Population structure of leaf pathogens of common spring wheat in the West Asian regions of Russia and North Kazakhstan in 2017

Листовые болезни яровой пшеницы – бурая ржавчина (возбудитель – Puccinia triticina), желтая пятнистость ( пиренофороз) (Pyrenophora tritici-repentis) и темно-бурая пятнистость (Сochliobolus sativus = Bipolaris sorokiniana) – относятся к группе распро страненных и потенциально опасных болезней в западноазиатских регионах России и Северном Казахстане. Для обоснования стратегий генетической защиты пшеницы необходимы популяционные исследования фитопатогенов. Цель работы – характеристика структуры популяций возбудителей бурой ржавчины и желтой пятнистости яровой пшеницы по признакам вирулентности и оценка распространенности возбудителя темно-бурой пятнистости в западноазиатских регионах Российской Федерации и Северном Казахстане в 2017 г. Источником инфекционного материала служили пораженные бурой ржавчиной и пятнистостями листья образцов яровой пше ницы, собранные в Челябинской и Омской областях и Северном Казахстане. Анализ вирулентности 109 изолятов P. triticina на 20 линиях-дифференциаторах показал, что все изученные монопустульные изоляты были авирулентны к ТсLr24. Изоляты, вирулентные к ТсLr19, выявлены только в челябинской популяции. Частоты вирулентных изолятов к ТсLr2a, ТсLr2b, ТсLr2c, ТсLr11, ТсLr15, ТсLr16, ТсLr20 и ТсLr26 были выше в омской и североказахстанской популяциях, а к ТсLr9 – в челябинской. При использовании 20 ТсLr-ли ний определено 27 фенотипов вирулентности P. triticina: 12 в омской, 19 в челябинской, 8 в ка захстанской. Феноти пы TLTTR (авирулентность (av) к ТсLr16, ТсLr19, ТсLr24, ТсLr26), TCTTR (av: TcLr9, TcLr16, ТсLr19, ТсLr24), TBTTR (av: ТсLr9, ТсLr16, ТсLr19, ТсLr24, ТсLr26) встречались во всех регионах. Фенотипы TQTTR (av: ТсLr19, ТсLr24, ТсLr26) и TGTTR (av: ТсLr9, ТсLr19, ТсLr24, ТсLr26) были общими для омской и североказахстанской популяций, а THPTR (av: ТсLr9, ТсLr11, ТсLr19, ТсLr24) и TCTTQ (av: ТсLr9, ТсLr16, ТсLr19, ТсLr20, ТсLr24) – для омской и челябинской. Определено высокое генетическое сходство омской популяции с североказахстанской и челя бин-

Fungus population studies are important for improving genetic strategies of wheat protection. With these studies, the researcher can characterize the race composition dynamics, effectiveness of resistance genes at the host plants and evaluate the influence of commercial wheat varieties on fungus population changes. The population biology of leaf rust pathogens is the most studied. By virulence and microsatellite analyses, the existence of a common P. triticina population in the Urals West Siberia, and Kazakhstan (Mikhailova, 2006;Kolmer, Ordoñez, 2007;Kolmer et al., 2015; was shown, which should be taken into account when disposing varieties with Lr-genes. Annual virulence surveillance of P. triticina populations conducted by the Chelyabinsk Scientific Research Institute of Agriculture and the Omsk State Agrarian University allows the dynamics of pathogen variability to be monitored and breeding programs to be improved. First studies of P. tritici-repentis in Russia were carried out by Mikhailova et al. (2010Mikhailova et al. ( , 2015. The existence of several P. triticirepentis populations in Russia (North Caucasian, Northwestern and West Siberian) was determined according to virulence frequencies in a special wheat differential set. An independent status of the Omsk P. tritici-repentis population was also confirmed by microsatellite markers (Mironenko et al., 2016).
In the world literature, data about differential interactions between the plant host and С. sativus are controversial. The absence of differential sets significantly limits the population studies of the spot blotch pathogen based on virulence (Mikhailova et al., 2002). Mycological analysis is usually used to assess the spread of this pathogen and to estimate the prevalence of C. sativus isolates.
