Savazzini Federica, Longa Claudia Maria Oliveira, Pertot Ilaria, Gessler Cesare
SafeCrop Centre - Istituto Agrario San Michele all'Adige, 38010 San Michele AA Trento, Italy.
J Microbiol Methods. 2008 May;73(2):185-94. doi: 10.1016/j.mimet.2008.02.004. Epub 2008 Mar 28.
Trichoderma (Hypocreales, Ascomycota) is a widespread genus in nature and several Trichoderma species are used in industrial processes and as biocontrol agents against crop diseases. It is very important that the persistence and spread of microorganisms released on purpose into the environment are accurately monitored. Real-time PCR methods for genus/species/strain identification of microorganisms are currently being developed to overcome the difficulties of classical microbiological and enzymatic methods for monitoring these populations. The aim of the present study was to develop and validate a specific real-time PCR-based method for detecting Trichoderma atroviride SC1 in soil. We developed a primer and TaqMan probe set constructed on base mutations in an endochitinase gene. This tool is highly specific for the detection and quantification of the SC1 strain. The limits of detection and quantification calculated from the relative standard deviation were 6000 and 20,000 haploid genome copies per gram of soil. Together with the low throughput time associated with this procedure, which allows the evaluation of many soil samples within a short time period, these results suggest that this method could be successfully used to trace the fate of T. atroviride SC1 applied as an open-field biocontrol agent.
木霉属（肉座菌目，子囊菌门）是自然界中广泛分布的一个属，几种木霉属物种被用于工业生产过程以及作为防治作物病害的生物防治剂。准确监测有意释放到环境中的微生物的持久性和扩散情况非常重要。目前正在开发用于微生物属/种/菌株鉴定的实时PCR方法，以克服传统微生物学和酶学方法在监测这些种群时遇到的困难。本研究的目的是开发并验证一种基于实时PCR的特异性方法，用于检测土壤中的深绿木霉SC1。我们开发了一种基于内切几丁质酶基因突变构建的引物和TaqMan探针组。该工具对SC1菌株的检测和定量具有高度特异性。根据相对标准偏差计算出的检测限和定量限分别为每克土壤6000和20000个单倍体基因组拷贝。再加上该方法的低通量时间，能够在短时间内评估许多土壤样品，这些结果表明该方法可成功用于追踪作为田间生物防治剂施用的深绿木霉SC1的去向。
