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

Document Type : Research Paper


1 Assistant Professor, Pistachio Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Rafsanjan, Iran.

2 Assistant Professor, Horticultural and Crop Sciences Research Department, Agricultural and Natural Resources Research and Education Center of Semnan Province (Shahrood), AREEO, Shahrood, Iran.


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).
Materials and methods
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 of
A. 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.


احمدی کریم، عبادزاده حمیدرضا، حاتمی فرشاد و همکاران (1400) آمارنامه کشاورزی، جلد سوم: محصولات کشاورزی. وزارت جهاد کشاورزی، معاونت برنامه­ریزی و اقتصادی، مرکز فناوری اطلاعات و ارتباطات. 157 صفحه.
اسماعیل­پور علی، امامی سیدیحیی، بصیرت مهدی و همکاران (1399) پسته ایران. انتشارات آموزش و ترویج کشاورزی. 424 صفحه.
حسینی­فرد سیدجواد، بصیرت مجید، صداقتی ناصر، اخیانی احمد (1396) دستورالعمل مدیریت تلفیقی حاصلخیزی خاک و تغذیه درختان پسته. انتشارات موسسه تحقیقات خاک و آب. 100 صفحه.
رحیمی پریسا، شریف­نبی بهرام، بهار مسعود (1386) گونه­های آسپرژیلوس جداشده از میوه­های پسته و بررسی تولید آفلاتوکسین در آنها. رستنیها، 8 (1)، 30-42.
صداقتی ناصر، محمدی محمدآبادی اکبر، حسینی­فرد سیدجواد (1387) بررسی اثر رژیم­های مختلف آبیاری بر روی زودخندانی پسته رقم اوحدی. پژوهش و سازندگی، 87، 14-158.
علیزاده امین (1390) رابطه آب و خاک و گیاه. انتشارات آستان قدس رضوی. 484 صفحه.
فانی سیدرضا، مرادی محمد، زمانی زاده حمیدرضا و همکاران (1392) پراکنش سویه­های غیرتوکسین­زای قارچ Aspergillus flavus در مناطق پسته­کاری ایران. آفات و بیماری­های گیاهی، 81 (2)، 179-190.
مرادی محمد، ارشاد جعفر، میرابوالفتحی منصوره، پناهی بهمن (1383) نقش بقایای گیاهی، خاک و کودهای حیوانی روی تراکم جمعیت قارچ­های گروه Aspergillus flavus و Aspergillus niger در باغ­های پسته استان کرمان. بیماری­های گیاهی، 40، 221-234.
مرادی محمد، تاج­آبادی پور علی، فانی سیدرضا (1395) زمان تشکیل و خصوصیات فیزیکی پسته­های زودخندان به عنوان منابع آلودگی به آفلاتوکسین در سه رقم تجاری پسته در استان کرمان. علوم و فناوری پسته، 2 (4)، 60-75.
مرادی محمد، حکم آبادی حسین، فانی سیدرضا (1393) بررسی عوامل موثر بر رشد قارچی و تولید آفلاتوکسین در انبارهای پسته استان کرمان. علوم غذایی و تغذیه، 12 (2)، 83-92.
Abdallah MF, Audenaert K, Lust L et al. (2020) Risk characterization and quantification of mycotoxins and their producing fungi in sugarcane juice: A neglected problem in a widely-consumed traditional beverage. Food Control 108, 106811.
Ahmadi K, Ebadzadeh HR, Hatami F et al. (2021) Agricultural statistics (2020). Volume III: Horticultural products. Ministry of Agriculture-Jahad, Deputy of Planning and Economics, Information and Communication Technology Center, 157 p.
Alizadeh A (2011) The relationship between water, soil and plants. Astan Quds Razavi Publications. 484 pages (In Persian).
Arzanlou M, Samadi R, Frisvad JC et al. (2016) Two novel Aspergillus species from hypersaline soils of The National Park of Lake Urmia, Iran. Mycol Progress 15, 1081–1092.
Atlas RM (2010). Handbook of Microbiological Media. CRC Press, Boca Raton.
