| Picea Dietr is an evergreen tree with straight trunk and excellent material,which can be used as landscape tree species and industrial raw materials.It has been planted in a large area in China.Therefore,diseases of spruce have been paid more and more attention by garden and forestry departments.In 2018,a new disease of P.crassifolia Kom.was found in the dead shoots of P.crassifolia Kom.of Nanhuashan National Nature Reserve of Ningxia.In this study,the pathogens of this new disease were identified by morphology,Koch’s Postulate and molecular biology,and the biological characteristics of the pathogen were studied.The antibacterial activity of Bacillus velezensis against Dothiorella gregaria and the antibacterial activity B.methylotrophicus against it were studied by confronting incubation.The grow curve of two kinds of antagonistic bacteria and the antibacterial activity of different incubation time and different concentration were measured.The antibacterial activity stability of two kinds of antagonistic bacteria was studied.The morphological changes of the mycelium of the pathogen after treatment with two kinds of fermentation broth were studied by microscope.The liquid fermentation conditions of two kinds of antagonistic bacteria were optimized.At the same time,six kinds of low toxic fungicide were selected to study the bacteriostasis of D.gregaria,the pathogen of Shoot blight of P.asperata Mast.In order to provide more theoretical basis for the prevention and control of the disease.Results:The pathogen of Shoot blight of P.asperata Mast was isolated and purified,and it was identified as D.gregaria by morphological identification and ITS gene sequence comparison.Verified by Koch’s Postulate it was finally determined that Dotl strain was the pathogen of Shoot blight of P.asperata Mast,and Shoot blight of P.asperata Mast was found for the first time in China.The optimum carbon source for mycelial growth of D.gregaria was sucrose,the optimum nitrogen source was yeast extract powder,the optimum temperature was 25℃,and the optimum pH was 6.0.Two kinds of antagonistic bacteria had significant inhibition effect against D.gregaria with the antibacterial rate of 57.50±1.44%、56.67±2.36%.The fermentation broth of B.velezensis and B.methylotrophicus had almost no inhibitory effect on pathogens in the period of 0～12 h,and the antibacterial rate was 0.The inhibition rate reached the maximum at 24 h,and decreased from 24 h to 72 h.The antibacterial activity of the fermentation broth of the two antagonistic bacteria increased with the increase of volume fraction.The bacteriostatic activity of the fermentation broth of the two antagonistic bacteria has good thermal stability.After the fermentation broth was treated with different pH,the bacteriostatic activity decreased.Through the observation of microscope,the fermentation broth of two kinds of antagonistic bacteria could make the mycelium of pathogenic mycelium thicker and dilated.In the Complete randomized design,the best carbon source of B.velezensis is glucose,the best nitrogen source is ammonium chloride,and the best inorganic salt is magnesium sulfate.The best carbon source of B.methylotrophicus was glucose,the best nitrogen source was ammonium chloride,and the best inorganic salt was potassium chloride.The optimum culture conditions of the two antagonistic bacteria were pH 4.2,culture time 24 h,culture temperature 35℃ and rotational speed 150 r/min.Response Surface Methodology results:the optimal medium ratio of B.velezensis was glucose 6.25 g/L,ammonium chloride 12.5 g/L and magnesium sulfate 12.5 g/L.The optimal medium ratio of B.methylotrophicus is glucose 6.25 g/L,ammonium chloride 12.5 g/L and potassium chloride 12.5 g/L.Carbendazim,thiophanate-methyl and pyrimethanil had strong inhibitory effect on the mycelial growth of D.gregaria,and the EC50 were 0.03 mg/L,0.04 mg/L and 0.04 mg/L,respectively.Chlorothalonil,triadimefon and pentachloronitrobenzene had weak inhibitory effect on the mycelial growth of D.gregaria. |
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