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Quantification of Pratylenchus penetrans in radishfields using a combination method of soil compactionand real-time PCR to determine the economicthresholdErika Sato a , Yuko Suga a , Chihiro Kisaki b , Koki Toyota b , Kazuto Miyake c , Atsushi Takadac , Koji Takeuchi d & Rie Matsuura da National Agricultural Research Center for Western Region, 200 Ueno, Uenocho, Ayabe,Kyoto 623-0035b Graduate School of Bio-Applications and Systems Engineering, Tokyo University ofAgriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588c Kanagawa Agricultural Technology Center, 3002 Shimomiyada, Hassecho, Miura, Kanagawa238-0111d Tokyo Development Foundation for Agriculture, Forestry and Fisheries, 3-8-1, Fujimicho,Tachikawa, Tokyo 190-0013, JapanPublished online: 25 May 2011.

To cite this article: Erika Sato , Yuko Suga , Chihiro Kisaki , Koki Toyota , Kazuto Miyake , Atsushi Takada , Koji Takeuchi& Rie Matsuura (2011) Quantification of Pratylenchus penetrans in radish fields using a combination method of soilcompaction and real-time PCR to determine the economic threshold, Soil Science and Plant Nutrition, 57:2, 213-220, DOI:10.1080/00380768.2011.574233

To link to this article: http://dx.doi.org/10.1080/00380768.2011.574233

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Soil Science & Plant NutritionSoil Science and Plant Nutrition (2011), 57, 213—220 doi: 10.1080/00380768.2011.574233

ORIGINAL ARTICLE

Quantification of Pratylenchus penetrans in radish fields using acombination method of soil compaction and real-time PCR todetermine the economic threshold

Erika SATO1, Yuko SUGA1, Chihiro KISAKI2, Koki TOYOTA2, Kazuto MIYAKE3,Atsushi TAKADA3, Koji TAKEUCHI4 and Rie MATSUURA4

1National Agricultural Research Center for Western Region, 200 Ueno, Uenocho, Ayabe, Kyoto 623-0035, 2Graduate School of

Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo

184-8588, 3Kanagawa Agricultural Technology Center, 3002 Shimomiyada, Hassecho, Miura, Kanagawa 238-0111 and 4Tokyo

Development Foundation for Agriculture, Forestry and Fisheries, 3-8-1, Fujimicho, Tachikawa, Tokyo 190-0013, Japan

Abstract

The objective of this study was to compare the economic threshold (ET) of Pratylenchus penetrans in radish

fields of andosols using the Baermann method and the combination method of soil compaction and real-time

polymerase chain reaction (PCR). Soil samples were collected from 26 plots at depths of 0 to 30 and 30 to

60 cm before seeding of radishes. The number of P. penetrans in each sample was estimated by the Baermann

method and the combination method. No P. penetrans was detected in 13 plots by the Baermann method,

while the number of plots in which no P. penetrans was detected was only two by the combination method.

The number of spots caused by P. penetrans on radishes harvested from the plots was also counted. It was

difficult to determine the ET of P. penetrans estimated by the Baermann method, based on whether severe

damage (more than 10 spots on average per radish) was seen. However, the ET of P. penetrans estimated by

the combination method was determined at 5.3 J2 equivalents per 20 g dry soil. In plots with P. penetrans

densities lower than the ET as evaluated by the combination method, the ratio of plots with no damage was

87%. The results suggest that the combination method has an advantage in the estimation of damage to

radish by nematode.

Key words: diagnosis, eDNA, real-time PCR, root-lesion nematode, soil compaction.

INTRODUCTION

The root-lesion nematode Pratylenchus penetrans infects

many crops, such as alfalfa, bean, cabbage, carrot, celery,

chickpea, clover, cowpea, cucumber, faba bean, ground-

nut, lentil, lettuce, pea, potato, radish, spinach, squash,

sorghum, strawberry and tobacco (Castillo and Vovlas

2007), and therefore is considered an important nema-

tode pest. In Japan, damage caused by P. penetrans is a

threat to radish production, and nematicides are fre-

quently used for prevention.

