Journal articles: 'Nematode quantification'

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Sayler, Ronald J., Courtney Walker, Fiona Goggin, Paula Agudelo, and Terrence Kirkpatrick. "Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes." Plant Disease 96, no. 12 (December 2012): 1757–62. http://dx.doi.org/10.1094/pdis-12-11-1033-re.

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Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.

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Min, Yu Yu, Koki Toyota, and Erika Sato. "A novel nematode diagnostic method using the direct quantification of major plant-parasitic nematodes in soil by real-time PCR." Nematology 14, no. 3 (2012): 265–76. http://dx.doi.org/10.1163/156854111x601678.

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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-parasitic nematodes was measured using both the Baermann funnel extraction method and real-time PCR methods. In some soils, P. penetrans and M. incognita were not found with the Baermann method but were detected with the real-time PCR method. At harvest, damage to crops was evaluated and its relationship with initial densities was investigated. The real-time PCR method more precisely predicted damage to radish and sweet potato by nematodes and was considered to be a powerful tool in the diagnosis of nematode diseases.

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Katsuta, Akane, Koki Toyota, Yu Yu Min, and The Thiri Maung. "Development of real-time PCR primers for the quantification of Meloidogyne graminicola, Hirschmanniella oryzae and Heterodera cajani, pests of the major crops in Myanmar." Nematology 18, no. 3 (2016): 257–63. http://dx.doi.org/10.1163/15685411-00002957.

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The rice root-knot nematode,Meloidogyne graminicola, and the rice root nematode,Hirschmanniella oryzae, are soil-borne pests causing damage to rice, and the pigeon pea cyst nematode,Heterodera cajani, is a pest to beans and sesame. Real-time PCR primers were designed for quantification. Relationships between the threshold cycle (Ct:y) values and number (no.) of nematodes inoculated () were:M. graminicola: ;H. oryzae: ; andH. cajani: .Meloidogyne graminicolaandH. oryzaewere detected in 25 and 38 out of 50 soils, collected from different fields in the lowland and central area of Myanmar, and their densities ranged from 1.0 to 4779 and from 0.4 to 787 (20 g soil)−1, respectively, whileH. cajaniwas detected only in two fields (3 and 268 (20 g soil)−1). The DNA-based method enables rapid and reliable quantification of the nematodes in soil.

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Huang, Danqiong, Guiping Yan, Neil Gudmestad, and Andrea Skantar. "Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan Probe and SYBR Green real-time PCR assays." Nematology 19, no. 8 (2017): 987–1001. http://dx.doi.org/10.1163/15685411-00003101.

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The ectoparasitic stubby root nematode,Paratrichodorus allius, transmits tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. A diagnostic method for direct quantification ofP. alliusfrom soil DNA using TaqMan probe and SYBR Green real-time PCR assays was developed to assist the potato industry in management of this important vector. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA ofP. alliuswas demonstrated byin silicoanalysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculatedP. alliusindividuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil. Moreover, the numbers ofP. alliusdetermined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented positive correlation relationships (). Although the quantification using TaqMan probe overestimated the target nematodes compared to using SYBR Green in eight out of ten field soil samples, results of the two methods correlated well (). This is the first report ofP. alliusquantification from soil DNA extracts using real-time PCR, providing a rapid and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification.

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Frankenberg, Andrea, Andreas Paffrath, Johannes Hallmann, and Harald Schmidt. "Occurrence and importance of plant-parasitic nematodes in organic farming in Germany." Nematology 9, no. 6 (2007): 869–79. http://dx.doi.org/10.1163/156854107782331261.

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AbstractIn an attempt to evaluate the occurrence and economic importance of plant-parasitic nematodes in organic farming in Germany, a survey was conducted with the main emphasis on vegetable and cereal production systems. For vegetables, the survey included quantification and identification of plant-parasitic nematodes in soil samples and a questionnaire for growers querying production factors and damage levels. For cereals, the survey focused on quantification and identification of plant-parasitic nematodes in soil and plant samples. Overall, Pratylenchus and Tylenchorhynchus were the most prominent nematode genera under both production systems with an incidence of over 90% of the samples. Meloidogyne was detected in 51% of the samples in both systems. Other nematode genera showed differences between the two production systems. In production systems with a high frequency of vegetables, Paratylenchus was detected in 56% of the samples and Heterodera in 15%, whereas in rotations with a high cropping frequency of cereals, incidences of plant-parasitic nematodes were 56% for Heterodera, 47% for Trichodorus and 45% for Paratylenchus. Yield losses could exceed 50% on carrots, onions and cereals and were most pronounced on sandy soils. In many cases, nematode problems started 5 to 10 years after conversion to organic farming. The survey indicated that plant-parasitic nematodes are widely spread in organic farming in Germany and can cause severe damage which may result in complete loss of the crop.

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Arora, Deepika, Guiping Yan, and Richard Baidoo. "Developing a real-time PCR assay for direct detection and quantification of Pratylenchus scribneri in field soil." Nematology 22, no. 7 (July 24, 2020): 733–44. http://dx.doi.org/10.1163/15685411-00003336.

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Summary The endomigratory root-lesion nematode, Pratylenchus scribneri, is one of the major plant-parasitic nematodes infecting potato. Accurate identification and quantification of this nematode are essential to develop management strategies but microscopic observations are particularly challenging and time consuming. In this study, a SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. scribneri from field soil DNA extracts. A primer set was designed from the internal transcribed spacer (ITS) region of the P. scribneri rDNA gene. Primer specificity to the target nematode was evaluated by both in silico analysis and qPCR and no detection or non-specific amplification was observed for other non-target nematode species/communities tested in this study. Standard curves were generated using DNA extracts from autoclaved soil infested with varying nematode numbers for calibration. The curves were supported by a high correlation between the P. scribneri numbers artificially added to soil or estimated from naturally infested field soils by traditional methods, and the numbers quantified using the qPCR assay. The assay was able to detect 1 out of 128 (0.0078) equivalents of the DNA of a single nematode in 0.5 g of soil. The qPCR assay developed in this study provides a specific and sensitive detection and quantification of P. scribneri from field soils and a rapid alternative to time-consuming traditional nematode identification and enumeration.

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Huang, Danqiong, Guiping Yan, Neil C. Gudmestad, and Jonathan Whitworth. "Assessment of Factors Associated with Molecular Quantification of Stubby Root Nematode Paratrichodorus allius from Field Soil DNA." Plant Disease 103, no. 12 (December 2019): 3265–73. http://dx.doi.org/10.1094/pdis-12-18-2240-re.

