Journal of Integrative Agriculture 2019, 18(9): 2072–2079

Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Evaluating effective Trichoderma isolates for biocontrol of Rhizoctonia

solani causing root rot of Vigna unguiculata

WANG Chao1, 2, ZHUANG Wen-ying1

1
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R.China
2
University of Chinese Academy of Sciences, Beijing 100049, P.R.China

Abstract
The highly diverse genus Trichoderma has provided many formulations that are alternatives to the chemical pesticides
in agriculture. The present study was undertaken to investigate the biocontrol potential of eight Trichoderma species,
T. atrobrunneum, T. guizhouense, T. paratroviride, T. pyramidale, T. rufobrunneum, T. simmonsii, T. thermophilum and
T. viridulum, against the phytopathogenic fungus Rhizoctonia solani. Trichoderma isolates were first evaluated in vitro by
dual culture tests for their antagonism, mycoparasitic ability and antifungal activity against R. solani. Their growth promoting
potential was further assessed in relation to phosphate solubilization, indole acetic acid and siderophore production. Five
of the isolates were selected and evaluated for their abilities to prompt plant growth and to control R. solani infecting
Vigna unguiculata (cowpea) seedlings in vivo. Two most effective isolates, T. guizhouense 9185 and T. simmonsii 8702,
significantly (P<0.05) reduced the disease severity incidences (36.6 and 45.0%, respectively) and promoted plant growth,
which have good prospects for application.

