Entomology and Nematology Department, Citrus Research and Education Center, University of Florida, IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA.
J Invertebr Pathol. 2011 Sep;108(1):30-9. doi: 10.1016/j.jip.2011.06.005. Epub 2011 Jun 23.
Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research validate several new tools for studying the ecology of EPNs and their phoretic bacteria.
实时荧光定量 PCR（qPCR）是一种强大的工具，可用于检测和定量土壤样本中细菌、真菌和线虫等隐生生物。qPCR 为研究土壤生境的生态学提供了新的机会，它提供了一种从 DNA 样本中描述不同生物群落的单一方法。在这里，我们描述了用于检测和定量两种与昆虫病原线虫（EPN）相关的细菌（Paenibacillus nematophilus 和 Paenibacillus sp.）的分子工具，这些细菌属于异小杆科和斯氏线虫科。我们还扩展了 EPN 的物种特异性引物和 TaqMan®探针的范围，包括广泛用于生物防治的异小杆属的嗜虫致病杆菌、斯氏线虫、嗜线虫致病杆菌和斯氏线虫。引物和探针是根据 EPN 的 ITS rDNA 区域和细菌的 16S rDNA 区域设计的。使用纯培养的 EPN 的 DNA 和细菌的质粒 DNA 建立了标准曲线。使用 qPCR 的 TaqMan 探针解决了 EPN 和一些细菌引物扩增的非特异性问题，而 Paenibacillus sp. 的引物也扩增了与线虫无关的 Paenibacillus thiaminolyticus 和 Paenibacillus popilliae。EPN 的引物-探针组能够准确检测到从土壤样本中回收的三种感染性幼虫 EPN。Paenibacillus sp. 的分子组检测到附着在 Steinernema diaprepesi 上的细菌，这些细菌悬浮在水中或添加到从土壤样本中回收的线虫中，但在土壤样本中的检测明显下降，即使使用嵌套 PCR 方案也是如此。使用 qPCR，我们在柑橘园检测到了低水平的 S. scapterisci，这表明这种外来物种的自然远距离传播，该物种被应用于牧场和高尔夫球场以管理地老虎（Scapteriscus spp.）。在同一调查中，也检测到了低量的 Paenibacillus sp.（但不是 P. nematophilus），但其与 S. diaprepesi 的空间模式无关。这项研究的结果验证了几种用于研究 EPN 及其携带细菌生态学的新工具。
