Study Of Kiwifruit Brown Leaf Spot

Recently,a brown leaf spot disease of kiwifruit emerges seriously in the major production areas of kiwifruit in Sichuan province,and breaks out as a disaster in some regions.This disease mainly infects leaves of kiwifruit,and at the initial stage of disease,a circle,dark-green,watery spot appeared on the infected leaves,and gradually formed small brown circular spot with the chlorotic halo around the lesions.Under the condition of high humidity,the disease spots rapidly expanded into the circular lesions with grayish white powdery centers surrounded by brownish concentric rings.Finally,multiple disease lesions combined leading to blade fracture,caduceus or dead.Repeated infection by pathogens in the field caused blade fracture of kiwifruit,and greatly inhibited the growth and development of kiwifruit trees.Kiwifruit brown leaf spot disease in the field can rapidly expand and widely spread resulting into a great difficulty of disease control.Until now,studies on kiwifruit brown leaf spot are just starting,and there are few reports on the causative pathogens,biological characteristics,pathogenicity mechanism,disease occurrence and disease control.This leads to the blindness of disease forecast,disease control and application of resistant varieties.In this study,we focus on the following six aspects of kiwifruit brown leaf spot.Firstly,the causative pathogen of kiwifruit brown leaf spot was identified,and genetic diversity was analyzed.Phenotype identification was performed basing on the colony characters,spore shape and so on,and then the sequencing,similarity and phylogenetic tree of the internal transcribed spacer(ITS),P-Tubulin and translation elongation factor EF-1 a genes was also analyzed.The pathogen of kiwifruit brown leaf spot was identified as Corynespora cassiicola(Berk.&Curtis)Wei according to the phenotype and molecular biology characteristic mentioned above.Eighty-four C.cassiicola strains from kiwifruit and 9 C.cassiicola strains from other host plants were collected from Sichuan to analyze genetic diversity of using the molecular markers of inter-simple sequence repeat(IS SR).A total of 188 fragments were amplified using 16 primer pairs of ISSR,and the polymorphism was 79.79%.Cluster analysis of 93 C.cassiicola strains using UPGMA.software showed that all strain was classified into four subgroups(Ⅰ,Ⅱ,Ⅲ,Ⅳ),and most strains belongs to Ⅲ group which can be divided into five sub-groups,indicating C.cassiicola,the pathogen of kiwifruit brown leaf spot in Sichuan,has rich genetic diversity.Genetic diversity analysis indicated C.cassiicola has a host-selective characteristic,whereas implied the genetic diversity was not correlated with geographical distributionSecondly,biological characteristics of kiwifruit brown leaf spot were investigated including growth conditions of mycelium,induced sporulation,conditions of spore germination and lethal temperature.The results showed that the optimal growth conditions of mycelium was cultured on the PSA culture medium with pH 6.0 at 25～30℃.Different illumination,carbon and nitrogen sources weakly affect mycelium growth.Sporulation was induced by UV-irradiation,high temperature,inoculation on leaf,lack of nutrition or nutrition inhibition,and results showed lack of nutrition stimulated sporulation.The optimal condition of sporulation was on the PDA medium(pH 6.0)at 25℃ with a alteration of light and dark.The spore germinates under the condition of the kiwifruit juice medium with glucose as carbon source(pH 6～7)at 25～27 ℃.Spore germination had no significant correlation with light,carbon source and nitrogen sources.Spore germination need high humidity,i.e.100%of relative humidity availed spore germination,whereas water drop wasn’t the necessary condition of spore germination.The lethal temperature was 52 ℃ for 10 min.Thirdly,pathogenic factors of kiwifruit brown leaf spot were studied.Inoculation of crude toxin on different host plants showed that cassiicolin was a host-selective toxin.the symptom showed that them could successfully infect kiwifruit leaf,and they had the same sopt on the leaf.the crude toxin is a factor in infection.Cassiicolin-encoding genes(Cas)were amplified from 84 kiwifruit C.cassiicola strains by six pairs of primer,and then their sequences were analyzed.The crade toxins of C.cassiicola were prepared,conditions for toxin producing was optimized,and subsequently pathogenicity of crude toxins and its host selection were tested.Results showed that Cas genes were amplified from only six strains using Cas2F17-Cas2R primer pair.The analysis of Blastn and phytogenetic tree suggested that the amplified Cas genes showed 100%similarity to Cas2 gene,and predicted protein sequence was also the same as Cas2 protein,which indicated that only 7%of C.cassiicola strains causing kiwifruit brown leaf spot in Sichuan had Cas2 genes.The toxin showed good thermostability,i.e.90 ℃ couldn’t affect toxin activity,and toxin still kept toxicity at 121 ℃ for 30 min.Conditions of toxin production were optimized to show the