Table 1.
Comparison of the sequences in the positions of the specific primer set for Paratylenchus dianthus (Pdia) with different taxonomic relationships.
| Sequence (5′ → 3′) | ||
|---|---|---|
| Nematode species for sequence comparison (accession no.) | Forward | Reverse |
| PdiaF/R | TGACTG---TCG--AAGGCATAGTGGTAGA | CGGCACCTAGAGCAA---GGTACTCA |
| The genus Paratylenchus | ||
| Paratylenchus aquaticus (KF242278) | ..G ...CCG .-.--.G.CGT .- .C.A .... | .......C.AGC.-----........ |
| P. guangzhouensis (KT725626) | ..G...ACAAT.--....GT.-........ | .......AC-.-.T----........ |
| P. hamatus (KF242258) | ......CGT.TT--.......T........ | .......AGT....C---........ |
| P. lepidus (EF126178) | ......TG-.T.--.CA..G.T........ | ..........C....---........ |
| P. leptos (KR270605) | ..G...-CG.T.--...CGT.-AC.TG.TT | .......AC-T-.C----........ |
| P. minutus (EF126180) | ..T...TG-.G.--.C.....T........ | ........C.C..T.---...G.... |
| P. nanjingensis (KM366103) | ..G..TGCAGT.--...CGT.-.......- | ........C-C-.T----........ |
| P. nanus (KY468906) | ......CGT.TT--.......T........ | .......AGT.T..C---........ |
| P. rostrocaudatus (KR270604) | ..G...-CG.T.--...CGT.-AC.AG..T | .......AC--..T----........ |
| The same subfamily Paratylenchinae | ||
| Gracilacus bilineata (EU247525) | ..G...ACAAT.--...CGT.-........ | .......AC-.-.T----........ |
| G. aculenta (EU247526) | ..G..TGCAAT .--...CGT .- .......- | ........C-T-.T----........ |
| The other subfamily Tylenchulinae | ||
| Meloidoderita kirjanovae (DQ768427) | ..G...C TGA-.--.... GT .-CC.AC. .G | ..A....A..T-.T----...GT... |
| Plant-parasitic nematode species frequently detected in Okinawa, Japan | ||
| Ditylenchus destructor (KX181647) | ..G...---.-..TG.A.G.A.AC....CG | .......A.-...C.----.TG.... |
| Helicotylenchus dihystera (LC030373) | ..G...---C- ..TG..A .GAC.C.AC ..G | ....G.AACAT..T---C..CG.... |
| Hoplolaimus columbus (AB933480) | ..G...---C-..TG..ATGAC.C.....G | ....T.TCTAC---.---C.AG.... |
| Meloidogyne arenaria (LC030354) | ..G...---.ATATGT ..TGACA..... .G | ....-.TC.-CTT..---.AGG.... |
| M. incognita (KY985255) | ..G...---.ATATGT ..TGACA. .....G | ....-...CACTT..---.AGG.... |
| M. javanica (AY438555) | ..G...---.ATATGT..TGACA... ...G | ....-.TC.-CTT..---.AGG.... |
| Mesocriconema xenoplax (FN433851) | ..G...---C..-TG.T.TG.T.-C.A.... | ......-.-...TCC---........ |
| Pratylenchus coffeae (FJ712906) | ..G...---.G- ATG..A ..AC.. .....G | ....C.TGG..CT..TTG C.GG.... |
| P. kumamotoensis (LC030317) | ..G...---.A-ATG.....ACAC.....G | ..T.C.AC--.T.T.---..GG.... |
| P. pseudocoffeae (LC030337) | ..G...---.G-ATG.....AT.C.....G | ..T.C.AC .C.CA.TA AAT.GG.... |
| P. penetrans (LC030333) | ..GT..---- .TATC..A.. A.CG.TAG .. | ....C.....-...---TT.GG.... |
| P. zeae (KY424187) | ..GT..---.-..TG....GACA......G | ....-..GT-...CGT--.-GG.... |
| Rotylenchulus reniformis (AY335191) | ..G...---.-..TG..A..AC.C.....G | ....G..A.ACCAG----..CG.... |
| Tylenchorhynchus annulatus (MG430283) | ..G...---.-..TG..ATGAC.C.....G | ....-..C...-...---C.GG.... |
| T. leviterminalis (AB933474) | ..G...---.-..TG..ATG AC.C.....G | .A ..-..A.-..AC.-- CA.GG..AT |
| T. zeae (J461599) | ..G...---.G-ATG..ATGAC.C.....G | .. ..C..-...-..---GCAGG.... |
Table 2.
