INVESTIGATION OF SPECIES COMPOSITION OF THE FUNGI OF THE FUSARIUM GENUS AND THE RESISTANCE OF THE CHORNOBYL RADIO-MUTANTS TO FUSARIUM HEAD BLIGHT FOR THE PURPOSES OF WINTER WHEAT BREEDING IN THE FOREST-STEPPE OF UKRAINE

Aim. To investigate the ﬁ eld resistance of collection spelt-like samples of the Chornobyl radio-mutants (RM) of winter wheat to Fusarium head blight and to determine the species composition of the fungi of the Fusarium genus in the Forest-Steppe of Ukraine. Methods. The resistance (in scores) of ten RM-samples of winter wheat to Fusarium head blight was determined in ﬁ eld conditions by visual inspection of plants using standard methods (Trybel et al, 2010). To determine the infection rate of the seeds, the biological method of seed germination in the rolls of ﬁ ltration paper was used. DNA was extracted using the reaction kit AGROSORB NK (Agrogen Novo, Ukraine), according to the manufacturer’s instructions. The molecular identi ﬁ cation of the species composition of the fungi of the Fusarium genus was conducted with the commercial test systems “Fuzarioz zlakiv” (AgroDiagnostica, RU) according to the manufacturer’s instructions, using the real-time PCR. Results. The results of evaluating the ﬁ eld resistance of ten spelt-like RM-samples of winter wheat to Fusarium head blight are presented. The species composition of the Fusarium fungi as of 2016–2018 in the Forest-Steppe of Ukraine, namely in Kyiv, Cherkasy, Sumy, Vinnytsia, Chernivtsi, Poltava, Kharkiv, Ternopil, and Khmelnytsky regions is presented. To study the species composition of the Fusarium fungi, 639 seed samples of winter wheat were taken from the farms located in the territory of nine regions in the Forest-Steppe zone. The results of molecular identi ﬁ cation of the infection rate in the seed material demonstrate the presence of seven out of eight fungi species under investigation: F. avena-ceum, F. culmorum, F. graminearum, F. langsethiae, F. poae, F. sporotrichioides, F. tricinctum during the years of studies. Yet, F. culmorum , F. langsethiae were not identi ﬁ ed in 2017, and F. cerealis was not found during the study years, in 2016–2018. It should be noted that the most frequent incidence was found for F. graminearum, F. avenaceum and F. tricinctum, which was within the range of 25.8–44.0 %, and according to (Zhdanova


INTRODUCTION
In recent years, the global community has frequently raised the issue of global safety of food products, which could be improved via the intensifi cation and implementation of modern technologies in plant cultivation, the enhanced level of effi ciency in using assortment of varieties and, as a result, the decrease of the negative impact on the environment (Miroshnychenko et al, 2014).
The phenotype of the variety is formed due to the interaction between the genotype and the environment. The imperfect agriculture and global climatic changes promote the increase in the harmfulness of weeds, diseases, and pests, which aggravates the phytosanitary state of agrophytocenoses Sanin et al, 2019). Among the biotic factors, decreasing the potential performance of the variety, the leading role is played by harmful organisms (Tsyliuryk, 2019), including fungal disease agents. Once spread, fungal diseases decrease the grain performance considerably along with the deterioration of food products, produced from the infected materials Foround et al, 2016).
The most harmful organisms on awned cereals are fungi of the Fusarium genus, whose manifestations may be diverse -wheat crown rot, root rots, head or ear blight, etc. (Gencheva et al, 2020). In recent years, the harmfulness of Fusarium head blight and the accumulation of inoculum of disease agents in the agrophytocenoses of Ukraine and the world has been increas-ing rapidly and becoming global. Fusarium head blight causes the infection of grain in the spike, which results in annual 20-30 % losses of harvest and in the epiphytotic years -up to 45-73 % Liew et al, 2018;Murashko et al, 2021).
