Molecular detection and quantification of slug parasitic nematodes from the soil and their hosts

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

Capture of nematodes using antiserum and lectin-coated magnetised beads

Nematology ◽

10.1163/156854101753389202 ◽

2001 ◽

Vol 3 (6) ◽

pp. 593-601 ◽

Cited By ~ 7

Author(s):

Lee Robertson ◽

Vivian Blok ◽

Qing Chen ◽

Derek Brown ◽

John Jones ◽

...

Keyword(s):

Expert Systems ◽

Magnetic Beads ◽

Nematode Species ◽

Globodera Rostochiensis ◽

Parasitic Nematodes ◽

Meloidogyne Arenaria ◽

Plant Parasitic Nematodes ◽

Second Stage ◽

Plant Parasitic ◽

Detection And Quantification

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.

Download Full-text

Molecular detection of Colletotrichum lindemuthianum in bean seed samples

Journal of Seed Science ◽

10.1590/2317-1545v40n4192761 ◽

2018 ◽

Vol 40 (4) ◽

pp. 370-377 ◽

Cited By ~ 2

Author(s):

Stélio Jorge Castro Gadaga ◽

Carolina da Silva Siqueira ◽

José da Cruz Machado

Keyword(s):

Common Bean ◽

Molecular Detection ◽

Seed Quality ◽

Colletotrichum Lindemuthianum ◽

The Other ◽

Inoculum Potential ◽

Bean Seed ◽

Detection Techniques ◽

Pcr Techniques ◽

Detection And Quantification

Abstract: Colletotrichum lindemuthianum is the causal agent of anthracnose in common bean, and infected seeds are the most typical propagation form of the disease. Thus, using common bean seeds free of C. lindemuthianum is crucial to managing this pest, as well as employing fast and accurate detection techniques to ensure high seed quality. In this study, both conventional and quantitative PCR techniques (cPCR and qPCR) were used for the detection and quantification of C. lindemuthianum in samples of common bean seeds. For that, seeds were inoculated by exposing them to fungal colonies for different periods of time, 0 h, 36 h, 72 h, 108 h and 144 h, each period corresponding to an inoculum potential. Then, they were mixed with healthy seeds, so incidences of 0.25%, 0.50%, 1%, 10%, and 100% of seeds with different inoculum potentials were obtained, in samples of 400 seeds. Both cPRC and qPCR techniques were effective in detecting the fungus. With the cPCR method, the highest sensitivity was recorded in those samples with 10% inoculated seeds with inoculum potential P36. On the other hand, with the qPCR technique, the highest sensitivity in detecting the fungus was observed in samples with 0.25% inoculated seeds with inoculum potential P36.

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