Identification Of Pathogen Causing Gray Blight On Camellia Sinensis And Their Sensitivity To Three Fungicides

Tea plant（Camellia sinensis（L.）O.Kuntze）is an important economic crop in China.Tea is the most economically valuable part of tea plant,and has the effects of health preservation and anti-aging.With the expansion of tea planting,the attacked area and harm degree of tea plant diseases have increased significantly.Among them,tea grey blight is one of the most serious diseases in the places where tea plants are planted.The disease is mainly harmful to mature leaves,old leaves and young shoots.Seriously,it causes a large number of leaves falling off and causes the whole plant death,which not only reduces tea yield,but also seriously affects tea quality.In this study,morphological characteristics,multilocus sequence analysis and Koch’s rules were used to identify the pathogens isolated from the disesed tissues of tea in main production areas.The sensitivity of the tested isolates to difenoconazole,tebuconazole and carbendazim was determined by using mycelium growth rate method,respectively.The main research results are as follows:1.Isolation and morphological characteristic of Pestalotiopsis spp.The diseased leaves with typical symptoms of gray blight disease were collected from tea producing areas in Fujian,Henan,Hunan,Sichuan,Anhui and Guangdong.The pathogen were isolated and purified by tissue separation method,a total of 54Pestalotiopsis-like isolates were obtained.According to the colony morphology and conidia characters of the isolates on PDA,the isolates were divided into six types.Type 1（24 isolates）:mycelium milk white villous,laminated growth,with groove;type 2（7isolates）:mycelium pure white felt,with concentric rings;type 3（9 isolates）:Mycelial pure white cotton flocculent,striated growth;type 4（7 isolates）:mycelium pure white,scattered growth,colony shape petal;type 5（3 isolates）:mycelium milk white cotton flocculent,colony front furrow concentric ring;type 6（4 isolates）:mycelium pure white,scattered growth,colony gear.The conidia of isolates from type 1 to type 4 were 5 cells,which were spindle-shaped,straight or slightly curved,and brown in the middle.The conidia of isolates from type 1 to type 4 were 5 cells,which were spindle-shaped,straight or slightly curved,and the middle three cells were brown.The conidia of isolates from type 5 and type 6 were 5 cells,which were spindle-shaped,straight or slightly curved,and the first and second cells of the middle three cells were brown,and the third cell was light yellow.2.Molecular identification of Pestalotiopsis spp.Sixteen representative isolates were selected from each type for further molecular identification.The target fragments were amplified by PCR,sequenced and analyzed using primers of ITS,TUB and TEF genes,and the phylogenetic tree was constructed using multi-locus sequences.The results showed that the representative isolates tested included four different species,namely,Pseudopestalotiopsis.camelliae-sinensis,Pseudopestalotiopsis.chinensis,Neopestalotiopsis.clavispora and Neopestalotiopsis.mesopotamica,among which Ps.camelliae-sinensis had high isolation frequency.3.Pathogenicity testThe pathogenicity of representative isolates on detached leaves of cv.Shuchazao was tested by needle-punched method.For strains that can produce spores,10⁷conidia per m L suspensions were prepared for pathogenicity test.The results showed that 7 isolates of Ps.camelliae-sinensis were pathogenic in leaves.Among the five tested Ps.chinensis isolates,four isolates were pathogenic on leaves,but isolate FMZ5 did not cause leaves of cv.Shuchazao disease.For the isolates that failed to produce conidia（CCY51,CCY31 and Y823）was tested by mycelium inoculation method.The results showed that two isolates CCY51 and CCY31 were pathogenic.4.Sensitivity of representative isolates to three fungicidesIn this study,11 representative isolates were selected and their sensitivity to three fungicides was tested.The results showed that the tested isolates were highly sensitive to difenoconazole,the EC₅₀value was 0.0350-0.2675μg/m L,followed by carbendazim,the EC₅₀value was 0.1046-0.1647μg/m L,and the sensitivity to tebuconazole was low,the EC₅₀value was 0.4662-1.5826μg/m L.