Effect of soil water content, sampling method and sample storage on the quantification of root lesion nematodes (Pratylenchus spp.) by different methods (original) (raw)
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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...
Japanese Journal of Nematology, 2007
Pratylenchus penetrans causes damage to different vegetables such as radish, carrot, burdock, lettuce, butterbur, and strawberry (Chikaoka, 1983) and is considered as one of the most important nematode pests for Japanese agriculture. Because it is known that there is a significant correlation between the population density of P. penetrans in soil and degree of damage on the host (Chikaoka, 1983; Ohbayashi, 1989), reliable and rapid identification and counts of the causal agent is critically important for successful management of the nematode pest. Mandani et al. (2005) also emphasized that precise identification and knowledge about the density of nematodes in field soil are necessary to develop effective integrated pest control. However, identification of nematodes based on their morphological characteristics is sometimes difficult, especially for inexperienced nematologists. In addition, to detect a single P. penetrans individual in a sample with an abundant number of nematodes is tedious and time-con
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...
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...
Plant Disease, 2019
Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (<359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatments showed that autoclaved field soil had significantly reduced DNA amount and quality. The air- or oven-dried soil yielded a lower amount of DNA with similar purity, compared with natural field soil. PCR inhibitors were detected in soil DNA substrates targeting pBluescript II SK(+)-plasmid DNA. Al(NH4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed ...
2018
Dry bean contains high fiber, minerals, and vitamins as well as protein. Although it has an important place for human nutrition, dry bean production is gradually decreasing. Biotic and abiotic factors affect dry beans productions. One of the important biotic effects is plant-parasitic nematodes. Each year, plant-parasitic nematodes have been estimated caused damage of 80 billion dollars. Root lesion nematodes are important root parasitic nematodes especially cereal and legumes growing areas. The aim of this study was to determine root lesion nematodes in Central Kizilirmak Valley which is one of the important dry bean growing areas in Turkey. In this study, 144 soil and root samples were collected from dry bean fields of 8 cities. Soil and root samples were extracted by modified baermann funnel method (pedri dish method). The survey result showed that Pratylenchus species is widespread in all areas. Most common species was found in Pratylenchus thornei (Sher et Allen).