Development and Application of a Multiplex qPCR Method for the Simultaneous Detection and Quantification of Pratylenchus alleni and P. penetrans in Quebec, Canada

Root lesion nematodes are very common plant-parasitic nematodes that affect a wide range of plants. More than one species can be found simultaneously in a field, and each has a different impact on crop yield. Unfortunately, identifying them using classical morphometric criteria is very difficult and time consuming. The species Pratylenchus alleni was recently observed for the first time in Canada, associated with severe damage in a soybean field in the province of Quebec. The major species, P. penetrans, is also known to be endemic in Quebec but no data exist on its distribution in field crops. This prompted the development of a multiplex quantitative polymerase chain reaction (PCR) assay for the simultaneous detection and quantification of P. alleni and P. penetrans. The method was found to be specific and sensitive, systematically detecting a single larva in a 100-cm3 soil sample with no cross-amplification with other species, even when they outnumbered the target species. An exogenous internal positive control was included in the test to avoid false negatives due to the presence of PCR inhibitors. This assay was used to study the distribution of P. alleni and P. penetrans in 185 soybean fields in the major soybean-producing areas of Quebec during a 3-year survey. Overall, P. penetrans was found in 42% of the fields, P. alleni in 8%, and both species in 4%. The population density of P. alleni in positive fields was still very low, with only a few larvae detected. However, densities of P. penetrans were much higher: the provincial mean was 51.7 nematodes per 100 cm3 of soil (in positive samples), and 8% of the fields (15 of 185) exceeded the theoretical economic threshold. The presence of P. penetrans was also strongly correlated with soil texture, with lighter soil being the most favorable.

Download Full-text

Expression of Oryzacystatin I and II in Alfalfa Increases Resistance to the Root-Lesion Nematode

Phytopathology ◽

10.1094/phyto.2003.93.7.799 ◽

2003 ◽

Vol 93 (7) ◽

pp. 799-804 ◽

Cited By ~ 49

Author(s):

Deborah A. Samac ◽

Ann C. Smigocki

Keyword(s):

Vascular Tissue ◽

Gus Expression ◽

Parasitic Nematodes ◽

Control Line ◽

Cysteine Proteinases ◽

Root Lesion Nematode ◽

Mesophyll Cells ◽

Lesion Nematodes ◽

Lesion Nematode ◽

Root Lesion Nematodes

Digestive cysteine proteinases have been isolated from plant-parasitic nematodes as well as coleopteran and hemipteran insects. Phytocystatins, inhibitors of cysteine proteinases, are found in a number of plants where they may play a role in defense against pathogens and pests. The cDNAs of the phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II (OC-II), were expressed in alfalfa (Medicago sativa) plants under the control of the potato protease inhibitor II (PinII) promoter and the plants were evaluated for resistance to the root-lesion nematode (Pratylenchus penetrans). A PinII-β-glucuronidase (GUS) gene was introduced into alfalfa to determine the pattern of gene expression from this promoter. Constitutive GUS expression was observed in leaf and root vascular tissue, and in some plants, expression was observed in leaf mesophyll cells. Mechanical wounding of leaves increased GUS expression approximately twofold over 24 h. Inoculation with root-lesion nematodes resulted in localized GUS expression. Populations of root-lesion nematodes in alfalfa roots from one line containing the PinII::OC-I transgene and one line containing the PinII::OC-II transgene were reduced 29 and 32%, respectively, compared with a transgenic control line. These results suggest that oryzacystatins have the potential to confer increased resistance to the root-lesion nematode in alfalfa.

Download Full-text

Comparing the efficiency of six common methods for DNA extraction from root-lesion nematodes (Pratylenchus spp.)

Nematology ◽

10.1163/15685411-bja10049 ◽

2020 ◽

pp. 1-9

Author(s):

Valeria Orlando ◽

Simon G. Edwards ◽

Roy Neilson ◽

Tom Prior ◽

David Roberts ◽

...

Keyword(s):

Dna Extraction ◽

Crop Yields ◽

Low Cost ◽

Dna Amplification ◽

Extraction Methods ◽

Parasitic Nematodes ◽

Potential Threat ◽

Accurate Identification ◽

Lesion Nematodes ◽

Root Lesion Nematodes

Summary Robust and accurate identification of root-lesion nematodes (Pratylenchus spp.) is an essential step for determining their potential threat to crop yields and, consequently, development of an efficient agronomic management strategy. It is recognised that DNA-based techniques provide rapid identification of a range of plant-parasitic nematodes including Pratylenchus spp. Efficient and repeatable DNA extraction is central to molecular methodologies. Here, six common DNA extraction protocols were compared to evaluate their efficiency to obtain quality DNA samples for Pratylenchus penetrans. Samples with five and ten individuals of P. penetrans were successfully extracted and amplified by all extraction methods tested, whereas samples with a single nematode presented challenges for DNA amplification. Among all methods tested, the DNA extraction protocol with glass beads proved to be efficient for P. penetrans and all other species tested (P. crenatus, P. neglectus and P. thornei), generating high quality DNA at comparatively low cost and with a rapid sample throughput.

