Design And Application Of Genus-specific Primers For PCR Detection Of Plant Pathogenic Bacteria Within The Family Pectobacteriaceae

Most bacteria belonging to the genera Pectobacterium,Dickeya,Brenneria and Lonsdalea within the family Pectobacteriaceae are plant pathogens.Pectobacterium and Dickeya cause bacterial soft rot diseases of crops,vegetables and ornamental plants;Brenneria and Lonsdalea cause tree diseases.Establishing accurate,sensitive,and effective methods to detect and identify these pathogenic bacteria is critical for effective control of these plant diseases.We found Dickeya and Pectobacterium could infect sweet potato plants and cause soft rot disease in fields in Zhejiang Province,and thus attempt to develop genus-specific primers and universal primers for PCR detection of Dickeya and Pectobacterium pathogens.We tested known Dickeya-or Pectobacterium-specific or Dickeya-and Pectobacterium-specific primers and revealed that they are nonspecific or non-universal.Therefore,this study aimed to find conserved signature genes for Pectobacterium,Dickeya,Brenneria,and Lonsdalea and develop genus-specific primers for PCR detection of Pectobacterium,Dickeya,Brenneria,or Lonsdalea.This study did a phylogenomic analysis on representative strains within the family Pectobacteriaceae based on their whole genome sequences and did an analysis on their genome relatedness indices,and clarified the members within the Pectobacterium,Dickeya,Brenneria,and Lonsdalea.Genus-specific primers were designed targeting the conserved signature genes of Pectobacterium,Dickeya,Brenneria or Lonsdalea by using the pangenomic analysis pipeline and screened out by PCR amplification from bacteria belonging to the four genera and some other close or distantly related plant-associated strains.Genus-specific primers were determined for Pectobacterium,Dickeya,and Lonsdalea.A Pectobacterium-specific primer pair PecCSP-F/PecCSP-R targeting the conserved signature gene encoding protein WP?039471229 was determined to amply a 171-bp fragment from only Pectobacterium.A Dickeya-specific primer pair DicCSP-F/DicCSP-R targeting the conserved signature gene encoding protein WP?012885041 was determined to amply a 124-bp fragment from only Dickeya.A Lonsdalea-specific primer pair Lon CSP-F/Lon CSP-R targeting the conserved signature gene encoding WP?139824007 was determined to amply a 122-bp fragment from only Lonsdalea.The PecCSP-F/PecCSP-R primer pair is not universal for Pectobacterium because the target 171-bp fragment could not be amplified from P.cacticida.A Pectobacterium-specific primer pair 23 SPecF/23 SPecR targeting the 23 S rRNA gene was determined to amply a 266-bp fragment from only Pectobacterium including P.cacticida.Because Pectobacterium has seven copies of the 23 S rRNA genes,the PCR detection of Pectobacterium using the 23 SPecF/23 SPecR primer pair is more sensitive than those primer pairs targeting the single-copy genes.Moreover,this study,for the first time,isolated a pectin-degrading bacterium from diseased sweet potato plants in fields,identified the pathogen belonging to P.brasiliense,and determined its pathogenicity to sweet potato and potato plants.In conclusion,this study designed and screened out genus-specific primer pairs for PCR detection of Pectobacterium,Dickeya and Lonsdalea and developed specific and sensitive PCR and qPCR methods to detect Pectobacterium,Dickeya and Lonsdalea,and thus provide effective methods for detection and quarantine inspection of these pathogenic bacteria and to control the plant diseases.