A combination method consisting of soil compaction and real-time PCR was developed to quantify Pratylenchus penetrans in soil. When five andosols and three gray lowland soils naturally infested with P. penetrans were compacted and used to quantify P. penetrans, the threshold cycle (Ct) values were always lower compared with those in non-compacted soils. Furthermore, Ct values were not obtained in some cases and standard errors were sometimes large. To avoid these problems, a DNA extraction method was newly developed and used to quantify P. penetrans in the two naturally infested soils. We obtained Ct values two to four times lower in the modified method than those in the original method. When known numbers of second-stage juvenile (J2) P. penetrans were added to 20 g samples of non-infested andosols and gray lowland soils, which were then compacted, and DNA was extracted by the modified method, there were highly significant correlations (r² = 0.993 and r² = 0.965, respectively) between the Ct values and the number of J2s added. The detection limits for P. penetrans in 20 g of soil were 10 and 25 individuals for gray lowland soil and andosol, respectively. To improve the detection limit, the DNA extraction method should be further modified.

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Caenorhabditis japonica is a bacterial-feeding nematode phoretically associated with the shield bug, Parastrachia japonensis. Caenorhabditis japonica dauer larvae (DLs) are mainly found on adult female bugs throughout the year, suggesting an intimate association between the nematode and the shield bug, but the mechanisms of the female bug-specific association, as well as the life history of C. japonica, are not well known. To understand the biology of C. japonica and the behavior of DLs, we developed a simple method to selectively collect phoretically active DLs using swarming behavior. We also demonstrated that the DLs obtained by this method were able to associate with the shield bug.

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Various methods for extracting DNA from single nematodes for PCR amplification have been developed by many authors. Such techniques generally do not include a purification step, and direct application of crude lysate to PCR amplification is usual. SDS is not used in DNA extraction methods for PCR amplification from single nematodes, although it is a common detergent used in DNA extraction. This is because SDS has an inhibitory effect on PCR (Gelfand, 1989). The author developed an SDS-based DNA extraction method from single nematodes, where the crude lysate can be applied directly to PCR amplification, and reports it here.

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[in Japanese]

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