Fig 1.
Symptoms of beech leaf disease in a forest, Pennsylvania, USA.
Table 1.
Samples of Litylenchus crenatae and other nematodes tested in the present study.
Table 2.
Species specific primers and probe for Litylenchus crenatae used in the present study.
Fig 2.
RPA products on agarose gel amplified with LcF1spec-mod and LcR5 using TwistAmp® Basic kit.
Lanes: 1: L. crenatae (Pen); 2: Anguina agrostis (CD4194); 3: A. graminis (CD4196); 4: Mesoanguina millefolii (CD4197); 5: Ditylenchus dipsaci (CD4198); 6: negative control (no DNA); M: 100 bp DNA marker (Promega, Madison, WI. USA). Species-specific amplicon are indicated by an arrow.
Fig 3.
The fragment of the ITS rRNA gene sequence alignment of Litylenchus crenatae and some related anguinid nematodes with positions of RPA primers (yellow) and probe used in the present assay.
Fig 4.
Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips.
Specificity assay with DNA samples of different nematodes. Strips: 1: Litylenchus crenatae (Pen); 2: L. crenatae (CD4200); 3: Anguina graminis (CD4196); 4: Mesoanguina millefolii (CD4197); 5: Ditylenchoides destructor (CD4199); 6: Ditylenchus dipsaci (CD4198); 7: Anguina agropyri (CD4588); 8: negative control (no DNA).
Fig 5.
Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips.
Sensitivity assay with crude nematode extract of Litylenchus crenatae (CD4200). Strips: 1: 1 nematode per tube reaction; 2: 0,5 nematode per tube; 3: 0.25 nematode per tube; 4: 0.125 nematode per tube; 5: 0.06 nematode per tube; 6: 0.015 nematode per tube; 7: 0.007 nematode per tube; 8: 0.002 nematode per tube; 9: negative control (no DNA).
Fig 6.
Lateral flow recombinase polymerase amplification (LF-RPA) assay with examples of lateral flow strips.
Sensitivity assay with DNA extracted from samples containing beach leaves and different numbers of Litylenchus crenatae (CD4200) specimens. Strips: 1: 0.01 g beech leaf without nematodes (negative control); 2: 1 µL from 30 µL DNA extract from 0.01 g beech leaf + 1 nematode; 3: 1 µL from 30 µL DNA extract from 0.01 g beech leaf + 5 nematodes; 4: 1 µL from 30 µL DNA extract from 0.01 g beech leaf + 15 nematodes; 5: 1 µL from 30 µL DNA extract from 0.01 g beech leaf + 30 nematodes; 6: nematode crude extract (positive); 7: negative control (no DNA).