Soil Science and Plant Nutrition

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Reduction of resting spore density of

Plasmodiophora brassicae and clubroot disease
severity by liming

Hiroharu Murakami , Seiya Tsushima , Yukiko Kuroyanagi & Yoshihiro

Shishido

To cite this article: Hiroharu Murakami , Seiya Tsushima , Yukiko Kuroyanagi & Yoshihiro
Shishido (2002) Reduction of resting spore density of Plasmodiophora�brassicae and
clubroot disease severity by liming, Soil Science and Plant Nutrition, 48:5, 685-691, DOI:
10.1080/00380768.2002.10409258

To link to this article: https://doi.org/10.1080/00380768.2002.10409258

Published online: 22 Nov 2011.

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Soil Sci. Plant Nutl:, 48 (5), 685 - 691, 2002 685

Reduction of Resting Spore Density of Plasmodiophora brassicae

and Clubroot Disease Severity by Liming

Hiroharu Murakami 1, Seiya Tsushima", Yukiko Kuroyanagi, and Yoshihiro Shishido

National Agricultural Research Center for Tohoku Region. Fukushima, 960-2156 Japan

Received February 7, 20m; accepted in revised form June 28. 2002

Relationships between the disease severity of clubroot caused by Plasmodiophora brassi-

cae, the soil pH value and the concentration of exchangeable calcium associated with liming
were investigated under controlled density of resting spores. Disease indices were lower in
the plots treated with lime than in the control plots without lime application. The disease
index was significantly lower when lime materials were mixed two weeks before sowing
compared with four weeks before sowing. The reduction rate of the disease index was
larger for a concentration of 2.0 g kg- 1 than 1.0 g kg- 1 of lime in soil. The density of the
resting spores in soil at the time of sowing was significantly reduced by liming. The reduc-
tion rate was 17-31 % for calcium cyanamide, 12-29% for dolomite, and 20-39% for calcium
carbonate compared with the control plot. It was suggested that the disease severity was
influenced by both the soil pH and the content of exchangeable calcium in soil based on the
analysis of covariance.

Key Words: exchangeable calcium, liming, Plasmodiophora brassicae, resting spore den-
sity, soil pH.

Clubroot disease is caused by Plasmodiophora brassi-
cae Woron., a soilborne pathogen, and results in a seri-
ous yield loss of crucifers, such as Chinese cabbage
(Brassica rapa var. pekinensis) and cabbage (B. olera-
cea var. capitata).
It is well known that liming used to be one of the
measures for the control of clubroot disease (Larson and
Walker 1934; Walker and Larson 1935; Haenseler 1939;
Motohashi et al. 1956; Kashiwakura et al. 1979; Hira-
sawa et al. 1983; Campbell et al. 1985). Nevertheless,
the effects of liming on the density of resting spores in
soil have never been investigated. We suggested that
previous experiments could not explain why liming was
not always effective because the disease severity de-
pends on both the resting spore density in the soil tested
and the dose-response relationship in a field, referred to
the dose response curve (DRC) , in which the pattern of
DRC is affected by the soil suppressiveness, crop sus-
ceptibility, and the virulence of the pathogen tested
(Murakami et al. 2002).
We analyzed here the effects of lime application on
the disease severity under controlled spore density and
Present addresses: 1 National Institute of Vegetable and Tea Sci-
ence, Tsukuba, 305-8666 Japan. :! National Institute for Agro-
Environmental Science, Tsukuba, 305-8604 Japan.
on the density of resting spores in soil.
MATERIALS AND METHODS
1. Preparation of infested soil. Samples of a
Haplic Andosol (Fukushima) were collected from a field
located at the National Agricultural Research Center for
Tohoku Region (NARCT) and passed through a 5 mrn
mesh sieve before use. Physical and chemical character-
istics of the soil samples are shown in Table 1.
The suspension of resting spores of P brassicae was
prepared with clubbed roots of Chinese cabbage in the
infested field at NARCT by the method of Takahashi
and Yamaguchi (1987). The soil samples were sprayed
with suspensions of resting spores and mixed thorough-
ly to provide a range of 1O~ to 107 resting spores g-l soil
(Murakami et al. 2000b).
2. Application of lime materials. Lime
materials used consisted of calcium cyanamide (pow-
dery type or granular type), dolomite (powdery type or
granular type), and calcium carbonate. Calcium hydrox-
ide was additionally used for the experiment to analyze
the factors related to the reduction of the disease severi-
ty. Granular type of calcium cyanamide and dolomite
were passed through a 2 mrn mesh sieve before use.
686 H. MURAKAMI et al.

