Pistachio Male Inflorescences as an Alternative Substrate for the Application of Atoxigenic Strains of Aspergillus flavus.

Aflatoxins are carcinogens mainly produced by Aspergillus flavus and A. parasiticus in susceptible crops, including pistachio. The primary inoculum sources of these pathogens are plant debris in the orchard soils. In Californian fields, one approach to controlling aflatoxin contamination is based on releasing the atoxigenic strain of A. flavus AF36 in inoculated (coated) sorghum grains (AF36 Prevail). However, this control method can fail due to poor sporulation of the AF36 strain or sorghum grain losses due to predation. In 2008 and 2018, we showed that toxigenic and atoxigenic isolates of Aspergillus spp. frequently colonized fallen inflorescences of male pistachio trees. Under controlled conditions, strain AF36 profusely colonized pistachio male inflorescences when humidity was higher than 90%. However, there were significant differences between types of inflorescence (aerial > fallen). In 2016, we considerably (P = 0.015) increased the population of AF36 on the canopies of trees when fallen inflorescences were inoculated with AF36, compared with untreated trees. In 2017 and 2018, these differences were not detected (P > 0.05) due to cross-contamination of strain AF36 between seasons and neighboring plots. In any case, the density of AF36 spores on the canopy of the inflorescence-treated trees was similar (P > 0.05) to that on trees treated with the commercial product. Here, we present a new method for applying strain AF36 based on using a natural, abundant, and uniformly distributed substrate in pistachio fields, and we discuss how it can be improved. Furthermore, our results indicate that, in pistachio orchards where biocontrol practices are not conducted, eliminating this important source of toxigenic Aspergillus inoculum is recommended.

Distribution and incidence of atoxigenic Aspergillus flavus VCG in tree crop orchards in California: A strategy for identifying potential antagonists, the example of almonds.

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.

Community Structure of Aspergillus flavus and A. parasiticus in Major Almond-Producing Areas of California, United States.

Several nut crops, including almond, pistachio, and walnut, can become contaminated with mycotoxins. Of greatest economic significance are aflatoxins, which are mainly produced by members of Aspergillus section Flavi. The distribution of the two sclerotial-size morphotypes of Aspergillus flavus (i.e., S and L strains) and A. parasiticus, the main species responsible for aflatoxin production among section Flavi, was monitored in the soil of almond orchards in California over a 5-year period from 2007 to 2011, excluding 2009. In total, 4,349 Aspergillus isolates were collected from 28 almond orchards located in the northern, central, and southern Central Valley in California. Overall, A. flavus L strain was the most frequent, followed by A. parasiticus and A. flavus S strain. However, variations in the spatial distribution of these three taxa were found between the three regions. Over the 5-year period, higher frequencies of L strain were more often observed in the southern region (79.9 to 95.1%, depending on year) compared with the northern region (21.4 to 47.1%). In the north, A. parasiticus was the most common strain, with frequencies of 28.5 to 61% for the various years. In addition, the frequency of aflatoxin-producing isolates among L strains fluctuated from year to year. A significant increase (P = 0.0001) was observed from 2008 (75% of the isolates produced aflatoxins) to 2007 (59%), and a decrease was observed from 2010 (61%) to 2011 (53%). Aflatoxin-producing L strain isolates were significantly more prevalent than atoxigenic isolates in each region during the 5-year survey, except in 2011 in the north, where more isolates were atoxigenic (56%) than aflatoxin-producing (44%). Our results indicate that the structure of A. flavus and A. parasiticus communities in the soil and the proportion of toxigenic isolates vary across regions and years. Such knowledge may help devise appropriate aflatoxin control strategies, including the use of atoxigenic isolates as biological control agents adapted to the soil environments in each region.

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.

Description of a distinctive aflatoxin-producing strain of Aspergillus nomius that produces submerged sclerotia.

A new distinctive strain of Aspergillus nomius that produces the potent mycotoxins, aflatoxins, is described from pistachio, pecan, and fig orchards in California. Similar to the typical strain of A. nomius (as represented by the ex-type), the O strain produced both B and G aflatoxins but not cyclopiazonic acid, had similar conidial ornamentation, and grew poorly at 42 degrees C. Furthermore, previous published DNA sequence supports that the new strain is very closely related to the ex-type of A. nomius. However, the O strain differs from the ex-type in several morphological characters. The ex-type was initially described as producing "indeterminate sclerotia" that appear as large (up to 3 mm long) elongated sclerotia on surfaces of media. The O strain produces only small spherical sclerotia (mean diameter <0.3 mm) submerged in the medium. In addition, the O strain has predominantly uniseriate conidial heads, whereas the typical strain of A. nomius has predominantly biseriate heads. The O strain colony color on both Czapek solution agar and Czapek yeast extract agar was more yellowish than the ex-type of A. nomius and other common aflatoxin-producing fungi. Isolates of the O strain reported here from several orchards represent the first report of A. nomius in California.

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.

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus.

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 degrees 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 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/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.

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.

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.