Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
ISSN: 2319-7706 Volume 3 Number 12 (2014) pp. 128-139
http://www.ijcmas.com
Original Research Article
Response of Coleus aromaticus to molecular characterised
Trichoderma harzianum isolated from different soil types of agro climatic
zones of Karnataka, India
C.Sandeep1*, R.Sowmya2, K.C.Shilpa2, L.Pallavi2, Sushmitha Nayak2 and C.K.Suresh3
1
Tree Improvement and Genetics Division, Institute of Wood Science and Technology,
Bangalore-560003, India
2
Department of Biotechnology, CMR Institute of Technology, Bangalore-560037, India
3
Department of Plant Biotechnology, University of Agricultural Sciences, GKVK Campus,
Bangalore-560065, India
*Corresponding author
ABSTRACT
Keywords
Trichoderma
harzianum,
Biocontrol
agent,
Coleus
aromaticus,
Chlorophyll and
Protein content
The aim of this project was to study the effect of molecularly characterized
Trichoderma harzianum isolated from different soil types of agroclimatic zones of
Karnataka on economically important medicinal plant Coleus aromaticus under
polyhouse conditions. Pure cultures of T. harzianum representing each zone were
separately grown in potato dextrose broth and molecular diversity of isolates was
characterized by molecular markers. The effect of isolated organisms on physical
and biochemical parameters of Coleus aromaticus was studied. The parameters
such as height, number of leaves, fresh and dry weight of the roots and shoots, were
measured along with control (uninoculated plants). Biochemical parameters like
chlorophyll, total sugar and protein content in root and shoot were estimated too.
The results showed that Trichoderma harzianum had profound effect on growth as
well as the nutrient content of the plants. The genetic relatedness of 10 isolates
studied, formed 2 groups based on phylogenetic tree obtained. The genetic
variations however had no relationship with the effect on plant growth and
nutrition. From the study, it could be concluded that T. harzianum isolates of zone5 showed maximum effect on growth and nutrient content of Coleus aromaticus
compared to control.
Introduction
Trichoderma is a genus of asexually
reproducing fungi that are often the most
frequently isolated soil fungi; nearly all
temperate and tropical soils contain 101 103
cultureable propagules per gram. They show
a high level of genetic diversity, and can be
used to produce a wide range of products of
Trichoderma species are widely distributed
all over the world and occur in nearly all
soils and natural habitats, especially in those
consisting of organic matter. Tichoderma is
also found on root surfaces of various plants,
on decaying bark, especially when it is
damaged by other fungi.
128
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
commercial and ecological interest. They are
prolific producers of extracellular proteins,
and are best known for their ability to
produce enzymes that degrade cellulose and
chitin although they also produce other
useful enzymes (Harman and Kubicek,
1998).
organisms in localized soil conditions in
promoting growth of plants and biochemical
characterization of plant growth promoting
fungi isolated from different soil types of
different zones is very much limited. Many
studies on interaction of plant growth
promoting microorganisms have been
conducted on different crop plants (Guojing
et al., 2001). Further, screening of the
isolates either individually or in combination
is needed to select efficient isolates to
improve the plant growth and biomass.
Trichoderma species have long been
recognized as agents for the control of plant
disease and for their ability to increase plant
growth and development. They are
becoming widely used in agriculture,
horticulture, forestry and the most useful
strains show a property that is known as
rhizosphere competence i.e, the ability to
colonize and grow in association with plant
roots (Harman, 2000). They produce or
release a variety of compounds that induce
localized or systemic resistance responses,
and this explains their lack of pathogenicity
to plants. This fungus root associations
cause substantial changes to the plant
proteome and metabolism.
India s biodiversity coupled with its vast
natural resources like natural flora and fauna
make our country the best choice for
growing medicinal plants. Among various
medicinal plants Coleus aromaticus has a
phenomenal impact on human health and are
highly valuable in terms of medicinal
properties holding a reputed position in
Ayurveda, Unani and Siddha system of
medicine. Coleus aromaticus belongs to
family Lamiaceae and is a large succulent
herb, fleshy and highly aromatic, much
branched, possessing short soft erect hairs,
with distinctive smelling leaves. Other uses
include as an ornamental, and for its
essential oils. C. aromaticus also has
antimicrobial activity, antioxidant activity,
and pharmacological activity.
Plants are protected from numerous classes
of plant pathogen by responses that are
similar to systemic acquired resistance and
rhizobacteria-induced systemic resistance.
