Virulence and Diversity of Blumeria graminis f. sp. tritici Populations in China

Journal of Integrative Agriculture2014, 13(11): 2424-2437 November 2014RESEARCH ARTICLE

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(13)60669-3

Virulence and Diversity of Blumeria graminis f. sp. tritici Populations in China

ZENG Fan-song1, 2, YANG Li-jun1, GONG Shuang-jun1, SHI Wen-qi1, ZHANG Xue-jiang1, WANG Hua1, XIANG Li-bo1, XUE Min-Feng1 and YU Da-zhao1, 2

1 Laboratory of Integrated Pest Management on Crop in Central China, Ministry of Agriculture/Institute for Plant Protection and Soil Science, Hubei Academy of Agricultural Sciences, Wuhan 430064, P.R.China

2 College of Life Sciences, Wuhan University, Wuhan 430072, P.R.China

Abstract

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important disease in China. To characterize the virulence and diversity of the pathogen, 1 082 isolates were obtained from 8 major wheat-growing regions during the spring growing season in 2011. The virulence test was performed by inoculation on detached leaves of 22 differential lines with known Pm genes. Frequencies of virulence on these genotypes ranged from 0 to 97.4%. None of the 1 082 isolates was compatible to Pm21 and less than 20.0% were virulent to the genotype carrying Pm13. In contrast, the virulence frequencies of each population was more than 50.0% to differentials carrying Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8. In total, 1 028 pathotypes were detected, of which 984 were unique. Phenotypic diversity indices revealed a high level of diversity within populations. Genetic distance between different populations correlated significantly with geographical distance (R2=0.494, P 0.001). In addition, isolates from Xinjiang appear to form a separate group. Significant positive or negative associations between alleles at pairs of virulence loci were detected in 57 allele pairs to Pm genes. Virulence and diversity of the 8 populations suggested that varieties with effective resistance gene combinations should be developed at a regional level.

Key words: Blumeria graminis f. sp. tritici, virulence, diversity, wheat, China

INTRODUCTION

Wheat powdery mildew, caused by Blumeria graminis (DC) E.O. Speer f. sp. tritici Em. Marchal (syn. Erysiphe graminis DC. f. sp. tritici Em. Marchal), is one of the most economically important diseases of wheat (Triticum aestivum) in many cool wheat-producing regions. Yield losses of up to 20% have been reported in Canada on highly susceptible cultivars without fungicide application (Conner et al. 2003). In China, the disease affected up to 12 million hectares in 1990 and resulted in estimated

grain yield loss of 14.4 million metric tonnes (Liu and Shao 1994). An average of more than 6 million hectares per year has been affected by this disease over the last decade (http://www.agri.gov.cn/).

Although fungicides can be utilized to control this disease, deployment of resistant cultivars is a more effective, economical and environmentally friendly method. Several race-specific major powdery mildew re-sistance genes (Pm genes), such as the alleles at the Pm3 locus, have been widely and successfully deployed in resistance breeding programmes (Yahiaoui et al. 2004). As of May in 2013, 61 resistance genes or alleles had been reported at 47 loci (Pm1a-Pm47) (Yahiaoui et al.

Received 3 July, 2013 Accepted 30 December, 2013ZENG Fan-song, E-mail: [email protected]; Correspondence YU Da-zhao, Tel/Fax: +86-27-87380681, E-mail: [email protected]

Virulence and Diversity of Blumeria graminis f. sp. tritici Populations in China 2425

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

2004; Xiao et al. 2013). However, the pathogen often develops virulence against commonly deployed genes, leading to an increase in the frequency of virulent isolates and increasing disease levels in the crop (McDonald and Linde 2002). Strategies that are thought to be effective in overcoming the cyclic breakdown of resistance genes include employing multi-genetic, adult plant resistance genes to lower diseases severity, breeding cultivars with pyramided Pm genes, or using multiline strategies to prolong effective periods of resistant cultivars (Brunner et al. 2011).

Monitoring virulence, diversity and dynamics of pow-dery mildew populations is a prerequisite for efficient use of race-specific Pm genes. Virulence data obtained from these populations can help in prioritizing Pm genes for utilization in breeding lines (Heun 1987). For example, if several characterized resistance genes have been over-come by the pathogen, then it would be of little use to combine these in a pyramid breeding strategy.

Virulence and diversity of B. graminis f. sp. tritici has been studied extensively throughout the world and investigations in Brazil (Costanilan 2005), the United States (Niewoehner and Leath 1998; Parks et al. 2008), Hungary (Szunics 2001), Slovakia (Slováková et al. 2004), Czech (Vechet 2012), Iran (Elyasi-Gomari and Lesovaya 2011) and Morocco (Imani et al. 2002) have been conducted. In China, Yu (2000a) analyzed the virulence structure of 1 750 isolates collected in 1996 and 1997 at different altitudes in central China. Chi et al. (2007) revealed the presence of two dominant races in growing regions of Shandong and Hebei provinces. The research by Zhu et al. (2012) who monitored B. graminis f. sp. tritici populations in the spring wheat-growing areas of Northeast China during 2004 to 2010 showed that the dominant race was different in each of the time periods of 2004-2006, 2007 and 2008-2010. Although different races were present in each period, virulence to Pm2, Pm4b, Pm2+6, Pm4+8, Pm12, Pm16, Pm18, Pm21, Pm22 and Pm23 occurred at relatively low frequencies of 0 to 26.5%. Despite these and other virulence structure studies in different growing regions of China, a nation-wide comparative analysis is difficult as many of the studies use different differential sets. A large-scale sur-vey to determine virulence structure, pathotype diversity and geographic patterns in the B. graminis f. sp. tritici population in China is essential to generate an accurate

understanding of the epidemiology of this pathogen. The objectives of this study were to (1) investigate the pathotype diversity and virulence of isolates to 22 Pm genes, (2) assess genetic distance among the 8 popula-tions in major wheat-growing regions and the correlation between genetic distance and geographic distance and (3) identify possible associations among 22 avirulence loci of isolates corresponding to respective Pm genes.