Keywords: antagonism, growth promoting, hydrolytic enzymes, mycoparasitism

and damping-off of young plants, and sore shin with lower

1. Introduction
Rhizoctonia solani is an important destructive soil-borne
phytopathogenic fungus that causes severe loss of
agricultural crops around the world. As a necrotrophic
pathogen, R. solani extracts nutrition from dead tissues after
killing plant cells, which causes seed decay, stem/root rot
Received 4 July, 2018 Accepted 7 December, 2018
WANG Chao, Tel/Fax: +86-10-64807326, E-mail: siraowang@
foxmail.com; Correspondence ZHUANG Wen-ying, Tel/Fax: +86-
10-64807326, E-mail: zhuangwy@im.ac.cn
© 2019, CAAS. Published by Elsevier Ltd. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
doi: 10.1016/S2095-3119(19)62593-1
stem root of older plants (Huang et al. 2011; Jaiswal et al.
2014). In addition, persisting as mycelium or sclerotia,
R. solani is able to survive in harsh condition for years.
The disease control relies primarily on chemical pesticides
belonging to the carboxamides, benzimidazoles, carbamates
and dicarboximides classes (Tredway and Burpee 2001).
Although these chemical compounds have given successful
results, they are surely not long-term solutions because of
the adverse effects to environmental and human health
(Monfil and Casas-Flores 2014). Therefore, exploring for
eco-friendly, safe, long-lasting and effective ways to protect
crops from pests and phytopathogens becomes imperative
(Boukaew et al. 2013), and biological control should be the
main focus.
Trichoderma species, especially those in the Viride and
Harzianum clades, are ubiquitous and easily isolated from
 WANG Chao et al. Journal of Integrative Agriculture 2019, 18(9): 2072–2079
soil, decaying wood, plant roots and leaves and other plant
organic matters (Howell 2003), even fruiting bodies of other
fungi (Chaverri and Samuels 2013). Trichoderma asperellum
and T. harzianum are among the most outstanding biological
control microbes in agriculture due to their benefits to plant
health, especially the high capacities against a wide range of
phytopathogenic microorganisms, including fungi, bacteria,
oomycetes and viruses (Harman et al. 2004; Woo et al.
2014; Samuels and Hebbar 2015; Sarrocco et al. 2017).
It has been well-demonstrated that Trichoderma species
display five main mechanisms including mycoparasitism
through secretion of hydrolytic enzymes, competition for
nutrient and space, antibiosis by production secondary
metabolites, promotion of plant growth, and inducing plant
systemic resistance reactions (Kubicek et al. 2011; Monfil
and Casas-Flores 2014).
So far, more than 340 Trichoderma species have been
described (Bissett et al. 2015; Qin and Zhuang 2015; Zhu
and Zhuang 2015; Chen and Zhuang 2016; Qin and Zhuang
2016a, b; Chen and Zhuang 2017a, b, c; Qin and Zhuang
2017), which constitute a vast pool of microbes with potential
to protect crops against phytopathogenic fungi. Up to now,
the most commonly used Trichoderma species as biocontrol
agents (BCAs) are T. asperellum, T. atroviride, T. harzianum
and T. polysporum (Woo et al. 2014; Samuels and Hebbar
2015), and only very few of them were reported with the
application potential against R. solani (Woo et al. 2014).
Therefore, exploration of effective Trichoderma resources
against R. solani is worthy and urgent. This study is aimed
at evaluation of biological control abilities of 25 isolates
belonging to eight Trichoderma species against R. solani.
The isolates were assessed in vitro for their antagonism
against R. solani and promotion of plant growth, and
then five selected isolates were further evaluated in vivo
for biocontrol effects against R. solani root rot of cowpea
seedlings.
2. Materials and methods
2.1. Fungal isolates and cultural media
In recent years, a large number of Trichoderma species have
been discovered from nature reserves in different regions
of China. Species identifications were based on integrated
studies of morphological data, culture characteristics and
phylogenetic information (TEF1-α and RPB2). Twenty-five
Trichoderma isolates, belonging to eight species deposited
in the State Key Laboratory of Mycology, Institute of
Microbiology, Chinese Academy of Sciences, were screened
(Table 1). The Trichoderma “harzianum” isolate T22 (correct
name T. afroharzianum, Chaverri et al. 2015), purchased
from BioWorks Inc., USA, was set as a reference isolate
2073
for in vivo test. The strain of R. solani was from the Beijing
Academy of Agriculture and Forestry (China) by the courtesy
of Prof. Qiu Jiyan. All fungal isolates were stored on potato
dextrose agar (PDA) slants at 25°C for 3 days, then kept at
4°C, and freshly sub-cultured on PDA plates for use.
2.2. Dual culture of R. solani and Trichoderma
isolates
Following the method of Zhang and Zhuang (2017),
direct confrontation assay of Trichoderma species against
R. solani were tested by dual culture method on PDA.
Mycelial blocks (5 mm diam.) cut from active-growing edges
of colonies of the freshly sub-cultured Trichoderma isolates
and R. solani were placed on PDA plates 5 cm apart with
three replications for individual treatments. Plate inoculated
with only R. solani was used as the control. The plates
were kept at 25°C for 9 days. Growth radius of R. solani in
each treatment was measured. Inhibition percentage (I%)
against the pathogen was calculated using the formula:
I%=(C–T)/C×100 (C=mean growth radius of R. solani in
control; T=mean growth radius of R. solani in dual culture).
2.3. Antifungal assay
For testing antifungal activity of Trichoderma isolates (You
et al. 2016), two mycelial blocks taken from active-growing
margin of colonies were inoculated in a 250-mL flask
containing 50 mL PDB (BD Dicfo, Sparks, Maryland) for
