aflatoxins ( afs ) are carcinogenic , mutagenic , and teratogenic metabolites mainly produced by aspergillus flavus and the very closely related species a. parasiticus ( 1 ) . these mycotoxins can grow on a wide range of food commodities , including cereals , peanuts , and crops in the eld , but also during postharvest operations and storage ( 2 ) . the group of afs includes b1 , b2 , g1 , and g2 , while afbs include afb1 and afb2 , and afgs include afg1 and afg2 ( 3 ) . afs are potent hepatotoxic , teratogenic , and mutagenic metabolites that are now well - recognized as a public health hazard ( 1 , 4 ) . although some synthetic fungicides have been improved to protect crops against af production , there are some proven indications that these are themselves hazardous to human health ( 5 ) . therefore , using natural sources is an affirmative approach to protecting from fungal infections in agriculture ( 6 ) . a medicinal herb , pistacia atlantica subsp . kurdica , has been traditionally used as a therapeutic agent for infections ( 7 - 9 ) . the genus pistacia belongs to the family anacardiaceae , and among 15 known species of pistachios , only three grow in iran : p. vera , p. khinjuk , and p. atlantica ( 10 , 11 ) . p. atlantica has three sub - species : mutica , kurdica , and cabulica ( 11 ) . this plant is an evergreen bush that grows native in some southern and central american and eastern mediterranean countries ( 10 ) . kurdica is obtained as an exudate after crushing the trunk and branches , and has numerous qualities . the gum of this plant is used in medicine , with many researchers reporting that it possesses considerable antimicrobial activity ( 12 , 13 ) . kurdica , as a natural compound , with regard to antifungal activity and inhibition of af production in a. parasiticus infections . kurdica was obtained from saghez sazi ( van ) ( kurdistan , iran ) in the summer of 2014 . the gum was obtained as an exudate of the trunk and branches . the a. parasiticus strain ( american type culture collection 15517 ) was incubated at 28c for 72 hours on sabouraud dextrose agar ( germany , merck ) . in vitro antifungal susceptibility testing for the determination of minimum inhibitory concentration ( mic ) was performed according to recommendations provided in the clinical and laboratory standards institute ( clsi ) m38-a2 document , with some modification in gum concentration ( 14 ) . rpmi 1640 medium ( sigma chemical co. ) was buffered to ph 7.0 with mops ( sigma ) , with l - glutamine and without bicarbonate . kurdica was dissolved in water to reach a concentration of 500 mg / ml . it was then diluted to achieve concentrations of 500 to 15.6 mg / ml ( 500 , 250 , 125 , 62.5 , 31.2 , and 15.6 mg / ml ) . the concentration of fungal spores was calculated using the hemacytometry method , and finally adjusted to 2x concentrated suspensions in such a way that each test well contained 5  10 colony - forming units ( cfus)/ml . negative ( only rpmi 1640 medium ) and positive controls were also run alongside each experiment . the mic endpoints were determined as the lowest concentrations that prevented any recognizable growth , mostly based on the antifungal capacity of the gum ( 100% inhibition ) . the amount of afs produced by a. parasiticus alone and in combination with different gum concentrations ( 62.5 mg / ml and 125 mg / ml ) was determined using the high - performance liquid chromatography ( hplc ) technique ( waters 474 scanning fluorescence detector ) ( 6 ) . pdb plates containing 62.5 mg / ml or 125 mg / ml of gum were inoculated with 5  10 fungal spores . then , 25 g of each fungal biomass with 2.5 g of sodium chloride was added into 100 ml of hplc - grade methanol and blended for 3 minutes at 18,000 rpm . cell - free filtrates were obtained using whatman filter paper no.4 and subjected to analysis by hplc . quantitative changes in expression of the aflr gene were analyzed by use of a quantitative real - time pcr assay . a. parasiticus was cultured in the presence of 62.5 mg / ml or 125 mg / ml of gum of p. atlantica var . equal amounts of rna ( 1 g in 20 l ) were subjected to cdna synthesis by using random hexamer primers , according to the manual protocol ( fermentase ) . the primers fafl ( 5'-cggaacagggacttccggcg-3 ' ) and rafl ( 5'-gggtggcgggggactctgat-3 ' ) were designed on the basis of the published sequence of the aflr gene of a. parasiticus ( ncbi , accession no : af441438 ) . the b - actin gene ( act1 ) with fact ( 5'-acggtattgtttccaactgggacg-3 ' ) and ract ( 5'- tggagcttcggtcaacaaaactgg-3 ' ) primers was used as an endogenous reference gene . quantitative real - time pcr was carried out using the sybr green master mix ( applied biosystems , foster city , ca , usa ) performed with a step one plus real - time pcr system ( applied biosystems , foster city , ca , usa ) . the aflr gene expression was analyzed with rest software ( 2008 v2.0.7 , corbett research , sydney , australia ) . kurdica was obtained from saghez sazi ( van ) ( kurdistan , iran ) in the summer of 2014 . the a. parasiticus strain ( american type culture collection 15517 ) was incubated at 28c for 72 hours on sabouraud dextrose agar ( germany , merck ) . in vitro antifungal susceptibility testing for