Characterization of wheat monogenic lines with known Sr genes and wheat cultivars for resistance to three new races of Puccinia graminis f.sp.tritici in China

WU Xian-xin ,ZANG Chao-qun ,ZHANG Ya-zhao ,XU Yi-wei ,WANG Shu ,Ll Tian-ya#,GAO Li

1 State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

2 College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, P.R.China

3 Institute of Plant Protection, Liaoning Academy of Agricultural Sciences, Shenyang 110161, P.R.China

4 Agronomy College, Shenyang Agricultural University, Shenyang 110866, P.R.China

Abstract Wheat stem rust,caused by Puccinia graminis f.sp.tritici (Pgt),is a potentially devastating fungal disease of wheat worldwide.The present study was to evaluate the resistance of 42 wheat monogenic lines with known stem rust resistance (Sr) genes and 69 wheat cultivars to three new Pgt races (34C0MRGQM,34C3MKGQM,and 34C6MTGSM)identified from aeciospores at the seedling and adult-plant stages.The phenotyping results revealed that monogenic lines harboring resistance genes Sr9e,Sr17,Sr21,Sr22,Sr26,Sr30,Sr31,Sr33,Sr35,Sr36,Sr37,Sr38,Sr47,SrTmp,and SrTt3 were effectively resistant to all three Pgt races at the seedling and adult-plant stages.In contrast,monogenic lines containing Sr5,Sr6,Sr7b,Sr9a,Sr9d,Sr9f,Sr9g,Sr9b,Sr16,Sr24,Sr28,and Sr39 were highly susceptible to these races at both seedling and adult-plant stages.The other lines with Sr8a,Sr10,Sr11,Sr13,Sr14,Sr15,Sr18,Sr20,Sr19,Sr23,Sr25,Sr27,Sr29,Sr32,and Sr34,displayed variable levels of resistance to one or two of the tested races.Seedling infection types (ITs) and adult-plant infection responses (IRs) indicated that 41 (59.4%) of the wheat cultivars showed high resistance to all the three races.Molecular marker analysis showed that four wheat culitvars likely carried Sr2,20 wheat culitvars likely carried Sr31,9 wheat culitvars likely carried Sr38,and none of the cultivars carried Sr24,Sr25,and Sr26.Our results provide a scientific basis for rational utilization of the tested Sr genes and wheat cultivars against these novel Pgt races.

Keywords: wheat stem rust,Puccinia graminis f.sp.tritici,wheat cultivars,resistance genes

1.lntroduction

Wheat is one of the three leading cereal crops grown worldwide.Despite the annual global wheat production exceeding 700 million tons,undernourishment caused by food shortage is still a widespread problem attributed to the substantial population growth (FAO 2017).Huge yield losses caused by wheat pathogens exacerbate this situation (Pardeyet al.2013;Moscou and van Esse 2017).In order to achieve the growth of global wheat production that is needed to feed the growing population,it is important to maximize wheat yield through effective control of wheat diseases (Pardeyet al.2013).Wheat stem rust,caused byPucciniagraminisf.sp.tritici,is one of the most devastating fungal diseases of wheat.Historically,wheat stem rust has erupted many times and has caused serious yield losses (Wu and Huang 1987;Pardeyet al.2013;Moscou and van Esse 2017;Peterson 2018).Worldwide,wheat stem rust has been effectively controlled since the 1970s attributed to the use and deployment of stem rust resistance (Sr) genes(in particular the use ofSr31gene) in commercial wheat cultivars (Wu and Huang 1987;Liet al.2016a).However,during the long-term natural evolution process,hosts and pathogens compete for dominance and pathogens adapt to the plant cultivars that are developed in modern agriculture.However,sooner or later a novel virulence gene will emerge in the pathogen that is not recognized by a resistance gene of the host plant (Flor 1971).Resistant cultivars that are widely grown often lose their resistance due to the emergence of new races.In recent years,the most typical example was the newPgtrace Ug99 and its related variants that overcame the most widely used resistance genes and spread rapidly throughout Africa and West Asia,threatening wheat production in large parts of the world (Singhet al.2011).Two additional newPgtraces,TTRTF (with combined virulence toSr9eandSr13) and TKTTF (with virulence toSrTmp),caused large field yield losses in wheat in Southern Ethiopia and Sicily(Oliveraet al.2015;Bhattacharya 2017).The emergence of new races has led to an in-depth global analysis of changes in population dynamics as well as the virulence spectrum ofPgt.