Table 1. Physical and chemical characteristics of the 5. Measurement of disease index. Roots of
Haplic Andosol soil sampled in Fukushima, Japan. each Chinese cabbage plant were removed from the pots
Total carbon 43.7 g kg-I and washed gently to determine the disease severity,
Total nitrogen 2.80 g kg-I which was classified into four categories (0 to 3) accord-
C/N 15.5 ing to the following criteria: no clubbing, category 0;
Organic matter content 75.2 g kg-I only lateral roots clubbed, category 1; less than half of
Maximum water holding capacity 681 g kg-I
tap root clubbed, category 2; more than half of tap root
Hydraulic conductivity 2.05 X 10- 3 em S-I
pH(HP) 6.01
clubbed, category 3.
Electric conductivity 0.04 mS em-I Disease index (Dr) for each pot was calculated by the
Cation exchange capacity 26.8 cmol, kg-I dry soil following formula: OJ = ~(i X nJ / (3 X N) X 100, N:
Nitrate-nitrogen 17.0 mg kg-I dry soil total number of plants, n i : number of plants classified
Phosphate (Bray No.2) 150 mg kg-I dry soil into category i.
K+ 0.29 cmol, kg-I dry soil Protective value (PV) was calculated as follows: PV =
Cac+ 8.09 cmo), kg-I dry soil
(CP - TP) / CP X 100, where CP and TP indicate the
Mg2+ 2.92 cmol, kg-I dry soil
Na+ 0.05 cmol, kg-I dry soil
mean of the disease indices for the untreated control plot
AI3+ 10.9 mg kg-I dry soil and treated plots, respectively (Murakami et al. 2001).
Phosphate absorption coefficient 18.2 g kg-I dry soil DIs were analyzed statistically by Tukey's multiple
range test (Sakal and Rohlf 1995). The effects of some
factors: soil pH value, the content of exchangeable calci-
Lime materials were added at a rate of 1.0 g kg-I, 2.0 um in soil and lime material, on Dr were analyzed statis-
g kg-I, and so on into the inoculated soil in a polyethyl- tically by ANalysis of COVAriance (ANCOVA) (Sakal
ene bag and mixed thoroughly. They were packed in and Rohlf 1995).
each of five replicate pots (115 mm dia., 110 mm high) 6. Effect of soil pH on root hair infection.
per treatment plot, put in each container (330 X 330 Chinese cabbage plants were sown two weeks after lim-
mm, 99 mm high) to avoid contamination between ing and were cultivated for 10 d at 23 DC in the soil inoc-
plots. Water was applied through the base of each pot ulated with 107 resting spores g-I (Murakami et al.
and the soil temperature in the pots was maintained at 2000b, 2001). Root hair infection was estimated as the
~25DC by placing them on a thermostatically controlled percentage of root hairs containing zoosporangia in sec-
mat until sowing. tions cut 2 to 3 cm from the hypocotyl according to the
3. Measurement of resting spore density in description given in previous reports (Samuel and Gar-
soil and soil analysis. Soil was sampled from each rett 1944; Channon et al. 1964). The difference in the
pot to measure the density of resting spores, pH, the root hair infection rate between the treatment plot and
content of exchangeable calcium (Ex.-Ca) at the time of control plot was examined statistically by t-test.
sowmg.
The resting spores were recovered from the soil sam-
ples inoculated initially with 106 resting spore g-l RESULTS
(Murakami et al. 2000a) and stained with 0.1 g kg- 1 cal-
cofluor white M2R solution (Takahashi and Yamaguchi 1. Reduction of disease severity by liming
1987). Numbers of resting spores were determined DIs were generally lower in the plots treated with
using fluorescence microscopy with differential phase lime (1.0 g kg-I) 2 weeks before sowing than in the con-
contrast. The reduction of the resting spore density com- trol plots without lime (Fig. 1).
pared with that of the control plot was examined statisti- For an inoculum concentration of lQ4 resting spores
cally by the t-test. g-l soil, DIs were 17 (PY: 64) for granular calcium
Soil pH(H 2 0) was measured, and exchangeable cat- cyanamide, 37 (PY: 23) for powdery calcium cyana-
ions were extracted from air-dried soil with ammonium mide, 31 (PY: 36) for powdery dolomite, and 32 (PY:
acetate prior to the analysis by ICP (Inductively Cou- 32) for calcium carbonate when the DI was 48 for the
pled Plasma). control. In contrast, for granular dolomite, no reduction
4. Plant cultivation. Thirteen seeds of Chinese of the DI was observed.
cabbage (B. rapa var. pekinensis, cv. Shin-Azuma) per For an inoculum concentration of 106 resting spores
pot were sown two or four weeks after the application of g-l soil, the DI values were 84, 56 (PY: 34), 75 (PY:
lime materials and grown for about 35 d using a thermo- 11), 84 (PY: 1), 64 (PY: 25), and 65 (PY: 23) for the
statically controlled mat in a greenhouse. Water or nutri- control, granular calcium cyanamide, powdery calcium
ents also were applied through the base of each pot, cyanamide, granular dolomite, powdery dolomite, and
when required, according to the plant growth. calcium carbonate, respectively.
 Effects of Liming on Clubroot Disease 687

