Using a selective fast turn-around bioassay for population density determination of Heterodera schachtii

In Central Europe, Heterodera schachtii is kept below threshold levels by cover-cropping with resistant crucifers and crop rotation with non-hosts. Determining population densities of H. schachtii in soil is critical when implementing resistant and tolerant sugar beet cultivars in integrated pest management (IPM) programmes. Soil extraction of the cysts followed by egg counts or extraction of the second-stage juveniles (J2) facilitated by the chemical stimulant acetox can be unsatisfactory in mixed field populations of cyst nematodes. In contrast to H. schachtii, nematodes typically present in sugar beet soils, e.g., Globodera pallida, G. rostochiensis, H. avenae, H. filipjevi, Meloidogyne hapla, M. incognita and Pratylenchus penetrans, rarely penetrated radish roots. In this bioassay, equivalents of 50 g of soil dry weight were adjusted to 10-20% moisture, seeded with Raphanus sativus cv. Saxa 3, and incubated at a day-night (16:8 h) cycle of 28/23°C for 4 days before J2 in radish roots were enumerated. In different soil types, penetration by H. schachtii reflected the inoculation levels. When inoculated with mixes of H. schachtii with H. avenae or H. filipjevi, counts of H. schachtii were similar to those in soils with H. schachtii only. When comparing three methods in three soils spiked with H. schachtii cysts, the bioassay and the extraction method were lightly impacted by the soil texture but results of the acetox method varied with texture. When implemented for field samples from Franconia, the radish bioassay and the acetox method provided results related to cyst and egg extraction data. The radish bioassay provided a quick and easy method for quantifying H. schachtii in the presence of other nematode species in a wide range of soil types. Including this assay in IPM programmes may serve as an alternative to standard methods and will improve the decision making in sustainable production systems.

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Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples

Life ◽

10.3390/life11121358 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1358

Author(s):

Chen Jiang ◽

Yingdong Zhang ◽

Ke Yao ◽

Sulaiman Abdulsalam ◽

Guangkuo Li ◽

...

Keyword(s):

Sugar Beet ◽

Cyst Nematode ◽

Soil Samples ◽

Heterodera Schachtii ◽

Cyst Nematodes ◽

Pcr Assay ◽

Beet Cyst Nematode ◽

Field Samples ◽

Species Specific ◽

Sugar Beet Cyst Nematode

Sugar beet cyst nematode (SBCN, Heterodera schachtii) is an important nematode that causes significant yield losses of 25–50% or more in most areas of sugar beet production worldwide. Rapid and accurate identification of this species is essential to support decisions on pest management. However, the difference between H. schachtii and other Heterodera spp. based on morphology is a challenging task. In the present study, a SCAR-PCR assay was developed to identify and differentiate H. schachtii in infected root and soil samples. H. schachtii-species-specific SCAR-PCR primers OPA06-HsF and OPA06-HsR were designed from the randomly amplified polymorphic DNA (RAPD) marker amplified with random primer OPA06. The developed primers specifically amplify a 922-bp fragment from the target populations but did not amplify DNA from non-target cyst nematodes including Heterodera, Globodera, Cactodera, and other related species tested in this study. The sensitivity detection indicated that 5 × 10−4 of a single cyst, 1/320 of a single second-stage juvenile (J2), or 10 pg of genomic DNA could be detected. The assay accurately identifies the different stages of H. schachtii in sugar beet and oilseed rape roots as well as a single J2 in 10 g of soil. Finally, the SCAR-PCR assay detected H. schachtii in seven samples out of the fifteen field samples. The assay will not only be useful for differentiating H. schachtii from mixed populations of Heterodera spp. but also for effective detection of the species directly from infested samples. The assay also requires no expertise in the taxonomy and morphology of the species but serves to improve the diagnosis of H. schachtii in infested fields.