The use of resistance genes to breed resistant cultivars is the most cost-effective and environmentally friendly strategy to control wheat stem rust (Linet al.2021).So far,more than 70Srgenes have been identified and located in specific chromosome regions (McIntoshet al.2016;Chenet al.2020).Sr loci can include seedling resistance genes (major genes) and adult-plant resistance genes (minor loci).The most knownSr2gene confers durable non-race-specific slow-rusting adultplant resistance (Spielmeyeret al.2003).Presently,several of thoseSrgenes are deployed in commercial wheat cultivars,either alone or in combination,all over the world.The utilization ofSrgenes should be based on changes in virulence of thePgtpopulation.For this reason,we,as researchers from Shenyang Agricultural University,China,continuously monitor the population ofPgteven though the occurrence of wheat stem rust has become infrequent in China.Recently,we have identified three new races,named 34C0MRGQM,34C3MKGQM,and 34C6MTGSM,from samples that were collected fromBerberisspp.(an alternate host ofPgt) (Caoet al.2019).Pucciniagraminisf.sp.triticican reproduce sexually on the alternate host barberry plants (Berberisspp.),produce new virulence races,and diversify the population structure ofP.graminisf.sp.tritici(Stakmanet al.1930;Jinet al.2011).So far,threeBerberisspp.(B.aggregata,B.brachypoda,andB.potaninii) have been proved to be the alternate host ofP.graminisf.sp.tritici(Zhaoet al.2015).These new races displayed virulence toSr5,Sr6,Sr7b,Sr10,Sr11,andSr24genes.We found that races 34C0MRGQM and 34C6MTGSM possess combined virulence toSr5andSr11genes that are widely deployed in wheat cultivars grown in the wheatproducting areas of China.Up until 2008,13 310 isolates ofPgthave been identified in China,but none of them had combined virulence toSr5andSr11(Wu and Huang 1987;Yaoet al.1997,1998;Liet al.2016b;Caoet al.2019).Therefore,the objectives of this study were to determine the effectiveness of the monogenic lines with knownSrgenes at the seedling and adult-plant stages against the three new ChinesePgtraces 34C0MRGQM,34C3MKGQM,and 34C6MTGSM,and to determine the resistance levels of the 69 wheat cultivars to these races to identify resistant cultivars.

2.Materials and methods

2.1.Wheat lines

A total of 42 monogenic lines with known resistance genes and Little Club (LC) were provided by College of Plant Protection,Shenyang Agricultural University and 69 commercial wheat cultivars (Appendix A) were provided by Dr.Liu Taiguo at the State Key Laboratory for Biology of Plant Disease and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences.These 69 wheat cultivars are the main commercial cultivars cultivated in different wheat-producting areas in China.

2.2.Races of Pgt

The races 34C0MRGQM (isolate Xn16 fromBerberis brachypoda),34C3MKGQM (isolate Xn13 fromBerberis oligodentata),and 34C6MTGSM (isolate Xn9-1 fromB.brachypoda) were identified by Dr.Li Tianya at the College of Plant Protection,Shenyang Agricultural University.The names,virulence/avirulence spectrums,and urediniospores produced method of races were described by Liet al.(2018).

2.3.Evaluation of seedling infection types in the greenhouse

Testing for infection types (ITs) at the seedling stage was conducted in the greenhouse at the College of Plant Protection,Shenyang Agricultural University in 2018.The wheat lines and cultivars were seeded in pots with a diameter of 10 cm (12-cm height) filled with sand and vermiculite mixture (2:3,v/v) levelled and 10 seeds of each line/cultivar planted.All the monogenic lines and wheat cultivars were planted in three replicates to determine their resistance or sensitivity to the three novel races.Wheat cultivar LC was used as a susceptible control.The 0.1 g urediniospores were mixed with talcum at a ratio of 1:20 (w/w) and inoculated onto the fully expanded primary leaves (approximately 8-to 10-day old).More details of this method can be found in a previous study (Liet al.2016a).Two weeks after inoculation,ITs were scored according to the 0–4 scale (Stakmanet al.1962).The IT values of 0,1,and 2 were considered resistant,while 3 and 4 were considered susceptible.

