Abstract
Clavibacter is an agriculturally important genus comprising a single species, Clavibacter michiganensis, and multiple subspecies, including, C. michiganensis subsp. nebraskensis which causes Goss's wilt/blight of corn, accounts for high yield losses and is listed among the five most significant diseases of corn in the United States of America. Our research objective was to develop a robust and rapid multiplex TaqMan real-time PCR (qPCR) to detect C. michiganensis in general and C. michiganensis subsp. nebraskensis with enhanced reliability and accuracy by adding non-complementary AT sequences to the 5’ end of the forward and reverse primers. Comparative genomic analyses were performed to identify unique and conserved gene regions for primer and probe design. The unique genomic regions, ABC transporter ATP-binding protein CDS/ABC-transporter permease and MFS transporter were determined for specific detection of C. michiganensis and C. m. subsp. nebraskensis, respectively. The AT-rich sequences at the 5’ position of the primers enhanced the reaction efficiency and sensitivity of rapid qPCR cycling; the reliability, accuracy and high efficiency of the developed assay was confirmed after testing with 59 strains from inclusivity and exclusivity panels–no false positives or false negatives were detected. The assays were also validated through naturally and artificially infected corn plant samples; all samples were detected for C. michiganensis and C. m. subsp. nebraskensis with 100% accuracy. The assay with 5’ AT-rich sequences detected up to 10- and 100-fg of C. michiganensis and C. michiganensis subsp. nebraskensis genome targets, respectively. No adverse effect was observed when sensitivity assays were spiked with host genomic DNA. Addition of 5’ AT-rich sequences enhanced the qPCR reaction efficiency from 0.82 (M = -3.83) and 0.91 (M = -3.54) to 1.04 (with optimum slope value; M = -3.23) for both C. michiganensis and C. michiganensis subsp. nebraskensis, respectively; an increase of 10-fold sensitivity was also obtained with C. michiganensis primer set. The methodology proposed here can be used to optimize reaction efficiency and to harmonize diagnostic protocols which have prodigious applications in routine diagnostics, biosecurity and microbial forensics.
Highlights
The gram-positive aerobic species Clavibacter michiganensis, which resides in the xylem vessels of the host, is a devastating bacterial pathogen of many economically important agricultural crops
Nine subspecies based on host specificity and bacteriological characteristics have been identified within the single species of C. michiganensis
Four subspecies infect two important plants in the Solanaceae family while the other five subspecies infect plant hosts from Fabaceae, Solanaceae and Poaceae families—C. m. subsp. michiganensis (Cmm; bacterial canker of tomato), C. m. subsp. capsici (Cmc; bacterial canker of pepper), C. m. subsp. insidiosus (Cmi; stunt and wilt of alfalfa), C. m. subsp. nebraskensis (Cmn; wilt and blight of maize), C. m. subsp. sepedonicus (Cms; ring rot of potato), C. m. subsp. tessellarius (Cmt; leaf freckles and leaf spots of wheat), C. m. subsp. phaseoli (Cmp; bacterial leaf yellowing of bean); two new subspecies C. m. subsp. californiensis (Cmcf) and C. m. subsp. chiloensis (Cmcl) were isolated from seed of tomato and pepper, respectively [1,2,4,5]
Summary
The gram-positive aerobic species Clavibacter michiganensis, which resides in the xylem vessels of the host, is a devastating bacterial pathogen of many economically important agricultural crops. The genus Clavibacter is known only for one species, C. michiganensis which belongs to family Microbacteriaceae within the phytopathogenic coryneform group, known for epi- or endophytic colonization of symptomatic as well as several asymptomatic plant species [1,2]. Virulence genes within this species are plasmid-borne (not essentially present), while host colonizing genes are chromosomal [3]. The leaf blight phase involves leaves and above-ground parts of the plant which exhibit small, dark and discontinuous water-soaked spots; orange shiny bacterial exudates are observed in advanced stages. Transmission of Goss’s disease usually occurs through open wounds on leaves, and C. m. subsp. nebraskensis can overwinter and remain in plant debris to serve as inoculum for the crop; transmission through seeds is minimal [2,8,9]
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