Figure 1.
Chickpea roots with galls induced by Meloidogyne artiellia. Arrows indicate females on roots.
Figure 2.
The fragment of alignment of the ITS rRNA gene sequences for several root-knot nematodes with the positions of RPA primers and probes for M. artiellia detection used in this study.
Figure 3.
RPA assays using real-time fluorescent detection with examples of amplification plots. (A) Specificity assay with DNA samples of Meloidogyne spp. and crude second-stage juvenile (J2) extract of M. artiellia. Plot - 1: M. artiellia (CD4108, extract); 2: M. artiellia (CD3477); 3: M. hapla (CD3384); 4: M. hispanica (CD2435); 5: M. arenaria (CD3093); 6: M. floridensis (CD3324); 7: M. floridensis (CD2452); 8: M. arenaria (CD2444); 9: M. enterolobii (CD3991); 10: M. baetica (CD3382); 11: M. javanica (CD2355); 12: M. christiei (CD1471); 13: M. paranaensis (CD3510); 14: M. hapla (CD2461); 15, 16: negative control (no DNA). (B) Sensitivity assay with a dilution series of crude J2 extracts of M. artiellia. Plot - 1, 1’: J2 specimen per reaction tube; 2, 2’: 0.5 J2 specimen per reaction tube; 3, 3’: 0.25 J2 specimen per reaction tube; 4, 4’: 0.125 J2 specimen per reaction tube; 5, 6 - M. artiellia (CD3962), 1/20 female (dried) per reaction tube; 7–8: negative control (no DNA). The vertical line on a graph: fluorescence ∆Rn. ∆Rn is calculated at each cycle as DRn (cycle) = Rn (cycle) - Rn (baseline), where Rn = normalized reporter. The horizontal line on a graph: cycles, each cycle = 20 s.
Table 1.
Samples of cyst-forming nematodes tested in the present study.
| Species | Sample code | Origin | Plant-host | RPA experiments |
|---|---|---|---|---|
| Meloidogyne artiellia | CD3476 | Italy, Bari | Wheat | + |
| M. artiellia | CD3477 | Spain, Córdoba, Montalbán | Chickpea | + |
| M. artiellia | CD3962 | Spain, Granada, Alomartes | Chickpea | + |
| M. artiellia | CD4107 | Spain, Granada, Guadix | Wheat | + |
| M. artiellia | CD4018 | Spain, Jaen, Jamilena | Wheat | + |
| M. arenaria | CD2444 | USA, Florida | Prunus persica | − |
| M. arenaria | CD3093 | USA, Florida | P. persica | − |
| M. baetica | CD3382 | Spain | Olea europaea ssp. silvestris | − |
| M. christiei | CD1471 | USA Florida | Turkey oak | − |
| M. floridensis | CD3324 | USA, California | P. dulcis | − |
| M. floridensis | CD2452 | USA, Florida | P. persica | − |
| M. enterolobii | CD3984 | USA, Florida | Guava | − |
| M. enterolobii | CD3991 | USA, Florida | Guava | − |
| M. hapla | CD3384 | Moldova, Tiraspol | Sweet pepper | − |
| M. hapla | CD2461 | USA, Florida | Strawberry | − |
| M. haplanaria | CD3446 | USA, California | Sarracenia sp. | − |
| M. hispanica | CD2435 | USA, Florida | Urena lobata | − |
| M. hispanica | CD3416 | USA, Florida | Caladium × hortulanum | − |
| M. incognita | CD3101 | USA, California | Solanum lycopersicum | − |
| M. javanica | CD3050 | USA, Florida, | P. persica | − |
| M. javanica | CD2355 | USA, Florida | Humulus lupulus | − |
| M. naasi | CD3381 | Germany | Grasses | − |
| M. paranaensis | CD3510 | USA | Caladium sp. | − |
| Meloidogyne sp. | CD3732 | USA, Florida | Unknown plant | − |
Table 2.
RPA primers and probes used for amplification of DNA of Meloidogyne artiellia.
| Primer or probe | Sequence (5′ – 3′) |
|---|---|
| M-artiel-RPA-ITS-F1 | T GGG GAC TGT CGA TTG TCG GTG CGT TCG |
| M-artiel-RPA-ITS-R1 | AGC GCC AGC CGT TCC ACA GCG GAA AGA |
| M-artiel-RPA-ITS-R1-biotin | [Biotin] AGC GCC AGC CGT TCC ACA GCG GAA AGA |
| Probe-Mart-ITS1-nfo1 | [FAM]* CTT TAC CAT GTG ATG GTT ACA TTT CGA AAG TGC [THF] AAA ACA CAC TAT TAT T [C3-spacer] |
| Probe-Mart-ITS1-exo | A TGG TTA CAT TTC GAA AGT GCC AAA ACA CAC [FAM-dT]AT [THF] AT [BHQ1-dT] TAT TAA GTG TTG TGT T [C3-spacer] |
Figure 4.
Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. (A) Specificity assay with DNA samples of different nematodes. Strips: 1 - M. hispanica (CD2435); 2 - M. hispanica (CD3416); 3 - M. baetica (CD3382); 4 - M. javanica (CD2355); 5 - M. christiei (CD1471); 6 - M. hapla (CD2461); 7 - M. paranaensis (CD3510); 8 - Meloidogyne sp. (CD3732); 9 - M. enterolobii (CD3984); 10 - M. artiellia (CD3962). (B) Sensitivity assay with crude M. artiellia extract (CD3962). Strips: 1: M. artiellia (DNA control, CD3962); 2 – 1 J2 specimen per reaction tube; 3: 0.5 of J2 specimen per reaction tube; 4: 0.25 of J2 specimen per reaction tube; 5: 0.125 of J2 specimen per reaction tube; 6: 0.06 of J2 specimen per reaction tube; 7: 0.03 of J2 specimen per reaction tube; 8: negative control (no DNA).
Figure 5.
Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips. Strips: 1 - M. artiellia (CD4108), 1 J2 per reaction tube; 2, 3 - M. artiellia (CD4107), 1/20 female (ethanol fixed) per reaction tube; 4 - M. artiellia (CD4107), 1 J2 per reaction tube, 5, 8 - M. artiellia (CD3962) dried females with plant root tissues; 6 - M. artiellia (CD3962), 1 J2 per reaction tube; 7 - M. artiellia (CD3962), 1/20 of female (dried) per reaction tube; 9: negative control (no DNA).