| Peanut is an important cash crop and occupies an important position in world agriculture.Sclerotium rolfii Sacc.is one of the major peanut diseases in China.The fungus overwinters in the soil or on the diseased plant with hyphae or sclerotia,which becomes the first source of infection in the next year.Due to the diverse culture characteristics and nutrient affinity of Sclerotium rolfii Sacc.,the complex population structure and the lack of disease-resistant varieties,chemical control is still the main method for the prevention of such disease.Studies on the biology and population characteristics of pathogens help reveal the population structure,genetic evolution,pathogenic mechanisms of the pathogens,and provide theoretical basis for disease resistance breeding,forecasting and comprehensive control.In this study,210 strains were collected from 451 peanut blight disease samples of five provinces in China.The phenotypic characteristics of the pathogenic population,nutritional compatibility of mycelia,genetic diversity and susceptibility to pesticides were systematically studied in order to provide a theoretical basis for the comprehensive prevention and control of this pathogen.The main results are as follows:1.Phenotypic characteristics of pathogen population from S.rolfii in peanut.(1)In these 210 strains,the growth rate of the tested strains was 0.32~2.56cm/d(mean value 1.83 cm/d),the number of sclerotia was 0~120(mean value 58.34),the size of the sclerotia was 0.3~3.28 mm(mean value 1.40 mm),the onset time of the sclerotial development was 6~13 d(mean value 7.78 d)and the maturation time of the sclerotia was 11~21 d(mean value 15.33 d).Among them,the mycelium growth rate of strains from Jiangsu province was the fastest,with an average value of 1.78cm/d.The largest number of sclerotia-producing strains came from Hebei province,with an average of 73.6.The sclerotium diameter of the test strain from Fujian was the largest,with an average value of 1.61 mm.The sclerotium formation period and maturity period of the test strains from Hebei were both earliest,with average values of 6.40 d and 14.6 d,respectively.(2)According to the results of cluster analysis,the mycelial growth rate of the tested strains was divided into four types,namely A1 type(growth rate range: 0~1.0 cm/d),A2 type(growth rate range: 1.0~1.5 cm/d),A3 type(growth rate range: 1.5~2.0 cm/d),A4 type(growth rate range: >2.0 cm/d)and the proportion of strains were 34.28%,30.95%,16.19% and 18.57%,respectively.The number of sclerotium production was divided into 3 types,B1 type(sclerotium number range: 0~50),B2 type(sclerotium number range: 50~100),B3 Type(sclerotium number range: 100~120),and the proportion of strains were 44.76%,37.62% and 17.62%,respectively.Sclerotium diameter was divided into 4 types,C1type(sclerotium diameter: 0 mm),C2 type(sclerotium diameter: 0~1.5 mm),C3 type(sclerotium diameter: 1.5~2.0),C4 type(sclerotium diameter: >2.0 mm),and the proportion of strains were 0.01%,49.52%,12.38%,37.61%,respectively.(3)The 210 test strains were divided into 22 mycelial affinity groups(MCGs).MCGs were highly correlated with the geographic origin of the strains.Except for MCG1,MCG4 and MCG12,other affinity groups all came from the same area.There was no obvious correlation between the biological characteristics of the strains and MCGs.2.Establishment and optimization of ISSR-PCR reaction system of S.rolfsii.Single factor and orthogonal design were used to establish and optimize the ISSR-PCR reaction system.The reaction system(25 ?L)included 2.0 U Taq DNA polymerase,2.0 mmol/L Mg2+,0.5 mmol/L d NTPs,1.3 ?mol/L primers and 20 ng template DNA.The eight optimal ISSR primers and annealing temperatures were selected as UBC809(52.7 ?),UBC810(50.4 ?),UBC812(55.6 ?),UBC834(52.7 ?),UBC842(55.6 ?),UBC844(52.7 ?),UBC880(52.7 ?)and UBC881(50.4?).The PCR reaction procedures included: pre-denaturation at 94? for 4 min,denaturation at 94? for 1 min,annealing for 1 min,extension at 72? for 1 min,35 cycles,extension at 72? for 10 min.3.Study on genetic diversity of S.rolfsii.Based on the optimized ISSR reaction system,the genetic diversity of 102 representative strains collected from 5provinces of China was analyzed,and the correlation between the genetic lineage and the phenotypic characteristics of pathogen was analyzed.The results showed that 57 DNA fragments were amplified by the 8 primers,mainly distributed in the 300-2000 bp interval,of which 48 were characteristic fragments,accounting for 84.21% of the total number of fragments.The population polymorphism loci were highest in Jiangsu province and lowest in Hebei province.The Nei's genetic diversity index(H)of S.rolfsii population in five provinces was 0.06,and the Shannon index(I)was 0.13.The population with the highest genetic diversity index was in the Jiangsu province(H =0.07,I = 0.15),and the population diversity in Hebei province was the lowest(H =0.01,I = 0.01).When the similarity coefficient was 0.92,the test strain could be divided into 10 genetic lineages.Although the biological characteristics of pathogens were different in different genetic lineages,but the genetic lineages of test strains had little correlation with the biological characteristics of pathogens.Excepted for the four mycelial affinity groups in genetic lineage 1,the other genetic lineages included one mycelial affinity group.4.Determination of susceptibility of S.rolfsii to tebuconazole.The sensitivity of 210 strains of S.rolfsii to tebuconazole was determined by hyphal growth rate method.The results showed that the average value of EC50 of tebuconazole to the 210 tested strains was 0.1932 ?g/m L and the total range fell on 0.0064~1.1292 ?g/m L,which had a 176 times' difference.Based on the principle of a normal distribution of the sensitivity of the wild sensitive pathogen population to the medicament,the EC50 value of 0.0630 mg/L was preliminarily set as the baseline of sensitivity of S.rolfsii to tebuconazole.The SPSS software was used for cluster analysis of test strains and divided into three categories.Test strains from different geographical sources appeared in different clustering groups.There was no obvious correlation relationship between the sensitivity of the test strains to tebuconazole and geographical sources. |
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