scholarly journals Multiplex Real-Time PCR Assays for the Identification of the Potato Cyst and Tobacco Cyst Nematodes

Plant Disease ◽  
2010 ◽  
Vol 94 (8) ◽  
pp. 959-965 ◽  
Author(s):  
Mark K. Nakhla ◽  
Kristina J. Owens ◽  
Wenbin Li ◽  
Gang Wei ◽  
Andrea M. Skantar ◽  
...  
Keyword(s):  
Real Time ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
Cyst Nematodes ◽  
Detection And Identification ◽  
Polymerase Chain ◽  
Probe Set ◽  
Internal Transcribed Spacer Rdna ◽  
Quantitative Pcr Assay

TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCNs) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time polymerase chain reaction (PCR). One tube contained a primer-probe set specific for G. pallida (pale potato cyst nematode) multiplexed with another primer-probe set specific for G. rostochiensis (golden potato cyst nematode). A second tube consisted of the G. pallida-specific primer-probe set multiplexed with a primer-probe set specific for G. tabacum (the morphologically similar tobacco cyst nematode). This internal transcribed spacer rDNA-based system was specific for the Globodera spp. of interest and successfully identified several populations of PCN. This rapid, sensitive, and specific quantitative PCR assay presents a useful tool for PCN regulatory response and management programs.

scholarly journals First Report of the Pale Potato Cyst Nematode Globodera pallida from Bosnia and Herzegovina

Plant Disease ◽  
2014 ◽  
Vol 98 (4) ◽  
pp. 575-575 ◽  
Author(s):  
B. Nježić ◽  
B. Gerič Stare ◽  
S. Širca ◽  
N. Grujić
Keyword(s):  
Bosnia And Herzegovina ◽  
Potato Production ◽  
Cyst Nematode ◽  
Soil Samples ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
Economic Losses ◽  
Cyst Nematodes ◽  
Seed Potatoes ◽  
Infested Field

Potato cyst nematodes (PCN), the golden cyst nematode Globodera rostochiensis (Woll.) Bahrens, and the pale potato cyst nematode G. pallida (Stone) stand out by their remarkable and efficient parasitic adaptations causing high economic losses in potato production worldwide. They are A2 quarantine pests in EPPO countries as well as in Bosnia and Herzegovina. Up to now, only G. rostochiensis was reported from Bosnia and Herzegovina in 2011 (1). A systematic survey on the presence of PCN on entire territory of Bosnia and Herzegovina started in 2011. During the PCN survey, each year 120 soil samples of 1.5 liters were collected in the Republic of Srpska, one of the two entities of Bosnia and Herzegovina. The samples were collected by soil auger 10 cm in length with a diameter of 1.5 cm consisting of 100 cores. Entire samples were processed by Seinhorst elutriator (4). In autumn of 2012, PCN viable cysts were found in two soil samples originating from one field. From one sample, 12 cysts were extracted, and 2 from another sample. The morphology of cysts and second stage juveniles and molecular analysis established the identity of this species as pale cyst nematode G. pallida (3). In addition, the sequencing of the ribosomal DNA region revealed unequivocal similarity to G. pallida (GenBank Accession No. HF968449), while PCR-RFLP analysis (2) showed European type of G. pallida. The infested field is located in Rogatica, 70 km east of Sarajevo, which is one of the main seed and potato production areas in Bosnia and Herzegovina. The field history revealed that farm-propagated, farm-saved seed potatoes could spread the nematodes to other fields as well. Therefore, 26 additional samples were taken from the fields that belong to the infested field owner and the surrounding fields, but no cysts were found in additional samples. To determine infestation focus and its size, the infested field (1.1 ha) was divided into 46 plots (25 × 10 m) and resampled by taking samples of 60 cores per plot. The detailed sampling of the field revealed a high infestation of 1 cyst per gram of soil in the infestation focus. The high infestation level and the propagation of farm-own seed potatoes suggest that the introduction of G. pallida might have occurred several years ago, probably with imported seed potatoes. The infested field was subjected to the phytosanitary measure of banning potato production for a period of 6 years with the possibility of its prolongation, if cysts with live content are found afterward. It is expected that the nematode is present in other fields due to the farmer's practices of propagating farm-saved seed potatoes and deficient field machinery hygiene. Therefore, the whole area will be intensively monitored for the presence of PCN in the future. An adequate pest management plan will be prepared after PCN pathotype identification. To our knowledge, only one field was found infested with G. pallida during the 3-year PCN survey in Bosnia and Herzegovina. Strict phytosanitary measures for preventing further PCN introductions and spreading should be intensified. References: (1) I. Ostojić et al. Plant Dis. 95:883, 2011. (2) S. Širca et al. Phytopathol. Mediterr. 49:361, 2010. (3) A. M. Skantar et al. J. Nematol. 39:133, 2007. (4) J. van Bezooijen. Methods and Techniques for Nematology, Wageningen University, 2006.

