4.1 Introduction
Fungi reproduce and disperse through spores produced sexually or asexually during
their life cycle and these structures are of immense value in identification and
classification. Traditionally fungi are also identified depending on the cultural
characteristics such as colony morphology in addition to sexual structures and spores.
Comparative morphological features and the development of sexual or asexual
reproductive structures remained a main stay in the identification and classification of
various groups of fungi (Sette et al., 2006; Crous et al., 2007). Special care is needed
particularly when identifying and classifying closely related or morphologically similar
endophytes because the morphological characteristics of some fungi are usually
medium-dependent and cultural conditions can substantially affect the vegetative and
sexual compatibility (Hyde and Soytong, 2007). It is much more difficult with non
sporulating filamentous fungi. Inspite of the development of various methods to
promote sporulation (Guo et al., 1998, 2000; Taylor et al., 1999), most of the isolates
(up to 54% of the total) do not sporulate in cultures (Petrini et al., 1982; Fisher et al.,
1993; Guo et al., 2000, 2008; Photita et al., 2001; Kumaresan and Suryanarayanan
2002; Wang and Guo 2007; Sun et al., 2008, 2011). Moreover, the conventional
methods cannot be applied for identifying fungal isolates that do not sporulate in
culture, hence are grouped as mycelia sterilia (Lacap et al., 2003). Guo et al. ( 2000)
described that various mycological media have been attempted to induce and promote
sporulation of these fungi, like potato dextrose agar (PDA), malt extract agar (MEA),
corn meal agar (CMA), potato carrot agar (PCA), and water agar (WA). Mycelia sterilia
have been reported to be very frequent and predominant group of endophytic fungi.
Although morphological characters represent the phenotype of the organism, these are
not always reliable as they are liable for change due to diverse environmental
conditions. In recent times, new molecular techniques have been developed for
identification of the organisms based on the analysis of the genome or ribosomal RNA.
Molecular analysis appears to be highly sensitive and specific for identification of
microorganisms and can be used for classifying microbial strains at diverse hierarchical
70
taxonomic levels. Recent studies have shown that genetic methods can be successfully
used in the studies of endophytic fungi a s w e l l ( Gao et al., 2005; Arnold et al.,
2007). Comparison of the ribosomal DNA sequences, especially the ITS regions,
helped to detect and identify most of the endophytic fungi. Guo et al. (2000) and Lacap
et al. (2003) assessed the endophytic fungal morphotype concept referring mycelia
sterilia. A high diversity of endophytic fungal communities was revealed from either
Heterosmilax japonica or Livistona chinensis using a cultivation-independent approach
by analyzing fungal DNA sequences extracted from plant tissues (Guo et al., 2001; Gao
et al., 2005).
The PCR-RFLP technique with specific oligonucleotide primers ITS 1 and ITS 4
(White et
al., 1990) and
restricting withdifferent
endonucleases, has
been
successfully used to analyze regions of ribosomal DNA of various groups of fungi
(Cai et al., 2006; Shenoy et al.,2007; Hyde and Soytong, 2008). Sequence analysis of
the ITS region of nuclear ribosomal DNA has been widely used for molecular
identification and to study phylogenetic diversity of fungi.
Some of the selected endophytic fungi isolated from the medicinal plants of
Bhadrachalam forests have been analysed by sequencing the ribosomal RNA with the
following objectives:
1. To confirm the traditional morphological identification of the selected species
2. To identify the sterile forms of the selected isolates
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4.2 Methodology:
Purification of fungal strains for the molecular identification
The endophytes isolated from the medicinal plants were selected for molecular
identification based on their importance, frequency of occurrence and the lack of
sporulation. One of the potential endophytic fungus identified on morphological basis
as Arthrinium phaeospermum was isolated from an important medicinal plant, A.
paniculata has been selected for molecular analysis. In addition to the identified one,
two more potential non sporulating endophytes were also selected for this study.
The cultures were purified by single spore isolation (for sporulating) and hyphal tip
culture methods (for non sporulating fungi).
4.2.1 Single spore isolation
Spore suspension of the fungus was prepared in 10 ml of sterile water containing 0.5 ml
of tween-20. Suspension was thoroughly mixed, serially diluted to obtain 104 and from
this 1 ml aliquots were withdrawn and poured into sterilized Petri plates followed by
water agar medium under aseptic conditions. Uniform spread of the medium was
ensured by gentle movement of the plates and incubated for one day.
After 24 h, the Petri plates were observed under microscope for the germinating spores.
Single germinating spores were selected and picked up aseptically with single spore
isolators fixed to the microscope. The isolated single spores were placed on PDA and
cultured. Hyphal tip cultures are developed by cutting the tip of hyphae at the growing
edge.