2.4.Evaluation of adult-plant infection responses in the field

The infection responses (IRs) at the adult-plant stage were tested in 2018 and 2021 as previously described by Linet al.(2021).The field nursery site was located at latitude 41°49′N,longitude 123°33′E,altitude 67 m in elevation,with an average daily maximum temperature of 27.9 and 26.0°C,and an average daily minimum temperature of 17.5 and 16.4°C (night) in 2018 and 2021,respectively.The change of daily average temperature after inoculation are approximately ±4°C.In brief,urediniospores (1 g)were mixed with talc powder at a ratio of 1:30 (w/w) and used to inoculate plants at the jointing growth stage.For detailed methods,refer to previously published study (Wuet al.2020).Disease severity and IRs were assessed 14 days after inoculation,when plants were at the heading and flowering stages.Stem rust severity was defined as the percentage of leaf area covered by rust pustules,with each percent representing 0.37% of leaf area covered with uredinia,and recorded as 1,5,10,20,30,40,50,60,70,80,90,or 100% (Petersonet al.1948).IRs were based on the size of stem rust pustules and amount of associated chlorosis and necrosis.IRs included resistant(R),moderately resistant (MR),moderately susceptible(MS),and susceptible (Roelfset al.1992).The disease data were recorded three times at 5-day intervals to ensure the accuracy of the field investigation results.The highest IR and severity were used to characterize the response of each line.Area under the disease progress curve (AUDPC) was calculated according to Donget al.(1986).

2.5.Marker screening

DNA was extracted from two leaves of 15-day-old seedlings using a Genomic DNA Extraction Kit (Sangon Biotech,China).The molecular marker primers used to detect stem rust resistance (Sr2,Sr24,Sr25,Sr26,Sr31,andSr38) genes were synthesized by by Shanghai Biotech Biotech Co.,Ltd.,China (Appendix B).PCR amplification conditions were as described in previous studies (Xuet al.2017).Fragments of the targeted genes were detected by electrophoresis using 2% (w/v) agarose gels and then gels were observed under UV light.

3.Results

3.1.Responses of the monogenic Sr gene lines to the novel Pgt races

All ITs and IRs data outlining 42 monogenic lines with knownSrgenes to the races 34C0MRGQM,34C3MKGQM,and 34C6MTGSM at the seedling and adult-plant stages are summarized in Tables 1 and 2.Seventeen monogenic lines,including W2619Sr9e(Sr9e),Combination VII (Sr17andSr13),CnS_T_mono_deri (Sr21),SwSr22T.B.(Sr22),Eagle (Sr26),BtS30Wst(Sr30),Sr31/6＊LMPG (Sr31),Federation＊4/Kavkaz(Sr31),Tetra Canthatch/Ae (Sr33),RL5405 (Sr33),Mq(2)5XG2919 (Sr35),W2691SrTt-1 (Sr36),W2691Sr37(Sr37),Trident (Sr38),DAS15 (Sr47),CnsSrTmp (SrTmp),and Fed/SrTt3 (SrTt3) exhibited resistant ITs (0 to 2) at the seedling stage,and R to MR at the adult-plant stage with relatively low severity (<40%) in 2018 and 2021(Table 1),indicating that theSrgenes in those lines were highly effective against each of the three tested races.In contrast,monogenic lines ISr5-Ra (Sr5),ISr6-Ra (Sr6),ISr7b-Ra (Sr7b),ISr9a-Ra (Sr9a),ISr9d-Ra(Sr9d),CnsSr9f (Sr9f),CnSr9g (Sr9g),W2691Sr9b (Sr9b),ISr16-Ra (Sr16),LcSr24Ag (Sr24),W2691Sr28 (Sr28),and RL6082 (Sr39) produced ITs 3 to 4 (susceptible) at the seedling stage and moderate to highly susceptible responses at the adult-plant stage with relatively high severity (>50%) (Table 2),indicating that theSrgenes in those lines were not effective against any of the three races.The remaining monogenic lines with stem rust resistance genesSr8a,Sr10,Sr11,Sr13,Sr14,Sr15,Sr18,Sr20,Sr19,Sr23,Sr25,Sr27,Sr29,Sr32,andSr34displayed variable resistance to one or two of the tested races.In general,the resistance or susceptibility of monogenic lines to the tested races at the adult-stagewas the same in 2018 and 2021,but the disease severity was generally higher in 2021 than in 2018.