ResUlTs

Virulence frequencies

In all, 1 082 single colony isolates from 8 major wheat-growing regions were obtained in 2011 (Table 1). Only 36 isolates from 58 single pustule samples were recovered from Xinjiang, probably due to the long distance in transporting the samples to Wuhan. The virulence structure of 1 082 isolates to 22 differentials was investigated. Virulence frequencies on these differ-entials varied from 0 to 97.4% (Table 2). None of the isolates was virulent to Yangmai 5-Haynaldia villosa translocation line (T6VS·6AL) which possessed Pm21. Isolates virulent to the genotype carrying Pm13 were detected in relatively lower frequencies with no more than 17.0% in any population. Virulence frequencies to Pm5b and mlxbd reflected that resistance spectra of both genes were moderate to broad in each population. In contrast, at least 56.0% isolates were virulent to Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8, and more than 84.0% of them were virulent to Pm5a and Pm6. The χ2 test demonstrated a divergent distribution of frequencies (P<0.05), according to the frequencies of each population to Pm1e, Pm2, Pm3a, Pm3d, Pm4a, Pm4b, Pm7, Pm17, Pm19 and Pm20 (Table 2). In detail, more than 80% of isolates from the North China (NC) growing region were virulent to genotypes carrying Pm4a or Pm4b, however, fewer than 27.0% of isolates from population in the midstream of the Yangtze Valley (MY) displayed compatibility to both genotypes. Similarly, the popula-tion from Xinjiang (XI) was virulent to the differential carrying Pm17 with a frequency of 80.6%, but all other populations had frequencies lower than 43.0%. Isolates from XI had virulence frequencies of not less than 51.0% to 14 Pm genes. Isolates from MY were compatible

2426 ZENG Fan-song et al.


with only 7 Pm genes with frequencies of over 51.0%. In addition, no differential line was susceptible to all the 1 082 isolates and no isolate was able to infect all differentials successfully.

Pathotype and virulence complexity

In total, the 1 028 distinct pathotypes among 1 082 isolates were detected, of which 984 were only found once (95.7% of all pathotypes), with 44 pathotypes being detected more than once (Table 3). The most abundant pathotypes (V1a,1e,3b,3c,3e,3f,4a,4b,5a,6,8 and V1a,1e,2,3a,3b,3c,3d,3e,3f,4a,4b,5a,5b,6,7,8) were found four times each. The former was found in

the populations from NC and the downstream of the Yangtze Valley (DY), and the latter from the Yellow River and Huaihe River (YH) and Shaanxi and Gansu region (SG). 36 pathotypes were found twice and 6 pathotypes were found three times. These pathotypes were mainly present in populations from NC, YH, DY, MY, SG or Sichuan (SI). Only 8 pathotypes that were found twice belonged to populations from Yunnan and Guizhou (YG) or XI. Pathotype comparison re-vealed that pathotype V1a,1e,3c,3f,5a,6 differed from pathotype V1a,1e,3c,3f,6 by possessing virulence to Pm5a and pathotype V1a,2,3b,3c,3e,3f,4a,4b,5a dif-fered from pathotype V1a,2,3b,3c,3e,3f,4a,4b,5a,6 by the absence of virulence to Pm6. Pathotype V1e,3b,3c, 3d,3f,5a,6,7,8,17,19,20,mlxbd lost virulence to Pm13 and gained virulence to Pm20 in comparison with pathotype V1e,3b,3c,3d,3f,5a,6,7,8,13,17,19,20,mlxbd and V1e,3b,3c,3d,3f,5a,6,7,8,17,19,mlxbd, respectively. Differentiations were also found at the virulence loci corresponding to Pm1e, Pm7 and Pm19 in pathotype pairs as follows: V1a,1e,3c,3f,4b,5a,6,8,20 and V1a,1e,3c,3f,4b,5a,6,7,8,20, V3b,3c,3d,5a,6,8,17,20 and V3b,3c,3d,5a,6,8,17,19,20 and V3c,5a,6 and V1e,3c,5a,6.

Virulence distribution of 8 populations was illustrated by the number of virulence genes (virulence complexity) as shown in Table 4. Complexity values ranged from 2 to 20 with an average of 10.9. There were 133 iso-lates (12.3%) where 13 virulence genes were detected, followed by 125 isolates with 12 virulence genes and 125 isolates with 10 virulence genes. Two isolates, belonging to pathotype V1a,1e,2,3a,3b,3c,3d,3e,3f,4a,4b,5a,5b,6,7,8,17,19,20,mlxbd (only avirulent to Pm13 and Pm21), were found with the highest complexity value of 20. One isolate, designated as pathotype V1a,1e,2,3a,3b,3c,3d,3e,3f,4a,4b,5a,5b,6,7,8,13,17,19, was only avirulent to Pm20, Pm21 and mlxbd. Both of these pathotypes originated from population XI. In contrast, 6 isolates from population NC, YH, MY, SG, SI, and YG were found with only two virulence genes.

Diversity and genetic distance

The Shannon normalized index and Simpson index of diversity were calculated in the 8 populations

Table 1 Locations of sample collections and population partitions of wheat powdery mildew in China, 2011Wheat-growing regions

LocationNo. of isolates from different locations

No. of isolates from different growing regions

Sichuan (SI) Mianyang 31 146Zhitong 52Yanting 52Santai 21

Shaanxi and Gansu (SG)

Meixian 81 200Qishan 38Zhouzhi 14Gangu 36

Wenxian 20Huixian 11

Yunnan and Guizhou (YG)

Dehong 28 126Kunming 20Xingyi 39Anshun 39

Middle stream of the Yangtze River (MY)

Zhaoyang 45 114Jingzhou 32Wuchang 28Lanzhang 9

Yellow River and Huaihe River (YH)

Yiyang 21 137Jiaozuo 8Jinan 31Qufu 21Heze 15Jining 41

Downstream of the Yangtze River (DY)

Yangzhou 15 191Xuzhou 19

Shangqiu 59Fuyang 42Huaibei 14Hefei 42

North China (NC)

Baoding 26 132Dingzhou 25

Shijiazhuang 31Xingtai 22Handan 28

Xinjiang (XI) Yining 21 36Lanati 8Fukang 7



(Table 4). Very high level of diversity was observed, with the Simpson index usually near the maximum value of 1.00, and the Shannon normalized index around 1.00 with the exception of the XI population. In terms of both indices, SG had the highest pathotype diversity, with XI having the lowest. The correlation coefficients between percentage of unique pathotypes and Shannon normalized index, percentage of unique pathotypes and Simpson index, Shannon normalized index and Simpson index were positive and highly significant, however, these parameters were not correlated with virulence complexity at a significance level of 0.05 (Table 5).

