Abstract
Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10−4 single cysts and single females, 4−3 single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.
Highlights
IntroductionMore than 4100 plant-parasitic nematode (PPN) species have been described [1], and the estimated annual loss caused by PPNs is more than 100 billion USD, mainly attributable to cyst nematodes (Heterodera spp. and Globodera spp.) and root-knot nematodes (Meloidogyne spp.) [2]
The blast results indicated that the sequence was similar to H. schachtii genomic sequences (JAHGVF010000211.1) and H. glycines genomic DNA sequences (VAPQ01000257.1)
Schachtii DNA template, while the test line was absent when DNA from another cyst nematode or sterile H2 O were used as template. These results suggest that recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) strips can distinguish H. schachtii from H. glycines and other closely related cyst nematode species
Summary
More than 4100 plant-parasitic nematode (PPN) species have been described [1], and the estimated annual loss caused by PPNs is more than 100 billion USD, mainly attributable to cyst nematodes (Heterodera spp. and Globodera spp.) and root-knot nematodes (Meloidogyne spp.) [2]. The sugar beet cyst nematode (SBCN), Heterodera schachtii, is an important plant parasite that greatly impacts sugar beet cultivation worldwide [3]. H. schachtii and its related species are usually distinguished by cyst morphology, which requires significant 4.0/). A series of DNA-based detection methods have been developed for the identification of SBCN. Polymerase chain reaction (PCR)-random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) have been used to determine the genetic diversity of SBCN populations [5,6]
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