Molecular characterization of Trichoderma longibrachiatum 21 PP isolated from rhizospheric soil based on universal ITS primers

Trichoderma species are morphologically very similar and were considered for many years as a single species. A new strain Trichoderma longibrachiatum 21PP was isolated from a rhizospheric soil sample collected from Kaushambi district of an Indian State (Uttar Pradesh). The universal primers (ITS-1& ITS4) were used for the amplification of 28S rRNA gene fragment that produced a sharp band of about 700 bp on the gel. The amplified gene fragment was then sequenced (664 bp) and then deposited in GenBank with the Accession No. JX978542. Thus, the molecular identification of the specified strain enabled us for further characterization as this strain of Trichoderma species can be used as a biocontrol agent against pathogenic fungi such as Fusarium, Alternaria, Sclerotinia, etc. It is proposed that the identified strain T. longibrachiatum 21PP be assigned as the type strain of a species of the genus together with the 28S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases.


INTRODUCTION
Accurate and definitive fungal identification is essential for correct disease diagnosis and treatment of associated fungal infections.Characterization of fungal species using classical methods is not as specific as the genotyping methods.Genotypic techniques involve the amplification of a phylogenetically informative target such as the smallsubunit (28S) rRNA gene (Woese and Fox, 1977).rRNA is essential for the survival of all cells, and the genes encoding the rRNA are highly conserved in the fungal and other kingdoms.The sequences of the rRNA and proteins comprising the ribosome are highly conserved throughout evolution because they require complex inter-and intramolecular interactions to maintain the proteinsynthesizing machinery (Sacchi et al., 2002;Hillis et al., 1991;Woese, 1987).Trichoderma spp. is common soil inhabitants and is effective in providing biocontrol of soil borne pathogens due to its antagonistic behaviour.The major aspect of successful biological control strategies includes the production, formulation and delivery system of bioagents.The Internal Transcribed Spacer (ITS) regions of the rDNA are perhaps the most widely sequenced DNA regions in fungi.It has typically been most useful for molecular systematic study at species level, and even within species (Ospina-Giraldo et al., 1998;Kubicek et al., *Corresponding author.Email: shahid.biotech@rediffmail.com.2000; Kulling et al., 2002;Lee and Hseu, 2002).Kindermann et al. (1998) attempted a first phylogenetic analysis of the whole genus of Trichoderma using sequence analysis of the ITS-1 region of rDNA.
The study aims at identifying a potential strain of Trichoderma species as it can help us in the commerciallization and eventually developing a better solution to the eradication of soil borne pathogens.Here, the method of isolation and identification of an unknown fungus from the Indian Kaushambi (25° 31′ 50.68″N, 81° 22′ 38.25″ E) district using 28S rRNA gene sequence is described (Srivastava et al., 2008;Yadav et al., 2009) to characterize the strain Trichoderma longibrachiatum 21PP as a member of the Trichoderma spp.We initiated a systematic screening program to catalogue the microbial composition of the Indian district Kaushambi.

Isolation of Trichoderma from soil sample
Rhizospheric soil samples were collected from various experimental fields of an Indian district Kaushambi (25° 31′ 50.68″N, 81° 22′ 38.25″ E).The strain was isolated and identified in Potato Dextrose Agar (PDA) with low sugar medium by serial dilution method (Johnson and Crul, 1972;Nirenberg, 1976).

DNA isolation of Trichoderma
Pure culture of the target fungal was grown overnight in liquid PDB for the isolation of genomic DNA using a method described by Hiney et al. (1992).

Identification of Trichoderma
The isolated strain was then identified at molecular level using PCR amplification of the specific gene sequence with universal ITS-1 (forward) and ITS-4 (reverse) primers.It is then deposited to the Indian Type Culture Collection (ITCC), IARI (New Delhi, India).

Molecular characterization
The total genomic DNA was extracted from isolate of Trichoderma based on Cetrimide Tetradecyl Trimethyl Ammonium Bromide (CTAB) mini extraction method as was described by Crowhurst et al. (1995) in their study.

Agarose gel electrophoresis
Ten microlitre of the reaction mixture was then analyzed by submarine gel electrophoresis using 1.0% agarose with ethidium bromide at 8 V/cm and the reaction product was visualized under a UV transilluminator.

