Academic literature on the topic 'Nematode quantification'

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The objective of this dissertation is to use modelling and statistical approaches to expand our knowledge of the immune responses against gastrointestinal nematode infections, to assess the impact of nematode infection, and to use our improved knowledge to examine novel means of selective breeding in farm animals (sheep) as a control strategy. To expand the knowledge of the host immune response against infection, Chapter 2 of this dissertation focuses on immunoglobulin A (IgA), an antibody that binds nematode molecules, and its transfer through the body from the abomasal mucus (i.e. site of infection, where it is produced) to the blood plasma, where it is typically measured. These findings have been published in Parasitology (Prada Jimenez de Cisneros et al., 2014a). The implications of low levels of infection in adult milking ewes, which are more resistant than lambs, were also studied. A relationship is generated between infection levels using parasitological data and production data. There were however limitations in the dataset, which are discussed at the end of Chapter 3. Parasite resistance in adults sheep at low levels of infection was also studied, especially since the most common parasitological marker of disease is the faecal egg count (i.e. number of nematode eggs in the animals faeces) which is subject to substantial measurement error, among other limitations. Chapter 4 analyses a dataset of adult animals with low infection levels using a zero inflated binomial model (ZINB) and extends the model by including other evidence of parasite resistance to discriminate between exposed and unexposed animals. To examine selective breeding, an individual-based data-driven immunogenetically explicit mathematical model was developed. One application of this model is to compare the efficacy of two selective breeding schemes, each based on a different marker for disease, namely faecal egg counts and plasma IgA. This work has been published in Journal of the Royal Society Interface (Prada Jimenez de Cisneros et al., 2014b). The model can be extended to create a distribution for the variation in larval intake that best fits the field data. This allows the partitioning of the variation in adult worm burden into different components. The purpose is to quantify the contribution of the immune response and larval intake to determine which of the two accounts for more of the variation in the level of infection. The model can be also extended to explore selection schemes in the two components of the immune response (i.e. namely the IgA mediated and IgE mediated immune response) and estimate animal size at the end of the grazing season.

La vigne (Vitis vinifera) est sensible à un grand nombre de maladies. Les politiques de limitation des traitements phytosanitaires font qu'aujourd'hui, les viticulteurs ne disposent d'aucun moyen de lutte contre certains bioagresseurs. C'est le cas des maladies du bois causées par des complexes fongiques nécrosant les ceps et du court noué, maladie virale transmise par des nématodes. La vigne est une espèce végétale connue pour sa production particulière de métabolites secondaires en réponse à des infections. Dans le cadre de cette thèse, nous nous sommes intéressés aux phytoanticipines, préformées dans les plantes et dont le potentiel de toxicité envers des agents pathogènes est intéressant, c’est le cas des triterpénoïdes. Les objectifs de cette thèse ont donc été dans un premier temps d'estimer la capacité de la vigne à produire de tels composés. Pour cela, la composition en triterpénoïdes de différents organes et de différents cépages a été analysée. Nous avons pu montrer que la composition générale en triterpénoïdes est caractéristique de chaque cépage et de chaque organe. Dans une deuxième partie, 9 gènes impliqués dans la synthèse de triterpènes chez la vigne ont été identifiés et leur expression a été évaluée dans différents organes, chez différents cépages et sous différentes conditions de stress biotique et abiotique. Cette étude exploratoire nous donne des pistes pour mettre en corrélation l'expression de certaines triterpène synthases avec la production différentielle de certains triterpènes à la surface des feuilles de différents cépages. Enfin, nous nous sommes intéressés aux triterpènes glycosylés, les saponines, afin d'évaluer leur potentiel dans la lutte contre certaines maladies majeures de la vigne pour lesquelles aucun traitement n'est actuellement disponible. Pour cela, l'efficacité de saponines issues de la gypsophile et du quillaja a été testée contre certains champignons associés aux maladies du bois ainsi que contre les nématodes vecteurs des virus du court noué. Nous avons pu mettre en évidence que les souches de champignons testées étaient capables de contourner la toxicité des saponines, tandis qu'un tel traitement était rapidement efficace pour lutter contre les nématodes. Afin de vérifier l'innocuité de ce traitement pour l'environnement, les doses efficaces ont été testées et n'ont pas eu d'impact significatif sur différents bioindicateurs
Vitis vinifera is susceptible to many pathogens. These past few years, treatment policies led to the withdrawal of many pesticides. Renee, no chemical treatments are available to treat some grapevine diseases such as the grapevine trunk diseases caused by fungi complexes and the grapevine fanleaf degeneration, a viral disease transmitted from grapevine to grapevine by vector nematodes. Grapevine is known for the production of secondary metabolites as a response to pathogen infections. In this work, we focused on phytoanticipins such as triterpenoids, that are found as preformed compounds and that confer a basal resistance level to plants. First, a chemical analysis was made on the triterpenoid composition of some grapevine cultivars and organs. This study revealed that the triterpenoid composition is specific to the V. vinifera cultivar and the organ. In a genomic approach, 9 candidate genes involved in the triterpene biosynthesis were identified and their expression was studied in different organs, varieties and biotic or abiotic stress conditions. This explorative study shows correlations between gene expression and differential triterpene production at the leaf surface of the different varieties. In the last part of this study, the use of glycosylated triterpenes, also called saponins, as a substitution solution to withdrawed treattnents against major grapevine diseases was tested. Therefore, the efficiency of saponins extracted from gypsophila and quillaja was tested against fungi associated to grapevine trunk diseases and some nematodes vector of the grapevine fanleaf degeneration. These tests evidenced that the fungi were able to avoid saponins toxicity, whereas such treatment was efficient to kill nematodes. In order to evaluate the effect of the treatment on the environment, the efficient doses were tested and bad no significant impact on some bioindicators

