Biocatalysis and Agricultural Biotechnology 1 (2012) 8588

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Biocatalysis and Agricultural Biotechnology

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A simple method for production of uniform inoculum of Rhizoctonia solani

with strong pathogenicity
Yun-Nung Tsai, Mei-Ju Lin, Wen-Hsiung Ko n
Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan

a r t i c l e i n f o

abstract

Article history:
Received 24 February 2011
Received in revised form
26 June 2011
Accepted 17 August 2011
Available online 30 August 2011

A simple method for production of uniform inoculum of Rhizoctonia solani AG 1-IC was developed by
colonization of autoclaved rape seeds with the fungus. When ve radish seedlings in a pot were
inoculated with two colonized seeds, R. solani was able to kill essentially all the seedlings. The pathogen
in one seed was able to cause death of more than two seedlings. Such colonized seeds have greater
infection potential than sclerotia on the basis of number and weight. Rape seeds colonized with AG 2-1,
AG 4 or AG 4 HG-1 were also effective in causing damping-off of radish seedlings. When colonized with
R. solani AG 1-IC, other seeds of brassica including mustard cabbage, cauliower, broccoli, spoon
cabbage, head cabbage and Chinese cabbage were as pathogenic as colonized rape seeds. The technique
is inexpensive and simple, and the material is readily available. Inoculum produced by this method is
uniform in shape and size, and is strong in pathogenicity. The result also shows that the method is
suitable for use in the detection of soils suppressive to the disease caused by R. solani.
& 2011 Elsevier Ltd. All rights reserved.

Keywords:
Uniform inoculum
Rhizoctonia solani
Colonized rape seed
Pathogenicity

1. Introduction
Rhizoctonia solani is an important soil-borne plant pathogen
causing diseases in a wide range of crops worldwide (Anderson,
1982; Adams, 1988). For ecological and pathological studies of a
fungal pathogen, it is important to use uniform and reliable source
of inoculum which closely resembles that existing in nature.
Sclerotia have been reported to be the main survival structure of
R. solani in soil (Baker and Martinson, 1972; Summer, 1996; Keijer,
1996). In culture, most sclerotia of R. solani are roughly spherical and
variable in size, ranging from barely visible to 8 mm in diameter
(Summer, 1996; Sneh et al., 1991) with most of them smaller than
2 mm in diameter (Naiki, 1978). It is, therefore, difcult to obtain
uniform sclerotia of R. solani for research. Moreover, some isolates of
R. solani are not capable of producing sclerotia in culture (Naiki,
1978; Luttrell, 1962). Grains of oat, wheat, barley and rice colonized
by R. solani have been used as inoculum (Luttrell, 1962; Sneh, 1986;
Grosch, 2006; Paula, 2008. However, these grains have elongated
shape and their size is relatively large in comparison with that of
sclerotia of R. solani. Mycelium discs and infected cornmeal-sands,
which do not resemble that existing in nature, also have been used
as inoculum of R. solani (Otten et al., 2003; Rollins et al., 1999).
Brassica seeds that are similar to sclerotia of R. solani in size, are
spherical and uniform. R. solani-colonized rape seeds were tested
and found to be an ideal source of inoculum for pathological and

Corresponding author. Tel.: 886 4 22840780x371; fax: 886 4 2285 0773.

E-mail address: kowh@dragon.nchu.edu.tw (W.-H. Ko).

1878-8181/$ - see front matter & 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bcab.2011.08.006

ecological studies. Similar results were obtained with different

kinds of brassica seeds and the technique was applicable to various
anastomosis groups of R. solani. Details of the study are reported
herein.

2. Materials and methods

2.1. Sources of R. solani and sclerotium formation
Isolates of R. solani used were described previously (Liu et al.,
2010, 2011). Cultures were maintained on potato dextrose agar
(PDA) and stored in sterile distilled water in test tubes at 24 1C
(Ko et al., 2010).
For production of sclerotia, four culture blocks of R. solani AG
1-IC (ca. 5  5  3 mm) were placed on a 7 cm cellophane laid on
the 1% V-8 agar (1% V-8 juice and 2% agar) plate. After inoculation
at 24 1C with light for 12 h, the culture blocks were removed with
a pair of forceps and colonies from two plates were transferred to
25 ml of 5% V-8 juice medium in a 250 ml ask. After 48 h, the
mycelium mat was transferred to a sterile 150 mm sieve and
washed with 200 ml sterile distilled water before being transferred to an empty Petri plate to induce sclerotium formation.
Sclerotia were used after 7 days of incubation.
2.2. Preparation of uniform inoculum
Approximately 0.3 g of rape seeds and 0.6 ml distilled water in
a test tube (1.5  5 cm2) was sterilized by 15 min of autoclaving.