Root colonization by Trichoderma species
also frequently enhances root growth and
development, crop productivity, resistance
to abiotic stresses and the uptake and use of
nutrients (Harman et al., 2004). The most
strongly rhizosphere competent strains can
be added to soil or seeds by any method and
once they come into contact with roots, they
colonize the root surface or cortex,
depending on the strain.
Materials and Methods
The present investigation was carried out to
isolate, identify, characterize and study the
effect of Trichoderma harzianum from
different soil types of various agro climatic
zones of Karnataka on medicinal plant
Coleus aromaticus. The study was
conducted at the Department of Plant
Biotechnology, University of Agricultural
Sciences, G.K.V.K. Campus, Bangalore,
Karnataka, India. The material used and
methods followed are described below.
Thus, if added as a seed treatment, the best
strains will colonize root surfaces even when
roots a meter or more below the soil surface
and they can persist at significant numbers
up to 18 months after application. The
information on the compatibility of these
129
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
were taken in to account and the bands were
not scored if they were faint or diffused, as
such fragments posses poor reproducibility.
The band sizes were determined by
comparing with the 500bp DNA ladder,
which was run along with the amplified
products. The Genetic distance was
computed as:
Sample collection and isolation of
Trichoderma harzianum
The soil samples were collected from the
different agro climatic zones of Karnataka.
Rhizosphere soil was collected in polythene
covers and stored in cool place.
Trichoderma harzianum was isolated by
serial dilution plate method on potato
dextrose agar media. The fungal isolates
were subjected to morphological tests as
listed in The Manual of Soil Fungi
(Gilman, 1961). Formation of characteristic
colonies by fungal isolates and other
characters were taken as a tool for
preliminary identification by comparing
with the standard.
n
=1
dj2
where dj = ( Xik Xjk )
Where X ik refers to binary code of the tree
for allele k and Xjk refers to the binary
code of the jth tree for allele k .
Dendrogram was computed based on
Ward s method of clustering, using
minimum variance algorithm (Ward, 1963).
Inoculum preparation
DNA isolation and amplification
T. harzianum isolates were grown
separately, in a 250 ml flask containing
100ml potato dextrose broth for 7 days. The
grown cultures were homogenized and 15
ml of each of the solution was given to plant
sapling.
DNA extraction protocol followed was
according to Raeder and Broda (1985).
DNA was isolated from fungal isolates
grown in potato dextrose broth (PDB) by
phenol, chloroform and isoamyl alcohol
mixture. The isolated DNA was amplified
by using five random primers (Table 1).
1.5% Agarose gel was used to resolve the
amplification product and the gel was
visualized under UV light and documented
using Hero Lab Gel Documentation unit.
Physical and Biochemical Parameters
Plant saplings were obtained from division
of horticulture, University of Agricultural
sciences, GKVK, Bangalore. Plant saplings
of the plant were planted on pots containing
sterile sand: soil mixture (1:1). The plants
were allowed to grow for 60 days by
watering as per the requirement. The
physical parameters namely plant height,
number of branches, root fresh and dry
weight and shoot fresh and dry weight were
determined on 15th, 30th, 45th and 60 days.
Root and shoot dry weights were calculated
by drying the harvested roots or shoots in an
oven at 60oC for 4 days to attain constant
weight and then the root dry weight were
recorded and expressed as grams/plant and
expressed as grams/plant. The biochemical
parameters namely chlorophyll content
(Hiscox and Israelstam, 1979), total soluble
Analysis of RAPD data
The bands were manually scored 1 for the
presence and 0 for the absence and the
binary data were used for statistical analysis.
The scored band data (Presence or absence)
was subjected to cluster analysis-using
STATISTICA. The dendrogram was
constructed by Ward s method of clustering
using minimum variance algorithm. The
dissimilarity matrix was developed using
Squared Euclidean Distance (SED), which
estimated all the pair wise differences in the
amplification product (Sneath and sokal,
1973). Only clear and unambiguous bands
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Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
proteins (Lowry et al., 1951), total sugars
(Dubois et al., 1996) were estimated on 60th
day.
pattern, colour of the spores, etc. All the
check isolates and the standard strains
formed white fungal colonies initially and
turned to complete dark green colour after
sporulation due to colour of the spores. All
the isolates exhibited the typical spore
arrangement on the conidial head as that of
the standard reference strain, in which the
spores are arranged linearly on the conidial
head and also spores. The 10 isolates
obtained from samples of different area have
been mentioned as either Zone -1 to Zone
10 or Z1 to Z10 in results.