Aydin S, Ulvi M (2019) Residue levels of pesticides in nuts and risk assessment for consumers. Qual Assur Saf Crops Foods 11, 539–548. 
Babaee R, Karami-Osboo R, Mirabolfathy M (2022). Evaluation of the use of Ozone, UV-C and Citric acid in reducing aflatoxins in pistachio nut. J Food Compos Anal 106, 104276.
Bensassi F, Rhouma A, Ghrab M et al. (2010) Evaluation of cultivar susceptibility and storage periods towards aflatoxin B1 contamination on pistachio nuts.Mycotoxin Res 26, 199–203. 
Cotty PJ, Jaime-Garcia R (2007) Influences of climate on aflatoxin producing fungi and aflatoxin contamination. Int J Food Microbiol 119(1), 109-115.
Davis ND, Iyer S K, Diener UL (1987) Improved method of screening for aflatoxin with a coconut agar medium. Appl Environ Microbiol 53(7), 1593–1595.
Donner M, Atehnkeng J, Sikora RA, Bandyopadhyay R, Cotty, PJ (2009) Distribution of Aspergillus section Flavi in soils of maize fields in three agroecological zones of Nigeria. Soil Biol Biochem 41(1), 37-44.
Doster MA, Cotty PJ, Michailides TJ (2014). Evaluation of the atoxigenic Aspergillus flavus strain AF36 in pistachio orchards. Plant Dis 98(7), 948-956.
Doster MA, Michailides TJ (1994a) Aspergillus molds and aflatoxins in pistachio nuts in California. Phytopathology 84(6), 583-590.
Doster MA, Michailides TJ (1994b) Development of Aspergillus molds in litter from pistachio trees. Plant Dis 78(4), 393-397.
Doster MA, Michailides TJ (1995a) The development of early split pistachio nuts and their contamination by molds, aflatoxins and insects. Acta Hort 419, 359-364.
Doster MA, Michailides TJ (1995b) The relationship between date of hull splitting and decay of pistachio nuts by Aspergillus species. Plant Dis 79, 766-769.
Doster MA, Michailides TJ (1999) Relationship between shell discoloration of pistachio nuts and incidence of fungal decay and insect infestation. Plant Dis 83(3), 259-264.
Doster MA, Michailides TJ, Goldhamer DA, Morgan DP (2001) Insufficient spring irrigation increases abnormal splitting pistachio nuts.California Agric 55(3), 27-30.
Ehrlich KC, Kobbeman K, Montalbano BG, Cotty PJ, (2007) Aflatoxin-producing Aspergillus species from Thailand. Int J Food Microbiol 114, 153–159.
Esmailpour A, Emami Y, Basirat M et al. (2020). Pistachio of Iran. Agricultural Education and Extension Press, 424p.
Fani SR, Moradi M, Probst C et al. (2014a) A critical evaluation of cultural methods for the identification of atoxigenic Aspergillus flavus isolates for aflatoxin mitigation in pistachio orchards of Iran. Eur J Plant Pathol 140(4), 631-642.
Fani SR, Moradi M, Tajabadipour A et al. (2014b) The Role of Early Splitting in Contamination of Pistachio Nuts by Aspergillus Species and Aflatoxin in Kerman Province. J Food Tech Nutr 11 (3), 97-105.
Fani SR, Moradi M, Zamanizadeh HR et al. (2014c) Distribution of Nontoxigenic strains of Aspergillus flavus throughout pistachio growing areas in Iran. Appl Entom Phytopath 81 (2), 179-190. (In Persian)
Frisvad JC, Hubka V, Ezekiel CN et al. (2019) Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins. Stud Mycol 91(1), 37-59.
Frisvad JC, Samson RA (2004) Polyphasic taxonomy of Penicillium subgenus Penicillium A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol 49, 1–173.
Frisvad JC, Skouboe P, Samson RA (2005) Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov. Syst Appl Microbiol 28,442–453.
Georgiadou M, Dimou A, Yanniotis S (2012) Aflatoxin contamination in pistachio nuts: A farm to storage study. Food Control 26, 580–586.
Giorni P, Battilani P, Pietri A, Magan N (2008) Effect of aw and CO2 level on Aspergillus flavus growth and aflatoxin production in high moisture maize post-harvest. Int J Food Microbiol 122(1-2), 109-113.
Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61(4), 1323-1330.
Heidarian R, Javan-nikkhah M, Ormaz B, Peyambari M (2005) Study of fungal contamination of pistachio seeds in Kerman Province, Iran and some new fungi for Iranian pistachio mycoflora.  IV International Symposium on Pistachio and Almond. P, 180. 
Heshmati A, Zohrevand T, Khaneghah AM et al. (2017) Co-occurrence of aflatoxins and ochratoxin A in dried fruits in Iran: Dietary exposure risk assessment. Food Chem Toxicol 106, 202-208.
Hosseinifard SJ, Basirat M, Sedaghati N, Akhyani A (2018) Guidlines for integerated soil fertility and plant nutrition management of pistachio trees. Soil Water Res Institute, 100 p.
Kabirian HR, Afshari H, Mohammadi Moghadam M, Hokmabadi H (2011) Evaluation pistachio contamination to Aspergillus flavus in Semnan Province. J Nuts 2, 1–6.
Khaneghah AM, Fakhri Y, Gahruie HH et al. (2019) Mycotoxins in cereal-based products during 24 years (1983–2017): A global systematic review. Trends Food Sci Technol 91, 95–105. 
Khaneghah AM, Fakhri Y, Raeisi S et al. (2018) Prevalence and concentration of ochratoxin A, zearalenone, deoxynivalenol and total aflatoxin in cereal-based products: A systematic review and meta-analysis. Food Chem Toxicol 118, 830-848.
Klich MA (2002) Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures, Utrecht.
Klich MA (2007) Aspergillus flavus:the major producer of aflatoxin. Mol Plant Pathol 8,713-722.
Mehrnejad MR, Panahi B (2006) The influence of hull cracking on aflatoxin contamination and insect infestation in pistachio nuts. Appl Entom Phytopath 73(2), 39-42.
Michailides TJ, Doster M,  Cotty PJ, et al. (2007) Aflatoxin control in pistachios: biocontrol using the atoxigenic strain AF36, survival of AF36, and EUP status. In Proceedings of the 2007 Annual Multicrop Aflatoxin/Fumonisin Elemination and Fugal Genomics Workshop. pp. 54-55.
Mohammadi AH, Banihashemi Z, Haghdel M (2009) Identification and prevalence of Aspergillus species in soils of Fars and Kerman Provinces of Iran and evaluation of their aflatoxin production. Rostaniha 10(1), 8-30.
Mohammadi Moghadam M, Rezaee S, Mohammadi AH et al. (2020) The Potential of Aflatoxin Production in the Aspergillus Section Flavi Isolates of Pistachio in Iran. J Fast Health 8(4), 254-263.
Moradi M, Ershad D, Mirabolfathi M, Panhi B (2004) The role of plant debris, soil and manure on population density of Aspergillus flavus and Aspergillus niger groups in pistachio orchards of Kerman province. Iran J Plant Path 40, 221-234. (In Persian)
Moradi M, Fani SR, Masoumi H (2014) Population Density of Aspergillus Species Belong to Section Flavi and Nigri on Pistachio Nut in Kerman Province. J Appl Res Plant Prot 3 (2), 79-91. (In Persian)
Moradi M, Hokmabadi H (2011) Control of Mycotoxin Bioactives in Nuts: Farm to Fork. pp: 253-273. In: Fruit and Cereal Bioactives Sources, Chemistry, and Applications.
Ö. Tokusoglu (ed). CRC Press.
Moradi M, Hokmabadi H, Fani SR (2015) A Study concerned with the factors affecting the fungal growth and aflatoxin production during storage of pistachio in Kerman province. J Food Tech Nutr 12 (2), 83-92. (In Persian)
Moradi M, Hokmabadi H, Mirabolfathi M (2010) Density fluctuations of two major Aspergillus species airborne spores in pistachio growing regions of Iran. Int J Nuts Related Sci 1, 54-64.
Moradi M, Javanshah M (2006) Distribution of aflatoxin in processed pistachio nut terminals. Acta Hort 726, 431-436.
