Identification and analysis of pathogens caused Cymbidium goeringii leaf spot

【Objective】To identify pathogens causing the leaf spot disease during introduction and domestication of wild Cymbidium goeringii，and to provide scientific basis for diagnosis，prevention and control of C.goeringii leaf spot disease.【Method】C. goeringii leaves with typical leaf spot disease were collected from Guangzhou City，Guangdong Province and Zunyi City，Guizhou Province，and the pathogens were isolated and purified by tissue separation；according to Kochʼs rule，their pathogenicity was tested by acupuncture inoculation and friction inoculation in vitro. Based on their morphological characteristics，pathogen classification and identification were carried out through multi-gene molecular systematic analysis of partial sequences of ITS，ACT，CAL，EF-1a，GAPDH，GS and TUB2.【Result】In the collected leaves with 3 typical leaf spot diseases Symptoms，4 strains of pathogens were detected，and were named as GZH1， GZH2，GZH3 and GZH4. Among them，GZH1 and GZH4 were isolated from diseased leaves collected in Guangzhou City，Guangdong Province；GZH2 and GZH3 were isolated from diseased leaves collected in Zunyi City，Guizhou Province. The results of pathogenicity tests showed that strain GZH1 showed strong pathogenicity through acupuncture inoculation in vitro，while strains GZH2，GZH3 and GZH4 could cause typical necrotic spots at the inoculation site by friction inoculation.On PSA medium，strain GZH1 showed colony morphology，morphology and size of macroconidia，microconidia，microconidia conidiophore and chlamydospores were consistent with those of Fusarium oxysporum，and partial sequences of ITS and EF-1A of strain GZH1 had a consistency of 98.23% and 97.24% with F. oxysporum strain UACH-217； combined with phylogenetic tree analysis by multi-gene sequences，strain GZH1 was identified as F. oxysporum. On PDA medium，colony morphology，conidia morphology，conidia size，appressoria morphology and appressoria size of strains GZH2，GZH3 and GZH4 were highly consistent with those of Colletotrichum cymbidiicola，C. fructicola and C. jiangxiense，respectively. Partial sequences of ITS，ACT，CAL，GAPDH and TUB2 of strain GZH2 were 99.00%，100.00%， 99.00%，99.00% and 99.60% consistent with those of C. cymbidiicola strain CBS:123757，respectively. Partial sequences of ITS，ACT，CAL，GAPDH and TUB2 of strain GZH3 were 99.00%，99.00%，99.00%，100.00% and 99.70% consistent with those of C. fructicola strain ICMP:18613，respectively. Partial sequences of ITS，ACT，CAL，GAPDH， TUB2 and GS of strain GZH4 were 99.60%，99.00%，99.00%，99.00%，100.00% and 97.10% consistent with those of C. jiangxiense strain LF687，respectively. Combined with phylogenetic trees by multi-gene sequences showed that GZH2 was identified as C. cymbidiicola，GZH3 as C. fructicola and GZH4 as C. jiangxiense.【Conclusion】Pathogens composition of C. goeringii leaf spot disease is different in different places. F. oxysporum and C. jiangxiense are the pathogens of C. goeringii leaf spot disease in Guangzhou City，Guangdong Province；C. cymbidiicola and C. fructicola are the pathogens of C. goeringii leaf spot disease in Zunyi City，Guizhou Province. Among them，C. fructicola and C. jiangxiense are reported as disease infecting orchid plants for the first time，which are newly-marked orchid diseases.