Identification And Genetic Diversity Analysis Of The Pathogen Causing Sugarcane Pokkah Boeng Disease

Sugarcane pokkah boeng is a fungal disease caused by Fusarium.At present,there are at least 7 pathogens that can cause sugarcane pokkah boeng.In order to understand the species and genetic diversity of sugarcane pokkah boeng in Guangxi,Yunnan and Fujian provinces,this study used morphology and polygenic molecular biology to identify the pathogenic bacteria of sugarcane pokkah boeng.According to the sequence of a Fusarium sacchari strain whose genome has been sequenced in our laboratory,development of a series of F.sacchari SSR primers,and screening of universal SSR primers for Fusarium,The genetic diversity of Sugarcane pokkah pathogen Fusarium were evaluated and analyzed by SSR,SRAP and ISSR Molecular markers.The results are as follows:1.Identification of sugarcane pokkah pathogens: Combined with morphology and polygenic molecular biology,the sugarcane pokkah pathogens from Fujian,Guangxi and Yunnan were identified,and a total of 6 Fusarium species were identified: F.sacchari,Fusarium andiyazi,Fusarium proliferatum,Fusarium fujikuroi,Fusarium miscanthi and Fusarium nisikadoi,and 42,32 and 8 strains of F.sacchari,F.andiyazi and F.proliferatum respectively,accounting for 49.5%、37.6%、9.4%,and one strain of the other three Fusarium species was identified respectively,among which F.sacchari and F.andiyazi were the dominant pathogens of sugarcane pokkah.A total of 78 mating types were identified,including 45 MAT-1 mating types and 33 mat-2 mating types.Six Fusarium species have pathogenicity,which is the first report of sugarcane pokkah caused by F.miscanthi and F.nisikadoi.2.SSR distribution characteristics and SSR primer development of F.sacchari: 615 sequences were scanned for SSR loci by MISA,and a total of 3718 SSR loci were scanned.The number of F.sachari repeat units from more to less is: pentanucleotide(149)> tetranucleotide(123)> hexanucleotide(97)> trinucleotide(59)> diglycoside(12)> monoglycoside(4).There are significant differences in the frequency of different repeat primitive types.The number of SSR loci of the same type decreases gradually with the increase of the number of SSR primitives.Primer 3 software was used to design SSR primers,by screening the best conditions,458 pairs of SSR primers were successfully designed,250 pairs were selected for synthesis and PCR amplification,247 pairs of primers were successfully amplified,and the power of primer development was 98.8%。3.SSR analysis of genetic diversity of sugarcane pokkah boeng pathogens: 21 pairs of SSR primers with polymorphism and stable amplification among Fusarium species were screened,and Fusarium were amplified by PCR.A total of 81 bands were amplified,78 bands were polymorphic,and the polymorphic rate was 96%.The results of cluster analysis showed that the genetic similarity coefficients of all strains were between 0.6～1,and the same species of Fusarium could be clustered in the same group at a certain genetic similarity coefficient.Cluster analysis of F.sacchari from different provinces,although F.sacchar strains in different regions had certain genetic diversity in SSR region,this difference was not related to geographical origin.Cluster analysis of F.andiyazi strains with different mating types showed that although the strains with different mating types had certain genetic diversity,this difference was not related to mating type.4.SRAP analysis of genetic diversity of sugarcane pokkah boeng pathogens: from 25 pairs of SRAP primers,16 pairs of primer combinations with clear bands,polymorphism and good stability were selected to analyze the genetic diversity of strains.175 bands were amplified,173 bands were polymorphic.The results of cluster analysis showed that the genetic similarity coefficients of all strains were between 0.56～0.93.When the same species of Fusarium were clustered at a certain genetic coefficient,they could be clustered in one SRAP group.Cluster analysis of 42 strains of F.sacchari from different provinces showed that most strains from the same geographical source were clustered together,and the genetic differentiation of each strain in sickle strain was related to the geographical source.SRAP cluster analysis of F.andiyazi strains with different mating types showed that although the strains with different mating types had certain genetic diversity,this difference was not related to the mating type.5.ISSR analysis of genetic diversity of sugarcane pokkah boeng pathogens: Fusarium strains were amplified by PCR with 11 ISSR primers.94 bands were amplified,the proportion of polymorphism was 98.9%.The results of cluster analysis showed that the genetic similarity coefficient of all strains was between 0.58～0.94.When the genetic coefficient was 0.85,six sickle strains such as F.sacchari and F.andiyazi could be separated.The cluster analysis of F.sacchari strains from three provinces showed that most of the strains with the same geographical origin were clustered together,indicating that the division of F.sacchari taxa had a certain correlation with the geographical origin.Cluster analysis was performed on F.andiyazi strains of different mating types.Although the strains of different mating types had a certain genetic diversity,this diversity was not related to the mating type.6.Comparative analysis of amplification results of SSR,SRAP and ISSR markers of pokkah boeng disease: the results showed that the three molecular markers developed in this study could be used to analyze the genetic diversity of Fusarium.The three markers can separate different species of Fusarium,but SRAP and ISSR markers are more suitable for the study of genetic differentiation among pokkah boeng pathogens than the SSR marker developed this time.For the study of geographical origin and intraspecific genetic differentiation,SRAP markers and ISSR markers can also better reveal the relationship between intraspecific genetic differentiation and geographical origin of F.sacchari.The three molecular markers can not separate mating types.In practical research,we should use one or more molecular markers to better reflect the genetic relationship and genetic diversity between or within Fusarium species,and then draw a more accurate conclusion.