To analyze the possible relationship and differenti-ation among the eight populations, 28 comparisons for all possible pairs were performed by computing indices of Nei’s distance and Kosman distance (Table 6). A significantly positive correlation (r=0.882; P<0.001) was detected between the Nei’s distance index (ranging from 0.014 to 0.104) and the Kosman distance index (ranging from 0.175 to 0.261). The distribution of gen-etic diversity was narrow between populations from the pairs of YH and DY, YH and SG, YH and SI, DY and MY, DY and SG, DY and SI, and SG and SI, but was wide between the populations of NC and MY, NC and XI, MY and YG, MY and XI, SG and XI, SI and XI,

and YG and XI. The population XI was most distant genetically from the others.

The regression analysis for geographic distance be-tween locations versus genetic distance between popula-tions revealed a significant linear relationship using both Nei’s genetic distance (R2=0.212, P=0.014) and Kosman distance (R2=0.495, P<0.001) (Fig. 1).

Associations among alleles at avirulence loci

Statistical significance of possible associations between avirulence loci was tested by χ2 tests (Table 7). Twenty-six pairs, such as AVR-Pm1a and AVR-Pm6, associated positively, and associations of 31 pairs, such as AVR-Pm1a and AVR-Pm13, were negative at P<0.05. Six-ty-nine pairs could not be determined as clearly positive or negative since their proportion of AA plus VV was less than 60% of the total sample size, despite statistical significance.

DIsCUssION

The population structures of B. graminis f. sp. tritici in

Table 2 Frequencies of virulent isolates (%) of Blumeria graminis f. sp. tritici populations on 22 differentials tested with 1 082 isolates at the seedling stage

No. Differentials Pm geneVirulence frequency (%)1)

P-valueNC (132) YH (137) DY (191) MY (114) SG (200) SI (146) YG (126) XI (36) Mean

01 Axminster/8cc Pm1a 75.8 71.5 76.4 60.5 70.5 61.6 66.7 83.3 70.1 0.007 02 Nc96BGTA3 Pm1e 51.5 69.3 58.1 47.4 62.0 63.0 58.7 50.0 58.8 0.010 03 Ulka/8cc Pm2 31.8 54.7 31.9 30.7 42.0 39.0 50.8 58.3 40.6 0.000 04 Assosan/8cc Pm3a 25.8 43.1 19.9 20.2 30.5 30.1 45.2 52.8 31.0 0.000 05 Chul/8cc Pm3b 56.1 68.6 76.4 71.9 66.0 58.9 61.9 66.7 66.2 0.003 06 Sonoara/8cc Pm3c 73.5 78.8 90.1 85.1 85.5 85.6 84.1 75.0 83.5 0.004 07 Kolibri Pm3d 34.9 40.2 35.6 21.1 42.5 36.3 35.7 52.8 36.5 0.006 08 W150 Pm3e 73.5 57.7 52.9 43.0 54.5 55.5 55.6 44.4 55.6 0.000 09 Michigen Amber/8cc Pm3f 90.9 73.7 75.4 81.6 84.5 77.4 74.6 69.4 79.4 0.002 10 Khapli/8cc Pm4a 81.8 43.8 41.4 26.3 39.5 49.3 48.4 33.3 46.3 0.000 11 Armada Pm4b 80.3 43.1 41.9 16.7 32.0 44.5 44.4 30.6 42.5 0.000 12 Hope/8cc Pm5a 87.9 89.1 95.3 97.4 92.0 88.4 85.7 88.9 90.9 0.012 13 Aquila Pm5b 14.4 34.3 4.7 9.7 24.5 17.1 39.7 30.6 20.4 0.000 14 Timgalen Pm6 84.9 91.2 89.5 86.8 89.0 88.4 84.1 94.4 88.2 0.468 15 CII4189 Pm7 31.1 51.1 31.4 29.8 65.0 48.6 57.1 63.9 46.3 0.000 16 Kavkaz Pm8 81.1 86.1 75.9 61.4 81.0 71.9 69.1 66.7 75.6 0.000 17 R4A Pm13 13.6 16.8 11.0 3.5 12.0 7.5 7.1 11.1 10.5 0.021 18 Amigo Pm17 38.6 37.2 42.4 28.1 45.5 29.5 24.6 80.6 37.8 0.000 19 Bounty Pm19 43.2 43.1 46.1 31.6 54.0 45.2 27.8 52.8 43.3 0.000 20 KS93WGRC28 Pm20 45.5 41.6 55.5 43.0 54.0 58.2 49.2 63.9 50.8 0.016 21 Yangmai5/sub.6v Pm21 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -22 Xiaobaidongmai mlxbd 12.1 19.7 16.2 24.6 23.0 19.2 16.7 27.8 19.1 0.114 23 Chancellor 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 -

1) ■ 50.0-100%; ■ 20.0-50.0%; □ 0-20.0%.



