Black germ of wheat in the conditions of the Krasnoyarsk forest-steppe

. Black germ is a ubiquitous disease of wheat, which, without particularly affecting crop yields, can affect the parameters of the technological evaluation of grain. Until now, there is no consensus on the nature of the occurrence of this disease. In our study, we studied the relationship between the visible symptoms of damage by a black germ and infection of seeds of spring soft wheat samples with fungi of the genera Alternaria and Biporalis in the conditions of the Krasnoyarsk forest-steppe. The phytopathological analysis of asymptomatic and black-embryonic grains revealed in the latter a significant excess of infestation with p. Alternaria fungi in two out of ten samples. There were no differences in seed infection with p. Biporalis micromycetes between the variants. Only in one sample, the germination of seeds with manifested symptoms of the disease was lower than in visually healthy ones. The percentage of germination of other varieties and lines in both variants did not differ statistically significantly.


Introduction
Black germ of wheat is a disease that occurs in all regions of its cultivation.Symptoms of disease progression are usually visible after harvest and are expressed as a darkening of the kernel embryo from brown to black and discoloration of the rest of the grain.There are different opinions about the cause of the disease.Some researchers consider the presence of mycelium of fungi of the genera Alternaria and Bipolaris in the grain of wheat to be the cause of the appearance of the black germ.Others are biochemical processes in it caused by abiotic stresses, since symptoms most often occur after exposure to extreme environmental conditions.And the presence of these phytopathogens in the grain is considered as a result of the secondary colonization of seeds by them, and not the cause of the disease [1][2][3].Some literature data indicate that, in addition to the above-mentioned micromycete genera Alternaria and Bipolaris, some representatives of Fusarium, Drechslera, Nigrospora, Penicillium, Cladosporium, and Stemphylium can cause a black embryo [4].Factors that contribute to the infection of wheat with these phytopathogens are abundant rainfall and high humidity during flowering and ripening periods, and excessive application of nitrogen fertilizers [5].
Despite the fact that the disease has almost no effect on crop productivity, grain color changes can affect its quality parameters and commercial value.Thus, Malaker et all determined that as the degree of damage to wheat by black germ increased, the dry matter and ash content in the grain decreased, while the protein and fat content increased.The content of macro-and microelements in black germ seeds, namely, the amount of nitrogen, phosphorus, calcium, magnesium, sulfur and boron increased, while the content of potassium, iron, zinc, copper and sodium, on the contrary, decreased [6].Flour obtained from high-grain wheat with black germ symptoms, discoloration and bad smell, is unsuitable for use in food production.The very quality of seeds is one of the main indicators that determine the competitiveness of a variety in the domestic market, and creates good prerequisites for entering the international market.
Also of concern is the fact that mushrooms p. Alternaria and p. Fusarium, as potential pathogens, produce mycotoxins dangerous for humans and animals, which accumulate in infected grain [7].The level of grain contamination with mycotoxins is quite high.Mushrooms p.Fusarium produce a wide range of toxic metabolites that have strong carcinogenic, mutagenic and even psychotropic effects.These are trichothecene mycotoxins, fumonisins, zearalenone, moniliformin, etc.Once in the human body, fusariotoxins can lead to the development of such dangerous diseases as alimentary-toxic aleukia and "drunk bread".Tenuazonic acid, tentoxin, altertoxins I, II and III, alternariol and its methyl ester are common toxic metabolites of p.Alternaria genus fungi that can be found in food products if raw materials contaminated with micromycetes of this genus are used to obtain them.
In addition to abiotic factors, the intensity of the development of the disease can also be influenced by the region of growth and the genotype of wheat.Thus, scientists from China found that seeds with visible symptoms of damage by a black embryo had a lower percentage of germination than healthy ones, and the predominant causative agent of the disease was p. Bipolaris [8].Researchers from Romania and Italy, in turn, attributed to the fungi that cause the disease, p. Alternaria and p. Fusarium [9][10].
Based on the foregoing, it is obvious that despite the rather high degree of knowledge of such a problem as the black germ of wheat disease, it is still relevant to conduct additional research in this direction.
The aim of our study was to study the contamination of seeds of spring soft wheat with symptoms of damage by a black germ and without fungi of the genera Alternaria and Bipolaris in the conditions of the Krasnoyarsk forest-steppe.

