Races CYR34 and Suwon11-1 of Puccinia striiformis f. sp. tritici Played an Important Role in Causing the Stripe Rust Epidemic in Winter Wheat in Yili, Xinjiang, China

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici is a destructive disease. Its pathogen frequently adapts to newly invaded regions and overcomes resistance in wheat cultivars. This disease is especially important in China due to its favorable conditions for the stripe rust epidemic and the recombination population structure of pathogens. Xinjiang is a vast epidemic region in China, but very limited research on this disease has been performed in this region. In this study, we identified 25 races from 129 isolates collected from winter wheat fields from five different regions (Nileke, Xinyuan, Gongliu, Huocheng, and Qapqal) of Yili, Xinjiang, using the Chinese set of 19 differential wheat lines. All isolates were virulent on the differentials Fulhad and Early Premium, but no isolates were virulent on Yr5. Among the 25 races, Suwon11-1 was the most prevalent, followed by CYR34. Both races were found in four out of the five locations. It is important to continue monitoring stripe rust and its pathogen races in this region, as it forms a pathway between China and Central Asia. Collaborative research is essential for controlling stripe rust in this region, other regions in China, and neighboring countries.


Introduction
Plant genetic resistance is an effective approach to minimizing disease damage. The concept of plant resistance (R) began in the early 20th century [1]. Later, it was advanced by Harold Henry Flor's groundbreaking research establishing the gene-for-gene model [2]. Hosts have multilayered defense mechanisms against pathogens [3]. On the other side, pathogens have evolved strategies to overcome the defense responses of their plant hosts [4]. Successful pathogens often evade detection by host R genes [5]. Thus, disease resistance conferred by a single R gene often lacks durability in the field because pathogens can evolve to evade recognition by mutating the corresponding avirulence (Avr) gene. Multiple R genes are often introduced simultaneously to improve durability and broaden the resistance spectrum [6,7].
Puccinia striiformis f. sp. Tritici (Pst) is one of the most destructive pathogens causing wheat stripe rust disease, and frequently mutates and overcomes resistance genes in major wheat cultivars [8][9][10]. The race identification of Pst in China was started in 1940 by

Wheat Stripe Rust Surveillance and Sample Collection
Wheat stripe rust surveillance was conducted in winter wheat fields in the Yili district, including Nileke, Gongliu, Xinyuan, Qapqal and Huocheng in the Xinjiang Autonomous Region of northwestern China from May to July 2021 following the protocol as described by Ali and Hodson [31]. The area of each of these counties ranges from 7850 to 70,650 square kilometers. Stripe rust samples were collected from five sites in each county. The sites were at least 10-15 km away from each other (Supplementary Table S1). Rust prevalence (percentage of plants infected), rust severity (percentage of leaf area infected), cultivar name, latitude, longitude, and altitude were recorded. Leaf samples with fresh single-stripe uredinia were collected from and packaged in a moisture-absorbent paper bag for drying for one to two days, and then the leaf samples were stored at 4 • C with a moisture-absorbing silica gel in an airtight bag.

Spore Multiplication and Preservation
For urediniospore multiplication, the leaf samples were first moisturized on a wet filter paper in a Petri dish at 10 • C for 10 h in darkness to induce fresh urediniospore production [32]. The 10-15-day-old seedlings of Mingxian 169, which is a susceptible wheat genotype, were inoculated with urediniospores from a single uredinium using a sterilized inoculating needle. Inoculated seedlings were sprayed with water mist and incubated in a dew chamber at 10 to 13 • C for 24 h in darkness. Then, the inoculated seedlings were maintained in a growth chamber with a day/night thermoperiod of 17/13 • C, a photoperiod of 16 h, relative humidity of 60%, and weekly watering. Fifteen days later, inoculated leaves began to sporulate. Urediniospores were collected in a test tube and used to inoculate new Mingxian 169 seedlings to produce an adequate amount of urediniospores. Urediniospores were collected in a clean glass tube and stored in a desiccator at 4 • C for a short period of time [33]. For long-term preservation, urediniospores were stored at −20 • C. Before use, urediniospores in the sealed glass tube were heat-shocked by submerging the tube in warm water (about 50 • C) for exactly 2 min. The urediniospore collection from a single leaf sample was treated as one isolate and used in virulence tests to identify the race.
Virulence phenotypes (previously identified races) were used to determine Pst races. Major virulence races were designated as CYR (Chinese Yellow Rust) races and some other races nominated with abbreviations of specific wheat differential genotypes, and new virulence patterns with a low frequency were temporary designated as P_1 series as described by Zhan et al. [16]. Frequencies and distribution, races and virulence factors on differential genotypes were analyzed. Cluster analyses were carried out using the Ward method [34] to assess the interrelationship of the location populations.
Race diversity varies from region to region. The maximum race diversity was fou in Xinyuan and Qapqal ( Figure 3); some races were found only in specific locations, su as P_20 and Suwon11-8, which were only detected in the Gongliu region. Races CYR Hy7, Suwon11-4, and Suwon11-7 were specific to Qapqal, and Hy-4 and P_23 were spec to Xinyuan. The predominant race in Gongliu was Suwon11-1 (22%), followed