scholarly journals First Report of the Potato Cyst Nematode (Globodera pallida) in the Czech Republic

Plant Disease ◽  
2003 ◽  
Vol 87 (1) ◽  
pp. 98-98 ◽  
Author(s):  
M. Zouhar ◽  
P. Rysanek ◽  
V. Gaar
Keyword(s):  
Czech Republic ◽  
Polymerase Chain Reaction Method ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Czech Republic ◽  
Second Stage ◽  
Complete Series ◽  
Polymerase Chain

Potato cyst nematode, Globodera pallida, was detected in several soil samples collected from various areas of the Czech Republic. Globodera rostochiensis pathotype Ro1 is known to be widespread in the Czech Republic. G. pallida was reported from the neighboring countries of Austria, Germany, and Poland, and also was suspected to be present in the Czech Republic, but it has never been unambiguously proved (1). Recently, nematode isolates have been recovered that multiply readily on Ro1 resistant potato cultivars. These isolates were identified on the basis of three tests: (i) microscopic observations of cyst vulval area morphology and stylets of second-stage larvae; (ii) enzyme-linked immunosorbent assay using a commercial kit; and (iii) a polymerase chain reaction method (2). A complete series of G. pallida and G. rostochiensis pathotypes from Scotland and Germany served as controls. Results were identical for all three methods used. One sample contained G. pallida only, five samples contained mixtures of G. rostochiensis and G. pallida, and one sample contained G. rostochiensis only. The origin of G. pallida contamination is unknown. Strict quarantine measures have been taken to prevent G. pallida from spreading into neighboring areas. To our knowledge, this is the first evidence of the occurrence of G. pallida in the Czech Republic. References: (1) J. Potocek et al. EPPO Bull. 21:81, 1991. (2) M. Zouhar et al. Plant Prot. Sci. 36:81, 2000.

scholarly journals First Report of the Pale Potato Cyst Nematode Globodera pallida from Slovenia

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 773-773 ◽  
Author(s):  
S. Širca ◽  
B. Geric Stare ◽  
P. Strajnar ◽  
G. Urek ◽  
I. Miklic Lautar
Keyword(s):  
Soil Sample ◽  
Ribosomal Subunit ◽  
Posterior Part ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
Infested Soil ◽  
Cyst Nematodes ◽  
First Report ◽  
Ware Potato

