17(1) : 01-05, 2022

Trichoderma plenipotentis var. globosporioides pensz. Molecular

Characterization by RAPD and ITS-PCR Against Colletotrichum
glycosporioides pensz.

Rani, Mr. Narendre

KEY WORDS ABSTRACT

Mango Genetic variability among the isolates of Trichoderma spp. against Colletotrichum gloeosporioides (Penz.)
Anthracnose Penz. and Sacc. was studied using molecular techniques like RAPD and ITS-PCR. The RAPD banding pattern
Colletotrichum reflected the genetic diversity among the isolates with the formation of three clusters. ITS region of rDNA
gloeosporioid amplification with genus specific ITS1 and ITS4 universal primers produced products that varied in size from
Trichoderma 566 to 657 bp in all the isolates. These results indicated polymorphism at ITS region of rDNA among the
RAPD, ITS-PCR isolates of Trichoderma.

Received on :
17.08.2009
Accepted on :
21.12.2009
*Corresponding
author

INTRODUCTION diagnosis of potential biocontrol agents in future. Thus, the

present study was undertaken to assess the significant genetic
Mango (Mangifera indica L.) is considered as one of the most variations by nucleic acid based marker techniques using
popular fruits among millions of people grown throughout RAPD and ITS-PCR to distinguish the genetic variability in
the tropics and subtropics worldwide. India is the world’s nine isolates of Trichoderma spp. evaluated against
largest producers, shares around 56 per cent of total global Colletotrichum gloeosporioides.
production (Chadha, 2001) and Andhra Pradesh ranks first in
production (45.89 lakh tonnes). Mango is affected by large
number of fungal pathogens. Among which, Colletotrichum
gloeosporioides causes anthracnose, which is the most The molecular variability among the isolates of Trichoderma
important biological constraint to mango production in spp. having different degrees of antagonistic activity against
Southeast Asia resulting in substantial yield loss (Dodd et al., C.gloeosporioides incitant of mango anthracnose was studied
1991). Trichoderma species have been investigated as by using random amplified polymorphic DNA (RAPD) and
biological control agents (BCAs) for over 70 years (Hjelijord et ITS region of rDNA.
al., 1998), but it is only recently that strains have become Fungal cultures
commercially available. Knowledge concerning the behaviour
Mycelial discs of 5 mm diameter Trichoderma isolates were
of these fungi as antagonists is essential for their effective use
cut from periphery of an actively growing 7 day old culture on
since they can act against target organisms in several ways
(Jeffries et al., 1994) by producing extracellular enzymes PDA and inoculated on 250 mL conical flask containing 100
mL of sterile potato dextrose broth. The flasks were kept rotated
(Haran et al., 1996) antifungal antibiotics (Ghisalberti et al.,
constantly at a speed of 125 rpm in an orbital shaker at 28±2
1993) and also be competitors to fungal pathogens (Simon et
C. The resultant growth of mycelial mat was harvested and
al., 1989). Of late, molecular techniques are gaining
excess moisture was completely removed through sterile
importance in characterization and diagnosis of microbial
blotting paper and used for DNA extraction.
population. Molecular characterization and identification of
biocontrol isolates of Trichoderma spp. has been reported by DNA extraction from Trichoderma isolates
several researchers (Hermosa et al., 2000 and Venkateswarulu The total genomic DNA from different isolates of Trichoderma
et al., 2008). These techniques are not influenced by was extracted from vegetative mycelium using the procedure
environment, independent of growth stage and reproducible adopted by Reader and Broda (1985). Mycelial mat of 200 mg
when compared to conventional methods and helps in was grounded in the pre-sterilized pestle and mortar with liquid
identification of antagonists which can be used as markers in nitrogen until fine powder was obtained. About 50 mg of

