Determination of relative proportions of Globodera species in mixed populations of potato cyst nematodes using PCR product melting peak analysis
SUMMARY
Using a novel approach based on melting peak analysis of PCR products, we have developed a semi-quantitative assay to measure the relative proportions of Globodera pallida and Globodera rostochiensis in a sample of potato cyst nematodes (PCN). The method depends on a competitive multiplex PCR where the products of each species can be separated by their distinct melting temperatures (Tm). The melting curves of the products are measured by continual fluorescence monitoring in the presence of the intercalating dye SYBR Green I whilst gradually increasing the temperature. Varying the proportion of cysts of each PCN species gave rise to melting curves with different peak heights, which reflected the relative amounts of each DNA in the sample. By calculating the ratio of the melting peak heights at the Tm of each product and comparing it with standards run under the same conditions, it was possible to estimate the proportion of each product in the mixture. Sensitivity was such that 2% of G. pallida cysts in a mixture could be detected. All results predicted by the analysis were confirmed by agarose gel electrophoresis of the PCR products. The method is rapid, reproducible and following DNA extraction, up to 25 samples can be analysed in an hour.
Potato cyst nematodes (PCN) Globodera pallida and Globodera rostochiensis cause serious damage to potato crops world-wide by restricting root growth and uptake of nutrients, leading to a loss in tuber yield. At present, PCN are thought to account for losses of more than 12% of the average world potato crop yield. In England and Wales, the number of PCN-infested potato production sites appears to be increasing, from an estimated 42% in 1996 to 64% in 2000. A recent survey in Scotland found 23% of sites tested to be infested.
Correct identification of the PCN species present in soil samples submitted for advisory testing is essential as part of an effective integrated pest management strategy. In addition to crop rotation schedules and use of nematicides, cultivar resistance plays an important role in managing PCN. There are many potato cultivars that are highly resistant to G. rostochiensis but none with full resistance to G. pallida. Those with partial G. pallida resistance reduce PCN multiplication but not completely. The use of G. rostochiensis resistant cultivars has led to an increase in the prevalence of G. pallida, with an estimated 92% of infested sites in England and Wales containing G. pallida, either alone or as part of a mixed population compared to 33% containing G. rostochiensis. An added problem is that the market demand is for varieties that either have no PCN resistance (e.g. Desiree), or G. rostochiensis resistance only (e.g. Maris Piper).
G. rostochiensis and G. pallida can be distinguished by morphological features of the cysts but this requires a high degree of skill and is time consuming. A method based on iso-electric focusing (IEF) of proteins extracted from cysts has been used as a diagnostic test, as have immunological methods. More recently, PCR-based test methods have become the method of choice for species. A recent comparison of techniques for detection and determination of PCN in field samples found PCR methods to have a greater sensitivity and ability to identify mixed species populations. However the existing PCR methods are not quantitative. Our aim was to develop a rapid quantitative PCR assay that could estimate the proportion of each PCN species present in a sample, even when relatively few cysts were available for testing.
We used the species-specific primers of Bulman and Marshall (1997) in combination with primer ITS5 and PCR in a LightCycler instrument to develop a quantitative assay for PCN. By using the intercalating fluorescent reporter dye SYBR Green I, we observed that the PCR products from each species could be separated by their distinct melting temperatures and identified without agarose gel electrophoresis. In addition, varying the proportion of DNA from each PCN species in the PCR set up a competitive reaction, giving rise to melting curves with different peak heights, which reflected the relative amounts of each DNA in the sample.
Correct identification of the PCN species present in soil samples submitted for advisory testing is essential as part of an effective integrated pest management strategy. In addition to crop rotation schedules and use of nematicides, cultivar resistance plays an important role in managing PCN. There are many potato cultivars that are highly resistant to G. rostochiensis but none with full resistance to G. pallida. Those with partial G. pallida resistance reduce PCN multiplication but not completely. The use of G. rostochiensis resistant cultivars has led to an increase in the prevalence of G. pallida, with an estimated 92% of infested sites in England and Wales containing G. pallida, either alone or as part of a mixed population compared to 33% containing G. rostochiensis. An added problem is that the market demand is for varieties that either have no PCN resistance (e.g. Desiree), or G. rostochiensis resistance only (e.g. Maris Piper).
G. rostochiensis and G. pallida can be distinguished by morphological features of the cysts but this requires a high degree of skill and is time consuming. A method based on iso-electric focusing (IEF) of proteins extracted from cysts has been used as a diagnostic test, as have immunological methods. More recently, PCR-based test methods have become the method of choice for species. A recent comparison of techniques for detection and determination of PCN in field samples found PCR methods to have a greater sensitivity and ability to identify mixed species populations. However the existing PCR methods are not quantitative. Our aim was to develop a rapid quantitative PCR assay that could estimate the proportion of each PCN species present in a sample, even when relatively few cysts were available for testing.
We used the species-specific primers of Bulman and Marshall (1997) in combination with primer ITS5 and PCR in a LightCycler instrument to develop a quantitative assay for PCN. By using the intercalating fluorescent reporter dye SYBR Green I, we observed that the PCR products from each species could be separated by their distinct melting temperatures and identified without agarose gel electrophoresis. In addition, varying the proportion of DNA from each PCN species in the PCR set up a competitive reaction, giving rise to melting curves with different peak heights, which reflected the relative amounts of each DNA in the sample.
Melting peaks of PCR products from various PCN mixtures. The multiplex PCR is a competitive reaction: changing the relative amounts of each PCN DNA in the reaction mixture gave a pattern of melting peaks that reflected the proportions of each species in the starting sample. PITSp4
This resulted in a quantitative diagnostic test for PCN, which can accurately predict the proportion of each Globodera species in mixed populations. This test has significant advantages over the current IEF test in that it is rapid, more reliable and easier to interpret, especially when few cysts are available. In addition, it has the advantage over other PCR tests, being faster than conventional PCR and with “in-tube” analysis, avoiding cross-contamination from PCR products and the need to run agarose gels.
Agarose gel showing the relative intensity of PCR product bands from different PCN mixtures. Numbers refer to the number of cysts of either species used in the extraction.
This resulted in a quantitative diagnostic test for PCN, which can accurately predict the proportion of each Globodera species in mixed populations. This test has significant advantages over the current IEF test in that it is rapid, more reliable and easier to interpret, especially when few cysts are available. In addition, it has the advantage over other PCR tests, being faster than conventional PCR and with “in-tube” analysis, avoiding cross-contamination from PCR products and the need to run agarose gels.
Agarose gel showing the relative intensity of PCR product bands from different PCN mixtures. Numbers refer to the number of cysts of either species used in the extraction.