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Y.-N. Tsai et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 8588

Excess water was drained and each tube was inoculated with a
piece of culture block of R. solani AG 1-IC. After 3 days, test tubes
were shaken by tapping with ngers and further incubated for
4 days to allow uniform seed colonization by the fungus. The
same protocol was used to colonize rape seeds with other
anastomosis groups of R. solani and to colonize other kinds of
brassica seeds with R. solani AG 1-IC.
2.3. Pathogenicity test
Radish (Raphanus sativus) seeds were placed on moistened
cotton and covered with two layers of cheesecloth in a 15 cm
Petri plate. After 24 h, ve germinated seeds were planted in a
5 cm pot containing a mixture of perlite, vermiculite and peat
moss (1:1:3, v/v/v) at equal distance away from each other and
about 6 mm from the center. Two colonized seeds were placed on
the center of each pot after seedling emergence 24 h later. Pots
were kept moist and damping-off incidence was recorded after
5 days. Pots similarly inoculated with autoclaved seeds were used
as the control. All the experiments were repeated at least twice
with similar results.

Table 1
Damping-off incidence of radish seedlings inoculated with
rape seeds colonized by Rhizoctonia solani AG 1-IC under
different temperatures.
Temperature (1C)

Damping-off incidence (%)n

16
20
24
28
Control

477 12b
877 23ab
937 12a
737 23ab
07 0c

n
Data7 standard deviation represents the means of
three replicates. Value followed by the same letter are not
signicantly different using Fishers protected least signicant difference test at P 0.05.

Table 2
Damping-off incidence of radish seedlings inoculated with
rape seeds colonized by Rhizoctonia solani for different
periods of time.
Colonization period
(days)

Damping-off incidence
(%)n

3
5
7
14
21
28
Control

70 7 25a
85 7 19a
90 7 10a
90 7 10a
90 7 10a
1007 0a
07 0b

2.4. Detection of disease suppressive soil

Soil samples from various locations in Taiwan were taken from
a depth of 010 cm, sifted, moistened to 65% water holding
capacity and stored in plastic bags at 24 1C until use. Soil pH
was determined with a pH meter after mixing soil with water
(1:1, w/v). The experiments were done at least twice for each soil.
2.5. Data analysis
Difference between damping-off incidence of radish seedlings
caused by sclerotia of R. solani and R. solani-colonized rape seeds
was compared using the Students t-test. The other results from
individual experiments were subjected to ANOVA. Means of treatments were separated by Fishers protected least signicant test at
P0.05. Analyses were conducted with SPSS software (SPSS 6.1.3
for Windows). For the relationship of pH and damping-off incidence caused by R. solani, curve tting by nonlinear regression was
performed using Sigma plot for Windows (version 10.0).

3. Results
To determine the temperature most suitable for colonization,
rape seeds inoculated with R. solani AG 1-IC were incubated at 16,
20, 24, or 28 1C for 7 days before pathogenicity tests. The result
showed that damping-off incidence of radish seedlings was
highest when seeds were colonized at 24 1C followed by 20 and
28 1C. The disease incidence was lowest when seeds were colonized at 16 1C (Table 1). The colonization temperature of 24 1C
was, therefore, selected for subsequent studies.
For testing the period needed for colonization, rape seeds were
inoculated with R. solani AG 1-IC and incubated at 24 1C for 3, 5, 7,
14, 21 or 28 days before pathogenicity tests. When rape seeds
were colonized for 7 days or longer, the disease incidence reached
90100%. Even when colonization period was only 3 or 5 days,
7085% radish seedlings were killed (Table 2). For convenience
and consistence, colonization period of 7 days was selected for
further studies.
The minimum number of colonized seeds needed for high
disease incidence was determined by inoculating ve radish
seedlings in each pot with 1, 2, 4 or 8 rape seeds colonized with
R. solani AG 1-IC. Ranging from 85% to 95% radish seedlings were
killed when each pot was inoculated with 2 or more colonized

n
Data7 standard deviation represents the means of
four replicates. Value followed by the same letter are not
signicantly different using Fishers protected least signicant difference test at P 0.05.