Download Full-text

Yield of `Superior' Potatoes following Nematode-suppressive Cover Crops

HortScience ◽

10.21273/hortsci.33.3.525a ◽

1998 ◽

Vol 33 (3) ◽

pp. 525a-525

Author(s):

Alan W. McKeown ◽

J.W. Potter

Keyword(s):

Cover Crops ◽

Management Practices ◽

Soil Management ◽

Parasitic Nematodes ◽

Plant Parasitic Nematodes ◽

Marketable Yield ◽

Lesion Nematodes ◽

Series Of Experiments ◽

Early Dying ◽

Root Lesion Nematodes

`Superior', the common early potato in Ontario, has developed early dying and low yield problems along the Lake Erie Counties. A series of experiments were planted in 1993 to 1996 to evaluate nematode-suppressive cover crops as a means of soil management, improving yield of potatoes, and reduction of plant parasitic nematodes. Sorghum NK557 as a nematode host, reported suppressive species Sordan 79, Trudan 8 sorghum-sudan hybrids, Domo and Cutlass mustard, Forge canola, and `Norlee' flax were compared to either Telone IIB or Vorlex-Cp (225 L/ha) fumigants applied with a deep shank applicator to 30 cm. Fertility and pest management practices followed Ontario recommendations. Northern root lesion nematodes, Pratylenchus pentrans Cobb, populations were monitored prior to planting potatoes, during the season, and after harvest. Fumigation resulted in the highest total yields in all 3 years and marketable yield in 1994 and 1995. There was no difference in marketable yield in 1996. Yield was similar among cover crops treatments. Suppressive crops, while useful in soil management, were not as effective as fumigants.

Download Full-text

Survey of Plant-Parasitic Nematodes in Kansas and Eastern Colorado Wheat Fields

Plant Health Progress ◽

10.1094/php-rs-14-0003 ◽

2014 ◽

Vol 15 (3) ◽

pp. 112-117 ◽

Cited By ~ 3

Author(s):

T. C. Todd ◽

J. A. Appel ◽

J. Vogel ◽

N. A. Tisserat

Keyword(s):

Great Plains ◽

Yield Loss ◽

Parasitic Nematodes ◽

Plant Parasitic Nematodes ◽

Population Densities ◽

Economic Thresholds ◽

Lesion Nematodes ◽

Maximum Root ◽

Root Lesion Nematodes ◽

Plant Parasitic

Observations on the prevalence and abundance of plant-parasitic nematodes were made from soil and root samples collected from 2,640 wheat fields in Kansas and Colorado during 2007-2010. Stunt nematodes (predominately Merlinius brevidens and Quinisulcius acutus), root-lesion nematodes (predominately Pratylenchus neglectus), and pin nematodes (Paratylenchus projectus) were the most commonly encountered taxa. Maximum soil population densities of 6,520 and 1,880 nematodes/100 cm3 soil were observed for pin and stunt nematodes, respectively, while a maximum root population density of 90,309 nematodes/g dry root was observed for root-lesion nematodes. Lower nematode densities were associated with wheat following corn, grain sorghum, or soybean for pin and stunt nematodes, and with wheat following fallow for root-lesion nematodes. Based on the results of this survey, 6% and 8% of wheat acreage in the central Great Plains are estimated to be at risk for significant (>5%) yield loss due to stunt nematodes and root-lesion nematodes, respectively. The number of fields with high population densities of both stunt and root-lesion nematodes was negligible (1%); therefore, the total wheat acreage with nematode populations above provisional economic thresholds is estimated to be ∼13%. Damage relationships with greater accuracy, precision, and relevance are necessary to establish reliable yield loss estimates for this region. 3 June 2014. 11 August 2014.

Download Full-text

Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR

Plant Disease ◽

10.1094/pdis-02-18-0310-re ◽

2018 ◽

Vol 102 (12) ◽

pp. 2487-2493 ◽

Cited By ~ 2

Author(s):

Jeremiah K.S. Dung ◽

Jeness C. Scott ◽

Qunkang Cheng ◽

Stephen C. Alderman ◽

Navneet Kaur ◽

...

