Are Current Aspergillus sojae Strains Originated from a Native Aflatoxigenic Aspergillus Species Population Also Present in California?

Aspergillus sojae has long been considered a domesticated strain of Aspergillus parasiticus. This study delineated relationships among the two species and an Aspergillus PWE36 isolate. Of 25 examined clustered aflatoxin genes of PWE36, 20 gene sequences were identical to those of A. sojae, but all had variations to those of A. parasiticus. Additionally, PWE36 developmental genes of conidiation and sclerotial formation, overall, shared higher degrees of nucleotide sequence identity with A. sojae genes than with A. parasiticus genes. Examination of defective cyclopiazonic acid gene clusters revealed that the PWE36 deletion pattern was identical only to those of A. sojae. Using A. sojae SMF134 genome sequence as a reference, visualization of locally collinear blocks indicated that PWE36 shared higher genome sequence homologies with A. sojae than with A. parasiticus. Phylogenetic inference based on genome-wide single nucleotide polymorphisms (SNPs) and total SNP counts showed that A. sojae strains formed a monophyletic clade and were clonal. Two (Argentinian and Ugandan) A. parasiticus isolates but not including an Ethiopian isolate formed a monophyletic clade, which showed that A. parasiticus population is genetically diverse and distant to A. sojae. PWE36 and A. sojae shared a most recent common ancestor (MRCA). The estimated divergence time for PWE36 and A. sojae was about 0.4 mya. Unlike Aspergillus oryzae, another koji mold that includes genetically diverse populations, the findings that current A. sojae strains formed a monophyletic group and shared the MRCA with PWE36 allow A. sojae to be continuously treated as a species for food safety reasons.

Genome Sequence of an Aspergillus flavus CA14 Strain That Is Widely Used in Gene Function Studies.

Aspergillus flavus produces aflatoxins that adversely impact human health and the economy. We report the genome sequence of A. flavus CA14 that has been widely used in gene function studies. The information will benefit A. flavus functional genomics studies on fungal development, secondary metabolite production, and fungus-host plant interactions.

Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction.

Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs, were downregulated to variable levels depending on paired strains of toxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.

Aspergillus flavus NRRL 35739, a Poor Biocontrol Agent, May Have Increased Relative Expression of Stress Response Genes.

Biocontrol of the mycotoxin aflatoxin utilizes non-aflatoxigenic strains of Aspergillus flavus, which have variable success rates as biocontrol agents. One non-aflatoxigenic strain, NRRL 35739, is a notably poor biocontrol agent. Its growth in artificial cultures and on peanut kernels was found to be slower than that of two aflatoxigenic strains, and NRRL 35739 exhibited less sporulation when grown on peanuts. The non-aflatoxigenic strain did not greatly prevent aflatoxin accumulation. Comparison of the transcriptomes of aflatoxigenic and non-aflatoxigenic A. flavus strains AF36, AF70, NRRL 3357, NRRL 35739, and WRRL 1519 indicated that strain NRRL 35739 had increased relative expression of six heat shock and stress response proteins, with the genes having relative read counts in NRRL 35739 that were 25 to 410 times more than in the other four strains. These preliminary findings tracked with current thought that aflatoxin biocontrol efficacy is related to the ability of a non-aflatoxigenic strain to out-compete aflatoxigenic ones. The slower growth of NRRL 35739 might be due to lower stress tolerance or overexpression of stress response(s). Further study of NRRL 35739 is needed to refine our understanding of the genetic basis of competitiveness among A. flavus strains.

Volatile 1-octen-3-ol increases patulin production by Penicillium expansum on a patulin-suppressing medium.

1-Octen-3-ol is one of the most abundant volatile compounds associated with fungi and functions as a germination and growth inhibitor in several species. By investigating its effect on the biosynthesis of patulin, a mycotoxin made by Penicillium expansum, it was found that a sub-inhibitory level of volatile 1-octen-3-ol increased accumulation of patulin on a medium that normally suppresses the mycotoxin. Transcriptomic sequencing and comparisons of control and treated P. expansum grown on potato dextrose agar (PDA; patulin permissive) or secondary medium agar (SMA; patulin suppressive) revealed that the expression of gox2, a gene encoding a glucose oxidase, was significantly affected, decreasing 10-fold on PDA and increasing 85-fold on SMA. Thirty other genes, mostly involved in transmembrane transport, oxidation-reduction, and carbohydrate metabolism were also differently expressed on the two media. Transcription factors previously found to be involved in regulation of patulin biosynthesis were not significantly affected despite 1-octen-3-ol increasing patulin production on SMA. Further study is needed to determine the relationship between the upregulation of patulin biosynthesis genes and gox2 on SMA, and to identify the molecular mechanism by which 1-octen-3-ol induced this effect.