the determination of minimum inhibitory concentration ( mic ) was performed according to recommendations provided in the clinical and laboratory standards institute ( clsi ) m38-a2 document , with some modification in gum concentration ( 14 ) . rpmi 1640 medium ( sigma chemical co. ) was buffered to ph 7.0 with mops ( sigma ) , with l - glutamine and without bicarbonate . kurdica was dissolved in water to reach a concentration of 500 mg / ml . it was then diluted to achieve concentrations of 500 to 15.6 mg / ml ( 500 , 250 , 125 , 62.5 , 31.2 , and 15.6 mg / ml ) . the concentration of fungal spores was calculated using the hemacytometry method , and finally adjusted to 2x concentrated suspensions in such a way that each test well contained 5  10 colony - forming units ( cfus)/ml . negative ( only rpmi 1640 medium ) and positive controls were also run alongside each experiment . the mic endpoints were determined as the lowest concentrations that prevented any recognizable growth , mostly based on the antifungal capacity of the gum ( 100% inhibition ) . the amount of afs produced by a. parasiticus alone and in combination with different gum concentrations ( 62.5 mg / ml and 125 mg / ml ) was determined using the high - performance liquid chromatography ( hplc ) technique ( waters 474 scanning fluorescence detector ) ( 6 ) . pdb plates containing 62.5 mg / ml or 125 mg / ml of gum were inoculated with 5  10 fungal spores . then , 25 g of each fungal biomass with 2.5 g of sodium chloride was added into 100 ml of hplc - grade methanol and blended for 3 minutes at 18,000 rpm . cell - free filtrates were obtained using whatman filter paper no.4 and subjected to analysis by hplc . quantitative changes in expression of the aflr gene were analyzed by use of a quantitative real - time pcr assay . a. parasiticus was cultured in the presence of 62.5 mg / ml or 125 mg / ml of gum of p. atlantica var . equal amounts of rna ( 1 g in 20 l ) were subjected to cdna synthesis by using random hexamer primers , according to the manual protocol ( fermentase ) . the primers fafl ( 5'-cggaacagggacttccggcg-3 ' ) and rafl ( 5'-gggtggcgggggactctgat-3 ' ) were designed on the basis of the published sequence of the aflr gene of a. parasiticus ( ncbi , accession no : af441438 ) . the b - actin gene ( act1 ) with fact ( 5'-acggtattgtttccaactgggacg-3 ' ) and ract ( 5'- tggagcttcggtcaacaaaactgg-3 ' ) primers was used as an endogenous reference gene . quantitative real - time pcr was carried out using the sybr green master mix ( applied biosystems , foster city , ca , usa ) performed with a step one plus real - time pcr system ( applied biosystems , foster city , ca , usa ) . the aflr gene expression was analyzed with rest software ( 2008 v2.0.7 , corbett research , sydney , australia ) . kurdica inhibited a. parasiticus growth at mic values of 125 mg / ml . according to hplc analysis results , afl - b2 and afl - g2 production was significantly decreased and even entirely inhibited in 125 mg / ml of gum ( p < 0.01 ) . however , the production of afl - b1 was not fully inhibited at this concentration . based on quantitative real - time pcr results , the rate of aflr gene expression was significantly decreased after treating the a. parasiticus with 125 mg / ml of p. atlantica subsp . the results of an analysis of the relative quantification of the aflr gene after treatment with the gum of p. atlantica subsp . aflatoxins are extremely toxic and carcinogenic metabolites that widely contaminate agricultural commodities and animal feed ( 15 ) . in recent years , research has been ongoing into the development of new anti - fungal agents or through the search for natural sources to control growth and toxin production of aspergillus species ( 8 , 9 , 16 ) . in several studies , ghalem et al . reported that the gum of p. atlantica has antimicrobial activity against gram - positive and -negative bacteria that are resistant to commonly used antimicrobial agents , and this was considerably dependent on concentration ( 17 ) . hesami et al . suggested the potential substitution of antifungal chemicals by p. atlantica subsp . kurdica as a natural inhibitor to control the growth of botrytis cinereain ( 19 ) . aflatoxin inhibition mainly occurs during the primary stages of cell growth , when the genes involved in aflatoxin biosynthesis are at maximum activity within the fungal cells ( 7 ) . our study revealed that the rate of aflr gene expression was significantly decreased after treating the fungus with 125 mg / ml of p. atlantica subsp . showed that licorice extract could efficiently inhibit aflr gene expression , and consequently , af production , in a. parasiticus ( 6 ) . in another study , . showed potent inhibition of a. parasiticus growth and af production with 1 mg / ml of ephedra major ( 20 ) . kurdica could efficiently inhibit aflr gene expression and the consequent af production in a. parasiticus . this gum may therefore be considered a potential anti - mycotoxic agent in medicine or industrial agriculture . further studies are recommended to determine the effective components of the gum of p. atlantica subsp . kurdica and to consider it as a potential candidate for controlling af contamination of crops in the field .