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Qpcr Assay ◽

Management Approach ◽

Conidial Suspension ◽

Claviceps Purpurea ◽

Spore Trap ◽

Grass Seed ◽

Wide Range ◽

Detection And Quantification

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = –0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = –0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = –0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

Download Full-text

Identification of common root-lesion nematode (Pratylenchus thornei Sher et Allen) loci in bread wheat

Genome ◽

10.1139/g06-090 ◽

2006 ◽

Vol 49 (10) ◽

pp. 1319-1323 ◽

Cited By ~ 16

Author(s):

Halil Toktay ◽

C. Lynne McIntyre ◽

Julie M. Nicol ◽

Hakan Ozkan ◽

Halil I. Elekcioglu

Keyword(s):

Middle Eastern ◽

Wheat Yield ◽

Synthetic Hexaploid Wheat ◽

Parasitic Nematodes ◽

Resistance Locus ◽

Natural Genetic Variation ◽

Pratylenchus Thornei ◽

Lesion Nematodes ◽

Synthetic Hexaploid ◽

Root Lesion Nematodes

Plant parasitic nematodes are a major biotic cause of wheat-yield loss in temperate wheat-growing regions. A major strategy to develop resistance to root-lesion nematodes (RLNs) in wheat is to assess and then exploit their natural genetic variation. This study examines RLN (Pratylenchus thornei) resistance in 1 Middle Eastern landrace (AUS4930 7.2) and 1 synthetic hexaploid wheat, CROC_1/AE.SQUARROSA (224)//OPATA (CROC), using F2 and F9 populations generated by crossing AUS4930 7.2 and CROC with the susceptible cultivar Pastor, and inoculating these crosses with P. thornei in greenhouse trials. Wheat microsatellite markers linked to previously identified quantitative trait loci (QTLs) for resistance to P. thornei and P. neglectus were used to screen both populations. In the AUS4930 7.2 × Pastor population, resistance loci on chromosomes 1B, 2B, and 6D were detected. Similarly, in the CROC × Pastor population, 2 resistance loci, located on chromosomes 1B and 3B, were identified. Interestingly, a resistance locus located on chromosome 6D was not detected. More detailed mapping is required in these 2 populations, developed using new RLN resistance sources, to determine whether the QTLs identified on these chromosomes are the same, are allelic, or are linked to different resistance loci from those previously identified, and to determine whether these 2 sources contain other novel resistance loci.

Download Full-text

ROOT-KNOT AND LESION NEMATODES IN COFFEE SEEDLINGS PRODUCED IN THE STATE OF MINAS GERAIS, BRAZIL

Coffee Science ◽

10.25186/cs.v13i2.1412 ◽

2018 ◽

Vol 13 (2) ◽

pp. 178 ◽

Cited By ~ 1

Author(s):

Willian César Terra ◽

Júlio Carlos Pereira da Silva ◽

Vicente Paulo Campos ◽

Sônia Maria De Lima Salgado

Keyword(s):

Coffea Arabica ◽

Control Strategies ◽

Minas Gerais ◽

Parasitic Nematodes ◽

Plant Parasitic Nematodes ◽

Rotylenchulus Reniformis ◽

Lesion Nematodes ◽

Minas Gerais State ◽

Root Lesion Nematodes ◽

Plant Parasitic

Understanding the mechanisms of plant-parasitic nematodes (PPN) dispersion is vital to improve control strategies aiming to restrict dissemination of these plant parasites. In the present work, we evaluated the presence of PPN in Arabic coffee (Coffea arabica) seedlings produced in commercial nurseries in Minas Gerais, state, Brazil. A total of 2830 samples obtained from 318 coffee nurseries, in 84 counties within the South and Zona da Mata regions in Minas Gerais, Brazil and representing more than 62 million coffee seedlings, were analyzed. Meloidogyne spp. was identified in 11 samples from four counties. Pratylenchus spp. and Rotylenchulus reniformis were detected in 281 and 47 samples, respectively. According to the Regulatory Instruction N° 35 from the Ministry of Agriculture, Livestock and Food Supply (MAPA), in Brazil, coffee seedlings infected by Meloidogyne spp. are prohibited for commercialization and/or planting. However, such restrictions do not apply to other PPN. Therefore, seedlings sold in Minas Gerais may constitute sources of dissemination for root-lesion nematodes (Pratylenchus spp.) and the reniform nematode (R. reniformis).

