Identification Of Pathogen Causing Root Rot Disease On Salvia Miltiorrhiza And Its Fungicides Screening

Salvia miltiorrhiza Bge.is a traditional Chinese medicinal herb used as an important drug to care cardiovascular disease in China.Root rot disease is one of the severe diseases in the culture areas of S.miltiorrhiza.The symptoms and incidence rate of disease in field of the root rot disease were investigated in this study.The fungal culture was isolated and purified and then identified based on pathogenicity test and morphological characteristics observation,as well as molecular biological identification.The biological characteristics of pathogens were studied by different cultivation conditions.The biocontrol microorganisms were screened by the dural culture method on PDA plates and the fungicides against pathogenic fungi causing root rot disease in S.miltiorrhiza were quantified by mycelial growth rate method.It aims to provide theoretical basis and technical support for the prevention and treatment of root rot disease in S.miltiorrhiza.The main results in this study are as follows:1.Symptoms and regularity of root rot disease in S.miltiorrhizaThe disease incidence of root rot disease in S.miltiorrhiza ranged from 3.5%to 85.0%in different S.miltiorrhiza planting areas in Henan province,which was serious after continuous cropping.In the early stage of the disease,the plants stop growing or grow slowly,and the color of the leaves turned from purplish red to yellow,the fibrous roots became brown and rotted,the color of the main roots turned from red to brown.Finally,the roots became completely rotted and the plants were dead.The root rot disease occurred in the first half of April,high-incidence season continues from June to August,and decreased in after September.2.Isolation,identification and pathogenicity test of pathogenic fungiA number of 145 strains were isolated from 30 root rot samples of S.miltiorrhiza in four culture areas（Song county,Yuzhou,Fangcheng county,Mianchi county）and then identified based on pathogenicity test and morphological characteristics observation,as well as molecular biological identification.The fungal isolats were identified as Fusarium solani,F.proliferatum,F.oxysporum and A.tenuissima.The F.solani culture was isolated in the highest percentage at26.21%,was the predominant pathogen causing root rot disease in S.miltiorrhiza.In pathogenicity test by inoculation,F.Solani showed the strongest pathogenicity,F.proliferatum and A.tenuissima were lower in the pathogenic ability,while F.oxysporum were the most weak.3.Biological characteristics of pathogensThe pathogenicity characteristic of the four pathogens isolated under different cultivation conditions shows that the best growing temperature for four pathogens is 28℃under the conditions of 12 h lighting and 12 h darkness with p H value ranged from 6 to 8.The lactose was the best carbon source and the ammonium nitrate and peptone as the best nitorgen source were suitable for the mycelial grows of F.proliferatum.For the mycelial grows of F.solani,the most carbon and nitorgen source were glucose and nitrate and peptone,the speed was 18mm/d.For the mycelial grows of F.oxysporum,the most carbon and nitrogen source were D（+）-sucrose and nitrate and peptone,the speed was 15 mm/d.For the spore production of F.proliferatum,F.solani and F.oxysporum,the optimum carbon and nitrogen sources were glucose and yeast extract.The result showed that the best growth conditions was under 28℃with the p H value ranged from 10to 11 on the media,and the soluble starch was the best carbon source and the peptone as the best nitrogen source for the mycelial grows of A.tenuissima.4.Screening of biocontrol microorganisms against pathogens causing root rot disease in S.miltiorrhiza and their growth promotion on the hostA number of 453 strains of bacteria were isolated from rhizosphere and intra-rhizome strains of S.miltiorrhiza,and a total of 90 strains of bacteria strains with antagonism against four pathogens were screened through the plate confrontation method.9 strains of bacteria were obtained by further screening,and showed good antagonistic effect on four pathogens causing root rot disease in S.miltiorrhiza.Among them,1-4、2-2、D2XJ、190730-1、190730-2、190730-3and 190801-1 showed a good biocontrol effect and the inhibition rates of all four pathogens was more than 54%.The biocontrol microorganisms were identified based on morphological characteristics observation and molecular biological,the bacteria isolats were identified as Pseudomonas aeruginosa,Paenibacillus polymyxa and Bacillus velezensis.The biocontrol microorganisms screened were tested for their growth-promoting on the host and they showed different growth-promoting abilities.strains 13-2 and 15-1 showed the abilities to dissolve phosphorus,nitrogen fixation and producing indole acetic acid（IAA）,with IAA contents of119.8mg/L and 11.4 mg/L after 48 h culture.In plant growth promoting experiment,the plant height,fresh weight and dry weight,root and stem of S.miltiorrhiza promoted by 13-2 or 15-1culture resuspension were significantly higher than those of the control.The eleven strains of biocontrol microorganisms were the candidates for further studies on the development of biobacteria-resistant agents and field application on the control of root rot disease in S.miltiorrhiza.5.The result of screening fungicide for the control of root rot disease in S.miltiorrhizaThe fungicides 43%tebuconazole suspension,50%carbendazim wettable powder and 10%difenoconazole water dispersion granules showed a significant inhibitory effect on mycelium growth of F.solani and F.proliferatum.The inhibition rate of mycelium growth was more the79%under the concentration was 5 mg/L of fungicide described above and the EC₅₀value was less than 1.28 mg/m L.The fungicides 43%tebuconazole suspension,25%pyraclostrobin suspension and 10%difenoconazole water-dispersible granules showed the highest growth inhibitory effect on A.tenuissima,the inhibition rate was over 66%which contained 5 mg/L of fungicide,and the EC₅₀value was 0.66,72.9 and 0.65mg/L,respectively.The results laid a foundation for the control of root rot disease in S.miltiorrhiza and the screening of effective and safe fungicides which can be used in the field.