100 / ~.
b
ab
...------::
~
b
-,'
ab ab
'f:
80 a ~ .........----:-
...------: d
x
"
-c
.'
.' i .'
.~ 60 cd
bc ,
~------
"rn ~.'
,. , ab . ~bc
"'"
rn
0
40
a
Fig. 1. Reduction of disease index of
: clubroot disease on Chinese cabbage by
20

a v- ~D-'_ .~

~
-
- . ~ ~(.:. J??o:l 10 4
liming. Control treatment, no liming.
*Resting spores g-I soil. Means with the
control granular powdery granular powdery calcium same superscript were not significantly dif-
Density of
calcium calcium dolomite dolomite carbonate ferent (p < 0.05 l. according to Tukey's
cyanamide cyanamide Inoculum *
multiple range test at each inoculum con-
Lime materials centration.

100
A 104 resting spores g-1 soil

80
f
x
Q)
-c 60 ,--- ef
.~ def
Q) ,--
-
''""
Q) cde bcd
40
0'" ,--- bcd bcd
abc ,-- ,--- ,--
abc
ab ,--
20 -

n,a
a
control granular powdery granular powdery calcium
calcium calcium dolomite dolomite carbonate
cyanamide cyanamide
Lime materials
Concentration of lime mixed; 0 1 g kg' , 0 2 g kg'
100
d B d
6
10 resting spores g-1 soil
,-- cd -
80 cd
- - cd
bc cd cd
x ,--- - -
Q)
-C 60 bc
.~ ,-- ,--
"rn b
'rn"
Q)
40 ,--
0

20
a Fig. 2. Effect of concentration of lime materials on
the disease index of clubroot disease on Chinese cab-
a II bage. A: 104 resting spores g-I soil, B: 106 resting
control granular powdery granular powdery calcium spores g-l soil. Control treatment. no liming. Means
calcium calcium dolomite dolomite carbonate with the same superscript were not significantly dif-
cyanamide cyanamide
Lime materials ferent (p < 0.05), according to Tukey's multiple
Concentration of lime mixed; 0 1 g kg' , 0 2 g kg' range test at each inoculum concentration.

2. Concentration of lime mixed with soil two weeks before sowing.

The reduction of the DI was more conspicuous at a
concentration of 2.0 g kg- 1 of lime than at 1.0 g kg-I 3. Interval between liming and sowing
both for an inoculum concentration of 104 and 106 rest- When the content of lime materials in the soil inocu-
ing spores g-l soil (Fig. 2A B) when lime was mixed lated with 105 resting spores g-l soil was 2.0 g kg-I, the
688 H. MURAKAMI et al.