Chlorophyll a
12.7 (O.D.663) 2.69 (O.D.645) x Volume
mg/g fresh weight = ------------------------------------------------------1000 g x weight of leaves (g)
Chlorophyll b
22.9 (O.D.645) 4.68 (O.D.663) x Volume
mg/g fresh weight = ------------------------------------------------------1000 g x weight of leaves (g)
Total Chlorophyll
In the present study, Trichoderma
harzianum was isolated from different
agroclimatic zones of Karnataka. This
showed that the microorganism is a common
inhabitant of rhizosphere of many plant
species in different areas. Previous studies
have shown that the Trichoderma species
are very common in many parts of the world
including tropical, temperate or subtropical
region and in association with different plant
(Harman, 2000). After the preliminary
screening 10 Trichoderma harzianum
isolates from each Zones viz., NETZ,
NEDZ, NDZ, CDZ, EDZ, SDZ, STZ, NTZ,
HZ and CZ and they were examined for
their performance under Green house
condition.
20.2 (O.D.645) + 8.02 (O.D.663) x Volume
mg/g fresh weight = ------------------------------------------------------1000 g x weight of leaves (g)
Quantitative
estimation
of
soil
microorganisms by dilution plate count
technique
The populations of different groups of
microorganisms in the soil samples were
assessed by standard dilution plate technique
(Jensen, 1968) and the results were
expressed in colony forming units (CPU s)
per gram of the soil sample.
Statistical analysis
The data obtained in the pot experiments
were subjected to one way analysis of
variance using MSTAT-C software.
Morphological and biochemical
parameters
The plant height was significantly increased
in the inoculated plants at 15, 45, and 60
days after planting, compared to control.
Maximum height of 36.1 cm and maximum
number of leaves was observed in the plants
inoculated with zone-5 (SCZ) isolate (Table
3 and 4). This indicated that the Zone-5
isolate is more efficient in enhancing the
plant height compared to others. Similar
results were observed by Earanna et al.
(2001) and Guojing et al. (2001). Increased
cell elongation and multiplication due to the
Results and Discussion
Isolation and identification
Isolation of Trichoderma harzianum was
made from soil from different agro-climatic
zones of Karnataka (Table 2) by growing in
the potato dextrose agar media by serial
dilution plate method. For isolation,
preliminary identification was carried out by
morphological observations of the fungal
colonies such as colour, mycelial growth
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Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
enhanced uptake of mineral nutrients
(Yedidia et al., 2003), production of plant
growth substances and enzymes by T.
harzianum may be the cause for increased
plant height and number of branches. This
indicated the superiority of Zone-5 isolates
as compared to others.
increased uptake of nutrients, which resulted
in higher chlorophyll content in treated
plants as compared to control. Similar result
was reported by Inbar et al. (1994) who
studied effect of T. harzianum applied to
cucumber and pepper seedling and found
higher chlorophyll content in treated plants
as compared to uninoculated plants.
Plant Biomass
Total sugar and reducing sugar content
The total fresh weight (shoot and root) of
Coleus aromaticus was significantly higher
in the plants inoculated with T. harzianum
isolates compared to uninoculated control.
In C. aromaticus, among different
treatments, the zone 5th (EDZ) isolate
treatment recorded significantly maximum
total biomass of 172.82 and 83.29 g fresh
and dry weights respectively. This suggested
that the zone-5 isolate is most efficient in
enhancing the biomass compared to others.
The lowest total biomass was recorded in
control of 106.09 and 28.46 g fresh and dry
weight respectively (Fig. 1).
There was significant increase in total sugar
and reducing sugars in zone 5(EDZ)
inoculated plants as compared to that of all
other treatments (Fig. 3). This shows that
zone 5 (EDZ) isolate is more efficient in
enhancing sugar content of C. aromaticus as
compared to other zone isolates. This may
be due to increased number of leaves and
chlorophyll content of leaves which leads to
increase in photosynthetic rates of
inoculated plants. Similar results were
observed in Eucalyptus, inoculated with
mycorrhizal fungi (Govindasamy, 2003).
Increased biomass may be due to enhanced
plant growth, number of leaves and
branches, increase in root growth which was
influenced probably by greater availability
of mineral nutrients, plant growth promoting
substances and enzymes. Arpana (2000)
studied influence of Glomus mosseae and T.
harzianum on medicinal plant Kalmegh. The
results showed maximum biomass of treated
plants as compared to control.