Moradi M, Tajababdipour A, Fani SR (2017) The time of occurrence and physical characteristics of pistachio early splitting as sources of contamination with aflatoxin in three commercial cultivars of pistachio in Kerman. Pista Sci Tech 2(4), 60-75. (In Persian)
Moss M (2004) Toxigenic fungi. pp. 479-488. In: Food-borne pathogens, hazards, risk analysis and control. C.W. Blackburn and P.J McClure (eds.), Boca Raton: Woodhead Publishing CRC Press.
Nabizadeh S, Shariatifar N, Shokoohi E et al. (2018) Prevalence and probabilistic health risk assessment of aflatoxins B1, B2, G1, and G2 in Iranian edible oils. Environ Sci Pollut Res 25, 35562–35570. 
Panahi B, Khezri M (2011) Effect of harvesting time on nut quality of pistachio (Pistacia vera L.) cultivars. Sci Hortic 129, 730–734.
Pildain MB, Frisvad JC, Vaamonde G, Cabral D, Varga J, Samson RA (2008) Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts. Int J Syst Evol Microbiol 58, 725–735.
Probst C, Bandyopahyay R, Proce LE, Cotty PJ (2011) Identification of atoxigenic Aspergillus flavus isolates to reduce aflatoxin contamination of maize in Kenya. Plant Dis 95, 212-218.
Rahimi P, Sharifnabi B, Bahar M (2007) Aspergillus species isolated from pistachio and determination of their aflatoxin production. Rostaniha 8(1), 30-42. (In Persian)
Rodrigues P, Venâncio A, Kozakiewicz Z, Lima N (2009) A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almonds. Int J Food Microbiol 129, 187–193.
Russell TE, Watson TF, Ryan GF (1976) Field accumulation of aflatoxin in cottonseed as influenced by irrigation termination dates and pink bollworm infestation. Appl Environ Microbiol 31(5), 711-713.
Samson RA, Frisvad JC (2004) Penicillium subgenus Penicillium:new taxonomic schemes, mycotoxins and other extrolites. Stud Mycol 49, 1–157 
Satio M, Machida S (1999) A rapid identification method for aflatoxin-producing strains of Aspergillus flavus and A. parasiticus. Mycoscience 40, 205-208.
Sedaghati N, Mohammadi Mohammadabadi A, Hosseinifard SJ (2008) The effect of irrigation regimes on occurrence of early split of pistachio (Pistacia vera L.) CV. Ohadi. Pajouhesh & Sazandegi 78,149-158. (In Persian)
Shakerardekani A, Karim R, Mirdamadiha F (2012) The effect of sorting on aflatoxin reduction of storage in Turkey with particular reference to aflatoxin contamination. J Sci Food Agric 27(11), 1021-1026.
Taghizadeh SF, Rezaee R, Davarynejad G et al. (2018) Risk assessment of exposure to aflatoxin B1 and ochratoxin A through consumption of different Pistachio (Pistacia vera L.) cultivars collected from four geographical regions of Iran. Environ Toxicol Pharmacol 61, 61-66.
Tajabadipour A, Afshari H, Hokmabadi H (2011). Recognition and determination of contaminated pistachios to aflatoxin in processing. J Nuts 2 (2), 27-30.
Torre LA, Bray F, Siegel RL et al. (2015) Global cancer statistics, 2012. CA: a cancer J Clinicians 65, 87-108.
Tsakiris IN, Tzatzarakis MN, Alegakis AK et al. (2013) Risk assessment scenarios of children’s exposure to aflatoxin M1 residues in different milk types from the Greek market. Food Chem Toxicol 56, 261-265.
Vaamonde G, Patriarca A, Fernandez pinto V et al. (2003) Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section Flavi from different substrates in Argentina. Int J Food Microbiol 88(1), 79-84.
Varga J, Frisvad JC, Samson RA (2011) Two new aflatoxin        producing species, and an overview of Aspergillus section Flavi. Stud Mycol 69, 57-80.
Wei D, Jong S (1986) Production of aflatoxins by strains of the Aspergillus flavus group maintained in ATCC. Mycopathologia 93,19–24.
Williams JGK, Kubelik AR, Livak KJ et al. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res 18, 6531–6535.