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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: [email protected] 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
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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.
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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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