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Rapid and Visual Detection of Heterodera schachtii Using Recombinase Polymerase Amplification Combined with Cas12a-Mediated Technology

International Journal of Molecular Sciences ◽

10.3390/ijms222212577 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12577

Author(s):

Ke Yao ◽

Deliang Peng ◽

Chen Jiang ◽

Wei Zhao ◽

Guangkuo Li ◽

...

Keyword(s):

Sugar Beet ◽

Visual Detection ◽

Direct Detection ◽

Recombinase Polymerase Amplification ◽

Detection Sensitivity ◽

Heterodera Schachtii ◽

Economic Losses ◽

Cyst Nematodes ◽

Effective Prevention ◽

Field Samples

Heterodera schachtii is a well-known cyst nematode that causes serious economic losses in sugar beet production every year. Rapid and visual detection of H. schachtii is essential for more effective prevention and control. In this study, a species-specific recombinase polymerase amplification (RPA) primer was designed from a specific H. schachtii sequence-characterized amplified region (SCAR) marker. A band was obtained in reactions with DNA from H. schachtii, but absent from nontarget cyst nematodes. The RPA results could be observed by the naked eye, using a lateral flow dipstick (LFD). Moreover, we combined CRISPR technology with RPA to identify positive samples by fluorescence detection. Sensitivity analysis indicated that 10−4 single cysts and single females, 4−3 single second-stage juveniles, and a 0.001 ng genomic DNA template could be detected. The sensitivity of the RPA method for H. schachtii detection is not only higher than that of PCR and qPCR, but can also provide results in <1 h. Consequently, the RPA assay is a practical and useful diagnostic tool for early diagnosis of plant tissues infested by H. schachtii. Sugar beet nematodes were successfully detected in seven of 15 field sugar beet root samples using the RPA assay. These results were consistent with those achieved by conventional PCR, indicating 100% accuracy of the RPA assay in field samples. The RPA assay developed in the present study has the potential for use in the direct detection of H. schachtii infestation in the field.

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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.

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Comparative genomics among three cyst nematode species reveals distinct evolutionary histories among effector families and an irregular distribution of effector-associated promoter motifs

10.1101/2021.11.01.466756 ◽

2021 ◽

Author(s):

Joris J.M. van Steenbrugge ◽

Sven van den Elsen ◽

Martijn Holterman ◽

Jose L. Lozano-Torres ◽

Vera Putker ◽

...

Keyword(s):

Host Plant ◽

Reference Genome ◽

Nematode Species ◽

Cyst Nematode ◽

Globodera Pallida ◽

Heterodera Schachtii ◽

Cyst Nematodes ◽

Promoter Regions ◽

Long Read ◽

Additional Reference

Potato cyst nematodes (PCNs), an umbrella term used for two species, Globodera pallida and G. rostochiensis, belong worldwide to the most harmful pathogens of potato. Pathotype-specific host plant resistances are an essential handle for PCN control. However, the poor delineation of G. pallida pathotypes hampers the efficient use of available host plant resistances. Long-read sequencing technology allowed us to generate a new reference genome of G. pallida population D383 and, as compared to the current reference, the new genome assembly is 42 times less fragmented. For comparison of diversification patterns of six effector families between G. pallida and G. rostochiensis, an additional reference genome was generated for an outgroup, the beet cyst nematode Heterodera schachtii (IRS population). Large evolutionary contrasts in effector family topologies were observed. While VAPs diversified before the split between the three cyst nematode species, the families GLAND5 and GLAND13 only expanded in PCN after their separation from the genus Heterodera. Although DNA motifs in the promoter regions thought to be involved in the orchestration of effector expression (DOG boxes) were present in all three cyst nematode species, their presence is not a necessity for dorsal gland-produced effectors. Notably, DOG box dosage was only loosely correlated with expression level of individual effector variants. Comparison of the G. pallida genome with those of two other cyst nematodes underlined the fundamental differences in evolutionary history between effector families. Re-sequencing of PCN populations with deviant virulence characteristics will allow for the linking of these characteristics with the composition of the effector repertoire as well as for the mapping of PCN diversification patterns resulting from extreme anthropogenic range expansion.

