Identification,Biological Characteristics Of Pathogen Of Salvia Miltiorrhiza Anthracnose And Fungicide Screening In Vitro

Salvia miltiorrhiza,a perennial erect herb belonging to the genus Salvia of Labiatae,also known as purple Salvia miltiorrhiza,red ginseng,blood ginseng,etc.,is one of the traditional Chinese medicine.In recent years,the market demand of Salvia miltiorrhiza has been increasing year by year,the artificial planting area has been expanding,and various diseases have also occurred frequently,which has seriously affected the yield and quality of Salvia miltiorrhiza and hindered the development of Salvia miltiorrhiza industry.In 2020,a new leaf disease of Salvia miltiorrhiza was discovered in the planting field of Liangba Village,Hechuan District,Chongqing.The upper and middle leaves of the infected Salvia miltiorrhiza first developed the disease.In the early stage,light brown spots appeared on the leaf tip and leaf margin.In the middle stage,the lesions continued to expand,the color changed from light brown to dark brown,and the shape was irregular and the junction of disease and health was obvious.In the later stage,the lesions began to heal and connect into a piece,resulting in dry and fragile leaves,ultimately withering and falling,and in severe cases,the whole plant died.At present,no relevant reports have been found on the leaf diseases of Salvia miltiorrhiza.When the field diseases occur,there is also a lack of reasonable countermeasures.In this paper,the pathogen of Salvia miltiorrhiza disease was isolated and identified,and the biological characteristics of the pathogen were clarified.Effective antagonistic bacteria and antagonistic actinomycetes were screened out.Effective chemical fungicides and botanical fungicides were screened out by indoor virulence determination,which provided a theoretical basis for the prevention and treatment of Salvia miltiorrhiza disease.The results are as follows:1.Identification of pathogens Thirty-six isolates were isolated from 10 disease samples by tissue isolation and single spore isolation.The pathogens were preliminarily divided into three categories by sequencing with universal primers ITS,which were numbered as DSL,DSN and DSM.Combined with the ITS,LSU and RPB2 gene sequences of the genomic DNA of the strains,the phylogenetic tree of DSM strains was constructed.It was found that 6 DSM strains and Boeremia exigua were clustered in the same branch,with a bootstrap value of 100%.The phylogenetic tree of DSL strains was constructed based on ITS,TUB and His 3 gene sequences.It was found that 23 DSL strains and Colletotrichum karstii clustered in the same branch,with a bootstrap value of 99%.The phylogenetic tree of DSN strains was constructed based on RPB2,TEF1and Alt al gene sequences.It was found that 7 DSN strains clustered in the same branch with Alternaria alternata,and the bootstrap value was 97%.One representative strain of each of the three types of strains was selected for pathogenicity determination.Only DSL01 strain could cause the disease of Salvia miltiorrhiza leaves,and the verification of Koch’s rule was completed.Based on morphological and molecular biological analysis,DSL01 strain was identified as Colletotrichum karstii.Therefore,we identified the pathogen causing the leaf disease of Salvia miltiorrhiza as Colletotrichum karstii,and named this Salvia miltiorrhiza disease as Salvia miltiorrhiza anthracnose.2.Study of biological characteristics The mycelial growth temperature was10～35°C,the suitable temperature was 20～25°C,and the optimum temperature was25°C.It can produce spores in the range of 15～33°C,and the maximum spore production was at 25°C.It had strong adaptability to light,and could grow and sporulate under three different light conditions.The mycelial growth and sporulation were better in 12 h light 12 h dark and 24 h dark,and there was no significant difference between them.The strains could grow at p H=4～11,and the strains could produce spores at p H=5～11.When the p H was 5～9,the mycelial growth and spore production were good and there was no significant difference.Biolog phenotypic analysis technique was used to determine the utilization of 190 carbon sources,95 nitrogen sources,59 phosphorus sources and 35 sulfur sources by pathogenic bacteria at 20°C,25°C and 30°C,as well as the adaptability of different osmotic pressure and ionic strength.The results showed that the utilization rate and utilization degree of the three temperatures were25°C>30°C>20°C.3.Screening and identification of antagonistic bacteria The antagonistic screening and rescreening of 22 strains of bacteria and 28 strains of actinomycetes were carried out respectively,and the effects of fermentation filtrate of strong antagonistic bacteria on mycelial growth and spore germination of Salvia miltiorrhiza anthracnose were determined.The results showed that the antagonistic bacteria Itb220 and antagonistic actinomycetes XJC2-1 had the strongest inhibitory effect on mycelial growth and spore germination of Salvia miltiorrhiza anthracnose.Strain Itb220 was identified as Bacillus velezensis by morphology and molecular biology,and strain XJC2-1 was identified as Streptomyces violaceusniger.4.Screening of chemical fungicides and botanical fungicides Seven fungicides with mycelial growth inhibition rate of more than 70%at a concentration of 10μg/m L were screened from 15 chemical fungicides,and their effects on mycelial growth and spore germination of pathogens were tested.Among the seven chemical agents,98%fluazinam TC had the best inhibitory effect on mycelial growth and spore germination,with EC₅₀ of 0.0725μg/m L and 0.0378μg/m L,respectively.The six botanical fungicides tested had a certain inhibitory effect on the mycelial growth and spore germination of the pathogen.Among them,the best inhibitory effect on mycelial growth was 1%osthole EW with EC₅₀ of 4.8984μg/m L,and the best inhibitory effect on spore germination was 80%ethylicin EC with EC₅₀ of 0.5541μg/mL.