Serological Differentiation of the Potato-Cyst Nematodes Globodera pallida and G. rostochiensis: II. Preparation and Characterization of Species Specific Monoclonal Antibodies

AbstractComparison of potato root leachates (PRL) collected from the roots of mycorrhizal (using the mixed-isolate inoculum, Vaminoc) and non-mycorrhizal potato cv. Golden Wonder confirmed that mycorrhization caused a significant increase in hatching activity towards Globodera pallida but not G. rostochiensis. After fractionating the leachates by low pressure molecular exclusion/anion exchange liquid chromatography, several potato cyst nematode (PCN) species-specific hatching factors (HF) were found only in PRL from mycorrhizal plants. Leachate from mycorrhizal plants also contained more of several of those HF common to PRL from both mycorrhizal and non-mycorrhizal plants. Significantly more hatching factor stimulants (HS) active towards both PCN species were found in the PRL from mycorrhizal than from non-mycorrhizal plants; several HS were specific to mycorrhizal plants. No differences (quantitative or qualitative) were observed in hatching inhibitor (HI) levels between PRL from mycorrhizal and non-mycorrhizal plants. Mycorrhization of potato plants resulted in a 20% increase in carbon but a 48% decrease in nitrogen concentrations of the PRL compared to that from the non-mycorrhizal plants.

Download Full-text

Immunochemical studies on pathotypes of the potato cyst nematodes, Globodera rostochiensis and G. pallida

Parasitology ◽

10.1017/s0031182000055475 ◽

1985 ◽

Vol 90 (3) ◽

pp. 471-483 ◽

Cited By ~ 4

Author(s):

P. C. Fox ◽

H. J. Atkinson

Keyword(s):

Nematode Species ◽

Cyst Nematode ◽

Globodera Pallida ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Crossed Immunoelectrophoresis ◽

Specific Antigens ◽

And Cluster Analysis ◽

Species Specific ◽

Micro Organisms

The potential of antigenic differences for discriminating pathotypes of the potato cyst nematodes Globodera pallida and G. rostochiensis has been examined by the use of an antiserum raised to a homogenate of potato cyst nematode larvae. Species-specific antigens were detected among reference pathotypes but more variability was detected among field populations, and cluster analysis was used to interpret the precipitation are pattern produced by Laurell crossed-immunoelectrophoresis. A division into species was seen with this analysis but no definite pathotype groupings were detected. Cross-reaction with other cyst-nematode species was limited to general non-specific precipitation. The antigens were all proteinaceous, did not arise from micro-organisms within the cyst and were mainly hydrophilic with an acidic isoelectric point. Peptidase and acid phosphatase activity was detected in some precipitation arcs but this was not species specific.

Download Full-text

Molecular characterization of PCN populations from Serbia

Genetika ◽

10.2298/gensr1201189o ◽

2012 ◽

Vol 44 (1) ◽

pp. 189-200

Author(s):

Violeta Oro ◽

Vesna Oro-Radovanovic

Keyword(s):

Molecular Characterization ◽

Globodera Pallida ◽

Its2 Region ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Molecular Analyses ◽

Morphological Studies ◽

Similarities And Differences ◽

Insight Into

The morphology of potato cyst nematodes (PCN) was until recently almost the only way to identify these quarantine organisms. In the last two decades, molecular analyses contributed to faster and more efficient identification of two Globodera species (Globodera pallida and G. rostochiensis) and allowed insight into the genetic structure of those parts that were practically inaccessible by morphological studies. Molecular characterization was performed in ITS1-5.8S-ITS2 region. The comparison was made with sequences of different foreign PCN populations via NCBI GenBank database. The results of molecular studies showed similarities and differences between local and foreign PCN populations in the part of genome that was studied.

