التحري عن بعض الفطريات المنتجة للسموم والمرافقة لحبوب المحاصيل المخزونة والمستخدمة كمواد أولية في تصنيع عليقة الأسماك

Detection of some Fungi producing mycotoxins associated with stored crop grains used in fish feed.

Abstract
The study was conducted with the aim of investigating the extent of fungal contamination in stored crop grains used as essential components of fish feed, focusing on dangerous fungi capable of producing mycotoxins aflatoxin B1 and ochratoxin A, and examining the effects of storage periods and moisture content levels on their production, as well as assessing the impact of contamination levels of aflatoxin B1 and ochratoxin A on fish farming and health in Iraq. The survey results indicated contamination in all studied samples of wheat, barley, yellow corn, soybeans, and both local and imported fish feed collected from four governorates (Baghdad, Karbala, Babylon, and Anbar) with various fungal strains, the most important of which were Aspergillus sp ,A.flavus. A.niger, Cladosporium sp, Alternaria sp, Fusarium sp, Mucor sp, Penicillium sp, Rhizoctonia sp, Rhizopus sp Cylindrocarpom , and Trichoderma sp .
The isolation process focused on the biodiversity of the fungal strains Aspergillus spp , with 1400 fungal isolates identified belonging to the fungus Aspergillus niger , which was the most prevalent and frequent, followed by isolates of the species Aspergillus flavus, with 802 fungal isolates among the isolated fungi. While the percentage of fungal occurrence and frequency recorded, the most prevalent was the fungus A.niger with an average occurrence rate of 91.67% and a frequency of 30.93% among the collected samples, followed by the fungus A.flavus with an occurrence rate of 89.58% and a frequency of 16.88
The results of chromatographic analysis using HPLC technology demonstrated the ability of all nine tested fungal isolates of Aspergillus niger to produce ochratoxin in varying proportions. Aspergillus niger K1 isolates had the highest production concentrations, reaching 80.90 micrograms/kg for the ANK1 isolate, while the lowest production levels were for the ANA1 isolate, at a concentration of 35.9 micrograms. /kg isolated from local fish feed. At the same time, the ability of all nine tested fungal isolates of Aspergillus flavus to produce Aflatoxin B1 in varying proportions was demonstrated, with Aspergillus flavus K2 topping the list. The highest production concentrations reached 226.9 µg/kg for isolate AFK2, while the lowest production levels were for isolate AFB2 at a concentration of 134.9 µg/kg, isolated from local fish feed.
Chromatographic analysis to detect contamination with ochratoxin A showed that five samples out of nine were free of contamination with the mycotoxin ochratoxin (the local Anbar samples (A1F and A2F), the local Babylon samples (H1F and H2F), and the imported Babylon sample (H1FF)), while four samples showed contamination. With ochratoxin in varying proportions, with the highest percentage of ochratoxin A contamination recorded, reaching 6.90 micrograms/kg for sample K1F, followed by B2F, K2F, and B1F at concentrations (6.20, 5.70, and 5.50 micrograms/kg, respectively. While contamination of the samples with the mycotoxin aflatoxin B1 was detected, all samples were 100% contaminated with Aflatoxin B1 at varying concentrations, with the highest contamination rate recorded at a concentration of up to 9.33 micrograms/kg for sample KF2, followed by sample KF1 with a concentration of 9.25 micrograms/kg, while the lowest concentration was recorded. 2.65 micrograms/kg for imported fish feed sample H1FF.
The results of the molecular diagnosis of mycotoxin-producing isolates, of which the first two isolates were characterized by the highest production of the two mycotoxins Aflatoxin B1 and Ochratoxin A, and the two isolates that produced the least in local fish feed samples, confirmed their diagnosis under different species. The results of the nucleotide sequence analysis of the isolates showed that two isolates belonging to the fungus Aspergillus niger were recorded. Under accession codes (OR452868.1 and OR449322.1), and two isolates belonging to the fungus Aspergillus flavus were registered under accession codes (OR449324.1 OR449323.1), the genomic sequences were preserved in the database of the National Center for Biotechnology Information and were compared with Pre-registered isolates
The results of the storage experiment showed that the highest level of Aflatoxin B1 production was recorded during the longest storage period of three months (the production of Aflatoxin B1 is directly proportional to the storage periods), as its concentration reached 42.12, 68.43, and 82.34 ppb within three humidity levels (10%, 15%, and 25%). respectively. While the highest level of mycotoxin ochratoxin production was recorded during the longest storage period of three months, as its concentration reached 46.32, 49.42, and 64.67 ppb within three humidity levels (10%, 15%, and 25%), respectively.
The results of the effect of the mycotoxins aflatoxin B1 and ochratoxin A on growth rates and the number of fish kills during the rearing periods showed an increase in the level of fish kills, with the percentage of kills reaching 19.4% and 10.80%, respectively, compared to the control treatment, which amounted to 3.5%. At the same time, an increase in the percentage of weight losses. For fish, the highest percentage of weight loss was recorded in the 180-day period, amounting to 36.6% and 27.20%, while the 120-day period recorded a weight loss percentage of 43.5% and 20.20%, and the lowest percentage of loss was in the first 60 days period, which amounted to 22.4% and 13.70% compared to the treatment. Control 0.00%. The results of physiological blood components showed different negative effects, due to continuous exposure to the mycotoxins aflatoxin B1 and ochratoxin A, by reducing WBC, RBC, HGB, MCHC and PLT compared to the control treatment, while causing a significant increase in the level of HCT compared to the control treatment.