7 days at 200 r min–1 at 27°C. Supernatant of the liquid
culture was prepared by filtering through a 0.22-µm filter
(Merck Millipore, Germany), then mixed to unsolidified PDA
at a ratio of 10% (v/v). Pure PBD added to PDA with a same
ratio was served as the control. Then, a mycelial block of
R. solani was inoculated on all PDA plates (9 cm Petri dish),
and the plates were kept for 3 days at 25°C. The inhibition
percentage (I%) of Trichoderma against R. solani was
calculated using the formula: I%=(C–R)/C×100 (C=mean
growth radius of R. solani in control; R=mean growth radius
of R. solani in different treatments).
2.4. Estimation of chitinase and β-1,3-glucanase
activities
Trichoderma isolates growing on PDA plates for 7 days
at 25°C were washed with 5 mL sterile water for spore
suspension. Concentration of the spore suspension
was adjusted to ≈(1.0±0.05)×107 spores mL–1 using a
hemocytometer. Half milliliter spore suspension was
added into a 250-mL flask with 50 mL TLE medium (Lopes
et al. 2012) supplemented with 0.5% (w/v) cell walls of
R. solani (see below for details) as carbon source. The
2074 WANG Chao et al. Journal of Integrative Agriculture 2019, 18(9): 2072–2079
culture was incubated at 180 r min–1 at 25°C for 3 days,
and then centrifuged at 10 000×g to collect the supernatant
for enzyme assay.
Rhizoctonia solani cell walls were prepared according to
Huang et al. (2011). Flasks containing 250 mL PDB were
incubated each with four mycelial blocks of R. solani. The
culture was incubated at 25°C for 7 days. Mycelium was
collected by filtration through 2 layers of sterile gauze and
washed with distilled water for five times, then oven-dried
and stored at –20°C before use.
Chitinase activity was measured using the method
described by Jlde et al. (2000), the amount of reducing
sugar was estimated by the 3,5-dinitrosalicylic acid (DNS)
method (Miller 1959) with N-acetyl-D-glucosamine as a
standard. Reaction lasted for 7 h at 37°C and was ended
by placing the tubes in a boiling water bath for 5 min. One
unit of enzyme was defined as the amount of enzyme in
1 mL culture supernatant that releases 1 µg N-acetyl-D-
glucosamine at 37°C h–1. β-1,3-glucanase activity was
estimated using 0.25% (w/v) laminarin as substrate (Marco
and Felix 2007). The reaction lasted for 30 min at 50°C, and
was ended as described above. One unit of enzyme was
defined as the amount of enzymes that requires to release
1 μg glucose at 50°C in 1 min.
2.5. In vitro plant growth promoting traits
The plant growth promoting (PGP) potential of Trichoderma
isolates was estimated by tricalcium phosphate solubilization
ability, as well as indole acetic acid (IAA) and siderophore
production. A total of 0.5 mL Trichoderma spore suspension
(described above) was inoculated in 50 mL NBRIP medium
(Nautiyal 1999) and kept at 180 r min–1 for 7 days at 27°C.
Culture supernatant was harvested by centrifugation at
10 000×g for 10 min and the phosphate solubilization was
determined quantitatively using colourimetric molybdate/
antimony method (Murphy and Riley 1962). Colorimetric
method was chosen for estimating the IAA producing ability
(Gravel et al. 2007), 0.5 mL spore suspension was inoculated
in PDB supplemented with tryptophan (200 µg mL–1) and
incubated at 180 r min–1 for 7 days at 27°C. Filtrates were
collected for IAA measurements using the Salkowski’s
reagent method (Gordon and Weber 1951). Siderophore
production was analyzed qualitatively by the chormeazural
S (CAS) assay using split plates with half of culture medium
PDA and half of CAS‑blue agar (Milagres et al. 1999). The
halves containing culture medium were inoculated with a
mycelial block each and the plates were incubated at 27°C
for 2 weeks. The CAS‑­­­­agar color change from blue to
orange was indicated as the positive siderophore production.
Moreover, based on the results, 10 promising isolates were
selected for further tests of PGP traits including solubilization
of AlPO4 and FePO4, 1-aminocyclopropane-1-carboxylate
deaminase (ACCD) activity and ammonia production (see
Methods in Appendix).
2.6. Pathogen, Trichoderma suspension and soil
mixture preparation
Mycelium of R. solani was prepared as described above with
minor adjustments. Flasks containing 250 mL PDB were
inoculated with four mycelial blocks of R. solani at 25°C for
7 days. The mycelium was collected by filtration through
gauze and washed with distilled water. After being physically
dried for 24 h at room temperature, R. solani mycelium
was homogenized in distilled water using a laboratory
homogenizer, and adjusted to a final concentration of 4 g L–1.
Trichoderma spore suspension was prepared as described
above, and spore concentration was adjusted to 4×107
spores mL–1. Soil mixture containing peat moss, vermiculite
and soil (5:3:2) was autoclaved for 2 h at 121°C for three
consecutive days. Each plastic pot (12 cm high×10 cm in
diam.) was filled in with 200 g soil mixture.
2.7. Pot experiment test
Based on the results of in vitro experiments, six Trichoderma
isolates including T22 were selected for in vivo biocontrol
assay. Experiments were set up as follows: (I) blank
treatment marked as CK1, soil mixture not inoculated with
any microbes but 100 mL sterile distilled water; (II) treatment
only with R. solani marked as CK2, soil mixture inoculated
with 50 mL pathogen suspension and 50 mL sterile
distilled water per pot; and (III) six Trichoderma treatments,
soil mixture inoculated with 50 mL pathogen biomass
suspension and 50 mL Trichoderma spore suspension per
pot. Four replicates were set up for each treatment. The
pots were covered with plastic film, and incubated for 6 days
at room temperature.
Cowpea seeds were surface sterilized (1% sodium
hypochlorite for 4 min and rinsed three times with sterilized