Influence of R and S enantiomers of 1-octen-3-ol on gene expression of Penicillium chrysogenum.

Inhibition of spore germination offers an attractive and effective target for controlling fungal species involved in food spoilage. Mushroom alcohol (1-octen-3-ol) functions as a natural self-inhibitor of spore germination for many fungi and, therefore, provides a useful tool for probing the molecular events controlling the early stages of fungal growth. In Penicillium spp., the R and S enantiomers of 1-octen-3-ol delayed spore germination and sporulation in four species of Penicillium involved in soils of fruit and grains, but to different degrees. Because of its well-annotated genome, we used Penicillium chrysogenum to perform a comprehensive comparative transcriptomic analysis of cultures treated with the two enantiomers. Altogether, about 80% of the high-quality reads could be mapped to 11,396 genes in the reference genome. The top three active pathways were metabolic (978 transcripts), biosynthesis of secondary metabolites (420 transcripts), and microbial metabolism in diverse environments (318 transcripts). When compared to the control, treatment with (R)-(-)-1-octen-3-ol affected the transcription levels of 91 genes, while (S)-(+)-1-octen-3-ol affected only 41 genes. Most of the affected transcripts were annotated and predicted to be involved in transport, establishment of localization, and transmembrane transport. Alternative splicing and SNPs' analyses indicated that, compared to the control, the R enantiomer had greater effects on the gene expression pattern of Penicillium chrysogenum than the S enantiomer. A qRT-PCR analysis of 28 randomly selected differentially expressed genes confirmed the transcriptome data. The transcriptomic data have been deposited in NCBI SRA under the accession number SRX1065226.

Transcriptional Regulation of Aflatoxin Biosynthesis and Conidiation in Aspergillus flavus by Wickerhamomyces anomalus WRL-076 for Reduction of Aflatoxin Contamination.

Aspergillus flavus is a ubiquitous saprophytic fungus found in soils across the world. The fungus is the major producer of aflatoxin (AF) B1, which is toxic and a potent carcinogen to humans. Aflatoxin B1 (AFB1) is often detected in agricultural crops such as corn, peanut, almond, and pistachio. It is a serious and recurrent problem and causes substantial economic losses. Wickerhamomyces anomalus WRL-076 was identified as an effective biocontrol yeast against A. flavus. In this study, the associated molecular mechanisms of biocontrol were investigated. We found that the expression levels of eight genes, aflR, aflJ, norA, omtA, omtB, pksA, vbs, and ver-1 in the aflatoxin biosynthetic pathway cluster were suppressed. The decreases ranged from several to 10,000 fold in fungal samples co-cultured with W. anomalus. Expression levels of conidiation regulatory genes brlA, abaA, and wetA as well as sclerotial regulatory gene (sclR) were all down regulated. Consistent with the decreased gene expression levels, aflatoxin concentrations in cultural medium were reduced to barely detectable. Furthermore, fungal biomass and conidial number were significantly reduced by 60% and more than 95%, respectively. The results validate the biocontrol efficacy of W. anomalus WRL-076 observed in the field experiments.

Biocontrol strain Aspergillus flavus WRRL 1519 has differences in chromosomal organization and an increased number of transposon-like elements compared to other strains.

Aflatoxins are toxic secondary metabolites produced by members of the genus Aspergillus, most notably A. flavus. Non-aflatoxigenic strains of A. flavus are commonly used for biocontrol of the aflatoxigenic strains to reduce aflatoxins in corn, cotton, peanuts and tree nuts. However, genomic differences between aflatoxigenic strains and non-aflatoxigenic strains have not been reported in detail, though such differences may further elucidate the evolutionary histories of certain biocontrol strains and help guide development of other useful strains. We recently reported the genome and transcriptome sequencing of A. flavus WRRL 1519, a strain isolated from almond that does not produce aflatoxins or cyclopiazonic acid due to deletions in the biosynthetic gene clusters. Continued bioinformatics analyses focused on comparing strain WRRL 1519 to the aflatoxigenic strain NRRL 3357. The genome assembly of strain WRRL 1519 was improved by anchoring 84 of the 127 scaffolds to the putative nuclear chromosomes of strain NRRL 3357. The five largest areas of extrachromosomal mismatches observed between WRRL 1519 and NRRL 3357 were not similar to any of the mismatches that were observed with pairwise comparisons of NRRL 3357 to other non-aflatoxigenic strains NRRL 21882, NRRL 30797 or NRRL 18543. Comparisons of predicted secondary metabolite gene clusters uncovered two other biosynthetic gene clusters in which strain WRRL 1519 had large deletions compared to the homologous clusters in NRRL 3357. Additionally, there was a marked overrepresentation of repetitive sequences in WRRL 1519 compared to other inspected A. flavus strains. This is the first report of detection of a large number of putative retrotransposons in any A. flavus strain, initially suggesting that retrotransposons may contribute to the natural occurrence of genetic variation and biocontrol strains. However, the transposons may not be significantly associated with the chromosomal differences. Future experimentation and continued bioinformatics analyses will potentially illuminate causes of the differences and may reveal whether transposon activity in A. flavus can lead to random natural occurrences of non-aflatoxigenic strains.

