Mark A. Doster

Ochratoxin Production by the Aspergillus ochraceus Group and Aspergillus alliaceus

ABSTRACT Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30°C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 μg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 μg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.

Aspergillus species and mycotoxins in figs from California orchards.

Although 23 different Aspergillus spp. decayed figs in California orchards, only A. niger occurred in more than 0.2% of the figs. The black-spored Aspergillus isolates that caused the disease fig smut were classified as A. niger var. niger, A. niger var. awamori, A. japonicus, and A. carbonarius. Different fungi differed in their association with Aspergillus Section Nigri (causal agents of fig smut) infections in figs. For example, most figs infected with Aspergillus Section Flavi (potential aflatoxin producers) also had infections by Section Nigri. For other fungi, there was either no significant relationship between fig infections by these fungi and Section Nigri or simultaneous infections by Section Nigri were fewer than expected. Insect damage to the fig fruit, predominantly by navel orangeworm (Amyelois transitella), did not significantly increase the colonization of figs by Aspergillus spp. The incidences of infection by Aspergillus (Sections Nigri, Aspergillus, Flavi, and Circumdati) in figs differed little for different harvests. Figs naturally infected with A. alliaceus, A. melleus, A. ochraceus, and A. sclerotiorum of Aspergillus Section Circumdati contained ochratoxin up to 9,600 ng/g, although only 40% of the figs with these fungi had more than a trace amount of ochratoxin. Aflatoxin contamination in figs naturally infected with Aspergillus Section Flavi varied according to the species involved. No aflatoxins were detected in all figs infected with A. tamarii and in most figs infected with A. flavus. High levels of aflatoxin (>100 ng/g) were detected in 83% of the figs infected by A. parasiticus, but in only 32% of the figs infected by A. flavus. Section Flavi isolates from fig orchard soils were tested for their ability to produce the mycotoxins aflatoxin and cyclopiazonic acid. Aspergillus parasiticus isolates always produced aflatoxin but never cyclopiazonic acid; A. flavus strain S (producers of small sclerotia) isolates always produced both aflatoxin and cyclopiazonic acid, but strain L (producers of large sclerotia) isolates frequently did not produce aflatoxin or cyclopiazonic acid; and A. tamarii isolates never produced aflatoxin but always produced cyclopiazonic acid. Aspergillus flavus was recovered from the soil, at fewer than 6 CFU/g of dry soil of every fig orchard assayed in 1992 and 1993. Although A. parasiticus was rarer in fig fruit than was A. flavus for each year, in orchard soil A. parasiticus was more frequent than A. flavus. Isolates of A. flavus strain L were much more common in the orchard soil and fig fruit than those of strain S. Figs in commercial orchards seem to be a favorable substrate for infection by and growth of Aspergillus spp.

Relationship between shell discoloration of pistachio nuts and incidence of fungal decay and insect infestation.

Shell discoloration of pistachio (Pistacia vera) nuts collected from commercial orchards and processing plants was related to fungal decay and insect infestation of the kernel. Nuts with ruptured hulls (early split nuts and nuts with cracked hulls) varied considerably in the amount of shell discoloration, ranging from none to extensive. For both types of hull rupture, as shell discoloration increased, kernel decay also increased. Nuts with no discoloration had little or no fungal decay and navel orangeworm (Amyelois transitella) infestation. Processed nuts with an oily-shell appearance had the highest incidences of fungal decay and navel orangeworm infestation; nuts with a crinkled shell, nuts with extensive dark brown discoloration, and nuts with moderate dark brown discoloration along the suture had relatively high levels of decayed and infested kernels; while nuts with yellow discoloration, nuts with moderate dark brown discoloration not along the suture, and nuts with no discoloration had little or no decay and infestation. Also, as shell discoloration of processed nuts increased, kernel decay increased. Our results suggest that shell characteristics may be used by processors to identify poor quality nuts and to improve the quality of pistachio nuts sold to consumers.

A MORPHOLOGICALLY DISTINCT STRAIN OF ASPERGILLUS NOMIUS

DNA polymorphisms were used to infer relationships of a morphologically distinct new strain of aflatoxin-producing Aspergillus to other members of Aspergillus sect. Flavi. The new strain produced b...

