOW to act as a vector of A. flavus on almond, NOW eggs were dusted with A. flavus and incubated in microchambers adjacent to but not touching agar plates or almond kernels. Following egg hatch, A. flavus colonies developed on agar along trails left by NOW larvae. Almond kernels damaged with A. flavus-carrying NOW showed higher incidence of A. flavus colonization and aflatoxin contamination than control treatments. Interestingly, levels of aflatoxin in NOW-damaged, A. flavus-infected almond were significantly higher than control treatments, even in the absence of visible fungal growth. Commercial almond orchards had a relatively low level of contamination with Aspergillus section Flavi in spring and early summer and a high level during summer, corresponding with the higher level of NOW infestation of the crop. Our study demonstrates that NOW is capable of vectoring A. flavus to almond, and that monitoring and sorting of almond kernels for insect damage is warranted to limit aflatoxin contamination potential both before and after harvesting.

Distribution and mycotoxigenic potential of Aspergillus section Nigri species in naturally contaminated almonds.

In a previous study, inedible almond pick-out samples were assayed for aflatoxin and aflatoxigenic Aspergillus species. These samples contained high populations of black-spored Aspergillus section Nigri species. To investigate whether these species may contribute to the total potential mycotoxin content of almonds, Aspergillus section Nigri strains were isolated from these samples and assayed for ochratoxin A (OTA) and fumonisin B2 (FB2). The majority of isolates (117 strains, 68%) were identified as Aspergillus tubingensis, which do not produce either mycotoxin. Of the 47 Aspergillus niger and Aspergillus awamori isolates, 34 strains (72%) produced FB2 on CY20S agar, and representative strains produced lower but measurable amounts of FB2 on almond meal agar. No OTA-producing strains of Aspergillus section Nigri were detected. Almond pick-out samples contained no measurable FB2, suggesting that properly dried and stored almonds are not conducive for FB2 production by resident A. niger and A. awamori populations. However, 3 of 21 samples contained low levels (<1.5 ng/g) of OTA, indicating that sporadic OTA contamination may occur but may be caused by OTA-producing strains of other Aspergillus species.

Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains.

To determine the genetic basis for loss of fumonisin B2 (FB2) biosynthesis in FB2-nonproducing Aspergillus niger and A. awamori strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified from FB2-producing A. niger and A. awamori strains; from FB2-nonproducing strains four amplification patterns arose in which one or more fum gene fragments were absent. Southern hybridization analysis of strains yielding patterns 2 and 3 confirmed that loss of FB2 production in A. awamori is associated with gene deletions within the fumonisin biosynthetic gene cluster. In addition, we observed a fifth multiplex amplification pattern in which all eight fum gene fragments appeared. Reverse transcription-PCR analysis of strains yielding pattern 5 showed that the expression of at least one fum gene was reduced relative to expression in FB2-producing A. niger. This suggests that in these strains loss of FB2 production is a result of structural or regulatory mutations that alter gene expression or function. These results demonstrate a diversity of genotypes within FB2-nonproducing A. niger and A. awamori populations and provide tools useful for identifying certain non-toxigenic strains for industrial or ecological applications.

Incidence of fumonisin B2 production within Aspergillus section Nigri populations isolated from California raisins.

Fungi belonging to Aspergillus section Nigri occur frequently and in high populations on grapes. Species within this section include Aspergillus niger, A. tubingensis, and A. carbonarius, and they are potential sources for mycotoxins including ochratoxin A and fumonisin B(2) (FB(2)) in grapes and grape products. Aspergillus section Nigri strains were isolated from California raisins to examine the frequency and extent of FB(2) production. Of 392 strains isolated, 197 strains were identified as A. niger, 131 of which produced FB(2). These strains produced from 1.2 to 27 µg/ml FB(2) in culture. PCR amplification of fum1 and fum19 gene fragments showed that all FB(2)-producing strains and nearly all nonproducing strains of A. niger contain these genes. An additional 175 strains were identified as A. tubingensis, none of which produced FB(2). PCR with fum1 and fum19 primers amplified gene fragments of 14 and 25% of A. tubingensis strains, respectively, suggesting that putative orthologs of A. niger fumonisin biosynthetic genes might occur in A. tubingensis. These results indicate that FB(2) production is common among field isolates of A. niger and suggest that the potential for FB(2) contamination of California raisins should be addressed further.