Kazakhstani material. The isolates of tan spot were identified in all the regions. Five races of P. tritici-repentis were identified among Chelyabinsk isolates based on the toxins produced by the following pathogens: race 1 (PtrToxA PtrToxС); race 2 (PtrToxA); race 7 (PtrToxA, PtrToxВ), race 8 (PtrToxA, PtrToxВ, PtrToxС), and race 4 (does not produce toxins). Three races were identified in the Omsk region (1 -3) and four, in North Kazakhstan (1 -4). A total of 26 P. tritici-repentis phenotypes were identified by virulence analysis using 11 differential lines: two were present in all the populations; two. in Chelyabinsk and North Kazakhstan; one, in Omsk and Chelyabinsk; and all the others were original. A high degree of similarity between the obligate pathogen P. triticina and the saprophytic pathogen P. tritici-repentis in the West-Asian region of Russia and in North Kazakhstan demonstrates that this is one epidemiological region across this wheat production area. The presence of common phenotypes suggests there is a the possibility of gene exchange between the populations and this shall be considered while releasing genetically protected wheat varieties.
Key words: leaf rust; tan spot; spot blotch; spring wheat; populations; virulence; Lr-genes. Most population studies of lead rust and tan spot pathogens have been carried out in independent experiments. It was relevant to conduct a comprehensive analysis of the structure of pathogens that differed in parasitic type (obligate vs. hemibiotrophic), using a similar infectious material collected in geographically remote regions. The objective of this study was to explore the population structure studies of the causative agents of leaf rust and tan spot on spring wheat based on virulence and to assess the distribution of the causative agent of spot blotch in the West-Asian region of Russia and North Kazakhstan in 2017.

Materials and Method
Wheat samples with leaf rust and leaf spot symptoms were collected from the Ural (Chelyabinsk) and the East Siberian (Omsk) region of Russia and North Kazakhstan in 2017. Leaf rust severity at the sampling locations ranged from moderate to strong and spots, from low to moderate.
In the Chelyabinsk region, leaves were collected from 30 spring wheat samples in the breeding nursery of the Chelyabinsk Scientific Research Institute of Agriculture. In the Omsk region, leaves were collected from 40 wheat samples growing in the experimental fields of the Omsk State Agrarian University and Cherlak and Pavlodar state variety test plots. In Kazakhstan, infectious material was collected from commercial fields at seven points of the North Kazakhstan region and at two in the Akmola region.
Leaf rust uredinia from dry leaves were renewed on a susceptible wheat variety and single pustule isolates were obtained. Isolates' multiplication for virulence analysis was carried out using a laboratory method of pathogen cultivation. Single uredinial isolates were tested for virulence to 20 near isogenic lines of Thatcher wheat that differed in single leaf rust resistance genes. Three seeds of each of these Thatcher lines were sowed to a pot filled with soil. Each set of 10-14 dayold differentials (the first leaf stage) was spray inoculated by urediniospores of each isolate (10 6 /ml) and kept in a Versatile Environmental Test Chamber (Sanyo) at optimal temperature (22 °С) and moisture (75 %) (Gultyaeva, Soloduhina, 2008). Virulent phenotypes were determined 10 days after inoculation using E.B. Mains and H.S. Jackson scale (1926), where 0 means no visible uredia; 0, hypersensitive flecks; 1, small uredia with necrosis; 2, small-to medium-sized uredia with green islands and surrounded by necrosis or chlorosis; 3, medium-sized uredia with or without chlorosis; 4, large uredia without chlorosis; Х, heterogeneous, similarly distributed over the leaves. The plants with infection types 0 to 2 were classified as resistant and infection types 3 to 4 and Х as susceptible.
A differential set of 20 near isogenic TcLr-lines was used for studying the leaf rust pathogen's population structure. Each isolate was given a five-letter code based on virulence or avirulence to each of the five subsets of four differentials as adapted from the North American nomenclature for virulence in P. triticina (Long, Kolmer, 1989). The following order of sets was used: 1, Lr1, Lr2a,Lr2c,and Lr3а;2,Lr9,Lr16,Lr24,and Lr26;3,Lr3ka,Lr11,Lr17,and Lr30;4,Lr2b,Lr3bg,Lr14a,and Lr14b;5,Lr15,Lr18,Lr19, and Lr20. The first three groups were similar to the original differential set (Long, Kolmer, 1989) widely used for P. triticina population studies (Kolmer, Ordoñez, 2007;Kolmer et al., 2015). Thatcher lines highly informative for differentiation of Russian populations were included in the other two groups . Five-letter phenotype codes, virulence frequencies, Nei (Hs) and Shennon (Sh) indexes of population diversity were determined using Virulence Analysis Tool (VAT) software package (Kosman et al., 2008).
