Studies On Identification Of A New Tea Disease And Its Detection Technology

Hubei is one of the main tea-producing provinces of,where many tea diseases occur and some diseases occur widely and have caused severe losses on tea output and tea quality.In this study,the diseases on tea leaves have been surveyed in main tea-planting regions in Hubei Province of China.The pathogens have been characterized based on morphological and molecular features.A rapid and sensitive detection technology for the selected pothogens has been developed.The results are summarized below.1.In 2013 and 2014,foliar diseases on tea have been surveyed in 160 tea gardens of five tea-planting regions in Hubei of China.The results showed that a total of fifteen diseases had been found in Hubei.Six diseases occurred in Enshi,ten diseases occurred in Shiyan,eight diseases occurred in Yichang,ten diseases occurred in Huanggang,and eleven diseases occurred in Wuhan.The main fungal diseases include brown-black spot,brown blight,blister blight,gray blight,anthracnose,Macrophoma stem blight,sooty mould,red leaf spot and white scab.Brown-blight and anthracnose are two foliar diseases which occurred most in five regions,and other diseases occurred in different regions with incidence ranging from 0 to 15.8%.Two regions(Enshi and Yichang)had low disease index values ranging from 9.3 to 19.8,whereas the other three regions(Shiyan,Huanggang,and Wuhan)had high disease index values ranging from 9.9 to 28.5.The high geographical association was likely concerned with the construction of quality safety demonstration plots for agricultural product export.During the surveys,brown-black spot was firstly described as a new tea disease,which is characterized with the symptoms being different from those of gray blight,white scab and red scab.2.During the surveys,a new foliar disease with small brown-black spots(1~2 mm in diameter)on tender tea leaves(Camellia sinensis)was characterized,but the pathogen(s)for this disease were not known.A total of fifty four fungal isolates were obtained from the leaf samples with brown-black spots collected from Xuan En County of Enshi,Zhuxi County of Shiyan,Yingshan County of Xinzhou district of Wuhan.Morphological characterization and phylogenetic analysis of ribosomal internal transcribed spacer(ITS)showed that twelve might belong to Phomopsis spp.,eight toColletotrichum spp.,and thirty four to Pestalotiopsis spp.Conbination of the phylogenetic analysis of ?-tubulin(TUB2)and translation elongation factor 1-?(TEF1),the thirty four isolates were further identified as Pestalotiopsis theae(14 isolates),P.camelliae(12 isolates),and P.clavispora(8 isolates).Among thirty four Pestalotiopsis isolates,seven(TP1-2O,TP2-1A,TP2-2W,LT3-2O,LT4-1O,LT4-2B,and PF2-8O)were in-depth examined.Pathogenicity tests on detached tea leaves showed that under on wounded conditions and no matter what mycelial discs or conidium suspensions were used,inoculation of the Pestalotiopsis fungi resulted in small brown-black spots(1~2mm in diameter)on wounded leaves,similar to those observed in the field in the sizes and colors and P.theae and P.clavispora showed significantly higher virulence than P.camelliae;under unwounded conditions,only P.theae had pathogenicity on tea leaves.Inoculation test with conidium suspension on intact tea leaves in the field also caused similar black spots and the pathogens could be re-isolated from the diseased leaves,confirming that the inoculation of Pestalotiopsis spp.caused the symptoms and P.theae was the pathogen of brown-black spot disease.The P.theae isolates such as TP1-2O and TP2-2W responsible for brown-black spot were obviously different from those for tea gray blight disease in median cell color,apical appendage morphology,growth rate,and pathogenicity,and also in phylogenetic relation.To our knowledge,this is the first report that the Pestalotiopsis spp.caused the brown-black spot disease on tender tea leaves.3.A total of five isolates were obtained from leaf samples with typical symptoms of brown blight,gray blight,anthracnose,or macrophoma stem blight collected from Zhuxi county of Shiyan and Huazhaong Agricultural University of Wuhan.Basing on the colony features and microscopic observations,isolate ZX3-1,HN-40 and HN-1 were characterized as Collectotrichum camelliae Massee,Pestalotiopisis theae(Sawada)Steyaert,and Colletotrichum gloeosporioides,respectively,and both HN-15 and HN-42 were as Phoma spp.According to the Koch's rule,all isolates were analyzed in isolation,pathogenicity test,re-isolation and morphological characterization,and suggested that these isolates are the pathogens for the original symptoms.Mating protein 2(MAT2)or ITS sequence analysis was consistent with the morphological characterization,confirming the identity of five isolates.In this study,Colletotrichum gloeosporioidesisolate HN-1 and Phoma spp.isolate HN-15 and HN-42 are firstly reported as pathogens associated with tea anthracnose and macrophoma stem blight,respectively.It remains unclear whether they can cause same symptoms on tea leaves as those observed in the field.4.Three pairs of specific primers and three specific Taq Man probes were designed basing on the ITS sequences of Collectotrichum camelliae,Pestalotiopisis theae and Phoma spp.and a Real-time fluorescent PCR technology was developed for rapid and sensitive detection of the pathogens for tea brown blight,gray blight,anthracnose,and macrophoma stem blight.Sensitivity analysis showed that 1~10 pg/?L of DNA from the pure culture was enough to generate strong fluorescence signals,showing much higher sensitivity than that of conventional PCR.Using this technology,Colletotrichum camelliae isolates ZX3-1 for tea brown blight was successfully detected,but the isolates of Colletotrichum gloeosporioides for mango anthracnose and C.fragariae for strawberry anthracnose were not detected,showing high specificity of the primers and probe;however,all the P.theae isolates TP1-2O,TP2-1A,and P.clavispora isolate TP2-2W for tea brown-black spot and P.theae isolate HN-40 for tea gray blight were also succesfully detected,showing no specificity of the primers and probe at a species level.Using this technology,direct detection of the pathogen P.theae in brown-black spot leaf was also achieved.These results laid a solid foundation on rapid detection and identification of pathogenic fungi on tea.