Total soluble protein content
In C. aromaticus, maximum total soluble
protein content of 0.416mg/2g of plant was
noticed in plant inoculated with zone 5
(EDZ) isolate (Fig. 4). The reason may be
attributed to increase in more nutrient uptake
and more chlorophyll content of leaves,
which leads to increase in photosynthetic
rates. Similar results were observed by
Gupta et al. (1995) who reported increased
total soluble proteins (34 to 54 g/ ml) in
Ocimum
carnosum
applied
with
Triacontanol as foliar spray which was
associated to promotory effect of
Triacontanol on this crop and by stimulation
of the enzyme activity associated with
protein metabolism.
Chlorophyll estimation
Significant variation in chlorophyll content
of leaves inoculated with T. harzianum
isolates was noticed as compared to control
(Fig. 2). The highest chlorophyll a, b and
total chlorophyll content were noticed in
zone 5 (EDZ) isolate of 0.436, 0.24 and
0.483 mg/0.1g fw (fresh weight) of leaves
respectively. This may be due to the
Microbial population studies
A
132
significantly
enhanced
microbial
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
population (that includes bacteria, fungi and
actinomycetes) were noticed in the root zone
soil of C. aromaticus inoculated with fungi
T. harzianum when compared with that of
control (Table 5). Similar kind of
observations were noticed by Meyer and
Linderman (1986) who reported an increase
in the population of bacteria in the root zone
soil of mycorrhiza inoculated sweet corn and
clover in contrast to the non-mycorrhizal
plants. Several other identical findings have
also been reported by many workers
(Mamatha, 2000; Lalitha et al., 2010; Rajesh
et al., 2011). Soil microorganisms are
known to play crucial role to benefit plant
growth and development (Handman et al.,
1991) thus enhanced microbial population
points towards an improved soil quality in
the Trichoderma colonized soil which
ultimately boost up plant health which is
also reflected by the results obtained in the
study.
Molecular identification of T. harzianum:
Five random primers were selected for
fingerprinting and diversity analysis of T.
harzianum isolates. A total of 125 bands
were scored, out of which 100 bands were
found to be polymorphic (80.0%) and it
seems to be quite high within species. The
bands were manually scored 1 for the
presence and 0 for the absence and the
binary data were used for statistical analysis
(Table 6).
The scored band data (Presence or absence)
was subjected to cluster analysis-using
STATISTICA. The dendrogram (Fig. 5) has
clearly depicted that all the 10 T. harzianum
isolates formed two major clusters.
Table.1 Random Primers used for RAPD
Primer No.
Random primer 1
Random primer 2
Random primer 3
Random primer 4
Random primer 5
Sequence (5 -3 )
5 GTCGATGTCG3
5 CGTCGCCCAT3
5 CGGTGACATC3
5 CTGTCCAGCA3
5 CAGCACCCAC3
Table.2 List of isolates and their samples
Isolate
Isolate 1
Isolate 2
Isolate 3
Isolate 4
Isolate 5
Isolate 6
Isolate 7
Isolate 8
Isolate 9
Isolate 10
Zone of Collection
North Eastern Transition Zone
North Eastern Dry Zone
Northern Dry Zone
Central Dry Zone
Eastern Dry Zone
Southern Dry Zone
Southern Transition Zone
Northern Transition Zone
Hill Zone
Coastal Zone
133
Name
NETZ or zone 1
NEDZ or zone 2
NDZ or zone 3
CDZ or zone 4
EDZ or zone 5
SDZ or zone 6
STZ or zone 7
NTZ or zone 8
HZ or zone 9
CZ or zone 10
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
Table.3 Effect of T. harzianum on the height of C. aromaticus
Treatments
th
15 Day
26.23
23.83
21.53
19.76
24.96
26.43
22.3
20.13
27.36
24.36
21.06
0.84
2.48
Control
Zone 1
Zone 2
Zone 3
Zone 4
Zone 5
Zone 6
Zone 7
Zone 8
Zone 9
Zone 10
SEM
CD
Plant height(cm)
30 Day
45th Day
26.33
26.63
29.1
31.96
32.2
33.06
32.9
33.26
30.56
32.16
29.53
33.66
29.1
32.7
26.43
31.2
31.46
32.43
30.73
33.26
26.53
26.33
0.73
0.72
2.15
2.13
th
60th Day
27.83
31.03
32.16
34.06
32.50
34.36
32.73
29.93
33.16
32.70
