Identification Of Barley Crown Rust In China And The Development Of Molecular Markers Specific For Its Pathogen

Rusts are fungal diseases of wheat and other cereal crops,which cause epidemics and outbreaks of diseases worldwide,leading to seriously yield losses.Barley is an important food crop,feed crop and raw material for brewing beer in China.So far,rusts on barley reported in China include stripe,leaf and stem rust.However,blarley crown rust has not been reported yet.In recent years,barley crown rust was discovered for the first time in Qilian county of Qinghai province of China by our laboratory.Therefore,objectives of this study were aimed at morphologicial characteristics,pathogenicity,and sexual reproduction of the barley crown rust by combined methods of morphological observation,molecular biological,pathogenicity tests.The main results were as follows:1.The occurrence of barley crown rust has been demonstrated using morphological and molecular biological methos in China.The causal pathogen was identified as Puccinia coronata var.hordei.Barley crown rust was proven to be a nes disease on barley in China.Two isolates,HZJ0004 from of the barley rust sample,and POR3 from naturally rusted buckthorn(Rhanmus sp.),were obtained by picking single uredium respectively,and their teliospores were induced to produce.Morphological observations showed that urediospores of P.coronata var.hordei(isolate HZJ0004)were bright yellow,nearly round,and their size were 29(24 ～ 32)μm × 23(21 ～ 25)μm,and that teliospores of the pathogen were brownish yellow and dual-nucleated,with very short and light brown stalks at the base.The middle parts of a teliospore were divided into bispores by a transverse septum.The sizes were 57.4(43.4 ～ 73.3)μm × 15.5(10.9 ～ 21.2)μm,with attached branches at the top the length was 4.3(3.0 ～ 7.1)μm ～ 9.6(5.2 ～ 14.6)μm.Phylogenetic tree analysis showed that identity of ITS(internal transcribed spacer)sequences of isolate HZJ0004 and Puccinia coronata var.hordei(accession No.: DQ355454)was 93%,and were clustered into a branch with 92% of the bootstrap value,indicating that the pathogen was Puccinia coronata var.hordei.2.Pathogenicity tests indicated that the pathogenicity of Puccinia coronata var.hordei(isolates HZJ0004 and POR3)and Puccinia coronata var.avenae(isolate POC8)to 170 cereals and grasses was significantly different.Of 84 barley cultivars tested,69(82.1%)were susceptible to isolate HZJ0004 of P.coronata var.hordei,60 showed susceptibility to isolate POR3,and all were highly resistant to isolate POC8 of P.of coronata var.avenae.Tested 4 oat cultivars were resistant to isolates of HZJ0004 and POR3,showing sporadic chlorotic spots or asymptomatic,but 3 oat cultivars were susceptible to isolate POC8.Three isolates of HZJ0004,POR3 and POC8 were avirulent to 63 tested wheat cultivars.Of17 gramineous grasses species tested,6,including 5 Elymus species and 1 Bromus species(B.himalaicus),were susceptible to isolate HZJ0004,and 2,including E.atratus and Aegilops tauschii,were susceptible to POR3.However,the isolate POC8 was not pathogenic to all grass species.3.Alternate host species of Puccinia coronata var.hordei were determined and the pathogen completes sexual reproduction on wild buckthorn(Rhanmus spp.)under natural conditions were demonstrated.In the present study,4 buckthorn cultivars,including R.davurica,R.utilis,R.cathartica and R.parvifolia,were tested using basidiospores of isolate HZJ0004.Among them,R.davurica and R.utilis were infected by basidiospores to produce pycnia and aecidia,and aeciospores infected a barley variety(Guoluo)to produce urediospores,indicating P.coronata var.hordei completed the sexual reproduction.However,R.cathartica and R.parvifolia can not be infected by basidiospores.Therefore,R.davurica and R.utilis can serve as alternate hosts for P.coronata var.hordei.The above results indicated that P.coronata var.hordei can complete sexual reproduction under laboratory conditions.Isolate POR3,collected from naturally rusted buckthorn plants,shared a high sequence identity of 97.42% with isolate HZJ0004 from barley based on sequence alignment using software DNAMAN.Phylogenetic analysis showed both isolates were clustered into a branch with 99% of the bootstrap value,suggesting that that P.coronata var.hordei can complete the sexual reproduction on wild buckthorn under natural conditions.4.A molecular marker specific for Puccinia coronata var.hordei was established,and can be used for molecular detection of the pathogen.Based on sequences produced PCR amplification using ITS1 Rust F10D/ITS4 primers,a pair of specific molecular marker A1/S1(5ˊ AATAGCACCACTCACCAT 3ˊ / 5ˊ TAAACTTCACCCAAACTC3ˊ)was designed.PCR amplification using genomic DNA of 13 rust fungal speices belonging to genus of Puccina showed that only P.coronata var.hordei isolates HZJ0004 and POR3 produced target positive bands,but other rust fungi were not.Therefore,molecular marker A1/S1 can specifically detect Puccinia coronata var.hordei,providing technique support for the forecast of barley crown rust.