Traditional method of classification and identification of fungi has relied upon microscopic features, colony characteristics on artificial media and biochemical reactions (1). Such approaches have served in past but these methods have major drawbacks as these methods cannot be applicable for non cultivable organisms and also occasionally biochemical characteristic of some organisms do not fit into patterns of any known genus and species, Hence specialist knowledge is needed to differentiate large and complex genera such as Penicillium, Aspergillus and Fusarium (2) Recently there have been worldwide interest on molecular techniques (3) Amplification and sequencing of target regions within the ribosomal DNA gene complex has emerged as a useful adjunctive tool for the identification of fungi and does not depend on mold sporulation for identification (4, 5, 6 and 7). The internal transcribed spacer (ITS) regions 1 and 2 located between the highly conserved small (18S) and large (28S) ribosomal subunit genes in the rRNA operon are known to have sufficient sequence variability to allow identification to the species level for many fungi (4, 5, 6, 7, 8 and 9).
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In the present studies we are reporting the isolation and identification of pigment producing isolate klmp51 with the help of partial 18S rRNA sequence analysis and determining its phylogenetic relationship.
MATERIALS AND METHODS
Sabouraud Dextrose Agar (SDA): The ingredient of the media was procured from Hi-media Mumbai, India. The composition of the media was (g L−1): Dextrose 40; Peptone 20; Agar 20; pH 5.6 + 0.2.
Isolation and screening
Various soil samples from different regions of Gulbarga, India were collected. The soil samples were serially diluted by serial dilution method. 0.1ml from 10-3, 10-4and 10-5 dilution tube were
transferred to SDA media and spread over the entire surface of the media using spreader and incubated for a week at 270C. 0ver 59 fungi were isolated and named as klmp1-59 of which klmp51 produced red pigment which was transferred on the fresh media and stored for further use.
Isolation of genomic DNA and 18S rRNA sequencing Genomic DNA Isolation
The genomic DNA was isolated by transferring 1.5 ml of the culture to a micro centrifuge tube and centrifuged for 2 min. Then the supernatant was discarded and the pellet was re suspended in 467μl TE buffer by repeated pipetting, then 30μl of 10% SDS and 3μl of 20 mg/ml proteinase K were added and incubated for 1 hr at 37°C. After incubation an equal volume of chloroform was added and mixed well by inverting the tube until the phases are completely mixed. Carefully the DNA/phenol mixture was transferred into a fresh tube and centrifuged for 2 min. The upper aqueous phase was transferred in to a new tube. An equal volume of chloroform was added again
mixed well and transfer to a new tube and centrifuged for 2 min. The upper aqueous phase was transferred to a new tube. Then 1/10 volume of sodium acetate and 0.6 volumes of isopropanol was added and mixed gently until the DNA precipitates. The DNA was spooled onto a glass rod (or Pasteur pipette with a heat-sealed end). The DNA was washed by dipping the end of rod into 1 ml of 70% ethanol for 30 sec. The DNA was re suspended in 100-200μl TE buffer.
PCR amplification of 18S rRNA
PCR amplification of 18S rRNA gene, from the purified genomic DNA was carried out using the primer sets. Forward primer ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC -3'). The PCR condition
PCR product purification
The unpurified DNA sample was dissolved (at least 10-15μl) in 50μl of PCR cleanup solution and incubate at 55 °C for 15-20 minutes. The mixture was Centrifuge at 12000 rpm for 15 minutes, during which time the contaminants was released into the supernatant and the supernatant was discarded at the end of the centrifugation. Further the DNA was precipitated by the addition of 600μl of 80% ethanol and centrifugation at the same conditions as before. The residual cleanup solution and the contaminants were removed along with ethanol by discarding the supernatant. Finally, the DNA pellet was dried and dissolved in 10-15μl of Milli Q water.
The sequencing of the target gene was done using BigDye Chemistry, and was performed as per the manufacturer's protocols (Applied Biosystems 3730xl DNA Analyzer) the tube was placed in
Always on Time
Marked to Standard
the thermal cycler. The thermo cycler was programmed as follows: 25 cycles of [96o C for 10 sec, 50o C for 5-10 sec, 600C for 4 min], then ramp to 40C.