Table 3 Distribution of pathotypes containing more than 1 isolate in 8 populations of B. graminis f. sp. tritici in China, 2011Pathotype NC YH DY MY SG SI YG XI TotalV1a,1e,3b,3c,3e,3f,4a,4b,5a,6,8 3 　 1 　　　　　 4V1a,1e,2,3a,3b,3c,3d,3e,3f,4a,4b,5a,5b,6,7,8 　 2 　　 2 　　　 4V1e,3b,3c,3d,3e,3f,4a,4b,5a,6,7,8,17,19,20 　　　　 3 　　　 3V1a,1e,3c,3f,5a,6 　 1 1 1 　　　　 3V1a,3b,3c,3f,5a,6,7,8 　　 1 　 2 　　　 3V1a,1e,3b,3c,3d,3e,3f,4a,4b,5a,6,8,17,20 2 　 1 　　　　　 3V1a,1e,2,3a,3b,3c,5a,5b,6,7,8 　 3 　　　　　　 3V1a,1e,2,3a,3b,3c,3e,3f,4a,4b,5a,5b,6,7,8,20 　　 1 　　 2 　　 3V3f,5a,6,8,20 　　 1 　 1 　　　 2V3c,3e 1 　　 1 　　　　 2V1e,3b,3c,3e,3f,5a,6,8 　 1 1 　　　　　 2V1e,3b,3c,3e,3f,4a,4b,5a,6,8 2 　　　　　　　 2V1e,3d,3e,3f,4a,4b,6,7,8,19 1 　　　　　　 1 2V1a,3c,3e,3f,5a,6,8,17,19,20 　　 1 　　 1 　　 2V1a,1e,3c,3e,3f,4a,4b,5a,6,7,8,20 　　　　 1 　 1 　 2V1a,1e,3b,3c,3f,5a,6,7,8 　　　 1 1 　　　 2V1a,1e,3b,3c,3f,5a,6,7,8,19,20 　　　 1 1 　　　 2V1a,1e,3b,3c,3f,5a,6,7,8,17,19,20 　　 1 　 1 　　　 2V1a,1e,3b,3c,3f,5a,6,7,8,17,19,20,mlxbd 　　　　 2 　　　 2V1a,1e,3b,3c,3e,3f,5a,6,7,8,17,19,20 　　 1 　 1 　　　 2V1a,1e,3b,3c,3f,4a,4b,5a,6,8 　　 1 　　 1 　　 2V1a,3b,3c,3e,3f,4a,4b,5a,6,8,20 　　　　　 1 　 1 2V1a,1e,3b,3c,3e,3f,4a,4b,5a,6,8,17,19 1 　 1 　　　　　 2V1a,1e,3b,3c,3d,3f,4b,5a,6,7,8,17,19,20,mlxbd 　　　　 1 1 　　 2V1a,1e,3b,3c,3d,3e,3f,5a,6,8,17,19,20 　　 2 　　　　　 2V1a,1e,3b,3c,3d,3e,3f,4a,4b,5a,6,8,19 　　 2 　　　　　 2V1a,3b,3c,3d,3e,3f,4a,4b,5a,6,8,17,19,20 1 　 1 　　　　　 2V1a,1e,3b,3c,3d,3e,3f,4a,4b,5a,6,8,13,17,19,20 1 　 1 　　　　　 2V1a,1e,3b,3c,3d,3e,3f,4a,4b,5a,6,7,8,17,20 　　 2 　　　　　 2V1a,3b,3c,3d,3e,3f,4a,4b,5a,6,7,8,17,19,20 2 　　　　　　　 2V1a,1e,2,3b,3c,5a,6,8,20 　　 1 　　　 1 　 2V1a,1e,2,3b,3c,3f,5a,6,20 　　 1 1 　　　　 2V1a,1e,2,3b,3c,3f,5a,6,8,20 　　　 1 　 1 　　 2V1a,1e,2,3b,3c,3f,5a,6,7,8,20 　　　　　 2 　　 2V1a,1e,2,3b,3c,3f,4a,5a,6,7,8 　　　　 2 　　　 2V1a,1e,2,3b,3c,3e,3f,4a,4b,5a,6,7,8 1 　　　 1 　　　 2V1a,2,3b,3c,3e,3f,4a,4b,5a,6,7,8,19 　　 1 1 　　　　 2V1a,1e,2,3b,3c,3d,5a,6,7,8,17,19,20 　　 1 　　 1 　　 2V1a,1e,2,3b,3c,3d,3f,5a,6,8,17,20 　 1 　　 1 　　　 2V1a,2,3b,3c,3d,3e,3f,4a,4b,5a,6,7,8,17,19,20,mlxbd 　　 1 　　　　 1 2V1e,3a,3b,3c,3d,3e,3f,4a,5a,6,7,8,17,19,20,mlxbd 1 1 　　　　　　 2V1a,1e,3a,3b,3c,3d,3e,3f,5a,6,7,8,17,20 　 1 　　　　 1 　 2V1a,1e,2,3a,3b,3c,3f,5a,5b,6,7,8,17,20 　 1 　　 1 　　　 2V1a,1e,2,3a,3b,3c,3d,3e,3f,4a,4b,5a,5b,6,7,8,17,19,20,mlxbd 2 2

16 11 25 7 21 10 3 5 98

Table 4 Pathotype diversity, virulence complexity and phenotypic diversity of populations of B. graminis f. sp. tritici in China, 2011

Population No. of isolates No. of pathotypes Unique pathotypes (%) Average virulence complexity per isolatePhenotypic diversity

Shannon normalized index Simpson indexNC 132 127 96.2 11.28 0.91 0.98 YH 137 134 97.8 11.55 0.91 0.98 DY 191 188 98.4 10.68 0.95 0.99 MY 114 114 100.0 9.20 0.90 0.98 SG 200 194 97.0 11.50 0.94 0.99 SI 146 144 98.6 10.75 0.92 0.98 YG 126 126 100.0 10.87 0.91 0.98 XI 36 35 97.2 11.97 0.74 0.96



8 major wheat-growing regions in China were surveyed on a large scale in 2011. Data from virulence monitor-ing uncovered the composition of these populations in light of frequencies of virulence to individual Pm genes.

