A COMBINED BIOLOGICAL AND ENZYMATIC AMPLIFICATION (BIO-PCR) TECHNIQUE TO DETECT PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA IN BEAN SEED EXTRACTS

Authors: SCHAAD N W - 1920-05-00; CHEONG S S - CHONBUK NATL UNIV., KOREA; TAMAKI S - UNIV. CALIF., BERKELEY; HATZILOUKAS E - UNIV. CALIF., BERKELEY; PANOPOULOS N J - UNIV. CALIF., BERKELEY

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/1994
Publication Date: N/A
Citation: N/A

Interpretive Summary: Halo blight of beans is an important seed-transmitted disease. To keep the disease out of seed-producing states such as Idaho, all imported seed must be assayed and found free of the pathogen. All seed fields are inspected and if halo blight is found, the field is destroyed. Current assay methods using agar media lack sensitivity and are time-consuming. A polymerase chain reaction (PCR) method is available, however, the method still lacks sensitivity and cannot distinguish dead cells from viable cells. This can result in false positives. This paper describes a novel, highly sensitive viable cell-PCR method for routine detection of Pseudomonas syringae pv phaseolicola in bean seeds

Technical Abstract: A polymerase chain reaction (PCR) method based on viable cells is described for the first time for assaying bean seed extracts for Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of beans. Seeds are soaked and the extracts plated on to a general growth medium following standard protocols. After 48 h incubation, the plates are washed and aliquots subjected to two consecutive rounds of PCR using "nested" primers derived from the organism's tox (phaseolotoxin) gene region. Detection of 5-13 viable cells of the pathogen per milliliter of seed wash was routinely obtained. This novel viable cell-PCR technique should prove useful for detection of other bacterial pathogens of quarantine importance