INRA, UR1115, Plantes et Systèmes de culture Horticoles, F-84914 Avignon CEDEX 9, France.
Int J Mol Sci. 2017 Sep 22;18(10):2031. doi: 10.3390/ijms18102031.
Biological control requires specific tools for the accurate detection and identification of natural enemies in order to estimate variations in their abundance and their impact according to changes in environmental conditions or agricultural practices. Here, we developed two molecular methods of detection based on PCR-RFLP with universal primers and on PCR with specific primers to identify commonly occurring larval parasitoids of the tortricid fruit pests and to estimate parasitism in the codling moth. Both methods were designed based on DNA sequences of the mitochondrial gene for a range of parasitoids that emerged from and caterpillars (102 parasitoids; nine species) and a range of potential tortricid hosts (40 moths; five species) damaging fruits. The PCR-RFLP method (digestion by AluI of a 482 bp fragment) was very powerful to identify parasitoid adults and their hosts, but failed to detect parasitoid larvae within eggs or within young caterpillars. The PCR method based on specific primers amplified fragments of different lengths (131 to 463 bp) for (Braconidae); (Ichneumonidae); (Ichneumonidae); and Perilampus tristis (Perilampidae), and demonstrated a higher level of sensibility than the PCR-RFLP method. Molecular estimations of parasitism levels in a natural population with the specific primers did not differ from traditional estimations based on caterpillar rearing (about 60% parasitism in a non-treated apple orchard). These PCR-based techniques provide information about within-host parasitoid assemblage in the codling moth and preliminary results on the larval parasitism of major tortricid fruit pests.
生物防治需要特定的工具来准确检测和识别天敌，以便根据环境条件或农业实践的变化来估计它们的丰度变化及其影响。在这里，我们开发了两种基于 PCR-RFLP 的分子检测方法，使用通用引物和基于特定引物的 PCR 来识别桃小食心虫幼虫的常见寄生蜂，并估计苹果蠹蛾的寄生率。这两种方法都是基于从桃小食心虫和幼虫中出现的一系列寄生蜂的线粒体基因的 DNA 序列设计的（102 种寄生蜂；9 种），以及一系列潜在的桃小食心虫宿主（40 种飞蛾；5 种）。PCR-RFLP 方法（用 AluI 消化 482 bp 的片段）非常强大，可以识别寄生蜂成虫及其宿主，但无法检测到卵内或幼龄幼虫内的寄生蜂幼虫。基于特定引物的 PCR 方法扩增了不同长度的片段（131 到 463 bp），用于 Braconidae、Ichneumonidae、Ichneumonidae 和 Perilampus tristis（Perilampidae），并且比 PCR-RFLP 方法具有更高的灵敏度。使用特定引物对自然种群中寄生率的分子估计与基于幼虫饲养的传统估计没有差异（在未处理的苹果园中约有 60%的寄生率）。这些基于 PCR 的技术提供了有关苹果蠹蛾体内寄生蜂组合的信息，并初步结果表明了主要桃小食心虫水果害虫的幼虫寄生率。