Includes Appendix 1 -- Appendix 2 -- Movie Clip 6.1
The root lesion nematode Pratylenchus thornei feeds on roots of wheat (Triticum aestivum) plants, causing significant damage to the roots at the cellular level, resulting in yield reduction. In a previous study, P. thornei resistance QTL, QRlnt.sk-6D and QRlnt.sk-2B were identified in a Sokoll/Krichauff wheat DH population. The current project was undertaken with the aim to dissect the genetic and biological basis of this resistance. To better define the genetic basis of resistance, both resistance loci were fine mapped using the Sokoll/Krichauff DH population and six newly developed RIL populations. Bulked segregation analysis with the 90K Wheat SNP array identified linked SNPs, which were subsequently converted to KASP assays for mapping in the DH and RIL populations. QRlnt.sk-6D was delimited to a 3.5 cM interval, representing 1.77 Mbp in the bread wheat cv. Chinese Spring reference genome sequence and 2.29 Mbp in the Ae. tauschii genome sequence. These intervals contained 42 and 43 gene models in the respective annotated genome sequences. QRlnt.sk-2B was delimited to 1.4 cM, corresponding 3.14 Mbp in the durum wheat cv. Svevo reference sequence and 2.19 Mbp in Chinese Spring. The interval in Chinese Spring contained 56 high confidence gene models. Intervals for both QTL contained genes with similarity to those previously reported to be involved in disease resistance, namely genes for phenylpropanoid-biosynthetic-pathway-related enzymes, NBS-LRR proteins and protein kinases. The potential roles of these candidate genes in P. thornei resistance are discussed. The KASP markers reported in this study could potentially be used for marker assisted breeding of P. thornei resistant wheat cultivars. To quantify P. thornei from wheat root, a qPCR-based assay was developed. A standard curve was produced to quantify P. thornei from wheat root samples. The standard curve was validated by estimating P. thornei from sixteen wheat lines with known levels of resistance. Overall, the assay was 2.4-fold less expensive compared to the commercial service (PreDicta B test, SARDI). The DNA extraction protocol was inexpensive as it works without using a commercial DNA extraction kit. In order to identify metabolites associated with resistance loci, the GC-MS based metabolic profiles of root exudates and root tissues from the resistant lines were compared with the susceptible lines. In root exudates, 21 metabolites were found to be associated with resistance QTL. Likewise, from root tissue, 15 metabolites were found to be associated with the resistance QTL. These metabolites were derived from diverse biochemical groups, including amino acids and amines, organic acids, sugars, sugar alcohols and sugar phosphates. The possible roles of these resistance compounds in P. thornei resistance is largely unknown. However, their nematotoxic properties against other plant parasitic nematodes were discussed. In response to P. thornei infection, the histological and histochemical responses of wheat roots were investigated. The use of the fluorescent dye PKH26 (for P. thornei labelling) and confocal microscopy enabled visualisation of live P. thornei both out and inside wheat root tissue. In response to P. thornei infection, secondary cell wall thickening (deposition of cellulose, callose, lignin and suberin) was observed in the P. thornei resistant cultivar, Sokoll. Secondary cell wall thickening might result in physical reinforcement of the cell wall restricting P. thornei migration in the resistant root tissues.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2020

