Genetic Diversity And Molecular Detection For Blumiria Graminis F.sp.Tritici In Northerneastern China

Wheat powdery mildew caused by Blumeria graminis f.sp.tritici is one of the most important wheat diseases in our country,esp.in Northeastern Spring Wheat Region.The most effective control way of wheat powdery mildew is to use resistant varieties in combination with right time emergency chemical control based on accurate prediction and forecast.In the present paper all the relevant studies conducted are around this disease control principle.Firstly,race population trend and virulence variation of Blumeria graminis f.sp.tritici in the Northeastern Spring Wheat Region,Shandong,Henan and Hubei Winter Wheat zones in the years 2007 and 2008,and the resistance to the disease contained in wheat varieties(lines) were studied.Secondly,the genetic diversity of wheat powdery mildew,molecular virulence clustering were analyzed and the correlation among molecular diversity,virulence diversity and epidemic zone was analyzed using ISSR method.Thirdly,aimed at the internal transcribed spacer(ITS) of ribosome of Blumeria graminis f.sp.tritici,molecular detection techniques using conventional PCR,DNA Dot-blot,Real-time PCR were studied,specific primers and probes were designed and repeatedly tested,the optimized conditions for detection and diagnoses were conducted.At last,PCR detection techniques to another three important diseases(wheat sheath blight,wheat loose smut and wheat scab) were studied.The main results were as it follows:1.The analyses of population dynamics of the pathogen and the resistance(gene) contained in wheat cultivars were carried out.For the population dynamics,the result was shown that 61 races were met with from 223 isolates tested.In northeastern spring wheat region,race 17 was dominant in the year 2007 with the occurrence frequency being 10.2%.In 2008,the race 411 became most common with an occurrence frequency of 20.9%,and the trend of occurrence frequency for races 11,415,611 and 631 appeared increasing.The prevailing race was race 11 with the frequency of 16.4%in Shandong winter wheat belt in the year 2008.The virulence gene analysis was shown that virulence frequencies for V2,V4b,V2+6,V4+8,V12,V13,V16,V18,V20, V21,V22and V23 were low,distributing between 0.0%~29.9%,indicating the corresponding genes Pm2,Pm4b,Pm2+6,Pm4+8,Pm12,Pm13,Pm16,Pm18,Pm20, Pm21,Pm22,Pm23 were effective and applicable in breeding.The virulence association of 147 isolates was studied and shown that the frequency of the single spore isolates that contain 9 virulent gene association was high,with its frequency being 15.64%.Powdery mildew resistance(gens)in 302 wheat cultivars(lines)was appraised and the effective resistant genes carried in 10 resistant cultivars were inferred.It was indicated that the cultivar 'Baofeng 104' contained resistant gene(s)Pm2+6,'Sunwon85' and 'WF 08-182', Pm5+Pm8,'Jingdog 8' and 'Xifeng 20',Pm2+Pm8,and 'Nanda 2419' and 'Chuanyu 55871', Pm24+Pm8.'Lantianl8' had the same resistant gene as 'Era','Lantianl7 'and 'Mianyang 28' carried resistant genes unknown. 2.The genetic diversity and molecular virulence clustering for the powdery mildew were studied.12 isolates from Northeastern Spring Wheat Region and 14 isolates from Shandong and Henan and Hubei Winter Wheat zones were tested with 18 ISSR primers.Clustering analysis was showed that these isolates were sorted into 4 groups at a similarity of 0.61.Group 1 consisted 13 isolates,races 7,11,17,411 from Liaoning,races 11 and 411 from Shandong, races 11,35,331 and 711 from Henan and races 11,21 and 31 from Hubei.Group 2:race 611 from Shandong and race 311 from Liaoning.Group 3 had 10 races including races 431,55, 611,631 and 731 and 77from Liaoning,races 431 and 731 from Shandong,races 631 and 731 from Hubei.Group 4 contained 1 race:Race 23 from Liaoning.The dendrogram was constructed based on virulence clustering.The similarity of 26 isolates was 0.52～1.00.They could be clustered into 3 groups at the point of similarity 0.70. Group 1 contained race 17,55 and 77 from Liaoning.Group 2,race 7 and 23 from Liaoning. Group 3 included the rest 21 isolates.The above results were indicated that the closed DNA relationship existed between the wheat powdery mildews from Shandong winter area and from Northeastern spring wheat region. 3.Three molecular detection and diagnose techniques based on internal transcribed spacer (ITS)of rDNA of Blumeria graminis f.sp.tritici were studied.The result was shown that by primers or probes designing and screening,and the detection and diagnose conditions optimizing,the primer pair XBFF/R of PCR,the probe of DNA Dot-blot,the real-time primer pair XBFTF/R with TaqMan probe were proven specific and validated with other 9 related plant pathoens as references.With conventional PCR primer pair XBFF/R,the same 352bp bands appeared by amplifying all the DNA samples from the eight races,and no bands appeared by amplifying all the DNA samples from the 9 plant pathogen species(Cucumber powdery mildew,wheat yellow rust,wheat leaf rust,wheat stem rust,wheat scab,wheat sheath blight,wheat loose smut,maize sheath blight and maize head smut)and control.With the probe of DNA Dot-blot,all the eight powdery mildew DNA samples were detected to be positive,and all the rest 9 plant pathogen DNA samples and control were shown negative.With the real-time primer pair XBFTF/R and TaqMan probe,the strong positive signals were obtained from all the eight powdery mildew DNA samples,with the values for dRn:0.35~0.45,Ct:15.92~18.53,and otherwise,no signals increased for all the 9 plant pathogen DNA samples and control.The detection sensitivity test for the three methods was conducted.It was indicated that the detection limits to DNAs of Blumeha graminis f.sp.tritici.were 10pg for the conventional PCR primer pair XBFF/R,1pg for probe of DNA Dot-blot and as low as 1fg for the real-time primer pair XBFTF/R with TaqMan probe.The comparision of the least time needed from powdery midew inoculation to disease dectected or seen was made under greenhouse condition among conventional PCR detection, Real time PCR detection and natural observation.It was shown that under greenhouse condition,the DNAs from powdery mildew infected wheat leaves were detected 3 days after inoculation with conventional PCR assay and only one day with real-time PCR,the disease lesions were seen with naked eyes 5days after inoculation.indicating that the disease after inoculation was detected two days earlier with conventional PCR and 4days earlier with real time PCR than natural observation,respectively. 4.Molecular detection techniques based on ITS were also used to detect wheat diseases; wheat sheath blight:Rhizoctonia cerealis,wheat loose smut:Ustilago nuda and wheat scab:Fusarium graminearum.3 primer pairs XWKF/R,XSHF/R and XCMF/R were designed and tested.It was shown that all the primer pairs were each unique and highly sensitive to the corresponding target pathogen.