Discussion
In the present study, we analyzed the virulence of stripe rust samples from the winter wheat region of Yili, Xinjiang in 2021. Xinjiang is the largest wheat producer in northwestern China, with an annual wheat growing area of about 1.13 million ha, accounting for 4% of the wheat area in China. It is classified as a separate wheat zone due to its unique geographic location and climatic conditions. This region is highly diverse and has different characteristics due to geographical subdivisions based on deserts, high mountains, and temperature fluctuations [36]. Furthermore, it is close to Central Asia. These spatial characteristics are highly influential for the dynamics of P. striiformis f. sp. tritici races [30]. There are approximately equal proportions of winter and spring wheat in Xinjiang. However, recent field surveys conducted in 2021 highlighted that winter wheat crops are severely affected by stripe rust. In this study, we found that CYR34 and Suwon11-1 were prevalent in Yili, with 14% and 18.6% frequencies, respectively. Previously, the race CYR34, which was virulent against Guinong 22 with Yr26, was absent in Xinjiang [16], but present in other epidemic regions of China [37]. This virulence against Yr26 was also found in other continents, although it had low frequencies [20,38]. In recent years, reports of CYR34 in the winter epidemic region of Xinjiang have presented an alarming situation for breeders. They need to develop resistant cultivars with high yield potential to replace the currently grown cultivars. Most recently, the breeding line 041133 was found to be highly resistant to CYR34 [39].
Epidemiologically, the Xinjiang region is an independent region where Pst can survive in some areas all year long. The Xinjiang Pst population is genotypically different from the rest of the epidemic regions of China. However, many multilocus genotypes (MLG) have been detected in this area, suggesting that this region's overall population was recombinant and could pose a potential threat to other regions [25]. Our study also detected 21 previously identified races and 4 new races. As the new races were observed at low frequencies, we assigned them temporary names in this study.
The deployment of resistance genes is the most economical and sustainable technique to control stripe rust [40,41]. So far, more than 80 resistance genes (Yr1-Yr83) have been discovered [12,42,43], and not all of them are widely used in wheat programs globally. Yr26 has become ineffective against CYR34 (V26) since 2011 [44]. Previous studies reported that 10% of Chinese winter wheat cultivars are resistant to CYR34. In our study, the CYR series races (CYR28-CYR34) were found in the Xinjiang region. CYR28 and CYR29 were first found in China in 1983-1985 [45] and belong to the Lv10/13 pathotype group. These races caused a massive epidemic in 1990 and affected 62% of the total planted area [12]. CYR30, which is virulent to Hybrid 46 (Yr3b, Yr4b, and YrH46), and CYR31, which is virulent against Hybrid 46 and Suwon 11, were first detected in 1991 in Sichuan and 1993 in Gansu, respectively, and became the predominant races in 1996 [46,47]. Our results revealed that in the overall population of Yili, Xinjiang, Vr2 and Vr7 had 100% frequency. In contrast, Yr5 is still effective in this region. However, recently, a new race, TSA-6, was found to be virulent to Yr5. This race has a similar virulence pattern to the CYR32 and CYR34 races. It can evolve and generate new races [48,49]. Nowadays, wheat cultivars with the Yr9 gene are susceptible to stripe rust worldwide. The breakdown of Yr9 has been reported since the late 1980s due to the widespread use of this gene in wheat production worldwide, resulting in major epidemics of stripe rust [50,51]. In China, the first cases of the Yr9-virulent races Lv10 and Lv13 were detected in Longnan in Gansu province [52,53]. Our study also reported virulence against Yr9 in the winter wheat crop in the Xinjiang region. As the genetic bases of resistance in most Chinese differentials used in the present study are not fully understood, it is difficult to compare Chinese Pst races with those in other countries.
There are several reasons for the variation in virulence in Xinjiang regions. One could be the presence of Berberis species, identified as potential alternate hosts for Pst in the Xinjiang region [28]. The long-distance dispersal of the pathogen by the wind may also be responsible for its racial diversity. The cultivars grown in this region, which are different from those grown in other regions, might have selected and maintained unique races. The growing of both winter and spring wheat crops in this region may also increase the pathogen's variation. More studies are needed to characterize Pst populations on spring wheat.

Conclusions
In this study, race identification was conducted in the Yili region of Xinjiang, and 25 races were identified. CYR34 and Suwon11-1 were the top two predominant races. The wheat differentials Fulhard and Early Premium lines were susceptible, whereas Yr5 in the differential T. spelta var. Album was still effective. It is essential to understand the population structure of Pst in Xinjiang and its genetic relationships with the populations in other regions, especially in neighboring Central Asian countries. Such information should provide the basis for developing effective strategies for the control of the disease.  Data Availability Statement: Original data presented in this paper are available within paper. If further detail needed, contact corresponding authors.