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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 by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.

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Waeyenberge, Lieven, Nancy de Sutter, Nicole Viaene, and Annelies Haegeman. "New Insights Into Nematode DNA-metabarcoding as Revealed by the Characterization of Artificial and Spiked Nematode Communities." Diversity 11, no. 4 (April 2, 2019): 52. http://dx.doi.org/10.3390/d11040052.

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Nematodes are ideal biological indicators to monitor soil biodiversity and ecosystem functioning. For this reason, they have been receiving increasing attention from a broad range of scientists. The main method to characterize soil nematode communities until at least genus level is still based on microscopic observations of nematode morphology. Such an approach is time-consuming, labor-intensive, and requires specialized personnel. The first studies on the potential use of DNA-metabarcoding to characterize nematode communities showed some shortcomings: under- or overestimation of species richness caused by failure to detect a number of nematode species or caused by intraspecific sequence variants increasing the number of OTUs (operational taxonomic units) or ‘molecular’ species, and flaws in quantification. We set up experiments to optimize this metabarcoding approach. Our results provided new insights such as the drastic effect of different DNA-extraction methods on nematode species richness due to variation in lysis efficacy. Our newly designed primer set (18S rRNA gene, V4-V5 region) showed in silico an improved taxonomic coverage compared with a published primer set (18S rRNA gene, V6-V8 region). However, results of DNA-metabarcoding with the new primer set showed less taxonomic coverage, and more non-nematode reads. Thus, the new primer set might be more suitable for whole soil faunal analysis. Species-specific correction factors calculated from a mock community with equal amounts of different nematode species were applied on another mock community with different amounts of the same nematode species and on a biological sample spiked with four selected nematode species. Results showed an improved molecular quantification. In conclusion, DNA-metabarcoding of soil nematode communities is useful for monitoring shifts in nematode composition but the technique still needs further optimization to enhance its precision.

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Yan, Guiping, Richard W. Smiley, and Patricia A. Okubara. "Detection and Quantification of Pratylenchus thornei in DNA Extracted from Soil Using Real-Time PCR." Phytopathology® 102, no. 1 (January 2012): 14–22. http://dx.doi.org/10.1094/phyto-03-11-0093.

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The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.

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Gorny, Adrienne M., Xiaohong Wang, Frank S. Hay, and Sarah J. Pethybridge. "Development of a Species-Specific PCR for Detection and Quantification of Meloidogyne hapla in Soil Using the 16D10 Root-Knot Nematode Effector Gene." Plant Disease 103, no. 8 (August 2019): 1902–9. http://dx.doi.org/10.1094/pdis-09-18-1539-re.

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The Northern root-knot nematode (Meloidogyne hapla) is an important soilborne pathogen of numerous agricultural crops in temperate regions. Accurate detection and quantification is vital to supporting informed pest management decisions. However, traditional methods of manual nematode extraction and morphology-based identification are time-consuming and require highly specialized training. Molecular methods may expand the diagnostician’s toolkit beyond those methods that rely on this disappearing specialized skillset. However, molecular assays targeting the internal transcribed spacer region may lead to inaccurate results because of intraspecific variability. The Meloidogyne spp. effector gene 16D10 was assessed as a target for a SYBR Green I quantitative PCR (qPCR) assay for detection and quantification of M. hapla. M. hapla-specific qPCR primers were developed and evaluated for specificity against five M. hapla isolates and 14 other plant-parasitic nematodes. A standard curve was generated by relating the quantification cycle (Cq) to the log of M. hapla population densities artificially introduced into soil. The influence of soil inhibitors on quantitative amplification was assessed by generating a dilution series from DNA extracted from pure nematode cultures and inoculated soil. Extracts from soil produced significantly higher Cq values than those produced from pure culture extracts. The utility of the qPCR was evaluated using soil samples collected from three naturally infested potato fields, resulting in a significant positive relationship between populations estimated using qPCR and populations derived from manual counting. The qPCR developed in this study provides a useful method for detecting and quantifying M. hapla in soil and demonstrates the utility of effector genes in plant-parasitic nematode diagnostics. The ability to use effector genes as targets for qPCR and other molecular detection and quantification methods may open additional avenues of novel research and support development of improved species-level diagnostics.

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Robertson, Lee, Vivian Blok, Qing Chen, Derek Brown, John Jones, and Mark Phillips. "Capture of nematodes using antiserum and lectin-coated magnetised beads." Nematology 3, no. 6 (2001): 593–601. http://dx.doi.org/10.1163/156854101753389202.

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AbstractPlant-parasitic nematodes are small and extremely difficult to identify. Previous studies have demonstrated the potential for lectin- or antibody-assisted identification of nematodes. We present an extension of this technology, using antibody- or lectin-coated magnetic beads (Dynabeads) to recover target nematodes from mixtures of specimens. Lectins and antisera that bound specifically and reproducibly to the whole surface of second stage juveniles of Globodera rostochiensis and Meloidogyne arenaria were identified. These were then used as probes bound to Dynabeads to recover nematodes from test suspensions. While both types of probe isolated nematodes from suspension, antibody-coated beads recovered them more efficiently than beads coated with lectins. Other factors that affected the efficiency of recovery, such as the age of the nematode samples, were analysed. This study revealed that Dynabeads coated with a probe of suitable specificity could be used to extract nematodes from mixtures of species. This technology may ultimately be useful in 'non-expert systems' for routine detection and quantification of nematode species.

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Gorny, Adrienne M., Frank S. Hay, Xiaohong Wang, and Sarah J. Pethybridge. "Isolation of nematode DNA from 100 g of soil using Fe3O4 super paramagnetic nanoparticles." Nematology 20, no. 3 (2018): 271–83. http://dx.doi.org/10.1163/15685411-00003140.

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An economical method for extracting nematode DNA from 100 g of soil was developed to facilitate nematode detection and quantification, and tested using the Northern root-knot nematode,Meloidogyne hapla. The method utilised enzymatic laundry detergent lysis, Fe3O4super paramagnetic iron oxide nanoparticle (SPION) capture, and polyvinylpolypyrrolidone (PVPP) purification. Resultant DNA from this SPION capture method was approximately 100-fold less but of similar quality to DNA obtained from a standard phenol procedure and a commercial DNA extraction kit. An addition of 10 mg of nanoparticles to the extraction lysate was identified to maximise DNA yield while minimising co-capture of contaminants. The detection limit of the SPION capture method was approximately 100 nematodes (100 g soil)−1. The SPION capture method extracted nematode DNA from mineral soils but requires further optimisation for extraction from high organic matter (i.e., ‘muck’) soils. The benefits of this method compared to alternative techniques are discussed.