Download Full-text

Molecular and morphological characterization of the root -lesion nematode, Pratylenchus neglectus, on corn from Henan Province of China

Helminthologia ◽

10.2478/helm-2021-0044 ◽

2021 ◽

Vol 58 (4) ◽

pp. 385-393

Author(s):

Y. H. Xia ◽

Y. K. Liu ◽

P. H. Hao ◽

H. X. Yuan ◽

K. Wang ◽

...

Keyword(s):

Control Strategies ◽

Phylogenetic Analyses ◽

Morphological Characters ◽

Henan Province ◽

Root Lesion Nematode ◽

Lesion Nematodes ◽

Wide Range ◽

Two Samples ◽

Lesion Nematode ◽

Root Lesion Nematodes

Summary Root-lesion nematodes, Pratylenchus spp., are economically important pathogens because of their detrimental and economic impact on a wide range of crops. In August 2018, two samples of both roots and rhizosphere soil were collected from a corn field in Liangyuanqu of Shangqiu city, Henan Province, China. Root-lesion nematodes were recovered from the roots and soil samples using the modified Baermann funnel extraction method. Both the morphological characters and molecular analysis of the internal transcribed spacer (ITS) and D2-D3 expansion region of 28S ribosomal RNA sequences confirmed that the root-lesion nematode population collected from corn in this study was P. neglectus. Phylogenetic analyses showed that this isolate formed a highly supported clade with other P. neglectus isolates. To the best of our knowledge, this is the first report of P. neglectus on corn in Henan Province of China. This study reports the first partial sequences of 28S D2-D3 region of P. neglectus on corn in China. Due to the great harmfulness of root-lesion nematodes to corn, care should be taken to prevent the spread of P. neglectus to other regions in China. At the same time, further study on the biological characteristics of P. neglectus is needed, which will be helpful to develop corresponding management and control strategies.

Download Full-text

Developing a real-time PCR assay for direct detection and quantification of Pratylenchus scribneri in field soil

Nematology ◽

10.1163/15685411-00003336 ◽

2020 ◽

Vol 22 (7) ◽

pp. 733-744

Author(s):

Deepika Arora ◽

Guiping Yan ◽

Richard Baidoo

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Qpcr Assay ◽

Parasitic Nematodes ◽

Field Soil ◽

Accurate Identification ◽

Soil Dna ◽

Field Soils ◽

Pratylenchus Scribneri ◽

Detection And Quantification

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.

Download Full-text

PLANT-PARASITIC NEMATODES ASSOCIATED WITH WALNUT IN THE SANANDEJ REGION OF WEST IRAN

Journal of Plant Protection Research ◽

10.2478/jppr-2013-0060 ◽

2013 ◽

Vol 53 (4) ◽

pp. 404-408 ◽

Cited By ~ 1

Author(s):

Jebraeil Bahmani ◽

Farhad Khozeini ◽

Shapour Barooti ◽

Saeed Rezaee ◽

Reza Ghaderi

Keyword(s):

Meloidogyne Incognita ◽

Parasitic Nematodes ◽

Routine Laboratory ◽

Plant Parasitic Nematodes ◽

Pratylenchus Neglectus ◽

Pratylenchus Vulnus ◽

Time Period ◽

Lesion Nematodes ◽

Root Lesion Nematodes ◽

Plant Parasitic

Abstract A survey of plant-parasitic nematodes associated with walnut was carried out in the Sanandej region, of the province of Kurdistan, in western Iran, during the 2011-2012 time period. After taking samples from fifty-four localities and then doing the routine laboratory work for processing, fixing, and mounting of the nematodes, twenty-one species of plant-parasitic nematodes were finally identified. Among the identified species: Cacopaurus pestis, Mesocriconema xenoplax, Pratylenchus vulnus and Meloidogyne incognita are likely to cause damage. Spiral (Helicotylenchus crassatus, H. digonicus, H. pseudorobustus and H. vulgaris), ring (Mesocriconema antipolitanum and M. xenoplax) and root-lesion nematodes (Pratylenchus neglectus, P. thornei, P. vulnus and P. delattrei) were the most predominant nematodes in the sampled area (frequencies in soil were 87.0, 77.8, and 72.2% and 46.3, 20.4 and 14.8% in root samples, respectively). In the present study, Pratylenchus delattrei, Trophurus lomus, Paratylenchus similis, Geocenamus stegus, Helicotylenchus crassatus, Scutellonema brachyurus and Meloidogyne incognita were reported as new species associated with walnut in Iran.

Download Full-text