100
d
d
80 cd

x
~ 60 bc bc
.s: b b
Q)

''""
_~ 40
o
a Fig. 3. Effect of duration of the period
a a
20 of liming on the disease index of clubroot
disease on Chinese cabbage. Control treat-
ment, no liming. Infested soil (l05 resting
o
control 2w 4w 2w 4w 2w 4w 2w 4w 2w 4w
spore g-l soil) with lime at 2 g kg- 1 con-
centration was used. Means with the same
granular calcium powdery calcium granular powdery ca:cium
cyanamide cyanamide dolomite dolomite carbonate superscript were not significantly different
(p < 0.05), according to Tukey's multiple
Lime materials and duration of application period before sowing range test.

100 10
/;'

80 rf- -+- .;; .~)

l' ~
0
~
x
-"i- ~
-+-
~
Q) '1- ~

.s: 60
-a
Q)
-+-
'" Fig. 4. Reduction of the density of resting
"'"
0'"
40 ~

spores in soil and disease index of clubroot

disease on Chinese cabbage. Control treat-
20 ment, no liming. Bars, S.E. Soil inoculated ini-
tially with 106 resting spore g-I soil was used.
r+l Effects of liming, except for I g kg- 1 powdery
0 o dolomite, on the spore density were signifi-
control 2 2 2 2 2
granular powdery granular powdery calcium cantly different (5% level) from the control
calcium calcium dolomite dolomite carbonate treatment. A high cOlTelation coefficient be-
cyanamide cyanamide tween disease indices and resting spore densi-
Lime materials and concentrations (g kg- 1) ties obtained in all the materials tested was not
D Disease index ~ Density of resting spores recognized.

DI showed a more appreciable decrease two weeks
before sowing than four weeks before sowing (Fig. 3).
When lime was applied four weeks before sowing, the
disease was inhibited in the case of powdery calcium
cyanamide application (PV: 77), powdery dolomite (PV:
35), and calcium carbonate (PV: 32), but not in the case
of granular calcium cyanamide (PV: 7) and granular do-
lomite (PV: -7). When lime was mixed to the soil two
weeks before sowing, the PV values were the highest;
78 for granular calcium cyanamide, 90 for powdery cal-
cium cyanamide, 30 for granular dolomite, 34 for pow-
dery dolomite, and 68 for calcium carbonate.
4. Reduction of spore density in soil
When the density of resting spores in soil was investi-
gated two weeks after lime was mixed with the soil (10 6
resting spores g-l), the reduction rates of the resting
spore density were 17-31 % for calcium cyanamide,
12-29% for dolomite, and 20-39% for calcium carbon-
ate compared with the control (Fig. 4).
For each material, whenever the increase in the mix-
ing rate of the lime materials and, consequently, the re-
duction in the resting spore density in soil were ob-
served, the disease index decreased (Fig. 4).
5. Effects of liming on root hair infection
The percentage of root hair infection did not show any
significant differences between the control (26%) and
lime-treatment plots; granular dolomite (23-27%) and
calcium carbonate (21-24%), but was lower for pow-
dery and granular calcium cyanamide (13-18%), and
powdery dolomite (19%) than for the control (Fig. 5).
6. Relationships between DI values, soil pH,
and the content of exchangeable calcium
The relationships between the DI values, soil pH, and
the content of exchangeable calcium (Ex.-Ca) in soil at
the time of sowing were investigated in inoculated soil
 Effects of Liming on Clubroot Disease 689

40

§
c
0
.+' 30
....."
Q)
~

.'·iii...." ~

.---- ,---
..<::
20 * * * * ~
+-'
.---- .----
0
e * - .----
..... * ~
0
Q)
.----
bIJ
+-'
C '"
Q)
10

f:Q)
CL

0 Fig. 5. Effects of liming on root hair infec-

control 2 2 2 2 2
tion. Control treatment. no liming. Soil inocu-
granular powdery granular powdery calcium
calcium calcium dolomite dolomite carbonate lated with 10 7 resting spore g-l soil was used.
cyanamide cyanamide Means of root hair infection with asterisks
were significantly different (5% level) from
Lime materials and concentrations (g kg- 1) the control treatment.

