Phylogenetic Study On Ergot Fungi And The Characteristics Of DmaW Gene Encoding Dimethylallyltryptophan Synthase

Claviceps spp.(Clavicipitaceae,Hypocreales,Ascomycota) are economically important fungi infecting gramineous plants grown in the world.Until now,about 45 teleomorph species of Claviceps have been described,and mainly distributed in tropic and subtropical regions.Many species exist only in forms of anamorphic(Sphacelial) and attracted less attentation,therefore,the species in genus Claviceps should be many more.In this study,abundant gramineous populations were found to be infected by Claviceps spp.in Jiangsu,Xinjiang and Qinghai.Five fungal isolates were obtained from Roegneria kamoji in Nanjing,and 18 from grasses in grasslands in Xinjiang and Qinghai. Basing on morphological characters and molecular phylogenetic evidences,taxonomic positions of these fungal isolates were determined.The characteristics of the dmaW (encoding dimethylallyltryptophan synthase) fragment were analyzed.Finally sensitivity of these fungal isolates to 2 popular fungicides were also investigated.Firstly,filed surveys were carried in 3 provinces(Jiangsu,Xinjiang and Qinghai). Ergotism on Roegneria kamoji population were found in Nanjing,and on grassed belonging to 5 genera within 3 subfamily,in west China.Sclerotia on R.kamoji were usually 0.5-1.0×5-10mm,cylindrical,straight or slightly curved.The surface was purple-black to black with several longitudinal grooves.Sclerotia on grasses grown in west China were purple-black or brown-black with several longitudinal grooves,ellipse or cylindrical, 0.5-1.8×5-21 mm.Honeydrews were observed from inflorescenses of these host grasses.All of these were typical morphological characters of Claviceps.From ergots harvested from infected plants,23 fungal isolates were obtained. Morphological characteristics and the growth rate of isolates from Xinjiang,Qinghai,were nearly identical to C.purpurea.Conidia colorless,transparent,ellipse, 2.5-6.25×4.5-13.75μm,the arrangement of conidia on phialides rather special,especially short phialides,1.1-2.3×3.5-20μm.Under the same condition,the Roegneria isolates grow more rapidly,up to 5 mm/d,conidia colorless,transparent,ellipse or cylindrical,the size of phialide was 2.5-3.3×6.8-10μm.The morphological features of the isolates were similarly to Claviceps sp.(Anamorph:Sphacelia sp.).Significant differences in morphological characters between these fungal Chinese isolates and other Claviceps species reported previously were observed.Genomic DNA of 12 isolates was extracted,and their rDNA-ITS,tubB and tefA fragments were cloned,sequenced and analyzed.Sequences from representative strains of Claviceps spp.obtained from GenBank were aligned with the sequences obtained in this study.Sequences of the 5 isolates obtained from R.kamoji were similar each other; sequences of the 2 isolates obtained from Leymus secalinus in Qinghai were different from the remaining 5 isolates.These 3 groups were totally different each other.In phylogenetic analysis,tubB and tefA sequences were more effective than rDNA-ITS sequences to clarify the relationships between the Claviceps isolates.The 5 isolates from R. kamoji and 2 from L.secalinus clustered into an exclusive clade respectively,while the remaining 5 isolates from other grasses in grasslands in Xinjiang closely clustered with C. purpurea,indicating these isolates were quite diverse.Based on conserved sequences of dmaW genes in NCBI,selective primers were designed and used for amplification of dmaW fragment of Claviceps from R.kamoji isolates CRN503.A 1260bp fragment was obtained and sequenced.By using iPCR with the primers oriented in the reversed direction of the usual orientation,2 franking regions(862 bp in total) were obtained and sequenced.Combining these regions,a 2122bp fragment of dmaW,including 2 supporsing intron regions(82bp and 58bp respectively),was obtained. These indicated that the full length of dmaW gene might exist in isolate CRN503 from R. kamoji.In addition,a low homology was observed when analysed with the DNA sequences of dmaW from other fungi,indicationg dmaW in these fungi should be not conserved.Finally,sensitivity of 7 Claviceps isolates to 2 of most popular chemical fungicides, Carbendazim and Mancozeb,were tested.Inhibition of hyphal elongation inhibition (inhibition of colony development) tests,Carbendazim was significantly effective than Mancozeb to all isolates.In conidia germination suppression tests,Carbendazim also exhibited higher activity.Therefore,application of Carbendazim should be more effective than Mancozeb if chemical control of ergot disease is necessary.