Faculty of Agricultural and Nutritional Sciences, Christian-Albrechts-University Kiel, Hermann-Rodewald-Str. 4, 24118 Kiel, Germany; e-nema GmbH, Klausdorfer Str. 28-36, 24223 Schwentinental, Germany.
Department of Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
J Invertebr Pathol. 2024 Mar;203:108048. doi: 10.1016/j.jip.2023.108048. Epub 2023 Dec 28.
Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.
基于昆虫病原线虫异小杆线虫的生物防治产品在毒力（质量）上可能有所不同。过去，它们共生细菌发光杆菌属在感染性持久幼虫（DJ）中的存在对 DJ 质量的影响并没有受到太多关注。DJ 中细菌的存在对于其生物防治潜力至关重要。本研究提供了一种基于 qPCR 技术定量 DJ 内细菌负荷的方法。利用发光杆菌 DE2 菌株的基因组信息，鉴定出与任何其他细菌序列无同源性的单拷贝基因。选择一个基因（命名为 CG2）用于引物设计和进一步的 qPCR 实验。与同样是异小杆线虫共生菌的 P. thracensis 和 P. kayaii 的交叉扩增测试呈阳性，而 P. temperata 或 Xenorhabdus nematophila 则没有扩增子产生。我们在携带特定比例无细菌（无菌）与带菌线虫的 DJ 种群中测试了我们的 qPCR 系统。随着种群中无菌 DJ 的比例增加，毒力下降，并且毒力与通过 qPCR 在种群中检测到的细菌 DNA 量成正比。随着长时间的液体储存，毒力也下降，并且该因素与各自 DJ 种群中细菌 DNA 的减少相关。我们观察到储存的 DJ 保持毒力长达 90 天，此后毒力以及细菌 DNA 的数量急剧下降。储存温度也会影响细菌的存活。在配方 DJ 中，当储存温度低于 7.5°C 时，DJ 种群中细菌 DNA 的损失加速，这表明当生长温度有利时，细菌细胞会繁殖。使用这种分子质量控制技术，现在可以充分解决储存 DJ 中细菌存活的问题。
