Survival of inoculated fungal strains in a new environment plays a critical role in functional performance, but few studies have focused on strain-specific quantitative PCR (qPCR) methods for monitoring beneficial fungi. In this study, the Trichoderma guizhouense strain NJAU 4742 (transformed with the gfp gene and named gfp-NJAU 4742), which exhibits a growth-promoting effect by means of phytohormone production and pathogen antagonism, was selected as a model to design strain-specific primer pairs using two steps of genomic sequence comparison to detect its abundance in soil. After a second comparison with the closely related species T. harzianum CBS 226-95 to further differentiate the strain-specific fragments that had shown no homology to any sequence deposited in the databases used in the first comparison, ten primer pairs were designed from the whole genome. Meanwhile, 3 primer pairs, P11, P12 and P13, were also designed from the inserted fragment containing the gfp gene. After verification testing with three types of field soils, primer pairs P6, P7 and P8 were further selected by comparison with P11, P12 and P13. A practical test using a pot experiment showed that stable colonization of gfp-NJAU 4742 in pepper rhizosphere soil could be detected using primer pairs P6 and P7, showing no significant difference from the results of primers P11 and P12. Hence, the strategy described here for designing fungal-strain-specific primers may theoretically be used for any other fungi for which the whole genome sequence is available in a database, and the qPCR methodology developed can also be used to further monitor the population dynamics of different strains based on the designed primers.

中文翻译：

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两步基因组序列比较策略设计木霉菌株特异性引物用于定量PCR

接种真菌菌株在新环境中的存活在功能性能中起着至关重要的作用，但是很少有研究集中在监测有益真菌的菌株特异性定量PCR（qPCR）方法上。在本研究中，选择了通过植物激素产生和病原菌拮抗作用表现出促生长作用的贵州木霉菌株NJAU 4742（经gfp基因转化，命名为gfp -NJAU 4742）作为设计菌株特异性模型的模型。引物对使用两步基因组序列比较来检测其在土壤中的丰度。在与密切相关的物种哈茨木霉进行第二次比较之后为了进一步区分CBS 226-95，该菌株特异的片段与第一次比较中使用的数据库中显示的任何序列均无同源性，从整个基因组中设计了十对引物。同时，还从含有gfp基因的插入片段中设计了3对引物P11，P12和P13 。在对三种类型的田间土壤进行验证测试后，通过与P11，P12和P13的比较进一步选择了引物对P6，P7和P8。使用盆栽实验的实际测试表明，稳定的gfp定植使用引物对P6和P7可以在辣椒根际土壤中检测到-NJAU 4742，与引物P11和P12的结果没有显着差异。因此，这里描述的设计真菌菌株特异性引物的策略在理论上可以用于数据库中可获得其全基因组序列的任何其他真菌，并且开发的qPCR方法学也可以用于进一步监测其种群动态。根据设计的引物选择不同的菌株。