Table 5 Correlation between unique pathotypes, complexity, phenotypic and genetic diversity of wheat powdery mildew populations in China, 2011

Mean Standard deviation Unique pathotypes (%) Virulence complexityPhenotypic diversity

Shannon normalized index Simpson indexUnique pathotypes (%) 123.00 44.61 1.000 -0.189 0.940** 0.979**

Virulence complexity 10.98 0.84 1.000 -0.385 -0.324Shannon normalized index 0.90 0.07 1.000 0.961**

Simpson index 0.98 0.01 1.000**, correlation is significant at the 0.01 level (2-tailed).

Table 6 Genetic distance values between different populations of wheat powdery mildew in China, 2011NC YH DY MY SG SI YG XI

NC - 0.214 0.194 0.222 0.211 0.207 0.220 0.261 YH 0.049 - 0.187 0.215 0.184 0.187 0.194 0.221 DY 0.042 0.028 - 0.175 0.176 0.178 0.204 0.221 MY 0.075 0.048 0.021 - 0.198 0.197 0.216 0.251 SG 0.057 0.015 0.021 0.034 - 0.183 0.197 0.220 SI 0.036 0.019 0.017 0.032 0.014 - 0.197 0.237 YG 0.053 0.015 0.038 0.044 0.023 0.018 - 0.242 XI 0.104 0.038 0.052 0.077 0.033 0.054 0.054 -

Significant difference between all population pairs was detected via both Kosman’s distance and Nei’s standard distance values. Nei’s standard distance values were shown on the bottom left corner. Kosman’s distance values were shown on the top right corner.

Fig. 1 Regression curves of genetic distance based on Kosman’s distance values (A) or Nei’s standard distance values (B) vs. geographic distance for 8 populations of wheat powdery mildew across China in 2011.

y=2E-05x+0.183R2=0.494P<0.001

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A

B

Pm21, a resistance gene transferred from Haynaldia villosa, has been notable for its excellent broad-spectrum resistance for more than one decade. In China, 12 com-mercial cultivars possessing Pm21 have been planted on more than 3.4 million ha since 2002 (Cao et al. 2011). In the present study, Yangmai 5 (T6VS·6AL), which contains Pm21, was immune or showing a hypersensi-tive reaction to all of the tested isolates, demonstrating that it is widely effective in controlling to powdery mildew. Pm13, mlxbd and Pm5b were also effective in the majority of wheat producing areas due to their com-paratively broad resistance spectra. Pm3a, Pm3d and Pm17 conferred resistance to most isolates except those in Xinjiang, suggesting that these genes will be useful in many breeding programmes for the immediate future. Resistance conferred by Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8 have been overcome by isolates from the 8 regions, which was consistent with previ-ous work of Chi et al. (2007) in the Yellow River and Huaihe River, China, of Shi et al. (2009) in the Shaanxi and Gansu growing region, China, and of Yang et al. (2009) who investigated populations in central China. This indicated that these genes are of little current use. Apart from Pm13 and Pm21, no other Pm genes provide broad resistance to the isolates from Xinjiang, identifying an urgent need for the development of novel resistance sources. A divergent distribution of frequencies to Pm1e, Pm2, Pm3a, Pm3d, Pm4a, Pm4b, Pm7, Pm17, Pm19 and Pm20 in the 8 populations (P<0.05) suggests that



Table 7 Pair wise association of virulence alleles in isolates of Blumeria graminis f. sp. tritici from 8 wheat-growing regions in China in 2011

Pm gene pairPathogen alleles1)

Proportion of VV2) Association type3) P-value4)

AA AV VA VV1a 2 236 88 407 351 0.32 +- <0.0011a 3b 127 197 239 519 0.48 +- 0.0181a 3c 70 254 109 649 0.60 + 0.0051a 3d 223 101 464 294 0.27 +- 0.0211a 3e 160 164 320 438 0.40 +- 0.0351a 4b 207 117 415 343 0.32 +- 0.0071a 6 56 268 72 686 0.63 + <0.0011a 13 306 18 662 96 0.09 - 0.0011a 17 223 101 450 308 0.28 +- 0.0041a mlxbd 236 88 639 119 0.11 - <0.0012 3a 517 126 230 209 0.19 + <0.0012 3e 305 338 175 264 0.24 +- 0.0162 4a 367 276 214 225 0.21 +- 0.0082 4b 391 252 231 208 0.19 +- 0.0092 5b 573 70 288 151 0.14 + <0.0012 6 87 556 41 398 0.37 +- 0.0452 7 370 273 211 228 0.21 +- 0.0033a 3b 269 478 97 238 0.22 +- 0.0283a 3e 348 399 132 203 0.19 +- 0.0333a 4a 417 330 164 171 0.16 +- 0.0423a 4b 446 301 176 159 0.15 +- 0.0323a 5b 657 90 204 131 0.12 + <0.0013a 6 103 644 25 310 0.29 - 0.0043a 7 430 317 151 184 0.17 +- <0.0013a 8 197 550 67 268 0.25 +- 0.0293b 3c 143 223 36 680 0.63 + <0.0013b 3d 256 110 431 285 0.26 +- 0.0023b 4b 183 183 439 277 0.26 +- <0.0013b 5a 71 295 27 689 0.64 + <0.0013b 6 79 287 49 667 0.62 + <0.0013b 17 263 103 410 306 0.28 +- <0.0013b 20 210 156 322 394 0.36 +- <0.0013b mlxbd 309 57 566 150 0.14 +- 0.0413c 3d 131 48 556 347 0.32 +- 0.0043c 4b 87 92 535 368 0.34 +- 0.0113c 5a 55 124 43 860 0.79 + <0.0013c 6 53 126 75 828 0.77 + <0.0013c 17 133 46 540 363 0.34 +- <0.0013c 20 104 75 428 475 0.44 +- 0.0113d 3f 157 530 66 329 0.30 +- 0.023d 5a 79 608 19 376 0.35 +- <0.0013d 6 96 591 32 363 0.34 +- 0.0053d 7 420 267 161 234 0.22 + <0.0013d 17 506 181 167 228 0.21 + <0.0013d 19 444 243 170 225 0.21 + <0.0013d 20 372 315 160 235 0.22 +- <0.0013d mlxbd 573 114 302 93 0.09 + 0.0073e 3f 136 344 87 515 0.48 + <0.0013e 4a 381 99 200 402 0.37 + <0.0013e 4b 404 76 218 384 0.35 + <0.0013e 5b 408 72 453 149 0.14 +- <0.0013e 7 288 192 293 309 0.29 +- <0.0013e mlxbd 369 111 506 96 0.09 +- 0.0043f 4a 163 60 418 441 0.41 +- <0.0013f 4b 173 50 449 410 0.38 +- <0.0013f 5a 35 188 63 796 0.74 + <0.0013f 7 137 86 444 415 0.38 +- 0.0123f 19 147 76 467 392 0.36 +- 0.0023f 20 124 99 408 451 0.42 +- 0.0373f 22 112 111 334 525 0.49 +- 0.0033f mlxbd 195 28 680 179 0.17 - 0.0074a 4b 520 61 102 399 0.37 + <0.001

