Miniprimer PCR assay targeting multiple genes: a new rapid and reliable tool for genotyping Pantoea stewartii subsp. stewartii.

AIM

Development of a 'miniprimer' PCR assay for genotyping Pantoea stewartii subsp. stewartii, the causal agent of the Stewart's bacterial wilt on maize.

METHODS AND RESULTS

Four 10-nucleotide (10-nt) 'miniprimer' sets were designed and evaluated in the presence of Titanium Taq DNA polymerase. Under optimal reaction conditions, the miniprimer pair Uni-BacF-10/Uni-BacR-10 reproducibly generated identical banding patterns among 10 strains of P. stewartii subsp. stewartii, different patterns from strains of P. stewartii subsp. indologenes, other Panteoa species, Clavibacter michiganensis, Pectobacterium spp., Pseudomonas spp. and other bacterial species. The amplicons of Pantoea stewartii subsp. stewartii were cloned and sequenced to identify genes or DNA fragments that are targeted by the miniprimer PCR assay. Of the 14 'clone types' identified, sequences of a 1.23-kb fragment had a 99.8% similarity to part of the Pantoea stewartii zeaxanthin diglucoside biosynthetic operon (AY166713). Other dominant cloned fragments included a 411-bp amplicon that exhibited 99.8% similarity to the psaU gene (syn:ysaU; GQ249669), a type III protein-secretion system complex of P. stewartii subsp. stewartii strain DC283, and a 548-bp fragment showed 63% homology to the Asp/Glu racemase encoding gene in Erwinia tasmaniensis strain ET1/99.

CONCLUSION

The miniprimer PCR assay reported here is highly discriminatory and reproducible in genotyping Pantoea stewartii subsp. stewartii.

SIGNIFICANCE AND IMPACT OF THE STUDY

This miniprimer PCR assay could be a new reliable and rapid tool for fingerprinting the Stewart's wilt pathogen of maize.