Race and virulence characterization of Puccinia graminis f. sp. tritici in China

Wheat stem rust was once the most destructive plant disease, but it has been largely controlled. However, to prevent future problems, the ongoing development of resistant wheat varieties requires knowledge of the changing virulence patterns for Pgt virulence of the fungus that causes wheat stem rust and the detection of new races. Surveys were conducted from 2013–2014 to determine the races of the Pgt present in China. Low levels of stem rust infections were found in China during this investigation and 11 Puccinia graminis f. sp. tritici (Pgt) samples were obtained. In addition, 22 Pgt samples collected from the alternate host (Berberis) were obtained and have been reported for the first time. Fifty-three isolates were obtained from all samples. Four race groups, including 13 physiological races, were identified. They included the most prevalent races, 34C3MTGQM and 34C6MRGQM, with 13.2% predominance, followed by 34C0MRGQM at 11.3%. Six new races that were virulent against the resistance genes, Sr5 + Sr11, were found for the first time in China, namely 34C0MRGQM, 34C3MTGQM, 34C3MKGQM, 34C3MKGSM, 34C6MTGSM, and 34C6MRGQM, with a predominance of 11.3, 13.2, 9.4, 9.4, 1.9, and 13.2%, respectively. Most of the genes studied were ineffective against one or more of the tested isolates, except Sr9e, Sr21, Sr26, Sr31, Sr33, Sr38, Sr47, and SrTt3. Genes Sr35, SrTmp, Sr30, Sr37, Sr17, and Sr36 were effective in 92.5, 86.8, 84.9, 84.9, 79.3, and 77.4% of the tested isolates, respectively. In contrast, all of the isolates were virulent against Sr6, Sr7b, Sr9a, Sr9b, Sr9d, Sr9g, and SrMcN. Our results indicate that remarkable differences exist among the categories of the races in this study (i.e., their known virulence gene spectra) and the Pgt races reported previously. In addition, the sexual cycle of Pgt may contribute to its diversity in China.


Introduction
Wheat stem rust caused by Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn. (Pgt) is a major disease of wheat. Heavy epidemics occurred frequently from the 1920s until the 1960s, causing huge yield losses. In recent years, because of the cultivation and utilization of resistant cultivars of wheat, and changes in the cultivation system in China after 1980, wheat stem rust now only occurs sporadically under natural conditions in the Yangtze River area [1]. However, it is undeniable that this disease had devastated wheat production in China and other parts of a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the world. Exploring resistance genes and growing resistant cultivars are effective measures for controlling this disease because of low cost, effectiveness, and environmental friendliness [2]. However, the changing virulence patterns and the evolution of fungal races constitute a threat to most wheat germplasm resources. For example, the new races, TTKSK (known as Ug99), TKTTF, and TTTTF, and their variants, threaten wheat production worldwide [3][4][5].
Maintaining dynamic information on pathogens is the basis for breeding disease resistance. Although wheat stem rust has not been common in recent years, understanding the population structure, virulence diversity, and distribution of Pgt at various times and in different locations is essential for selecting effective resistance genes to control this disease [6]. Furthermore, work on detecting and identifying races of Pgt has not stopped. Particularly, the emergence and rapid propagation of Ug99 and its variants have attracted increasing attention worldwide, and 28 trapping plots or nurseries have been established yearly in China under the unified arrangement of the wheat rust and powdery mildew cooperative group [7]. Simultaneously, wheat breeding and disease researchers have focused on the investigation and collection of Pgt to identify and analyze wheat stem rust. Particularly, aecia from the wheat stem rust alternate host (Berberis) were collected from 2013-2014. The objective of the present study was to analyze the pathotypes and characterization of Pgt races.

Ethic statement
Samples in this study were obtained from public areas that no specific permissions were required for these locations. Fields sites are public access and P. graminis f. sp. tritici is not an endangered or protected species.

Collection of wheat stem rust samples
In 2013-2014, eleven samples of rust-infected stems were collected from wheat fields in Hubei, Heilongjiang, Liaoning, Guizhou, Sichuan, Yunnan, and Gansu provinces (Fig 1), and 22 samples previously collected from the alternate host, Berberis, alternate host for many Puccinia spp. including graminis and striiformis, were isolated and identified.

Isolation and multiplication of spores
Seedlings of the universally rust-susceptible wheat variety, Little Club (LC), were raised in 12-cm diameter porcelain pots for the propagation of strains. Infected samples were removed from the refrigerator (4˚C), and single pustules from the wheat stem rust samples were isolated in order to inoculate leaves of LC collected from seven-day-old seedlings or seedlings with fully expanded primary and secondary leaves. We followed the procedure of Huang [8]. LC leaves were cut into small pieces (6-7 cm long) and were placed between two pieces of filter paper kept in 15-cm petri dishes. The two ends of each leaf segment were pressed with glass (length 120 mm, width 10 mm, thickness 3 mm) and 40 mgÁL -1 6-benzylaminopurine (BA) was added as a preservative solution to soak the filters. The uredia were scraped from the rustinfected stem samples using flat toothpicks and were then sprayed on the abaxial surface of the lamina. Each leaf was inoculated at two spots by daubing gently, preventing scratching of the foliage. Thereafter, the inoculated leaves were moistened with 0.05% Tween 20 using an atomizer, and were placed in an incubation chamber for 20 h in the dark at 16-20˚C. Subsequently, they were transferred from the dew chamber to glass compartments in an artificial culture room, where the conditions were controlled with a 14 h:10 h (light:dark) photoperiod and temperature of 18-22 ± 1˚C. When the uredinia (diameter of 0.5-0.7 mm) appeared after 5-6 days, single pustules were isolated and used for re-inoculation. The spore multiplication procedure was repeated 2-3 times until sufficient numbers of spores were produced for experimentation. Twenty-three and 30 single pustules were identified from 11 samples obtained from wheat and 22 samples obtained from Berberis, respectively. All materials used in the experimental set-up had been sterilized.