(Continued on next page)



regional deployment of specific genes could give short to medium term protection.

Several changes of virulence frequencies happened along the Yellow River and the Huaihe River covering parts of Shandong, Hubei, Henan, Shaanxi and Gansu provinces. Comparison with a 2004/2005 survey (Chi et al. 2007) indicated that in these river regions, the virulence frequency to Pm17 decrease greatly over time whereas that of Pm19 slightly increased. In comparison to Yang (2009), the virulence frequencies to Pm3a, Pm3d, Pm3e, Pm7, Pm17 and Pm19 displayed a clear decline in central China, while virulence to Pm3f, Pm4a,

Pm4b and Pm20 increased. The most likely explanation to this change would be through the changed deployment of specific resistance genes. For example, if Pm3a, Pm3d and Pm3e are no longer used in cultivars taking up most of the area in a given region, genetic drift will see a de-cline in virulence frequency. If these Pm3 alleles were replaced in the most commonly grown varieties by Pm3f, then it would be expected that virulence to this allele would increase. It is therefore important to monitor the virulence frequencies to these genes continuously in the future as once virulence is achieved, it can be maintained in the population at very low levels, and start to increase

Pm gene pairPathogen alleles1)

Proportion of VV2) Association type3) P-value4)

AA AV VA VV4a 5b 486 95 375 126 0.12 +- <0.0014a 6 84 497 44 457 0.42 +- 0.0054a 7 333 248 248 253 0.23 +- 0.0124a mlxbd 437 144 438 63 0.06 +- <0.0014b 5b 525 97 336 124 0.11 +- <0.0014b 7 357 265 224 236 0.22 +- 0.0064b 8 174 448 90 370 0.34 +- 0.0024b mlxbd 469 153 406 54 0.05 +- <0.0015a 6 41 57 87 897 0.83 + <0.0015a 17 81 17 592 392 0.36 +- <0.0015a 20 70 28 462 522 0.48 +- <0.0015a mlxbd 88 10 787 197 0.18 - 0.0265b 7 531 330 50 171 0.16 + <0.0015b 19 471 390 143 78 0.07 +- 0.0095b 22 386 475 60 161 0.15 +- <0.0016 7 81 47 500 454 0.42 +- 0.0266 8 42 86 222 732 0.68 + 0.0246 17 95 33 578 376 0.35 +- 0.0046 19 93 35 521 433 0.40 +- <0.0016 20 78 50 454 500 0.46 +- 0.0066 mlxbd 113 15 762 192 0.18 - 0.0317 8 168 413 96 405 0.37 +- <0.0017 13 538 43 430 71 0.07 +- <0.0017 17 390 191 283 218 0.20 +- <0.0017 19 374 207 240 261 0.24 +- <0.0017 22 286 295 160 341 0.32 +- <0.0017 mlxbd 490 91 385 116 0.11 +- 0.0028 17 186 78 487 331 0.31 +- 0.0028 20 149 115 383 435 0.40 +- 0.00813 17 619 349 54 60 0.06 + 0.00113 19 574 394 40 74 0.07 +- <0.00117 19 438 235 176 233 0.22 + <0.00117 20 429 244 103 306 0.28 + <0.00117 mlxbd 570 103 305 104 0.10 + <0.00119 20 338 276 194 274 0.25 +- <0.00119 22 282 332 164 304 0.28 +- <0.00119 mlxbd 523 91 352 116 0.11 +- <0.00120 22 241 291 205 345 0.32 +- 0.00920 mlxbd 463 69 412 138 0.13 +- <0.0011) Data are number of isolates virulent (V) or avirulent (A) at respective Pm loci.2) Proportion of isolates virulent to both Pm genes.3) +, a positive association with either AA or VV; -, a negative association with AV or VA; +-, the association was not clear.4) Pairs with significant variations at P=0.05 based on χ2 test were listed.

Table 7 (Continued from preceding page)



once the corresponding resistance genes are redeployed. Pathogen virulence alteration and diversity can be

attributed to mutations, selections, sex recombination and migration (Burdon and Silk 1997). A closer look into the composition of pathotypes via comparison between virulence patterns present in different isolates identified single or several alterations at virulence loci that confirmed the high adaptability of the pathogen. The AVRk1 powdery mildew-specific gene family, an effector gene family required for virulence in the pow-dery mildew fungus has coevolved with TE1a, a class of LINE-1 retrotransposon (Sacristan et al. 2009). In this context, one possible mechanism for mutations at these loci might be associated to a major invasion of transposable elements in the B. graminis f. sp. tritici genome (Parlange et al. 2011).

Sexual recombination may affect pathotype compos-ition, leading to high variation during ascospore trans-mission from the crop to the volunteers (Bousset and Vallavieille-Pope 2003). In Hubei Province of central China, cleistothecia play an important role in transmitting powdery mildew by infecting volunteers via ascospores in highlands at low temperature, but not in lowlands at high temperature (Yu 2000b). In the population MY, there were fewer diseased leaves of volunteers and this was likely due to the higher temperature (data not shown). In result, populations in this region have to be re-built every year. This is an important factor that likely contributed to the relatively lower phenotypic diversity as indicated by Shannon normalized index than in other populations.

