Detection of Globodera pallida directly from soil sample using mt-COI region based LAMP assay

Abstract Potato cyst nematodes (PCN), Globodera rostochiensis (Golden/yellow) and G. pallida (White), are economically important and relatively specialized pest of potato (Solanum tuberosum L.). Both the species are being identified based on cyst colour after 55-60 days after planting (DAP) however, after 65 DAP, we cannot differentiate based on cyst colour as both species turns brown. Moreover, the molecular techniques available to detect the PCN at species level is laborious, time consuming and costly. Therefore, development of rapid, accurate and economically cheap technique for detection of PCN at species level from the field is important to device effective management strategies for sustainable potato production. Accordingly, in the first instance, loop-mediated isothermal amplification (LAMP) assay was developed to detect G. pallida directly from soil by using the mitochondrial (mt-COI) gene specific primer. The LAMP assay was completed within 60 min at 60 °C isothermal conditions and the primer, efficiently detects the G. pallida without any cross reaction with G. rostochiensis, Meloidogyne incognita, M. javanica, Heterodera avenae, H. carotae, and Cactodera spp. In analytical sensitivity tests, the assay was able to detect G. pallida with 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl) and the LAMP product was visualized by using SYBR Gold nucleic acid dye and the assay can be highly useful in detection of G. pallida.

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Taxonomic diversity of fungi associated with some PCN populations from Serbia

Pesticidi i fitomedicina ◽

10.2298/pif1201041o ◽

2012 ◽

Vol 27 (1) ◽

pp. 41-47

Author(s):

Violeta Oro ◽

Jelena Boskovic ◽

Slobodan Milenkovic ◽

Solveig Tosi

Keyword(s):

Biological Control ◽

Food Chain ◽

Bacterial Species ◽

Taxonomic Diversity ◽

Species Level ◽

Globodera Pallida ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Antagonistic Fungi ◽

Taxonomic Categories

Increased content of pesticides in food chain resulted in using microorganisms as agents of biological control. The potato cyst nematodes (PCN) - Globodera pallida and G. rostochiensis belong to the group of the most important parasites - the quarantine organisms. The external and internal area of a cyst harbor numerous fungal and bacterial species. The aim of this study was to identify antagonistic fungi associated with some PCN populations from Serbia. Fungal antagonists of potato cyst nematodes have not been previously investigated in our country. The diversity of PCN fungal antagonists is not reflected only at the species level but also at the level of higher taxonomic categories.

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Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

Malaria Journal ◽

10.1186/s12936-021-03717-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Claire Y. T. Wang ◽

Emma L. Ballard ◽

Zuleima Pava ◽

Louise Marquart ◽

Jane Gaydon ◽

...

Keyword(s):

Clinical Trials ◽

Plasmodium Falciparum ◽

18S Rrna ◽

Drug Efficacy ◽

18S Rrna Gene ◽

Molecular Techniques ◽

Qpcr Assay ◽

Rrna Gene ◽

Analytical Sensitivity ◽

Blood Samples

Abstract Background Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. Methods A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. Results The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. Conclusions The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

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Taxonomy, Morphological and Molecular Identification of the Potato Cyst Nematodes, Globodera pallida and G. rostochiensis

Plants ◽

10.3390/plants10010184 ◽

2021 ◽

Vol 10 (1) ◽

pp. 184

Author(s):

John Wainer ◽

Quang Dinh

Keyword(s):

Life Cycle ◽

Molecular Identification ◽

Soil Sampling ◽

Globodera Pallida ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Primary Focus

The scope of this paper is limited to the taxonomy, detection, and reliable morphological and molecular identification of the potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis. It describes the nomenclature, hosts, life cycle, pathotypes, and symptoms of the two species. It also provides detailed instructions for soil sampling and extraction of cysts from soil. The primary focus of the paper is the presentation of accurate and effective methods to identify the two principal PCN species.

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Occurrence and molecular characterization of cyst nematode species (Globodera spp.) associated with potato crops in Colombia

PLoS ONE ◽

10.1371/journal.pone.0241256 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0241256

Author(s):

Daniela Vallejo ◽

Diego A. Rojas ◽

John A. Martinez ◽

Sergio Marchant ◽

Claudia M. Holguin ◽

...

