The primers SCN44f and SCN124r were designed for the quantitative detection of the soybean cyst nematode (SCN) Heterodera glycines using real-time PCR. An andosol naturally infested with SCN was put into a 100 ml core and compacted to various degrees, i.e. 0.93 to 1.4 g cm^-3, using a manually-operated compactor. Then, DNA was extracted from the compacted soils as well as non-compacted soil (0.66 g cm^-3) and used as templates for real-time PCR. Ct values were the lowest at 1.4 g cm^-3, the maximum physical compaction of the andosol in the compactor, and the difference in the Ct values between compacted and non-compacted soil was four cycles at the largest, suggesting that 16 times more DNA derived from SCN was detected by the compaction. Then, different numbers (10 to 3,000) of SCN eggs were added to 20 g of a non-infested andosol and the soils were compacted to 1.4 g cm^-3. There was a significant correlation (r² = 0.8615, P < 0.001) between the Ct values and the number of eggs added. These results demonstrated that the present method using a combination of soil compaction and real-time PCR enabled rapid and sensitive quantification of SCN eggs in soil.

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A new diagnostic and quantitative real-time PCR assay was developed for the pine wood nematode, Bursaphelenchus xylophilus, which causes a serious epidemic wilt disease of susceptible landscape and forest pine trees. One, 10, 50, 100, 200, and 400 individuals of B. xylophilus in 100 mg of pine tissues were detected and quantified with two sets of primers coding the rDNA-ITS region and β-tubulin (tbb) gene of B. xylophilus, avoiding the need to extract nematodes with Baermann funnels. The assay was efficient, specific for pine wood nematode and sensitive enough to detect a single individual in pine tissues. To test the applicability of the assay for infested hosts, seedlings of the susceptible host species, Pinus thunbergii, were inoculated with B. xylophilus, and the dispersal and multiplication of B. xylophilus inside the seedlings were monitored by both Baermann funnel extraction and realtime PCR. Quantification by real-time PCR gave similar trends in estimates of the nematode population changes inside the host to those obtained by Baermann funnel extraction, although the size of the population estimated was larger for the new method than for the Baermann funnel extraction. The method developed in this study, which was expected to detect and quantify all life stages of the pine wood nematode should be useful for quarantine and field diagnosis.

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A survey to detect three Pratylenchus species, P. kumamotoensis, P. penetrans and P. pseudocoffeae, was conducted in chrysanthemum fields in the Kyushu-Okinawa region of southern Japan from 2005 to 2007. PCR-RFLP analysis was used for identification of extracted root lesion nematodes. The three species could be distinguished from one another by digestion patterns of Alu I, Hha I, Hinf I, or Taq I in a preliminary experiment and as a result Alu I and Hinf I were used for species identification in the field survey. A total of 86 soil samples were collected, and 38 samples (44%) were infested with root lesion nematodes. Pratylenchus kumamotoensis was detected most frequently (29%) and considered the most important root lesion nematode in this region. Detection rates of P. penetrans and P. pseudocoffeae were 22% and 9%, respectively. Pratylenchus penetrans was detected from Okinawa Prefecture for the first time.

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We tested the utilization of waste rootstocks of asparagus forcing culture as a control agent of Pratylenchus penetrans in lettuce production. When asparagus waste rootstocks used in forcing culture were mixed with P. penetrans naturally infested soil, population densities of P. penetrans declined. Subsequently cultivated lettuce plants of incorporated plots had better growth and higher yield than those of the non-treated control. Waste rootstock mixed with P. penetrans naturally infested soil in glass jars at an incorporation rate of 1% (w/w) rootstock significantly lowered P. penetrans densities. Rootstocks were incorporated at 11 kg/m² (equivalent to 1% of plowed soil) into nematode infested field plots in late April of 2006, and at rates of 5.5 kg/m² or 11 kg/m² into other plots in early May of 2007. At the beginning of September in 2006, P. penetrans population densities in the 11 kg/m² incorporated plot declined to 16% of the control and in July 2007 to 23% and 11% of the control in the 5.5 kg and 11 kg/m² plots, respectively. Lettuce seedlings were then transplanted to the field plots and the head weights and above-ground weights at the harvest were higher in the incorporated plots than the control in both years. Lettuce seedlings were again transplanted to the same field in March of the next year, and the 11 kg/m² plots gave effective control results.

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In a pot test with soil infested with the northern root-lesion nematode, Pratylenchus penetrans, conducted to examine nematode development in plant root tissue and nematode populations in soil, no nematode eggs were found in the root tissues of 7 cultivars of rye and triticale among 35 cultivars tested. In a spring seeded experiment, 5 of the 7 cultivars which had no nematode eggs in root tissue were grown for 2 months from the end of May in nematode-infested field plots. The experiment was repeated twice in two successive years with each year having a different population level of 92 and 52 nematodes/25 g of soil, respectively. A nematode suppressive effect was found in the“BILBAO” and “WIANDI” plots, but the effect was lower than that in the control plots planted with a commercially available antagonistic plant, Avena strigosa “Hay oats”. Proportions of marketable radish grown after rye for two successive years were respectively 43% and 73% in the “BILBAO” plot, 3% and 30% in the “WIANDI” plot, and 100% for both years in the A. strigosa plot. In the autumn seeded experiment, rye plants were grown from 27 September to 12 June the following year in nematode-infested field plots (45 nematodes/25 g of soil) with or without a fungicide application for snow mold control. The nematode Pf/Pi ratios were 10% for “SBR601” (newly tested breeding line), 23% for “BILBAO” and 24% for “WIANDI” in the plots where crop growth was protected by the fungicide application. Proportions of marketable radish grown after rye were 90% in the “SBR601” plot, 60% in the “BILBAO” plot and 67% in the “WIANDI” plot. The suppressive effect on nematode populations was found to be higher in “SBR601” than in the other two cultivars.

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