Host selection has a major impact on pathogen divers-ity. The most frequently planted cultivar in the MY area is Zhengmai 9023, whereas cultivars in the DY region are made up of Yangmai, Ningmai, and Wanmai groups, which likely contain a greater diversity of resistance loci (Yang et al. 2013). Divergent resistance patterns to all isolates used in this study further implies different Pm gene compositions among Zhengmai 9023, Yangmai 11, Ningmai 13 and Wanmai 50 (Yang et al. 2013). The use of different cultivars often results in the deployment of different resistance genes, therefore enhancing the di-vergence in phenotypic diversity observed between the populations. The most likely cause in the divergence in phenotypic diversity between these populations is based on directional selection favouring specific genotypes. This has also been shown to result in a reduction in

genotype diversity (Burdon and Silk 1997). Migration may serve as another factor influencing the

genetic difference among populations in China. Analysis of Kosman genetic distance and Nei’s standard genetic distance showed the lowest genetic distance was between populations DY and MY. This suggests that these two regions composed a large uniform epidemiological area for the migration of population from central to eastern China without geographic barriers to disrupt the dispersal of pathogen conidia. Population SG and YH likely can be defined as another large epidemiological unit along the Yellow River due to their near genetic distance. Formation of the two large epidemiological units has been attributed to two major phases of powdery mildew expansion (Liu and Shao 1994). The first expansion in China occurred during the late 1970s and early 1980s from Yunnan and Guizhou provinces to the Yangtze Valley and the second happened in the mid 1980s from south to north. Data from the distribution of powdery mildew and leaf rust showed that virulence complexity of the two pathogens increased along the route of migration from an interaction of migration and selection (Limpert and Bartos 2002). In the current report, an increase in virulence complexity was also found from the Yangtze River population to the Yellow River population in the direction of powdery mildew migration (Table 4).

It was noticeable that population XI, which neigh-bours Eastern Europe, appeared to be an isolated group distinguishing from the other populations in China, and was shown by the greater genetic distance and virulence complexity from the other regions. This region is geo-graphically isolated from the other regions by deserts that inhibit the passage of powdery mildew.

Regression analysis showed that the genetic difference between populations of wheat powdery mildew ascended with the increase of geographic distance between loca-tions. At least three possible reasons for this relationship can be considered. The first is resistance composition of local cultivars specified the pathogen populations. Secondly, the longer distance between different locations makes it more difficult for the spores to spread. Thirdly, geographic separation prevents the genetic flow across the different locations.

Virulence to Pm4b and Pm8 were significantly asso-ciated with each other in the pathogen population. This could suggest linkage disequilibrium between these virulence loci within the pathogen. However, as Pm8 and



Pm4b are the most commonly used resistance genes in 56 Chinese wheat cultivars (Wang et al. 2005), it is possible that cultivars carrying the two genes are co-selecting this virulence composition. If so, such a case will also increase the proportion of isolates carrying both virulence loci. In contrast, the associations between virulence pairs to Pm3b and Pm20, as well as pairs to Pm8 and Pm20 were not definite. High frequencies of virulence to several Pm genes individually were also detected in pairs with high proportions of VV in all tested isolates. For example, the VV proportion of virulence pairs to Pm1a and Pm3c, Pm3b and Pm5a, Pm3c and Pm8, Pm3f and Pm5a were always more than 60%. Therefore, pyramiding of these specific Pm genes will not result in more durable resistance. On the contrary, virulence to Pm2 and Pm3a, Pm2 and Pm5b, Pm3a and Pm5b, Pm3d and mlxbd, Pm17 and mlxbd were positively associated with a lower VV proportion (Table 7) than virulence frequencies of these genes used alone, suggesting that pyramiding these genes might be a candidate strategy to prolong the effective period of resistant cultivars.

CONClUsION

Regional populations of Blumeria graminis f. sp. tritici in China sampled in 2011 differ both in phenotypic diversity and in pathotypic composition, indicating that cultivars with effective resistance gene combi-nations should be deployed according to virulence complexity of various populations. Pm21, Pm13, mlxbd and Pm5b are effective and deserve to be used for breeding programme for wheat resistance. A large uniform epidemiological area is present in downstream and midstream of the Yangtze Valley, however, the population composed of isolates from Xinjiang region is likely segregated from the other populations due to geographic barriers.

MATeRIAls AND MeThODs

sample collection

Isolates of B. graminis f. sp. tritici were collected from 8 epidemic regions (Fig. 2) of wheat powdery mildew between

March and May, 2011. These regions were North China (NC), Yellow River and Huaihe River (YH), downstream of the Yangtze valley (DY), midstream of the Yangtze valley (MY), Shaanxi and Gansu region (SG), Sichuan (SI), Yunnan and Guizhou (YG) and Xinjiang (XI). Diseased leaf pieces carrying single pustules were sampled at random and placed in plastic boxes with small wells filled with 5 g L-1 agar containing 50 mg L-1 benzimidazole. The plastic boxes were transported from the sampling locations to the laboratory using a cooler box containing ice to avoid high temperatures. Single colony isolates were obtained using the method described by Persaud and Lipps (1995).

Multiplication and preservation of isolates

A highly susceptible line, Chancellor (abbreviated as CC) which possessed no Pm genes (Srni et al. 2005), was used for isolate multiplication and preservation. Conidia from each isolate were shaken onto healthy leaf segments in 60 mm Petri dishes containing 5 g L-1 agar amended with 50 mg L-1 benzimidazole. Subsequently the Petri dishes were incubated in a (17±1)°C growth chamber in the dark for the first 12 h followed by a period of 10 d at constant light of 72 µmol m-2 s-1.

Differential sets

Virulence of isolates was tested on a differential set listed in Table 2. Genotypes containing Pm1a, Pm2, Pm3a, Pm3b, Pm3c, Pm4a and Pm5a were near-isogenic lines in the genetic background of CC developed by Briggle (1969) and 14 lines carrying single Pm gene were kindly provided by Professor Zhou Yilin, Institute of Plant Protection, China Academy of Agricultural Sciences. Xiaobaidongmai, a Chinese landrace, contains a single recessive Pm gene, mlxbd (Zhai et al. 2008).

