Tropical Plant Pathology, vol. 39(5):401-406, 2014
Copyright by the Brazilian Phytopathological Society.
www.sbfito.com.br
SHORT COMMUNICATION
Supression of seed borne Cladosporium herbarum on
common bean seed by Trichoderma harzianum and promotion
of seedling development
Gesiane R. Guimarães1, Faber S. Pereira1, Fabio S. Matos2, Sueli C. M. Mello3 & Daniel D. C. Carvalho1
Laboratório de Fitopatologia, Universidade Estadual de Goiás. Rodovia GO 330, km 241, Anel Viário, Setor Universitário,
75780-000, Ipameri, GO, Brazil; 2Laboratório de Produção Vegetal, Universidade Estadual de Goiás, Rodovia GO 330,
km 241, Anel Viário, Setor Universitário, 75780-000, Ipameri, GO, Brazil; 3Embrapa Recursos Genéticos e Biotecnologia,
Parque Estação Biológica, Final W-5 Norte, Cx. Postal 02372, 70770-900, Brasília, DF, Brazil
1
Author for correspondence: Daniel D. C. Carvalho, e-mail: daniel.carvalho@ueg.br
ABSTRACT
Trichoderma harzianum isolates have been broadly used for biocontrol of plant diseases caused by fungi. Cladosporium herbarum
is a common saprophyte and seed borne fungus, which is easy to manipulate under controlled conditions. It was chosen as a model to
test the effectivity of seed treatments with T. harzianum. Common bean seeds (cv. Pérola) contaminated with C. herbarum were treated with
conidial suspension (CS) and autoclaved filtrate (AF) of five isolates of T. harzianum and subsequently submitted to health and germination tests.
The proportion of normal seedlings formed, the length of roots, hypocotyls and leaves, and total plantlet length, total plantlet biomass, root mass
ratio (RMR), stem mass ratio (SMR), leaf mass ratio (LMR), aerial part/root system ratio (AP/RS) and leaf area were also evaluated. Isolates
CEN289 and CEN290 (CS and AF) provided 66 to 77% of supression of C. herbarum on seeds and a higher number of normal seedlings
as compared with control. It also yielded a higher total biomass of plantlets. Moreover treatment with isolates CEN289 and CEN290
increased root and stem length in the experiments with CS. Such results indicate the potential of T. harzianum for seed treatment and
suggest that it should be further tested as control for seed borne fungal diseases and as a plant growth promoter. The better performance
found for CEN289 and CEN290 confirms the variability in terms of biocontrol activity among strains of T. harzianum.
Key words: Phaseolus vulgaris, antagonism, biological control, seed pathology.
Common bean (Phaseolus vulgaris L.) is the most
important source of protein for human nutrition in several
countries, especially in Latin America and Africa. Brazil is
the largest producer and consumer of this legume worldwide
(Alwathnani et al., 2012). The incidence of pathogens in
common bean seeds can reduce the physiological quality
of the seeds, with consequences on the germination and
initial stand establishment of the crops (Carvalho et
al., 2014). Besides, seeds are important vehicles for the
introduction and dissemination of pathogens into new
areas. When seeds are exposed to inadequate moisture and
temperatures during storage, growth of noxious fungi may
result (Costa & Scussel, 2002), resulting in loss of seed
quality and viability. Cladosporium spp. are commonly
found associated with seeds of crop and native plants (e.g.
Dhingra et al., 2002) and Cladosporium herbarum (Pers.)
Link is one of the most common species in this genus.
Treatment of the seeds with fungicides can eliminate
pathogens and protect seedlings against diseases and may
be fundamental to produce an adequate plant stand and crop
establishment (Carvalho et al., 2011). Although fungicides
are used to control seed pathogens, the results are often not
persistent (Salman & Abuamsha, 2012). Moreover, the use
of fungicides may have negative consequences to human
health and the environment (Carvalho et al., 2007) and lead
to the development of fungicide-resistant populations of
plant pathogens (Maketon et al., 2008). Biological control
of plant diseases with antagonists (alone or in combination
with chemicals) is an important component of integrated
plant disease management (Salman & Abuamsha, 2012).
