Identification And Control Of The Pathogen Causing Phyllosticta Leaf Spot Disease In Polygonatum Cyrtonem

Polygonatum cyrtonema,also known as King Solomon’s seal,is a perennial herbaceous plant belonging to the Asparagaceae family.It was included in the 2015 edition of the Chinese Pharmacopoeia and is a medicinal and edible herb with the effects of tonifying the spleen and lungs,nourishing qi and yin,and possessing antiviral and anticancer properties.It plays an important role in healthcare,daily diet,and economic development.With the continuous development of its medicinal and culinary values,the cultivation area of Polygonatum cyrtonema has grown rapidly.However,in 2020–2021,a leaf disease was found in a poorly managed extensive Polygonatum cyrtonema planting base in Wanzhou District,Chongqing City.The disease mainly affects the old leaves,initially appearing as nearly circular or circular yellow-brown spots,which then enlarge and become irregularly-shaped large spots with multiple small spots.In severe cases,multiple spots can connect and cause the entire plant to wither and die.Therefore,the identification and separation of leaves with typical symptoms were carried out to determine the type of pathogenic causing the disease.Based on this,the biological characteristics of the pathogenic were studied,and chemical and biological pesticides were screened.The specific research results of the screening of biocontrol bacteria are as follows:1.The pathogenic bacteria were isolated and purified from plants with typical symptoms of leaf spot disease in Polygonatum cyrtonema.Based on pathogenicity determination,the pathogenic were found to be pathogenic.Combined with morphological characteristics and multi-gene analysis of ITS,ACT and TEF,the pathogenic were determined to be Phyllosticta capitalensis.This is the first report of P.capitalensis infecting Polygonatum cyrtonema.2.In the biological study,the J2 strain of pathogenic showed optimal growth on a PDA medium at 25°C and p H 7.0 with alternating light and dark cycles.The optimal carbon and nitrogen sources were sucrose and peptone,respectively.The optimal conditions for sporulation were PDA medium at 25°C,p H 7.0,and full light,with glucose as the carbon source,and peptone as the nitrogen source.3.Among the 9 chemical pesticides selected in this study,25%triazole had the strongest inhibitory effect on the J2 strain of pathogenic,with an EC₅₀of 0.292 mg·L^-1.Among the 5 biopesticides,10%polyoxin had the strongest inhibitory effect on the J2strain of pathogenic,with an EC₅₀of 0.205 mg·L^-1.4.Two strains of bacteria with stronger control effects on P.capitalensis were screened using the plate confrontation method from the healthy soil of P.multiflorum.Through morphological characteristics,physiological and biochemical tests,and molecular biology（16S r DNA and gyr A）identification,strain ZU-6 was identified as Pseudomonas fluorescens,and strain ZU-17 was identified as Pseudomonas bijieensis.The optimal carbon sources for strains ZU-6 and ZU-17 were glucose and soluble starch,respectively.The optimal nitrogen source was yeast.The optimal inorganic salts were Fe SO₄and Mg Cl₂,respectively.The optimal culture media for strains ZU-6 and ZU-17 were NYBD and LB,respectively.5.Through single-factor and orthogonal experiments analysis,the optimal fermentation conditions for strain ZU-6 are a fermentation time of 18 hours,a temperature of 35°C,p H of 7.0-8.0,an inoculum size of 3%,an inoculum age of 20 hours,a liquid volume of 75 m L,and shaking speed of 160 r/min;the optimal fermentation conditions for strain ZU-17 are fermentation time of 20 hours,the temperature of 32℃,p H of 7.0,inoculum size of 3%,inoculum age of 20 hours,the liquid volume of 75 m L,and shaking speed of 160 r/min.6.A pot experiment was conducted to determine the biocontrol effect of the fermentation broth of strains ZU-6 and ZU-17.It was found that the sprayed broth of ZU-6and ZU-17 on diseased plants showed good biocontrol effects compared to the control group,and the disease spots did not continue to expand.7.Two biocontrol strains were tested for their antibacterial activity in sterile filtrates at different temperatures,p H values,and storage times.The results showed that the antibacterial activity of the sterile filtrate was high and had good stability.In the study of the seed-promoting effect of sterile fermentation broth,the fermentation broth of both strains showed significant promotion effects on four types of seeds.