Inhibition of Aspergillus flavus in soil by antagonistic Pseudomonas strains reduces the potential for airborne spore dispersal.

Pseudomonas chlororaphis strain JP1015 and P. fluorescens strain JP2175 were previously isolated from Mississippi cornfield soil samples and selected for their growth inhibition of Aspergillus flavus in laboratory culture. In this study, the antifungal activity of these bacterial strains against A. flavus in soil coculture was determined. Growth of A. flavus was inhibited up to 100-fold by P. chlororaphis strain JP1015 and up to 58-fold by P. fluorescens strain JP2175 within 3 days following soil coinoculation. A. flavus propagule densities after 16 days remained 7- to 20-fold lower in soil treated with either bacterial strain. Using a bench-scale wind chamber, we demonstrated that treatments of soil with P. chlororaphis strain JP1015 and P. fluorescens strain JP2175 reduced airborne spores dispersed across a 1 m distance by 75- to 1,000-fold and 10- to 50-fold, respectively, depending on soil type and inoculum level. These results suggest that application of these bacterial strains may be effective in reducing soil populations of mycotoxigenic fungi, thereby reducing fungal spore formation, and ultimately reducing the potential for crop plant infection via airborne transmission.

Strain of Fusarium oxysporum isolated from almond hulls produces styrene and 7-methyl-1,3,5-cyclooctatriene as the principal volatile components.

An isolated strain of Fusarium oxysporum from the hulls of Prunus dulcis (sweet almond) was found to produce relatively large quantities of the hydrocarbons styrene and two isomers of 7-methyl-1,3,5- cyclooctatriene (MCOT). Production of styrene and MCOT was reproduced on a small scale using potato dextrose agar as a growth medium and scaled up using 1 L of inoculated potato dextrose broth. The compounds were trapped as volatile organic compounds (VOCs) onto solid-phase microextraction (SPME) for small scale and Tenax for large scale and then isolated using standard high-performance liquid chromatography (HPLC) methods. Styrene was authenticated by a comparison to the retention times, fragmentation patterns, and calculated retention indices of a commercially available sample. The identity of MCOT was verified by a short chemical synthesis and a comparison of spectroscopic data to the isolated sample. A biosynthetic scheme of styrene is proposed on the basis of a (13)C-labeling study. This is the first report of MCOT isolated as a natural product.

Inhibition of ochratoxin A production and growth of Aspergillus species by phenolic antioxidant compounds.

The phenolic antioxidants, gallic acid, vanillic acid, protocatechuic acid, 4-hydroxybenzoic acid, catechin, caffeic acid, and chlorogenic acid were studied for their effects on ochratoxin A (OTA) production and fungal growth of ochratoxigenic Aspergilli. Of the 12 strains tested, which included A. alliaceus, A. lanosus, A. ochraceus, A. albertensis, A. melleus, A. sulphureus, A. carbonarius, A. elegans, and A. sclerotiorum, the greatest inhibition of OTA production was seen in A. sulphureus, A. elegans, and A. lanosus. Vanillic acid and 4-hydroxybenzoic acid were the most inhibitory to both OTA production and growth of most of the strains tested. However, A. ochraceus was not inhibited by either compound, and A. carbonarius was not inhibited by vanillic acid. The effect of each compound on OTA production and growth differed among strains and generally was variable, suggesting that species-specific OTA production and response to phenolic compounds may be influenced by different ecological and developmental factors. In addition, inhibition of OTA production by antioxidant compounds may be useful in determining biosynthetic and regulatory genes involved in both OTA production and stress response in ochratoxigenic Aspergilli.