Download Full-text

Serological differentiation of the potato-cyst nematodes Globodera pallida and G. rostochiensis: partial purification of species-specific proteins

Parasitology ◽

10.1017/s003118200005784x ◽

1987 ◽

Vol 95 (2) ◽

pp. 421-428 ◽

Cited By ~ 8

Author(s):

A. Schots ◽

J. Bakker ◽

F. J. Gommers ◽

L. Bouwman-Smits ◽

E. Egberts

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Protein Composition ◽

Globodera Pallida ◽

Partial Purification ◽

Heat Denaturation ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Specific Proteins ◽

Mixed Field ◽

Species Specific

SUMMARYTwo major groups of heat-stable proteins have been purified by heat denaturation from homogenates of eggs of the potato-cyst nematodes Globodera rostochiensis and G. pallida. SDS-polyacrylamide gel electrophoresis of protein homogenates from 6 G. rostochiensis populations and 7 G. pallida populations revealed 2 bands specific for G. rostochiensis and 3 bands specific for G. pallida. Two-dimensional electrophoresis showed that the 2 bands specific for G. rostochiensis consisted of 2 polypeptides differing slightly in isoelectric point, as did one of the bands specific for G. pallida. Conventional antisera made against protein homogenates of either Globodera species showed a complete cross-reaction with the species-specific proteins. The perspectives of the differences in protein composition between G. rostochiensis and G. pallida, established in this study, for a quantitative differentiation of mixed field populations of the two Globodera species, involving monoclonal antibodies, are discussed.

Download Full-text

Integrative Morphometric and Molecular Approach to Update the Impact and Distribution of Potato Cyst Nematodes Globodera rostochiensis and Globodera pallida (Tylenchida: Heteroderidae) in Algeria

Pathogens ◽

10.3390/pathogens10020216 ◽

2021 ◽

Vol 10 (2) ◽

pp. 216

Author(s):

Aouicha Djebroune ◽

Gahdab Chakali ◽

Eugénia de Andrade ◽

Maria João Camacho ◽

Leidy Rusinque ◽

...

Keyword(s):

Nematode Species ◽

Globodera Rostochiensis ◽

Globodera Pallida ◽

Morphological Identification ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Additional Information ◽

Second Stage ◽

Species Specific ◽

The Impact

Morphological and molecular studies were conducted to characterize the specific identity of 36 isolates of potato cyst nematodes (PCNs) recovered from soil samples collected in several potato producing areas of Algeria. Morphometric data revealed that 44% of isolates contained Globodera pallida alone, 28% contained Globodera rostochiensis alone and 28% mixtures of the two species. Morphometric values of cysts and second-stage juveniles were generally distributed with slight differences in the expected ranges for both Globodera species. Inter- and intraspecific morphometric variability in nematode isolates was noted. Molecular analysis using conventional multiplex PCR with species-specific primers and TaqMan real-time PCR confirmed the morphological identification. In addition, the distribution of both potato cyst nematode species throughout various parts of the country was investigated. In the central areas, the isolates of G. pallida alone dominate, whereas isolates of G. rostochiensis alone are more frequent in the southern areas. In the eastern regions, mixed isolates are more representative. Most isolates examined in the western areas are mixtures of the two species or G. rostochiensis alone. Comparatively, G. pallida remains the most widely distributed species in its geographic range. This study confirms the presence of two PCN species, G. pallida and G. rostochiensis, in Algeria and provides additional information on their biogeographic distribution.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Variability of D2/D3 segment sequences of several populations and pathotypes of potato cyst nematodes (Globodera rostochiensis, Globodera pallida)

Plant Protection Science ◽

10.17221/1/2010-pps ◽

2010 ◽

Vol 46 (No. 4) ◽

pp. 171-180 ◽

Cited By ~ 3

Author(s):

O. Douda ◽

M. Zouhar ◽

E. Nováková ◽

J. Mazáková ◽

P. Ryšánek

Keyword(s):

