Genetic Diversity Analysis Of Ustilaginoidea Virens And Molecular Detection Of U.virens And Rhizoctonia Solani From South China Crop Breeding Area

South China crop breeding area,located in southern Hainan island,plays an irreplaceable role in the scientific breeding of rice and other food crops,is an important base for the breeding and seed production of crops in China.With a large number of breeding materials from other provinces in China in and out of Hainan island,while taking advantage of the advantages of South China crop breeding area,exotic organisms including insect pests,pathogens and genetically modified organisms from other places have caused potential damage to the agricultural ecological environment and production safety in this Area.In order to clarify the genetic diversity of Ustilaginoidea virens?Patou.?Bref.and develop a rapid detection technique for U.virens and Rhizoctonia solani Kühn respectively so as to provide a theoretical basis for the formulation of control measures for rice diseases.The cultural characteristics,morphological characteristics and AFLP polymorphisms of DNAs of U.virens from South China crop breeding area?Sanya,Ledong and Lingshui?were analyzed in this study;at the same time,a loop-mediated isthermal amplication?LAMP?assay for the rapid detection of Ustilaginoidea virens and Rhizoctonia solani was established and applied to the detection of thiese two pathogens form rice seeds,repectively.The main results are reported as follows:?1?Cultural characteristics observation and molecular identification of U.virens.In this study,the disease samples of rice false smut were collected from South China crop breeding area twice,and a total of 57 isolates of U.virens strains were obtained.These isolates grew slowly on the PSA medium,the cultural characteristics of these isolates were not completely consistent,showing a certain diversity;by amplification and identification with ITS-specific primers,these isolates were able to produce a 380 bp specific band,and indicating that all isoaltes are U.virens.?2?Analysis of AFLP polymorphisms of U.virens DNA.10 pairs of primers with rich amplification bands and high polymorphism were selected from 256 pairs of primers.The results showed that a total of 545 DNA bands were amplified,including 500 polymorphic bands,and the percentage of polymorphism was 91.74%.Among the three populations of U.virens,the percentage of polymorphic sites in the population of Lingshui county?PPL?was81.75%,which was higher than 74.60%of the population of Ledong county and 49.21%of the population of Sanya city;the Shannon's Information Index?I?of the Lingshui county population is 0.3062,which is higher than 0.2656 of Ledong County population and 0.1997of Sanya City population;Nei's gene diversity index?H?of Lingshui county was 0.1934,which was higher than that of Ledong county population of 0.1662 and Sanya city group of0.1273.Lingshui county population showed greater genetic diversity;the results of cluster analysis showed that 57 isolates were divided into 11 lineages,Lingshui population isolates were distributed in 8 lineages,and the other two populations were distributed in 7 lineages,indicating a high level of genetic differentiation among the isolates in Lingshui county.?3?The genetic structure analysis of U.virens.The gene differentiation degree Gst of the total species was 0.0541,and the gene flow Nm was 8.7460,indicating that the genetic variation mainly came from within each population,there was a certain gene exchange between different populations in the two regions;the cluster analysis based on genetic consistency showed that the genetic similarity of three population was very high.?4?The establishment and application of LAMP rapid detection technique for the pathogens of rice false smut and sheath blight.In this study,based on the sequences of the flanking of Uvt3277 gene of U.virens and the acetolactate synthase gene AG1IA₀0222of R.solani,the detection technology of loop-mediated isothermal amplification?LAMP?of U.virens and R.solani was established.The optimal concentration of Mg²⁺in the LAMP reaction system of U.virens was 4 mmol/L,and that of betaine was 0.2 mol/L,that of dNTPs was 1.2 mmol/L,and the reaction at 61?C for 60 min,and then inactivate the Bst DNA polymerase to end the reaction at 80?C for 10 min.the optimal concentration of Mg²⁺in the LAMP reaction system of R.solani was 4 mmol/L,and that of betaine was 0.2 mol/L,that of dNTPs was 0.8 mmol/L,and the reaction at 63?C for 50 min and then inactivate the Bst DNA polymerase to end the reaction at 80?C for 10 min.The results of this test can be determined by adding 1?L of SYBR Green I,observing colour changes under black background conditions or analyzing whether or not ladder bands appear on agarose gel electrophoresis.?5?Sensitivity and specificity of LAMP primers of U.virens and R.solani.The results showed that only the DNAs of U.virens and R.solani was positive,while the other tested strains DNA were negative,indicating that both of them had good specificity of LAMP primers;the DNA concentrations of U.virens and R.solani were both 100 fg/?L,the sensitivity of the LAMP assay were 10² and 10³ higher than that of conventional PCR,respectively.?6?Application of LAMP technology in the detection of targeted pathogens from rice seeds.Through routine PCR of fungal ITS universal primers,only one band was absent in 22 tested samples,indicating that the remaining 21 samples contained targeted fungi;in the LAMP detection reactions of U.virens and R.solani,14 and 2 tested samples showed positive results,respectively,indicating that the LAMP assay can accurately detect the presence of these two pathogenic fungi in rice seeds.This study provides a rapid,simple,specific,and sensitive method for the early diagnosis of fungal pathogens of rice smut and sheath blight,whichi was suitable for the use in field and grassroots departments.