Determination of population density of Aspergillus flavus and Aspergillus parasiticus in the last irrigation treatments before harvest in soil and fruits of pistachio trees

Contamination of pistachio with Aspergillus and aflatoxin is one of the most important problems in the production and export of this valuable product. In this research, in addition to molecular identification of Aspergillus flavus and A. parasiticus and determination of the frequency of their toxigenic and atoxigenic isolates, the population of Aspergillus flavus clade was measured in soil, intact and cracked fruits of cv. Ohadi in 6 treatments of last irrigation before harvest (5, 10, 15, 20, 25 and 30 days).

Isolation of Aspergillus flavus clade fungi from soil, intact and cracked fruits was done by serial dilution method on DRBC culture medium. Aspergillus flavus and A. parasiticus were identified by macromorphological and molecular (calmodulin primer) features and screening of toxigenic and atoxigenic isolates was performed using of coconut-agar medium (CAM) and thin layer chromatography (TLC). This experiment was carried out in a complete randomized block design with four replications during the years 2012 to 2014.

Out of 233 collected isolates of Aspergillus flavus clade, 221 and 12 isolates belonged to Aspergillus flavus and A. parasiticus, respectively. Frequency of toxigenic isolates ofA. flavus and A. parasiticus was 86.5, 87.5, 88.7% and 80, 75, 100% in cracked, intact fruits and soil, respectively. Based on the results of the combine analysis, the last irrigation treatments 30 and 25 days before harvest have the lowest and the last irrigation treatments 10 and 5 days before harvest have the highest population of Aspergillus flavus clade in the soil, intact and cracked fruits which showed significant difference with each other. Using of manure significantly increased the population of Aspergillus flavus clade, so that this increase in cracked, healthy fruits and soil in 2013 was 66, 83 and 114%, respectively.

The results of the present research showed that reduction of the interval between the last irrigation and the time of fruit harvesting (high moisture percentage of the surface soil of the orchard) and the using manure can increase the population of Aspergillus flavus clade in the soil, intact and cracked pistachio fruits. Increasing of the fungal population is accompanied by the frequency of toxigenic isolates of Aspergillus flavus and A. parasiticus. Adjusting the irrigation cycle, so that the last irrigation is 25 to 30 days before the fruit harvesting, can reduce the possibility of contamination of the pistachio kernels to Aspergillus and aflatoxin.