distilled water). Three cowpea seedlings, pre-germinated in
sterile soil mixture for 5 days, were transferred into a pot for
each treatment. The pots were incubated in a green-house
for 42 days with a photoperiod of 16 h light/8 h dark interval,
at a brightness of 3 500 lux and 24–26°C. The pots were
watered every 2 days. Half-strength Hoagland solution (Lei
et al. 2017) was added at the end of the 2nd week, then
full-strength Hoagland solution was further added at the end
of the 4th and 5th week, respectively.
2.8. Evaluation of disease severity
After 42 days, the plants were carefully removed from the
 WANG Chao et al. Journal of Integrative Agriculture 2019, 18(9): 2072–2079
soil and washed with tap water. Disease severity was
scored later using a 5-class scale as described by Wen
et al. (2005): 0=no necrotic lesion; 1=root necrosis ≤5 mm in
length; 2=necrosis 5 to 10 mm in length; 3=necrosis ≥10 mm;
4=lesion emerged on the crown and shoots; and 5=seedling
damping off. Disease severity index (DSI) was calculated
using the formula (Keshavarz-Tohid et al. 2017): DSI=[(1n1+
2n2+3n3+4n4+5n5)/5N], where, ni (n1, n2, … n5)=number of
plants with disease severity score i; N=the total number
of plants used in each treatment. The wet weight and dry
weight (72 h in an oven at 70°C of root system) and the aerial
part of the plants were measured separately.
2.9. Statistical analysis
All the experiments were repeated three times using a
completely randomized design. The SPSS 20 software
(SPSS, Chicago, Illinois) was used for analyses. Means
and standard deviations of the data were calculated, and
all data were subjected to analyses of variance (ANOVA)
and Duncan tests (P<0.05).
3. Results
3.1. Mycoparasitic and antifungal abilities of Tricho-
derma isolates
The dual culture assay was used to detect growth inhibition
ability of the Trichoderma spp. against R. solani. Most
Trichoderma isolates distinctly inhibited the growth of
R. solani (Table 1), in which T. simmonsii 8702 and
T. pyramidale 7921 showed the highest inhibition rate
(82%), followed by T. pyramidale 8991 (75%). Trichoderma
atrobrunneum 9926 and T. paratroviride 8997 had very
low inhibitions (<60%). Hyphal interaction and sporulation
of Trichoderma spp. in dual cultures were also recorded
(Appendix A). The antifungal test was applied to investigate
the potential of metabolites produced by Trichoderma
isolates to inhibit the growth of R. solani. Among the 25
isolates tested, 11 were able to inhibit the growth of R. solani
(>10%) with six of them showing relatively strong inhibitory
effect (>30%). The inhibition rate of the T. guizhouense
isolate 9185 was the highest (63%), while that of T. viridulum
was the lowest (2%) (Table 1).
3.2. Activities of enzymes related to mycoparasitism
All Trichoderma isolates were grown in TLE medium
supplemented with R. solani cell walls as carbon source.
Five of them showed high chitinase activities, and the isolate
9926 appeared to be the highest (339.6 U mL–1), which was
followed by 8991 (201.4 U mL–1), 8084 (188.3 U mL–1), 8987
2075
(186.1 U mL–1) and 8881 (119 U mL–1). Nine of them were
at a moderate level (>50 U mL–1), while the rest displayed
low activities (Table 1).
Most of the Trichoderma isolates showed high-yield
production of β-1,3-glucanase (Table  1). The enzyme
activities of 18 isolates were more than 100 U mL–1, and the
highest was attributed to the isolate 9026 (361.5 U mL–1),
followed by 8155 (316.7 U mL–1), 8702 (299.6 U mL–1) and
9103 (289.6 U mL–1). The rest of the isolates were poor
(<50 U mL–1).
3.3. Plant growth promoting potential in vitro
Twenty-five Trichoderma isolates were subjected to the tests
of plant growth promoting abilities as indicated by production
of phosphate solubilization, IAA and siderophore. As the
result, the phosphate solubilization ranged from 2.9 to 23.6 U
mL–1, wherein the isolate 10127 was the highest, followed
by 10130 and 8991. The IAA production varied from zero
to 23.4 U mL–1, and the isolate 9767 received the highest
score followed by 9185. Eighteen of the isolates were able
to produce siderophore (Table 1). As the results of the
preliminary tests, 10 promising isolates were selected for a
further investigation of PGP traits, in which the isolates 8702,
8991 and 9288 were the best in AlPO4 solubilization, 8084
was the best for FePO4, and 8702 had the highest ACCD
activity and NH4 production ability (Appendix B).
3.4. Biocontrol potential of Trichoderma isolates
against R. solani infection of cowpea in vivo
According to the results of the above multiple-criterion
assays in vitro, five Trichoderma isolates (8084, 8702,
8991, 9185, 9288) were selected for in vivo test because
their inhibition percentages against R. solani growth were
more than 65%, they expressed high activities of both
chitinase and β-1,3-glucanase, and all had good potential
for promoting plant growth. The seedlings were removed
from soil in pots after 42 days, and the weight of aerial parts
and root system were measured separately. The disease
severity index of different treatments was calculated.
In the case of weight of aerial parts, plants treated by five
of the six Trichoderma isolates were significantly different
from CK2 excluding the isolate 8991 which did not improve
dry or wet plant weights (Fig. 1-A), and the treatment with
9288 resulted in the highest weights. However, plants
treated with 8084, 8702, 9185 and 9288 did not differ
significantly from the blank control CK1 and that of the
commercial isolate T22. In view of the impact of the isolates
on root system, significant differences were not found in wet
weight (Fig. 1-B), but their dry weight for the isolates 8702,
8084 and 9288 in comparison with CK2 were significant
2076 WANG Chao et al. Journal of Integrative Agriculture 2019, 18(9): 2072–2079