The wheat spikes are infected with Fusarium head blight during the phases of blossoming-kernel ripening. The characteristic symptoms of spike infection are the occurrence of watery brown spots at the base of the spike glume and the early aging of some spikes. Under the conditions favorable for the development of the disease, a layer of mycelium and spores of the fungus appear on the spike glume; its color depends on Fusarium species. From the surface glume, the infection spreads along the tissues of the generative organs. Also, the macroconidia and chlamydospores may penetrate the spike along with the drops of rain and dew and promote the infection of Fusarium head blight (Scherm et al, 2013;Vughan et al, 2016). The specifi city of Fusarium head blight is its particular etiology -the participation of several species of Fusarium spp. in the pathogenic process (Burkyn et al, 2015;Oryna et al, 2017).
The infection of wheat spikes and grain with fungi species of Fusarium genus has a negative effect on the energy of seed germination, seedling, thousand-grain weight, chemical and technological properties of grain , and the products of processing plant residues into the feeds for cattle (Foround et al, 2016;Gagkaeva et al, 2013;Scherm et al, 2013).
Also, the fungi of Fusarium genus are capable of producing mycotoxins -secondary metabolites of micro-evaluated the fi eld resistance in ten spelt-like RM-samples of winter wheat to Fusarium head blight, whose resistance fl uctuated within 7.4-8.4 scores in the study years. Under the climatic conditions most favorable for the disease development, in 2016 (from the time of spring vegetation restoration on March 01, to full ripeness of grain on July 09, there was 404.2 mm of precipitation which exceeded the perennial indices by 175 %) the samples under investigation: RM-1, RM-3, RM-4, RM-5, RM-6, RM-9, RM-10 exceeded the resistance of the standard variety, Lisova Pisnia, -7.0 scores. Conclusions. The fi eld and laboratory studies related to the species composition and geographic distribution of the Fusarium fungi give grounds for the assumption of the presence of an expansive species complex of Fusarium fungi in the territory of nine regions in the Forest-Steppe of Ukraine: F. avenaceum, F. culmorum, F. graminearum, F. langsethiae, F. poae, F. sporotrichioides, F. tricinctum. A close association between the biology of the group of Fusarium fungi and weather conditions of their environment was used by us to investigate the resistance of ten collection spelt-like samples of the Chornobyl radio-mutants in 2016-2018 and to isolate fi ve RM-samples of winter wheat (RM-9, RM-5, RM-6, RM-10, RM-4), which had higher resistance to Fusarium head blight than the standard variety, Lisova Pisnia, on average for three years of studies. It should be noted that in 2016, under weather conditions favorable for pathogen development, RM-3 and RM-4 were found to be reliably more resistant compared to the standard. The fi ve spelt-like RM-samples of winter wheat (RM-9, RM-5, RM-6, RM-10, RM-4) with high resistance indices on average for the years of studies (2016)(2017)(2018) are introduced by us to the breeding programs as the initial material to obtain varieties, resistant to Fusarium head blight.

INVESTIGATION OF SPECIES COMPOSITION OF THE FUNGI OF THE FUSARIUM GENUS
scopic fungi with expressed toxic properties, which are considered to be among the most harmful substances for the health of humans and animals in recent half a century. The products of processing cereals, including wheat, are the main sources of Fusarium mycotoxins.
In addition to causing direct diseases, mycotoxins can promote delayed negative effects on living organisms: teratogenic, embryotoxic, carcinogenic, mutagenic. Some metabolites are the reason for food poisoning with different degrees of complications for humans and animals (Kaminska et al, 2020). Some of the most widespread and harmful mycotoxins of Fusarium fungi, which get accumulated in winter wheat grain, are deoxynivalenol, T-2 toxin, zearalenone, and afl atoxin. They are notable for their immunosuppressive effect and no sensibilization of the organism (Miedaner et al, 2021). Their specifi city lies in the ability to have a negative impact in minimal doses Foround et al, 2016;Nesic, 2018). The contamination of agrophytocenoses with mycotoxins decreases the global crop production and animal products considerably (Liew et al, 2018;Alshannaq et al, 2017).
The breeding of genetically resistant material is the most effective and well-known method of crop protection in the world (Morgun et al, 2016;Kovalyshyna et al, 2017). Therefore, it is rather urgent now to search for and investigate the sources of resistance for the purpose of creating new varieties of winter wheat.
The aim of our studies was to determine the species composition of Fusarium fungi in the territory of nine regions of the Forest-Steppe of Ukraine and to evaluate the fi eld resistance of ten spelt-like collection samples of the Chornobyl radio-mutants (RM) of winter wheat (Triticum aestivum L.) to Fusarium head blight.

MATERIALS AND METHODS
The studies were conducted in 2016-2018. The species composition of Fusarium fungi in the grain was identifi ed in the laboratory conditions of the Bila Tserkva Diagnostic Center of "Syngenta" LLC (26/74, Liudmyla Pavlichenko Str., Bila Tserkva, Ukraine). To determine the grain infection, 639 samples of seed material of soft winter wheat were obtained from the farms in nine regions (Kyiv, Cherkasy, Sumy, Vinnytsia, Chernivtsi, Poltava, Kharkiv, Ternopil, Khmelnytsky) in the Forest-Steppe of Ukraine. For further determination of the infection rate, the grain was germinated in wet fi ltration paper at 25 °С for seven days. The seeds with evident signs of Fusarium head blight infection were selected for further molecular identifi cation of the species composition of Fusarium fungi using real-time PCR. For the purposes of molecular-biological studies, DNA was individually isolated from 639 samples of seed material of winter wheat.
The isolation of total DNA from the infected grain was conducted using the commercial test kit "AGRO-SORB NK" (Agrogen Novo, Ukraine), according to the manufacturer's instructions. The DNA concentration from the obtained samples was determined using the spectrophotometer "NanoDrop 1000" (Thermo Fisher Scientifi c, USA) by the optic density at λ = 260 nm, and the purity of the preparation was determined by the ratio of А260/А280 and А260/А230.
The identifi cation of the species composition of Fusarium fungi in the investigated samples involved the use of commercial test systems for PCR-amplifi cation of DNA of phytopathogens in real time, "Fuzarioz zlakiv" (AgroDiagnostica, RU) according to the manufacturer's instruction. PCR-amplifi cation was conducted with the Real-time PCR cycler "CFX 96 TOUCH" (Bio-Rad Laboratories Ltd., USA). The results of PCR-RT were automatically registered and interpreted using the software Bio-Rad CFX Manager 3.1 (Bio-Rad Laboratories, Inc., USA).
In the conditions of the scientifi cally substantiated crop rotation of the Bila Tserkva Breeding Research Station (BTBRS) of the Institute of Bioenergy Crops and Sugar Beet of the NAAS (Mala Vilshanka village, Bila Tserkva district, Kyiv region), we studied the resistance of spelt-like samples of winter wheat from the Bila Tserkva collection of the Chornobyl radio-mutants (samples RM1-10) ( Fig. 1-2).
The collection originates from the wheat plants, selected in 1987 under the guidance of D.M. Hrodzinsky, O.D. Kolomiets, P.K. Shkvarnikov, and M.F. Batyhin in the agricultural fi elds in the 20-km Chornobyl exclusion zone and for two vegetation periods -in the fi eld, sown in 1986 and self-sown fi eld of 1987, which were under chronic radiation on all the stages of organogenesis. As of the time of the Chornobyl catastrophe, the following varieties of soft winter wheat were cultivated in a radius of 20 km from the ruined reactor: Bilotserkivska 47, Myronivska 808, Poliska 70, Kyianka. In 1988, 239 samples in the form of kernels of individually thrashed spikes were transferred to the BTBRS for further studies. The mutated forms are re-sown by the pedigree method; the backcrosses, reciprocal, and analyzing crosses of the obtained mutants with initial varieties are conducted. As a result of non-stable mutagen-esis, the spectrum of mutations has got extended from generation into generation for 34 years. At present, the collection covers about three thousand mutants and the obtained wide spectrum of mutations: with a different degree of spike sterility manifestation, squarehead, spelt with crack duramen of the spike and aggravated threshing, dwarfs with the plant height of 25 cm and high plants of over 150 cm, with twisted stem under the spike, and complicated shooting of the spike from the leaf sheath, with two spikes on the stem, and many other plant forms, ugly in their morphology, which are usually not present in nature. There were also systemic mutations with the features of other hexaploid species: Triticum spelta, Triticum compactum, Triticum Vavilovi (Am agai et al, 2017).