DEVELOPMENT OF ASPERGILLUS MOLDS IN LITTER FROM PISTACHIO TREES

Aspergillus molds frequently infested, infected, and sporulated on pistachio litter such as fallen pistachio fruit and male inflorescences throughout summer in commercial pistachio orchards in California. A. niger was isolated much more frequently from pistachio litter than any other species, but the aflatoxin producers, A. flavus and A. parasiticus, also developed in pistachio litter. Two distinct strains of A. flavus were isolated: strain L (a few large sclerotia) and strain S (abundant small sclerotia). Strain L occurred substantially more often than strain S. All isolates of strain S and A. parasiticus were aflatoxin producers, but only 43% of the isolates of strain L produced aflatoxins

Production of Bright Greenish Yellow Fluorescence in Figs Infected by Aspergillus Species in California Orchards

The relationship of bright greenish yellow fluorescence (BGYF) of dried figs under longwave UV light to colonization by Aspergillus fungi was determined. BGYF in naturally infected figs was associated with decay by only four fungal species: the aflatoxin-producing species Aspergillus flavus (both L and S strains) and A. parasiticus, and the aflatoxin nonproducers A. tamarii and A. alliaceus. BGYF was more likely to be visible internally (after cutting open the fig) than externally. For all four species associated with BGYF, some infected figs did not show BGYF. The absence of fluorescence is probably not associated with the fungal strain or isolate involved, since isolating Aspergillus spp. from nonfluorescent figs followed by inoculating other figs with these isolates resulted in BGYF. Many of the nonfluorescent figs had small fungal colonies (<7 mm in diameter), even though some figs with large colonies were also nonfluorescent. The additional colonization of figs by other fungi did not affect the occurrence of BGYF in figs colonized by fungi in Aspergillus section Flavi. Figs infected with A. flavus or A. parasiticus and showing no BGYF were occasionally contaminated with aflatoxin, while other figs showing BGYF and infected with A. flavus or A. tamarii had no aflatoxins. Although not as promising as originally hoped, BGYF might be of use to remove aflatoxin-contaminated figs for certain specific situations in California.

Quantification of conidial density of Aspergillus flavus and A. parasiticus in soil from almond orchards using real-time PCR.

Aims:  To design the Aspergillus flavus and Aspergillus parasiticus‐specific primers and a real‐time PCR assay for quantification of the conidial density in soil.

Automated detection of pistachio defects by machine vision

Pistachio (Pistacia vera) nuts with shell and kernel defects detract from consumer acceptance and, in some cases, may be more prone to insect damage, mold decay, and/or aflatoxin contamination. The objective of this study was to develop imaging algorithms to improve sorting of nuts with the following shell defects: oily stains, dark stains, adhering hull, and the following kernel defects: navel orangeworm (NOW) damage, fungal decay, and Aspergillus molds, all of which indicate risk of aflatoxin contamination. Imaging algorithms were developed to distinguish normal nuts from those nuts with oily stains, dark stains, and adhering hull as well as nuts having kernel defects. Image algorithm testing on a validation data set showed that nuts having oily stain, dark stain, or adhering hull could be distinguished from normal nuts with an accuracy of 98%. Removing nuts with oily stain, dark stain, and adhering hull will also remove 89.7% of nuts with kernel decay, 93.8% of nuts with Aspergillus molds present, and 98.7% of NOW positive nuts.

Evaluation of the Atoxigenic Aspergillus flavus Strain AF36 in Pistachio Orchards

The atoxigenic strain Aspergillus flavus AF36, which has been extensively used as a biocontrol agent in commercial corn and cotton fields to reduce aflatoxin contamination, was applied in research pistachio orchards from 2002 to 2005 and in commercial pistachio orchards from 2008 to 2011. AF36 was applied as hyphae-colonized steam-sterilized wheat seed (the same product and same application rate as used in cotton fields). In all orchards, applying the wheat-AF36 product substantially increased the proportion of vegetative compatibility group (VCG) YV36, the VCG to which AF36 belongs, within A. flavus soil communities. Application of the AF36 product in additional years further increased YV36 in the soil until it composed 93% of the A. flavus isolates in treated commercial orchards. Nonetheless, application of the AF36 product did not result in increased incidence of kernel decay of the nuts. For nuts harvested from commercial orchards, reductions in percentages of samples contaminated with aflatoxin from treated orchards (relative to that for untreated orchards) ranged from 20 to 45%, depending on the year. Because of the high value of pistachio nuts and the costs associated with rejection of shipments due to aflatoxin contamination, these reductions are significant and valuable to the pistachio industry.

Fungal Decay of First-Crop and Main-Crop Figs

Fig cultivars grown in California typically have two crops, although the first crop may be unimportant commercially. The first crop, also known as the breba crop, ripens in late spring and early summer, whereas the main or second crop ripens in late summer. For both cultivars studied, Conadria and Calimyrna, the first-crop figs typically are left in the orchard unharvested. First-crop figs had relatively high levels of fungal decay and tended to have more fungal decay than main-crop figs, especially Alternaria rot (caused by Alternaria alternata and Ulocladium atrum). At least 16 different Aspergillus spp. were found decaying first-crop figs. Fig smut, a serious disease caused by Aspergillus niger and related fungi, usually was present at approximately the same level in first-crop and main-crop figs. Aspergillus spp. known to produce the mycotoxins aflatoxin or ochratoxin were found decaying first-crop figs. Aflatoxin was detected in first-crop figs at low levels similar to those detected in the main-crop figs. Because the abundant spores produced on the first-crop figs can infect main-crop figs, the fungal decay of first-crop figs might result in higher levels of decay for main-crop figs.