Keyword(s):

Management Strategies ◽

Nematode Species ◽

Globodera Pallida ◽

Rrna Gene ◽

Potato Cyst Nematodes ◽

28S Rrna ◽

Cyst Nematodes ◽

Nucleotide Substitutions ◽

Internal Transcribed Spacer Region ◽

The Status

Potato cyst nematodes (PCN) from the genus Globodera spp. cause major losses in the potato (Solanum tuberosum) industry worldwide. Despite their importance, at present little is known about the status of this plant pathogen in cultivated potatoes in Colombia. In this study, a total of 589 samples collected from 75 geographic localities in nine potato producing regions of Colombia (Cundinamarca, Boyacá, Antioquia, Nariño, Santander, Norte de Santander, Tolima, Caldas and Cauca) were assayed for the presence of potato cyst nematodes. Fifty-seven percent of samples tested positive for PCN. Based on phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene, all populations but one were identified as Globodera pallida. Sequences of G. pallida from Colombia formed a monophyletic group closely related to Peruvian populations, with the lowest average number of nucleotide substitutions per site (Dxy = 0.002) and net nucleotide substitutions per site (Da = 0.001), when compared to G. pallida populations from Europe, South and North America. A single sample formed a well-supported subclade along with G. rostochiensis and G. tabacum from Japan, USA and Argentina. To our knowledge this is the first comprehensive survey of Globodera populations from Colombia that includes genetic data. Our findings on species diversity and phylogenetic relationships of Globodera populations from Colombia may help elucidate the status and distribution of Globodera species, and lead to the development of accurate management strategies for the potato cyst nematodes.

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Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences

Nematology ◽

10.1163/156854102765216731 ◽

2003 ◽

Vol 5 (1) ◽

pp. 99-111 ◽

Cited By ~ 112

Author(s):

Zahra Tanha Maafi ◽

Sergei Subbotin ◽

Maurice Moens

Keyword(s):

Phylogenetic Analyses ◽

Species Level ◽

Its Rdna ◽

Heterodera Avenae ◽

Published Data ◽

Rdna Sequences ◽

First Time ◽

Group Species ◽

High Support ◽

Different Parts

Abstract RFLP and sequences of ITS-rDNA of 45 populations of cyst-forming nematodes collected from different parts of Iran were analysed and identified as representatives of 21 species. Eight enzymes generated RFLP for all studied populations. Comparison of RFLP profiles and sequences of the ITS regions with published data confirmed the presence of Heterodera avenae, H. filipjevi, H. glycines, H. hordecalis, H. latipons, H. schachtii and H. trifolii in Iran. RFLP patterns and ITS sequences for H. elachista, H. turcomanica, H. mothi and C. cacti were obtained for the first time in this study. Heterodera humuli, H. goettingiana, H. fici, H. elachista, H. turcomanica and Cactodera cacti are recorded for the first time in Iran. These results correspond with morphological and morphometric identification of the populations. Several populations were not identified at the species level and are attributed to Heterodera sp.; some of these may correspond to new species. Twenty-one new sequences from Iranian cyst-forming nematodes and 36 known sequences were used for the phylogenetic analyses. The cyst-forming nematodes formed several clades corresponding to their morphological features. Heterodera mothi and H. elachista clustered with high support with other Cyperi group species and H. turcomanica formed a moderately to highly supported clade with the Humuli group.

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Phylogenetic relationships among species of Ganoderma (Ganodermataceae, Basidiomycota) from Cameroon

Australian Journal of Botany ◽

10.1071/bt12011 ◽

2012 ◽

Vol 60 (6) ◽

pp. 526 ◽

Cited By ~ 8

Author(s):

T. R. Kinge ◽

A. M. Mih ◽

M. P. A. Coetzee

Keyword(s):

Plant Species ◽

Phylogenetic Relationships ◽

Internal Transcribed Spacer ◽

New Records ◽

Small Subunit ◽

Species Level ◽

Molecular Techniques ◽

Tropical Species ◽

The Tropics

Ganoderma is an important genus of the Polyporales in the tropics. Identification of tropical species has mainly been based on morphology, which has led to misidentification. This study aimed to elucidate the diversity and phylogenetic relationships of Ganoderma isolates from different hosts in Cameroon using morphological and molecular techniques. Analyses of basidiocarp morphology and the internal transcribed spacer and mitochondria small subunit were undertaken for 28 isolates from five plant species. The results show that the isolates belong to eight species. Three of the species were identified to species level; of these only G. ryvardense has been previously described from Cameroon while G. cupreum and G. weberianum are new records. The five remaining species did not match with any previously described species and have been designated as Ganoderma with different species affinities.

