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United States Department of Agriculture

Agricultural Research Service

Title: Specific Detection of Pseudomonas Syringae Pv. Phaseolicola DNA in Bean Seed by Polymerase Chain Reaction-Based Amplification of a Phaseolotoxin Gene Region

item Hatziloukas, E - UNIV. CA, BERKELEY
item Schaad, Norman
item Panopoulos, N - UNIV. CA, BERKELEY

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 5, 1993
Publication Date: N/A

Interpretive Summary: Halo blight of beans, caused by Pseudomonas syringae pv phaseolicola, is a serious seedborne disease which is controlled only by pathogen-free seeds. Seed producing states such as Idaho have strict quarantine programs. All foreign seed lots entering Idaho must be assayed for the pathogen and, if positive, are rejected. Current methods being used to assay seeds are either too time consuming, expensive, or lack the sensitivity needed to detect low numbers of the pathogen. A DNA based method called Polymerase Chain Reaction (PCR) was developed using genetic information from the unique toxin gene of the organism. Tests with naturally infected seeds showed that as few as 1,000 to 2,000 cells could be detected in seed washings. The test is 10 times more sensitive than current methods.

Technical Abstract: The polymerase chain reaction (PCR) is described for the specific detection of bean halo blight pathogen Pseudomonas syringae pv. phaseolicola in bean seed. The method involves the amplification of a segment of the tox (phaseolotoxin) gene cluster. Two oligonucleotide primers, designed according to the sequence of this segment, allowed the exclusive amplification of a 1.9-kb fragment from total DNA extracted from pure cultures of the pathogen and from water extracts of soaked bean seed. DNA's from 57 strains of other bacteria, including the bean pathogens P. s. pv. syringae and Xanthomonas campestris pv. phaseoli, and presumptive saprophytes associated with bean seed did not contain sequences homologous to the 1.9-kb fragment and did not produce this or other discrete bands upo amplification. The detection threshold in water extracts from soaked bean seeds was 10-20 cfu/ml. Presumptive epiphytes present at concentrations of f10,000 cfu/ml or more did not interfere with detection even when the pathogen was present at concentrations of 30 cfu/ml or less. In control experiments, heterologous DNA increased the intensity of the 1.9-kb band. The method was used to detect the pathogen in a commercial seed lot that failed to yield the pathogen by conventional methods. The method is highly specific because no other bacterium contains the unique toxin.

Last Modified: 4/19/2015
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