The investigated samples RM-1-10 were obtained as a result of chronic radiation in the fi eld of 1986 and the self-sown fi eld of 1987 in agrophytocenoses of the agricultural farms in the 20 km Chornobyl exclusion zone. After the specialists of BTBRS received morphologically different forms from the initial varieties (1988), every year they re-sow them for 26 generations  by the pedigree method. At the start of the breeding work, the corresponding codes depending on the origin were obtained (Table 1).    (2014), RM-1-10 samples were sown in the control nursery to study their biological, morphological, and economically valuable traits.
The Chornobyl radio-mutants (10 samples: RМ1-10) were sown in the control nursery of the scientifi cally substantiated crop rotation of the BTBRS. The fungicidal treatment was not used during the crop vegeta-  tion; all the registrations were done with the natural infectious background. The area of the experimental plot for one sample is 10 sq.m., there were three repeats, and the number of plants of each sample, selected for the analysis (N) -75 it., the ones selected in the period of 71-85 stages of development (BBCH Growth Stage Keys for cereals). The predecessor was a pea, the seeds were sown in the terms, optimal for the cereal, using the grain seeder СЗ-3.6. For comparison, the standard variety for the Forest-Steppe of Ukraine, Lisova Pisnia, was used, which is considered to be resistant to Fusarium head blight according to the data of the originator and the state variety trial. The resistance of plants to Fusarium head blight was evaluated in the period of maximal development of the disease using the 9-pointscale (Table 2) (Fig. 3) (Trybel et al, 2010). The hydrothermal coeffi cient of Selyaninov was used for a comprehensive evaluation of the humidity conditions (Opalko et al, 2017).
In 2016-2018, the weather conditions were notable for considerable fl uctuation of indices, which affected the growth and development of winter wheat and the investigated pathogen in particular. The conditions of 2016 were more favorable for the development of Fusarium head blight. There were 404.2 mm of precipitation from the time of spring vegetation restoration (March 01) to the complete ripeness of grain (July 09). The amount of precipitation in 2017 and 2018 was less for a similar period of 2016 by 241.3 and 276.1 mm respectively. In 2016, the hydrothermal coeffi cient up to the complete ripeness of grain was 2.33. In 2017 and 2018, the restoration of wheat vegetation occurred on March 06 and April 04, respectively. In 2017, the complete ripeness of grain was registered on July 12, and in 2018 -on July 14, according to the hydrothermal coeffi cient of Selyaninov, 0.86 and 0.66, respectively, from the time of air temperature transition every 10 °С until the completion of wheat ontogenesis (Table 3).
The incidence of the fungi of Fusarium spp. in the experimental samples was calculated by the formula: where С -the incidence, %; А -the number of samples, where this species was found; В -the total number of the investigated samples (Beznosko et al, 2022).
If the registered frequency of microscopic fungi of one species was found to be over 50 %, this species was considered dominant, 30-50 % -rather widespread, 10 % and less -a rare species (Zhdanova, 2002).

RESULTS
To understand the species diversity of the disease agent, we conducted the monitoring of eight species of fungi of the Fusarium genus in the territory of nine regions (Kyiv, Cherkasy, Sumy, Vinnytsia, Chernivtsi, Poltava, Kharkiv, Ternopil, Khmelnytsky) in the Forest-Steppe of Ukraine.
The results of the molecular study demonstrate the presence of the following species of Fusarium fungi on the seed material of soft winter wheat, selected in the agrophytocenoses of the Forest-Steppe of Ukraine in 2016-2018: F. avenaceum, F. culmorum, F. graminearum, F. langsethiae, F. poae, F. sporotrichioides, F. tricinctum. The species of F. cerealis was not found during the studies, and in 2017 there were no F. culmorum and F. langsethiae (Table 4).
The molecular identifi cation of the species of Fusarium fungi demonstrated that in the population of the fungi, on average for the years of studies, the most frequently observed fungus was F. graminearum which is especially dangerous due to its ability to form mycotoxins DON-deoxynivalenol and ZEA-zearalenone (Perczak A et al, 2019). F. avenaceum and F. tricinctum produce MON-moniliformin, which is lethal for mammals and birds. The average incidence of these agents during the years of studies was 25.3, 36.5, and 16.7, respectively.
