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

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.

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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.

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Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of Vitis vinifera L.

OENO One ◽

10.20870/oeno-one.2013.47.4.1559 ◽

2013 ◽

Vol 47 (4) ◽

pp. 227 ◽

Cited By ~ 1

Author(s):

Gemma Marsal ◽

Núria Boronat ◽

Joan Miquel Canals ◽

Fernando Zamora ◽

Francesca Fort

Keyword(s):

Vitis Vinifera ◽

Dna Extraction ◽

Woody Plants ◽

Extraction Method ◽

Low Cost ◽

Handling Time ◽

Extraction Methods ◽

Original Method ◽

Dna Extraction Method ◽

Functional Dna

Aim: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of Vitis vinifera woody plants and propose a modification of a previously published method to reduce the time and cost of extraction.Methods and results: DNA was extracted from young and mature leaves as well as from stems and seeds using some of the most common methods of DNA isolation and two commercial kits. Another commercial kit, which does not require DNA extraction prior to PCR, was also used. Only two methods provided adequate results in all tissues. Other methods were only applicable to some tissues and some did not yield any functional DNA in any tissue. A modification of the method reported by Marsal et al. (2011) is proposed to reduce handling time and cost.Conclusion: All of the methods studied here use a surfactant to improve the extractions. For DNA extraction from recalcitrant tissues to be optimal, it is best to use a combination of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The changes made to the protocol reported by Marsal et al. (2011) enable functional DNA to be obtained from leaves in only 90 minutes and at very low cost (17 €/8 samples). However, this method cannot adequately isolate DNA from recalcitrant tissues (stems and seeds) and so, for this type of sample, we would recommend using the original method.Significance and impact of the study: Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method. This study compares not only the effectiveness of the various methods but also the handling time and cost. It also proposes a modification of the fastest and most economic DNA extraction method for leaves so that handling time and processing cost will be reduced even further.

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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.

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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.

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Direct RAPD evaluation of bacteria without conventional DNA extraction

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132004000300006 ◽

2004 ◽

Vol 47 (3) ◽

pp. 375-380 ◽

Cited By ~ 12

Author(s):

Welington Luiz Araújo ◽

Derlene Attili de Angellis ◽

João Lúcio Azevedo

Keyword(s):

Dna Extraction ◽

Bacillus Pumilus ◽

Population Analysis ◽

Random Amplified Polymorphic Dna ◽

Dna Amplification ◽

Extraction Methods ◽

Genetic Population ◽

Rapd Profile ◽

Phenol Extraction ◽

Template Dna

The present work reports successful DNA amplification of Pantoea agglomerans and Bacillus pumilus through Random Amplified Polymorphic DNA (RAPD). For this, template DNA was obtained without conventional DNA extraction. The procedure was as follows: cultures grown for 20 hours in 5 mL LB medium were centrifuged and the resulting preparation was suspended in TE buffer. After boiling, the cell suspension was diluted and 2.0 µl were used in reactions of 15 µl. The results showed no significant differences among the RAPD profile of the PCR reactions derived from the boiling and phenol extraction methods, suggesting the utilization of this method for genetic population analysis.

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

Plant Disease ◽

10.1094/pdis-08-17-1222-re ◽

2018 ◽

Vol 102 (5) ◽

pp. 970-976 ◽

Cited By ~ 3

Author(s):

Nathalie Dauphinais ◽

Myriam Vandal ◽

Annie-Ève Gagnon ◽

Guy Bélair ◽

Pierre-Yves Véronneau ◽

...

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Simultaneous Detection ◽

Positive Control ◽

Parasitic Nematodes ◽

Strongly Correlated ◽

Lesion Nematodes ◽

Wide Range ◽

Root Lesion Nematodes ◽

Single Larva ◽

Detection And Quantification

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.

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Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples

Microorganisms ◽

10.3390/microorganisms8091450 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1450

Author(s):

Nicolas Valeix ◽

Damien Costa ◽

Louise Basmaciyan ◽

Stéphane Valot ◽

Anne Vincent ◽

...

Keyword(s):

Comparative Study ◽

Real Time ◽

Dna Extraction ◽

Cryptosporidium Parvum ◽

Real Time Pcr ◽

Sample Pretreatment ◽

Dna Amplification ◽

Extraction Methods ◽

Mechanical Pretreatment ◽

Stool Samples

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

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Influence of previous crops and nematicide treatments on root lesion nematode populations and crop yields

Phytoprotection ◽

10.7202/706015ar ◽

2005 ◽

Vol 73 (1) ◽

pp. 3-11 ◽

Cited By ~ 6

Author(s):

J. Kimpinski ◽

L.M. Edwards ◽

C.E. Gallant ◽

H.W. Johnson ◽

J.A. MacLeod ◽

...

Keyword(s):

Prince Edward Island ◽

Trifolium Pratense ◽

Crop Yields ◽

Red Clover ◽

Dry Weight ◽

Phleum Pratense ◽

Root Lesion Nematode ◽

Lesion Nematodes ◽

Lesion Nematode ◽

Root Lesion Nematodes

A field study assessing the influence of the previous crop, the crop sequence, and aldicarb treatments on root lesion nematode populations and crop yields was carried out in Prince-Edward-Island, Canada. The most recent crop had the greatest impact on nematode numbers. The crop sequences had an influence in some cases on root lesion nematode populations (primarily Pratylenchus penetrans) and on crop yields. In commercial barley (Hordeum vulgare) fields, root lesion nematodes in roots were greatest when barley followed a red clover (Trifolium pratense) timothy (Phleum pratense) ley, and stunt nematodes (Tylenchorhynchus spp., primarily T. dubius, and Merlinius spp.) were more common when barley followed barley. The combined dry weight of foliage and grain was larger when barley was planted after potato (Solanumtuberosum) and smaller when barley followed barley or a red clover-timothy mixture. Under experimental field conditions, root lesion nematode populations were largest in barley roots when barley followed potato and grain yields were smallest when barley followed barley. Changes in nematode populations in potato were not associated with crop sequences. Potato tuber yields were higher in the sequences that began with wheat (Triticum aestivum) or barley than in the sequences that began with potato or soybean (Glycine max). Aldicarb reduced the numbers of root lesion nematodes in roots with concomitant yield increases in potato and soybean.

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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.

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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).

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