Purification of sequencing extension product by isopropanol precipitation method
The tube was spin and transferred by pipetting entire sequencing reactions into 1.5 ml micro centrifuge tube. Then 40 ml of 75% isopropanol, or 10 ml of de ionized water and 30 ml of 100%
isopropanol was added and mixed by vortexing and left at room temperature for >15 min to precipitate products. The tube was centrifuged for a minimum of 20 min at maximum speed in a micro centrifuge. The supernatant was aspirated completely with a separate pipette tip for each sample, being careful not to disturb the DNA pellet, and then it is discarded. About 125 to 250 ml of 75% isopropanol was added to the tube and vortex briefly and centrifuged for 5 min at maximum speed, and the supernatant was aspirated as in above step. The sample was dried for 10 - 15 minutes and stored at -200C until ready for electrophoresis. The purified extension products were separated in the ABI 3730xl DNA Analyzer by Capillary Electrophoresis. Sequence data analysis was done using ChromasPro and Sequencing Analysis software. The isolation of genomic DNA and sequencing Fig.1, Fig.2.was performed at Ocimum Bio Solution, Hyderabad, India.
The analysis of nucleotide sequence was done in Blast-n site at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST).The alignment of the sequences was done by using CLUSTALW (www.ebi.ac.uk/clustalw) Fig.3.
Fig 3. Partial 18S forward and reverse assembled sequence of isolate klmp51
A phylogentic analysis of the isolate klmp51 was performed to determine how the 18S rRNA sequence of the isolate klmp51 and related strain might have been derived during evolution. The evolutionary relationships among the sequences were depicted by placing them as outer branches on a phylogenetic tree. The branching relationships on the inner part of the tree reflect the degree to which different sequences are related. Sequences that were very much alike were located as neighboring outside branches and joined to a common branch beneath them. The objective of phylogenetic analysis is to find out all of the branching relationships in the tree along with branch lengths. For this phylogenetic tree was constructed using the aligned sequences by the neighbor joining method using Kimura-2-parameter distances in MEGA5 software . Distances between the studied sequences helps in understanding the evolutionary distances among the species.
Criteria for species identification
Identification of species through sequence similarity basis was performed according to criteria used by  which states the following selection parameters: (a) when the percentage similarity of the query sequence and the reference sequence is 99% or above, the unknown isolate would be assigned to reference species; (b) when percentage similarity is between 95 - 99 %, the unknown
isolate would be assigned to the corresponding genus; (c) when percentage similarity is less than 95 %, the unknown isolate would be assigned to a family.
Results and discussion
Identification and phylogenetic position of isolate klmp51 The identification was done based on 18S rRNA gene sequencing. The 18S rRNA sequence of the isolate klmp51was compared with the data present in NCBI. The BLASTn of the isolate klmp51 was showing 100% homology with the Lasiodiplodia spp. The sequence was submitted to the Gene Bank under the accession number JQ073734. To analyze the phylogenetic position of the 18S rRNA sequence. The phylogenetic tree was constructed using Mega5 by neighbor-joining tree using Kimura-2-parameter with 1000 bootstrap replication. The phylogentic relation was determined Fig. (4).Shows the phylogenetic relationship between the isolate klmp51 and other related fungi. The homology assay result indicated the isolate klmp51 were in the phylogenetic branch of Lasiodiplodia spp. The isolate showed a remarkable red pigment on SDA (Plate.1), which may be used in foodstuff, dyestuff, cosmetics and pharmaceutical manufacturing processes. Fig 4. Phylogenetic tree of Isolate klmp51 showing homology with Lasiodiplodia spp. Numbers near the nod region represents the bootstrap value, while Aspergillus niger as a outer group. Plate 1. a) Control b) Red pigment production by Lasiodiplodia klmp51 in SDA
From this study we have concluded that the isolate klmp51 belongs to Lasiodiplodia spp. based on Molecular taxonomy and phylogeny. Lasiodiplodia klmp51 produced a red pigment which may be used as a bio-color in dyestuffs, foodstuffs and in cosmetic industries.
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