Sensitive and Direct Detection of <i>Heterodera filipjevi</i> in Soil and Wheat Roots by Species-Specific SCAR-PCR Assays

Peng, Huan; Qi, Xiaoli; Peng, Deliang; Long, Haoyu; He, Xufeng; Huang, Wenkun; He, Wei

doi:10.1094/pdis-02-13-0132-re

Cited by 31 publications

(15 citation statements)

References 27 publications

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“…The direct detection of plant nematodes using DNA from infected plant tissues has been published2731323334, although those methods for preparing nucleic acids require toxic reagents and expansive instruments. In this study, direct extraction of DNA from plant root galls greatly improved the efficiency when using the FTA technology.…”

Section: Discussionmentioning

confidence: 99%

Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

Peng

Long

Huang

et al. 2017

Sci Rep

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The northern root-knot nematode (Meloidogyne hapla) is a damaging nematode that has caused serious economic losses worldwide. In the present study, a sensitive, simple and rapid method was developed for detection of M. hapla in infested plant roots by combining a Flinders Technology Associates (FTA) card with loop-mediated isothermal amplification (LAMP). The specific primers of LAMP were designed based on the distinction of internal transcribed spacer (ITS) sequences between M. hapla and other Meloidogyne spp. The LAMP assay can detect nematode genomic DNA at concentrations low to 1/200 000, which is 100 times more sensitive than conventional PCR. The LAMP was able to highly specifically distinguish M. hapla from other closely related nematode species. Furthermore, the advantages of the FTA-LAMP assay to detect M. hapla were demonstrated by assaying infected root galls that were artificially inoculated. In addition, M. hapla was successfully detected from six of forty-two field samples using FTA-LAMP technology. This study was the first to provide a simple diagnostic assay for M. hapla using the LAMP assay combined with FTA technology. In conclusion, the new FTA-LAMP assay has the potential for diagnosing infestation in the field and managing the pathogen M. hapla.

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Section: Discussionmentioning

confidence: 99%

Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

Peng

Long

Huang

et al. 2017

Sci Rep

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“…Studies with other nematode species such as cyst nematode and root-knot nematodes also found that a single nematode could be used for nematode species detection by real-time PCR assay (Madani et al 2005;Toyota et al 2008). In a sensitivity analysis for H. filipjevi detection, Peng et al (2013) detected 1/160 of a juvenile, 1/5,120 of a female, and 5 × 10 −4 of a cyst by a speciesspecific sequence-characterized amplified region PCR assay.…”

Section: Discussionmentioning

confidence: 99%

Specific Detection of the Root-Lesion Nematode Pratylenchus scribneri Using Conventional and Real-Time PCR

Huang

Yan

2017

Plant Disease

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Pratylenchus scribneri is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological methods is an arduous task requiring extensive training on nematode taxonomy and years of experience. Thus, molecular methods for P. scribneri detection and identification were developed. Conventional and real-time polymerase chain reaction (PCR) assays with new species-specific primers were used in this study, which exclusively amplified DNA of P. scribneri but not DNA from other Pratylenchus spp. or non-Pratylenchus spp. tested. Compared with conventional PCR that was able to detect an equivalent to 1/4 of the DNA of a single nematode, real-time PCR was more sensitive and could amplify an equivalent to 1/128 of the DNA of one nematode. Both conventional and real-time PCR assays successfully identified P. scribneri and distinguished it from P. penetrans and P. neglectus isolated from field samples collected from various locations in North Dakota and Minnesota. The Blast-search based on the sequence information confirmed the reliability of the PCR assays for species identification. This is the first report of P. scribneri identification using a real-time PCR assay. The developed PCR assays are suitable for use in diagnostic laboratories and detection of field infestations with this nematode species.

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“…Cereal cyst nematodes (CCNs, Heterodera spp.) are sedentary plant-parasitic nematodes that infect cereal food crops such as wheat ( Triticum aestivum) ), barley ( Hordeum vulgare ) and oat ( Avena sativa ) [ 1 , 2 ]. CCNs occur in most wheat-growing regions worldwide and cause serious yield losses globally, thereby being major pests affecting the world’s food supply [ 3 , 4 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

et al. 2016

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Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.

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Sensitive and Direct Detection of Heterodera filipjevi in Soil and Wheat Roots by Species-Specific SCAR-PCR Assays

Cited by 31 publications

References 27 publications

Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

Rapid, simple and direct detection of Meloidogyne hapla from infected root galls using loop-mediated isothermal amplification combined with FTA technology

Specific Detection of the Root-Lesion Nematode Pratylenchus scribneri Using Conventional and Real-Time PCR

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

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