Leaf segments with one infection spot surround by an area of green tissue were cut out for tan spot and spot blotch studies and put on agar medium V4 (Mikhailova et al., 2012). Dishes with leaf segments were incubated in a thermostat with UV lamps (LE-30) and at a temperature of 20 to 22 °С for three days and were then placed in a refrigerator (5-8 °С) for one day for stimulation of P. tritici-repentis conidia development. The frequency of P. tritici-repentis and С. sativus colonies obtained from different geographic populations was used as a criterion of the distribution of these pathogens.
Reproduction of P. tritici-repentis fungus culture was carried out according to L.A. Mikhailova et al. (2012). Virulence analysis was carried out using methods of cutting leaves placed on the benzimedazole solution (0.004 %).
The racial identity revealed by the ability of P. triticirepentis isolates to form the toxins Ptr ToxA, Ptr ToxB and Ptr ToxC was determined by inoculation of the cultivar Glenlea, lines 6B662 and 6B365, by the presence of necrotic and chlorotic spots on wheat leaves (Lamari, Bernier, 1989;Lamari et al., 1998).
The virulence of P. tritici-repentis isolates was studied using the following set of cultivars: Allies (France); Norin 58, Satsukei 86, Hokkai 252, Komadi 3 (Japan); Riley 67, Clark (USA); Asiago (Italy); Salamouni (Egypt); and M3 (Canada), which differentiate the fungus isolates for their ability to produce necrosis and chlorosis. The type of infection caused by isolates was assessed using a five-point scale corresponding to the size of necrotic and chlorotic spots, according to Mikhailova et al. (2012). A comparison of the population samples on the basis of virulence was carried out according to the index of the average score of infection per isolate (the ratio of the sums of the points exhibited by isolates on tan spot wheat differential sets to the number of isolates). For the determination of phenotypes, only the indicator of the necrotic reaction evaluation was used, since it characterizes the result of the action of one (Ptr ToxA), and not two toxins, as in the case of a chlorotic reaction, when two independent traits appear that are identical in phenotype (Mikhailova et al., 2010). The results of the virulence evaluation of P. triticirepentis isolates were presented as a binary matrix: 1, virulence (scores 3-5); 0, avirulence (scores 0-2).
The degree of genetic similarity between the Omsk, the Chelyabinsk and the North Kazakhstani populations of P. triticina and P. tritici-repentis for virulence was evaluated using Nei (Nei genetic distance, Nei D) and Fst indexes calculated by GenAlEx (Genetic analysis in Excel, 6.5 http:// biology.anu.edu.au/GenAlEx) software package.

Results and Discussion
One hundred and nine single-pustule isolates -30 from Chelyabinsk, 45 from Omsk and 34 from North Kazakhstanwere characterized during the leaf rust population studies. All single-pustule isolates studied were avirulent to TcLr24.
Isolates virulent to TcLr19 were detected in the Chelyabinsk population. The prevalence of isolates virulent to TcLr2a, TcLr2b, TcLr2c, TcLr11, TcLr15, TcLr16, TcLr20 and TcLr26 was higher in the Omsk and the North Kazakhstani population and of those virulent to TcLr9, in the Chelyabinsk population (Table 1). A high virulence to Lr9 in the Chelyabinsk population in comparison to the other populations studied was due to a high prevalence (10 %) of varieties with this gene in the infectious material (3 % in the Omsk population).
A high efficiency of the Lr24 gene in the regions of Russian Federation is due to the absence of commercial varieties with this gene. Nevertheless, at present, Lr24 donors are used in breeding in Russia (Tyunin et al., 2017). The world practice of cultivating varieties with the Lr24 gene shows that mass cultivation is rapidly followed the emergence of virulent races and the gene loses its effectiveness. Virulence to Lr24 occurs in P. triticina populations across North America and Australia, where wheat varieties protected by this gene are widely grown (McIntosh et al., 1995).
