Bio-PCR - An important tool for detection of Phytopathogenic bacteria
Author: Raj Kiran, A. Kandan, Pardeep Kumar and J. Akhtar
Division of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), Pusa Campus, New Delhi - 110 012
Bio-PCR is a technique that combines biological and enzymatic amplifications of PCR targets. The biological amplification is used to increase the pathogen numbers to detectable levels in asymptomatic tissue while PCR is used to increase the number of copies of target DNA. Hence, sensitivity of detection of bacterial pathogen increase many fold. It has reported that Bio-PCR is 100 times more sensitive than conventional PCR (Schaad et al. 1999).
For Bio-PCR, maceration of seed, leaf, stem or asymptomatic leaf tissue from diseased plants are crushed and allow for 45 minutes to ooze the bacterial cells. One ml aliquot of bacterial ooze is taken for serial dilution up to 10-7 with distilled water, a 100 μl aliquot of bacterial suspension from each sample is inoculated with nutrient sucrose broth and incubated for 24 hours in shaker incubator (at 150 rpm). Two microliter of bacterial broth cultures are used for PCR assay (Singh et al. 2014). In another other method, aliquot of water extract from infected seed is plated on agar media and aliquot collected after washing of agar plates are used as template for PCR (Schaad et al. 1995).
Target cell enrichment followed by PCR improves the efficacy and sensitivity of PCR by allowing target pathogen population to increase in a pre-enrichment phase. Bio-PCR have several advantage over classical PCR. Bio-PCR provides improved sensitive detection and estimation of viable cells of bacterial pathogens present in seed whereas PCR assay is unable to discriminate between the viable and dead propagule of the pathogens. Selective pre enrichment increases pathogen population relative to non-target micro-organism and results in high quantity of target DNA, which ultimately results in sensitivity. Additionally, during incubation and enrichment on artificial media, inhibitory compounds are adsorbed or diluted during cell harvest and do not interfere with DNA amplification. Therefore, Bio-PCR eliminates the false positives and negatives in detection. A major disadvantage of Bio-PCR is that bacteria, which were not able to grow on media, were fail to detect by Bio-PCR.
Bio-PCR has been successfully used for the detection of many phytopathogenic bacteria like Pseudomonas syringae pv. phaseolicola (Schaad et al. 1995) in bean seed extract, specific detection of the strains of race 3, biovar 2 of Ralstonia solanacearum in asymptomatic potato tubers (Ozakman and Schaad 2003), Acidovorax avenae ssp. avenae in rice seeds (Song et al 2004), Xanthomonas arboricola pv.pruni in Prunus sp. (Ballard et al. 2011), Xanthomonas compestris pv. compestris in black rot infected seed (Singh et al. 2014) .
Greater sensitivity, detection of only viable cells and helps in early detection of disease by Bio-PCR plays an important role in detection of quarantine bacterial pathogens and for future epidemiological studies of bacterial diseases
Ballard E L, Dietzgen R G, Sly L I, Gouk C, Horlock C, and Fegan M 2011. Development of a Bio-PCR protocol for the detection of Xanthomonas arboricola pv. pruni. Plant Disease 95:1109-1115
Ozakman M and Schaad N W (2003) A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3, biovar 2, in asymptomatic potato tubers. Can J Plant Patho 25: 232-239
Schaad N W, Cheong S S, Tamaki S, Hatziloukas E, and Panopoulos N J (1995) A combined biological amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts. Phytopathology 85: 243-248.
Schaad N W, Berthier-Schaad Y, Sechler A, and Knorr D (1999) Detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers by BIO-PCR and an automated real-time fluorescence detection system. Plant Disease 83:1095-1100.
Singh D, Raghavendra B T, Rathaur P, Singh H, Raghuwanshi R and Singh R P (2014) Detection of black rot disease causing pathogen Xanthomonas campestris pv. campestris by bio-PCR from seeds and plant parts of cole crops. Seed Science & Technology 42: 36-46
Song W Y, Kim H M, Hwang C Y and Schaad N W (2004) Detection of Acidovorax avenae ssp. avenae in rice seeds using BIO-PCR. J Phytopathol 152: 667-676
About Author / Additional Info:
Scientist, Plant Quarantine Division, ICAR- National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi