Experiments were conducted in 2007 and 2008 in a farmer's field (Miura, Kanagawa Prefecture) that was naturally infested with the lesion nematode. Some properties of the soil (Andisol) were as follows: soil texture: loam (clay 6.0%, silt 62.2%, sand 31.8%), maximum water holding capacity 0.937 g g-1
, pH (H2
O) 6.2 (Min et al., 2007
). There were two treatments (nematicide treated and untreated control), and each treatment had three plots (2 m x 2 m each). These two treatments were set in two different locations ca. 20 m away from each other depending on the year.
Nematicide treatment: A granular nematicide imicyafos (Nemakick, Agro-Kanesho Co., Ltd., Tokyo, Japan; 1.5% a. i.) was applied to the soil surface at the rate of 3 kg a. i./ha and plowed in 0 - 10 cm. In 2007, imicyafos was applied on 27 September, and radish (Raphanus sativus L.) seeds (cv. ‘T465’) were sown on 28 September with a 20-cm inter-plant distance and 40-cm inter-row space. In 2008, imicyafos was applied on 24 September, and radish (R. sativus L.) seeds (cv. ‘T465’) were sown on 24 September with a 20-cm inter-plant distance and 50-cm inter-row space. In both years, radish cultivation was carried out by the farmer using conventional techniques.
Soil sampling: Soil at 0 - 10 cm and 10 - 30 cm was collected from three locations within each plot using a root auger (4-cm diameter) and bulked to make a composite sample per plot for each depth. The first soil sampling was done before the nematicide treatment (20 September 2007 and 9 September 2008), and then soil sampling was done 12, 36, 70 and 89 (harvest) d after treatment in 2007. In 2008, soil sampling was done 91 (harvest) d after treatment.
Nematode number: Nematodes were extracted from 20 g of soil (fresh basis, in triplicate) by the Baermann funnel method for 48 hr at room temperature. The nematodes extracted from three Baermann funnels per plot were mixed to make a composite sample. The numbers of free-living nematodes and P. penetrans were separately counted under a microscope (x80).
Nematode community structure:
We used PCR-DGGE (denaturing gradient gel electrophoresis) to evaluate the impact of the nematicide on nematode community structure, since this procedure allows assessment of the presence and relative abundance of different species and thus communities can be profiled in both a qualitative and a semi-quantitative way (Muyzer et al, 1993
). PCR-DGGE is currently widely used to study soil microbial communities (Oros-Sichler et al., 2007
). Recently, this technique has been applied to the analysis of nematode community structure (Sato and Toyota, 2006
; Okada and Oba, 2008
). Some limitations are known to the interpretation of PCR-DGGE results. For example, bands of different species may partially or completely overlap on gel (Oros-Sichler et al., 2007
). Nonetheless, PCR-DGGE remains an effective technique for assessing whether changes in nematode community structure or diversity are associated with treatments.
A suspension of the nematodes extracted above was adjusted to a concentration of 200-250 individuals per 150-200 μl of distilled water and stored at -20°C until use. DNA was extracted from the nematode suspension by the method of Sato et al. (2009)
. One hundred fifty to two hundred microliters of a nematode suspension was put into a 2-ml tube with 0.2 g of zirconia beads (0.1 mm in diameter), and 20 μl of 10x TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0) was then added. The nematode suspension was treated with bead beading at 2,770 x g for 90 sec two times (Bead-smash 12; Wakenyaku, Kyoto, Japan), and the tube was placed on ice. Fifty microliters of a skim-milk solution (200 mg/ml) was added to the tube, along with 40 μL of 3M sodium acetate, 200 μl of an extraction buffer (5M NaCl, 0.5M Tris, 0.5M EDTA, pH 8.0) and 500 μl of chloroform and mixed well. After centrifugation for 15 min at 4°C at 20,350 x g (3740; Kubota, Osaka, Japan), 400 μl of the supernatant was transferred to a new tube with 8 μl of a glycogen solution (5 mg/ml), 80 μl of 3M sodium acetate, and 600 μl of isopropanol. Four hundred microliters of distilled water was added to the tube, mixed well, and then centrifuged for 15 min at 4°C at 20,350 x g. Four hundred microliters of this supernatant was combined with the first 400 μl of the supernatant. The DNA was precipitated by centrifugation for 15 min at 4°C at 20,350 x g, washed with 70% ethanol once and air-dried. Finally, the DNA was dissolved in 200 μl of TE buffer.
PCR amplification for DGGE was performed using the template DNA extracted above in a 25 μl volume containing 5 μl template DNA, 5 μl of 5 × PrimeSTARTM buffer (Mg2+
plus; Takara, Otsu, Japan), 200 μM each dNTP, 0.5 μM each primer (SSU18A and SSU9RGC; Okada & Oba 2008
) and 0.24 U of PrimeSTARTM HS DNA polymerase (Takara, Otsu, Japan). The temperature program included a denaturing step at 98°C for 3 min followed by 30 cycles of 98°C for 10 sec, 54°C for 15 sec, 72°C for 40 sec, and final extension step of 72°C for 10 min. The PCR products were loaded in an agarose gel (0.7%, w/v) and stained with ethidium bromide. Based on the band intensity determined visually post-PCR in an agarose gel, the volume of PCR product loaded onto the DGGE gel was adjusted (ca. 200 ng/lane). DGGE was carried out using a Bio-Rad DCode mutation analysis system (Bio-Rad Laboratories, Inc., Tokyo, Japan). Electrophoresis was done using a 6% (w/v) polyacrylamide gel in 1X TAE buffer (40 mmol/l Tris, 20 mM acetic acid and 1 mM EDTA, pH 8.0) under 75V at 60°C for 16 hr. The polyacrylamide gels were made with parallel denaturing gradients 15 - 50%; 100% denaturant contained 7 M urea and 40% formamide (Sato et al.,
2009). The marker lane consisted of known clones of soil fauna including nematodes. The fourth band from the top in the marker corresponds to Pratylenchus
sp. (Okada and Oba, 2008
). Band intensity was measured using a BioNumerics version 4.5 (Infocom Corporation, Tokyo, Japan), and the Shannon-Wiener index (H’)
was calculated based on the intensity data. The primers used in this study (SSU18A and SSU9RGC) amplify protozoa as well as nematodes. In our previous study (Sato and Toyota, unpub. data), results of random cloning of the PCR products showed that the clones with band positions below the second band of marker were all nematode species, while some of those above this band were nematode species and others were protozoan species. Therefore, to analyze the nematode community, we included only the bands below the second band of marker.
Estimation of damage to radish caused by P. penetrans:
Ten to 16 radishes per plot were harvested, washed with running tap water, and damage caused by P. penetrans
was evaluated based on 5 levels of 0 (no damage) to 4 (most severe) according to Mizukubo (2004)
Statistical analyses: Nematode density was compared between treatments with t-test. Two-way repeated measures ANOVA was also used to analyze the 2007 data. The Shannon-Wiener Index calculated from DGGE data was analyzed with t-test. Damage to radish caused by P. penetrans was analyzed with Mann-Whitney U-test. Excel statistics 2006 software for Windows (Social Survey Research Information Co., Ltd., Tokyo, Japan) was used for analysis of all data.