There is a significant correlation between the popula-

tion density of Pratylenchus penetrans in the soil and the

degree of damage on the host (e.g. Castillo and Vovlas

2007). Therefore, the economic threshold (ET) has been

studied based on whether damage to radish by nematode

is seen; e.g. ETs of one second-stage juvenile (J2) 50 g�1

(Nishizawa 1973), 10 J2 50 g�1 (Ohbayashi 1989) and

2.5 J2 20 g�1 of soil (Sato et al. 2009) have been

reported. However, due to problems with calculating the

ET, nematode diagnosis is not widely performed. Firstly,

damage by nematode to radish has frequently been

observed even in fields in which P. penetrans was not

detected by the Baermann method (Mihira 2002). One

reason might be the sampling depth since radish roots

elongate into a soil layer 30 to 40 cm deep, but the soil

sampling depth for nematode diagnosis is generally 15

Correspondence: E. SATO, National Agricultural ResearchCenter for Western Region, 200 Ueno, Uenocho, Ayabe, Kyoto623-0035, Japan. Email: erikasato@affrc.go.jpReceived 25 May 2010.Accepted for publication 18 March 2011.

� 2011 Japanese Society of Soil Science and Plant Nutrition

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to 20 cm. We found that damage by nematode was not

observed in fields in which no P. penetrans was detected

by the Baermann method when the soil was sampled

from a depth of 0 to 60 cm, although the number of fields

tested was small. Based on this result, we proposed that it

is preferable to collect soil samples from a depth of 0 to

60 cm for nematode diagnosis. However, damage to

radish may be seen even in fields in which no

P. penetrans is detected at a soil depth of 0 to 60 cm,

as shown in the current study. Secondly, a large variation

is often seen in the relationship between nematode

density and damage to radish, especially at a low density

of P. penetrans such as 0—10 J2 20 g�1 of soil (e.g. Sato

et al. 2009). Thirdly, the nematode extraction efficiency

is as low as 50% (Ingham 1994). Fourthly, some forms

of live nematode are not extracted by the Baermann

method (Ingham 1994).

The development of a precise method of identifying

nematodes and their density in the soil is needed in order

to initiate control of the species (Madani et al. 2005). For

this purpose, we developed a direct quantification

method, consisting of soil compaction and real-time

polymerase chain reaction (PCR), for the soybean cyst

nematodes Heterodera glycines (Goto et al. 2009) and

Pratylenchus penetrans (Sato et al. 2010) in soil. In the

proposed method, nematodes at any stage, including

cyst, egg and vermiform, are destroyed by compaction to

release their DNA and their numbers are quantified by

real-time PCR with primers specific to the nematodes.

The objectives of this study were to collect examples

of the relationship between the initial densities of

Pratylenchus penetrans, estimated by the Baermann

and the combination methods, and damage to radish

caused by P. penetrans and to compare the ET, which is

the minimum density at which damage to radish is

observed and control measures are required to prevent

damage to radish, between the two methods.

MATERIALS AND METHODS

Study sites and experimental plot management

Eighteen plots, each 4 m�3 m in size, were set in a field

of andosol at the Kanagawa Agricultural Technology

Center (KATC) in Miura city, Kanagawa Prefecture. Six

plots, each 4 m�6 m in size, were set in a field of

andosol at the Tokyo Development Foundation for

Agriculture, Forestry and Fisheries (TDFAFF) in

Tachikawa city, Tokyo. Two plots, each 1.5 m�1 m,

were set in TDFAFF in Hachioji city, Tokyo.