Table 3
Damping-off incidence of radish seedlings inoculated with
different amounts of rape seeds colonized by Rhizoctonia
solani AG 1-IC.
Colonized seed (no.)

Damping-off incidence (%)n

1
2
4
8
Control

537 19b
957 10a
857 10a
907 12a
07 0c

n
Data7 standard deviation represents the means of
four replicates. Value followed by the same letter are not
signicantly different using Fishers protected least signicant difference test at P 0.05.

seeds. Even when each pot was inoculated with one colonized
seed, 53% radish seedlings were killed (Table 3), indicating that
one colonized rape seed can cause death of more than two radish
seedlings in 5 days.
For comparison of infection potential of colonized seeds with
sclerotia, ve radish seedlings in each pot were inoculated with
one sclerotium of R. solani AG 1-IC or one rape seed colonized by
this fungus. The results showed that colonized seeds have much
higher infection potential than sclerotia. Three days after inoculation, only 2% radish seedlings were killed by a sclerotium, while
48% were killed by a colonized seed (Table 4). After 9 days, 48%
radish seedlings were killed by a sclerotium and 94% were killed
by a colonized seed, indicating that in 9 days a sclerotium can kill
an average of 2.4 radish seedlings and a colonized seed can kill
4.7 seedlings. Colonized seeds were relatively uniform in size,

Y.-N. Tsai et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 8588

when compared with sclerotia. The size of sclerotia of R. solani AG

1-IC ranged from 0.40.9 mm in diameter, while that of rape
seeds colonized with this fungus was 1.52.0 mm in diameter
(Fig. 1). The average weight of a colonized rape seed was 3 mg.
When the infection potential of a colonized seed was compared
with that of 3 mg sclerotia, the former caused 42% damping-off of
radish seedlings, while the later caused only 14% seedling death
in 3 days (Table 4). On the 5th and 9th day, the disease incidence
caused by these two types of inoculum was about the same.
To determine if the rape seed colonization method is applicable to other anastomosis groups of R. solani, rape seeds were
colonized with three other anastomosis groups of R. solani and
subjected to pathogenicity tests on radish seedlings. Like AG 1-IC,
all the other groups were able to cause damping-off of radish
seedlings using this method. When each pot was inoculated with
two colonized seeds, AG 2-1 was most effective in causing the
disease followed by AG 1-IC, AG 4, and AG 4-HG-I (Table 5). When
each pot was inoculated with eight colonized seeds, the dampingoff of radish seedlings caused by all the four anastomosis groups
ranged from 80% to 100%.
Other kinds of brassica including mustard cabbage, cauliower, broccoli, spoon cabbage, head cabbage and Chinese
cabbage also produced small and uniform seeds (Table 6). When
they were colonized with R. solani AG 1-IC, all of them were able
to cause damping-off of radish seedlings ranging from 85% to 95%
(Table 6).
Radish seedlings planted in 18 soil samples were tested by
inoculating each pot with two rape seeds colonized with R. solani
AG-1 IC, The damping-off incidence in these soil samples ranged
from 0% to 100% (Fig. 2), indicating that the colonized rape seed

Table 4
Comparison of damping-off incidence of radish seedlings caused by sclerotia of
Rhizoctonia solani and R. solani-colonized rape seeds.
Treatment

Damping-off incidence (%)n

3

9 (days)

One individual/pot
Sclerotium
Colonized rape seed

2 76b
48 721a

147 19b
767 18a

48 733b
94 79a

3 mgnn/pot
Sclerotium
Colonized rape seed

14 726a
42 719b

407 21a
547 16a

70 723a
68 719a

n
Data 7 standard deviation represents the means of 10 replicates. Value
followed by the same letter are not signicantly different using Students t-test
at P 0.05.
nn
Average weight of a colonized seed.