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K. GIRIDHAR KUMAR et al.,

freeze-dried ground mycelium in an eppendorf tube was pattern of each isolate was evaluated, assigning character state
resuspended in 500 µL of extraction buffer. To this 350 µL of ‘I’ to all the bands that could be reproducible and detected in
phenol and 150 µL of chloroform was added and thoroughly the gel and ‘O’ for the absence of band. The data matrix thus
mixed. The suspension was centrifuged at 13000 rpm for 1 generated was used to calculate Jaccard similarity coefficient
hr. The upper aqueous phase was transferred to a new for each pair wise comparison. The coefficients were calculated
eppendorf tube. To this, 25 µL of RNAse (10%) was added In Silico following Jaccard (1908). The similarity coefficients
and incubated to 37°C for 30 min. The solution was extracted were subjected to Unweighted Pair-Group Method on
with chloroform isoamylalchohol (24:1). The upper phase Arithmetic Average (UPGMA) cluster analysis to group the
was transferred to an eppendorf tube and 0.54 volume (270 isolate based on their overall similarities. Statistical Package
µL) of isopropanol was added and mixed thoroughly by for Social Science (SPSS) package was used for the cluster
inverting the tubes. DNA precipitated visibly into lump was analysis and subsequent dendogram preparation.
observed. The tube was spun at 12,000 rpm for 10 min to PCR amplification of ITS region
pellet the DNA and the remaining liquid was removed. The
pellet was rinsed with 70 percent ethanol two times, dried PCR amplification of ITS (Internal Transcribed Spacers) region
of rDNA was performed using universal primers ITS-1 (5' –
under vaccum and resuspended in 100 µL TE buffer (pH 8.0).
The DNA samples were stored at -20 C for further studies. TCC GTA GGT GGA CCT GCG G – 3') as forward primer and
ITS-4 (5' – TCC TCC GCT TAT TGA TAT GC – 3') as reverse
Qualitative and quantitative verification of DNA primer (White et al., 1990) in eppendorf PCR master cycler.
The quality and quantity of DNA was analyzed by running 2 Amplification was carried out in 0.2 mL eppendorf tubes with
µL of each sample mixed with 2 µL of 10x loading dye in 25 µL reaction mixture containing 2.5 µL of 10 x Taq buffer, 2
0.8% agarose gel. The DNA from all isolates produced clear µL of 25 mM Mgcl2, 0.5 µL of ITS-1 primer (25 picomolar/ µL),
sharp bands in one percent agarose gel indicating the good 0.5 µL of ITS-4 primer (25 picomolar/ µL), 0.5 µL of 100 mM
quality of DNA. The DNA has been quantified by comparing dNTP mix, 0.5 µL of Taq polymerase (3 U/µL) and 15.5 µL of
with the marker (Genei, Bangalore). sterile PCR water (Genei, Bangalore) and 3 µL (30-40ng) of
DNA sample. The PCR amplification was carried out by 35
RAPD profiles through polymerase chain reaction (PCR)
cycles, of which denaturation at 94°C for 1 minute, annealing
Five different random primers viz., OPA-03, OPA-05, OPA- at 56°C for 1 minute and extension at 72°C for 1.5 minutes
08, OPA-09 and OPA-10 (Operon technologies Inc.,) were with initial denaturation at 94°C for 4 minutes before cycling
screened for generating polymorphism among the isolates and final extension at 72°C for 6 minutes after cycling.
under the study. The experiment was repeated thrice and Amplified PCR products were observed in 1.0 per cent agarose
results were reproducible. The primer sequence used in RAPD gel in 1% TBE buffer and visualized under trans-illuminator
technique are given as below: with ethidium bromide staining. The size of the PCR product
S.No. Name of the primer Sequence was estimated by comparison with known DNA marker of 1
kb DNA ladder. The banding profiles of ITS-PCR products
1. OPA-03 5' – AGT CAG CCA C – 3'
were documented in gel documentation system (Alpha
2. OPA-05 5' – AGG GGT CTT G – 3'
Innotech).
3. OPA-08 5' – GTG ACG TAG G – 3'
4. OPA-09 5' – GGG TAA CGC C – 3'
5. OPA-10 5' – GTG ATC GCA G – 3'
PCR amplifications were carried out in 0.2 mL eppendorf tubes Total genomic DNA extracted from the isolates produced clear
with 25 µL reaction mixture which consists of 2.5 µL of 1 x sharp bands indicating the good quality of DNA.
Taq buffer, 2 µL of 25 mM Mgcl2, 4 µL of primer (25 picomolar Characterization of Trichoderma isolates by RAPD
/ µL), 0.5 µL of 100 mM dNTP mix, 0.6 µL of Taq polymerase Five random primers viz., OPA-3, OPA-5, PA-8, OPA-9 and
enzyme (conc. 3U µL-1) and 13.4 µL of sterile PCR water (Genei,
OPA-10 generated reproducible polymorphism among the
Bangalore) and 2 µL (20-25 ng) of DNA sample. Amplification nine isolates of Trichoderma. Amplified products with all the
was carried out by 5 minutes of initial denaturation of 94°C
primers showed polymorphic and distinguishable banding
followed by 45 cycles (Sreenivasaprasad et al., 1992) of
pattern indicating the genetic diversity among all isolates of
denaturation of 94°C for 1 minute; annealing at 37°C for 1 Trichoderma. A total of 119 reproducible and scorable
minute; extension at 72°C for 2 minutes with final elongation
polymorphic bands ranging approximately as low as 75 bp to
at 72°C for 5 minutes. Amplified PCR products were subjected
as high as 2500 bp were generated with five primers among
to 1.0 per cent agarose gel electrophoresis with 1.0 x TBE as the nine Trichoderma isolates characterized.
running buffer. The banding patterns were visualized under
UV trans-illuminator with ethidium bromide (1 mg mL-1) The Trichoderma isolates T1 and T7 were identified as
staining. The DNA banding profiles were documented in the Trichoderma koningii (T1) (accession no. 6623) and
gel documentation system (Alpha Innotech) and compared Trichoderma fasciculatum (T7) (accession no. 6624)
with 1 kb DNA ladder (Genei, Bangalore). respectively at IARI, New Delhi. All the five primers produced
unique bands in case of T.koningii (Isolate T1). Primer OPA-3
Scoring and data analysis amplified a single fragment of approximately 300 bp in case
Each amplified band was considered as RAPD marker and of T2 isolate and 710 in case of T6 isolate (Fig 1A). All the five
recorded for all samples. Data were entered using a matrix in primers amplified similar fragments in case of T and T isolates,
3 4
which all observed bands or characters were listed. The RAPD except for an 850 bp fragment by OPA-3 in case of T4 indicating

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 MOLECULAR CHARACTERIZATION OF TRICHODERMA SPP.