On August 18, 2009 (one month before seeding), in

the KATC plots, 1,3-dichloropropene (1,3-D) was

applied to three [plot identification (ID) M5, M9 and

M15] out of 18 plots. The chemical was injected, at

33 cm intervals, at a 20 cm depth to result in a total of

2 L a�1 in each plot. These plots were covered with vinyl

sheets to retain the chemical in the soil. Liming and

0.1 t a—1 of cow manure were applied on September 2,

2009. On September 11, chemical fertilizers were applied

at dosages of nitrogen/phosphorus/potassium (N:P:K)¼

0.4:1.85:0.48 kg a�1. Soil samples were collected from

two depths, 0 to 30 cm and 30 to 60 cm, in the center

area (1.5 m2: 1.5 m� 1 m) of each plot with a root auger

(3 cm diameter, Daiki Rika Kogyo, Co., Ltd, Kounosu,

Japan) on September 14 (one week before sowing). A

composite soil sample was made by combining soils

collected from four sites in the center area. On September

24, radish seeds (Fuyunoura, Kaneko Seeds Co., Ltd,

Maebashi, Japan) were sown with a 24 cm inter-plant

distance and a 50 cm inter-row space. On October 19,

chemical fertilizers were applied at dosages of N:P:K¼

0.64:0:0.64 kg a�1. Fifteen radishes cultivated in the

center area were harvested on December 4.

On August 13, 2009, 1,3-D was applied to four (plot

ID T1, T2, T4 and T5) out of six TDFAFF plots. The

chemical was injected at 30 cm intervals and 30 cm deep

to result in a total of 2 L a�1 in each plot. Plots T1 and T2

were covered with vinyl sheets to retain the chemical in

the soil. Soil sampling was done on August 24 (eight days

before sowing), as at KATC. Chemical fertilizers

(N:P:K¼1.5:1.3:1 kg a�1) and liming were applied on

August 31. On September 1, 2009, radish seeds (Natsu-

tsukasa, Tohoku Seed Co., Ltd, Utsunomiya, Japan)

were sown at a 30 cm inter-plant distance and a 45 cm

inter-row space. Ten radishes cultivated in the center

area from which soil had been collected were harvested

on November 3.

Nematode extraction and counting

Soil samples were firstly sieved (5 mm aperture sieve),

and nematodes were extracted from three subsamples

(20 g, a total of 60 g) using the Baermann funnel method

(two days at room temperature) (Ingham 1994). The

number of Pratylenchus penetrans was counted under a

microscope (�100).

Estimation of the density of Pratylenchuspenetrans by soil compaction andreal-time PCR

The sieved soils were air-dried as soon as possible after

sampling and were stored at room temperature for three

to four weeks and then in a freezer until measurement

(total storage period was two to three months). Soil

compaction was done following the method of Sato et al.

(2010). Twenty grams of air-dried soil put in a 50-mL

soil sampling core cylinder (2.5 cm height) was

214 E. Sato et al.

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compacted to 1.4 g cm�3 by a manually operated soil

compactor (Daiki Rika Kogyo Co., Ltd) (three replica-

tions). The compacted soils were removed from the

cylinder and mixed well with 17.5 mL of TE buffer using

a homogenizer (Auto Cell Master CM-200, As One

Corporation, Tokyo, Japan) at 15,000 rpm for 10 min.

Then, DNA was extracted from 0.5 g of the mixed soil by

the method of Sato et al. (2010). At the DNA extraction,

a solution of a PCR-amplified fragment using the specific

primers (Toyota et al. 2008) to potato cyst nematode,

Globodera rostochiensis, was added into the soil samples

as an internal standard. The extracted DNA was used as

a template in real-time PCR after 10-fold dilution

(Griffiths et al. 2000).

