| Grey mould of strawberry is a kind of important disease in strawberry's production.lt occurs every year,especially strawberry happens to meet low-temperature and rainy climate in the flowering period,the occurrence of this disease become more serious.During this period,If strawberry were not managed well,production could reduce by 10-30%at light level,and even total crop failure at severe level.Because of fast growth speed of Botrytis cinerea,giant propagation,strong adaptability,large resource and easy variation in the effect of external environment,all of this bring much difficulties for disease's prevention and control work.Therefore,it is essential enough to research origin of Botrytis cinerea,genetic structure,genetic differentiation,genetic variation,population's trends,relationship with external environment and screen new kind of fungicide.Since Hunan is located in south of the Changj iang River,its climate characteristic and geographical characteristic are obviously different from other areas,and its climates are distinguished from the other areas around the province,it is complex in topography,with a variety of planted strawberrys,all of its natural condition provide natural endowments for variation and changing of population's structure of Botrytis cinerea.At present,many institutions pay close attention to the study of Botrytis Cinerea and the screening work of new fungicide,such as Jiangsu,Hubei,3eijing,Henan and so on,but the study of Botrythis cinerea's genetic diversity in Hunan has not been seen yet.Therefore,the study is going to research genetic diversity of Botrytis cinerea of Hunan and screen new fungicide,which aims to learn the richness of Botrytis cinerea's diversity in different areas of Hunan province,clear the relationship between diversity and geographical location,and answer genetic structure and differentiation,population's category and related law of evolution,and provide examples for reasonable using of drugs to control grey mould of strawberry in the future,the main research results are as follows:1)The separation of a Botrytis cinerea from Hunan provinceThe separation study of grey mould of strawberry collected 234 strains samples from Chenzhou,Hengyang,Xiangtan,Zhuzhou,Changsha,Yueyang,Changde,Zhangjiajie and Shaoyang of Hunan province,in the end 223 strains were separated from all the samples.The average separated rate is 95.30%,but the number of samples from every place is not totally equal.Observing growth characteristics of separated strains,it indicates that all of these strains could grow on the PSA medium,though growth rate vary from each other of different places.Botrytis cinerea mainly have 3kinds of types in Hunan,the type of mycelium,the type of sclerotium and the type of spore,among which,the type of mycelium takes up the largest proportion with 54.71%,the type of sclerotium takes up 30%in the Second place,the type of spore takes up the smallest proportion.Besides,one strain of grey mould of strawberry in perfect stage was separated During the research,it was named HXzH01,it can grow on the PSA or PDA medium with a low growth rate,its mycelia presents villiform and white,with short length and slightly red in final stage,It produces red substance without any spore or sclerotium on PSA or PDA medium.Template for genome's DNA of HXzH01,after amplification of PCR and electrophoresis's detection,a piece of specific band about 510bp was produced.The sequencing result shows that the amplified ITS 514bp long,compared with NCBI database,it was founded that HXzH01 belongs to perfect stage of Botrytis cinerea-Sclerotinia fuckeliana.2)Genetic diversityBotrytis cinerea is one of the most devastating diseases occurred in most strawberry production areas.The genetic diversity of 180 isolates of B.cinerea,collected from 9 locations of Hunan province in China from 2013 to 2015,was investigated by simple sequence repeat(SSR)analysis.A total of 56 alleles(Na)for 9 SSR loci were detected,and the effective number of alleles(Ne)ranged from 1.27 to 4.89.The Nei's gene diversity(Nei's)varied from 0.21 to 0.80.The Shannon's information index(?)varied from 0.37 to 1.83.The polymorphism information content(PIC)for 9 loci ranged from 0.13 to 0.91,the variation law of which was coincident with the Shannon's information index and gene diversity index.The denote gene diversity in the species(Ht)of different SSR loci ranged from 0.21 to 0.77.The gene diversity within populations(Hs)varied from 0.19 to 0.50.The population genetic differentiation(GST)varied from 0.10 to 0.46.The gene flow(Nm)ranged from 0.58 to 4.47.At geographical population level,the observed number of alleles(Na)and the effective number of alleles(Ne)were 2.93 and 2.07,the average I and Nei's gene diversity(Nei's)were 0.84 and 0.4,the number of polymorphic loci(NP)and the proportion of polymorphic loci(PIC)were 6.78 and 75%.Based on Nei's genetic distance,ranging from 0.2797 to 1.9225,9 geographical populations of MLP isolates from different regions in Hunan province could be initially grouped into 4 clusters:cluster I included isolates CS,SY,YY,HY,ZZJ and XT,cluster II included isolates CZ,cluster III included isolates ZZ,and cluster IVcomprised CD.The genetic diversity of Botrytis cinerea population from Hunan province is rich,but 9 populations has small difference between different geographical population.Meanwhile,some Botrytis cinerea existed move between different area.3)Fungicide screeningTo discover the synergism of Boscalid and Fluazinam and find out a new reagent against Botrytis cinerea,we used growth rate method to measure their indoor toxicity and use Wadley to evaluate its synergism potential ability.The results showed that when the mass ratios of Boscalid and Fluazinam were 1:1,1:5,5:1,3:1,2:3,they showed additive effects and the synergistic coefficients were 1.09,0.67,0.73,0.56,0.56;when the mass ratios of Boscalid and Fluazinam were 1:2,1:3,2:1,3:2,they showed antagonistic effects and the synergistic coefficients were 0.44,0.42,0.41,0.48.The results showed that Boscalid and Fluazinam compound can improve the control effect of Botrytis cinerea under certain conditions,but the uncoordinated proportion will reduce to the control effect of the pathogen. |
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