Developing a real-time PCR diagnostic method for a potential threat to chrysanthemum, Paratylenchus dianthus

Soil samples used were collected at Ch01, Ch02 and Ch03 from Uruma, Ch04, Ch05, Ch06 and Ch12 from Yomitan in the mainland Okinawa, Japan. Soil was collected from each field at 0 to 25 cm depth and at or around a base of post-harvest chrysanthemum plants with a spade, passed through a 5 mm aperture sieve and kept at room temperature until use. For Experiment 1 (a nematode survey), 1.5 kg of soil of each sampling site (Ch01–Ch06) was well mixed independently for further analysis. For Experiment 2, the soil samples (ca. 10 kg each) from each site (Ch04: the same timing of the soil collection for Experiment 1 in 2017; Ch05 and Ch12 in 2018) were also well mixed individually for the pot experiments.

Nematodes were extracted in triplicate from 20 g subsamples of the soil samples from each field using the Baermann funnel nematode extraction method (room temperature, 72 hr) followed by the sugar-flotation nematode extraction method (Jenkins, 1964; Kawanobe et al., 2014).

DNA was extracted following Iwahori et al. (2000) with minor modifications. A single nematode crushed with a sterilized filter paper chip (1×1 mm) was placed into a 200 µl Eppendorf tube containing 10 µl lysis buffer (10 mmol/liter Tris-HCl (pH 8.0), 0.1 mmol/liter EDTA, 10 g/liter of IGEPAL^® CA-630 (nonionic detergent; MP Biomedicals, Solon, OH), 100 µg/ml Proteinase K). After 1 hr in −85°C, the sample was thawed and incubated at 65°C for 1 hr to degrade the nematode’s body, and at 98°C for 10 min to inactivate proteinase K, and was then used as the DNA template (Kawanobe et al., 2015).

DNA from soil was extracted following Kawanobe et al. (2015) with minor modification (10 g soil in duplicate were homogenized in a 15-ml Falcon tube with two stainless steel balls (3/8 inch in diam.) using FastPrep 24 (MP Biomedicals, Irvine, CA) at 4.5 m/sec for 60 sec twice, instead of in a ball mill (MM400, Retsch, Germany) for 2 min at a frequency of 20/sec in the original method). In the preliminary experiment, soil samples processed with FastPrep 24 showed equivalent results obtained with MM400 under the real-time PCR analysis (data not shown). The DNA solutions dissolved in 100 µl Tris-EDTA buffer (10 mmol/liter Tris-HCl, 1 mmol/liter EDTA, pH 8.0) were diluted 10-folds with RNase-free water and used for real-time PCR assay.

Real-time PCR assays were performed with a Step One Real-Time PCR System (Life Technologies, Tokyo, Japan). Final sample volumes of 10 µl contained 5 µl of Fast SYBR Green Master Mix (Life Technologies, Tokyo, Japan), 0.4 µmol/liter of each primer, 2.2 µl of RNase-free water, and 2 µl of template DNA. The manufacturer’s recommended conditions were used with slight modifications (95°C for 10 sec, 40 cycles of (95°C for 5 sec, and 62°C for 20 sec), and melting curve profiles were generated). A negative control was prepared with RNase-free water instead of the DNA template, and a positive control (a DNA template extracted from each target nematode) was also included.

Real-time PCR assays were conducted using a primer set “Pdia” newly developed for P. dianthus, NEG (NEGf (5′- ATT CCG TCC GTG GTT GCT ATG-3′) and NEGr (5′-GCC GAG TGA TCC ACC GAT AAG-3′); Sato et al., 2007) for P. penetrans, Pkuma (PkumaF (5′-CGT GAA ACC GAT GAG ATG GAA AC-3′) and PkumaR (5′-CAA TGG GAG TGC GGA TGA ATA C-3′); Koyama et al., 2016) for P. kumamotoensis, and RKN (RKNf (5′-GCT GGT GTC TAA GTG TTG CTG ATA C-3′) and RKNr (5′-GAG CCT AGT GAT CCA CCG ATA AG-3′); Toyota et al., 2008) for M. incognita.

