Sexual Genetic Recombination Between Puccinia Striiformis F.sp.tritici And P.achnatheri-sibirici

Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most important diseases of wheat in almost all wheat production regions worldwide.The disease mainly spreads by wind-borne urediniospores to cause damage,causing a huge yield loss in a severely epidemic year.Previous studies have been shown that some ways,including mutations,heterokaryosis(somatic recombination)and sexual genetic recombination,can cause virulence variation.Recently,with the finding of barberry(Berberis)as the alternate host for Pst,some studies demonstrated that under natural conditions Pst infects wild susceptible barberry complete sexual cycle in China,and that sexual genetic recombination can by conducted through the intra-species selfing,intraspecific(race to race)hybridization of Pst,and sexual hybridization between Pst and formae speciales(f.sp.)of P.striiformis.However,so far there has been no report with regard to interspecific hybridization between Pst and Puccinia species.Gramineous grasses are a large group of species,many of which act as accessory(secondary)host for a large number of rust fungi,including devastating rust pathogens of cereals.Among the rust fungi,some are known to be heteroecious and have a complete life cycle,but for many others their life cycles are not clear with knowing only asexual stage and unknowing sexual stage.Puccinia achnatheri-sibirici(Pas)is a rust fungus that infects the grasses of Achnatheherum spp.Thus,whether sexual hybridization can occur between Pst and Pas through sexual genetic recombination,producing new races(pathotypes)to cause stripe rust infection on wheat.Therefore,sexual hybridization between both rust species of Puccinia was carried out.The main results were as follows.1.The rust species infecting the grass Achnatheherum extremiorientale were identified as P.achnatheri-sibirici(Hara)Keng ex P.C.Kuo based on combined methods of traditional biology and molecular biology.Leaves bearing urediniospores and teliospores of Pas were collected from A.extremiorientale growing in Guancun Village,Baoji City in western Shaanxi Province.Single uredinium isolate was established and P.achnatheri-sibirici was confirmed by identification of pathogenicity on hosts,observation of morphologic characteristics of urediniospores and teliospores,sequence alignment,and phylogenetic tree analysis.The ITS sequence of Pas did not match with those submitted in the NCBI database,and had the highest sequence homology with 94% compared to P.brachypodii and P.aizazii.However,Phylogenetic tree analysis indicated that Pas was clustered into an independent clade rather than one clade with other related rust species in genus Puccinia,showing a scaled branch of 92% at branch node?2.Barberry(Berberis spp.)were identified to serve as alternate hosts for P.achnatheri-sibirici and the rust pathogen is macrocyclic.The results completed life cycle of the rust speices.Morphological characteristics of the rust during the development of sexual stage on barberry were observed.Some of Berberis species,including B.shensiana,B.aggregata,B.cavaleriei,B.soulieana,and B.phanera,were identified whether they could be alternate hosts for the Pas through inoculation with basidiospores produced from teliospores of the rust pathogen.The results showed that B.shensiana,B.aggregata,and B.cavaleriei serve as alternate hosts for the rust pathogen,demonstrating that Pas is heteroecious,and macrocyclic and determining that the rust pathogen completes the whole life cycle on the grass as primary host and barberry as alternate host.At the same time,B.shensiana is determined as shared alternate host for Pas and Pst,providing a biological basis for conducting sexual hybridization between both rust fungi.Observations of morphological characteristics of Pas during the development of sexual stage on B.shensiana were performed using light and scanning electronic microscopes.3.Sexual hybridization between Pst and Pas successfully confirmed,showing a high virulence variation and producing new races(pathotypes)among progenies.The results indicated interspecific hybridization between Pst and Pas is a new way of sexual reproduction and resulting in virulence variation of Pst.Barberry(B.shensiana)seedlings were inoculated with basidiospores produced from teliospores of Pst(CYR32)and Pas respectively.As nectar appeared on barberry leaves,fertilization was conducted by transferring individual nectar of Pas into one of Pst and,conversely,by also transferring individual nectar of Pst into one of Pas.After production of aecia on the backside of leaves,single aeciospore isolate was established by picking individual.The results showed that a total of 85 progenies were obtained from 19 aecia produced from two ways of hybridizations of Pst and Pas,producing 56 pathotypes based on tests on the differential host consisting of 28 single Yr gene lines and a rate of 65.8% in virulence variation compared to the parental isolate CYR32.Of 85 progenies,82 were obtained from the hybridization(Pas to Pst)and 3 from reverse hybridization(Pst to Pas).The 82 progenies produced 53 pathotypes and the number of progenies virulent to Yr10,Yr24(=Yr26)accounted form 20%.The 3 from reverse hybridization were virulent to Yr8.All of 85 progenies were not match with CYR32 in virulence patterns and were not detected to be virulent to Yr5 and Yr15.Altogether,virulence spectrum of progenies became narrow compare to the parental isolate CYR32.In this study,sexual hybridization of Puccinia striiformis f.sp.tritici and P.achnatheri-sibirici revealed that interspecies hybridization resulted in a distinct virulence variation among the progeny and is a new way of virulence variation of Puccinia striiformis f.sp.tritici.The results provide an important theoretical basis for revealing the causes of genetic diversity of Pst and for making a new strategy for management of wheat stripe rust in China.