The potato cyst nematodes (PCN) Globodera rostochiensis (Woll.) Behrens and G. pallida (Stone) Behrens are considered the most important nematode threat to potato production worldwide, and they are subject to strict quarantine regulations in many countries. The first report of the PCN in Slovenia dates back to 1971, when a single cyst of G. rostochiensis was detected (3). In the last decade, G. rostochiensis was detected several times, mainly in the central and northern parts of the country (2). Interceptions of G. pallida in imported consignments of ware potato were made several times, but had not been detected in soil in Slovenia. Therefore, the country was declared as a protected zone for G. pallida in the European Union by the national authorities in 2003. During the official PCN systematic survey in autumn 2011, the pale potato cyst nematode, G. pallida, was found in a soil sample. Two viable cysts were extracted and the nematode species was identified. The posterior part of the cysts containing eggs and juveniles were used for morphometrical analysis, while the anterior parts were used for DNA extraction and molecular analyses. The ribosomal internal transcribed spacer (ITS) region was amplified using ITS5 and PITSp4 primers and detected in real-time PCR using ABI7500 (Applied Biosystems, Life Technologies, Carlsbad, CA,) (1). Also, the ribosomal DNA region that extends from the 3′ end of the 18S ribosomal subunit and includes all of ITS1, 5.8S, and ITS2, to the 5′ end of the 28S ribosomal subunit, was used to generate a DNA sequence. The sequence obtained was compared with those from several Globodera species, revealing unequivocal similarity to G. pallida. The infested soil sample originated from a ware potato field near Ivancna Gorica, central Slovenia. Subsequently, an additional 69 samples were taken from the surroundings and viable G. pallida cysts were found in another five samples taken from two neighboring fields (one of grassland and the other of clover). Three fields, totaling 1.9 ha, were declared as G. pallida-infested. The eradication of the pest will take place by enforcing strict phytosanitary measures. Ware potatoes originating from areas where the pests occur is considered to be the most probable pathway for the introduction of G. pallida in Slovenia. A ware potato processing facility is situated in very close proximity to the infested fields. The waste waters from potato tuber washing were discharging onto the grassland, never used for potato or other field crop production in which the G. pallida infestation was found. The facility processes imported ware potato from several European and non-European countries. This case demonstrates that ware potato may pose a serious risk for the introduction of such pests, and should be therefore subjected to more intensive phytosanitary inspection. References: (1) J. Bacic et al. Russ. J. Nematol. 16:63, 2008. (2) S. Širca et al. Phytopathol. Mediterr. 49:361, 2010. (3) G. Urek et al. Nematology 5:391, 2003.

scholarly journals Identification and Characterization of Pale Potato Cyst Nematode (Globodera pallida) in Teplá, the Czech Republic

Plant Disease ◽  
2012 ◽  
Vol 96 (9) ◽  
pp. 1386-1386 ◽  
Author(s):  
O. Douda ◽  
M. Zouhar ◽  
J. Urban ◽  
V. Čermák ◽  
V. Gaar
Keyword(s):  
Czech Republic ◽  
Plant Protection ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
The European Union ◽  
Pcr Cloning ◽  
Cyst Nematodes ◽  
The Czech Republic ◽  
Mediterranean Plant