Table 1 Infection types (ITs) on seedlings and infection responses (IRs) on adult plants of wheat lines carrying Sr genes to novel races (34C0MRGQM,34C3MKGQM,and 34C6MTGSM) of Puccinia graminis f.sp.tritici in China1)

Table 2 Resistance and susceptibility proportion of 69 wheat cultivars to three races at seedling stage

3.2.Evaluation of the infection responses of wheat cultivars to Pgt races 34C0MRGQM,34C3MKGQM,and 34C6MTGSM

The ITs and IRs of 69 wheat cultivars to the races 34C0MRGQM,34C3MKGQM,and 34C6MTGSM are listed in Tables 3 and 4.The cultivars displayed a wide range of ITs and IRs to the tested races.Forty-one(59.4%) wheat cultivars (No.1–41) displayed ITs 0 to 2+at the seedling stage,and 0 to MR at the adult-plant stage both in 2018 and 2021 in the field nursery (Table 3),indicating these cultivars have all stage resistance to the tested races.Moreover,these wheat cultivars have lower AUDPC values.Seven (10.1%) wheat cultivars (No.42–48) produced ITs 3– to 4 (susceptible) at the seedling stage,and MS to S responses at the adult-plant stage,indicating that these cultivars were not effective against the tested races (Tables 3 and 4).The remaining wheat cultivars,including Jingshuang 16,Wenmai 14,Yimai 14,and Zhongyu 6 displayed ITs 1 to 2 and IRs M to S to race 34C0MRGQM,while the cultivars Chuanmai 107 and Jinmai 47 had similar ITs and IRs to race 34C3MKGQM,indicating that those cultivars only possess resistance to the tested races at the seedling plant stage.The IRs remained relatively stable in the field nursery between 2018 and 2021 while for many of the wheat cultivars disease severity was lower in 2018 than in 2021.This reason is attributed to the frequent rainfall in 2021,which makes many diseases,including wheat stem rust,more serious than in recent years.

Table 3 Resistance and susceptibility proportion (%) of 69 wheat cultivars to three races at adult-plant stage1)

3.3.Molecular detection results of tested stem rust resistance genes

Six molecular markers closely linked with resistance genesSr2,Sr24,Sr25,Sr26,Sr31,andSr38were detected in 69 wheat cultivars.The results showed that marker Iag95 amplified an 1 100-bp PCR fragment (Fig.1)inSr31-carrying lines “Sr31/6＊LMPG” and 20 tested wheat cultivars (Appendix A).A 262-bp PCR fragment of 2NS-specific primer VENTRIUP-LN2 were amplified in nine wheat cultivars and positive control,indicating that these wheat cultivars carriedSr38.Four wheat cultivars Jimai 20,Shangnong 15,Zhongyu 6,and Fan 6 may carry the resistane geneSr2.The molecular markers closely linked withSr24,Sr25,andSr26can only amplify the specific fragment in the positive control,but there is no PCR fragment in the tested wheat cultivars,indicating that these cultivars lackedSr24,Sr25,andSr26.

Fig.1 Amplification result for parts of wheat cultivars with markers Iag95.

4.Discussion

Berberisspp.,the alternative host ofPgt,plays a key role in epidemics of wheat stem rust and the virulence variation ofPgt.The aeciospores produced byBerberiscan represent the initial inoculum of wheat stem rust,playing a ‘bridge’ role in the life cycle ofPgt.In addition,new pathogenic races may emerge through sexual hybridization.New races may cause disease epidemics when the susceptible hosts are planted in large areas and the environmental conditions are favorable.In a previous study,three newPgtraces (34C0MRGQM,34C3MKGQM,and 34C6MTGSM) fromBerberiswere identified (Liet al.2018).Therefore,we measured the ITs of 42 monogenic lines,each with a knownSrgene and evaluated the resistance level of main wheat cultivars in China to these three emerging races.Monogenic lines containing resistance genesSr9e,Sr17,Sr21,Sr22,Sr26,Sr30,Sr31,Sr33,Sr35,Sr36,Sr37,Sr38,Sr47,SrTmp,andSrTt3and 41 wheat cultivars were highly resistant to the tested races both at the seedling and adult-plant stages.Among those genes,Sr17,Sr31,andSr38are commonly used in Chinese wheat cultivars (Heet al.2008;Liet al.2016a;Xuet al.2017,2018),which is confirmed by our molecular detection results with 20 and 8 of 69 cultivars carringSr31andSr38,respectively.Up to now,noPgtrace virulent toSr31andSr38has been found in China (Liet al.2016b;Caoet al.2019;Wuet al.2020).Therefore,these two genes have provided excellent resistance to allPgtraces in China and can be used in combination with other resistant genes (especially against the Ug99 race group) in future wheat resistance breeding programs to keep long-term disease resistance through gene pyramiding.TheSr33gene,originating from the wild relativeAegilopstauschii,still provides resistance to all ChinesePgtand Ug99 race groups,even though it has been used in wheat breeding programs for more than 70 years in combination with the adult resistance geneSr2(Periyannanet al.2013).GenesSr9e,Sr26,Sr47,andSrTt3,effective against Ug99 and thePgtpopulation in China,are sparsely used in the production of wheat cultivars (Caoet al.1996).In this study,no cultivars containingSr26were detected in the 69 wheat cultivars tested.Sr22,Sr35,andSr37,conferring resistance to Ug99 and TKTTF and moderately effective against ChinesePgtraces,were postulated in some commercial wheat cultivars (Jinet al.2007;Liet al.2016b).

