Detection,identification and differentiation of corn pathogen pantoea stewartii subspecies by membrane-based multi-gene oligonucleotide array

Pantoea stewartii subsp. stewartii (pss) is mostly known as the stewart’s wilt pathogen of sweet corn and it is classified as a quarantine bacterium,requiring certification of grain shipments to over 60 countries. it is found throughout the corn belt in the united states,canada as well as other countries in south and central america,europe,and asia. the conventional molecular methods of identification,reported to date,are not specific enough to differentiate pss from pantoea stewartii subsp. indologenes (psi) in infected corn leaves and seeds. in this study,we have developed the first multi-gene array for accurate detection,identification and differentiation of pss from psi and pantoea. the technique consisted of multiplex (16s rrna,leus,gyrb,rpob,cps) pcr amplifications using universal and specific primers in a single reaction,followed by the hybridization of the digoxigenin-labeled amplicons to 22 specific oligonucleotide probes (19- to 24-mers) immobilized on a nylon membrane. the reliability of the array was tested on 50 bacterial strains consisting of pantoea species,closely- and distantly-related bacterial genera and species. the hybridization signals were consistent and reproducible and the specificity for the identification of pure cultures was 100%. in growth chamber-inoculated and naturally-infested corn leaves and seeds,the array detected the pathogen in all infected plants. we conclude that the reported multi-gene dna array is a reliable tool for routine identification and differentiation of pss from psi and other pantoea species in pure cultures and infected corn leaves and seeds. © 2015 penerbit utm press. all rights reserved.