28.67
0.75
2.20
(SEM- Standard error mean: CD-Critical difference)
Table.4 Effect of T. harzianum on number of leaves of C. aromaticus
Treatments
th
Control
Zone 1
Zone 2
Zone 3
Zone 4
Zone 5
Zone 6
Zone 7
Zone 8
Zone 9
Zone 10
SEM
CD
15 Day
95.66
61.66
47.66
48.66
65.33
69.33
53.00
47.00
65.33
58.00
54.00
4.72
13.84
Number of leaves
30 Day
45th Day
100
110.66
106.33
111.33
72.00
83.00
75.00
85.66
101.00
119.00
118.00
130.66
96.33
107.33
79.00
97.00
115.33
127.00
101.33
110.66
65.33
72.66
5.68
6.96
16.67
20.42
th
(SEM- Standard error mean: CD-Critical difference)
134
60th Day
113.66
112.00
82.33
87.333
123.33
132.00
114.00
97.00
135.66
126.66
79.33
7.30
21.41
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
Table.5 Effect of fungal inoculation on population of bacteria, fungi and actinomycetes in
root zone soil of Coleus aromaticus
Root zone microbial population (CFU s/g soil)
Treatments
Bacteria
Fungi
Actinomycetes
Control
51.66
3.00
21.33
T1
65.00
5.33
30.66
T2
45.33
6.66
32.33
T3
41.66
6.66
26.00
T4
69.66
7.00
24.66
T5
71.00
8.00
25.00
T6
57.33
9.00
25.33
T7
66.66
9.66
26.00
T8
60.33
9.00
27.00
T9
68.00
6.33
26.00
T10
56.33
5.33
24.00
SEM ±
5.11
0.72
0.80
CD at 5%
15.00
2.12
(SEM- Standard error mean: CD-Critical difference)
2.37
Table.6 List of primers used for genetic variation among the T. harzianum isolates
Primers
Random primer 1
Random primer 2
Random primer 3
Random primer 4
Random primer 5
Total
Percentage
No. of
amplified
fragments
23
25
24
33
20
125
No. of polymorphic bands
Shared
unique
18
22
21
24
15
05
03
03
09
05
100
80%
25
20%
135
No. of
Monomorpic
bands
00
00
00
00
00
00
0%
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
Figure.1 Effect of inoculation of T. harzianum isolates on biomass of C. aromaticus
Figure.2 Effect of T. harzianum on chlorophyll content of C. aromaticus
136
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
Figure.3 Effect of T. harzianum on sugar content of C. aromaticus
Figure.4 Effect of T. harzianum on Protein content of C. aromaticus
137
Int.J.Curr.Microbiol.App.Sci (2014) 3(12) 128-139
Tree Diagram for 10 Variables
Ward`s method
Squared Euclidean distances
110
100
90
(Dlink/Dmax)*100
80
70
60
50
40
30
20
10
ZONE9
ZONE4
ZONE7
ZONE6
ZONE8
ZONE10
ZONE2
ZONE3
ZONE5
ZONE1
Figure.5 Dendrogram based on RAPD profile of 10 T.harzianum isolates from different
agroclimatic zones of Karnataka
Kalmegh (Andrographis paniculata
Nees.) to VAM and a plant growth
promoting rhizo-microorganism. M.Sc.
(Agri.)
Thesis,
University
of
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Dubois, R.N., Giardiello, F.M., Smalley, W.E.
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D.J., Suresh, C.K. 2001. Response of
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aromaticus
to
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Gilman, J.C. 1961. A manual of soil fungi,
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Govindasamy, V. 2003. Biological and
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and
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Guojing, L., Benoit, F., Ceustermans, N., Li,
Among the two major groups the isolates
from zone1 (NETZ), zone 2 (NEDZ), zone
5 (EDZ), zone 3 (NDZ) and zone10 (CZ)
isolates were grouped together. Isolates
from, zone 8 (NTZ), zone 6 (SDZ), zone 7
(STZ) and zone 9 (HZ) were grouped
separately and isolate from zone 4 (CDT)
was quite, distinct forming a separate
entity. There was no correlation between
RAPD and geographical origin of isolates.
Similar results obtained by Veena (2005)
who carried out AFLP marker analysis in
Aspergillus awamori isolated from
different agroclimatic zones of Karnataka,
mainly to study the molecular variability
among the isolates. Results of the cluster
analysis revealed that all the isolates
grouped in to 3 major clusters and 33 per
cent polymorphism was found among the
isolates.
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