| Rice False Smut(green smut)is a fungal disease at the panicle of rice,which occurs in the north and south rice regions and has become one of the main obstacles that restrict the high and stable yield of rice in China.In this test,the morphological observation and r DNA-ITS sequence analysis were used to determine the species,homology and intra-species population differentiation of the pathogenic bacteria.The mycelial growth rate method,spore germination method and correlation analysis were used to study the biological characteristics of Ustilaginoidea virens in different regions.We used repetitive element-based PCR(rep-PCR)technology and field inoculation methods to explore genetic diversity and pathogenicity differences of Ustilaginoidea virens.The single-factor and orthogonal design were used to establish the optimal Sequence-Related Amplified Polymorphism(SRAP)reaction system for Ustilaginoidea virens and to screen SRAP polymorphic primers.The optimal SRAP reaction system was used to further clarify the genetic diversity level of Ustilaginoidea virens population.The above research aims to provide a theoretical basis for the comprehensive control of rice false smut(green smut)and distribution of disease-resistant rice varieties.The research results were as follows:Isolation and identification of r DNA-ITS from rice false smut pathogen Ustilaginoidea virens in Liaoning Province: a total of 159 tested strains were isolated from 273 disease samples from chlamycladospore strains of Ustilaginoidea virens collected from eight cities of Liaoning Province in 2017 by body tissue or agricultural streptomycin washing method and single-spore isolation.A total of 63 representative strains were selected and PCR-amplified with universal primers ITS1 and ITS4 all showed a single band and the ITS size was within 500 bp-750 bp;BLAST comparison on NCBI revealed that the similarity with Ustilaginoidea virens was 99%-100%;Phylogenetic trees constructed by MEGA 7 were found to be in the same branch.Therefore,combining morphological observation and r DNA-ITS technology can confirm that all tested strains were rice false smut pathogen Ustilaginoidea virens.The genetic distance between the tested strains ranged from 0.000 to 2.000,that is,the r DNA-ITS sequences of different Ustilaginoidea virens strains varied during evolution.Comparison of biological characteristics of rice false smut pathogen Ustilaginoidea virens in different areas from Liaoning Province: the optimal hypha growth conditions of the 18 representative strains selected in this experiment were different.Under the same culture conditions,the mycelial growth rates of the tested strains were significantly different.The most suitable culture medium for the mycelial growth of the tested strains were PDA(1 strain),PSA(1 strain),XBZ(1 strain),Rice(2 strains)and OA(13 strains);the most suitable carbon sources were starch(14 Strains)and sucrose(4 strains);the most suitable nitrogen sources were yeast extract(17 strains)and peptone(1 strain);the most suitable temperature were 25?(4 strains),28?(11 strains)and 30?(3 strains);the most suitable p H were 4(8 strains),5(1 strain),6(5 strains)and 7(4 strains);the most suitable light conditions were light(3 strains),dark(12 strains)and 12 h Light/12 h dark(3 strains).The optimal conditions for germination of thin-walled conidia of all tested strains were different.Under the same conditions,the germination rates of thin-walled conidia of different strains were significantly different.The most suitable temperatures for germination of thin-walled conidia of the tested strains were 25?(12 strains)and 28?(6 strains);the most suitable p H were 6(3 strains),7(13 strains)and 8(2 strains);the most suitable light conditions were light(4 strains),dark(8 strains)and 12 h light/12 h dark(6 strains).The lethal temperatures of mycelia and thin-walled conidia of all tested strains were 55? and 78?,respectively.The mycelial growth rate of Ustilaginoidea virens were moderately and lowly correlated with different carbon sources and temperatures;the color of Ustilaginoidea virens were moderately,lowly,none,highly and lowly with different culture medium,carbon sources,nitrogen sources,temperature and p H;the rest were not statistically significant.Analysis of genetic diversity and pathogenicity of rice false smut pathogen Ustilaginoidea virens in Liaoning Province by rep-PCR: based on the genetic diversity of the three primer pairs of BOX1/BOX2,ERIC1/ERIC2 and REP1/REP2,the DNA fingerprints amplified with BOX1/BOX2(0.764)and ERIC1/ERIC2(0.707),respectively,which were greater than 0.7.The DNA fingerprints amplified by these two primers were thus selected for genetic diversity analysis.When the similarity coefficient of DNA fingerprints was 0.78,the amplified fingerprints using BOX1/BOX2 and ERIC1/ERIC2 as the primers divided the tested strains into 12 and ten genetic groups,respectively.In August 2008,an experiment was conducted to inoculate the rice variety Yanfeng 47,a main rice variety in Liaoning Province,into Ustilaginoidea virens in the experimental field,the tested strains were divided into three pathogenicity types,among which weak pathogenicity type,moderate pathogenicity type and strong pathogenicity type accounted for 33.33%,58.82% and 7.85%,respectively.The strong pathogenicity type only appeared in Shenyang,Anshan and Dalian cities.All dominant groups contained three pathogenicity types.The results indicated that the genetic structure of Ustilaginoidea virens was complicated in Liaoning Province.There were some differences in pathogenicity of the strains of Ustilaginoidea virens from different geographical sources.The same pathogenicity type of Ustilaginoidea virens belonged to different genetic groups and the same genetic group contained different pathogenic types.Establishment of SRAP system and analysis of genetic diversity of rice false smut pathogen Ustilaginoidea virens in Liaoning Province: three representative strains(SY-19,AS-3 and DD-8)from different geographical locations were selected,and eight primers(Me1/Em6,Me2/Em2,Me2/Em4,Me2/Em6,Me3/Em7,Me4/Em3,Me5/Em6,Me6/Em5)with high polymorphism were selected from 100 pairs of SRAP primers.Accordingly,a SRAP system suitable for Ustilaginoidea virens was established(3.5 mmol/L Mg2+,3.5 U/?L TaqTM,200 ng/?L DNA template,0.4 ?mol/L primers,0.4 mmol/L d NTPs).According to the values of p were less than 0.05 in the analysis of variance,each factor has a significant effect on the reaction system.According to the order of the F values in the analysis of variance,Mg2+ has the largest effect on the reaction system and the DNA template has the smallest effect.The optimal SRAP reaction system of Ustilaginoidea virens was used to analyze the genetic diversity of 78 representative strains.The results showed that a total of 179 obvious bands were amplified by 8 pairs of primers,which 93 bands were polymorphic.For each primer pair,18-27 alleles were detected with an average of 22.On the overall level,observations number of alleles,effective number of alleles,diversity index of Nei's gene and Shannon information index were 2.000,1.4886,0.3248 and 0.5047,respectively.Genetic diversity of Shenyang population was the maximal and the minimal genetic diversity were found in Anshan population.The genetic similarity coefficient was 0.88,a total of 78 strains of Ustilaginoidea virens were divided into 14 groups.The results indicated that the pathogen population of Ustilaginoidea virens in Liaoning Province had rich genetic diversity and there was no obvious correlation between SRAP genetic group division and geographical origin. |
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