100 100
A B
80 80

x
Q)
"0
C
60 .... • xQ)
"0

.'"
60 • •
Q) A

••
Q)

'"ell '" 4)
Q)

0'" 40 0
ell
Q)

0'" 40
• 0
>f >s /':,
2 X
/':, /':,
0 0 0
20 /':,
•
0 20 /':,
•
0
• 0
0
.0

5.5 6.0 6.5 7.0 7.5 6.0 6.5 7.0 7.5 8.0 8.5
Concentration of exchangeable calcium
Soil pH at the time of sowing 1
at the time of sowing (cmol c kg- dry soil)

Fig. 6. Effects of the soil pH and the content of exchangeable calcium on the disease index. A: soil pH. B: exchangeable calcium.
Soil inoculated with 10" resting spore g-l soil was used. The value of the disease index was significantly influenced by both the pH
and the content of exchangeable calcium according to ANCOVA. ., control; ., granular calcium cyanamide; 0, powdelY calcium
cyanamide; &, granular dolomite; "'-, powdery dolomite; x, calcium carbonate.

(104 resting spores g-l soil) treated with lime materials. rate, though the increase of the soil pH and the content
The reduction of the disease severity was found to be of Ex.-Ca was negligible. For calcium carbonate, the 01
influenced not only by the pH but also by the content of values were almost the same in spite of the increase of
Ex.-Ca based on ANCOVA (p = 0.05) (Fig. 6A, B). the soil pH and the content of Ex.-Ca along with the
The relationships between the 01 values, soil pH, and increase of the mixing rate. On the contrary, for calcium
the content of Ex.-Ca, additionally, were investigated in hydroxide, the 01 decreased with the increase of the soil
soil 006 resting spores g-l soil) treated with the four pH and the content of Ex.-Ca. In these results, the
lime materials and three concentrations of each material ANCOVA shown in Fig. 7 revealed a significant interac-
(Fig. 7). For calcium cyanamide, both powdery and tion between the lime materials and the soil pH / Ex.-Ca
granular types, the soil pH and the content of Ex.-Ca (p = 0.05), implying that the effectiveness of the soil
increased with the increase of the mixing rate, whereas pH / Ex.-Ca ratio on the suppression of the clubroot dis-
the 01 decreased with the increase of the rate. For dolo- ease may differ among these materials (Table 2).
mite, the 01 decreased with the increase of the mixing
690 H. MURAKAMI et al.

100 8.5
• • •
II

80 • 8.0
Oil
4- .~
o ~

f "
7.5 ..3 ~
0

6
:l
""0
.~
:l]
nl
Ql
60

40
•
! 7.0
nl

g '0
0

" "
""0

~~
0

~Ql :JE
n;
Ql

6.5 :r:0. "~

=-= [IJ
0'<:
C/J C)
X
Fig. 7. Relationships between the
20 Ql
disease index of clubroot disease, soil
6.0
pH. and the content of exchangeable
calcium at the time of sowing. Soil
a inoculated with 106 resting spore g-l
oontrol 0.5 1 2 0.5 1 2 2 3 2 2 3 0.5 1
granular powdery granular powdery calcium calcium soil was used. Bars, S.E. Significant
calcium calcium dolomite dalomrr.e carbonate hydroxide
cyanamide cyanamide interactions between lime materials,
Lime materials and oonoentrations (g kg-I) soil pH and exchangeable calcium
o Soil pH 0 Conoentration of exohangeable oaloium ComO" kt dry soil) • Disease index were recognized by ANCOVA.

