3.3.33
POLYMERASE CHAIN REACTION AND ELISA FOR THE IDENTIFICATION OF BURKHOLDERIA CEPACIA AND PSEUDOMONAS VIRIDIFLAVA, BACTERIAL PATHOGENS OF ONION

RD GITAITIS, FH SANDERS and RR WALCOTT

Department of Plant Pathology, Coastal Plain Experiment Station, University of Georgia, P.O. Box 748, Tifton, GA, USA 31793-0748

Background and objectives
Sourskin, caused by Burkholderia cepacia, and bacterial streak and bulb rot, caused by Pseudomonas viridiflava, have been responsible for onion losses in the field and in storage in Georgia, USA. Both pathogens are endemic in Georgia. Although both bacteria are cultured easily on nutritionally complex as well as semi-selective media, methods less laborious and timeconsuming were required to rapidly diagnose these diseases to better assess the effect of chemical treatments and cultural practices on reducing disease in the field or in storage. Both ELISA and PCR were developed and evaluated for that purpose.

Materials and methods
Polyclonal antisera were developed for both bacteria by injecting New Zealand white rabbits with antigen prepared from heat-treated (100 C for 2 hr) whole bacterial cells. Antigen was adjusted to pH 7.0 and precipitated with ammonium sulfate overnight. The precipitate was harvested by centrifugation and dialyzed (50,000 mw cutoff) in distilled water. Final antigen was adjusted to 300 ug of protein 1 mi. Indirect ELISA using alkaline phosphatase was used. Sequences from the lipase A gene of B. cepacia, pectate lyase gene from P. viridiflava and ice nucleation gene from P. syringae distributed by GenBank [1] were selected for primer development. PCR was performed on a DNA thermocycler and the amplicons were visualized by electrophoresis (1.5% agarose gel with 0.05% ethidium bromide in 0.5 M TE-buffer).

Results and conclusions
Primers (5'-CAACCGATTAGAGAACCG-3' and 5'-TAAGCGAGCAACTGTTCG-3') for the lipase A gene produced an amplicon of 494 bp for only 60% of the B. cepacia strains isolated from onions with sourskin symptoms, whereas ELISA reacted with 100% of the strains tested. Primers (5'-GTATTGCTGGTGTTACCC-3'and 5'-GGTATCCAGAAACGACAC-3') for the pectate lyase gene produced an amplicon of 606 bp with 80 % of all P. viridiflava strains tested. However, that increased to 92% when only P. viridiflava strains from onions and weeds from the onion growing areas in Georgia were tested and data from strains from bean, pepper, parsnip, tomato, and watermelon either from Georgia or other geographical areas of the U.S. were removed. When combined with primers (5'-GGAATGGTCAAGTTTCCG-3'and 5'-CCTGTACTTCCATAACCG-3') from the ice nucleation gene, which produced an amplicon of 395 bp, PCR could detect over 95% of the P. viridiflava strains normally associated with bacterial streak and bulb rot in Georgia. It was concluded that ELISA and PCR could be used to rapidly diagnose bacterial rots of onions.

References
1. Burks C, Cinkosky MJ, Fischer WM, Gilna P, Hayden JE-D, Keen GM, Kelly M, Kristofferson D, Lawrence J, 1992. GenBank. Nucleic Acids Research 20 (Suppl.): 2065-2069.