Isolation of Cold Tolerant Antifungal Strains of Trichoderma
sp. From Glacial Sites of Indian Himalayan Region
Three species of Trichoderma viz., T. harzianum,
T. konengii and T. viride have been isolated
from the soil samples collected from forest sites in higher altitudes
of Indian Himalayan Region. The species could grow between 9 to 35°C
temperature and 4 to 12 pH on agar plates; the optimum requirement being
24°C and 5.5 pH, respectively. Further incubation of the agar plates
showing normal growth of Trichoderma sp. at 4°C, induced heavy
sporulation in three weeks of time. Induction of sporulation on exposure
to low temperature appeared to be a strategy for survival of these species
in extreme cold environment experiencing sub zero temperatures. Antifungal
activities were demonstrated between Trichoderma sp. and phytopathogenic
fungi in dual cultures. The antifungal metabolites produced by Trichoderma
sp., diffusible as well as volatile, caused abnormalities in fungal structures
of pathogenic fungi. Plant growth promotion abilities of Trichoderma
sp. was also demonstrated through a plant based bioassay in greenhouse.
The study is important for documentation of microbial diversity of Indian
Himalayan Region (IHR) and determination of the associated biotechnological
Trichoderma species are free living fungi that occur in nearly
all the soils and other natural habitats. They can be easily isolated
from soil and decomposing organic matter. Most of the Trichoderma
sp. grow rapidly in artificial culture media and produce large numbers
of green or white conidia from conidiogenous cells. Their abundance in
soil under diversified climatic conditions is mainly due to their ability
to degrade a variety of organic substrates in soil, their metabolic versatility
and their resistance to microbial inhibitors. Certain strains of Trichoderma
sp. including T. viride are known to be restricted to the
areas identified by low temperature, T. harzianum is mostly found
in warmer climate and strains of T. hamatum and T.
konengii are widely distributed in areas of diverse climatic conditions.
Diversity in various ecosystems, including forest and mountains and molecular
taxonomy of Trichoderma sp. have received importance in the recent
literature (Grondona et al., 1997; Kullnig-Gradinger et al.,
2002; Fu-Qiang et al., 2004; Woo et al., 2006).
Several species of the genus Trichoderma received attention mainly
due to their importance in biological control of soilborne plant pathogens.
Antibiosis, mycoparasitism and competition for nutrients are the mechanisms
involved in biological control. Recent studies have shown that they are
opportunistic, avirulent plant symbionts, as well as the parasites of
other fungi. Many species of the genus Trichoderma have also been
recognized for their plant growth promotion abilities (Vizcaíno
et al., 2005). In the present study, three species of Trichoderma
isolated from soil samples collected from forest sites in high altitudes
of IHR have been investigated in vitro for growth characters and
biocontrol properties. Bioassay was also performed to demonstrate the
effect of these species on plant growth.
MATERIALS AND METHODS
Soil samples were collected from various locations in high altitudes
(beyond 3000 m above mean sea level) of Pindari Glacier region of Uttarakhand
in IHR (Pandey et al., 2008).
Isolation and Growth Characters of Fungi
Serial dilutions of soil samples were made for isolation of fungi.
Appropriate dilutions were plated (pour plate method) using Czapek, Potato
Dextrose (PD), Sabouraud Maltose and Malt Extract agar. The agar plates
were incubated at 21°C for 7 days. Morphologically distinct colonies
were subjected to purification following subculturing. The pure cultures
were maintained on PDA slants at 4°C in a refrigerator. Based on colony
morphology and microscopic observations (smears made in lactophenol cotton
blue) the frequently occurring species of genus Trichoderma were
selected for further experiments. The species level identification was
carried out with support of Indian Type Culture Collection, Indian Agricultural
Research Institute, New Delhi and Agharkar Research Institute, Pune, India.
For colony morphology and microscopic observations, fresh cultures were
grown on PD agar at 21°C for 5 days. After taking observations on
growth characters, the plates were kept in refrigerator. The temperature
and pH requirements of the Trichoderma sp. were determined by incubating
the fungal isolates at different temperatures (4, 9, 14, 21, 28, 35 and
42°C) for 7 days and by inoculating the fungal isolates on PD agar
plates set at different pH levels, i.e., 3.0-13.0 with an interval of
0.5 unit. For salt tolerance, the cultures were inoculated on PD agar
plates supplemented with 2.0, 5.0 and 7.0% salt, respectively.
Plate Assays for Evaluation of Biocontrol Properties
Five phytopathogenic fungi viz., Alternaria alternata, Cladosporium
oxysporum, Fusarium oxysporum, Pythium afertile and
a non-sporulating dematiaceous fungus were selected for biocontrol experiments.
These fungi were isolated from the same study locations and are known
to cause minor or major disease symptoms in a range of plant species.
