Detection of the root-lesion nematode, Pratylenchus penetrans (Cobb), in a nematode community using real-time PCR (original) (raw)
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Nematology, 2012
We have developed a direct quantification method using real-time PCR for various plant-parasitic nematodes. Firstly, specific primers were designed for the root-knot nematode Meloidogyne incognita, the root-lesion nematode Pratylenchus penetrans, the potato cyst nematode Globodera rostochiensis and the soybean cyst nematode Heterodera glycines. A DNA extraction method was then developed beginning with 20 g of soil, a relatively large amount of soil but a necessary amount in the consideration of heterogeneous distribution of nematodes in soil. To estimate the density of the target nematode in soil, calibration curves for each plant-parasitic nematode were obtained by inoculating different numbers of the target nematode and then extracting DNA from the soils. The detection limit was 4-5 nematodes (20 g soil)−1. This method was applied to nematode diagnostics. Soil sampling was done when transplanting of radish and sweet potato in fields was taking place, and the density of plant-paras...
Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR
European Journal of Plant Pathology, 2013
Pratylenchus penetrans is one of the most economically damaging plant-parasitic nematodes and is found on a wide variety of crops. Correct identification and quantification of this nematode are necessary for providing advice to farmers, but are not easily obtained with the traditional way of microscopic observation. We developed a qPCR assay to detect and quantify P. penetrans in a short but accurate manner. A qPCR primer set, including two primers and a TaqMan probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimized by using the primers in a qPCR assay with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 60°C to 64°C. Based on the Ct-values, we retained the program with an annealing temperature of 63°C. The assay with the probe was very sensitive as it was able to detect a single individual of P. penetrans, even when mixed with up to 80 individuals of P. thornei. The specificity of the reaction was confirmed by the lack of amplification of DNA from 28 populations of 18 other Pratylenchus species and from plant-parasitic nematodes from nine other genera. DNA from 21 different isolates from P. penetrans was amplified. DNA extraction from 80 individuals and quantification by qPCR was repeated four times; Ct-values showed consistent results (Ct=24.4±0.4). A dilution series from DNA of P. penetrans resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R 2 =0.99; slope=−3.23; E=104 %). The tests showed a high correlation between the real numbers of nematodes and the numbers detected by the qPCR. The developed qPCR assay provides a sensitive means for the rapid detection and reliable quantification of individuals of this pest. This method does not require expertise in nematode taxonomy and morphology, and can be used as a rapid diagnostic tool in research, as well as in diagnostic labs and extension services advising farmers for pest management.
Phytopathology, 2006
The vertical distribution of Pratylenchus penetrans was monitored in four fields cropped with maize, black salsify, carrot, or potato. Soil samples were collected at 21-day intervals from May 2002 until April 2003 from five plots (2 × 5 m2) per field. Per plot, 15 cores were taken to a depth of 70 cm and split into seven segments of 10 cm each. Within the plots, segments from corresponding depths were pooled. After mixing, 200-g subsamples were taken and nematodes were extracted by zonal centrifugation from the root fraction and the mineral soil fraction separately. In most crops, the root fraction contained more than 50% of the total number of P. penetrans. Because the ratio between the numbers of nematodes in the root fraction and mineral soil fraction changes during the growing season, numbers of P. penetrans found in the mineral soil fraction cannot be used to estimate the total number in the soil. Therefore, both fractions have to be processed to obtain a reliable estimate of t...
Australasian Plant Pathology, 2003
Quantification of root lesion nematodes (Pratylenchus thornei and P. neglectus) was evaluated using three different methods; the Whitehead tray method, the mister method and the commercially available quantitative DNA assay. These methods were compared to determine the effect of soil water content, sampling method and soil storage conditions on estimates of pre-sowing densities of nematodes. The Whitehead tray method, which is reliant on extraction of live nematodes, recovered fewer nematodes from dry soil than from moist soil and fewer from soil dried before storage. By contrast, the DNA assay was not influenced by soil water content at the time of sampling or drying of the soil after sampling. A P 0 2 0 7 1 Q u a n t i f i c a t i o n o f r o o t l e s i o n n e m a t o d e s ( P r a t y l e
European Journal of Plant Pathology, 2010
The polyphagous obligate parasites Meloidogyne spp. devastate a wide range of crop plants including bananas and plantains. Their infestations impact agriculture worldwide. Therefore, an effective combating regime against this nematode species and an in-depth understanding of plant-nematode interaction are essential. Early detection of infection by visual inspection is not possible. This hampers early control strategy efforts and makes in-depth research of the early infection and plant defence unfeasible. A simple and robust in planta PCR-based nematode detection method is described here as the first crucial step. This PCR-based detection assay exploits the existence of the Internal Transcribed Spacer 1 (ITS 1) region of the ribosomal DNA (rDNA) gene family in the nematodes for early detection of nematode penetration into the roots. The results demonstrate that this detection assay is suitable to serve as a molecular screening tool for plant root diagnostic purposes.
Nematology, 2020
Summary Robust and accurate identification of root-lesion nematodes (Pratylenchus spp.) is an essential step for determining their potential threat to crop yields and, consequently, development of an efficient agronomic management strategy. It is recognised that DNA-based techniques provide rapid identification of a range of plant-parasitic nematodes including Pratylenchus spp. Efficient and repeatable DNA extraction is central to molecular methodologies. Here, six common DNA extraction protocols were compared to evaluate their efficiency to obtain quality DNA samples for Pratylenchus penetrans. Samples with five and ten individuals of P. penetrans were successfully extracted and amplified by all extraction methods tested, whereas samples with a single nematode presented challenges for DNA amplification. Among all methods tested, the DNA extraction protocol with glass beads proved to be efficient for P. penetrans and all other species tested (P. crenatus, P. neglectus and P. thornei...