optimal conditions of producing cassiicolin as on the improved Frise3 medium for 12 d at 25 ℃,150 r/min on a shaker under the light.Pathogenicity comparison of the crude toxins of C.Fourthly,the occurrence and epidemiology of kiwifruit brown leaf spot were investigated.Both major kiwifruit cultivar of Hongyang and Jinyan harmed by brown leaf spot disease in Sichuan.Hongyang was more susceptible to brown leaf spot than Jinyan.Hongyang showed 99.1%of the average percentage of diseased leaves and 49.66 of average disease index in Sichuan province,whereas Jinyan was a little susceptible with 62.09%of average percentage of diseased leaves and 9.14 of average disease index.It was found that brown leaf spot disease developed in summer less seriously at high altitude sites than low altitude regions.Dynamic of single leaf lesion extension induced by brown leaf spot disease fitted Cubic curve.Single leaf lesion had good correlation with temperature and humidity,and extension rate of leaf lesion showed a significant positive correlation with the accumulated temperature of 25～30℃,average humidity,as well as accumulated humidity of 85-95%.Dynamics of disease index in field fitted Quadratic model and Cubic curve with R2=0.922.The increasing value of disease index in field showed significant correlation with average temperature,accumulated temperature of 25～30 ℃,average humidity and 85-95%of relative humidity.The amount statistics of spore release suggested that the amount of spore release at day was much higher than night.The amount of spore release at day showed significant negative correlation with the average temperatures of the continuous three days before capturing spore but significant positive correlation with the average humidity of the continuous three days before capturing spore.However,the amount of spore release at night showed significant positive correlation with the accumulated temperature of lower than 20 ℃ of the three days before capturing spores.At 27 ℃,spore started to germinate into leaf at 4h,sprout elongate at 4-8 h,appressorium and invasion structure formed at 8-12 h,and finally invaded into leaf tissue at 16h,the symptoms appeared on leaf at 48h.Pathogens overwintered mainly by disease residues on land surface,and the mycelium prepared a new cycle infection in March of the following year.Fifthly,disease resistance of kiwifruit germplasm resources was evaluated.In this study,disease resistance of 17 commercial varieties,17 wild varieties and 8 combined varieties were investigated by nature disease in the field or indoor detached inoculation with pathogen.Results of testing in field demonstrated that 58.8%of commercial kiwifruit varieties showed high resistance to the pathogen of kiwifruit brown leaf spot disease,and both varieties of Llongshan and Miliang 1 were high-resistant varieties.A total of 17%of major cultivated kiwifruit showed little or weak resistance,among which Hongyang was high susceptible to the pathogen while Jinfeng and Jinnong were susceptible varieties.For 17 wild varieties,only one was high resistant variety,and other 11 were resistant varieties.Five of eight combined varieties had good resistance against the disease,and three of them showed mid-resistance.Resistance analysis by indoor detached inoculation indicated that extension speed of disease lesions was closely correlated with the different varieties investigated in the field.The lesion extension speed at 6 days after inoculated on detached leaves with mycelium showed correlation with disease index in field.Therefore,the lesion extension speed can be used as a evaluation index for disease resistance of kiwifruit germplasm resources.Sixthly,exploration of botanical pesticides and screening of chemical agent were done for controlling the brown leaf spot disease.Biological activities of 192 botanical ingredients and 11 chemical agents against kiwifruit brown leaf spot disease were tested in vitro and in vivo.Results of botanical ingredients screening showed that both Glabridin and Psedolaric acid had high inhibition of spore germination under the inoculation concentration of 2.9057 μg-mL-1and 4.8072 μg-mL-1.When the two botanical ingredients were inoculated to leaves,Glabridin showed higher effect on disease control of kiwifruit brown leaf spot with 89.33%of protection effect and 87.87%of therapeutic effect.Screening of chemical agents demonstrated that Cyprodinil and Tetraconazole showed inhibiting effect against kiwifruit brown leaf spot disease,i.e.they inhibited mycelium growth with the EC50 value of 3.9869 μg-mL-1 and 4.2571μg-mL-1,respectively,and their EC50 of inhibiting spore germination respectively were 5.6376pg·mL-1 and2.1753μg·mL-1.Inoculation on leaves showed that Cyprodinil and Tetraconazole defended against kiwifruit brown leaf spot disease with a protection effect of 91.16%and 92.37%and with a therapeutic effect of 87.43%and 87.52%,respectively.Field experiments indicated that Cyprodinil and Tetraconazole controlled the kiwifruit brown leaf spot with the controlling effect of 85.83%and 83.89%,respectively.