Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight.
Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight.
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开发用于监测荧光假单胞菌 EPS62e（一种新型火疫病生物防治剂）的菌株特异性定量方法。
DOI：
10.1016/j.femsle.2005.06.029
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发表时间：
2005
影响因子：
2.1
通讯作者：
E. Montesinos
中科院分区：
文献类型：
--
作者：
M. Pujol;E. Badosa;J. Cabrefiga;E. Montesinos
荧光假单胞菌EPS62e因其对花卉、未成熟果实和幼龄梨树上的淀粉欧文氏菌的高效防治而被选入筛选程序。我们开发了两种监测方法，将经典微生物学技术与分子工具相结合，实现了对EPS62e的特异性检测和定量。利用随机扩增多态性DNA和非特异性PCR指纹图谱对EPS62e菌株和其他菌株进行了鉴别。荧光菌株。以EPS62e的差异扩增片段为SCAR标记，设计并筛选出两对特异的引物。采用平板法和最大可几数法对EPS62e在温室条件下梨树上的种群动态进行了评价和验证。这两种技术在监测生防剂方面都很有用。治疗后EPS62E的群体水平为7logCFU(gF.W.)−1，17天后逐渐下降至4-5logcfu(gF.W.)−1，然后保持稳定，直到11天后检测结束。这两种监测方法的检出限都在3logcfu(g F.W.)−1左右，因此为温室或田间试验中分析EPS62E提供了可靠的工具，并为有效生物防治火灾疫病的阈值种群水平评估提供了可靠的工具。
Pseudomonas fluorescensEPS62e has been selected in a screening procedure for its high efficacy controllingErwinia amylovorainfections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from otherP. fluorescensstrains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU (g f.w.)−1, which in turn decreased progressively to 4–5 log CFU (g f.w.)−1after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU (g f.w.)−1, thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.