Trichoderma species are widely used in agriculture as
biopesticides, being efficient mycoparasites and prolific
producers of secondary metabolites (Mukherjee et al., 2013).
Seed treatment with Trichoderma spp. is an option that has
been investigated as a substitute to chemical treatments and
shown to have great potential (Carvalho et al., 2011; Maciel
et al., 2014). Additionally, it is known that certain isolates
of Trichoderma can form colonies on plant roots (HoyosCarvajal et al., 2009) and stimulate plant development
by producing substances that promote plant growth and
solubilize nutrients which may be assimilated by plants
(Jha et al., 2013). In the present study selected isolates of
Trichoderma harzianum Rifai were tested on common bean
seeds for their effect on suppression of a common seedborne fungus C. herbarum and on early seedling growth
promotion.
Tropical Plant Pathology 39 (5) September - October 2014
401
G.R. Guimarães et al.
Five T. harzianum isolates (CEN287, CEN288,
CEN289, CEN290 and CEN316) belonging to the collection
of fungi for biological control of plant pathogens of Embrapa
Genetic Resources and Biotechnology (Brasília, DF,
Brazil) selected in previous studies (Carvalho et al., 2011;
Carvalho et al., 2014) were evaluated. The isolates had been
previously identified on the basis of their morphological
traits (Samuels et al., 2014) and sequencing of ITS-1 and
ITS-4 regions of rDNA. The experiments were carried out
at the Universidade Estadual de Goiás, Ipameri, State of
Goiás, Brazil.
In order to produce inoculum, seven-day-old agar
plugs (5 mm) of T. harzianum were transferred to 250 mL
Erlenmeyer flasks (four plugs flask-1), containing parboiled
rice (15 g flask-1), previously moistened (60% w v-1) and
autoclaved (121ºC for 40 min). After seeding flasks were
kept at 25ºC and 12h/12h darkness/light. After six days,
spores were collected by agitating colonized rice grains with
distilled water and filtering through sterile cheesecloth. The
conidial concentration for each T. harzianum isolate was
adjusted at 2.5 x 108 conidia mL-1.
Metabolites of each T. harzianum isolate were also
tested. These isolates were separately grown in 250 mL
of potato dextrose broth (PDB) in 500 mL Erlenmeyer
flasks at 120 rpm on an orbital shaker (Quimis® Aparelhos
Científicos Ltda., Q225K) at 25ºC and 12 h/12h darkness/
light, for six days. After that period, filtrates of the five
isolates were obtained and autoclaved at 121°C for 21 min.
Common bean seeds (cv. Pérola) naturally
contaminated externally with C. herbarum were treated
with (a) 2 mL of a 2.5 x 108 conidia mL-1 suspension of T.
harzianum per 100 g of seeds or (b) 2 mL of autoclaved
filtrate of T. harzianum per 100 g of seeds. Subsequently a
seed health test was performed through the blotter method,
whereby 25 treated seeds were placed into a gerbox (11 x
11 cm) containing two moistened paper blotter sheets. The
gerbox were kept at 25ºC with a light regime of 12 hours of
light exposure, for seven days (light provided by a Philips
daylight fluorescent lamp). After that, seeds were examined,
individually, under a stereomicroscope. To confirm the
presence of C. herbarum, semi-permanent microscope slides
were prepared and examined under a Leica DM500 light
microscope. The experiment was carried out in a completely
randomized design, with eight replicates. Each replicate
consisted of one gerbox containing 25 seeds. Seeds treated
with carboxin+thiram (300 mL per 100 kg of seeds; 200 g
L-1 carboxin and 200 g L-1 thiram) and non-treated seeds
were used as positive and negative controls, respectively.
Autoclaved PDB medium was used as negative control in
the test with AF.
