Occurrence, pathogen identification and laboratory screening of efficient fungicides of kiwifruit brown leaf spot in Jiangx

Abstract: 【Objective】Kiwifruit brown leaf spot disease is the second largest disease in many places after the bacterial canker disease, which often causes a large number of fallen leaves, resulting in bare branches and yield losses. This study aims to understand the occurrence of kiwifruit brown leaf spot disease in Jiangxi province, clarify the pathogens of kiwifruit brown leaf spot disease, and screen of fungicides that can effectively inhibit the pathogens, helping for the prevention and control of kiwifruit brown leaf spot disease in Jiangxi.【Methods】A survey was conducted on the occurrence of kiwifruit brown leaf spot disease in kiwifruit experimental stations in four regions including east, west, south, and north of Jiangxi. Twenty-one orchards distributed in 4 counties named Wuning, Fengxin, Yushan, and Xunwu were included in this research, a 0 to 9 scale evaluation criterion of kiwifruit brown leaf spot was used to evaluate the disease severity of individual leaves, and a parallel line sampling method with 5 lines in each orchard was used to evaluate the disease severity of different orchards. Then, tissueisolation of pathogens was conducted on diseased leaves collected from the epidemic area of kiwifruit brown leaf spot disease in Jiangxi province. Leaf segments (approximately 1 cm2 ) were excised from the margins of lesions were washed in sterile water, surface sterilized in 75% ethanol for 45 s, washed again in sterile water, dried on sterilized filter papers, and placed on PDA medium containing streptomycin. Cultures were incubated at 25 ℃ in dark until colons grown out from leaf tissues, and isolates were purified from the hypha edge of colons. The pathogenicity was validated by Koch’s postulates, the surface of leaves of kiwifruit cultivar Jinguo was sterilized with 75% ethanol for 1 min, washed with sterilized water, wiped with clean soft paper, wounded with sterilized needles, and inoculated with mycelial plugs from PDA plates, inoculated leaves were incubated at 25 ℃ in dark for 5 days. The morphology of pathogen was preliminarily identified through colony, hyphae, and spores. The molecular identification of the strains was carried out using ITS1/ITS4 primers. The PCR amplicons were sequenced at Sangon Biotech and aligned to NCBI database. Fourteen ITS sequences of 12 species under genus Corynespora, ten ITS sequences of 5 species under genus Didymella, Alternaria, Diaporthe, Fusarium, and Colletotrichum were used to construct molecular phylogenetic tree by MEGA 7.0.26 with the neighbor-joining method, ITS sequence of Verticillium dahliae was used as out group in the molecular phylogenetic tree. Using the mycelial growth rate method, the indoor toxicity of 9 chemicals that have not been used in kiwifruit brown leaf spot disease was determined.【Results】Kiwifruit brown leaf spot disease occurred seriously in western and northern Jiangxi, with a Median Disease Rating (MDR) of grade 5 or above in 5 out of 11 survey sites; the incidence was relatively mild in eastern and southern Jiangxi, with a median disease rating of 0 in all 10 survey sites. The average Disease Indices (DI) of Wuning and Fengxin were 31.94 and 37.16, respectively, and the incidence rates of kiwifruit brown leaf spot diseased leaves were 100% in epidemic orchards. The average DI of Yushan and Xunwu were 0.24 and 3.09, respectively, which were significantly lower than that in Wuning and Fengxin. By Koch’s postulates, isolates of KBLS-1 could successfully infect leaves and cause necrosis lesion symptoms, re-isolation of pathogen from diseased leaves proved that the isolates of KBLS-1 were the pathogen of kiwifruit brown leaf spot. By morphological observation, the spores of KBLS-1 were of rod-shaped, and several of diaphragms existed in spores, which was in accordance with features of Corynespora cassiicola. By molecular identification of the isolates, the ITS sequence of isolate of KBLS- 1 was homologous with C. cassiicola with 100% identity. According to phylogenetic analysis, ITS sequence of isolate of KBLS- 1 was clustered together with C. cassiicola on molecular phylogenetic tree, and was distinguished from other Corynespora sp. or common pathogens in orchards such as Didymella sp., Alternaria sp., Diaporthe sp., Fusarium sp., and Colletotrichum sp. Thus, the pathogen of kiwifruit brown leaf spot disease in the main epidemic area of Jiangxi is C. cassiicola. The indoor toxicity test results showed that prothioconazole, cyproconazole, captan, and myclobutanil had extremely highly inhibitory effects on kiwifruit brown leaf spot pathogen. These four fungicides could inhibit the growth of pathogen at low concentrations, with EC50 values (μg · mL- 1 ) of prothioconazole, cyproconazole, captan, and myclobutanil being 17.36, 6.97, 5.30, and 2.50, respectively. Fenbuconazole, ningnanmycin, pyrimidine nucleoside antibiotics, and fosetyl aluminum had highly inhibitory effects only at high concentrations, with EC50 values of 86.12, 106.07, 304.46, and 509.62, respectively. Picoxystrobin had no obvious inhibitory effect on C. cassiicola in this research.【Conclusion】Kiwifruit brown leaf spot disease occurs seriously in northern and western Jiangxi, and is relatively mild in eastern and southern Jiangxi, with the pathogen being identified as C. cassiicola. Laboratory research showed that prothioconazole, cyproconazole, captan, and myclobutanil can be used as potentially efficient agents for the prevention and con-trol of kiwifruit brown leaf spot disease.