A high throughput membrane BIO‐PCR technique for ultra‐sensitive detection of Pseudomonas syringae pv. phaseolicola

Abstract

Molecular-based methods such as PCR have greatly improved detection of bacteria in environmental samples. However, the sensitivity of PCR is not high when compared to agar plating assays, and inhibitors from plants are often a problem. Pre-enriching bacteria on agar media (BIO-PCR) can increase the sensitivity of PCR by more than 100% and reduce the effects of inhibitors. To further increase the sensitivity and also reduce the labour needed for BIO-PCR, a high throughput 96-well membrane BIO-PCR technique is described for ultra-sensitive detection of Pseudomonas syringae pv. phaseolicola (PSP) (syn. P. phaseolicola ) in washings of seeds and leaves of Phaseolus vulgaris , using available classical PCR primers and newly designed real-time primers and probe. The primers and probe, designed from a tox-argK chromosomal cluster of the PSP-specific phaseolotoxin gene, were confirmed to be specific to PSP. Samples (1·2 mL) were filtered under vacuum in 96-well membrane plates. After incubating on soft agar medium for 48 -52 h, each well is washed with 200 µ L of sterile water and used immediately for nested (two-step) PCR or real-time PCR or stored at − 20 ° C. Results of assaying spiked seed washings showed that classical PCR was unable to detect PSP at mean concentrations of 40 colony forming units (cfu) mL . The sensitivity of detection from leaf washings was lower but the results were similar, classical and BIO-PCR were negative from all three levels of inoculum while membrane BIO-PCR detected three out of three samples at 80 mean cfu mL − 1 and one out of three at 40 mean cfu mL − 1 .

Development of a nested-PCR assay for the rapid detection of Pilidiella granati in pomegranate fruit

“…Molecular-based methods such as PCR have greatly improved the detection of microbes present in the environment28. PCR based assays are more rapid, sensitive, specific and accurate and have been often implemented for the routine diagnostics of a variety of pathogens24252930313233.…”

Detection of Bacterial and Phytoplasmal Pathogens

“…In contrast, the membrane real-time BIO-PCR format detected Psp at a concentration of 80 cfu/ml of bacterial cell suspension. The sensitivity of detection from leaf washings was lower compared with seed washings (Schaad et al 2007).…”

“…In contrast, membrane BIO-PCR detected the pathogen in all six samples tested containing as few as 0.4 mean cfu/ml. When large number of samples has to be handled, the 96-well plate allows two replications of 40-45 samples including controls (Schaad et al 2007). …”

Population dynamics of Pseudomonas savastanoi pv. phaseolicola in bean, throughout the epiphytic and pathogenic phases

“…The effect of rainfall on populations of P. syringae pv. tomato on tomato (Solanum lycopersicum L.) plants was determined by a rain simulator, showing that epiphytic populations are affected by rain events depending on the diameter of the rain drops, and, that, when the host-pathogen relation was already established, high rain intensity increased bacterial growth (Pietrarelli et al, 2006). Although it is essential to have open stomata for bacterial penetration, Melotto et al (2008) argue that these surface openings may play an active role in limiting bacterial invasion, as part of the plant's innate immune system (Baker et al, 2010).…”

“…Watering was done using micro-sprinklers, and rainfall was registered when it occurred. Plants were examined weekly for the presence of symptoms and to detect the qualitative presence of bacteria by Bio-PCR (Schaad et al, 2007). Plot II, sown with the susceptible cultivar Capitole, inoculated with the CFBP 4847 strain, which is more aggressive, was chosen for analysis of disease dispersion.…”