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A. A., Galal. "An improved protocol for quantification of root-lesion nematode infection in Barley." Pakistan Journal of Nematology 36, no. 1 (January 1, 2018): 49–58. http://dx.doi.org/10.18681/pjn.v36.i01.p49-58.

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Takada, Noboru, Sayaka Sutoh, Masuru Toyota, Yoshihisa Yamazaki, Nozomi Kitano-Yamashita, Chisato Ushida, and Kazuo Yamashita. "Methiin as a nematode attractant in Allium sativum." Canadian Journal of Chemistry 98, no. 3 (March 2020): 164–68. http://dx.doi.org/10.1139/cjc-2019-0338.

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Damage to garlic (Allium sativum L.) caused by nematodes (Ditylenchus destructor Thorne) is becoming a serious agricultural hazard, leading to a great loss in garlic production. Once the garlic bulbs are invaded, the pathogenic nematode drastically increases in number along with the rotting of bulbs. It was therefore conceived that nematode attractants are present in the bulbs. Based on this hypothesis, chemical investigations were performed to explore a nematode attractant in A. sativum bulbs, which resulted in the identification of methiin (S-methyl-l-cysteine S-oxide) as an attractant. Bioassay and quantification experiments of methiin in extracts of A. sativum bulb led to the conclusion that methiin possesses sufficient potential to attract D. destructor into A. sativum bulbs. Moreover, an activity comparing study of methiin with its analogs showed that the sulfoxide functionality is essential for attractant activity. Moreover, methiin was revealed to attract Caenorhabditis elegans. Further investigation of methiin will help to elucidate the neuronal system of D. destructor.

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Dossin, Mariana Fernanda, Nariane de Andrade, Lisiane Sobucki, Valéria Ortaça Portela, Regis Felipe Stacke, Cristiano Bellé, Rodrigo Ferraz Ramos, Maiara Figueiredo Ramires, Jansen Rodrigo Pereira Santos, and Zaida Inês Antoniolli. "Pratylenchus brachyurus Suppression by Organic Fertilizers and the Development of Soybean Plants." Research, Society and Development 11, no. 6 (May 8, 2022): e52611629301. http://dx.doi.org/10.33448/rsd-v11i6.29301.

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The root lesion nematode, Pratylenchus brachyurus (Godfrey) Filipjev & Schuurmans Stekhoven, has been caused significant losses in soybean crops in Brazil. The addition of organic fertilizers may suppress the population of these plant-parasitic nematodes. The aim of this work was to evaluate the action of cattle manure, cattle vermicompost, cattle compost, and slaughterhouse swine compost on the reduction of P. brachyurus population and the development in soybean plants (cv. NR 5909). The experiment was conducted for 90 days after P. brachyurus inoculation (DAI) in the pots, in greenhouse. At 90 DAI, shoots, roots and soil were collected from all experimental units to determine shoot dry matter, root fresh mass and nematodes quantification present in roots and soil. All the organic fertilizers promoted the increase of shoot dry matter and root fresh mass in relation to control, even under nematode infestation in soybean plants (cv. NR 5909). However, organic fertilizers did not promote a suppressive effect on P. brachyurus population in soybean plants.

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STORMO, SVEIN K., AGNAR H. SIVERTSEN, KARSTEN HEIA, HEIDI NILSEN, and EDEL ELVEVOLL. "Effects of Single Wavelength Selection for Anisakid Roundworm Larvae Detection through Multispectral Imaging." Journal of Food Protection 70, no. 8 (August 1, 2007): 1890–95. http://dx.doi.org/10.4315/0362-028x-70.8.1890.

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The occurrence of parasites in fillets of commercially important fish species affects both food quality and safety. Presently, the detection and removal of nematode parasites is done by inspection on a light table (candling) and manual trimming of the fillets. This operation is costly and time-consuming and is not effective for detecting and removing all the nematodes in the fillets. In the last decades, several alternative methods have been proposed, but these methods have failed to replace the candling method. A newly described method called imaging spectroscopy has produced promising results because the operator can record both spectral and spatial information from an object. In this work, we studied single-wavelength bands from a spectral image. Discrimination between nematodes and other objects in the fillets is dependent on the level of contrast. Quantification of the contrast in such images revealed that the level of contrast varied when different wavelengths were selected, and these variations are correlated with the absorption properties of the nematode. Visible light scatters greatly in fish muscle, generally complicating the detection of nematodes. In this study, light scattering was used in a way that reduces the background complexity in spectral images. When light scattering properties were used in a wavelength range different from the bulk of the nematode light absorption, spectral images with significantly higher contrast were produced.

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Büttner, Hannah, Sarah P. Niehs, Koen Vandelannoote, Zoltán Cseresnyés, Benjamin Dose, Ingrid Richter, Ruman Gerst, et al. "Bacterial endosymbionts protect beneficial soil fungus from nematode attack." Proceedings of the National Academy of Sciences 118, no. 37 (September 9, 2021): e2110669118. http://dx.doi.org/10.1073/pnas.2110669118.

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Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn. necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.

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Griesser, M., and F. M. W. Grundler. "Quantification of tomato expansins in nematode feeding sites of cyst and root-knot nematodes." Journal of Plant Diseases and Protection 115, no. 6 (December 2008): 263–72. http://dx.doi.org/10.1007/bf03356275.

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Ji, Hongli, Tina Kyndt, Wen He, Bartel Vanholme, and Godelieve Gheysen. "β-Aminobutyric Acid–Induced Resistance Against Root-Knot Nematodes in Rice Is Based on Increased Basal Defense." Molecular Plant-Microbe Interactions® 28, no. 5 (May 2015): 519–33. http://dx.doi.org/10.1094/mpmi-09-14-0260-r.