A separate assay aimed at evaluating the effect of
treatment with CS and AF of T. harzianum, on germination
and early seedling development was performed. The
same treatments and doses mentioned above were used,
except for the fungicide treatment. After being treated, the
seeds were subjected to the germination test, which was
carried out in a randomized block design (RBD) with four
replicates of 50 seeds per treatment. Seeds were placed on
germitest rolls (50 seeds per roll), incubated in a germinator
(Logen Scientific®) at 25ºC, for nine days. After incubation,
the following were evaluated: (a) percentage of abnormal
seedlings (seedlings exhibiting necrosis along the roots,
hypocotyls and cotyledons; seminal and secondary stunted
and malformed roots, or absence of secondary roots),
(b) percentage of normal seedlings (absence of necrosis
and pathogen, seminal and secondary roots without
deformations) and (c) percentage of dead seeds. All normal
seedlings of each treatment were measured, separately,
as follows: (a) root length, (b) hypocotyl length, (c) leaf
length and (d) total length (a+b+c). Additionally, leaf area
(cm2) was calculated from leaf length and width data using
the equation according to Figueiredo et al. (2012). Leaves,
roots and stems were then detached and dried at 72oC until
reaching a constant dry mass and the values (in grams) of
root dry mass (rdm), stem dry mass (sdm) and leaf dry mass
(ldm) were recorded. The total biomass (rdm+sdm+ldm),
the leaf mass ratio [LMR=(ldm)/(total biomass)] and the
aerial part/root system ratio (AP/RS) [AP/RS=(sdm+ldm)/
(rdm)] were then calculated.
The data were subjected to analysis of variance and
to the Scott-Knott test (P≤0.05), using the SISVAR software
(Ferreira, 2011).
The fungicide treatment was the most effective
leading to complete eradication of C. herbarum from
seeds (Table 1). Levels of control of C. herbarum on seeds
reached with CS treatments with T. harzianum varied
between 67 and 77% for the five isolates of T. harzianum.
For AF treatments CEN289 and CEN290 had the best
performance reaching a control higher than 70% (75 and
73%, respectively). Additionally, isolates CEN289 and
CEN290 yielded the highest number of normal seedlings
for both CS and AF treatments, respectively, 62% and 36%
more normal seedlings than control.
As for the length of seedlings, it was observed that the
only CS treatments which significantly increased root and
hypocotyl length were those involving isolates CEN289 and
CEN290 (Table 2). Total length of seedling was statistically
superior for CEN287, CEN289 and CEN290 CS-treated
seeds. Conversely, in AF-treated seeds no significant
statistic distinction was found between treatments (Table
2). CS and AF application on seeds yielded significantly
higher values for leaf length for isolates CEN287, CEN289
and CEN316 as compared with other isolates and control.
As for total plantlet length only CS applications had
a statistically significant result and isolates, CEN287,
CEN289 and CEN290 yielded higher values as compared
to other isolates and control.
Root length values were higher in the CS experiment
for all isolates of T. harzianum as compared to AF. Total
length values were higher in the CS experiment for all
isolates of T. harzianum, except for CEN316, compared to
AF.
402
Tropical Plant Pathology 39 (5) September - October 2014
Supression of seed borne Cladosporium herbarum on common bean seed by Trichoderma harzianum...
TABLE 1 - Incidence of Cladosporium herbarum on common bean seeds cv. Pérola treated with conidial suspension (CS) of Trichoderma
harzianum and its autoclaved filtrate (AF), and the respective effect of each treatment on normal seedling formation(1).
31.0 c
Means followed by the same letter within the same column do not differ by Scott Knott test (P≤0.05).
Seedlings originated from seeds naturally contaminated by C. herbarum.
Carboxin+thiram (300 mL 100 kg-1 of seeds; 200 g L-1 carboxin and 200 g L-1 thiram)
(4)
Potato dextrose broth (200 g cooked potato, 20 g dextrose, 1000 mL distilled water).
(1)
(2)
(3)
TABLE 2 - Root length (cm), hypocotyl length, leaf length and total length of common bean seedlings cv. Pérola originated from seeds
treated with conidial suspension (CS) of T. harzianum and its autoclaved filtrate (AF)(1).