Nematode (per 20 g soil) survey using microscopic method in chrysanthemum fields and nematode estimation (per 20 g soil) using real-time PCR method.
| Microscopic method | Real-time PCR estimation | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Samplea | Nematode species | Primer and calibration curveb | ||||||||
| NEGc | Pkumad | Pdiae | RKNf | |||||||
| Pratylenchus penetrans | P. kumamo- toensis | Paratylenchus dianthus | Meloidogyne sp. | Soil | Lysate | Lysate | Soil | Lysate | Lysate | |
| Ch01 | 151 | nd | nd | nd | 151 | 110 | nd | nd | nd | nd |
| Ch02 | nd | 7 | 10 | nd | nd | nd | 8 | 30 | 40 | nd |
| Ch03 | nd | 22 | 47 | nd | nd | nd | 42 | 219 | 240 | nd |
| Ch04 | nd | nd | 119 | nd | nd | nd | nd | 595 | 589 | nd |
| Ch05 | nd | nd | 38 | nd | nd | nd | nd | 324 | 341 | nd |
| Ch06 | nd | nd | nd | 315 | nd | nd | nd | 13 | 19 | 731 |
1 Notes: nd indicates non-detected. aCh01: 26° 22′ 12′′N 127° 50′ 21′′E, Ch02: 26° 22′ 19′′N 127° 50′ 02′′E, Ch03: 26° 22′ 15′′N 127° 50′ 00′′E, Ch04: 26° 25′ 02′′N 127° 43′ 07′′E, Ch05: 26° 24′ 52′′N 127° 43′ 05′′E, Ch06: 26° 22′ 12′′N 127° 50′ 21′′E; bSoil and lysate are based on the calibration curves and primer sets developed in each primer set; c Sato et al. (2007) (primer) and Kawanobe et al. (2015) (calibration curve) for Pratylenchus penetrans; d Koyama et al. (2016) for P. kumamotoensis; ethis study for Paratylenchus dianthus; f Toyota et al. (2008) (primer) and this study (calibration curve) for Meloidogyne sp.
Figure 1:
Results of pot experiments. A: Plant height; B: Plant dry weight of above-ground parts; and C: SPAD value (only for Ch05 and Ch12); and D: Pin-nematode densities with soil treated with imicyafos (3 kg a.i./ha), or non-treated as a control in triplicates, after 2-month growth period. Asterisks indicate significant difference from a control group by Student’s t-test (*p < 0.05 and **p < 0.01). Different alphabet letters indicate significant difference by a multiple comparison (Tukey–Kramer’s test, main effect (treatment): p < 0.05; interaction effect (treatment × soil: p > 0.05) following two-way ANOVA (alphabet letters in Figure 1D are only for reference; interaction effect (treatment × soil): p < 0.05).
Table 3.
Calibration curves to estimate nematodes in soil.
| Calibration curve | ||||
|---|---|---|---|---|
| Nematode species | Primer set | Typea | Equation | Reference |
| Pratylenchus penetrans | NEG | Soil | y = −3.1449x + 34.834 (R 2 = 0.97***) | Kawanobe et al. (2015) |
| Lysate | y = −3.3664x + 34.858 (R 2 = 0.9019***) | |||
| P. kumamotoensis | Pkuma | Lysate | y = −3.2173x + 34.178 (R² = 0.9987***) | Koyama et al. (2016) |
| Meloidogyne incognita | RKN | Lysate | y = −3.4585x + 33.798 (R² = 0.9985***) | This study |
Figure 2:
Relationship between calibration curves of soil DNA samples and nematode lysate for Paratylenchus dianthus. Solid line: a calibration curve derived from DNA samples extracted from Ch12 experiment at two-month after planting. Dotted line: a calibration curve derived from DNA samples extracted from nematode lysate. The curve was related to the DNA concentrations equivalent to the ones obtained by using DNA samples from soils. ***p < 0.001 and **p < 0.01.