A TaqMan real-time PCR assay targeting the cytochrome o ubiquinol oxidase subunit II gene for detection of several pathovars ofPseudomonas syringae

Abstract

The identification and detection of eight pathovars of Pseudomonas syringae, bacterial pathogens of several important agricultural plants, was achieved by TaqMan real-time polymerase chain reaction of a specific DNA fragment of the cytochrome o ubiquinol oxidase gene. Under optimal real-time PCR conditions, the selected primers and probe were specific for the detection of pathovars syringae, tomato, maculicola, tabaci, atropurpurea, phaseolicola, pisi and glycinea. Two pathovars (coriandricola and morsprunorum) tested could be differentiated from the other eight due to a single nucleotide mismatch. Thirty other Pseudomonas strains and 20 non-Pseudomonas strains were negative. The real-time PCR assay detected 100 fg of DNA and 4.5 × 10 3 P. syringae colony forming units per millilitre (four cells per reaction). In growth chamber experiments, tomato plants were inoculated using strain DC3000 and assayed by TaqMan real-time PCR. Serial dilution of leaf extracts spiked with lambda DNA and processed by real-time PCR indicated the presence of inhibitors. A 1:10 dilution of the crude extract reduced threshold cycles to those of milliQ water spiked with the same amount of lambda DNA. The TaqMan real-time assay consistently detected the pathogen in inoculated tomato leaves after a 1:10 dilution of crude extracts. TaqMan real-time results were validated by dilution-plating of leaf extracts and conventional PCR using the same primer set. This assay offers real-time monitoring of the targeted amplicon with high specificity and sensitivity, with no post-amplification analysis needed. This reduces opportunity for contamination of the reaction mixtures with target DNA, making this real-time PCR assay more reliable than conventional PCR. However, for routine diagnosis of the detected pathovars under greenhouse or field conditions, optimization of the assay might be required.Résumé: L'identification et la détection de huit pathovars de Pseudomonas syringae, agents pathogènes bactériens qui s'attaquent à plusieurs variétés de plantes économiquement importantes, ont été effectuées par réaction en chaîne de la polymérase (PCR) quantitative en temps réel d'un fragment précis de l'ADN du gène cytochrome o ubiquinol oxydase. Dans des conditions idéales de PCR en temps réel, les amorces et les sondes choisies étaient typiques de la détection des pathovars syringae, tomato, maculicola, tabaci, atropurpurea, phaseolicola, pisi et glycinea. Deux pathovars testés (coriadricola et morsprunorum) ont pu être différenciés des huit autres à cause de la disparité d'un seul nucléotide. Trente autres souches de Pseudomonas et 20 de non-Pseudomonas étaient négatives. Les analyses effectuées par PCR en temps réel ont permis de détecter 100 fg d'ADN et 4.5 × 10 3 unités formatrices de colonies de P. syringae par millilitre (quatre cellules par réaction). Au cours d'expériences en chambre de culture, des plants de tomate ont été inoculés avec la souche DC3000 et analysés par PCR en temps réel. Une dilution en série d'extraits de feuilles, enrichie d'AD...

Evaluating molecular diagnostic techniques for seed detection of Pseudomonas savastanoi pv. phaseolicola, causal agent of halo blight disease in mungbean (Vigna radiata)

“…Schaad et al 1995 reported an assay sensitivity of 3-15 cells per reaction using a nested PCR. The sensitivity of the hydrolysis probe qPCR used here was stated to be 4.5 × 10 3 CFU ml − 1 , or approximately 4 cells per reaction if using 1 µl of bacterial suspension as template (Xu and Tambong 2011). There is a one-to-one relationship between amplification and bacterial number as there is only a single copy for the cytochrome o ubiquinol oxidase subunit II gene for P. savastanoi pv.…”

“…With a 6 Mbp genome with 58% G + C (Noble et al 2020), 100 fg DNA represents 15.5 copies of the genome, which suggests a lower sensitivity for qPCR on extracted DNA versus direct bacterial suspensions used as template. However, as Xu and Tambong (2011) failed to state the template volumes or develop a risk assessment of a seed lot's potential for disease outbreak and severity. Once implemented, molecular diagnosis could be further leveraged to include other diseases such as tan spot in a dual diagnostic qPCR to transform the way clean seed is delivered to growers, leading to cleaner planting seed, higher yields and economic gains for growers and the industry.…”

MinION Nanopore-based detection of Clavibacter nebraskensis, the corn Goss’s wilt pathogen, and bacteriomic profiling of necrotic lesions of naturally-infected leaf samples

“…The locus tag for this fragment is CMN_00274. Primers were designed and tested for specificity, as reported previously [ 5 , 32 ]. The primers were checked for melting temperature (Tm), dimer or hairpin formation using an oligonucleotide properties calculator [ 33 ].…”

Diagnosis and management of halo blight in Australian mungbeans: a review

“…Seed crops should be grown in climates and locations non-conducive to pathogens, under drip irrigation to limit the amount of free moisture, and far removed from commercial crops (Grogan and Kimble 1967;Webster et al 1983;Gitaitis and Walcott 2007). Confirming the presence of pathogens in planting seed by using diagnostic assays such as serology, culturing and PCR further reduces the risk of epidemics (Vuurde et al 1991;Prosen et al 1993;Marques et al 2000;Rico et al 2006;Xu and Tambong 2011). Ultimately, managing the transmission of seed-borne diseases relies on precise identification of the target pathogens in planting seed and the development of targeted resistance to the pathogens within the host species (Bastas and Sahin 2017).…”