Genome sequence and comparative analyses of atoxigenic Aspergillus flavus WRRL 1519.

Aflatoxins are toxic secondary metabolites produced by Aspergillus flavus and a few other closely related species of Aspergillus. These highly toxigenic and carcinogenic mycotoxins contaminate global food and feed supplies, posing widespread health risks to humans and domestic animals. Field application of nonaflatoxigenic strains of A. flavus to compete against aflatoxigenic strains has emerged as one of the best management practices for reducing aflatoxins contamination, yielding successful commercial products for corn, cotton seed, and peanuts. In this study, we sequenced the genome and transcriptome of atoxigenic (does not produce aflatoxin or cyclopiazonic acid) A. flavus strain WRRL 1519 isolated from a tree nut orchard to define the genetic characteristics of the strain in relation to aflatoxigenic and other nonaflatoxigenic A. flavus strains. WRRL 1519 strain was similar to other strains in size (38.0 Mb), GC content (47.2%), number of predicted secondary metabolite gene clusters (46), and number of putative proteins (12 121). About 87.4% of the predicted proteome had high shared identity with protein sequences derived from other A. flavus genomes. However, the atoxigenic A. flavus strain WRRL 1519 had deletions, or low shared identity, for many genes in the clusters required for aflatoxins and cyclopiazonic acid (CPA) synthesis. Over half of the aflatoxin synthesis gene cluster was missing, and none of the components of the CPA gene cluster were identified with high sequence similarity. Importantly, the strain appeared to maintain functional sequences of several genes thought to be required for high infectivity. Since the ability to grow on target crop is an important attribute for a successful biocontrol agent, these results indicate that the nonaflatoxigenic A. flavus strain WRRL 1519 would be a good candidate as a biocontrol agent for reducing aflatoxin and CPA accumulation in high-value nut crops.

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.

First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae.

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using ß-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B1, B2, G1, and G2 and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153-1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.

Genome Sequencing and Analysis of the Postharvest Fungus Penicillium expansum R21.

Blue mold is the vernacular name of a common postharvest disease of stored apples, pears, and quince that is caused by several common species of Penicillium This study reports the draft genome sequence of Penicillium expansum strain R21, which was isolated from a red delicious apple in 2011 in Pennsylvania.

Characterization of Blue Mold Penicillium Species Isolated from Stored Fruits Using Multiple Highly Conserved Loci.

Penicillium is a large genus of common molds with over 400 described species; however, identification of individual species is difficult, including for those species that cause postharvest rots. In this study, blue rot fungi from stored apples and pears were isolated from a variety of hosts, locations, and years. Based on morphological and cultural characteristics and partial amplification of the ß-tubulin locus, the isolates were provisionally identified as several different species of Penicillium. These isolates were investigated further using a suite of molecular DNA markers and compared to sequences of the ex-type for cognate species in GenBank, and were identified as P. expansum (3 isolates), P. solitum (3 isolates), P. carneum (1 isolate), and P. paneum (1 isolate). Three of the markers we used (ITS, internal transcribed spacer rDNA sequence; benA, ß-tubulin; CaM, calmodulin) were suitable for distinguishing most of our isolates from one another at the species level. In contrast, we were unable to amplify RPB2 sequences from four of the isolates. Comparison of our sequences with cognate sequences in GenBank from isolates with the same species names did not always give coherent data, reinforcing earlier studies that have shown large intraspecific variability in many Penicillium species, as well as possible errors in some sequence data deposited in GenBank.

Genome Sequencing and Analysis of the Filamentous Fungus Penicillium sclerotiorum 113, Isolated after Hurricane Sandy.

Penicillium sclerotiorum is a distinctive species within the genus Penicillium that usually produces vivid orange to red colonies, sometimes with colorful sclerotia. Here, we report the first draft genome sequence of P. sclerotiorum strain 113, isolated in 2013 in the aftermath of Hurricane Sandy from a flooded home in New Jersey.

Draft Genome Sequence of the Fungus Penicillium solitum NJ1.