Materials and methods
As objects of study, 10 varieties and lines of spring soft wheat grown in the conditions of the central part of the Krasnoyarsk forest-steppe, competitive variety testing (CVT) of the Krasnoyarsk Research Institute of Agriculture in 2022 were selected: Svirel, Altaiskaya 75, Altaiskaya 70, Kuraginskaya 2, Uyarochka, Krasnoyarskaya 12, K-696-6, K-229-9, KSI-21 and K-613-2.The growing season of 2022 was characterized by unstable weather with frequent precipitation in June, air temperature fluctuations during the day and night, and heavy showers in July.Unstable weather conditions were also observed in August with fogs and heavy morning dews.
To carry out a phytopathological analysis for the presence of fungi of the genera Alternaria and Bipolaris, an average sample of 100 asymptomatic grains (control, K), as well as those with visible symptoms of a black germ (BG) infection, was taken from several batches of each studied sample (Figure 1).Grain samples were washed for 2 hours with the addition of surfactants, under running water, to remove impurities and contaminants.Then, in a microbiological box, they were surface sterilized in 70% ethanol for 3 minutes.The grains pre-washed from the sterilizing agent and dried were laid out in Petri dishes on the surface of potato-sucrose agar (PSA), 10 pieces each, and placed in a thermostat at a temperature of 24°C.After 7 days, we took into account the germination, the total infection of the average sample with fungi, as well as the infection of the average sample with fungi p. Alternaria and p. Bipolaris (Figure 2).The generic affiliation of micromycetes was determined by the totality of macro-and micromorphological features.

Results and Discussion
The phytopathological analysis carried out showed that all studied wheat samples were infected with phytopathogens.The total infection of seeds varied from 44.0 to 100% in the control and from 78.0 to 98.0% in the variant with a black embryo.Statistically significant differences between the control and experimental variants were found only in the Altaiskaya 70 variety and the K-696-6 line (Table 1).The total infection of the average sample of black germ seeds was higher in them than in healthy ones.It is known that seeds are one of the main sources of infection of plants with diseases.And during the phytopathological examination, a large number of pathogens of seed infections are isolated from them.The dominant position in the infection of seeds of grain crops with pathogenic mycoflora is occupied by fungi of the genera Alternaria, Bipolaris, the so-called storage molds, as well as pathogens of bacterioses.Mold spores p.Penicillium and Aspergillus fall on the grain during harvesting, its mechanical processing and storage.On injured grains at high humidity, fungi develop even more intensively.In our work, we focused on the study of the colonization of seeds of wheat accessions by representatives of the genera Alternaria and Bipolaris, which, according to many researchers, are the cause of the black germ of cereal crops.
The hyphae of B. sorokiniana penetrate the endosperm, pericarp, and, in most cases, the seed embryo, which prevents its development.When using B. sorokiniana infected seed, plants can develop root rot and thin crops.The infectious process caused by helminthosporiasis manifests itself in the partial browning of some germinal roots during grain germination or the defeat of all roots and the base of the coleoptile.Mycelium of mushrooms p. Alternaria, when infected, does not penetrate into the embryo, but develops in the fruit coat and endosperm, usually above the embryo.If the defeat of the grain is large, then during germination in favorable conditions for the pathogen, root rot may develop.
As can be seen from table 1, in terms of the percentage of infection of seeds, micromycetes of p. Alternaria prevailed over p. Bipolaris.Helminthosporium was not found at all in the seeds of the variety Svirel, both in the control and experimental variants.In other varieties and lines, the infestation of p. Bipolaris ranged from 0.0 to 10.0%.Differences in the presence of helminthosporiasis infection in asymptomatic and blackembryonic seeds were not revealed.
But infection with fungi p. Alternaria varied from 40% (line KSI-21) to 94% (line K-613-2).However, only two samples out of ten (Altaiskaya 70 and K-696-6) had p. Alternaria in the experimental variant with visually affected grains statistically significantly exceeded the control by 30 and 40%, respectively.
Most likely, wheat varieties and lines of different genotypes, in combination with unstable weather conditions during the growing season in the growing region, may react differently to grain contamination with the alleged black germ pathogens.
The germination of wheat seeds in both variants of the experiment was at a fairly high level, only in the Altaiskaya 75 variety with a black embryo this indicator was below 75% (Table 1).The maximum value of this indicator (100%) was recorded in the blackembryonic seeds of the Uyarochka variety, regardless of the visible symptoms of embryo damage.
It is known that completely resistant varieties to black wheat germ do not exist [4].However, it is necessary to evaluate the created breeding material for the degree of resistance to the disease in specific growing regions, taking into account abiotic environmental factors.After all, when cultivating varieties that showed resistance to black germ in a certain period of the study, under favorable conditions for the development of phytopathogens, additional phytosanitary measures may be required.

Conclusion
The results of the study showed that there is no clear evidence of the influence of contamination of grains of spring soft wheat with micromycetes of the genera Alternaria and Bipolaris on the manifestation of black germ symptoms in them.Only in two out of ten samples, the total infection with fungi in general, as well as with p. Alternaria fungi, was higher in grains with visible symptoms of black embryo damage than in asymptomatic ones.Differences in p. Bipolaris infestation of black-embryonic and visually healthy grains were not found.

Fig. 2 .
Fig. 2. Phytopathological analysis of wheat grain with symptoms of black germ damage on potatosucrose agar.

Table 1 .
Infection of spring soft wheat of competitive variety testing, 2022.