It should be noted that in the territory of Kyiv region, where the resistance of spelt-like samples of the Chornobyl radio-mutants was evaluated, in addition to the abovementioned species of F. graminearum and F. avenaceum, in 2016, F. sporotrichioides (13.3 %) -a producer of highly toxic Т-2 toxin and F. poae with the share of 6.7 %, mainly forming trichothecenes of B group, were identifi ed. In the following years, the ratio of species changed considerably, and we found F. tricinctum with a frequency of 40.0 % in 2017 and 11.6 % (2018) and in 2018 F. culmorum (5.8 %) -a producer of trichothecenes, was found. In 2017-2018, F. sporotrichioides and F. langsethiae were not found.
It was determined that under conditions, most favorable for the development of Fusarium head blight in 2016, when HTC coeffi cient during the spring-summer vegetation of winter wheat in the studies was 1.97, and the sum of air temperature indices was 170.8 °С, seven investigated samples (RM-1, RM-3, RM-4, RM-5, RM-6, RM-9, RM-10) exceeded the standard variety in terms of resistance (7.0 scores) (Table 5). Yet, only RM-3 and RM-4 were reliably resistant. RM-7 sample Table 4. The incidence of Fusarium fungi in the seed material of winter wheat in the territory of nine regions of the Forest-Steppe of Ukraine (the incidence, % from the total number of cases) Note. n/s * -not studied.  (Table 5). DISCUSSION Ukraine is a part of the region of intense agricultural production in Europe. The cultivation of quality agroproducts depends on weather conditions during the period of plant vegetation. At present, global climatic changes on the planet and the effect of unfavorable environmental factors become more tangible and determinative for a country on the global agrarian market (Batsmanova et al, 2014).

INVESTIGATION OF SPECIES COMPOSITION OF THE FUNGI OF THE FUSARIUM GENUS
Noteworthy are fl uctuating weather conditions, observed in recent years, when each phase of crop vegetation is affected by non-favorable conditions -from drought in autumn to rapid temperature changes in winter, and, as a result, unstable snow cover, which results in the change of the phytopathological situation in the fi elds of winter cereal (Vozhehova et al, 2018).
Perennial studies determined that the damage to the spike and grain by Fusarium head blight occurs under meteorological conditions, favorable for the development of micromycetes, the sensitivity of a host plant, and the presence of a virulent pathogen (Battilani et al, 2016;Moretti et al, 2019). The intensity of Fusarium head blight depends directly on the amount of precipitation in the phases of blossoming-kernel ripening of wheat. Under an excessive amount of precipitation, the damage is serious, and under dry conditions, it is less signifi cant (Kovalyshyna et al, 2017).
Environmental conditions have a considerable effect on the species composition of Fusarium fungi. Every particular species has its specifi c distribution and its niche in the agrocenosis (Xu et al, 2009;Gruber-Dorninger et al, 2017;Medina et al, 2015).
The rapid development of Fusarium head blight of cereals and contamination of food products with fusariotoxins is a problem which has become global now Liew et al, 2018). The wide distribution of Fusarium fungi, their fl exibility, and scientifi cally grounded facts of major hazards, coming from mycotoxins for the health of humans and animals, trigger a considerable interest of the scientifi c community in Fusarium head blight. The scientists' efforts are directed at the study of the life cycle of Fusarium fungi, and the search for ways of effective control of their number in global agrophytocenoses (Gencheva et al, 2020).
A widespread distribution of some species and local distribution of others, constant outbreaks of epiphytoties in some regions and the insignifi cant isolated manifestation of diseases in others demonstrate different ecological needs of Fusarium fungi within the genus. Frequently, an average sample of grain contains 10-15 species of fungi of the Fusarium genus. Yet, simultaneous domination of up to four species of fungi is possible in some territories. Some species are notable for narrow specialization and geographic limitations (Zhang X-X et al, 2015). The species diversity of the population of Fusarium fungi on the seed material of winter wheat in 2016-2018 in the territory of Kyiv, Cherkasy, Sumy, Vinnytsia, Chernivtsi, Poltava, Kharkiv, Ternopil, and Khmelnytsky regions is rather rich ( Table 2). The presence of seven species (F. avenaceum, F. culmorum, F. graminearum, F. langsethiae, F. poae, F. sporotrichioides, F. tricinctum.) out of eight species under investigation may be explained by a large territory and environmental conditions. In Kyiv region, the Right-Bank Forest-Steppe of Ukraine, where fi eld experiments were launched for the evaluation of the resistance of speltlike samples of the Chornobyl radio-mutants, in 2016 under high moisture provision (  (Beukes et al, 2017;Harris et al, 2016;Shen et al, 2012;Hrytsev et al, 2021). It was found that F. poae and F. langsethiae did not manifest visible symptoms of being affected, and the rest of the abovementioned agents had massive or possible manifestations (Table 6) (Kotowicz et al, 2015;Beukes et al, 2017).
