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

in Nematology
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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.

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

in Nematology

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References

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Figures

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    The standard curve describing the relation between known numbers of viable eggs and the Δ absorbance = (A2 − A1) − ΔAblanco of trehalose of glasshouse reared Globodera pallida Pa3 Rookmaker and G. rostochiensis Ro1 Mierenbos populations in three experiments (exp1-exp3). The solid line represents the linear regression, y=0.00088+0.030345x (R2=0.996) valid up to 10 eggs μl−1. To obtain the number of eggs in the original sample of 10 μl the number of viable eggs μl−1 has to be multiplied by 26.2.

  • View in gallery

    Trehalose-based absorbance of viable eggs of Globodera pallida and G. rostochiensis from field samples plotted on the standard curve (solid line), including its 95% confidence limits for single observation (dotted lines).

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    The relation between trehalose-based absorbance of the cyst contents of whole cysts and crushed cysts (cysts μl−1), both dry and pre-soaked, of Globodera pallida and G. rostochiensis. Both dry intact cysts and the soaked intact cysts had a significantly lower measured absorbance than when the cysts were crushed, with respectively P=0.002 and <0.001. The crushed dry cysts were not significantly different from the crushed soaked cyst, P=0.05. Bars indicate standard error of mean.

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    The relation between the Ct-values of a RT-PCR detecting elongation factor 1-α-encoding mRNA extract of Globodera pallida (original sample 31 eggs in 25 μl) and virus PepMv at different concentrations (ng).

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    The relation between Ct-values of a RT-PCR detecting elongation factor 1-α-encoding mRNA and the natural logarithm of the known number of viable eggs of Globodera pallida and G. rostochiensis. Two RNA extractions were carried out with the Kingfisher robot: i) extraction kit Epicentre (Duplos A, B); ii) the extraction kit Ambion (Duplos C, D); and iii) the control; RNA extraction with the normal method without robot (Duplos E, F).

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    The relation between natural logarithm of the number of known viable eggs of Globodera pallida and the Ct values of a RT-PCR detecting elongation factor 1-alpha-encoding mRNA. The solid line presents the linear regression y=36.43.027ln(x) with 95% confidence limits for single observations as dotted lines. Black symbols (◆): data used to develop standard curve (G. pallida Pa3 Rookmaker) and (□) field samples are included. To obtain the number of eggs in the original sample of 25 μl, the number of viable eggs 5 μl−1 has to be multiplied by 7 (5 μl subsample from 35 μl TE buffer).

  • View in gallery

    The relation between natural logarithm of the number of known viable eggs of Globodera rostochiensis and the Ct values of a RT-PCR detecting elongation factor 1-alpha-encoding mRNA. The solid line presents the linear regression y=27.41.798ln(x) with 95% confidence limits for single observations as dotted lines. Black symbols (◆): data used to develop the standard curve (G. rostochiensis Ro1 Mierenbos) and (□): field samples of Ro1 populations. To obtain the number of eggs in the original sample of 25 μl, the number of viable eggs 5 μl−1 has to be multiplied by 7 (5 μl subsample from 35 μl TE buffer).

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