Table 1 Trichoderma isolates studied and in vitro assay of their antagonism and growth promoting potential
Antifungal Hydrolytic enzymes Growth promotion
Dual culture
Species Isolate activity Chitinase β-1,3-Glucanase P-solubilized IAA Siderop-
(% inhibition)
(% inhibition) (U mL–1) (U mL–1) (µg mL–1) (µg mL–1) hore
T. atrobrunneum 9926 58.9±1.8 j 5.3±3.5 defgh 339.6±37.0 a 154.9±26.7 g 12.5±4.2 cdefgh 6.6±1.7 ghi +
T. guizhouense 8881 67.0±2.8 cdefgh 44.2±8.2 b 119.0±23.6 c 208.2±12.9 f 14.2±8.0 bcdefgh 21.8±6.8 ab +
9185 70.7±2.4 bcd 63.2±5.5 a 51.7±24.4 efg 163.6±23.0 g 7.6±1.4 fghi 22.1±2.3 ab +
9288 67.9±3.1 cdefg 3.0±1.5 efgh 71.8±13.0 def 111.3±9.9 hi 11.3±5.6 cdefgh 10.3±3.6 efg +
10025 71.6±3.2 bc 25.4±5.8 c 10.2±4.1 h 106.9±13.0 i 15.5±5.2 bcdefg 12.9±1.6 de +
T. paratroviride 8942 62.5±3.2 ghij 6.3±2 defgh 7.0±5.3 h 32.7±5.2 j 6.9±4.3 hi 7.1±0.5 fghi –
8997 59.0±2.7 j 5.6±0.4 defgh 3.1±4.4 h 12.0±1.3 j 15.2±4.2 bcdefgh 4.1±0.5 hijk –
9766 63.4±4.1 fghij 24.7±6.3 c 24.8±7.0 gh 24.2±7.2 j 17.2±1.3 abcde 4.8±0.7 hij –
10095 61.9±4.1 hij 4.3±2.6 defgh 3.1±5.8 h 18.0±5.2 j 11.5±5.4 cdefgh 8.5±0.2 fgh –
T. pyramidale 7921 82.1±3.6 a 15.2±5.5 d 93.2±27.1 cd 230.1±15.9 ef 15.1±3.4 bcdefgh 10.3±0.3 efg +
8987 64.3±1.5 efghij 42.1±8.3 b 186.1±19.9 b 252.6±10.6 de 13.8±3.8 bcdefgh 17.6±2.3 ef +
8991 75.0±4.2 b 30.1±8.9 c 201.4±19.6 b 220.7±26.4 f 21.3±4.3 ab 18.8±2.3 bc +
9155 64.3±1.6 efghij 10.0±4.7 def 40.5±24.7 fgh 112.5±13.0 hi 14.2±3.0 bcdefgh 7.5±1.3 fgh +
T. rufobrunneum 8084 66.7±2.9 cdefgh 7.6±4 defg 188.3±22.6 b 281.9±8.6 cd 8.2±2.0 fghi 2.9±0.6 ijk +
8155 69.5±3.6 cde 6.7±4 defgh 22.1±15.2 gh 316.7±36.3 b 12.2±0.6 cdefgh 1.5±0.4 jk +
T. simmonsii 8702 81.9±2.1 a 30.8±5.6 c 59.6±16.9 defg 299.6±13.1 bc 17.9±1.0 abcd 7.2±0.5 fghi +
9049 65.7±2.4 defgh 8.6±10.9 defg 29.0±17.3 gh 206.3±30.8 f 18.2±8.1 abc 6.6±2.1ghi +
9103 68.6±2.7 cdef 8.2±6.8 defg 69.8±28.2 def 289.6±23.0 bc 9.7±3.2 defghi 6.0±1.2 hi +
T. thermophilum 10127 65.7±4.0 defgh 12.1±9.7 de 50.0±37.9 fg 37.8±1.0 j 23.6±2.4 a 0.8±0.4 jk –
10129 60.0±3.1 ij 31.9±8.6 c 71.5±32.7 def 17.8±5.4 j 15.7±4.2 abcdef 0.0±0.0 k –
10130 61.9±1.7 hij 3.5±1.3 efgh 8.5±6.7 h 11.2±2.9 j 21.8±2.3 ab 1.4±0.5 jk –
T. viridulum 8707b 65.2±2.2 defghi –4.0±0.0 h 9.2±5.8 h 139.1±24.6 gh 7.2±0.4 ghi 14.4±4.7 cd +
9026 67.9±2.5 cdefgh 1.8±1.4 efgh 38.2±18.2 fgh 361.5±17.0 a 2.9±2.6 i 4.1±1.8 hijk +
9767 66.1±2.8 cdefgh 1.8±5.4 efgh 91.1±33.0 cde 209.6±31.0 f 9.1±7.2 efghi 23.4±3.4 a +
10068 64.0±1.7 efghij –1.8±3.9 gh 70.7±5.8 def 42.2±5.5 j 7.9±4.0 fghi 11.2±2.8 e +
+ indicates positive siderophore production and – indicates no siderophore production. Different letters indicate significant differences
among isolates according to Duncan test at P<0.05.