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Evaluation of rhizobacterial colonisation and the ability to induce Globodera pallida hatch

Nematology ◽

10.1163/15685411-00002863 ◽

2015 ◽

Vol 17 (2) ◽

pp. 203-212 ◽

Cited By ~ 3

Author(s):

Eoin P. Lettice ◽

Peter W. Jones

Keyword(s):

Sugar Beet ◽

Dry Weight ◽

Coarse Sand ◽

Globodera Pallida ◽

Cyst Nematodes ◽

Beet Root ◽

Shoot Dry Weight ◽

Sugar Beet Root

Three bacterial isolates, SB13 (Acinetobacter sp.), SB14 (Arthrobacter sp.) and SB15 (Bacillus sp.), were previously isolated from the rhizosphere of sugar beet (Beta vulgaris ssp. vulgaris) plants and shown to increase hatch of potato cyst nematodes in vitro. In this study, the three isolates were assayed for rhizosphere competence. Each isolate was applied to seeds at each of four concentrations (105-108 CFU ml−1) and the inoculated seeds were planted in plastic microcosms containing coarse sand. All three isolates were shown to colonise the rhizosphere, although to differing degrees, with the higher inoculation densities providing significantly better colonisation. The isolates increased sugar beet root and shoot dry weight. Isolates SB14 and SB15 were analysed for their ability to induce in vivo hatch of Globodera pallida in non-sterile soil planted with sugar beet. After 4 and 6 weeks, both isolates had induced significantly greater percentage hatch compared to controls.

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THE PROBLEM OF SUGAR BEET CYST NEMATODES IN MODERN CONDITIONS OF MACROECONOMIC AND CLIMATIC CHALLENGES

10.31016/978-5-6046256-1-3.2021.22.409-413 ◽

2021 ◽

pp. 409-413

Author(s):

Perevertin ◽

Belolubtsev ◽

Vasiliev

Keyword(s):

Population Density ◽

Sugar Beet ◽

Raw Materials ◽

Import Substitution ◽

Economic Consequences ◽

Heterodera Schachtii ◽

Fourth Generation ◽

Cyst Nematodes ◽

Systemic Crisis ◽

Important Challenge

In this work, the current state of infection of the cultivated areas of sugar beet farms with the sugar beet cyst nematode Heterodera schachtii is considered. After the systemic crisis of agricultural production in the early 90th, the practice of operating Russian sugar factories not using domestic (sugar beet) but imported (cane) raw materials was widespread. For many years, in the absence of a host plant in the fields, the population density of highly specialized phytohelminths naturally decreased to an economically imperceptible level. However, in accordance with the principle of irreversibility of soil biocontamination of agroecosystems, the nematode retained its presence due to the mechanisms of mesobiosis and the maintenance of the population on reserve weeds. After the strategic course taken in 2014 towards import substitution, sugar beet areas (usually around sugar factories) returned to the main crop, and in 2018, for the first time in its history, Russia even became a sugar exporting country. However, the technological regulations of the USSR, mostly crop rotations were not observed and the nematode problem was again actualized, first of all, in the Chernozem and Krasnodar region. The proposed model of the dynamics of the population density of the nematodes, depending on the cultivated crop, makes it possible to assess the ecological and economic consequences of disturbed crop rotation, taking into account the conjuncture of the world food market. Climate change is another important challenge to be considered in the assessment of the nematode problem. Along with the noted increase in the bioclimatic potential of a number of agricultural lands in the Russian Federation, the development of the parasite during the growing season can occur not in three generations with a reduced fourth, but with a full cycle for the fourth generation.