Potato cyst nematode poses a significant threat to potato producers in the Czech Republic. Both species of potato cyst nematodes (Globodera rostochiensis and G. pallida) are listed as quarantine pests in the Czech Republic and also by the European Union, European and Mediterranean Plant Protection Organization, and North American Plant Protection Organization. To date, G. rostochiensis was responsible for all damage to potatoes caused by cyst nematodes in the Czech Republic, while G. pallida was recorded only once in the Czech Republic (2) 8 years ago. It is important to note that this occurrence of G. pallida was not located in the free-cultivation area. In July 2011, soil samples from a potato field located in the area of Teplá (Karlovy Vary Region) were collected, and the cysts extracted were identified as G. pallida according to microscopic observation of cyst fenestra and morphology of juveniles (1). Cyst morphometrics (means from 10 cysts) included: fenestra diameter 21.2 μm, distance fenestra to anus 56.8 μm, Granek's ratio 2.7, number of cuticular ridges between fenestra and anus 14; while second stage juvenile morphometrics (means of 13 specimens) were: L 466.7 μm, stylet 24.2 μm, tail 53.2 μm, body width at anus 13.2, h 28.6, c 8.8, c′ 4.0. Terminus of juvenile tails was rounded, and stylet knobs possessed distinct forward projections. Total DNA was extracted from single cysts using the TriPure reagent (Roche), and the DNA samples were used to amplify cistron rDNA with the following primers: 18S, 5′-TTGATTAGGTCCCTGCCCTTT-3′, and 21S, 5′-TTTCACTCGCCGTTACTAAGG-3′. The amplified region contains the 3′ end of the 18S gene, ITS1, 5.8S, ITS2, and the 5′ end of the 28S gene. Pfu DNA polymerase (Fermentas) was used for accurate amplification. A PCR product of approximately 1.0 kb was amplified from three individual cysts. The PCR amplicons were cloned into pJET1.2 using the CloneJET PCR Cloning Kit (Fermentas) and sequenced in both directions. The sequences of representative isolates were deposited in GenBank (Accession Nos. JQ692592, JQ692593, and JQ692594). The resultant 1.0 kb sequences showed 99% nucleotide identity to sequences of G. pallida from Canada (GenBank Accession Nos. GQ294522.1, GQ355975.1, and GQ294523.1), thus confirming the results of the morphological analyses. To our knowledge, this is the first detection of G. pallida in the free-cultivation area of Karlovy Vary Region, and only the second in the Czech Republic since the first report in 2003 (2). References: (1) M. W. Brzeski. Page 237 in: Nematodes of Tylenchina in Poland and Temperate Europe. Muzeum I Instytut Zoologii Polska Akademia Nauk, Warszawa 1998. (2) M. Zouhar et al. Plant Disease, 87:98, 2003.

Development and validation of real-time PCR assays based on novel molecular markers for the simultaneous detection and identification of Globodera pallida, G. rostochiensis and Heterodera schachtii

Nematology ◽  
2017 ◽  
Vol 19 (7) ◽  
pp. 789-804 ◽  
Author(s):  
Sylvie Gamel ◽  
Aude Letort ◽  
Didier Fouville ◽  
Laurent Folcher ◽  
Eric Grenier
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Nematode Species ◽  
Globodera Pallida ◽  
Heterodera Schachtii ◽  
Cyst Nematodes ◽  
Detection And Identification ◽  
Template Dna

Considering the growing trade of seed potato, reliable diagnostic protocols are required for the detection of regulated nematode species. In this study, a specific and sensitive multiplex Taqman-based real-time PCR method was developed in order to detect and identifyGlobodera pallida,G. rostochiensisandHeterodera schachtii. The newly designed primers and probes enabled the detection of all the target populations tested and with no cross-reaction for closely related non-target species (55 populations tested). The limit of detection (LOD) was one juvenile forG. rostochiensisandG. pallidaand five juveniles forH. schachtii. For monitoring potato cyst nematodes, this analytical tool would extend the number of cyst investigated as five juveniles can be detected among 50 cysts in a sample. Furthermore, this multiplex assay detects DNA of the three targeted species in template DNA obtained directly from float material after nematode extraction from soil.

scholarly journals Molecular Assessment of the Introduction and Spread of Potato Cyst Nematode, Globodera rostochiensis, in Victoria, Australia

2019 ◽  
Vol 109 (4) ◽  
pp. 659-669 ◽  
Author(s):  
Mark J. Blacket ◽  
Arati Agarwal ◽  
John Wainer ◽  
Maggie D. Triska ◽  
Michael Renton ◽  
...  
Keyword(s):  
Market Access ◽  
Allelic Variation ◽  
Sampling Strategy ◽  
Cyst Nematode ◽  
Globodera Pallida ◽  
Published Data ◽  
Cyst Nematodes ◽  
Internal Transcribed Spacer Region ◽  
Molecular Assessment ◽  
Polymerase Chain