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The nonprotein amino acid β-aminobutyric acid (BABA) is known to protect plants against various pathogens. The mode of action is relatively diverse and specific in different plant-pathogen systems. To extend the analysis of the mode of action of BABA to plant-parasitic nematodes in monocot plants, we evaluated the effect of BABA against the root-knot nematode (RKN) Meloidogyne graminicola in rice. BABA treatment of rice plants inhibited nematode penetration and resulted in delayed nematode and giant cell development. BABA-induced resistance (BABA-IR) was still functional in mutants or transgenics defective in salicylic acid biosynthesis and response or abscisic acid (ABA) response. Pharmacological inhibition of jasmonic acid (JA) and ethylene (ET) biosynthesis indicated that BABA-IR against rice RKN likely occurs independent of JA and ET. However, histochemical and biochemical quantification in combination with quantitative real-time reverse transcription-polymerase chain reaction data suggest that BABA protects rice against RKN through the activation of basal defense mechanisms of the plant, such as reactive oxygen species accumulation, lignin formation, and callose deposition.

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Min, Yu Yu, Keita Goto, Koki Toyota, and Erika Sato. "A multiplex real-time PCR assay for the simultaneous quantification of the major plant-parasitic nematodes in Japan." Nematology 13, no. 6 (2011): 713–20. http://dx.doi.org/10.1163/138855410x543175.

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AbstractMultiplex real-time PCR assays were developed to quantify multiple species of Meloidogyne incognita, Pratylenchus penetrans, Globodera rostochiensis and Heterodera glycines in soil. The probes specific for P. penetrans and H. glycines are labelled with a fluorescence molecule, FAM, and those for M. incognita and G. rostochiensis with ROX. The primers and probes are species-specific to P. penetrans, but group-specific to the other species. DNA was extracted from suspensions containing each nematode and multiplex Cycleave® PCR assays were done for pairs of P. penetrans and M. incognita, P. penetrans and G. rostochiensis, or G. rostochiensis and H. glycines. The results revealed that the target nematode, except for H. glycines, was quantified in the presence of less than 100 times that of the other nematode (competitor), but underestimated in the presence of 1000 times the competitor. Such underestimation was solved by the use of SYBR Green I real time PCR assays targeting a single species. Multiplex PCR assay for P. penetrans and M. incognita was done using environmental DNA (eDNA) extracted from a soil naturally infested with the nematodes. Results quantified both species. Multiplex assay using eDNA may enable a sensitive and simultaneous detection of P. penetrans and M. incognita or P. penetrans and G. rostochiensis in soil although caution is needed in case the existing ratio is biased to one of the species.

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Yan, Guiping, Richard W. Smiley, Patricia A. Okubara, Andrea M. Skantar, and Catherine L. Reardon. "Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil." Plant Disease 97, no. 6 (June 2013): 757–64. http://dx.doi.org/10.1094/pdis-08-12-0729-re.

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Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R2 = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R2 = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.

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Baidoo, Richard, Guiping Yan, Seenivasan Nagachandrabose, and Andrea M. Skantar. "Developing a Real-Time PCR Assay for Direct Identification and Quantification of Pratylenchus penetrans in Soil." Plant Disease 101, no. 8 (August 2017): 1432–41. http://dx.doi.org/10.1094/pdis-01-17-0117-re.

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The root-lesion nematode Pratylenchus penetrans is a major pathogen of potato worldwide. Yield losses may be exacerbated by interaction with the fungus Verticillium dahliae in the potato early dying disease complex. Accurate identification and quantification of P. penetrans prior to planting are essential for developing effective integrated pest control measures. However, distinction between P. penetrans and other Pratylenchus spp. based on morphology is a tedious task. A SYBR Green I-based qPCR assay was developed to discriminate, identify, and quantify P. penetrans in field soil. P. penetrans-specific qPCR primers were designed from the D2-D3 region of the 28S rDNA. The specificity of the assay was evaluated using eight isolates of P. penetrans populations and 31 isolates of other nematode species. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. There was a high correlation between the P. penetrans numbers artificially added to soil or estimated from naturally infested field soils by conventional methods, and the numbers quantified using the qPCR assay. Grinding the field soil prior to DNA extraction improved P. penetrans detection from soil. The qPCR assay will not only be useful for differentiating P. penetrans from mixed populations of Pratylenchus spp., but also for efficient detection and quantification of P. penetrans from field soil. The assay requires no expertise in nematode taxonomy and morphology, and may serve as a useful diagnostic tool in research, diagnostic labs, and extension services for pest management.

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Banakar, Prakash, Amita Sharma, Catherine J. Lilley, Nagavara Prasad Gantasala, Mukesh Kumar, and Uma Rao. "Combinatorial in vitro RNAi of two neuropeptide genes and a pharyngeal gland gene on Meloidogyne incognita." Nematology 17, no. 2 (2015): 155–67. http://dx.doi.org/10.1163/15685411-00002859.

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Root-knot nematodes are the most economically important group of plant-parasitic nematodes. In the present study, functional validation using in vitro RNAi was carried out on Meloidogyne incognita with two FMRFamide-like peptide genes, flp-14 and flp-18, and a subventral pharyngeal gland specific gene, 16D10. It was found that RNAi silencing of each gene reduced the attraction of M. incognita at different time intervals both in combination and individually. Silencing of the genes reduced nematode infection by 23-30% and development as indicated by a reduction in the number of females by 26-62%. Reproduction was decreased by 27-73% and fecundity was decreased by 19-51%. In situ hybridisation revealed the expression of flp-18 in cells associated with the ventral and retro vesicular ganglia of the central nervous system. qRT-PCR supported the correlation between phenotypic effects of silencing with that of transcript quantification.

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Pun, Top Bahadur, Arjun Neupane, and Richard Koech. "Quantification of Root-Knot Nematode Infestation in Tomato Using Digital Image Analysis." Agronomy 11, no. 12 (November 23, 2021): 2372. http://dx.doi.org/10.3390/agronomy11122372.

