Some data on prevalence of microscopic fungi in conditions of the Republic of Tatarstan

. Research on plant feedstock for farm animals and poultry is now a pressing issue. Seeding with field isolates occurs in the soil layer in the autumn period. The infestation of isolates remains in the form of spores on crop residues during the winter period. The research aimed to conduct a mycological assessment of the field isolates prevalence in soil, green mass, and agricultural products in the farms of the Agricultural Production Cooperative [APC] “Tukaya” of the Baltasinsky district, the Agricultural firm “ Arch a” of the Arsky district, and the APC “Kubnya” of the Kaibitsky district of the Republic of Tatarstan. The mycological screening was carried out in the specialized laboratories of the Federal Center for Toxicological, Radiation and Biological Safety. To determine the toxicity, a biotesting method based on the survival rate of ciliated cultures was used after one hour of exposure in the extract of the studied product. The climatic conditions of the spring-summer period of 2020 were characterized by increased humidity in combination with temperature changes. In the research, a spread of fungi of the genus Fusarium (F. oxysporum, F. graminearum F. sporitrichioides), Penicillium (P.notatum, P.glaucum, P.chusodepim, P. expansium) was detected in crops . And fungi of the genus Aspergillus were more often sown in grain feeds ( A.fumigatus, A. flavus, A. niger ). During the determination of toxicity, it was found that all the objects studied: soil, green mass, and feed are non-toxic, the survival rate of infusoria was 86% – 100%. The authors recommend conducting regular feed screening and taking preventive measures that will make it possible to prevent the spread of aggressive field isolates in the agricultural complexes of the Republic of Tatarstan.


Introduction
Plants of grain and vegetable crops, from which food raw materials of plant origin are obtained, undergoing a long process of growth and development, can be contaminated with microscopic fungi both in any phase of the growing season from the moment of sowing to harvesting and during storage of finished products [1][2][3][4][5][6][7][8][9].Phytopathogens, preserved in soil, water bodies, parasitize the roots, causing vascular diseases of plants [2; 3; 8].Moldy micromycetes affect the nutrition of cereals and vegetable biological objects, hampering crops' normal growth and development [10; 11].
The micromycete species of the genus Fusarium are very common in all cultivated areas of the Republic of Tatarstan.Seeding with field isolates occurs in the soil layer in the autumn period.The infestation of isolates remains in the form of spores on crop residues during the winter period.
During the vegetation period, the isolates of micromycetes are localized along the plant tiers with the help of hyphae and spores, affecting most plant crops.
Today, more than 100 thousand mycelial isolates varieties are known, 250 varieties of which produce mycotoxins and secondary metabolites that are dangerous for farm animals and humans [12].

Materials and methods
The purpose of the research is a mycological assessment of the spread of field isolates in soil, green mass, and agricultural products on individual farms of the Republic of Tatarstan.
Mycological studies and sampling were carried out according to the Guidelines for Sanitary and Mycological Assessment and Improvement of Feed Quality.
The mycological screening was carried out in the specialized laboratories of the Federal State Budgetary Scientific Institution "Federal Center for Toxicological, Radiation and Biological Safety" (Kazan).Feed samples were received from the farms of the Republic of Tatarstan: of the Agricultural Production Cooperative [APC] "Tukaya" (Baltasinsky district), Agrofirm "Archa" (Arsky district), APC "Kubnya" (Kaibitsky district ) (Fig. 1).Under laboratory conditions, Czapek and Sabouraud agars were used to isolate fungi.Isolates were isolated by obtaining a suspension from feed, soil, and green mass samples with seeding on Czapek and Sabouraud agarized environments [13].
Differentiation and determination of genus and species of fungi were carried out according to atlas-determinants of microscopic fungi [5].
The type and genus of field isolates were determined in the initial sowing.Still, certain difficulties arose, and methods of direct replanting and separation were used to identify the pure culture of the isolate.
Direct replanting was used for the primary sowing of grown isolated colonies [13].This method was done using a needle bent at an obtuse angle, with which a piece of mycelium was carefully captured and placed on the surface of the nutrient environment.Clumping, twisting, or other deformations of the picked-up piece of mycelium were avoided.In the case of abundant sporulation in the fungus, a minimum number of spores were transferred with a dry needle.
Cultivation of crops was carried out at 22-25 °C in a BINDER RI 115 Solid.Line incubator thermostat (natural convection, 118 l, ambient temp.from +5 to +70 °C).
The degree of toxicity was determined by the survival rate of infusoria after 1 hour of exposure in the extract of the studied product (Table 1).Source: [1].
The obtained data were systematized and subjected to analytical processing.