Potato cyst nematodes (PCN) are damaging soilborne quarantine pests of potato in many parts of the world. There are two recognized species, Globodera pallida and G. rostochiensis, with only the latter species—the golden cyst nematode—present in Australia. PCN was first discovered in Australia in 1986 in Western Australia, where it was subsequently eradicated and area freedom for market access was reinstated. In Victoria, PCN was first detected in 1991 east of Melbourne. Since then, it has been found in a small number of localized regions to the south and east. Strict quarantine controls have been in place since each new detection. It has previously been speculated that there were multiple separate introductions of PCN into Victoria. Our study utilized a historic (years 2001 to 2014) PCN cyst reference collection to examine genetic variability of Victorian PCN populations to investigate potential historical origins and subsequent changes in the populations that might inform patterns of spread. DNA was extracted from single larvae dissected from eggs within cysts and screened using nine previously described polymorphic microsatellite markers in two multiplex polymerase chain reaction assays. Sequence variation of the internal transcribed spacer region of the DNA was also assessed and compared with previously published data. A hierarchical sampling strategy was used, comparing variability of larvae within cysts, within paddocks, and between local regions. This sampling revealed very little differentiation between Victorian populations, which share the same microsatellite allelic variation, with differences between local regions probably reflecting changes in allele frequencies over time. Our molecular assessment supports a probable single localized introduction into Victoria followed by limited spread to nearby areas. The Australian PCN examined appear genetically distinct from populations previously sampled worldwide; thus, any new exotic incursions, potentially bringing in additional PCN pathotypes, should be easily differentiated from existing established local PCN populations.

Allyl isothiocyanate shows promise as a naturally produced suppressant of the potato cyst nematode, Globodera pallida, in biofumigation systems

Nematology ◽  
2017 ◽  
Vol 19 (4) ◽  
pp. 389-402 ◽  
Author(s):  
Claire Wood ◽  
David M. Kenyon ◽  
Julia M. Cooper
Keyword(s):  
Allyl Isothiocyanate ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Egg Mortality ◽  
Juvenile Mortality ◽  
Viability Staining

The ability of isothiocyanates to suppressGlobodera pallidawas evaluated throughin vitroassays. Several isothiocyanates increased juvenile mortality, the most effective being allyl isothiocyanate, which caused 100% mortality at both 25 and 50 ppm after 72 and 24 h exposure, respectively. In a hatching assay, allyl isothiocyanate was able to suppress hatch; in addition, replenishing allyl isothiocyanate every 3 days increased hatch suppression, and viability staining indicated that egg mortality was increased. Allyl isothiocyanate above concentrations of 50 ppm significantly affected both hatch suppression and mortality. Differing effects of isothiocyanates onG. pallidasuggest that their toxicity depends on the pest of interest and this study shows that allyl isothiocyanate is a good candidate for the control of potato cyst nematodes using biofumigation.

An investigation of resistance to the white potato cyst-nematode

1982 ◽  
Vol 99 (2) ◽  
pp. 325-328 ◽  
Author(s):  
M. F. B. Dale ◽  
M. S. Phillips
Keyword(s):  
Solanum Tuberosum ◽  
Major Gene ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Globodera Pallida ◽  
Screening Methods ◽  
Inheritance Of Resistance ◽  
White Potato

SUMMARYThe inheritance of resistance to Globodera pallida was studied in seedling progenies derived from Solanum tuberosum ssp. andigena CPC 2802 (H3) and S. vernei and compared with resistance to G. rostochiensis derived from S. tuberosum ssp. andigena CPC 1673(H1). The resistance of CPC 2802 was originally thought to be due to a major gene (H3), but results presented here demonstrate that it and that derived from S. vernei are inherited in a similar manner quite distinct from the major gene inheritance from CPC 1673 (HI). It is concluded that the resistances derived from CPC 2802 and S. vernei are both polygenic in nature. These findings are discussed in relation to breeding policy and screening methods.

Sign in / Sign up

Export Citation Format

Share Document