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Variation in in vitro hatch of potato cyst nematodes in response to different potato cultivars inoculated with isolates of arbuscular mycorrhizal fungi

Nematology ◽

10.1163/138855410x538144 ◽

2011 ◽

Vol 13 (6) ◽

pp. 661-672

Author(s):

Patrick Haydock ◽

Peter Jones ◽

Thomas Deliopoulos

Keyword(s):

Mycorrhizal Fungi ◽

Glomus Intraradices ◽

Arbuscular Mycorrhizal ◽

Potato Cultivars ◽

Globodera Pallida ◽

Cyst Nematodes ◽

Commercial Mixture ◽

The Individual ◽

Shoot Emergence

AbstractSix potato (Solanum tuberosum) cultivars (Home Guard, Bintje, British Queen, Maris Piper, Pentland Dell and Saturna) were inoculated with Vaminoc (a commercial mixture of three selected arbuscular mycorrhizal fungal (AMF) isolates) and with two of the individual AMF isolates present in Vaminoc, Glomus intraradices (BioRize BB-E) and Glomus mosseae (isolate BEG 12). Root length colonisation by AMF at 6 weeks after shoot emergence ranged from 49 to 54%, with Vaminoc exhibiting the highest percentage. In comparison with control plants, AMF-inoculated plants accelerated the in vitro hatch (21% mean increase) of the potato cyst nematode (PCN) species Globodera pallida (but not of G. rostochiensis) in potato root leachate collected 3 weeks after shoot emergence. The effects of mycorrhization on PCN hatch were broadly similar across the six potato cultivars. This consistency supports the potential use of AMF inoculation of potato plants as part of an integrated pest management strategy for G. pallida.

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K-ras Point Mutation Detection in Lung Cancer: Comparison of Two Approaches to Somatic Mutation Detection Using ARMS Allele-specific Amplification

Clinical Chemistry ◽

10.1093/clinchem/46.12.1929 ◽

2000 ◽

Vol 46 (12) ◽

pp. 1929-1938 ◽

Cited By ~ 53

Author(s):

Simon J Clayton ◽

Frank M Scott ◽

Jill Walker ◽

Kay Callaghan ◽

Kemal Haque ◽

...

Keyword(s):

Point Mutation ◽

Mutation Detection ◽

Lung Tumor ◽

Molecular Techniques ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Real Time Quantitative Pcr ◽

Allele Specific ◽

Specific Amplification ◽

Tumor Dna

Abstract Background: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMSTM allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. Methods: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. Results: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04–100%. Conclusions: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

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Study of European and Czech populations of potato cyst nematodes (Globodera rostochiensis and G. pallida) by RAPD method

Plant Soil and Environment ◽

10.17221/3683-pse ◽

2011 ◽

Vol 50 (No. 2) ◽

pp. 70-74 ◽

Cited By ~ 6

Author(s):

P. Sedlák ◽

M. Melounová ◽

S. Skupinová ◽

P. Vejl ◽

J. Domkářová

Keyword(s):

Animal Species ◽

Molecular Genetic ◽

Globodera Rostochiensis ◽

Globodera Pallida ◽

Genetic Identification ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Genetic Dissimilarity ◽

Solanaceous Plants ◽

Molecular Genetic Identification

Potato cyst nematodes (PCN) are the big problem in worldwide planting of potatoes and another Solanaceous plants. Identification of individual pathotypes according to international scheme is very demanding but a very important part of the phytosanitary process to control these pests. Molecular genetic identification of different plant and animal species or individuals is a very interesting way at the present time and let’s hope that it will be important in future. This report presents results of the RAPD study of nine different real PCN populations. There were five Globodera rostochiensis populations and four G. pallida populations. Pathotypes Ro2, Ro2/3, Ro4, Ro5, Pa2 and Pa3 were from European populations; population Ro1 and X were of Czech provenance. Genetics variable of these populations was described by a set of six decameric primers (OPA 07, OPG 03, OPG 05, OPG 08, OPG 10 and OPG 13). Genetic dissimilarity was by Gel Manager for Windows evaluated. Detectable differences behind all populations were found and the dendrogram was compiled. The unknown population X was sorted into group of Globodera pallida species subgroup of Pa2 consequently.

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