Table 2. Analysis of covariance of disease index as a in the granular type than the powdery type on the whole.
function of lime materials, soil pH. and the content of ex- It was reported that the disease severity decreased when
changeable calcium. the particle size of calcium carbonate decreased, result-
Degree of Sum of ing in a pH increase (Dobson et al. 1982). The current
Source freedom squares F p experiment suggested that the effects of dolomite were
(df) (SS)
caused by the same factors as those of calcium carbon-
Treatment 3 2,936 4.49 0.Q25 ate, but in the case of calcium cyanamide, factors other
pH I 7,018 34.75 0.000 than the effects of particle size were considered to be
Treatment X pH 3 3,246 5.36 0.019
2,299
involved.
Error 10
As the DI showed a reduction exceeding the estimate
Treatment 3 1,937 5.07 0.022
Ex.-Ca 1 6.195 48.68 0.000 based on DRC and the spore density in the case of calci-
Treatment X Ex.-Ca 3 3,241 8.49 0.004 um cyanamide, it was considered that not only the
Error 10 1,273 reduction of the density of resting spores but also other
factors; e.g. effect of pH increase, fungistatic factor, etc.,
affected the reduction of the disease severity. The soil
DISCUSSION pH was suggested to be one of the factors as mentioned
above, because the percentage of root hair infection
The reduction of the DI by liming was influenced by decreased with increasing pH (Murakami et al. 2002),
the density of resting spores in soil, for instance, the PV and, moreover, the percentage of root hair infection in
decreased when the concentration of the inoculum in- the soil treated with calcium cyanamide was slightly
creased. Though the density of spores in soil had never lower than that for other materials. These results are in
been measured directly in field experiments previously agreement with those reported by Naiki and Dixon
reported in which liming was not always effective, these (1987) who showed that the germination of resting
results were ascribed to the differences in the density of spores was inhibited by calcium cyanamide in vitro.
resting spores in soil. This fact suggests that the spore However, the relationship between the percentage of
density should be measured when not only liming but root hair infection and the DI was not clearly observed,
also other control measures are planned. because the DI was not always proportional to the per-
The reduction of the density of resting spores by lim- centage of root hair infection (Naiki et al. 1978, 1984).
ing was recognized for the first time in this report. It was Moreover, cyanamide, contained in calcium cyanamide,
obvious that liming was a useful control method not could have influenced P brassicae directly, for instance,
only for the inhibition of the disease but also for the by the inhibition of germination of resting spores. These
reduction of the spore density. facts suggest that the mechanism of disease suppression
In the case of dolomite, the reduction of the DI was by calcium cyanamide should be further elucidated.
larger in the powdery type than in the granular type, Cyanamide is used as a herbicide, insecticide, nemati-
whereas in the case of calcium cyanamide it was larger cide, and fungicide (Cornforth 1971) and could be a
 Effects of Liming on Clubroot Disease
suitable control material for IPM of clubroot disease.
The relationships between the pH. the calcium con-
tent, and the disease severity were also reported previ-
ously in vitro or in the field. In the sand culture method,
root hair infection and club formation were reduced
when the value of the soil pH exceeded 7.2. For a low
density of resting spores, the germination of resting
spores was suppressed by calcium (Myers and Campbell
1985). At pH 7.2, the reduction rate of the DI increased
with the increase of the concentration of calcium, while
only a high concentration of calcium suppressed the dis-
ease at pH 6.2. Root hair infection was more suppressed
by the increase of the pH and the concentration of calci-
um (Webster and Dixon 1991). In field experiments.
Fletcher et al. (1982) reported that though calcium was
related to the disease severity. the pH played a major
role in the severity of the disease. These results suggest-
ed that calcium and the pH were associated with the
development of the symptoms.
Base on our results, it was suggested that the reduc-
tion of the disease severity was influenced by both the
soil pH and the content of Ex.-Ca and moreover, their
effects on the suppression were different among the
materials when the interaction between lime materials
and soil pH / Ex.-Ca was subjected to ANCOVA.
Acknowledgments. We thank Dr. K. Yamamura (National In-
stitute for Agro-Environmental Science. Tsukuba, Japan) for
suggesting the use of ANCOVA and Dr. N. Matsumoto (National
Institute for Agro-Environmental Science, Tsukuba. Japan) for
the critical reading of the manuscript.
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