Identification And Detection Of The Pathogen Associated With Basal Stem Rot Disease On Carya Cathayensis

Chinese hickory,Carya cathayensis,is an important economic nut plant in China,This plants are mainly distributed in the Tianmu mountainous area at the junction of Zhejiang and Anhui.As plantation areas of C.cathayensis continues to expand,the outbreak of certain diseases on Carya cathayensis occurred and caused huge economical and ecological loss.Recently,a new disease on C.cathayensis,called as basal rot disease,was found in Linan County of China and resulted in large amount of plant dealth.In this study,the pathogen causing basal rot disease was isolated and identified.The molecular detection of this pathogen was conducted for early diagnosis.The antagonistic organisms against the pathogen were screened.The main research results are as follows:1.Investigations of Chinese hickory basal rot were conducted in Tuankou and Longgang by field survey.It was found that the average incidence of symptomatic plants increased from 1.6%and 1.2%in 2018 to 2.4%and 2.2%in 2019 in Tuankou and Longgang,respectively.In both surveyed orchards,yellowing and wilting of leaves initiated to occur in the May,2018,followed by defoliation and dieback of young shoots.Root systems appeared reduced,showing extensive necrosis of feeder roots.Necrosis extended to the trunks and caused base trunk rot and canker.In some severe cases,the necrotic symptoms were further expended to one meter from the ground.2.According to morphological characteristics and phylogenetic tree,the isolated fungus was preliminarily identified as P.cinnamomi.Eight representative isolates was randomly selected and used to confirm the morphological identification by phylogenetic analysis.PCR with primers ITS4/ITS6and COⅠ-Levup/COⅠ-Levlo gave the products of approximately 940 bp and 680 bp and the ITS and COⅠsequences of the isolateST402 were selected for deposition in Gen Bank with the accession numbers MT675107 and MT683171,respectively.A phylogenetic tree comprising the isolate ST402and other Phytophthora species was constructed based on the concatenated sequences of the ITS and COⅠr egions.This isolate ST402 was distinguished and grouped into the P.cinnamomi.Based on the phylogenetic analyses,the fungal pathogen was identified as P.cinnamomi.The isolate ST402 studied in pathogenic tests caused significantly necrosis and bark discoloration around the wounded site 7days in the greenhouse.No symptoms were observed on the inoculation mock.Re-isolations resulted in colony cultures on PARP medium identical to the morphological and phylogenetic analysis of P.cinnamomi such as the isolate ST402,fulfilling Koch’s postulates.3.The sensitivity and specificity test of the Loop-mediated isothermal amplification primers detection system established in this experiment shows that the sensitivity and specificity of P.cinnamomi detected by the Loop-mediated isothermal amplification primers method is very high.The sensitivity of P.cinnamomi was 0.189 pg·μL^-1,about 100 times than the conventional PCR assay.By testing the isolate ST402 and four reference fungi,the results of the isolate ST402 is positive and other reference fungi were negative,which proved the good specificity of the method.In practical applications,P.cinnamomi from diseased plant tissues and rhizosphere soil can be detected by this method,which proved that the Loop-mediated isothermal amplification can be well applied to the field due to the higher sensitivity and specificity.4.Screening of antibacterial for Phytophthora cinnamomi,including chemicals and biocontrol microorganisms.Among three chemicals,the best effect is oxathiapiprolin with the EC₅₀ of about0.069μg·mL^-1,following by phosethy-Al with the EC₅₀ of about 48μg·mL^-1,but potassium phosphate no effect for P.cinnamomi.This study has screened out chemicals and biocontrol microorganisms with high antibacterial activity on P.cinnamomi.It was found that Trichoderma atroviride,Streptomyces spp.,Paenibacillus polymyxa,and other bacillus spp.have an antibacterial activity on P.cinnamomi.In summary,this study confirmed that Phytophthora cinnamomi is the pathogen of Chinese hickory basal rot threatening the ecosystem of native plants and established a feasible Loop-mediated isothermal amplification primers detection method for the basal rot on Chinese hickory caused by Phytophthora cinnamomi.It has been screened out chemicals and biocontrol microorganisms with high antibacterial activity on Phytophthora cinnamomi,which provides a theory and method for the integrated management and control for Chinese hickory basal rot.