Development of a rapid, sensitive, and field-deployable razor ex BioDetection system and quantitative PCR assay for detection of Phymatotrichopsis omnivora using multiple gene targets

Abstract

A validated, multigene-based method using real-time quantitative PCR (qPCR) and the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton, alfalfa, and other dicot crops in the southwestern United States and northern Mexico, leading to significant crop losses and limiting the range of crops that can be grown in soils where the fungus is established. It is on multiple lists of regulated organisms. Because P. omnivora is difficult to isolate, accurate and sensitive culture-independent diagnostic tools are needed to confirm infections by this fungus. Specific PCR primers and probes were designed based on P. omnivora nucleotide sequences of the genes encoding rRNA internal transcribed spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II (RPB2). PCR products were cloned and sequenced to confirm their identity. All primer sets allowed early detection of P. omnivora in infected but asymptomatic plants. A modified rapid DNA purification method, which facilitates a quick (∼30-min) on-site assay capability for P. omnivora detection, was developed. Combined use of three target genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a multigene-based, field-deployable, rapid, and reliable identification method for a fungal plant pathogen and should serve as a model for the development of field-deployable assays of other phytopathogens.

Fig 1

Bar codes generated to operate the Razor Ex BioDetection system. (A) Pouch protocol bar code; (B) PCR cycling program bar code.

Fig 2

Standard curves were obtained for qPCR amplification of 10-fold serially diluted plasmid DNA and genomic DNA using probes PO4, Pobt1, and PoRPB2-2. Probe PO4 amplified with plasmid DNA (A), genomic DNA (B), genomic DNA mixed with cotton extract (C), and genomic DNA mixed with soil extract (D). Probe Pobt1 amplified with plasmid DNA (E), genomic DNA (F), genomic DNA mixed with cotton extract (G), and genomic DNA mixed with soil extract (H). PoRPB2-2 amplified with plasmid DNA (I), genomic DNA (J), genomic DNA mixed with cotton extract (K), and genomic DNA mixed with soil extract (L). The unit of concentration is ng/reaction mixture; CT, cycle threshold; R², linear correlation; Ex, reaction efficiency; Y, slope.

Fig 3

Razor Ex BioDetection system graph obtained with primer and probe set PO4, Pobt1, and PoRPB2-2 amplification of plasmid DNA (positive control) and P. omnivora-infected symptomatic alfalfa samples POM1 (positive by real-time qPCR). The results were reproduced using a second symptomatic alfalfa sample POM2 (data not shown). Curves A, B, and C are for positive controls and show estimated C_T values of 21, 23, and 23 for primer/probe sets PO4, Pobt1, and PoRPB2-2, respectively; curves D, E, and F are for P. omnivora-infected sample POM1 tested in two replicates with primer/probe sets PO4, Pobt1, and PoRPB2-2 and show estimated C_T values of 36, 40, and 40, respectively; curve N is that of nontemplate controls (water) for each primer/probe set. Both POM1 and POM2 samples were collected from fields at the Samuel Roberts Noble Foundation, Ardmore, OK, in July 2010.