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Tomato is the most popular vegetable globally. However, in certain conditions, the vegetable is susceptible to plant parasites such as the root-knot nematode (RKN; Meloidogyne spp.). A proper detection method is required to identify RKN and eliminate related diseases. The traditional manual quantification of RKN using a microscope is a time-consuming and laborious task. This study aims to develop a semi-automated method to discern and quantify RKN based on size using an image analysis method. The length of RKN was assessed using three novel approaches: contour arc (CA), thin structure (TS), and skeleton graph (SG) methods. These lengths were compared with the manual measurement of RKN length. The study showed that the RKN length obtained by manual measurement was highly correlated to the length based on this method, with R2 of 0.898, 0.875, and 0.898 for the CA, TS, and SG methods, respectively. These approaches were further tested to detect RKN on 517 images. The manual and automated counting comparison revealed a coefficient of determination R2 = 0.857, 0.835 and 0.828 for CA, TS, and SG methods, respectively. The one-way ANOVA test on counting revealed F-statistic = 4.440 and p-value = 0.004. The ratio of length to width was investigated further at different ranges. The optimal result was found to occur at ratio range between 10–35. The CA, TS, and SG methods attained the highest R2 of 0.965, 0.958, and 0.973, respectively. This study found that the SG method is most suitable for detecting and counting RKN. This method can be applied to detect RKN or other nematodes on severely infected crops and root vegetables, including sweet potato and ginger. The study significantly helps in quantifying pests for rapid farm management and thus minimise crop and vegetable losses.

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Mauchline, T. H., B. R. Kerry, and P. R. Hirsch. "Quantification in Soil and the Rhizosphere of the Nematophagous Fungus Verticillium chlamydosporium by Competitive PCR and Comparison with Selective Plating." Applied and Environmental Microbiology 68, no. 4 (April 2002): 1846–53. http://dx.doi.org/10.1128/aem.68.4.1846-1853.2002.

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ABSTRACT A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A γ-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in γ-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.

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Fould, S., A. L. Dieng, K. G. Davies, P. Normand, and T. Mateille. "Immunological quantification of the nematode parasitic bacterium Pasteuria penetrans in soil." FEMS Microbiology Ecology 37, no. 3 (November 2001): 187–95. http://dx.doi.org/10.1111/j.1574-6941.2001.tb00866.x.

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Fenn, Katelyn, and Mark Blaxter. "Quantification of Wolbachia bacteria in Brugia malayi through the nematode lifecycle." Molecular and Biochemical Parasitology 137, no. 2 (October 2004): 361–64. http://dx.doi.org/10.1016/j.molbiopara.2004.06.012.

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Berry, Shaun D., Mireille Fargette, Vaughan W. Spaull, Serge Morand, and Patrice Cadet. "Detection and quantification of root-knot nematode (Meloidogyne javanica), lesion nematode (Pratylenchus zeae) and dagger nematode (Xiphinema elongatum) parasites of sugarcane using real-time PCR." Molecular and Cellular Probes 22, no. 3 (June 2008): 168–76. http://dx.doi.org/10.1016/j.mcp.2008.01.003.

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Gaspard, P. G., and J. Schwartzbrod. "Helminth eggs in wastewater: quantification technique." Water Science and Technology 31, no. 5-6 (March 1, 1995): 443–46. http://dx.doi.org/10.2166/wst.1995.0656.

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In the framework of agricultural wastewater reuse, the W.H.O. has defined a parasitological quality for sewage with less than one nematode egg per liter. The purpose of this work is to define an effective method to detect helminth eggs in wastewater. Seven techniques have been applied to waste water analysis, with a comparison of their respective results, varying from 26 to 74 %. Be it in the framework of artificial contamination or on site, the best results were obtained with the diphasic technique perfected at the laboratory including a treatment with antiformine at 8 % + ethylacetate followed by a flotation with zinc sulphate at 55%. The validation in the laboratory of the methods performance on treated wastewater allowed us to show that the yield of the method is significantly independent of the egg concentration as well as giving good homogeneity of results with a concentration of 1 egg/litre.

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Onkendi, Edward, and Lucy Moleleki. "Development of a real-time PCR assay to detect and quantify the tropical root-knot nematode (Meloidogyne arenaria) in latently infected potato seed." African Phytosanitary Journal 3, no. 1 (February 28, 2022): 1–14. http://dx.doi.org/10.52855/ikem3740.

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Tropical root-knot nematodes (M. incognita, M. arenaria and M. javanica) are a serious problem to most crops, both cultivated and non-cultivated. In the recent past, accurate detection and discrimination of tropical Meloidogyne spp. has been achieved through the development of DNA-based diagnostic assays among others real-time polymerase chain reaction (qPCR) to complement results obtained using morphological and biochemical-based diagnostic methods. However, despite the developments, most of the current methods have not been optimized to quantify and characterize the tropical Meloidogyne spp. particularly in latent infections. Currently, this is a big problem in potato seed production where latently infected tubers often get distributed resulting in the distribution of root-knot nematodes to new areas. In this study, we sought to develop and validate a diagnostic method of quantifying root-knot nematode, M. arenaria in latently infected potato seed tubers. Study findings indicated that there is a high (R2 > 0.953) and significant (P < 0.05) positive correlation between target DNA concentration and Ct values, and the assays can be used to quantify as low as 1.53/100th of DNA associated with individual juvenile nematodes. Using high resolution melting curve analysis, Meloidogyne arenaria samples produced specific melting peaks (79.32 ± 0.029°C, P < 0.05) clearly distinguishing themselves from other tropical Meloidogyne species (M. incognita 79.50 ± 0.022°C and M. javanica 79.96 ± 0.046°C). The development of the high-resolution melting curve (HRMC) analysis method for quantification and characterization of M. arenaria in this study will greatly improve on accurate screening of this pathogen during latent infections to curb potato losses often associated with distribution of infected seed. Key words: Root-knot nematode, real-time PCR, high resolution melting curve analysis, South Africa, Meloidogyne spp. and potato seed

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ZINGER, FERNANDO DOMINGO, LILIAN KATIANY CASTELLO RABELLO ZINGER, WILLIAN BUCKER MOARES, GUILHERME DE RESENDE CAMARA, and FABIO RAMOS ALVES. "QUANTIFICATION OF DAMAGE AND YIELD LOSSES AND MANAGEMENT OF ROOT-KNOT NEMATODES IN CONILON COFFEE." Revista Caatinga 34, no. 2 (June 2021): 287–97. http://dx.doi.org/10.1590/1983-21252021v34n205rc.