Pathogenicity assay at the seedling stage

Seeds of differentials and CC were sown in plastic pots (20 cm×20 cm) in rows in a greenhouse under a cellophane bag and grown at (17±0.5)°C, 70% relative humidity, on 16 h light (72 µmol m-2 s-1), 8 h dark cycle for 10-12 d. 3-cm segments were cut from the middle of primary leaves and placed in 9-cm Petri dishes containing benzimidazole agar. Inoculation for each isolate was carried out in separate plastic towers (35 cm high and 10 cm in diameter) with the differential set at the bottom of the tower. To evaluate the uniformity of inocula distribution, leaf segments of CC were placed randomly four times in each Petri dish. Multiplied inocula from individual isolates was blown into the tower at a density of 2×103-4×103 conidia cm-2 and the Petri dishes were incubated for 12 d using the conditions described above. Three replicates of each line



were tested per isolate. All the tests were performed at the Institute for Plant Protection, Hubei Academy of Agricultural Sciences, China, during 2011 to 2012

.Virulence determination

Infection type was scored using the scale of Si et al. (1987): 0=immune, no visible sign of infection or necrosis and no mycelium; 0;=hypersensitive reaction, visible necrosis but no mycelium; 1=resistant, increasing from no mycelium to little mycelium, sometimes with necrosis; 2=moderately resistant, with an increased amount of mycelium, little conidiospore production, no necrosis; 3=moderately compatible, with large amount of mycelium, moderate conidiospore production, no necrosis or chlorosis; 4=completely compatible, with a large amount of mycelium and substantial conidiospore production. To ensure reliability of data, all batches were scored independently by two people before crosschecking the results. Ratings of 0, 0;, 1 and 2 were designated as incompatible (avirulent) to a given genotype while ratings of 3 and 4 were scored as compatible (virulent).

Pathotype designation

Pathotypes were described as virulence formulae (V ineffective genes) according to Namuco et al. (1987). The virulence formula expresses the virulence spectrum of isolates to the differential set. A unique pathotype was defined as one whose virulence pattern was detected only once.

Virulence complexity and virulence frequency calculations

Virulence complexity of each isolate identified the number of virulence genes based on the virulence spectrum to the given deferential set and gives insight into the ability of the isolate to overcome host Pm genes. Virulence frequency signifies the proportion of virulent isolates in a population to an individual differential line. Virulence complexity and virulence frequency were analyzed using HaGis 3.1 software (Hermann et al. 1999) on the basis of the virulence pattern to the 22 differential lines.

Fig. 2 Locations of samples collection and population partition of wheat powdery mildew in China, 2011.



Classification of effectiveness of Pm genes

Effectiveness of Pm genes was classified into three groups according to the virulence frequencies using the resistance grouping system of Felsenstein and Jaser (2000) with minor modifications. The resistance level was classified as high, moderate or low as determined by the respective virulence frequencies of 0-20, 20-50 and 50-100%.

Data analysis

To examine the diversity of populations within different growing regions, the KOIND package (Kosman 2002) was used to calculate several descriptive parameters of populations. The Shannon normalized index (Shannon 1948) utilizes the similarities of the virulence frequencies. The Simpson index (Simpson 1949) describes the concentration of different pathotypes in a set of isolates. The Nei’s standard genetic distance (Nei 1972) measures the degrees of similarity among distinct pathotype. The Kosman genetic distance (Kosman 1996) takes into account both pathotype frequencies and dissimilarity degrees to estimate diversity among populations from geographic regions. The bootstrap method (Efron and Tibshirani 1993) was applied to examine the reliability of all diversity and distance parameters by randomly selecting 200 isolates, 100 times from the original population. Values were calculated for each bootstrap-generated pair of samples independently and averaged.

Possible associations of alleles at pairs of avirulence loci were determined using the method described by Parks et al. (2008). For pair-wise virulence comparisons, four possible categories of isolates at two avirulence loci were calculated: isolates avirulent to both Pm genes (AA); isolates virulent to both Pm genes (VV); and isolates avirulent to one Pm gene but virulent to the other (AV or VA). According to the proportion in each category, associations with more than 60.0% of the total isolates belonging to the AA or VV categories were considered positive, however, associations with greater than 60.0% of the isolates belonging to the AV or VA categories were considered negative. If the proportion of the AA plus VV was in the interval between 40.0 and 60.0%, the association was rated unclear.

Two-tailed Pearson correlation tests were performed with SPSS19.0 software to validate the relationship between indices such as unique pathotype, complexity and phenotypic diversity. Also, chi-square (χ2) test P values were used to determine whether at least one population differed significantly from others in the light of their virulence frequencies to 22 Pm genes. Independence tests were also conducted with χ2 test P values to estimate the statistical significance of the interaction between different avirulence alleles in pairs. A regression analysis was conducted to test whether the genetic distance among populations would increase as the geographic distance increased.

AcknowledgementsWe would like to thank Professor Zhou Yilin, Institute of Plant Protection, China Academy of Agricultural Sciences, for kindly providing wheat differential lines. We thank Dr. T A J van der Lee, Plant Research International Department Bio-interactions and Plant Health in Wageningen University, the Netherlands, and Dr. Garry Rosewarne, Centro Internacional de Mejoramientode Maizy Trigo, Mexico, for revising the paper carefully. We also acknowledge statistics assistance for data analysis by Ph D Wan Peng, Institute for Plant Protection and Soil Science, Hubei Academy of Agricultural Sciences and Professor Dong Rui, Huazhong University of Science and Technology, China. This work was supported by the National Basic Research Program of China (2013CB127700) and the Special Fund for Agro-Scientific Research in the Public Interest, China (201303016).

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(Managing editor ZHANG Juan)

Virulence and Diversity of Blumeria graminis f. sp. tritici Populations in China

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