Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed
Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed
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DOI：
10.1111/j.1365-3059.2011.02586.x
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发表时间：
2012-10-01
期刊：
影响因子：
2.7
通讯作者：
Munkvold, G. P.
中科院分区：
文献类型：
--
作者：
He, Y.;Munkvold, G. P.
轴足黄单胞菌致病变种菜豆疫霉（Phaseoli，Xap）是菜豆（Phaseolus vulgaris）的重要种传病原菌。准确的种子健康检测方法对于保护种子质量和满足植物检疫要求至关重要。目前采用的基于选择性介质的方法包括提取程序的几种变化。为了优化种子中病原菌的提取方法，研究了不同提取步骤对Xap检测灵敏度的影响。通过用Xap真空渗透接种种子，以达到每粒种子101至105 CFU的接种水平;将一粒受污染的种子与1000粒未受污染的P. vulgaris种子子样品混合。使用每种不同的浸提程序对1000粒种子的30个子样本进行了试验。这些方法包括将整个种子浸泡在无菌的磷酸盐缓冲液中，在4 ° C下过夜或在室温下3小时，进行或不进行真空提取，以及进行或不进行通过离心浓缩种子提取物。在半选择性琼脂培养基MT和XCP 1上培养种子提取物稀释液。比较阳性子样品的百分比，以测量每个提取步骤对检测灵敏度的影响。真空提取和离心的种子提取物增加的灵敏度，获得最高的灵敏度与3小时的真空提取，然后离心。这些结果与自然感染的种子得到证实; Xap检测到48的70个样品使用3小时真空提取离心，而只有35的70个现场样品测试阳性使用过夜浸泡，一个显着的差异。结果表明，这些步骤将是对国际种子检验协会（ISTA）批准的现行方法的有价值的修改。
Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media-based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 101 to 105 CFU per seed; one contaminated seed was mixed into 1000-seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4 degrees C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).