Biological Characteristics,Molecular Detection And Genetic Diversity Of Ustilaginoidea Virens In Heilongjiang Province

In recent years,the occurrence of rice false smut has become increasingly serious and the area of damage has expanded year by year,so the false smut has become one of the diseases which has seriously affected rice yield and quality in Heilongjiang Province.However,the basic research on false smut in Heilongjiang Province is relatively weak.In order to further clarify the occurrence and genetic background of false smut in Heilongjiang Province,this paper studied the isolation methods,biological characteristics and genetic diversity of the pathogens of false smut in Heilongjiang Province,which provides a theoretical referenc e for early identification,scientific control measures,molecular detection and disease resistance breeding of false smut.In this paper,108 samples were collected from five main rice-planting areas in Heilongjiang Province.Three isolation methods were used to compare the isolation rates of ustilaginoidea virens.The representative strains were selected from each sampling area for biological characteristics analysis,also the specific primers were designed and tested and then the genetic diversity of 89 ustilaginoidea virens strains which were successfully isolated was studied to analyze the genetic diversity of ustilaginoidea virens strains in Heilongjiang Province by using SSR molecular marker technique.The main results are as follows :(1)The improved tissue isolation method significantly improved the success rate of ustilaginoidea virens isolation.This method has low pollution and high isolation success rate and the isolation rate was up to 76%.This method was superior to the spore suspension and the sclerotium germination method,and the isolation rates of these two methods were 6.67% and 30%,respectively.Using the above three methods,89 strains of ustilaginoidea virens were successfully isolated from five main rice-planting areas in Heilongjiang Province.(2)The average growth rate of the isolated strain of ustilaginoidea virens was 0.1875 cm/d.Among them,15 strains with growth rate greater than the average growth rate accounted for 60% of all representative strains,mainly from strains in Harbin,Mud anjiang and Suihua areas;10 strains with growth rate less than the average growth rate accounted for 40% of all representative strains,mainly from strains in Jiamusi and Qiqihar.The growth rate of strains in Harbin and Mudanjiang and Suihua areas was higher than that in Qiqihar and Mudanjiang.(3)The results of culture conditions and nutritional requirements of ustilaginoidea virens showed that potato sucrose agar medium(PSA)is the optimum medium for growth of ustilaginoidea virens,26? is the optimum temperature for mycelial growth,and the optimum p H value is 7.0,sucrose is the most suitable carbon source,and KNO 3 is the most suitable nitrogen source.(4)A pair of specific primers UV-A/UV-B was designed and a specific fragment of 260 bp in length was amplified only for the ustilaginoidea virens,while the other tested strains did not amplify the target band,so it can be used for the detection of ustilaginoidea virens.This primers detect genomic DNA of ustilaginoidea virens in conventional PCR reactions and the sensitivity is 1 pg,which establishes a PCR detection system for ustilaginoidea virens with high sensitivity and specificity.The ustilaginoidea virens can be detected as early as 48 h after inoculation of false smut.(5)9 pairs of SSR primers selected form a total of 89 tested strains isolated were screened for amplification,and the amplified bands of each pair of primers were between 2 and 7.A total of 43 bands were obtained,and each primer was expanded for 4.8 band s.Among them,25 polymorphic bands accounted for 58.1% of the total.The polymorphism of primer S318 was the best,the polymorphic band ratio was 85.7%,the S509 polymorphism was the worst,and the polymorphic band ratio was 33.3%.The genetic similarity coefficient between strains by using SSR molecular marker analysis showed that the GS(genetic similarity coefficient)of the strain of ustilaginoidea virens was 0.613~0.955,with an average of 0.741 and a difference of 0.342.Among them,the GS minimum between the ustilaginoidea virens in Jiamusi and Qiqihar areas is 0.613,the genetic distance is the farthest,the genetic similarity is the lowest;the GS of the ustilaginoidea virens strain between Harbin and Mudanjiang areas is the largest,0.955,the genetic distance is the closest,and the genetic similarity is the highest.According to the results of SSR analysis,when the genetic distance is 0.73,89 strains are divided into 3 groups,5 strains of rice smut strains are distributed in 3 populations,and strains in Harbin are mainly distributed in group III.In the Mudanjiang area,the strains are evenly distributed in the group I,II and III,the strains in the Suihua area are mainly distributed in the group I and II,and the strains in the Jiamusi area are evenly distributed in the group I and II,and the strains in the Qiqihar area are mainly distributed in group II and ?.The results show that the genetic background of ustilaginoidea virens in Heilongjiang Province is relatively low in complexity,and the overall inheritance of the strain is relatively stable.