Pathogen Identification、Biological Characteristics And Fungicides Screening In Vitro Of Cypress Canker

Cypress,an excellent evergreen landscaping tree species,contains a variety of application value.Cypress canker is a fungal disease,which has been prevalent in many countries of the world and caused serious economic losses.Many studies have been carried out abroad,but this disease has not been reported in China.In March 2021,a certain number of dead shoots or crowns were found on cypress trees in the Yangtze River Forest Farm,Yunyang County,Chongqing City.The symptoms,Suspected cypress canker disease,of yellow burnt or even gray black death were found on cypress trees,What’s more,according to the local forestry department,the disease has been spreading continuously.In order to reduce to the risk of disease occurrence and prevent further spread of the disease,this paper studied the isolation and identification,biological characteristics and indoor fungicide screening of the pathogen of cypress canker,which laid a foundation for the subsequent disease control and pathogenic mechanism research,and also provided a theoretical basis for the risk assessment of this disease in the future.1.Pathogen identification.Collecting samples with typical symptoms,isolated 51strains by tissue isolation and single spore isolation.Colony characteristics,conidial sizes and colors of each strain were observed by morphology,which were divided into four types of isolates and marked as strain BA（1～8）,strain BB（1～18）,strain BC（1～13）and strain Y（1～12）.Using molecular biology techniques,the RPB2,TUB and TEF gene sequences representing the genomic DNA of strain BA1,the ITS,TUB and TEF gene sequences representing the genomic DNA of strain BB1 and BC3,and the CAL,TUB and TEF gene sequences representing the genomic DNA of strain Y1 were combined to construct phylogenetic trees.Finally,strains BA1,BB1,BC3 and Y1 were identified as Seiridium unicorne,Pestalotiopsis kenyana,Neofusicoccum salixmatsudana and Diaporthe pseudophoenicicola by morphology and molecular biology.Through Koch’s rule verification,the results showed that the four strains were the pathogenic bacteria of cypress canker,among which strain BB1 had the strongest pathogenicity,followed by BC3,BA1 and Y1 were weak.2.Biological characteristics.The effects of different temperatures and light conditions on the mycelial growth rate and sporulation of four pathogens were determined by mycelial growth rate method and germinating spore.The results showed that BA1 and Y1 had the highest sporulation at 20℃～25℃,and BB1 and BC3 had the highest sporulation at 25℃.The optimum temperature for mycelial growth of strains BA1 and BB1 was 25℃～30℃,and 30℃ was the optimum temperature for mycelial growth of strains BC3 and Y1.A certain amount of light can promote the mycelial growth of strains BC3 and Y1 and the sporulation of strains BA1,BB1 and BC3,and the alternation of light and dark can promote the sporulation of strain Y1.Use Biolog phenotype chip technology to examine the metabolism of four strains under different sources of carbon,nitrogen,phosphorus,sulfur,pH and biosynthesis pathway related substances,osmotic pressure and ionic strength.The results showed that the utilization rates of carbon sources of strains BA1,BB1,BC3 and Y1 were55.26%,71.58%,56.32%and 55.79%,respectively.The most utilized substance of the four strains was arbutin.Among BA1,BB1,BC3 and Y1,BB1 had the strongest metabolic capacity,whose utilization rates of nitrogen sources were 44.21%,89.47%,65.26%and 89.47%,respectively,and the utilization rates of phosphorus sources were22.03%,10.17%,52.54%and 25.42%,respectively.The utilization rates of sulfur sources were 34.29%,60.00%,57.14%and 77.14%,respectively.The four strains obtained strong metabolic capacity for the biosynthesis pathway-related substance m-inositol,and ranked as BB1,BC3,BA1 and Y1 from strong to weak.The metabolic trends of the four strains under different ionic strength and osmotic pressure conditions were similar.The four strains had strong metabolic utilization ability under the conditions of 1%sodium chloride,sodium sulfate,1%sodium lactate and sodium nitrate,while the metabolic ability was weak under the conditions of urea,sodium benzoate and sodium nitrite.The overall metabolic ability of BB1 was stronger and Y1was weaker.The metabolic trends of the four strains under different pH conditions were basically the same.Under the condition of pH=4.5～7,the four strains had strong metabolic utilization ability,and the order from strong to weak was BB1,BC3,BA1 and Y1.3.Indoor medicament screening of cypress canker.Use mycelium growth rate method to examine the indoor toxicity of strains BB1,BC3 and Y1.The results showed that 96%prochloraz manganese salt and 98%fludioxonil had the strongest antibacterial effect on BB1,with EC₅₀of 0.0213μg/mL and 0.0331μg/mL,respectively.The 96%prochloraz manganese salt had the strongest bacteriostatic effect on BC3,with 0.0482μg/mL EC₅₀.The 98%fludioxonil and 98%pyraclostrobin had the strongest antibacterial effect on Y1,and the EC₅₀ was 0.0263μg/mL and 0.1952μg/mL.The effect of each fungicide on the spore germination of strain BA1 was tested by spore germination method.The results showed that 98%pyraclostrobin and 97%azoxystrobin had the strongest inhibitory effect on the spore germination of BA1,and the EC₅₀ was0.0012μg/mL and 0.0122μg/mL,respectively.