Results
The results of mycological studies of water bodies, soil, and feed from some regions of the Republic of Tatarstan are presented in Table 2.

Aspergillus niger, Fusarium graminearum
The object of the study is non-toxic; the survival rate of infusoria was 82% Forage 1 3.6 x 10 3

Aspergillus flavus, Fusarium
The object of the study is non-toxic; the survival rate of infusoria was 78%

Fusarium graminearum
The object of the study is non-toxic; the survival rate of infusoria was 90% Forage 2  2.7 x 10 3

Fusarium graminearum
The object of the study is non-toxic; the survival rate of infusoria was 97% Kaibitsky district, APC "Kubnya" Water 1 x 10

Fusarium poae, Penicillium notatum
The object of the study is non-toxic; the survival rate of infusoria was 96% Green mass 2.9 x 10 3

Penicillium expansium, Fusarium sporotrichioides
The object of the study is non-toxic; the survival rate of infusoria was 68% Alfalfa, green mass from the feeder 2.9 x 10 3

Aspergillus niger, Fusarium poae, Fusarium graminearum
The object of the study is non-toxic; the survival rate of infusoria was 95% Compound feed

Aspergillus niger, Fusarium poae, Fusarium graminearum
The object of the study is non-toxic; the survival rate of infusoria was 82% Forage 3  3.1 x 10 3

Aspergillus flavus, Fusarium graminearum
The object of the study is non-toxic; the survival rate of infusoria was 88% Source: Compiled by the authors.
During the mycological analysis of the forage where the isolates of Aspergillus flavus, Fusarium graminearum were isolated, the total number of fungi was 3.6 x 10 3 , forage was 2.7 x 10 3 , where one isolate type of Fusarium graminearum was isolated, in the forage with more aggressive micromycetes identical to the forage of the first test of Aspergillus flavus, Fusarium graminearum, the total number of fungi was 3.1 x 10 3 .The mycological picture of field isolates is given in Fig. 2-5.The total number of fungi ranged from (1x10 3 ) to (4.2x10 3 ) CFU/g.Mycological analysis of the green mass of the Kaibitsky district from APC "Kubnya" showed weak toxicity on Paramecium caudatum (68%).Testing on paramecia showed the toxicity of the isolated Fusarium sporotrichioides isolate (death of infusoria was 50%).The application of mycelial isolate extract to the skin of rabbits caused redness of the skin, indicating the first degree of the lesion.

Discussion
Mycotoxin contamination is considered one of the most important economic problems of animal farming and feed production.
The research results are supported by the data of the study of fish food by Egyptian scientists, who found that the most common isolated fungi were Aspergillus spp.(86.66%), followed by Penicillium spp.(23.33%),Fusariam spp.(10%), Mucor spp.(6.66%), followed by Rhizopus spp.(3.33%).The most predominant genus was Aspergillus niger (43.33%), followed by Aspergillus flavus (30%) and Aspergillus fumigatus (6.66%), Aspergillus versicolor (3.33%), and Aspergillus terrus (3.33%).Some mycotoxins were produced by more than one species of fungi [6].Italian scientists conducted mycological and toxigenic screening of 36 batches used in animal feed collected in 2008, 2009, and 2010.A study of soybean seeds (n = 6) showed that soybean seeds are often colonized by Aspergillus spp.and Fusarium spp.Aflatoxins, fumonisins, and deoxynivalenol were detected in 88.9%, 72.2%, and 30.6% of samples, respectively.The simultaneous appearance of at least two toxins was observed in 72% of cases [4].Thus, the study of the contamination of feed with pathogenic fungi is an urgent area of research.

Conclusion
The seasonal weather conditions of the summer period, with a sharp drop in humidity and temperature spikes, created conditions in which the Fusarium fungi dominated the crops and green mass of the 2020 harvest.
Thus, the increased humidity of the spring-summer period, combined with temperature changes, contributed to the development of fusariosis.Only regular feed screening and preventive measures will make it possible to prevent the spread of aggressive field isolates in the agricultural complexes of the Republic of Tatarstan.

Fig. 1 .
Fig. 1.Map of the Republic of Tatarstan (the sampling points are marked).Source: Compiled by the authors.

Table 1 .
Toxicity of the studied product.

Table 2 .
Isolation of field isolates from the studied objects.