Real-time PCR was performed to estimate the density of

Pratylenchus penetrans using a Step One Real-Time PCR

System (Life Technologies Japan, Tokyo, Japan) at a final

volume of 10 ml containing 5 ml of the Fast SYBR Green

Master Mix (Life Technologies Japan, Tokyo, Japan),

0.1 mgml�1 of BSA (bovine serum albumin), 5 mM of each

primer (NEGf: 50-ATTCCGTCCGTGGTTGCTATG-30,

NEGr: 50-GCCGAGTGATCCACCGATAAG-30; Sato

et al. 2007) and 2 ml of template DNA under the

manufacturer’s recommended conditions [95�C for

20 sec, (95�C for 3 sec and 62�C for 30 sec)�40 cycles].

The density of P. penetrans (x: individual equivalent to

J2 20 g�1 of air-dried soil) was calculated based on the

detected threshold cycle (Ct: y) values and the modified

standard curve (y¼�0.89 xþ37.6, R2¼ 0.981)

described by Sato et al. (2010). The original curve was

obtained using the Ct values at densities of P. penetrans

ranging from 25 to 1000 J2 20 g�1 of soil (Sato et al.

2010). In this study, the Ct values in soil at densities of

four and 10 J2 20 g�1 were added in the modified curve.

The slope value was nearer to �1.000 (theoretical value)

in the modified curve (�0.89) than in the original one

(�0.757), suggesting an improvement in detection.

To detect Globodera rostochiensis as an internal

standard, real-time PCR was done using the primer set

of PCN280f (50-GCGTCGTTGAGCGGTTGTT-30)

and PCN398r (50-CCACGGACGTAGCACACAAG-30)

(Toyota et al. 2008). Real-time PCR was performed at a

final volume of 10 ml containing 5 ml of a Fast SYBR

Green Master Mix (Life Technologies Japan, Tokyo,

Japan), 0.1mgml�1 of BSA, 5 mM of each primer and 2 ml

of template DNA under the manufacturer’s recom-

mended conditions [95�C for 20 sec, (95�C for 3 sec

and 60�C for 30 sec)�40 cycles].

Estimation of damage to radish caused byPratylenchus penetrans

Harvested radishes (10 to 15 per plot) were washed with

running water, and the number of spots caused by

Pratylenchus penetrans infection was counted by visual

inspection. When many spots (more than 50) were

observed, the number was regarded as 100.

Comparison of the ratio of plots with nodamage to radish among different densitylevels of Pratylenchus penetrans estimated bythe Baermann and combination methods

Radishes with fewer than 10 spots are judged as

marketable according to the standard set by the Japan

Agricultural Cooperatives in Miura, Kanagawa

Prefecture. The density of Pratylenchus penetrans esti-

mated by the Baermann and the combination methods

was graded. The total number of plots and the number of

plots with no severe damage (less than 10 spots per

radish) were determined for each density level of

P. penetrans.

Data analysis

The correlation analysis between the density of

Pratylenchus penetrans and the number of spots per

radish, as well as a t-test, was conducted using Excel

Statistics 2002 for Windows (Social Survey Research

Information, Tokyo, Japan).

RESULTS

Comparison of the density of Pratylenchuspenetrans estimated by the combination andBaermann methods

In the case of the Baermann method, the number of plots

without Pratylenchus penetrans was 15 out of 26, using

soil samples collected from a depth of 0 to 30 cm. The

number was 18 when soil samples from a depth of 30

to 60 cm were used. In contrast, in the case of the

combination method, the number of plots without

P. penetrans was six for soil samples from a depth of

0 to 30 cm and five for soil samples from a depth of 30 to

60 cm. Putting together the results of soil samples from

0 to 30 cm and from 30 to 60 cm, P. penetrans was not

detected in 13 plots by the Baermann method and in only

two plots by the combination method.

The densities of Pratylenchus penetrans estimated by

the two methods were compared (Fig. 1). The density

estimated by the combination method was higher than

that by the Baermann method in most of the soil samples,

39 among 52, in which P. penetrans was detected. In nine

samples, no P. penetrans was detected by either method.