Au Canada, les pertes de rendement en agriculture attribuées aux nématodes sont généralement associées aux nématodes des lésions du genre Pratylenchus. En 2011, la découverte d’une nouvelle espèce exotique au Canada et qualifiée de rare dans le Nord-Est américain, soit Pratylenchus alleni Ferris, a soulevé de nouvelles inquiétudes. Afin de déterminer si cette espèce représente une menace pour les productions agricoles du Québec, mon projet de maîtrise visait à recueillir des informations sur sa virulence. Dans un premier temps, le spectre d’hôtes de P. alleni a été étudié et les résultats ont montré que ce nématode se développe très bien sur la pomme de terre, mais non sur la luzerne et le trèfle rouge. Ensuite, la reproduction de P. alleni dans un contexte de changements climatiques a été étudiée. L’augmentation prévue des températures et CO2 devrait favoriser le développement de P. alleni puisqu’il possède un meilleur taux de reproduction sur le soya lorsque soumis à un régime de températures de 17/28 ˚C et à une concentration en CO2 de 1200 ppm comparativement à 12/23 ˚C (400 ppm) et 15/26 ˚C (800 ppm). Dans cette même étude, une réduction de 19 à 58 % du poids sec racinaire des plants de soya inoculés avec P. alleni a été observée comparativement aux plants témoins. De plus, une méthode moléculaire de détection et de quantification simultanée de P. alleni et P. penetrans, l’espèce de Pratylenchus la plus répandu dans l’Est canadien, par qPCR a également été développée. Pour chacune des deux espèces, une sonde TaqMan associée avec le fluorophore CY5 pour P. alleni et FAM pour P. penetrans ciblant la région D2/D3 de la grande sous-unité ribosomale (28S) ont été développées et celles-ci se sont avérées spécifiques à chaque espèce. Ces résultats amènent de nouvelles connaissances sur ce ravageur et mettent en lumière sa pathogénicité.
In Canada, yield losses attributed to nematodes are generally associated with root-lesion nematodes from the genus Pratylenchus. In 2011, a new exotic species was detected in Canada and identified as Pratylenchus alleni Ferris. Pratylenchus alleni is rare in the Northeastern U.S. and its discovery has raised new concerns. To determine whether this species is a threat to agricultural production in Québec, my project aims to collect information about its pathogenicity. First, the host range of P. alleni was studied and the results showed that the nematode was developing well on potato, but poorly performed on alfalfa and red clover. The reproduction of P. alleni has also been studied in a context of climate change. The results showed that anticipated temperature and CO2 increases should favor P. alleni since it has a better reproduction rate on soybeans subjected to a night/day temperature regime of 17/28°C and a CO2 concentration of 1200 ppm compared to 12/23˚C (400 ppm) and 15/26°C (800 ppm) regimes. In the same study, a reduction of 19 to 58 % of roots dry weight of soybeans inoculated with P. alleni was observed compared to control plants. A simultaneous molecular detection and quantification method by qPCR of P. alleni and P. penetrans, the most widespread Pratylenchus species in Eastern Canada, was also developed. For each species, a TaqMan probe associated with the CY5 fluorophore for P. alleni and FAM for P. penetrans targeting the D2/D3 expansion segments of the large ribosomal subunit (28S) were developed and proved to be specific to each species. These results bring new insights into this new pest and highlight its pathogenicity.

Hyperspectral imaging technology is able to provide useful information about the interaction between electromagnetic radiation and matter. This information makes possible chemical characterization of materials in a non-invasive manner. For this reason, the technology has been of great interest for the food industry in recent decades. In this book chapter, we provide a survey of the current status of the use of hyperspectral technology for seafood evaluation. First, we provide a brief description of the optical properties of tissue and an introduction to the instrumentation used to capture these images. Then, we survey the main applications of hyperspectral imaging in the seafood industry, including the quantification of different chemical components, the estimation of freshness, the quality assessment of seafood products, and the detection of nematodes, among others. Finally, we provide a discussion about the current state of the art and the upcoming challenges for the application of this technology in the seafood industry.