Penicillium solitum is one of the most prevalent species causing postharvest decay of pomaceous fruits during storage. Here, we report the draft genome of P. solitum strain NJ1, received as a transfer of a strain originally identified as P. griseofulvum by classical means.

Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids.

The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 µL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids OPEN ACCESS Toxins 2015, 7 3888 were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in branched-chain amino acid degradation. Since secondary metabolism occurs after active growth has ceased, this growth stimulation resulted in suppression of expression of aflatoxin biosynthesis genes. On the other hand, increased activities in degradation pathways for branched-chain amino acids probably are required for the activation of the aflatoxin pathway by providing building blocks and energy regeneration. Metabolic flux in primary metabolism apparently has an important role in the expression of genes of secondary metabolism.

Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae.

Aspergillus oryzae and Aspergillus flavus are closely related fungal species. The A. flavus morphotype that produces numerous small sclerotia (S strain) and aflatoxin has a unique 1.5 kb deletion in the norB-cypA region of the aflatoxin gene cluster (i.e. the S genotype). Phylogenetic studies have indicated that an isolate of the nonaflatoxigenic A. flavus with the S genotype is the ancestor of A. oryzae. Genome sequence comparison between A. flavus NRRL3357, which produces large sclerotia (L strain), and S-strain A. flavus 70S identified a region (samA-rosA) that was highly variable in the two morphotypes. A third type of samA-rosA region was found in A. oryzae RIB40. The three samA-rosA types were later revealed to be commonly present in A. flavus L-strain populations. Of the 182 L-strain A. flavus field isolates examined, 46%, 15% and 39% had the samA-rosA type of NRRL3357, 70S and RIB40, respectively. The three types also were found in 18 S-strain A. flavus isolates with different proportions. For A. oryzae, however, the majority (80%) of the 16 strains examined had the RIB40 type and none had the NRRL3357 type. The results suggested that A. oryzae strains in the current culture collections were mostly derived from the samA-rosA/RIB40 lineage of the nonaflatoxigenic A. flavus with the S genotype.

The major volatile compound 2-phenylethanol from the biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus.

Aspergillus flavus is a ubiquitous saprophyte that is able to produce the most potent natural carcinogenic compound known as aflatoxin B1 (AFB1). This toxin frequently contaminates crops including corn, cotton, peanuts, and tree nuts causing substantial economic loss worldwide. Consequently, more than 100 countries have strict regulations limiting AFB1 in foodstuffs and feedstuffs. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. Pichia anomala strain WRL-076 is a biocontrol yeast currently being tested to reduce AF contamination of tree nuts in California. We used the SPME-GC/MS analysis and identified the major volatile compound produced by this strain to be 2-phenylethanol (2-PE). It inhibited spore germination and AF production of A. flavus. Inhibition of AF formation by 2-PE was correlated with significant down regulation of clustering AF biosynthesis genes as evidenced by several to greater than 10,000-fold decrease in gene expression. In a time-course analysis we found that 2-PE also altered the expression patterns of chromatin modifying genes, MYST1, MYST2, MYST3, gcn5, hdaA and rpdA. The biocontrol capacity of P. anomala can be attributed to the production of 2-PE, which affects spore germination, growth, toxin production, and gene expression in A. flavus.

Characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates from pistachio.

Pistachio is a popular snack food. Aflatoxin contamination of pistachio nuts is a serious problem for many producing countries. The development of biological control methods based on ecological parameters is an environmentally friendly approach. Thirty-eight Aspergillus flavus isolates collected from a pistachio orchard in California (CA) were analyzed for production of aflatoxin (AF), cyclopiazonic acid (CPA), vegetative compatibility groups (VCGs), and mating types. All aflatoxigenic isolates produced both AFB1 and CPA. The most toxigenic one was CA28 which produced 164 µg AFB1 per 5 ml PDA fungal culture and small sclerotia (S strain, sclertoium size less than 400 µm). The other aflatoxigenic strains produce AFB1 ranging from 1.2 µg to 80 µg per 5 ml fungal culture. Twenty-one percent of the CA isolates produced AFB1, 84% produced CPA and half formed sclerotia on at least one of three tested media. The 38 CA isolates formed 26 VCGs, 6 of which had two or more isolates and 20 contained single isolates. The S strain isolates belong to 4 different VCGs. Genomic profiling by a retrotransposon DNA probe revealed fingerprint patterns that were highly polymorphic. The predicted VCGs (Pred-VCGs) based on a similarity coefficient >80% matched the VCGs of multiple isolates determined by complementation. All isolates within a VCG had the same mating-type gene of either MAT1-1 or MAT1-2. Uncorrected and VCG-corrected MAT1-1 and MAT1-2 among the isolates were equally distributed.