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ABSTRACT Commercial exploitation of the coffee crop is one of the most important activities in the world’s agricultural sector. One of the main phytosanitary problems affecting the crop is the presence of Meloidogyne incognita. Several measures have been tested for the management of this pathogen, albeit with low efficiency. The objective of this work was to quantify the damage and losses and to manage M. incognita race 1 with Pochonia chlamydosporia and Trichoderma harzianum biological nematicides, comparing them to a chemical nematicide. The experiment was carried out in a commercial area naturally infested by the nematode and cultivated with the conilon coffee variety ‘Vitória INCAPER 8142’, clone V02. The treatments were tested with Carbofuran nematicide and with biological nematicides composed of P. chlamydosporia Pc-10 and T. harzianum ESALQ 1306. The biological products were applied alone or in combination. The lowest NPF (final nematode population) occurred in plants treated with P. chlamydosporia and Carbofuran. P. chlamydosporia was the most effective biological agent in the management of M. incognita. There was a reduction in production with an increase in the nematode population. The highest application costs of management methods for M. incognita race 1 per hectare were for three and two applications of Carbofuran and three applications of P. chlamydosporia + T. harzianum. The treatments with lowest application costs were one application of T. harzianum and one application of P. chlamydosporia. It was concluded that all treatments were efficient for the management of M. incognita race 1, causing a decrease in the roots and soil population.

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McNally, Jody, David Callan, Nicholas Andronicos, Nathan Bott, and Peter W. Hunt. "DNA-based methodology for the quantification of gastrointestinal nematode eggs in sheep faeces." Veterinary Parasitology 198, no. 3-4 (December 2013): 325–35. http://dx.doi.org/10.1016/j.vetpar.2013.09.014.

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MORGAN, M., J. M. BEHNKE, J. A. LUCAS, and J. F. PEBERDY. "In vitro assessment of the influence of nutrition, temperature and larval density on trapping of the infective larvae of Heligmosomoides polygyrus by Arthrobotrys oligospora, Duddingtonia flagrans and Monacrosporium megalosporum." Parasitology 115, no. 3 (September 1997): 303–10. http://dx.doi.org/10.1017/s0031182097001297.

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The influence of nutrient level, temperature and larval density on the trapping of Heligmosomoides polygyrus L3 by the nematophagous fungi Arthrobotrys oligospora, Duddingtonia flagrans and Monacrosporium megalosporum were investigated by quantification of trapped nematodes. All 3 factors were found to have a significant effect on the number of larvae trapped by A. oligospora and M. megalosporum. Decreased nutrient concentrations resulted in increased trapping for these 2 fungi, but nutrient availability was not found to have a significant effect on trapping by D. flagrans. The 3 fungi were found to have similar responses to temperature, with peak trapping occurring at or near the optimum growth temperatures. Nematode trapping was found to be density dependent for all 3 fungi, with increased percentage trapping at increased larval densities. Comparison in a single experiment of the relative importance of these factors to each fungus showed that nutrient level was the main factor influencing trapping by A. oligospora, whereas D. flagrans and M. megalosporum were more dependent on larval density.

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Atkins, S. D., I. M. Clark, D. Sosnowska, P. R. Hirsch, and B. R. Kerry. "Detection and Quantification of Plectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4788–93. http://dx.doi.org/10.1128/aem.69.8.4788-4793.2003.

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ABSTRACT Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

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Koyama, Yuki, So Pyay Thar, Chihiro Kizaki, Koki Toyota, Eiji Sawada, and Naruhito Abe. "Development of specific primers to Hirschmanniella spp. causing damage to lotus and their economic threshold level in Tokushima prefecture in Japan." Nematology 15, no. 7 (2013): 851–58. http://dx.doi.org/10.1163/15685411-00002723.

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Nematode diseases caused by Hirschmanniella spp. are becoming a major threat to lotus production in Tokushima Prefecture, a primary production area in Japan. The objectives of this study were: i) to design specific primers to Hirschmanniella spp. causing damage to lotus; ii) to evaluate the relationship between pre-plant density of Hirschmanniella spp. in soil and damage index of lotus; and iii) to establish an economic threshold level. Since two species, Hirschmanniella imamuri and H. diversa, are known as nematode pests on lotus, three real-time PCR primers were designed based on the ITS regions specific to H. imamuri (designated as imaF and imaR), H. diversa (divF and divR), and both species (HdiF and HdiR). The primers imaF and imaR did not react to DNA of nematodes extracted from lotus fields in Tokushima but the primers divF and divR did, suggesting that the damage seen in Tokushima is mainly caused by H. diversa. A calibration curve was made to evaluate the relationship between the number of Hirschmanniella spp. inoculated to soil and the threshold cycle values. The quantification of Hirschmanniella spp. in soil was conducted by real-time PCR method with the primers HdiF and HdiR as well as the Baermann method. The economic threshold level was estimated as ten individuals of Hirschmanniella spp. (100 g fresh soil)−1 with the Baermann method and 50 individuals (100 g oven-dried soil)−1 with real-time PCR.

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Subbotin, Sergei A., Mehrdad Madani, Eino Krall, Dieter Sturhan, and Maurice Moens. "Molecular Diagnostics, Taxonomy, and Phylogeny of the Stem Nematode Ditylenchus dipsaci Species Complex Based on the Sequences of the Internal Transcribed Spacer-rDNA." Phytopathology® 95, no. 11 (November 2005): 1308–15. http://dx.doi.org/10.1094/phyto-95-1308.

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The stem nematode Ditylenchus dipsaci is of great economic importance worldwide as a parasite of agricultural crops and horticultural plants. The internal transcribed spacer (ITS) of rDNA from 23 populations of the D. dipsaci complex from various host plants were amplified and sequenced. Seven previously studied populations were also included in the study. The phylogenetic analysis of the full ITS and ITS2 sequence alignments using minimum evolution, maximum parsimony, and Bayesian inference under the complex model of DNA evolution revealed trees with two main clades: (i) D. dipsaci sensu stricto with diploid chromosome numbers and comprising most isolates from agricultural, ornamental, and several wild plants, and (ii) Ditylenchus spp. with polyploid chromosome numbers, reproductively isolated from diploid populations, and subdivided into six subclades (“giant race” from Vicia faba, Ditylenchus species parasitizing various Asteraceae, and a Ditylenchus sp. from Plantago maritima). Using the energy minimization approach and comparative sequence analysis, it has been found that the secondary structure of ditylenchid ITS2 is organized in three main domains. The importance of knowledge on the RNA structure for phylogenetic analysis is discussed. Conventional polymerase chain reaction (PCR) and real-time PCR with SYBR green dye I with a species specific primer have been developed for detection and quantification of D. dipsaci sensu stricto Validation tests revealed a rather high correlation between real numbers of fourth-stage juveniles of the stem nematodes in a sample and expected numbers detected by real-time PCR. Problems of accuracy of quantification are discussed.