Inoculation of differential lines and designation of races
Differential wheat were planted in 12-cm diameter porcelain pots [9]. Each pot contained four genotypes (5-6 seeds per genotype) separated from each other. The susceptible wheat variety, LC, was used as the control to ascertain the viability of the spores. The leaves of seven-day-old seedlings were moistened with 0.05% Tween 20 using an atomizer. Thereafter, purified singlepustule isolates were sprayed/inoculated evenly onto the back of the leaves with flat toothpicks and then moistened again with 0.05% Tween 20. After inoculation, the plants were placed in a dew chamber with 16-20 h of darkness at 18-22˚C. Then, they were transferred to a greenhouse at a temperature of 18-22 ± 1˚C and relative humidity (RH) of 65-75%. The infections were assessed 14 days after inoculation using the 0-4 Infection Types (ITs) scale, as described by Stakman et al. [10]. The infections were grouped into two types, where values of 0, 1, 1+, 2, and 2+ were considered low infection types (indicating resistance) and values of 3-, 3, 3+, and 4 were considered high infection types (indicating susceptibility). Race names were designated according to the methods described by Li et al. [9]. In this study, the isolates were purified and assessed in three replicates to obtain uniform data regarding infection type.

Occurrence and frequency of Pgt races
From the twenty-three single pustules obtained from the 11 samples, we identified six races, 21C3CTHTM, 21C3CTQSC, 21C3CTTSC, 21C3CTTTM, 34C0MKGQM, and 34C0MRGSM that can be included in the race groups, 21C3 and 34C0. Races 21C3CTQSC, 21C3CTTSC, 21C3CTTTM, and 34C0MRGSM are new physiological races. To the best of our knowledge, this is the first report of the virulence of 34C0MRGSM to Sr5 + Sr11 in China. Race 21C3CTTTM was virulent to the 20 North American single-gene differentials, except Sr5, Sr9e, Sr31, and Sr38 (Table 1). Thirty single pustules were identified from the 22 samples obtained from Berberis. Three race groups, 34C0, 34C6, and 34C3, including seven physiological races, were identified. Six of the seven races are novel and virulent against Sr5 + Sr11, namely 34C0MRGQM, 34C6MTGSM, 34C6MRGQM, 34C3MTGQM, 34C3MKGQM, and 34C3MKGSM, whereas the other race (34C0MTGSM) has been previously identified.
Among the 13 races identified from the 53 isolates, the most predominant races were 34C3MTGQM and 34C6MRGQM, each with a frequency of 13.2%. The next predominant race was 34C0MRGQM, with a frequency of 11.3%. ( Table 2).
The virulence information of 13 physiological races against 46 genes revealed that the races, 21C3CTTTM, 21C3CTTSC, and 21C3CTQSM, included in the 21C3 race group and the race, MKGSM, in the 34C3 race group have broader spectra than do the other races. Race 21C3CTTTM and 34C3MKGSM have the broadest spectra and rendered 34 of 46 stem rust resistant genes ineffective. Race 21C3CTTSC and 21C3CTQSM have second broadest spectra, which demonstrated virulence against 31 of 46 stem rust resistance genes (Table 4).

Discussion
Due to the potential threat of new emerging races of Pgt such as Ug99 and their potential threat to global wheat production, this study aimed to identifying and characterising Pgt races in China from 2013-2014. The results revealed race groups that had been documented earlier, such as 34C1 and 34C2, and rare race groups (e.g., 34C4 and 116), all of which were not detected in the present study. In the 2013-2014 study, race groups 21C3 and 34C3 were predominant in China [11,12]. Previous studies suggested that this observation was related to the strong competitiveness of this pathogen [13,14]. Its wide spectrum of virulence may be another reason [11]. This was confirmed in our study by testing the virulence of 53 different strains against 46 resistance genes. For instance, race CTTTM, which has the widest virulence spectrum in group 21C3, was virulent to 33 of the 46 resistance genes, including Sr17, Sr24, SrTmp, and Sr36. Only 15 resistance genes, specifically Sr5, Sr9e, Sr14, Sr19, Sr22, Sr23, Sr26, Sr27, Sr31, Sr35, Sr37, Sr38, Sr47, SrGT, and SrTt3, were found to be effective against CTTTM. Race group 21C3 was dominant, occurring with high frequency, and significant changes in its population structure have occurred. For example, races CTHTM, CTQSM, CTTSC, and CTTTM are new to the 21C3 group.
Berberis is the alternate host of Pgt and plays a vital role throughout the sexual life cycle of Pgt [15]. Sexual reproduction of Pgt is completed on the leaves of Berberis, resulting in the diversity of Pgt races [16,17]. Since the 1950s, wheat stem rust has been effectively controlled  response; Symbols + and-indicate slightly larger and smaller pustule sizes, respectively; N was used for cases with significant necrosis [10]. C Symbol '+?' means the cultivar carry other unknown resistance gene.