87

method is suitable for use in screening of disease suppressive

soils. There was a direct relationship between damping-off
incidence and soil pH among these soil samples. The regression
equation was Y 17.554X 41.413 and the correlation coefcient
was 0.704, which was signicant at Po 0.0001.

4. Discussion
Sclerotia of R. solani vary in size ranging from o1 mm to 8 mm
in diameter with most of them smaller than 2 mm in diameter
(Summer, 1996; Sneh et al., 1991; Naiki, 1978). Although some
Table 5
Damping-off incidence of radish seedlings inoculated with rape seeds colonized by
Rhizoctonia solani of different anastomosis groups.
Strains

AG
AG
AG
AG
n

1-IC
2-1
4
4 HG-I

Damping-off incidence (%)n

2

8 (no. of colonized seeds)

737 12
1007 0
537 12
277 12

87 7 12
1007 0
807 20
407 20

877 12
1007 0
937 12
807 20

Data 7 standard deviation represents means of three replicates.

Table 6
Damping-off incidence of radish seedlings inoculated with different kinds of seeds
colonized by Rhizoctonia solani AG1 IC.
Scientic name (common name)

Size of seeds
(mm diam.)n

Damping-off
incidence (%)nn

Brassica juncea (mustard cabbage)

B. napus (rape)
B. oleracea ssp. botrytis subgroup
cauliora (cauliower)
B. oleracea ssp. botrytio subgroup
cymosa (broccoli)
B. oleracea ssp. chinensis (spoon
cabbage)
B. oleracea ssp. capitata subgroup
alba (head cabbage)
B. rapa ssp. pekinensis (Chinese
cabbage)
Control

1.52.5
1.52.0
1.82.5

90 720a
90 711a
85 710a

2.02.8

90 711a

1.52.3

90 720a

1.82.5

95 710a

1.52.5

90 711a

0 70b

For each kind of seeds, 50 seeds were measured.

Data 7standard deviation represents the means of four replicates. Value
followed by the same letter are not signicantly different using Fishers least
signicant difference test at P 0.05.
nn

Fig. 1. Comparison of two different kinds of inoculum of Rhizoctonia solani AG 1-IC: (A) Sclerotia, bar 1 mm; and (B) colonized rape seeds, bar 5 mm.

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Y.-N. Tsai et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 8588

due to the presence of free Al ion (Kobayashi and Ko, 1985).

Whether the same mechanism is responsible for suppression of
the disease in the soils obtained in this study remains to be
investigated.

120
Y = 17.554X-41.413
r = 0.704

Damping-off incidence (%)

100
80
60

Acknowledgments

40

We thank Drs. J.W. Huang and L.C. Chen for supplying isolates of
R. solani. This research was supported in part by a grant from the
National Science Council of Taiwan (NSC 97-2321-B-005007-MY3).

20
0

References

5
pH value

Fig. 2. Relation between soil pH and damping-off incidence of radish caused by

Rhizoctonia solani AG 1-IC in 18 soil samples collected from various locations in
Taiwan.

anastomosis groups of R. solani are relatively round, other groups

are irregularly shaped (Sneh et al., 1991). Rape seeds colonized
with R. solani are small like sclerotia of this fungus, and are round
and uniform in size (Fig. 1). They are, therefore, a suitable source
of inoculum for ecological and pathological studies. The uniform
size of colonized rape seeds also makes them ideal for use in the
breeding and genetic engineering for crop resistance to R. solani.
For R. solani AG 1-IC, colonized rape seeds are more pathogenic
than sclerotia on the basis of number and weight, indicating that
the formers have stronger infection potential than the latters.
Since rape seeds colonized with different anastomosis groups of
R. solani and different kinds of brassica seeds colonized by
R. solani AG 1-IC are all capable of causing damping-off of radish,
the technique developed in this study should be useful for
preparation of inoculum for other plant pathogenic fungi. The
advantages of the technique are inexpensive, simple and high
availability of the material. Inoculum produced by this method is
uniform in shape and size, and is strong in pathogenicity.
The result also shows that colonized rape seeds are suitable for
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