Nematode surveys were conducted in six chrysanthemum fields in April (Ch01–Ch05) and September (Ch06) 2017. All the fields were possibly damaged by plant-parasitic nematodes after examination by field specialists, yet, the nematode species in the fields were not known. The nematodes were classified under a stereo-microscope (SZX10, Olympus, Tokyo, Japan) based on their morphological characters and using a real-time PCR method for P. penetrans, P. kumamotoensis, and M. incognita. Pin-nematodes (Paratylenchus sp.) found in this study were identified as P. dianthus based on DNA sequence data (Accession numbers: LC462227-LC462228) of the ITS regions. For obtaining sequence data, DNA templates extracted from single pin-nematodes were amplified following Kawanobe et al. (2014) with slight modifications (a PCR primer set: TW81 (Joyce et al., 1994: 5′-GTT TCC GTA GGT GAA CCT GC-3′); rDNA26S (Vrain et al., 1992: 5′-TTT CAC TCG CCG TTA CTA AGG-3′)). The amplified PCR products were sequenced by a commercial sequencing service provider (FASMAC, Japan).

Chrysanthemum seedlings (cv. Okinootome, one of the commonly grown cultivars in Okinawa) of ca. 5 cm height with four leaves were used for pot experiments in 2017 and 2018. In total, 18 seedlings were used for each pot experiment. A subsample (1.2 kg) of each soil (Ch04 collected in April 2017, and Ch05 and Ch12 collected in April 2018) was put into a plastic pot (15 cm upper diameter; 10 cm bottom diameter; 15 cm height) together with imicyafos (0.35 g of Nemakick in a granular form (corresponding to its conventional dosage of 200 kg/ha), Agro-Kanesho, Tokyo, Japan, 1.5% a.i.) and 3.5 g chemical fertilizer (corresponding to N-P-K: 132-70-133 kg/ha, Kasei888, Omiya Green Service, Saitama, Japan). Pots without imicyafos were also prepared as non-treated controls. Experimental pots were prepared for each treatment in triplicates. Three chrysanthemum seedlings were transplanted to each pot and placed in the open air and watered when necessary during the two-month growth period.

After two-month growth in both 2017 and 2018 experiments, plant height of each seedling was measured. Then, the above-ground parts of seedlings were cut and dried at 70°C for longer than 72 hr, and plant dry weight (biomass) was measured after cooling down in a tightly sealed plastic bag. After removing the seedlings, the soil remaining in each pot was passed through a 5 mm aperture sieve and mixed well. Nematodes were extracted and counted based on their morphological character under a stereo-microscope (SZX10). For the pot experiments in 2018, single-photon avalanche diode (SPAD) values were also measured for leaves (the 6th to the 8th leaves from top) of each seedling using a SPAD meter (SPAD-502, Konica Minolta, Tokyo, Japan).

A specific primer set (Pdia, Table 1) was designed based on the ITS region of rRNA gene sequences of P. dianthus. Sequence comparisons were performed to design a specific primer set using the sequence data (comparisons with different taxonomic relationships within the same family and with plant-parasitic nematode species frequently detected in Okinawa, Table 1) collected from GenBank (http://www.ncbi.nlm.nih.gov/).

For generating calibration curves (lysate calibration curves), P. dianthus and M. incognita were collected from chrysanthemum field soils and DNA was extracted from a single nematode, in six replicates. Then, each 10 µl DNA solution was serially diluted (1:10, 1:40, 1:160, 1:640, 1:2560, and 1:10240, equivalent in DNA concentrations to soil DNA sample of 0.7 to 680 nematodes per 20 g soil) with RNase-free water. The diluted DNA samples were used as templates in real-time PCR assays to generate lysate calibration curves using the primer sets of Pdia and RKN. DNA was also extracted, in duplicates, from the subsample of the same soil used for nematode extraction at the end of the pot experiment (Ch12 only: the imicyafos treated and non-treated pots, in triplicates) in 2018. The DNA extracts were diluted 10-folds with RNase-free water and used for a real-time PCR assay to develop a soil calibration curve for Pdia. Lysate calibration curves for NEG (P. penetrans; Kawanobe et al., 2015) and Pkuma (P. kumamotoensis; Koyama et al., 2016) were adjusted to make the DNA dilution rate equivalent to soil calibration curves. This conversion reflected the difference in DNA concentrations between soil DNA samples (ca. 0.015 nematode in a 100 µl DNA solution, based on the extraction from 0.25 g oven-dried soil (equivalent to 0.29 g fresh soil with 15% water content) out of 20 g fresh soil containing one nematode) and single nematode DNA samples (one nematode in a 10 µl DNA solution).

