Japanese Journal of Phytopathology
Online ISSN : 1882-0484
Print ISSN : 0031-9473
ISSN-L : 0031-9473
The immunological diagnostic assay of watermelon bacterial fruit blotch by dot immuno-binding assay (DIBA) and tissue printing immunoassay (TPI)
Y. KOMIYAT. SHIRAKAWAK. ABIKO
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2002 Volume 68 Issue 3 Pages 291-296

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Abstract

We investigated DIBA and TPI techniques to detect Acidovorax avenae subsp. citrulli (Aac), the causal agent of watermelon bacterial fruit blotch from diseased plant materials. Direct DIBA used conjugate that was a conjugant of anti-Aac-IgG and alkaline phosphatase. The detectable limit of direct DIBA was about 106cfu/ml, when cultured Aac suspension was used as antigen. Strong positive reactions were obtained for only Aac by direct-DIBA and TPI techniques. Weak positive reaction was obtained for 1 or 2 strains of A. avenae subsp. avenae and Pseudomonas cichorii with a bacterial suspension of about 108cfu/ml. Strains of other plant pathogenic bacteria did not react. To detect Aac from symptomatic plant tissue, sections from symptomatic areas were macerated with phosphate-buffered saline in micro test tubes, and the result out supernatants tested by direct DIBA technique. When detection was carried out with the TPI technique, the sections from diseased plants were applied directly to the membrane filter. When diseased nursery plants obtained from infested seeds were tested by the direct DIBA and TPI techniques, positive reactions were obtained from all symptoms caused by Aac (isolated by dilution plate technique with selective medium AacSM). Moreover, positive reactions were obtained from old symptoms over 28 days after inoculation, and Aac was not isolated by the dilution plate technique using AacSM. Only about 30min/sample was needed to detect Aac by the direct-DIBA technique and 60min/50 samples was needed. From the results mentioned, direct-DIBA and TPI techniques are rapid and simple techniques for detecting of Aac from diseased plant materials.

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