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Shirai, Sayo, and Koki Toyota. "Optimisation of a species-specific primer set to quantify the soybean cyst nematode, Heterodera glycines, in soil using real-time PCR." Nematology 21, no. 10 (2019): 1037–42. http://dx.doi.org/10.1163/15685411-00003273.

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Summary We previously reported a real-time PCR primer set (SCN) that is specific to the soybean cyst nematode Heterodera glycines, a major nematode pest in soybean production in Japan. However, the primer set also amplified the related species H. trifolii and H. schachtii, whose presence was recently reported in Japan. The objective of this study was to optimise a primer set to be more specific for quantification of H. glycines. The newly optimised primer set (SCNnew) amplified H. trifolii and H. schachtii at amplification efficiencies less than 1% of H. glycines. Surveys for H. glycines in different green soybean fields in Japan demonstrated that most fields judged to contain low densities of H. glycines based on the SCN primer set were not actually infested with H. glycines. The SCNnew primer set quantifies H. glycines in soil more precisely.

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Boa, M. E., S. M. Thamsborg, N. C. Kyvsgaard, and A. A. Kassuku. "Comparison of Two Techniques Used for Quantification of Ovine Gastrointestinal Nematode Larvae in Herbage." Acta Veterinaria Scandinavica 39, no. 1 (March 1998): 141–44. http://dx.doi.org/10.1186/bf03547816.

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Piza, Marina, Fabiana Alves de Almeida, Cristiano Magalhães Pariz, Ciniro Costa, and Alessandro Francisco Talamini do Amarante. "Comparison of two methods for the quantification of gastrointestinal nematode infective larvae from pasture." Semina: Ciências Agrárias 40, no. 2 (April 15, 2019): 712. http://dx.doi.org/10.5433/1679-0359.2019v40n2p712.

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The economic losses caused by gastrointestinal nematodes are one of the biggest obstacles in the small ruminants production. Understanding the population dynamics of the infective larvae (L3) in the pasture is the key point to develop control programs, and reliable results depend on the used methodology to quantify L3 numbers. The use of the sampling directly from the pasture appears as a viable option, since it is not required the use of animals with an esophageal fistula or tracer animals, decreasing the costs involved in the study. Therefore, the present project, which had as objective evaluate the efficiency of two collection methods for quantification of L3 in the pasture, utilized 64 lambs (n = 16) allocated to four integrated crop-livestock systems (treatments) with 12 paddocks each. Pasture samples were collected every nine days. The W method consists in traversing the area in the form of a W and again an inverted W, forage samples being collected every 10 steps, and the Square method, in tossing a 0.16 m2 square to four random points within the area, the forage within the square being collected after each toss. After the forage samples had been processed, the L3 were recovered and identified. Cohen’s Kappa coefficient (k) was determined. The W-transect and Random-plot methods did not differ (p ? 0.05) with respect to the number of L3 recovered from the pasture, and a positive correlation was found between them, suggesting agreement with one another, being that when the number of L3 recovered by the W-transect method increases, the same occurs in the Random-plot technique. The Random-plot method, which is already used to collect samples of forage for chemical analyses, can also be employed to estimate the pasture contamination by L3. The W-transect and Random-plot methods showed to be important in the epidemiological study of gastrointestinal nematodes in sheep. Therefore, the use of both on the same occasions and with different purposes, with one complementing the information that is not provided by the other, may be more effective in the investigation of environmental contamination by L3 of gastrointestinal nematodes.

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Wang, Hai-Hua, Can Yin, Jie Gao, Ran Tao, Chun-Yan Wang, Yong-Xia Li, Lan-Ping Guo, Zhen Wang, and Chang-Keun Sung. "Development of a Real-Time TaqMan PCR Method for Absolute Quantification of the Biocontrol Agent Esteya vermicola." Plant Disease 104, no. 6 (June 2020): 1694–700. http://dx.doi.org/10.1094/pdis-10-19-2076-re.

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Esteya vermicola has been used as an effective biocontrol agent for the management of the pinewood nematode, Bursaphelenchus xylophilus. Tools for monitoring the colonization and parasitism patterns of E. vermicola are required for the development of highly effective biocontrol strategies. Because the TaqMan PCR technique is effective for quantification of species in environmental samples, a real-time PCR-based methodology was developed for absolute quantification of E. vermicola via internal standard addition and extrapolation of DNA quantity to hyphal length. Primers and a probe for the 28S ribosomal RNA gene of E. vermicola were designed, and nested TaqMan real-time PCR-based quantification was performed. In addition, internal standard-based yield measurement was correlated to the absolute quantity of target genomic DNA. Moreover, an extrapolation curve obtained by optical microscopy and image analysis of the mycelia was constructed for the measurement of fungal hyphal length. The absolute quantification method developed in the present study provides a sensitive and accurate technique to quantify fungal density in either wood or other substrate samples and can be used as an effective tool for future studies of biocontrol agents.

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CHEN, BAOZHEN, ALEX DEUTMEYER, JOHN CARR, ALAN P. ROBERTSON, RICHARD J. MARTIN, and SANTOSH PANDEY. "Microfluidic bioassay to characterize parasitic nematode phenotype and anthelmintic resistance." Parasitology 138, no. 1 (July 21, 2010): 80–88. http://dx.doi.org/10.1017/s0031182010001010.

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SUMMARYWith increasing resistance to anti-parasitic drugs, it has become more important to detect and recognize phenotypes of resistant isolates. Molecular methods of detecting resistant isolates are limited at present. Here, we introduce a microfluidic bioassay to measure phenotype using parameters of nematode locomotion. We illustrate the technique on larvae of an animal parasite Oesophagostomum dentatum. Parameters of sinusoidal motion such as propagation velocity, wavelength, wave amplitude, and oscillation frequency depended on the levamisole-sensitivity of the isolate of parasitic nematode. The levamisole-sensitive isolate (SENS) had a mean wave amplitude of 135 μm, which was larger than 123 μm of the levamisole-resistant isolate (LEVR). SENS had a mean wavelength of 373 μm, which was less than 393 μm of LEVR. The mean propagation velocity of SENS, 149 μm s−1, was similar to LEVR, 143 μm s−1. The propagation velocity of the isolates was inhibited by levamisole in a concentration-dependent manner above 0·5 μm. The EC50 for SENS was 3 μm and the EC50 for LEVR was 10 μm. This microfluidic technology advances present-day nematode migration assays and provides a better quantification and increased drug sensitivity. It is anticipated that the bioassay will facilitate study of resistance to other anthelmintic drugs that affect locomotion.