Dual cultures were performed for determination of production of antifungal
metabolites, both diffusible and volatiles. For determination of diffusible
metabolites individual Trichoderma sp. and phytopathogenic test
fungus were grown on PD agar plates. Five millimeter disc of both the
fungi (Trichoderma sp. and phytopathogenic test fungus) were placed
on PD agar plate about 2.0-2.5 cm away from each other. The plates were
incubated in inverted position at 21°C for one week. Inhibition of
the pathogenic fungal growth was measured by using the formula:
where, R1 control value represents the largest distance grown
by the test fungus in the direction of maximum radius, R2 represents
the distance between the inoculum of pathogen and Trichoderma sp.
For determination of production of volatile substances dual cultures
were performed in sealed plates. Five millimeter disc of Trichoderma
sp. and test fungus were placed in center of two separate PD agar plates
of same size and sealed with parafilm and incubated at 21°C for one
week. Control was taken without Trichoderma sp. in the bottom plate.
Observation was recorded after one week and percent inhibition was calculated
where, r1 was the radial growth of pathogen without Trichoderma
sp., r2 represents the radial growth of pathogen inoculated
with Trichoderma sp. All the experiments were conducted in triplicates.
Fungal smears were prepared in lactophenol cotton blue taking the growth
from the inhibition area. Fungal smears were also prepared from the control
plates to make the comparison.
Bioassay for Evaluation of Plant Growth Promotion Ability of Trichoderma
Wheat (Triticum aestivum L.) was chosen as a test species for
conducting the bioassay under greenhouse conditions. The four treatments
under consideration were: 1-Control (seeds without any inoculum) and 2,
3 and 4-seeds inoculated with T. harzianum, T. koningii
and T. viride, respectively. Trichoderma inoculum
(for each species, individually) was grown on PD agar plates (incubated
at 21°C for one week). Three mycelial discs (5 mm) of Trichoderma
sp. were cut from the agar plates and used as inoculum for one seed at
the time of sowing. Seeds were grown in trays (32x32x10 cm) containing
16 cups. The trays were kept inside the greenhouse (24.0±2°C)
of the Institute. Each treatment was taken in triplicate (48 seeds for
each treatment). After 42 days of growth, 10 plants from each treatment
were selected randomly and fresh weight of roots and shoots were taken.
Dry weight was taken after drying the roots and shoots in oven at 65°C
for 48 h. Data was statistically analyzed using ANOVA. Analysis of rhizosphere
soil was done for determining the effect of inoculation on rhizosphere
microflora. Soil samples (in triplicate) were collected from all the four
treatments and serial dilution technique was carried out for bacteria
and fungi on tryptone yeast extract and PD agar, respectively. Enumerations
were made after 5 days of incubation at 21°C.
RESULTS AND DISCUSSION
The frequently occurring fungal species in soil samples of glacier sites
in high altitudes of Indian Himalaya mainly belonged to the genera Alternaria,
Aspergillus, Chrysosporium, Cladosporium, Epicoccum,
Fusarium, Gangronella, Myrothecium, Paecilomyces,
Phoma, Phytophthora and Trichoderma (Pandey et
al., 2008). Trichoderma was observed as one of the most frequently
occurring genus in dilution agar plates throughout these experiments.
The species were often observed covering the entire area of the culture
plates, not allowing the other species to grow. The colony morphology
and microscopic features of three species of Trichoderma are presented
in Table 1. Based on these characters, the Trichoderma
sp. were identified as T. harzianum, T. konengii
and T. viride. These species could grow between 9 to 35°C
temperature and 4 to 12 pH on agar plates. All the three species of Trichoderma
could tolerate salt concentration upto 5% (w/v). The optimum temperature
and pH requirement of these species was observed to be 24 and 5.5°C,
respectively. It was interesting to note that all the three species showing
mycelial growth with moderate sporulation after 5 days of incubation at
24°C resulted in heavy sporulation in about three weeks when shifted
to 4°C. Induction of heavy sporulation due to low temperature might
be a strategy of these species for their survival under low temperature
environments including the sub zero temperatures.
||Growth characters of Trichoderma spp.
||Biocontrol related properties of Trichoderma sp.