Means followed by the same small letter within the same column and the same capital letter in the same line do not differ by Scott Knott test
(P≤0.05).
(2)
Potato dextrose broth (200 g cooked potato, 20 g dextrose, 1000 mL distilled water).
ns
Not significant.
(1)
CS applications on seeds of CEN289 and CEN290
resulted in the highest values of total biomass (Table 3).
AF applications yielded a total biomass of 133.43 g for
CEN289, 128.13 g for CEN290 and 70.39 g for control,
respectively.
Values of root-mass ratio for seeds treated with
isolates of T. harzianum (0.07-0.16) were lower than those
of the control for CS (0.31) and AF (0.28) treatments. On
the other hand, values of stem mass ratio of the seedlings
for seed treated with CS and AF (0.8-0.89) were higher
than those of control.
Treatments involving most isolates of T. harzianum
(either CS or AF) yielded lower values of leaf mass ratio
as compared to control. The sole exception was CEN287.
The aerial part/root system ratio (9.02-13.42) of seedlings
resulting from CS and AF treated seeds was higher than that
of control. The leaf area of the seedlings produced from
CS and AF-treated seeds was higher than that observed for
control for isolates CEN287, CEN289 and CEN316. No
differences were observed between CS or AF treatment for
the development variables under evaluation (Table 3).
According to Barbosa et al. (2001), the antagonistic
action of Trichoderma spp. against C. herbarum happens
through direct mycoparasitism, including the sporulation
of the antagonist within the pathogen’s hyphae. Moreover,
possible explanation for the effect of AF may be: (1)
antibiotic production by Trichoderma such as trichodermin,
trichodermol, harzianum A and harzianolid; (2) fungal cell
wall-degrading enzymes, such as lipase, NAGase, β-1,3glucanase, β-glucosidase and protease which are noxious
to hyphae of various plant pathogens (Dickinson et al.,
1995; Geraldine et al., 2013).
Carvalho et al. (2011) also reported that
carboxin+thiram completely eradicated Cladosporium sp. in
common bean seeds. These authors found that the biological
treatment (isolates of T. harzianum: CEN287, CEN288,
CEN289, CEB290 and CEN316) reduced the fungus
incidence in seeds at 14 to 41%. In the present study, the
Tropical Plant Pathology 39 (5) September - October 2014
403
404
TABLE 3 - Total biomass (g), root mass ratio (RMR), stem mass ratio (SMR), leaf mass ratio (LMR), aerial part/root system ratio (AP/RS) and leaf area (cm2) of common bean seedlings
cv. Pérola originated from seeds treated with conidial suspension (CS) of Trichoderma harzianum and its autoclaved filtrate (AF)(1).
G.R. Guimarães et al.
Tropical Plant Pathology 39 (5) September - October 2014
Means followed by the same small letter within the same column do not differ significantly by the Scott Knott test (P≤0.05).
Total biomass (rdm+sdm+ldm).
(3)
Root mass ratio [RMR=(rdm)/(total biomass)].
(4)
Stem mass ratio [SMR=(sdm)/(total biomass)].
(5)
Leaf mass ratio [LMR=(ldm)/(total biomass)].
(6)
Aerial part/root system ratio [AP/RS=(sdm+ldm)/(rdm)].
(7)
Potato dextrose broth (200g cooked potato, 20g dextrose, 1000 mL distilled water).
(1)
(2)
Supression of seed borne Cladosporium herbarum on common bean seed by Trichoderma harzianum...
reduction rates of C. herbarum incidence in contaminated
seeds were higher than those reported by Carvalho et al
(2011). Reduction rates ranged from 67 to 77% for CS and
from 54 to 75% to AF. Since the antagonist isolates involved
in those two studies were different, the performance
differences were possibly due to variations in the C.
herbarum population involved which were those naturally
occurring on the seeds in both cases (Narayanasamy, 2011).