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The effectiveness of zinc fertilizers as measured by DTPA soil extractable zinc, dry matter production and zinc uptake by subterranean clover in relation to soil properties of a range of Australian soils

Soil Research ◽

10.1071/sr9920045 ◽

1992 ◽

Vol 30 (1) ◽

pp. 45 ◽

Cited By ~ 1

Author(s):

RF Brennan

Keyword(s):

Organic Carbon ◽

Dry Matter ◽

Subterranean Clover ◽

Soil Types ◽

Dry Matter Production ◽

Soil Extraction ◽

Zn Uptake ◽

Matter Production ◽

Wide Range ◽

Zinc Fertilizer

The effectiveness of zinc fertilizer (Zn) on a wide range of Australian soils was examined using subterranean clover (Trifolium subterranean cv. Nungarin) as a test crop in a glasshouse experiment. The initial effectiveness (IE) of zinc fertilizer as measured by dry matter production (DMP), and zinc content (uptake) of subterranean clover (clover) was found to vary markedly among the soil types. No simple linear retationship between the initial effectiveness measured by either dry matter production or uptake and any one soil property was found. IE values were found to be related to the pH (1 : 5 soil :water) (pHw) and the level of DTPA soil extractable zinc measured in the unfertilized soil (Zno). IE based on Zn uptake by clover tops was also related to the organic carbon (OC) (%) content of the soils. The model for IE measured by DMP in a stepwise linear regression was IEDMP = 2.682 - 0.107 pH,-4.852 Zn, (n = 45; r2 = 0-86). IE based on Zn uptake by clover tops was: IEuptake = 10.842 - 0.882 pH, - 0.310 OC (%) - 1.349 Zn, (n = 54; R2 = 0.85). The IE of zinc fertilizer measured by DTPA soil extraction (IEDTPA-zn) was also found to vary markedly among soil types. The level of zinc extracted by DTPA after the addition of Zn fertilizer was found to be affected by clay (%), organic carbon (%) and calcium carbonate (CaCO3) (%) content of the range of Australian soils. This relationship could be described by: IEDTPA - Zn = 0.178 + 0.0.002 Clay (%) + 0.014 OC (%) + 0.018 CaCO3 (%) (N = 54, r2 = 0.84)

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Fungi associated with cysts of Globodera rostochiensis, G. pallida, and Heterodera schachtii; and egg masses and females of Meloidogyne hapla in Belgium

Phytoprotection ◽

10.7202/706135ar ◽

2005 ◽

Vol 79 (2) ◽

pp. 63-69 ◽

Cited By ~ 9

Author(s):

Q. Yu ◽

J. Coosemans

Keyword(s):

Solanum Tuberosum ◽

Lycopersicon Esculentum ◽

Sugar Beet ◽

Fusarium Oxysporum ◽

Beta Vulgaris ◽

Globodera Rostochiensis ◽

Heterodera Schachtii ◽

Meloidogyne Hapla ◽

Egg Masses ◽

Predominant Species

Cysts of Heterodera schachtii from sugar-beet (Beta vulgaris) fields and cysts of Globodera rostochiensis and G. pallida from potato (Solanum tuberosum) fields in northern Belgium, as well as egg masses and females of Meloidogyne hapla from a tomato (Lycopersicon esculentum) field in the Flemish-Brabant province, Belgium, were collected and examined for the presence of fungi. Of the total of 374 cysts of H. schachtii, 57.7% were colonized by one or more of 18 different species of fungi, all of which were from the genra Acremonium, Chaetomium, Cylindrocarpon, Fusarium, Gliocladium, Humicola, Mariannaea, Nematophthora, Periconia, Phoma, and Verticillium, and 45.3% of the 726 cysts of Globodera spp. were colonized by one or more of 18 different species, from the same gene. Of the 160 egg masses of M. hapla, 32% were colonized by one or more of 18 species of the genra Arthrobotrys, Cylindrocarpon, Fusarium, Monacrosporium, Paecilomyces, Phoma, Plectosphaerella, and Verticillium, while 31% of the 160 females were colonized by 12 species, from the same gene except Paecilomyces and Plectosphaerella. Fusarium oxysporum was by far the predominant species in both the cyst and root-knot nematodes. A black yeast-like fungus was found in cysts.

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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 ◽

10.1163/15685411-00003086 ◽

2017 ◽

Vol 19 (7) ◽

pp. 789-804 ◽

Cited By ~ 5

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.

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