Amplification of isolated DNA using ITS
The internal transcribed spacer (ITS) regions of the rDNA repeat from the 3′ end of the 28S and the 5' end of the 28S gene were amplified using the two primers, ITS-1 and ITS-4 which were synthesized on the basis of conserved regions of the eukaryotic rRNA gene (White et al., 1990).The PCR-amplification reactions were performed in a 50 µl mixture containing 50 mM KCl, 20 mM Tris HCl (pH 8.4), 2.0 mM MgCl2, 200 µM of each of the four deoxynucleotide triphosphates (dNTPs), 0.2 µmM of each primer, 40 ng/µl of template and 2.5 U of Taq polymerase (Fermentas).The cycle parameters included an initial denaturation for 5 min at 94°C, followed by 40 cycles of denaturation for 1 min at 94°C, primer annealing for 2 min at 55°C, primer extension for 3 min at 72°C, and a final extension for 10 min at 72°C.Amplified products were separated on 1.2% agarose gel in TAE buffer, pre-stained with ethidium bromide (1 µg/ml) and electrophoresis was carried out at 60 V for 3 h in TAE buffer.One Kb ladder (MBI, Fementas) was used as a marker.The gel was observed in a transilluminator over ultraviolet light.The desired bands were cut from the gel with minimum quantity of gel portion using QIAGEN gel extraction kit.

Purification of PCR product
The PCR product was purified by QIAGEN gel extraction kit using the protocol described in the manufacturer's manual.

DNA sequencing of the 28S rDNA fragment
A pair of universal ITS primers ITS-1 (forward) and ITS-4 (reverse) was used for sequencing of the amplified product and this step was carried out at the Merck Laboratory (Bangalore, India).The details of the primers are mentioned in Table 1.

Sequence analysis and phylogeny
A comparison of 28S rRNA gene sequence of the test strain against nucleotide collection (nr/nt) database was done using BLAST program (Zhang et al., 2000).Sequences that shared about 90% similarity with the test sequence were selected for a multiple sequence alignment that was carried out using ClustalW (Thompson et al., 1994).Subsequently, an evolutionary distance matrix was generated from these nucleotide sequences in the dataset.A phylogenetic tree was then constructed using the Neighbor Joining method (Saitou and Nei, 1987).Phylogenetic and molecular evolutionary analyses were conducted using MEGA (Molecular Evolutionary Genetics Analysis) version 4.0 (Tamura et al., 2007).The 16S rRNA gene sequence of test strain was compared with a different set of sequence database such as small subunit ribosomal RNA (SSU rRNA) and large subunit ribosomal RNA (LSU rRNA) using Ribosomal RNA BLAST (Altschul et al., 1997).28S rRNA gene sequence of the test strain was also compared against those sequences, in Ribosomal Database Project (Cole et al., 2009) by using the RDP Classifier check program (Wang et al., 2007).The annotated information for the sequence in the database to which 28S rRNA aligns is used for fungal identification.

RESULTS
Rapid identification of microorganisms is necessary in the pathological laboratory in an attempt to proceed for the preparation of a bioformulation that can be useful for the farmers.The rRNA based analysis is a central method in pathology used not only to explore microbial diversity but also to identify new strains.The genomic DNA was extracted from isolated fungal strain T. longibrachiatum 21PP and universal ITS-1 (19F) and ITS-4 (20R) primers were used for the amplification and sequencing of the 28S rRNA gene fragment.A total of 664 bp of the 28S rRNA gene was sequenced (Figure 1).
The sequenced gene fragment of the specific strain was then searched for other closely related and similar templates using BLAST program.Sequences that shared 90% similarity to the test sequence were selected for a multiple sequence alignment.Figure 2 shows the phylogenetic relationship of the test strain (T.longibrachiatum 21PP) with others strains of Trichoderma.Comparison of test strain against known sequences of SSU and LSU rRNA databases showed that the gene sequence of the isolate 21PP has 90% sequence similarity (Score=664 bits, Expect=0.0)with 28S rRNA gene sequence of Trichoderma (GenBank Acc.No.: JX978542).Thus, it can be concluded from the above data that the isolate 21PP is a member of the genus T. longibrachiatum.

Conclusion
The study is focused on the identification of bioagent T. longibrachiatum 21PP at species level.Biocontrol activity of fungal strains in laboratory is generally determined by biological assays.To check the presence or absence of these biocontrol agents is a need to develop some efficient method.In past, serial dilution method had some disadvantages as it cannot distinguish between indigenous and artificially introduced strains that is why the first step of utilizing the full potential of fungal strains is identified at molecular level.

ACKNOWLEDGEMENT
The authors are grateful for the financial support granted

Figure 1 .
Figure 1.Shows the amplified PCR product of the Trichoderma longibrachiatum 21PP.

Figure 2 .
Figure 2. Shows the phylogenetic relationship of the test strain with other strains of Trichoderma.

Table 1 .
Gene sequence with universal primers.