However, in four samples, M12, M13, M14 and T7, the

density estimated by the combination method was lower

than that by the Baermann method (Fig. 1B).

The economic threshold of P. penetrans 215

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The moisture contents of air-dried soil samples of plots

M12, M13, M14 and T7 were 14.6� 0.1% [mean�

standard deviation (SD)], 12.7� 0.2%, 12.6�0.1% and

12.6� 0.4%, respectively, while those of T1, T3 and T4,

in which the density of Pratylenchus penetrans detected

by the combination method was the same as or higher

than that by the Baermann method (Fig. 1A and B), were

11.7� 0.2%, 10.7�0.1% and 10.9� 0.1%, respectively

(Table 1). The average moisture contents of M12, M13,

M14 and T7 were significantly (t-test, P < 0.01) higher

than those of T1, T3 and T4.

Relationships between the density ofPratylenchus penetrans and damage to radish

There were no significant correlations between the

density of Pratylenchus. penetrans at a depth of 0 to

Figure 1 Comparison of the density of Pratylenchus penetrans estimated by the combination method or the Baermann method.(A) All data, (B) macrograph of the gray area in Fig. 1A. T1, T3, T4, T7, M12, M13 and M14 in the graph are the plots in which themoisture contents of air-dried soils were determined, as shown in Table 1. Dotted line in the graph shows the 1:1 ratio of thecombination method and the Baermann method.

Table 1 Comparison of the moisture contents of air-driedsoils

Group Soil IDMoisture

content (%) t-test

C < By M12 14.6� 0.1 P < 0.01M13 12.7� 0.2M14 12.6� 0.6T7 12.6� 0.4

C�Bz T1 11.7� 0.2T3 10.7� 0.1T4 10.9� 0.1

yC < B indicates that the density of Pratylenchus penetrans esti-

mated by the combination method was less than that by the Baermann

method.

zC�B indicates that the density of P. penetrans estimated by the combination

method was not less than that by the Baermann method.

216 E. Sato et al.

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60 cm estimated by both methods and the number of

spots per radish (Fig. 2A).

The ratio of the plots with no serious damage was

dependent on the density of Pratylenchus penetrans

(Tables 2 and 3). The highest ratio was 86% and 87% at

the density of P. penetrans below 0.2 individuals and 5.3

J2 equivalent 20 g�1 of soil by the Baermann method and

the combination method, respectively. The ratio

decreased as the density of P. penetrans increased in

the Baermann method. In contrast, the ratio was more

than 80% in the density range of less than 2 to less than

5.7 in the combination method.

DISCUSSION

Recently, we reported a direct quantification method for

Pratylenchus penetrans in soil. The method, which we

call the combination method, consists of soil compaction,

subsequent DNA extraction and real-time PCR (Sato

et al. 2010). In the current study, densities of

P. penetrans in the soils of radish fields were estimated

by this novel detection method and the Baermann

method before radish cultivation, and the relationship

between density of P. penetrans and damage to radish

caused by P. penetrans was evaluated in order to

determine the ET in radish cultivation.In 75% of soil samples [39 out of 52 soil samples

(26 plots�2 depths)], the densities of Pratylenchus

Figure 2 The relationship between the initial density of Pratylenchus penetrans in 0—60 cm deep soil estimated by the Baermann orcombination method and the number of spots per radish at harvest. (A) All data, (B) macrograph of the gray area in Fig. 2A. Dottedline in Fig. 2B shows the standard (10 spots/radish) for radish of marketable quality set by the Japan Agricultural Cooperatives inMiura.

Table 2 Number and ratio of plots with no serious damage(fewer than 10 spots per radish) in plots with each populationrange of Pratylenchus penetrans as estimated by the Baermannmethod

Density of P. penetrans/dry 20 g soil

�0.2 �0.3 �0.5 �0.8 �1.8 �2.4 �4.5

0� d < 10y 12 14 14 15 15 15 16Total 14 18 19 21 23 25 26Ratio (%) 86 78 74 71 65 60 16

yThe number of spots on a radish caused by P. penetrans is represented as d.

