Location of Acidovorax citrulli in watermelon seeds affects efficiency of pathogen detection by seed health testing
The influence of the location of Acidovorax citrulli in watermelon seeds on the efficiency of pathogen extraction and detection was investigated using seed lots generated by inoculating watermelon stigmas (pistil inoculation) or ovary pericarps (pericarp inoculation) with A. citrulli strain AAC00-1. Pistil inoculation results primarily in bacterial localisation in the seed embryo, whereas pericarp inoculation results in bacterial deposition under the seed coat. Acidovorax citrulli was extracted from watermelon seed samples (120 seeds per lot and inoculation method) by maceration or washing with phosphate-buffered saline. Total genomic DNA was purified from samples after the application of each extraction technique, and subjected to quantitative real-time PCR with A. citrulli-specific primers. To confirm PCR results, bacterial isolations were attempted from seed macerates and seed washes onto a semi-selective medium. Seed washing facilitated detection of A. citrulli from 80.0 and 82.5% of pericarp-inoculated seed lots by PCR and plating, respectively, as compared with 12.0 and 14.0% for pistil-inoculated seed lots. In contrast, maceration facilitated the detection of A. citrulli from ≥ 94% of the seed samples, regardless of inoculation method. Based on a seedling grow-out assay, 100% of the seed lots transmitted BFB to resulting seedlings. Additionally, A. citrulli populations from pericarp- and pistil-inoculated seed lots were enumerated to determine bacterial recovery from seed washes and seed macerates by real-time PCR and plating on semi-selective agar plates. The frequency distribution of A. citrulli populations (log10 CFU g−1) in seed wash vs. seed macerate was not significantly different for pericarp-inoculated lots (P = 0.070). However, for pistil-inoculated lots, the difference between frequency distributions was statistically significant (P < 0.001). These results indicate that the ability to extract A. citrulli from watermelon seeds was influenced by the location of the pathogen. Irrespective of pathogen location, maceration was more effective than washing for extracting A. citrulli from infected watermelon seeds.
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Document Type: Research Article
Publication date: October 1, 2012
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