Dna Analysis ◽

Pcr Amplification ◽

Globodera Rostochiensis ◽

Globodera Pallida ◽

Potato Cyst Nematodes ◽

Cyst Nematodes ◽

Routine Diagnostics ◽

Pair Comparisons ◽

Single Fragment ◽

Direct Management

Potato cyst nematodes (Globodera rostochiensis, Globodera pallida) remain a key pest in the main potato growing regions of the Czech Republic. Due to difficult direct management and presence of diverse pathotypes attacking different potato cultivars the rapid and reliable diagnostics is of crucial importance. Currently, efforts are aimed at a description of different pathotypes based on DNA analysis. The main objective of this study was to evaluate the homogeneity of sequences of D2/D3 segments of the 28S rDNA gene obtained from 3 populations of G. rostochiensis and 5 populations of G. pallida and estimate their value for diagnostic purposes. PCR amplification yielded a single fragment of the length of 700 bp approximately in all populations. The alignment score of the vast majority of all pair comparisons of G. rostochiensis and G. pallida populations varied from 98 to 99. In total 14 point deletions and 3 substitutions were observed. The variability of D2/D3 segments of potato cyst nematodes is rather low and this DNA region can be used for diagnostics on a species level because more differences were found after comparing with G. tabacum and G. millefolii sequences obtained from Gene Bank; however the applicability of D2/D3 sequences to routine diagnostics of potato cyst nematodes could be complicated by its similarity to corresponding sequences of the nematode G. artemisiae.

Download Full-text

Quantification of viable eggs of the potato cyst nematodes (Globodera spp.) using either trehalose or RNA-specific Real-Time PCR

Nematology ◽

10.1163/15685411-00002848 ◽

2014 ◽

Vol 16 (10) ◽

pp. 1219-1232 ◽

Cited By ~ 6

Author(s):

Johanna E. Beniers ◽

Thomas H. Been ◽

Odette Mendes ◽

Marga P.E. van Gent-Pelzer ◽

Theo A.J. van der Lee

Keyword(s):

Real Time ◽

Visual Inspection ◽

Membrane Integrity ◽

Globodera Pallida ◽

Potato Cyst Nematodes ◽

Original Sample ◽

Rt Pcr ◽

Cyst Nematodes ◽

Pcr Assay ◽

Field Samples

Two novel methods for the quantitative estimation of the number of viable eggs of the potato cyst nematodes (Globodera pallida and G. rostochiensis) were tested and compared with visual inspection. One is based on the loss of membrane integrity upon death and uses trehalose (a disaccharide) as a marker, the second test exploits the rapid degeneration of mRNA upon decease with a RNA-specific Real-Time Polymerase Chain Reaction (RT-PCR) assay. The viability of eggs in suspensions with different numbers of eggs was determined morphologically and was compared with both trehalose and elongation-factor-1-alpha (EF1α) mRNA measurements. The trehalose assay provided results that were close to those of the visual assessment using a microscope but only when samples contained low numbers of eggs. The lowest detectable value is 1.1 egg in the original sample and small differences in the number of viable eggs can be detected. Unfortunately, trehalose measurements reached a saturation limit at 1 cyst 10 μl−1; therefore, samples with nematode numbers above 262 eggs have to be diluted. The presence of dead cysts did not have a negative effect on the trehalose measurements. However, the use of egg suspensions instead of encysted eggs improved both the trehalose absorbance and the reliability of the measurements. When cysts were exposed to a treatment with allylisothiocyanate, the trehalose measurement detected the presence of more viable eggs than a hatching assay. The RT-PCR assay required a minimum of 30 eggs before detection occurred but can detect up to 8000 eggs in a 25 μl sample, which is an advantage when samples with high PCN infestations have to be processed. However, the confidence intervals (CI) of the RT-PCR assay are larger than those of the trehalose assay, which results in a high variation of single measurements. For example, at a density of 210 eggs in the original sample the 95% CI for the trehalose assay covers 191-228 eggs, and the 95% CI for the RT-PCR assay for G. pallida lies between 73 and 602 eggs and for G. rostochiensis between 59 and 745 eggs. Trials with field samples using both methods supported the laboratory tests. 95% of the field samples tested with the trehalose assay lie within the CI of the standard curve compared to 58% of the RT-PCR tested samples for G. pallida. The measurements of the field samples of G. pallida and G. rostochiensis populations using both methods resulted in larger numbers of viable eggs being detected compared to a hatching test. Neither of the investigated methods in their current state of development is optimal for use as a substitute for the visual inspection used in monitoring labs. The variance of the RT-PCR assay is too high if used for quantitative monitoring; the density range of eggs that can be detected using the trehalose assay is too small.

Download Full-text