The economic threshold of P. penetrans 217

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penetrans estimated by the combination method were

higher than those estimated by the Baermann method

(Fig. 1). There were only 11 soil samples in which no

P. penetrans was detected by the combination method,

while no P. penetrans was detected in 33 soil samples by

the Baermann method. This result indicates that the

combination method enables the detection of nematodes

not extracted by the Baermann method. Some forms of

live nematode, such as P. penetrans eggs, are not

extracted by the Baermann method; in fact, the extrac-

tion efficiency of the Baermann method is roughly 50%

(Ingham 1994). In contrast, in the combination method,

all nematode bodies are destroyed by compaction, and

their DNA is released, extracted and quantified by real-

time PCR (Goto et al. 2009; Sato et al. 2010). Therefore,

the detection efficiency of the combination method is

higher than that of the Baermann method.

There is a significant correlation between the popula-

tion density of Pratylenchus penetrans in soil and the

degree of damage on the host (Castillo and Vovlas 2007;

Sato et al. 2009). However, there were no significant

correlations between the number of spots per radish and

the density of P. penetrans in soil as estimated by both

methods in the current study (Fig. 2). The maximum

density of P. penetrans in the Baermann method was 4.3

individuals 20 g�1, and thus the range (0—4.3) of

nematode density was very narrow compared with that

found in the previous study (0—90) (Sato et al. 2009).

The number of spots per radish also differed between the

previous study (0—600) and the current study (0—120).

There was no significant relationship between the nem-

atode density and damage to radish in the density range

of zero to four individuals 20 g�1 even in the previous

study. Thus, the main reason for the lack of a significant

correlation observed in the current study may be the

narrow range of P. penetrans density in soil and damage

to radish. The soil nematode community structure can

affect the dynamics of plant-parasitic nematodes

(Sanchez-Moreno and Ferris 2007) and damage to

radish (Sato et al. 2009). In addition, climate, topogra-

phy and physicochemical properties of soil have intricate

effects on crop damage, which might have resulted in the

lack of a significant correlation between the nematode

density and damage to radish in the current study.

In our previous report (Sato et al. 2009), no damage to

radish was observed in soils with 0—2.5 individuals of

Pratylenchus penetrans 20 g�1 of 0 to 60 cm soil as

estimated by the Baermann method. However, according

to the ET (less than 2.5 P. penetrans: Sato et al. 2009),

the ratio of plots with no damage to radish was only

60% (15 out of 25 plots) in this study. This result was

considered low, suggesting that the ET of P. penetrans

calculated by the Baermann method must be much lower

than 2.5 J2.

According to the empirical standard set by the Japan

Agricultural Cooperatives in Miura, Kanagawa

Prefecture, a radish of marketable quality must have

fewer than 10 spots. Based on this standard, the ratio of

the plots with no serious damage (fewer than 10 spots)

was calculated for each density range of Pratylenchus

penetrans to evaluate the reliability of the ET (Tables 2

and 3). The ratio of plots with no serious damage was

86% when the ET was set tentatively at 0.2 individuals

based on the Baermann method, 78% at 0.3 individuals,

74% at 0.5 individuals and 71% at 0.8 individuals of

P. penetrans 20 g�1 dry soil. Densities of P. penetrans

with 0.2 to 0.5 individuals may not be suitable as an ET,

since these densities are too low to count with reliability.

In the case of plot M8, heavy damage (53 spots) was

observed on a radish even though no P. penetrans was

detected by the Baermann method. This result suggests

that it is difficult to set an ET with low density for

P. penetrans based on the Baermann method.

In the combination method, the ET was determined at

5.3 J2 equivalents of Pratylenchus penetrans per 20 g soil

based on the ratio of plots with no serious damage (87%,

13 out of 15 plots). In the exceptional two plots in which

serious damage was observed, the density of P. penetrans

could have been underestimated due to technical errors

in the combination method. One possible cause of the

underestimation of nematode density would be the

degradation of nematode DNA after soil sampling.

