| Tobacco wildfire is an epidemic bacteriosis caused by Pseudomonas syringae pv.tabaci.It has considerable negative impact on the yield and quality of tobaccos.Physiological differentiation of the pathogenic bacteria as well as the single disease control method,especially the long-term use of single chemical pesticides may change the pathogenicity and drug resistance of the pathogen population.This study conducts molecular identification and pathogenicity determination to obtained P.syringae pv.tabaci,and screens 7 housekeeping genes applicable to MLST of P.syringae pv.tabaci.Meanwhile,it conducts MLST based genetic diversity analysis to the pathogen population,detects the pathogenicity differentiation of the bacteria as well as the carbon and nitrogen sources utilization of different pathogenicity bacterial strains of major pathogenic groups for the purpose of determining the population genetic diversity of P.syringae pv.tabaci in different regions so as to provide theoretical basis and guidance for seed selection of disease resistance breeding in practical production and integrated control of tobacco wildfire.In this study,180 typical tobacco wildfire samples were collected from tobacco production areas of Chongqing,Sichuan,Hunan,Heilongjiang,Jilin and Shandong in 2016~ 2018,and 180 suspected P.syringae pv.tabaci strains were separated.Later,the 180 strains were tested by Koch’s rule and 144 inoculated strains were confirmed to be the pathogens of tobacco wildfire;the screened 144 strains received DNA sequencing by using the 16s-r DNA universal primer 27F/1492 R.After comparing the sequencing results with the data in Genbank by BLAST,the sequences of the closest relatives were acquired.The results suggest: the gene sequences of the 144 strains share 99% of similarity with P.amygdali(CP020351.1)and P.syringae(CP000075.1)in GenBank.Combined with the pathogenicity identification,it is determined that the tested strains are Pseudomonas syringae pv.tabaci.MLST technology was used to type the tobacco wildfire strains collected from the 5 provinces/cities.Using the whole genome sequence of the P.syringae pv.tabaci strain JWJF01000000,we acquired 7 housekeeping genes after screening: rpoD,gyrB,acnB,Gap,Pgi,Pfk and Cts.The 7 housekeeping genes were used to type the 144 strains,and 92 ST were obtained.By referring to three types of shared gene standards4/7,5/7 and 6/7,we got such results: when 4/7 standard is chosen,all the tested strains can be divided into 1 subgroup and 1 single group,which is too general;when 6/7 standard is chosen,all homologous compounds belong to a subgroup,which does not reflect the genetic relationship between the 4 subgroups and 30 single groups and not embody the diversity,so the standard is too strict;when 5/7 standard is chosen,all tested strains are divided into 5 subgroups and 4 single groups.Two of the subgroups(group1 and gruop2)cover most of the strains.The last typing result can better reflect the genetic relationships between strains than the first two typing standards,so this experiment mainly chose the group typing results of 5/7 gene standard.Group1 and group 2 cover all the strains collected from the 5 provinces/cities.There is no considerable difference between the ST of P.syringae pv.tabaci strains of southern and northern China.And the core ST of group 1 is from Jilin,Sichuan and Hunan.It can be seen that group 1 strains are evenly distributed in China,without any dominant geographic source.The sampled tobacco varieties were Yuyan 87,Jiyan 9 and Longjiang 911,all widely bred varieties throughout China,but there is no rule between tobacco variety and group typing.MLST can better uncover the genetic relationship and genetic difference among the geographical groups of tobacco wildfire pathogens,verifying that the P.syringae pv.tabaci groups in different regions of China see rich genetic diversity.The live plant acupuncture and needle free injection method was adopted to detect the pathogenicity of 144 tobacco wildfire pathogen strains to Yunyan 87 tobacco leaves.The results suggest: the pathogenicity is considerably varied among the strains from different regions,which can be divided into strong,relatively strong,medium,relatively poor,and poor.The tobacco zones of different provinces/cities have strains with similar pathogenicity,and the same tobacco zone of the same province/city has strains with different pathogenicity.The 6 strains of Group 4 all come from northeast China: 3 of strong pathogenicity and 1 of relatively strong pathogenicity from Heilongjiang Province,1 of strong pathogenicity and 1 of relatively strong pathogenicity from Jilin Province,which suggests certain relations between pathogenicity and genetic diversity.The strains from different provinces/cities are unevenly distributed in the 5 types of pathogenicity,but most belong to “relatively strong” and “medium”.That shows the dominant populations of China?s P.syringae pv.tabaci have relatively strong pathogenicity,and may easily cause severe disease outbreak.As for the pathogenicity of China?s P.syringae pv.tabaci,there are both similarities and differences among the provinces/cities,and the pathogenicity is obviously varied,which further verifies the rich genetic diversity of China?s P.syringae pv.tabaci.Biolog phenotypic analysis technology was used to determine the utilization of the 180 carbon substances in PM1-2 and the 90 nitrogen substances in PM3 by the strongly pathogenic strains JL-23 and HN-6(group 1),the poorly pathogenic strains SC-7 and CQ-17(group 2)and the medium pathogenic strains JL-7(group 3),HL-15(group 4)and SC-35(group5).According to the results,7 pathogen strains generally show similarity in utilization of carbon and nitrogen substances of PM1-3 microwell plate.The carbon source utilization rate is about 32.77%;the nitrogen source utilization rate is about 91.58%.However,strains of different pathogenicities display some differences: in carbon source utilization,the strains of three types of pathogenicity are remarkably varied in the utilization of PM1 carbon source,and strains of medium pathogenicity have higher utilization rate to PM2 plant than strains of strong or poor pathogenicity;In utilization of nitrogen source,strains of strong pathogenicity have higher utilization rate than strains of medium or poor pathogenicity;strains of poor pathogenicity have higher utilization rate to certain substances than strains of strong or medium pathogenicity.That suggests phenotypic differences among individuals in P.syringae pv.tabaci populations,which further proves that China?s P.syringae pv.tabaci has rich genetic diversity. |
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