Curiously, the isolates CEN289 and CEN290 differed from
the others in terms of percentage of normal seedlings in
both CS and AF treatments. The coincidence of this result
with those obtained in the evaluation of the incidence of
C. herbarum on seeds, when AF was used, suggests that
metabolites produced by these two isolates play some role in
yielding high rates of normal seedlings. In the specific case,
those metabolites were applied directly (AF experiment)
and indirectly (CS experiment), resulting in similar rates of
normal seedlings.
According to Saito et al. (2011), the factors involved
in the promotion of plant development by Trichoderma
species are still poorly understood. When CS or AF treatments
were compared (Table 2), it was found that, for all isolates,
root length values were higher for CS treatments than for
AF treatments. This suggests that thermolabile substances
produced by T. harzianum such as hormones and vitamins
and other plant growth-stimulating substances, only
available for CS-treated seeds and resulting plantlets may
be involved. In other words, active fractions present in the
metabolites produced in PDB may have been decomposed
into inactive fractions after autoclaving of the metabolites.
Thus, the increase in the root length follows an increase
in the total length in crops treated with Trichoderma spp.
(Saito et al., 2011; Carvalho et al., 2011). According to
Harman (2000) Trichoderma harzianum is favored by
the presence of abundant root systems which it can easily
colonize. Some isolates are highly capable of colonizing
and growing in roots just following their development.
Isolates of T. harzianum which have good performance at
colonizing roots may be added to the soil or to seeds and
after coming into contact with the roots, they colonize the
root surface and/or the cortex, showing rhizocompetence, as
demonstrated for the isolates CEN287, CEN288, CEN289,
CEN290 and CEN316 by Carvalho et al. (2011).
Surprisingly the short period of exposure of the
seedlings to CS and AF of T. harzianum was enough to
trigger significant alterations in the leaves and, in particular,
in the total biomass of the common bean seedlings. Thus,
development analysis is an efficient tool for identifying
promising materials, besides identifying traits that, in the
initial development, may promote increased yields in mature
plants (Peixoto et al., 2006). The seedlings treated with CS
and AF of T. harzianum showed high biomass accumulation
and higher development of the aerial part, especially the
stem. Total biomass is an important variable to select
promising isolates to be applied under field conditions
(Nobre et al., 2011; Matos et al., 2012). Thus, the higher
Tropical Plant Pathology 39 (5) September - October 2014
biomass accumulation in seedlings treated with CEN289
was in line with higher photosynthesizing leaf area.
Carvalho et al. (2011) conducted experiments with
the same isolates of T. harzianum in common bean plants cv.
‘Jalo Precoce’ under greenhouse and field conditions; they
found that CEN289 and CEN290 promoted increased root
length as compared with control. On the contrary, reduced
biomass allocation in the root system of plants treated with
CS and AF of T. harzianum was interpreted as resulting
from high water availability for the seedlings which were
irrigated daily during the germination test. Thus, without
water restriction, the allocation of resources to root system
formation was not justified. Consequently, the high AP/RS
ratio of the seedlings treated with CS and AF of T. harzianum
was likely to result from strong allocation of resources to
the formation of aerial part tissues.
As a conclusion to this study, it can be said that the
treatment of seeds of common bean cv. Pérola with CS
and AF of T. harzianum can reduce the population of the
model seed-borne fungus C. herbarum, and improve the
development of seedlings. Root growth promotion was
only possible by using CS. Seedlings from seeds treated
with the isolates of T. harzianum (either through CS or AF
seed treatment), especially with CEN289 and CEN290,
showed high biomass accumulation and vigorous vegetative
development.
ACKNOWLEDGEMENTS
The authors express their thanks to the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
for providing a fellowship to G. R. Guimarães, the Fundação
de Amparo à Pesquisa do Estado de Goiás - FAPEG
for providing financial support for the research (Proc.
201310267001026) and to Embrapa Genetic Resources
and Biotechnology for loan of the isolates of Trichoderma
harzianum (TRTM 02/2012), and the Universidade Estadual
de Goiás - UEG.
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Submitted: 20 May 2014
Revisions requested: 22 June 2014
Accepted: 15 July 2014
Section Editor: Trazilbo José de Paula Junior
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