even though small. The isolate 8084 expressed the highest
dry weight of root system, which were followed by CK1 and
8702, whereas, application of T22 did not obviously affect
root weight.
No symptoms were found in the seedlings in CK1,
while the plants exposed only to R. solani in CK2 was of
the highest disease incidence ((68.3±14.0)%) with severe
necrosis and even damping-off (Figs. 2 and 3). The plants
treated with the isolates 8702, 8991 and 9185 had much less
lesions compared with CK2. Difference was also shown by
the significantly reduced DSIs. For example, the seedlings
protected by 8702 had the lowest DSI. But no significant
differences were found between CK2 and the plants treated
with 8084, 9288 and T22.
4. Discussion
The results of dual culture assay indicate that all the
Trichoderma isolates tested are able to inhibit the growth
of R. solani. The highest inhibition percentage reaches
82% as expressed by the isolate 8702, and those of the
others range from 59 to 70%, which is apparently better
than 24–47% reported by Mayo et al. (2015) using the
same method. Hyperparasitic ability varies among the
Trichoderma isolates, which was congruent with the previous
reports testing with other pathogens (Saxena et al. 2015;
You et al. 2016). Variations in different treatments are no
doubt resulted from abilities of growth, sporulation or specific
biocontrol factors in individual isolates (Ruano-Rosa et al.
2010). To eliminate effect of nutrients from culture medium
rather than parasite in dual culture test, the pre-colonized
assay method is recommended for future study (Samuels
and Hebbar 2015).
As an ancestral life style, mycoparasitism is one of
the most salient mechanisms for Trichoderma against
plant pathogenic fungi (Howell 2003; Kubicek et al. 2011),
in which hydrolytic enzymes, especially chitinases and
β-1,3-glucanases, play a crucial role. These enzymes
were induced efficiently by pathogen cell walls (Almeida
et al. 2007). In our study, six of the eight Trichoderma
species were able to produce high level chitinases and/
or β-1,3-glucanases (Table 1) except T. paratroviride and
T. thermophilum showing low enzyme activities, which
suggests that mycoparasitic ability might be varied among
species but relatively stable in isolates within species,
and which is opposite to the previous result (Anees et al.
2010). Further investigations based on a rich sampling of
Trichoderma species are necessary.
Trichoderma species produce tremendous numbers of
water-soluble metabolites including pyrenes, terpenoids,
 WANG Chao et al. Journal of Integrative Agriculture 2019, 18(9): 2072–2079 2077