|ND = Not detected
||(A-C) Plate assays for antagonistic interactions between
T. harzianum, T. konengii and T.
viride and the dematiaceous fungus, A. alternata
and F. oxysporum, respectively; D-F: Abnormalities in
the respective phytopathogenic fungi as shown in plate assays- A,
B and C, respectively (Bar = 10 μm)
In plate based experiments, all the three species of Trichoderma
showed antagonistic interactions against the five test phytopathogens, Alternaria
alternata, Cladosporium oxysporum, Fusarium oxysporum,
Pythium afertile and a non-sporulating dematiaceous fungus (Fig
1A-C). Table 2 presents the results
on the effect of diffusible and volatile metabolites produced by Trichoderma
sp. in terms of reduced radial growth of the test fungus. The inhibition
of test pathogens due to production of diffusible metabolites ranged from
53.33 to 66.66%, 47.82 to 63.33% and 57.77 to 73.90% by T. harzianum,
T. konengii and T. viride, respectively. While
inhibition due to production of volatile metabolites ranged from 39.63 to
55.55%, 38.26 to 64.16% and 39.63 to 74.99%, by T. harzianum,
T. konengii and T. viride, respectively. None
of the species of Trichoderma showed visible inhibition of pathogenic
fungi Pythium afertile, probably due to the relatively fast growing
nature of pathogen. Microscopic observations revealed that diffusible as
well as volatile substances induced morphological abnormalities in fungal
structures. Deformation in mycelial, hyphal, or conidial structure was common
in all the test fungi (Fig 1D-F). In
general, the diffusible substances appeared to be more effective as compared
to volatiles. Induction of deformities was probably due to the ability of
Trichoderma sp. to develop direct interaction with pathogens and
to produce antimicrobial substances as the mycoparasitism involves physical
contact and synthesis of hydrolytic enzymes, toxic compounds, or antibiotics
(Benitez et al., 2004).
Studies have been conducted on effect of low temperature on spore germination
and germ tube growth of Trichoderma strains. In earlier studies,
performed on cold tolerant species of Trichoderma, out of 360 Trichoderma
strains investigated, 14 (identified as strains of T. aureoviride,
T. harzianum and T. viride) could grow well
at 5°C; all the cold tolerant strains produced appresoria and antagonized
the plant pathogens, Rhizoctonia solani and Fusarium oxysporum
f. sp. Dianthi, in dual culture tests performed at 10°C (Antal et
Knowledge of the prevalence of environmental conditions, both climatic
and edaphic, in the habitat of a given organism may be useful for exploitation
of the potential applications associated with the organism. The habitat
of the species of Trichoderma in present study was forest locations
mainly dominated by species of Abies, Rhododendron and Betula.
Dominance of Trichoderma species in tea gardens and temperate forest
locations of Indian Himalayan Region has been reported (Pandey et al.,
2001). Species of Trichoderma are known for their competence for
rapid colonization of plant roots. Due to their nature of colonizing in
presence of healthy roots they have evolved numerous mechanisms for attacking
other fungi and for enhancing plant and root growth (Harman et al.,
Observations taken on wheat based bioassay were indicative of plant growth
promotion abilities of the Trichoderma sp. Biomass of root and
shoot of wheat in inoculated plants was higher after 42 days of growth
as compared to control, statistically significant (p<0.05) in most
cases. Maximum benefit was observed in case of T. koningii.
The inoculated Trichoderma sp. colonized the rhizosphere of test
plant species and increased the plant biomass. Further, Trichoderma
sp. inoculation stimulated the bacterial and suppressed the fungal population
in the rhizosphere of test plant, i.e., wheat, indicative of associated
antifungal properties. In an earlier study, T. viride (same
strain that is used in the present study) was found to provide protection
to the young seedlings of Cedrus deodara against mortality due
to cutworms attack and wilting caused by Fusarium sp. Improvement
in plant health mainly due to improved nutrient uptake was also recorded
in the cited study (Bisht et al., 2003).
Trichoderma sp. have got attention due to their role in biological
control. Mycoparasitism has been proposed as the major mechanism supporting
the antagonistic activity of Trichoderma sp. Strains of Trichoderma
sp. inhibit pathogens through production of antifungal antibiotics and
or/hydrolytic enzymes. Ability for plant growth promotion and induced
resistance in plants by Trichoderma sp. has also been reported
(Monte, 2001; Vizcaíno et al., 2005; Harman, 2006). The
present study is important in view of the documentation of soil microbial
diversity in Indian Himalayan Region. It is mainly based on the isolation
of three most frequently occurring species of Trichoderma and demonstration
of their biocntrol abilities. Isolation and screening of cold tolerant
strains of Trichoderma sp. is important in order to select biocontrol
agents for application in colder regions. The species of Trichoderma
used in the present study have been deposited in Indian Type Culture Collection
(ITCC), Indian Agricultural Research Institute, New Delhi and Agharkar
Research Institute Fungus Culture Collection (ARIFCC), Agharkar Research
Institute, Pune, India; the accession numbers are presented in Table
1. The species should be used for further studies on production of
hydrolytic enzymes related to biocontrol as well as the strategies adopted
for their survival under cooler regimes.
Director, GBPIHED is thanked for extending the facilities. Department
of Biotechnology is thanked for partial financial support. Two anonymous
reviewers are gratefully acknowledged for providing suggestions on the
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