In addition to the 16Srgenes discussed above,the remaining genes displayed varying degrees of susceptibility to the three tested races.GeneSr5was effective against the 21 race group (e.g.,21C0 and 21C1)but ineffective against the 34 race group (e.g.,34C0 and 34C2).Some wheat cultivars (Minn 2761,Alondra‘S’,Rulofen,and Orofen) containSr5andSr6,alone or in combination,which are the second most commonly deployed resistance genes in China afterSr31(Zhanget al.1987).TheSr11gene has been used worldwide as a major resistance source towards wheat stem rust.The isolates that were virulent toSr11(such as 21C3CTR)were originally identified in 1993 from samples collected from Leshan,Sichuan Province,China.There had never been an isolate with combined virulence toSr5andSr11in China until the emergence of the 34C0MRGQM and 34C6MTGSM races.However,these two races have virulence to widely used wheat cultivars and their derivatives,including those harboringSr5,Sr11,orSr5+Sr11.Therefore,this situation should be given urgent attention.

The results of resistance identification of races 34C0MRGQM,34C3MKGQM,and 34C6MTGSM to 69 wheat cultivars showed that the Chinese main cultivars have a high level of resistance to these tested races.This may be related to the utilization of resistant germplasms.For example,since the 1950s,a number of resistant materials (Kavkaz and Avrora introduced from the Soviet Union;Lovrin 10 and 13 from Romania;Orofen and Rulofen from Chile;Mexipak 66 and Tanori from Mexico)have been introduced into the Chinese Wheat Breeding Program (Liet al.2016b).The responses of adult plants to the tested races are direct measurements of the effectiveness of resistance genes in wheat cultivars.The present study identified a group of wheat cultivars that are resistant to the emergingPgtraces 34C0MRGQM,34C3MKGQM,and 34C6MTGSM,which provides a scientific basis for rational utilization of wheat cultivars.

5.Conclusion

In this article,we evaluated the resistance levels of 42 monogenic lines containing known disease resistance genes and 69 commercial cultivars at seedling and adultplant stage to three races 34C0MRGQM,34C3MKGQM,and 34C6MTGSM identified from the alternate hostBerberis.Overall,the lines carryingSr9e,Sr17,Sr21,Sr22,Sr26,Sr30,Sr31,Sr33,Sr35,Sr36,Sr37,Sr38,Sr47,SrTmp,SrTt3,and 41 (59.4%) commercial wheat cultivars were effective against races 34C0MRGQM,34C3MKGQM,and 34C6MTGSM at the seedling and adult-plant stages.In contrast,the lines carryingSr5,Sr6,Sr7b,Sr9a,Sr9d,Sr9f,Sr9g,Sr9b,Sr16,Sr24,Sr28,Sr39,and 7 (10.1%)commercial wheat cultivars were highly susceptible to races 34C0MRGQM,34C3MKGQM,and 34C6MTGSM.The remaining 15 lines and 21 commercial wheat cultivars displayed variable levels of resistance to one or two of the tested races.This information provides resistant material and theoretical basis for the rational distribution and utilization of disease resistance genes and sustainable disease resistance breeding.

Acknowledgements

We are extremely grateful to Dr.Liu Taiguo at the State Key Laboratory for Biology of Plant Disease and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,for providing the 69 wheat cultivars.This study was supported by the Natural Science Foundation of Liaoning Province,China (2020-MS-204)and the National Natural Science Foundation of China(31701738).The funding sources had no role in the study design,data collection and analysis,and preparation of the manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.08.125