Wet weight Dry weight

A 3.5

3.0 a
a a
ab ab
2.5 ab
Aerial part (g)

c bc
2.0
1.5
1.0
ab ab ab ab a ab CK1 CK2 8084 8702 8991 9185 9288 T22
0.5 c bc

0
CK1 CK2 8084 8702 8991 9185 9288 T22
Treatment
B 0.200
0.175
0.150 a a a a
a CK1 CK2 8084 8702 8991 9185 9288 T22
Root system (g)

0.125 a a Treatment
ab a
a
0.100 abc
bcd cd abc
cd
0.075 Fig. 2 Growth of cowpea seedlings treated with Trichoderma
d isolates and Rhizoctonia solani. Arrows show necrotic lesions
0.050
in roots and steams of cowpea seedlings.
0.025
0
CK1 CK2 8084 8702 8991 9185 9288 T22
100
Treatment
c
Disease severity index (%)

80 bc
Fig. 1 Weights of aerial parts and roots system of cowpea bc
b bc
treated with Rhizoctonia solani and Trichoderma isolates.
60 b
CK1, blank treatment; CK2, seedlings treated with R. solani;
8084–T22, seedlings treated with R. solani and Trichoderma b
isolates. Data are mean±SD (n=12). Different letters indicate 40
significant differences among treatments according to Duncan
test at P<0.05. 20
a
0
steroids and polyketides (Monfil and Casas-Flores 2014) to CK1 CK2 8084 8702 8991 9185 9288 T22
Treatment
inhibit growth of plant pathogens. It is clearly demonstrated
in our study that antifungal metabolites produced by
Fig. 3 Biocontrol efficiency of the selected Trichoderma isolates
Trichoderma isolates are not stable within individual species, against disease caused by Rhizoctonia solani. CK1, blank
and vary among isolates of the same species (Table 1). treatment; CK2, seedlings treated with R. solani; 8084–T22,
In view of the above results, there might be a correlation seedlings treated with R. solani and Trichoderma isolates.
Data are mean±SD (n=12). Different letters indicate significant
between antifungal ability and biocontrol efficiency. For differences among treatments according to Duncan test at
instance, plants treated with the isolates 8702, 8991 and P<0.05.
9185 significantly reduced the DSIs and caused many fewer
lesions compared with CK2 (Figs. 2 and 3), and exhibited
also high antibiosis activities (Table 1). On the contrary, the isolate 8084 is up to 46.4% which is similar to the control
isolates 8084 and 8288 showed high DSIs and were of low efficiency (45%) shown by T. harzianum SQR-T37 for
antifungal abilities. This may be due to direct antibiosis by R. solani disease control of cucumber (Huang et al. 2011).
metabolites produced by Trichoderma or induced systemic Mayo et al. (2015) stated that T. harzianum T019 controls
resistance (ISR) of plant by enzymes or antibiotics released R. solani by increasing of the seedling size and stimulating
by Trichoderma (Anees et al. 2010). Understanding plant- expression level of defense-related genes of bean.
Trichoderma interaction mechanisms should be emphasized In the results of the wet and dry weight tests of plants
in future research. and DIS data, four of our Trichoderma isolates (8084,
As to the results from the in vivo assays, all the five 8702, 9185, and 9288) do not have significant differences
selected Trichoderma isolates efficiently protect the from CK1 (Fig. 1), which might be attributed to their growth
cowpea seedlings from the infection of R. solani (Fig. 2). promoting ability, such as phosphate solubilization ability
For example, the inhibition percentage of T. rufobrunneum and that for IAA and siderophore production. Nevertheless,
2078 WANG Chao et al. Journal of Integrative Agriculture 2019, 18(9): 2072–2079
the previous studies (Huang et al. 2011; Mayo et al. 2015;
Keshavarz-Tohid et al. 2017) showed that the plant weights
were significantly decreased with Trichoderma added in the
presence of R. solani.
Most of the commercial Trichoderma products in the
world are mainly concentrated on the limited well-known
species (Woo et al. 2014; Samuels and Hebbar 2015).
Many species of the genus remain uninvestigated for their
availabilities in biological control. Sixty-one new species
of Trichoderma have been recently reported from China
(Qin and Zhuang 2015; Zhu and Zhuang 2015; Chen
and Zhuang 2016; Qin and Zhuang 2016a, b; Chen and
Zhuang 2017a, b, c; Qin and Zhuang 2017). Exploiting new
resources as BCAs is surely worthy. In this work, six of the
eight Trichoderma species were investigated for the first
time, i.e., T. atrobrunneum, T. paratroviride, T. pyramidale,
T. rufobrunneum, T. thermophilum and T. viridulum. To
our knowledge, this is the first report that T. guizhouense
and T. simmonsii possess the capacity of effective control
of disease caused by R. solani. In summary, five of the
selected Trichoderma isolates tested in vivo either prompt
growth or decrease DSI of cowpea seedlings upon invasion
of R. solani. Our results suggest that T. guizhouense
9185 and T. simmonsii 8702 have a promising application
prospect for the purpose of biocontrol.
5. Conclusion
This study was performed to investigate the biocontrol
potential of eight Trichoderma species against the
phytopathogen R. solani in vitro and in vivo. Among them,
the isolates T. guizhouense 9185 and T. simmonsii 8702
exhibited excellent commercial exploitation prospect for
biocontrol in agriculture. A more comprehensive insight
into the biocontrol potential of the genus needs larger-scale
sampling of different species.
Acknowledgements
The authors would like to express their sincere thanks to Dr.
Qin Wentao and Dr. Zhang Yubo (Institute of Microbiology,
Chinese Academy of Sciences) for their preliminary
screening of Trichoderma isolates and Ms. Song Xia
(Institute of Microbiology, Chinese Academy of Sciences)
for technical assistance. This project was supported by the
National Natural Science Foundation of China (31570018)
and the National Key Research and Development Program
of China (2017YFD0200600).
Appendices associated with this paper can be available on
http://www.ChinaAgriSci.com/V2/En/appendix.htm
References
Almeida F B, Cerqueira F M, Silva R N, Ulhoa C J, Lima A L.
2007. Mycoparasitism studies of Trichoderma harzianum
strains against Rhizoctonia solani: Evaluation of coiling
and hydrolytic enzyme production. Biotechnology Letters,
29, 1189–1193.
Anees M, Tronsmo A, Edel-Hermann V, Hjeljord L G, Héraud
C, Steinberg C. 2010. Characterization of field isolates
of Trichoderma antagonistic against Rhizoctonia solani.
Fungal Biology, 114, 691.
Bissett J, Gams W, Jaklitsch W, Samuels G J. 2015. Accepted
Trichoderma names in the year 2015. Ima Fungus, 6,
263–295.
Boukaew S, Klinmanee C, Prasertsan P. 2013. Potential for
the integration of biological and chemical control of sheath
blight disease caused by Rhizoctonia solani on rice. World
Journal of Microbiology and Biotechnology, 29, 1885–1893.
Chaverri P, Brancorocha F, Jaklitsch W, Gazis R, Degenkolb
T, Samuels G J. 2015. Systematics of the Trichoderma
harzianum species complex and the re-identification of
commercial biocontrol strains. Mycologia, 107, 558.
Chaverri P, Samuels G J. 2013. Evolution of habitat preference
and nutrition mode in a cosmopolitan fungal genus with
evidence of interkingdom host jumps and major shifts in
ecology. Evolution, 67, 2823–2837.
Chen K, Zhuang W Y. 2016. Trichoderma shennongjianum and
Trichoderma tibetense, two new soil-inhabiting species in