Figure 1: A-E. Random Amplified Polymorphic DNA (RAPD) patterns

of nine Trichoderma isolates with five random primers viz., A – OPA 3;
B – OPA 5; C- OPA 8; D – OPA 9; E – OPA 10; M – 1 kb DNA ladder;
Lanes 1 to 9 represent Trichoderma isolates T1 to T9 respectively

the homogenicity of their genomes. primer OPA-8 amplified a Squared Equalidean Distance method. Based on the results
unique band of 800 bp in case of T.fasciculatum (Isolate T7) obtained, all the nine isolates were grouped into three main
and 1050 bp in case of T9 and 850 bp in case of T6 and T8 clusters. Cluster I contains four isolates viz., T 3, T ,4
isolates (Fig 1C). Primer OPA-10 amplified a unique band of T.fasciculatum and T9 of which the first two and last two forms
approximately 1400 bp in case of T5 and an 1800 bp band in two separate sub-clusters viz., Ia and Ib. Clusters II contains
case of T6 and T8 (Fig 1E). four isolates viz., T 2, T 5, T 6and T ,8 of which the first three forms
Relationship among the isolates were evaluated by cluster a separate sub-cluster viz., Ia and T8 isolate alone forms a
analysis of data based on similarity matrix. The dendogram separate sub-cluster Ib. Cluster III contains only one isolate T.
(Fig 2) was generated using UPGMA package based on Ward’s koningii indicating its genetic difference with other isolates.

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K. GIRIDHAR KUMAR et al.,

T3 3

T4 4

T6 6

T8 8

T2 2

T5 5

T7 7

T9 9

T1 1
Figure 2: Dendogram generated using UPGMA analysis showing polymorphism between isolates of Trichoderma spp. using RAPD markers

ITS 1

18 S 5. 8 S 28 A

ITS 4
ITS 1

ITS 4
Figure 3: Structure of rDNA cluster and the position of ITS primers used in the PCR amplification of Trichoderma isolates

Jaccard’s similarity co-efficient among the nine Trichoderma Characterization of Trichoderma isolates by ITS-PCR
isolates were calculated to establish their genetic relationships. Genus specific target primers viz., ITS-1 and ITS-4 were used
The similarity index values ranged from 0.00 to 100% for PCR amplification of ITS region of rDNA cluster which
indicating the presence of a high range of variability at nucleic includes ITS-1, 5.8S and ITS-2 regions of all the nine isolates.
acid level among the nine Trichoderma isolates. It is worthwhile The structure of rDNA and the expected amplified products
to screen more number of RAPD primers belonging to different with primers are shown in Fig 3. Amplified products size varies
operon series in order to generate more polymorphism among approximately in the range of 566 to 657 bp in all the isolates
the isolates. This study may help in tagging of the RAPD under the study. However, in case of T.koningii (Isolate T1)
fragment to its antagonistic activity. and T.falsicatum (Isolate T7) the size of the band obtained was
657 bp and 603 bp respectively (Fig 4). The results were in
accordance with Latha et al., (2002) who reported the size of
bands obtained with ITS-1 and ITS-4 primer pairs in case of all
Trichoderma isolates was around 600 bp. These results
confirmed that all the isolates belong to the genus Trichoderma.
Similar results were reported by Muthumeenakshi et al.,
(1994), Ospina (1999) and Lieckfeldt (1999), who observed
that the amplified rDNA fragment of approximately 500 to
600 bp by ITS-PCR in Trichoderma.
The ITS-PCR has helped to detect polymorphism at ITS region
of rDNA among the Trichoderma isolates. But based on these
results it is not possible to conclude that all the isolates are the
same. Digestion analysis of amplified ITS region with different
Figure 4: Amplification product of Internal Transcribed Spacer (ITS) restriction enzymes may reveal more polymorphism. Finally,
with ITS-1 and ITS-4 ribosomal DNA primers. M–1 kb DNA Ladder; sequencing of the amplified ITS region will help to know the
Lanes 1 to 9 represent Trichoderma isolates T1 to T9 respectively. The differences at rDNA level among the isolates under study. The
arrows indicate the ITS-PCR bands specific for T.koningii and T. sequence information allows in developing a SCAR marker
fasciculatum for Trichoderma koningii and Trichoderma fasciculatum, the

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 MOLECULAR CHARACTERIZATION OF TRICHODERMA SPP.

potential native antagonist against Colletotrichum JACCARD, P. 1908. Nouvelles recherches sur la distribution florale.
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