Among the most widespread species of Fusarium fungi, high aggressiveness to the spikes of cereals is shown by F. avenaceum, F. culmorum, F. graminearum; medium aggressiveness -F. poae, F. verticillioides, and low aggressiveness -by all the rest of species (Kotowicz et al, 2015;Beukes et al, 2017).
Fusarium head blight is one of the types of infecting wheat plants by the fungi of Fusarium genus; according to different data, this disease may result in a sharp decrease in grain harvest and quality (Malihipour et al, 2017). The character of the disease agent's infecting a plant allows for investigating fi eld resistance depending on the species composition of the pathogen in the agrophytocenosis. The obtained results of the evaluation of fi eld resistance of spelt-like RM samples (Table  5) confi rm the presence of selective RM-samples with high resistance to Fusarium head blight. Under favorable climatic conditions of 2016 and the presence of F. graminearum and F. avenaceum, aggressive to the spike of cereals, we isolated samples RM-3 and RM-4, reliably more resistant compared to the standard. During this period, the resistance of the standard variety Lisova Pisnia was at the level of seven scores, which was under the indices of samples RM-1, RM-5, RM-6, RM-9, RM-10 by 0.3 scores. The high resistance of spelt-like RM samples, confi rmed by the studies, will be used in the selection work of the BTBRS. The analysis of the scientifi c literature demonstrates that the use of genetic resistance is considered to be the most effective and ecologically safe measure of controlling the diseases of crops (Morgun et al, 2016;Kovalyshyna et al, 2017).

CONCLUSIONS
The analysis of the literature data on the geographic distribution, species composition, and the character of damage done by the fungi of the Fusarium genus, con-  Kotowicz et al, 2015;Beukes et al, 2017) Note. ++ -massive manifestation; + -possible manifestation; -no manifestation.
fi rms the global distribution and harmfulness, caused for cereals by the species complex of Fusarium fungi. Field and laboratory studies of species diversity in the territory of nine regions in the Forest-Steppe zone confi rm the presence of the following species of Fusarium fungi in the agrobiocenoses: F. avenaceum, F. culmorum, F. graminearum, F. langsethiae, F. poae, F. sporotrichioides, F. tricinctum. The dependence of Fusarium fungi on the environmental conditions is evident in the results of the studies on the species ratio in conditions of the Right-Bank Forest-Steppe of Kyiv region. In 2016, high indices of HTC and the sum of temperatures promoted the distribution of F. graminearum -46.6 % and in the following years, 2017-2018, in the same period, the amount of precipitation was lower by 241.3 and 276.1 mm respectively, which provoked the occurrence of more drought-stable F. culmorum in 2018 -5.8 % and F. avenaceum in 2017 -25 %, and in 2018 -47.4 %. A close association between the biology of the group population of Fusarium fungi and weather conditions and specifi c character of the manifestation of visual traits of their damage was used by us to investigate the resistance of ten collection spelt-like samples of the Chornobyl radio-mutants to Fusarium head blight in 2016-2018 and to isolate fi ve RM-samples of winter wheat (RM-9, RM-5, RM-6, RM-10, RM-4), which had higher resistance to Fusarium head blight than the standard variety, Lisova Pisnia, on average for three years of studies. The abovementioned RM-samples were introduced by us into the breeding programs of Bila Tserkva BRS to create the varieties, resistant to Fusarium head blight.
Our studies confi rmed the need for permanent monitoring of the dynamics of distribution for Fusarium fungi and the search for the initial material, resistant to one of the most harmful diseases of cereals -Fusarium head blight.

ACKNOWLEDGMENTS
We would like to express our gratitude and appreciation to "Syngenta" companies in Ukraine for fi nancing the molecular part of the studies and the BTBRS for fi nancing some part of the fi eld studies.