the strictipile clade. Mycoscience, 57, 311–319.
Chen K, Zhuang W Y. 2017a. Discovery from a large-scaled
survey of Trichoderma in soil of China. Scientific Reports,
7, 9090.
Chen K, Zhuang W Y. 2017b. Seven soil-inhabiting new species
of the genus Trichoderma in the viride clade. Phytotaxa,
312, 28.
Chen K, Zhuang W Y. 2017c. Three new soil-inhabiting
species of Trichoderma in the stromaticum clade with test
of their antagonism to pathogens. Current Microbiology,
74, 1049–1060.
Gordon S A, Weber R P. 1951. Colorimetric estimation of
indoleacetic acid. Plant Physiology, 26, 192–195.
Gravel V, Antoun H, Tweddell R J. 2007. Growth stimulation
and fruit yield improvement of greenhouse tomato plants
by inoculation with Pseudomonas putida or Trichoderma
atroviride: Possible role of indole acetic acid (IAA). Soil
Biology and Biochemistry, 39, 1968–1977.
Harman G E, Howell C R, Viterbo A, Chet I, Lorito M. 2004.
Trichoderma species - Opportunistic, avirulent plant
symbionts. Nature Reviews Microbiology, 2, 43.
Howell C R. 2003. Mechanisms employed by Trichoderma
species in the biological control of plant diseases: The
history and evolution of current concepts. Plant Disease,
87, 4–10.
Huang X, Chen L, Ran W, Shen Q, Yang X. 2011. Trichoderma
harzianum strain SQR-T37 and its bio-organic fertilizer
could control Rhizoctonia solani damping-off disease in
cucumber seedlings mainly by the mycoparasitism. Applied
Microbiology & Biotechnology, 91, 741–755.
 WANG Chao et al. Journal of Integrative Agriculture 2019, 18(9): 2072–2079
Jaiswal A K, Elad Y, Graber E R, Frenkel O. 2014. Rhizoctonia
solani suppression and plant growth promotion in cucumber
as affected by biochar pyrolysis temperature, feedstock and
concentration. Soil Biology and Biochemistry, 69, 110–118.
Jlde M, Lhc L, Mvde S, Felix C R. 2000. A Trichoderma harzianum
chitinase destroys the cell wall of the phytopathogen
Crinipellis perniciosa, the causal agent of witches’ broom
disease of cocoa. World Journal of Microbiology &
Biotechnology, 16, 383–386.
Keshavarz-Tohid V, Taheri P, Muller D, Prigent-Combaret C,
Vacheron J, Taghavi S M, Tarighi S, Moënne-Loccoz Y.
2017. Phylogenetic diversity and antagonistic traits of root
and rhizosphere pseudomonads of a bean from Iran for
controlling Rhizoctonia solani. Research in Microbiology,
168, 760–772.
Kubicek C P, Herrera-Estrella A, Seidl-Seiboth V, Martinez D A,
Druzhinina I S, Thon M, Zeilinger S, Casas-Flores S, Horwitz
B A, Mukherjee P K, Mukherjee M, Kredics L, Alcaraz L
D, Aerts A, Antal Z, Atanasova L, Cervantes-Badillo M G,
Challacombe J, Chertkov O, McCluskey K, et al. 2011.
Comparative genome sequence analysis underscores
mycoparasitism as the ancestral life style of Trichoderma.
Genome Biology, 12, R40.
Lei G J, Yokosho K, Yamaji N, Fujii-Kashino M, Ma J F.
2017. Functional characterization of two half-size ABC
transporter genes in aluminium-accumulating buckwheat.
New Phytologist, 215, 1080.
Lopes F A C, Steindorff A S, Geraldine A M, Brandão R S,
Monteiro V N, Júnior M L, Coelho A S G, Ulhoa C J, Silva R
N. 2012. Biochemical and metabolic profiles of Trichoderma
strains isolated from common bean crops in the Brazilian
Cerrado, and potential antagonism against Sclerotinia
sclerotiorum. Fungal Biology, 116, 815–824.
Marco J L D, Felix C R. 2007. Purification and characterization
of a beta-glucanase produced by Trichoderma harzianum
showing biocontrol potential. Brazilian Archives of Biology
& Technology, 50, 21–29.
Mayo S, Gutiérrez S, Malmierca M G, Lorenzana A, Campelo
M P, Hermosa R, Casquero P A. 2015. Influence of
Rhizoctonia solani and Trichoderma spp. in growth of
bean (Phaseolus vulgaris L.) and in the induction of plant
defense-related genes. Frontiers in Plant Science, 6, 685.
Milagres A M, Machuca A, Napoleão D. 1999. Detection of
siderophore production from several fungi and bacteria by
a modification of chrome azurol S (CAS) agar plate assay.
Journal of Microbiological Methods, 37, 1–6.
Miller G L. 1959. Use of dinitrosalicylic acid reagent for
determination of reducing sugar. Analytical Biochemistry,
31, 426–428.
Monfil V O, Casas-Flores S. 2014. Molecular mechanisms of
biocontrol in Trichoderma spp. and their applications in
agriculture. In: Gupta V G, Schmoll M, Herrera-Estrella A,
Upadhyay R S, Druzhinina I, Tuohy M, eds., Biotechnology
and Biology of Trichoderma. Elsevier Press, Amsterdam.
pp. 429–453.
2079
Murphy J, Riley J P. 1962. A modified single solution method for
the determination of phosphate in natural waters. Analytica
Chimica Acta, 27, 31–36.
Nautiyal C S. 1999. An efficient microbiological growth medium
for screening phosphate solubilizing microorganisms. FEMS
Microbiology Letters, 170, 265–270.
Qin W T, Zhuang W Y. 2015. Three new species of Trichoderma
with hyaline ascospores from China. Mycologia, 107,
328–345.
Qin W T, Zhuang W Y. 2016a. Four new species of Trichoderma
with hyaline ascospores from central China. Mycological
Progress, 15, 811–825.
Qin W T, Zhuang W Y. 2016b. Seven wood-inhabiting new
species of the genus Trichoderma (fungi, Ascomycota) in
Viride clade. Scientific Reports, 6, 27074.
Qin W T, Zhuang W Y. 2017. Seven new species of Trichoderma
(Hypocreales) in the harzianum and strictipile clades.
Phytotaxa, 305, 121.
Ruano-Rosa D, Moral-Navarrete L D, Lopez-Herrera C J.
2010. Selection of Trichoderma spp. isolates antagonistic
to Rosellinia necatrix. Spanish Journal of Agricultural
Research, 8, 1084–1097.
Samuels G, Hebbar P. 2015. Trichoderma Identification and
Agricultural Applications. American Phytopathological
Society Press, St. Paul.
Sarrocco S, Wu Q, Sun R, Ni M, Yu J, Li Y, Yu C, Dou K, Ren J,
Chen J. 2017. Identification of a novel fungus, Trichoderma
asperellum GDFS1009, and comprehensive evaluation of
its biocontrol efficacy. PLoS ONE, 12, e0179957.
Saxena A, Raghuwanshi R, Singh H B. 2015. Trichoderma
species mediated differential tolerance against biotic stress
of phytopathogens in Cicer arietinum L. Journal of Basic
Microbiology, 55, 195.
Tredway L P, Burpee L L. 2001. Rhizoctonia diseases of
turfgrass. The Plant Health Instructor, doi: 10.1094/
PHI-I-2001-1109-011
Wen K, Seguin P, St-Arnaud M, Jabaji-Hare S. 2005. Real-time
quantitative Rt-pcr of defense-associated gene transcripts
of Rhizoctonia solani-infected bean seedlings in response
to inoculation with a nonpathogenic binucleate Rhizoctonia
isolate. Phytopathology, 95, 345–353.
Woo S L, Ruocco M, Vinale F, Nigro M, Marra R, Lombardi
N, Pascale A, Lanzuise S, Manganiello G, Lorito M. 2014.
Trichoderma-based products and their widespread use in
agriculture. The Open Mycology Journal, 8, 71–126.
You J, Zhang J, Wu M, Yang L, Chen W, Li G. 2016. Multiple
criteria-based screening of Trichoderma isolates for
biological control of Botrytis cinerea on tomato. Biological
Control, 101, 31–38.
Zhang Y B, Zhuang W Y. 2017. First step evaluation of
Trichoderma antagonism against plant pathogenic fungi in
dual culture. Mycosystema, 36, 1251–1259. (in Chinese)
Zhu Z X, Zhuang W Y. 2015. Trichoderma (Hypocrea) species
with green ascospores from China. Persoonia, 34, 113–129.
Executive Editor-in-Chief WAN Fang-hao
Managing editor ZHANG Juan