DNA was extracted, in duplicates, from the subsample of the same soil sample used for the nematode extraction of the nematode survey (Ch01–Ch06). After extraction, DNA samples were diluted 10-folds with RNase-free water and used as templates. Specific primer sets for P. dianthus (Pdia), P. penetrans (NEG), P. kumamotoensis (Pkuma), and M. incognita (RKN) were used for real-time PCR assays. Soil calibration curves were applied for quantifying P. dianthus and M. incognita, and so were lysate calibration curves for all the nematode species. Nematode density of each plant-parasitic nematode species was estimated using each calibration curve by applying the C_q (quantification cycle) value measured in the real-time PCR assay.

The statistical difference was determined by Student’s t-test comparing control and test groups or analyzed by ANOVA followed by Tukey–Kramer’s test for multiple comparisons. Analysis of covariance (ANCOVA) was used to test the difference between the two regression slopes and intercepts following Ichihara (1990). Statistical analyses were conducted using Microsoft Excel and its add-in software Statcel (3rd ed., OMS, Tokyo, Japan).

The survey of the six locations revealed four different plant-parasitic nematode species (Table 2). P. penetrans was detected only in Ch01, while P. kumamotoensis was found in Ch02 and Ch03. P. dianthus was the most widely distributed species found in four locations (Ch02–Ch05). M. incognita was only present in Ch06.

Plant height of chrysanthemum seedlings grown with imicyafos was significantly (a multiple comparison with the Tukey–Kramer’s test, main effect (treatment): p < 0.05; no interaction effect (treatment × soil): p > 0.05) higher than of the non-treated seedlings after two-month growth (Fig. 1A). Dry weight of the imicyafos treated seedlings was also significantly (main effect (treatment): p < 0.01; no interaction effect (treatment × soil): p > 0.05)) higher than of the non-treated seedlings (Fig. 1B). SPAD value of imicyafos treated seedlings for both Ch05 and Ch12 in 2018 was significantly (main effect (treatment): p < 0.01; no interaction effect (treatment × soil): p > 0.05) higher than of the non-treated seedlings (Fig. 1C). P. dianthus densities of imicyafos treated seedlings were significantly (Student’s t-test: p < 0.05) lower than of the non-treated seedlings in all the three soils (Fig. 1D). A multiple comparison showed significantly different P. dianthus densities between the imicyafos treated and non-treated seedlings, yet the result should be treated as reference only due to the existence of interaction effect (main effect (treatment): p < 0.01; interaction effect (treatment × soil): p < 0.05). No other plant-parasitic nematodes were observed in all soil samples.

Figure 1:

The calibration curves for P. dianthus and M. incognita developed by serially diluted DNA templates from single nematode lysate (y-axis: C_q values, and x-axis: log₁₀ (the number of nematodes equivalent per 20 g soil) were y = − 3.6816x + 34.216 (R ²  = 0.9827, p < 0.001, Fig. 2) and y = − 3.4585x + 33.798 (R ²  = 0.9985, P < 0.001, Table 3), respectively. The calibration curve for P. dianthus developed using DNA extracted from soil (y-axis: C_q values, and x-axis: log₁₀ (the number of nematodes per 20 g soil) was y = −3.3105x + 33.202 (R ²  = 0.9233, p < 0.01, Fig. 2). The slopes and the intercepts of the calibration curves were not different (ANCOVA, p = 0.05) between calibration curves using serially diluted DNA and soil DNA for each of P. dianthus and P. penetrans (Table 3), respectively.

Figure 2:

All the real-time PCR assays using nematode species-specific primer sets detected the relevant nematode species, which were recorded from the nematode survey using the Baermann and sugar-flotation methods (Table 2). The estimated numbers of each nematode species were equivalent to or exceeded (×0.7–×9.0) those in the nematode survey with the microscopic method. The real-time PCR approach was also sensitive to detect P. dianthus in Ch06 where the microscopic approach did not detect P. dianthus.