Isolates virulent to the Lr19 gene were observed only in the Chelyabinsk population and were isolated from the line protected by this gene. They were not detected on any wheat sample carrying Lr19 and Lr26 at once -not, for example, on cv. Omskaya 37 or cv. Omskaya 38. Virulence to Lr19 gene is more often noted in the Volga region, where varieties with this gene are grown, but it can also occur in other regions (Kovalenko et al., 2012;Tyunin et al., 2017). All P. triticina isolates studied virulent to TcLr19 were avirulent to TcLr26. Similar observations were made for isolates virulent to TcLr9. Expanding areas with varieties carrying Lr9 provides for increase in the frequency of isolates with virulence to Lr9 in the West Asian regions of Russia, which are as powerful accumulators of infection (Meshkova et al., 2012, Tyunin et al., 2017. To stabilize the phytosanitary situation in the Urals and West Siberia, a strategy of pyramiding the Lr9 and Lr19 genes with Lr26 and other Lr-genes may be useful, because their effective combination will help prolong the "useful life" of new varieties. Twenty-seven virulent phenotypes (12 from Omsk, 19 from Chelyabinsk and 8 from Kazakhstan) were determined using 20 TcLr lines ( Table 2). The phenotypes TLTTR, TCTTR and TBTTR were found in all the populations studied. The phenotypes TQTTR and TGTTR were common in the Omsk and the North Kazakhstani population, while THPTR and TCTTQ were common in the Omsk and the Chelyabinsk population. A high degree of similarity by the virulence phenotypes indicates gene flow between pathogen populations in the study area in 2017. In general, no significant changes in the phenotypic composition of the Omsk and the Chelyabinsk population were observed in 2017 as compared to 2014-2016 (Tyunin et al., 2017).
The Nei (Ns) and Shannon (Sh) indices, which characterize the in-population genetic diversity, showed that the Chelyabinsk population was more heterogeneous for virulence (Ns = 0.21) and phenotypic composition (Sh = 0.82) compared to the Omsk and the North Kazakhstani population (Ns = 0.09 and 0.06, Sh = 0.51 and 0.49, respectively).
Nei's genetic distance (N) indicated a high similarity between the Omsk, the North Kazakhstani (N = 0.03) and the Chelyabinsk population (N = 0.05) and a moderate similarity between Chelyabinsk and North Kazakhstan (N = 0.13). The results obtained in 2017 suggest there had been no changes in the structure of the populations studied compared to the previous time (Kovalenko et al., 2012;Gultyaeva et al., 2017;Tyunin et al., 2017).
A total of 466 infected samples (segments of leaves with separate spots) were studied: 125 from Omsk, 215 from Chelyabinsk, and 126 from North Kazakhstan. The prevalence of C. sativus and P. tritici-repentis isolates was 12 % and 14 %, respectively, in Omsk samples; 3 % and 25 %, in Chelyabinsk; and 0 % and 43 % in North Kazakhstan. Thus, the presence of the causative agent of spot blotch disease of wheat was stronger in the Omsk than in Chelyabinsk region. In North Kazakhstani leave samples, C. sativus was not observed. P. tritici-repentis was noted in all regions. The prevalence of P. tritici-repentis isolates was higher in North Kazakhstani and Chelyabinsk samples and lower in Omsk.
Nineteen Chelyabinsk, 8 Omsk and 27 North Kazakhstani isolates of P. tritici-repentis were used to analyze the population structure on the basis of virulence and toxicity. P. tritici-repentis races identified in the three populations by toxicity are presented in Table. 3. Five races were found in the samples of the Chelyabinsk population; three, in Omsk; and four, in North Kazakhstan.
The racial structure of Omsk P. tritici-repentis isolates was characterized by a higher diversity in 2017 than 2007, when two races were found: race 2 and race 7 (Mikhailova et al., 2010). Races 1 to 4 of P. tritici-repentis, which predominate in the study populations, are also widely distributed in other Russian regions (Central European and North Caucasian) (Mikhailova et al., 2010(Mikhailova et al., , 2012. In general, a high incidence of isolates producing PtrToxA (87-95 %) was noted (see Table 3), which indicates a potential harmfulness of yellow spot in the West Siberian and the Ural region of Russia and North Kazakhstan.
Twenty-six phenotypes of P. tritici-repentis were identified by virulence analysis using 11 differential cultivars (on the