| Trichosanthes kirilowii Maxim.namely snakegourd fruit,is a perennial vine with wide distribution.It is a traditional Chinese medicinal material,cultivated in many provinces including Anhui,Shandong,Jiangsu of China.Its fruits,roots and seeds can be used as medicinal products of Trichosanthes kirilowii Maxim,Trichosanthis radix and Trichosanthis semen respectively,with the function of expectorant,hypoglycemic,anti-tumor,and treatment of cough,chest pain,heartache,and constipation.Recently,Trichosanthes kirilowii Maxim.has been found as a medicinal plant,but also as new medicinal value,health care and edible value.The cultivation of Trichosanthes kirilowii has been enlarged year by year because of its high medical and economic value.Trichosanthes kirilowii leaf spot was first reported in 2003,which did not attract much attention at that time.In this study,the pathogens of Trichosanthes kirilowii leaf spot were isolated using tissue separation and identified based on molecular methods.Its physiological and biochemical characteristics were also studied.At the same time,the method of distinguishing the pathogenic bacteria and non-pathogenic bacteria of Trichosanthes kirilowii leaf spot using REP-PCR technology was discussed to provide technical support for the monitor of Trichosanthes kirilowii leaf spot and theoretical basis for the effective prevention and treatment of this pathogen.The research progress is summarized as follow:1、Isolation and identification of Trichosanthes kirilowii leaf spot related bacteria From 2019 to 2020,280 Trichosanthes kirilowii strains were isolated from Trichosanthes kirilowii leaves collected from different habitats in Dabie Mountains.All strains were divided into three types,with 231 strains producing yellowish,round and convex colony in type Ⅰ,16 strains producing white,round and convex colony in type Ⅱ,33 strains producing yellowish,round and convex colony in type Ⅲ.The representative strains were amplified based on 16 S rDNA sequence.Results showed that Sphingomonas spp.strains accounted for 86% in type Ⅰ,and the rest included Microbacterium spp.and Curtobacterium spp.strains.At the same time,51 endophytic strains were recovered from healthy tissues.According to the colony separation above,stains belonging to type Ⅰ,type Ⅱ,and type Ⅲ accounted for 9.8%,41.1% and 39.2%,respectively.The representative endophytic strains were also amplified based on 16 S rDNA sequence.It was found that the main strains of type Ⅰ were Curtobacterium spp.,and the other strains were Agrobacterium spp.,etc..The results showed that some endophytic bacteria were isolated from the diseased leaves,which indicated that some endophytic bacteria were also isolated from the diseased leaves.A total of228 unsequenced strains isolated from diseased leaves were detected by Sphingomonas sp.with specific primers,179 strains were identified as Sphingomonas sp.with a fragment length of 500 bp.A total of 21 strains were selected(5 strains from Qianshan,9 strains from Shuihou,3 strains from Pineum,and 4 strains from Dongpu)for sequence and cluster analysis of 16 S rDNA.The results showed that 7 strains belonged to Sphingnomonas sanguinis,4 strains belonged to S.panni,1 strain belonged to S.azotifigens,1 strain belonged to S.zeae,1 strain belonged to S.yabuuchiae,and 1 strain belonged to S.carotinifaciens.And there were six strains of bacteria were isolated into one single clade,suggesting a new species in this clade.2、Pathogenicity determination The pathogenicity tests were conducted on Trichosanthes kirilowii seedlings using 52 representative strains.The leaves were initially stabbed by sterile needle.Results showed that S.sanguinis and S.panni strains can cause leaf disease,and the symptom is similar to the field symptom,but other types of strains have no obvious disease symptom.The infected leaves were re-isolated and the colony morphology of the isolates was similar to that of the inoculated strains.The pathogen of Trichosanthes kirilowii corner spot was identified as S.sanguinis and S.panni.3 、 Growth characteristics of pathogenic bacteria The physiological,biochemical,and growth characteristics of the pathogen were studied.It turns out that tween 80 hydrolysis,citrate utilization for S.sanguinis(QS8,QS17,QS19),S.panni(QS24,QS31)were all positive;oxidase,methyl red test,indole test were all negative,but starch hydrolysis and acetic acid oxidation of S.sanguinis strain were positive,but gelatin liquefaction was negative.S.panni is the exact opposite.S.sanguinis strain QS17 and S.panni strain QS24 were selected to test the effects of temperature,pH,salt concentration and antibiotics on growth.The optimum growth temperature were 28 ～ 32℃.The range of Acid and alkali tolerance were 5.5 ～ 8.5.Strains can not survive in weak acid and strong alkali environment.Salt concentration had a great effect on the growth of the strain.When the salt concentration was higher than 5%,the strain did not grow.Both strains could not grow on tetracycline plate,but could grow on carboxypenicillin,kanamycin and streptomycin sulfate plate.Meanwhile,it was found that S.sanguinis QS17 could not grow on chloramphenicol plate,while S.panni QS24 could grow on chloramphenicol plate.4 、 Molecular identification of bacteria associated with Trichosanthes kirilowii Diverse strain types were obtained from the diseased leaves of Trichosanthes kirilowii.The molecular identification of Sphingomonas spp.was only based on 16 S rDNA sequence analysis and cluster analysis.In order to determine the species of Sphingomonas spp.more accurately,this study primers were developed and used to amplify related sequences.The multi-gene sequences analyse based on 16 S rDNA and 16S-23 S rDNA sequences were carried out.The results showed that the genus Sphingomonas spp.was well distinguished,which was consistent with the previous results of 16 S rDNA cluster analysis.At the same time,REP-PCR was used to analyze the genomic fingerprints of the pathogenic bacteria and non pathogenic bacteria of Trichosanthes kirilowii.The results showed that the PCR amplification and electrophoresis showed obvious differences between the pathogenic bacteria and non pathogenic bacteria using REP,BOX and ERIC primers.At the same time,we found that their maps also showed certain differences between the two species of pathogenic bacteria,which provided us with a good technical support for the monitoring of the pathogen of Trichosanthes kirilowii in the further study. |
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