| Tobacco is an annual herb,and its leaves are the most economically valuable in production.At present,leaf spot is an important factor affecting the yield and quality of tobacco leaves in the process of tobacco planting.Identifying the type of pathogen is the key to conducting control research.This study investigated a tobacco leaf spot disease discovered during a disease investigation in Hezhou City,Guangxi Zhuang Autonomous Region,from 2021 to 2022.To better control the disease,the pathogen were isolated from the disease samples,mitochondrial genome sequencing,biological characteristics testing,laboratory screening tests on fungicide,ontrol effects of fungicides in field,and pathogenicity testing.The results are as follows.1.Seven strains were isolated and purified from 70 tobacco leaf disease samples by tissue isolation method,including three strains from Fuchuan County,numbered GHFC63,GHFC54,and GHFC2;Four strains from Zhongshan County,numbered GHZS80,GHZS21,GHZS8,and GHZS3.According to morphological characteristics observation and molecular biological methods of multiple genes(ITS,LSU,TUB,RPB2),all 7 strains were identified as Stagonosporopsis vannaccii.Using the strain GHZS80 as the material,the circular genome sequence of the pathogen was determined and analyzed using the Illumina+Pacbio technology.The total length was 48994 bp,and the AT% content was 70.95%.It contained 48 genes,including27 transport RNAs and 18 protein-coding genes(nad5,cox1&2,orf446,orf344,orf295,orf744,nad1,orf335,cob,orf298,nad4,nad6,cox3,orf311,nad2,nad3,nad4L),Two ribosomal RNAs(rnl,rns)and one rnp B gene.The results of this study lay the foundation for the mitochondrial genomics study of S.vannaccii.2.The biological characteristics of pathogen were studied using the method of mycelial growth rate.The results showed that the optimal temperature range for mycelial growth of pathogen was 25～28℃,the optimal carbon source was soluble starch,the optimal nitrogen source was peptone and beef extract,and the optimal p H was 6～9;The suitable culture media were potato glucose medium(PDA),oat medium(OA),and pumpkin juice agar medium(PA).The mycelium grew fastest under full illumination,with a minimum lethal temperature of 50℃ and continued water bath treatment for 10 minutes.Pathogens grow faster in environments with faintly acid to alkalescence,sufficient sunlight,and abundant nutrients.The biological characteristics of the same pathogen are not significantly affected by different geographical sources.The results of field plant pathogenicity testing showed that S.vannaccii was pathogenic to tobacco,pumpkin,potato,garland chrysanthemum,cabbage,and chili.3.The inhibitory effects of eight fungicides on pathogen were determined by using the mycelial growth rate method in the laboratory.The results showed that boscalid WG 50% had the strongest antibacterial activity,with an EC50 value ranging from 0.016 to 0.018 mg/L,and an average value of 0.017 ± 0.001 mg/L;Followed by fluopyram SC 41.7%,prochloraz EW 450 g/L,and difenoconazol ME 10%,with EC50 values ranging from 0.080 to 0.114 mg/L,0.325 to 0.376mg/L,and 0.844 to 1.084 mg /L,respectively;The antibacterial activities of azoxystrobin SC 250g/L and pyrazolidinolate SC 25% were poor,and the average range of EC50 was greater than 100mg/L.Under the same fungicide treatment,the sensitivity difference of mycelial growth among different strains was small.4.Four fungicides,boscalid WG 50%,fluopyram SC 41.7%,prochloraz EW 450 g/L,and difenoconazol ME 10%,were selected for field control efficacy testing.The results showed that the control effect of different fungicides on the disease showed an upward trend with the increase in the number of application times.Among them,boscalid WG 50% and fluopyram SC 41.7% had the best control effect,and the control effect after the third application of 7 days was 71.11% and68.67%,respectively,while the control effect of difenoconazole ME 10% was weak,57.93%. |
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