Quantifying potato pathogen DNA in soil

Brierley, J. L.; Stewart, Jennifer A.; Lees, A. K.

doi:10.1016/j.apsoil.2008.11.004

Cited by 59 publications

(49 citation statements)

References 23 publications

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“…An improved method for direct extraction of DNA from soil using a relatively large sample was developed for quantifying potato pathogens (Brierley et al 2009). Use of larger samples could also be tested for soil samples from carrot fields to obtain more reliable detection and quantification of soil borne carrot pathogens in 'bulk' soil.…”

Section: Discussionmentioning

confidence: 99%

Detection and prediction of post harvest carrot diseases

et al. 2011

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Specific PCR primers were developed for identifying two post harvest pathogens, Mycocentrospora acerina and Fibularhizoctonia carotae, which cause liquorice rot and crater rot respectively, during prolonged low temperature storage of carrots. The methods allow routine detection of less than 0.3 pg of M. acerina DNA and less than 0.03 pg F. carotae DNA, even in the presence of large excess of plant or soil DNA. Standard PCR and quantitative PCR gave similar results and either method could be used in a practical situation. Experiments were carried out testing these methods on different types of carrot tissue-and soil-samples. Soil was sampled before sowing, and soil adhering to the roots or root tissue was sampled at different times during the growing season or at harvest. Soil adhering to the carrots at harvest had the best predictive ability for liquorice rot development during storage (R 2 predicted 74.9% using standard PCR), but samples taken during the growing season also gave reasonably good predictive ability values. PCR data from soil samples taken in the spring were not as good as a predictor for this disease. A dense sampling strategy using 20 m between sampling points generally gave better correlation between PCR data and disease data than using 40 m between the sampling points. Use of the developed methods in an IPM strategy for liquorice rot is discussed. For crater rot the correlation between PCR data and disease data was generally poor for all types of samples. These results are discussed in relation to the biology of F. carotae.

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Section: Discussionmentioning

confidence: 99%

Detection and prediction of post harvest carrot diseases

et al. 2011

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“…Direct soil DNA extraction for microbes has been conducted using very small amounts of soil, up to maximum of 10 g and many of the commercially available kits use less than 1 g of soil (Brierley et al, 2009). Waite et al (2003) extracted DNA directly from 1 g soil for the analysis of nematode community.…”

Section: Discussionmentioning

confidence: 99%

“…However, such low amounts of soil are considered too small for nematode analysis (Donn et al, 2008). In addition, Brierley et al (2009) pointed out that the method of homogenising soil samples after soil collection from the field is important for accurate determination of pathogen population in the soil. For quantification of nematodes in soil, a volume of 60 g (20 g in triplicate using the Baermann funnel method) to 100-200 g (tray method) is typically used by nematologists carrying out morphological analyses (Ingham, 1994), because of the comparatively low abundance of soil nematodes and their patchy spatial distribution across a field, which can cause imprecise estimates of the population.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, extraction of DNA from a larger soil volume is desirable to achieve a representative sample. However, to extract nematode DNA directly from soil using, for example, 100 g soil (Brierley et al, 2009) is not practicable in terms of the availability of special apparatus, labour intensity and cost of consumables.…”

Section: Discussionmentioning

confidence: 99%

“…The possible reason may be that after milling samples all particles of sandy soil become fine powders (Brierley et al, 2009), which may provide a more uniform soil sample and better DNA extraction efficiency rather than mixing by a mortar and pestle.…”

Section: Discussionmentioning

confidence: 99%

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Development of a direct quantitative detection method for Meloidogyne incognita in sandy soils and its application to sweet potato cultivated fields in Tokushima prefecture, Japan

Min

Toyota

Goto

et al. 2011

Nematol

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A real-time PCR (quantitative PCR: qPCR)-based detection method of the root-knot nematode Meloidogyne incognita was developed for sandy soils, the major soil type in sweet potato cultivated fields in Tokushima prefecture, Japan. Different numbers (5, 20, 80, 200 and 500) of second-stage juveniles (J2) were artificially added into 20 g of an air-dried sandy soil not containing M. incognita. To make homogenous samples, soil was homogenised by two different ways (ground with either a mortar and pestle or ball mill) and then 0.5 g of the soil was used for DNA extraction. There was a strong negative correlation in each homogenisation method between the cycle threshold number (Ct) and inoculated numbers of M. incognita J2. The Ct values were consistently lower and their variations among replicates were smaller in the samples ground with ball mill, suggesting that grinding with ball mill may be suitable for the preparation of soil for DNA extraction. Sandy soils were collected from sweet potato fields in Tokushima prefecture at the transplanting and harvesting times. Damage to sweet potato caused by M. incognita was also evaluated in some of the fields. At the transplanting time, no M. incognita was extracted in all the soils by the Baermann funnel method, while detection in the qPCR method ranged from zero to 4 210 000 J2 equivalent (20 g soil) −1 . Heavy damage was observed in fields with more than 500 equivalent M. incognita J2 (20 g soil) −1 . By contrast, very few galls were observed in fields with fewer than four individuals (20 g soil) −1 . At harvest, zero to >1000 individuals of M. incognita was counted by the Baermann method and there was a significant correlation in estimated numbers of M. incognita between the two methods. However, the estimated numbers were 15 times higher in the qPCR method than in the Baermann method. These results indicate that direct quantification of M. incognita based on the qPCR method might enable a sensitive diagnosis to predict damage by the nematode.

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