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HARVEY, S. C., S. PATERSON, and M. E. VINEY. "Heterogeneity in the distribution of Strongyloides ratti infective stages among the faecal pellets of rats." Parasitology 119, no. 2 (August 1999): 227–35. http://dx.doi.org/10.1017/s0031182099004588.

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The distribution of helminth parasites within their host population is usually overdispersed and can be described by the negative binomial distribution. The causes of this overdispersion are poorly understood, but heterogeneity in the distribution of infective stages within the environment has been implicated as a possible factor. Here we describe the distribution of infective stages of the rat intestinal nematode parasite Strongyloides ratti among the faecal pellets of its host. The distribution of infective stages between faecal pellets is overdispersed and well described by the negative binomial distribution. This overdispersion increases during the course of infection and occurs over a range of infection intensities. Overdispersion of nematode infective stages among faecal pellets may result in increased spatial heterogeneity of the infective stages in the environment and thus may contribute to the generation of overdispersion of adult parasitic stages. In addition, these findings raise important issues regarding the accurate quantification of helminth egg counts from faecal samples.

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Kawanobe, Masanori, Naoko Miyamaru, Koichi Yoshida, Takeshi Kawanaka, and Koki Toyota. "Quantification of lesion nematode (Pratylenchus zeae), stunt nematode (Tylenchorhynchus leviterminalis), spiral nematode (Helicotylenchus dihystera), and lance nematode (Hoplolaimus columbus), parasites of sugarcane in Kitadaito, Okinawa, Japan, using real-time PCR." Nematological Research (Japanese Journal of Nematology) 45, no. 1 (2015): 35–44. http://dx.doi.org/10.3725/jjn.45.35.

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Rabello, Lilian Katiany C., Angelo Oliveira Gongalves, Tatiane Paulino da Cruz, Fernando Domingo Zinger, Waldir Cintra de Jesus Júnior, Lilian Lagen Rodrigues, Antonio Fernando de Souza, Willian Bucker Moraes, and Fábio Ramos Alves. "Quantification of damage and yield losses caused by Root-knot nematode in lettuce in Brazil." Idesia (Arica) 39, no. 2 (June 2021): 121–30. http://dx.doi.org/10.4067/s0718-34292021000200121.

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Persson, Yvonne, and Erland BÃ¥Ã¥th. "Quantification of mycoparasitism by the nematode-trapping fungusArthrobotrys oligosporaonRhizoctonia solaniand the influence of nutrient levels." FEMS Microbiology Letters 101, no. 1 (June 1992): 11–16. http://dx.doi.org/10.1111/j.1574-6968.1992.tb05756.x.

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MacMillan, Keith, Vivian Blok, Iain Young, John Crawford, and Michael J. Wilson. "Quantification of the slug parasitic nematode Phasmarhabditis hermaphrodita from soil samples using real time qPCR." International Journal for Parasitology 36, no. 14 (December 2006): 1453–61. http://dx.doi.org/10.1016/j.ijpara.2006.08.005.

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Buonicontro, Dalila Sêni, David Mark Roberts, Claudio Marcelo Gonçalves Oliveira, Vivian Carol Blok, Roy Neilson, and Rosângela D’arc De Lima Oliveira. "A Rapid Diagnostic for Detection of Aphelenchoides besseyi and A. fujianensis Based on Real-Time PCR." Plant Disease 102, no. 3 (March 2018): 519–26. http://dx.doi.org/10.1094/pdis-08-17-1160-re.

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Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.

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Kawanobe, Masanori, and Koki Toyota. "Application of a simple and high-throughput DNA extraction method to real-time PCR quantification of target plant-parasitic nematodes in nematode communities." Nematological Research (Japanese Journal of Nematology) 48, no. 1 (July 25, 2018): 1–10. http://dx.doi.org/10.3725/jjn.48.1.

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Grego, M., M. Stachowitsch, M. De Troch, and B. Riedel. "CellTracker Green labelling vs. rose bengal staining: CTG wins by points in distinguishing living from dead anoxia-impacted copepods and nematodes." Biogeosciences 10, no. 7 (July 9, 2013): 4565–75. http://dx.doi.org/10.5194/bg-10-4565-2013.

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Abstract. Hypoxia and anoxia have become a key threat to shallow coastal seas. Much is known about their impact on macrofauna, less on meiofauna. In an attempt to shed more light on the latter group, in particular from a process-oriented view, we experimentally induced short-term anoxia (1 week) in the northern Adriatic Sea (Mediterranean) and examined the two most abundant meiofauna taxa – harpacticoid copepods and nematodes. Both taxa also represent different ends of the tolerance spectrum, with copepods being the most sensitive and nematodes among the most tolerant. We compared two methods: CellTracker Green (CTG) – new labelling approach for meiofauna – with the traditional rose bengal (RB) staining method. CTG binds to active enzymes and therefore colours live organisms only. The two methods show considerable differences in the number of living and dead individuals of both meiofauna taxa. Generally, RB will stain dead but not yet decomposed copepods and nematodes equally as it does live ones. Specifically, RB significantly overestimated the number of living copepods in all sediment layers in anoxic samples, but not in any normoxic samples. In contrast, for nematodes, the methods did not show such a clear difference between anoxia and normoxia. RB overestimated the number of living nematodes in the top sediment layer of normoxic samples, which implies an overestimation of the overall live nematofauna. For monitoring and biodiversity studies, the RB method might be sufficient, but for more precise quantification of community degradation, especially after an oxygen depletion event, CTG labelling is a better tool. Moreover, it clearly highlights the surviving species within the copepod or nematode community. As already accepted for foraminiferal research, we demonstrate that the CTG labelling is also valid for other meiofauna groups.

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Bell, Amy, Jody McNally, Daniel V. Smith, Ashfaqur Rahman, Peter Hunt, Andrew C. Kotze, Sonja Dominik, and Aaron Ingham. "Quantification of differences in resistance to gastrointestinal nematode infections in sheep using a multivariate blood parameter." Veterinary Parasitology 270 (June 2019): 31–39. http://dx.doi.org/10.1016/j.vetpar.2019.05.007.

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Journal articles on the topic 'Nematode quantification'

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