Therefore, the ET based on the combination method

Table 3 Number and ratio of plots with no serious damage (fewer than 10 spots per radish) in plots with eachpopulation range of Pratylenchus penetrans as estimated by the combination method

Density of P. penetrans/dry 20 g soil

�1 �2 �3 �4 �5.3 �5.7 �8 �10 �20 �60

0� d < 10y 7 10 11 12 13 13 13 13 14 16Total 9 12 13 14 15 16 17 19 23 26Ratio (%) 78 83 85 86 87 81 76 68 61 62

yThe number of spots on a radish caused by P. penetrans is represented as d.

218 E. Sato et al.

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could most probably be estimated at 5.3 J2 equivalent

20 g�1 dry soil in the present study.

To avoid such degradation of nematode DNA, soil was

air-dried immediately after sampling. However, part of

the nematode DNA could have degraded during the

process of air-drying. In four soil samples, the estimated

densities of Pratylenchus penetrans were lower in the

combination method than in the Baermann method. The

moisture contents of these four air-dried soil samples

were significantly higher than those in samples in which

the densities of P. penetrans estimated by the combina-

tion method were not less than those by the Baermann

method (Table 1). Although all the soils were air-dried

and then stored similarly, their moisture contents differed

depending on the samples. For the air-drying process, we

spread the soil as thinly as possible on trays. However,

our preliminary study showed that the Ct values were 1.4

cycles higher when soil with a high moisture content

(40%) was air-dried for four days compared with the Ct

values obtained using the same soil dried in an oven at

40�C for one day (data not shown). This result suggests

that part of the P. penetrans DNA was degraded in the

process of air-drying, causing the Ct values to become

higher. According to Min et al. (2011), c. 80% of

Meloidogyne incognita DNA degraded in a sandy soil

when the dead nematodes were inoculated and incubated

for two days. Thus, part of the DNA from P. penetrans

that died during the air-drying process might have been

decomposed by surviving soil microbes when soils with

high moisture contents were air-dried insufficiently.

Another possibility is that the storage condition caused

the underestimation of nematode density. Sato et al.

(2010) reported that DNA originating from P. penetranswas stably detected in soil samples which had been stored

at room temperature for no less than five months,

suggesting that nematode DNA may remain stable in air-

dried soil even at room temperature. However, the

moisture contents of the soils used in this study ranged

from 10.7% to 14.6% (Table 1), higher values than

those (9.2%) found in our previous study. It is possible

that the DNA degradation rate is higher under higher

moisture conditions even if all the soils have been air-

dried in advance. The fact that a significant difference

was observed in the moisture contents between two

sample groups (Table 1) supports this speculation. In

the current study, the soils were air-dried as soon as

possible after sampling, and air-drying was continued for

two to three weeks at room temperature. Thus, we

cannot deny the possibility of the degradation of

nematode DNA during this process. The time and

temperature of air-drying and storage might not have

been suitable. In a future analysis, soil will be dried in an

oven immediately after sampling and then stored in a

freezer.

In conclusion, the combination method enabled more

sensitive detection of Pratylenchus penetrans in soil. The

ET of P. penetrans was estimated at 5.3 J2 equivalent

20 g�1 of soil based on the combination method consist-

ing of soil compaction, DNA extraction and real-time

PCR. The method will be a powerful tool for the

diagnosis of P. penetrans in radish fields.

ACKNOWLEDGMENTS

The authors thank radish farmers in Tokyo forallowing them to do soil sampling and damagecheck. This work was supported by Research andDevelopment Projects for Application in PromotingNew Policy of Agriculture, Forestry and Fisheries(21008).

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