4.2.2 Molecular Identification of endophytic fungi
Fungal strains were identified using a molecular biological protocol by amplifying the
rDNA using the internal transcribed spacer (ITS) primers. This was carried out by
Macrogen. The amplified 18S rDNA fragments were analyzed (18S rRNA genes) using
bioinformatics tools. The sequencing result obtained was pasted in Basic Local
Alignment Search Tool (BLAST) software supported by National Institute of Health
72
(NIH) using the National Centre for Biotechnology Information (NCBI) database
(http//: www. NCBI.NLM.NIH.GOV) to compare and align with the sequence of
known fungi that helps in identification at molecular level. It also helps in establishing
the phylogenetic relationships of the fungus in question.
Isolation of genomic DNA, PCR amplification and sequencing
The isolation of genomic DNA, PCR amplification and sequencing was carried out by
Macrogen. The procedure is as follows: The fungus was cultured on potato dextrose
agar medium and small amount of mycelium from the growing edge was suspended in
40 μl MQ (Make: Bio Rad) water. The suspended culture was added with 160 μl of
NaOH (0.05M) and mixed well. The samples were incubated on dry bath for 45 min at
60 C and vortexed intermittently. Then 12 μl of Tris-HCl (0.01M) was added and the
mixture was diluted up to 100 fold. From the diluted extract 6μl was used for PCR.
The PCR was set up using the following components: 2.5 μl Buffer (10x), 1.5 μl MgCl2
(25 mM), 2.5 μl dNTPs (2 mM), 0.2 μl pro mega Taq Polymerase (5 U/μl), 1.0 μl
primer F (5 pm/μl) and 6.0 μl DNA from diluted extract. The PCR was run in such a
way, where initial denaturation was at 94 C for 3 min. Denaturation, annealing and
extension were done at 96C for 10 sec, 55C for 10 sec and 72 C for 30 sec respectively
in 45 cycles. Final extension was done at 72 C for 10 min and hold at 4 C for infinite
time. After the PCR cycle, 2 μl of the product was used to check on 1% agarose gel. It
was then purified using quick spin column and buffers (washing buffer and elution
buffer) according to the manufacturer’s protocol (QIA quick gel extraction kit Cat
No.28706).
DNA sequencing was performed using an Applied Biosystem 3130xl analyzer. For
amplification of ITS- rDNA region PCR reactions for sequencing were carried with
universal primers ITS 1 (with base sequences TCCGTAGGTGAACCTGCGG) and
ITS 4 (with base sequences TCCTCCGCTTATTGATATGC), as primers, (White et
al., 1990).
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DNA sequence assembly and alignment
Sequence similarity searches were performed or each fungal sequence against the nonredundant database maintained by the National Center for Biotechnology Information
using the BLAST algorithm (http://www.ncbi.nlm.nih.gov).
4.3 Results
Molecular characterization of selected isolates OU E 38, OU E 203, OU E 211, OU E
208 was done using respective sequences of 18S rRNA and amplified with universal
primers
ITS1
(TCCGTAGGTGAACCTGCGG)
and
ITS
4
(TCCTCCGCTTATTGATATGC). Sequences of 18S rDNA gene were analysed by
Basic Local Alignment Search Tool (BLAST) and compared with the available
reference nucleotide sequences from data base sequences of National Center for
Biotechnology Information (NCBI) and European Molecular Biology Laboratory
(EMBL). Based on similarity percentage, taxonomic position was determined.
Four isolates of endophytes from different medicinal plants exhibiting morphological
variation were selected for molecular identification viz., OU E 38, OU E 203, OU E
211, and OU E 208. These were isolated from Andrographis paniculata, Pupalia
lappacea, Terminalia bellerica and Emblica sp. All the four endophytic fungi were
single spored or developed from the hyphal tips before molecular characterization.
OU E 38
The selected endophytic fungus OU E 38 was isolated from Andrographis paniculata
and identified on the basis of morphological features as Arthrinium phaeospermum (OU
E 38). The identity of the endophyte was further subjected to molecular characterization
by analyzing 18S rRNA.
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The forward and reverse sequences of the fungus O_U_E38 are:
18S rRNA forward sequence
GGGGGAATCGAGTTTACACTCCACACCATTTGCCACTTTACTCAGTTATG
CCTCGGCGTAAGCTCCGTACGGGGCTGCCGGGTTGCGCTGCGGGCGACAG
CTACCCTGTAGCTTACCCTGTAGCGCTACCCTGTAGCGTTACCCTGCGGC
GGCCCGCCGGTGGAAACGAAACTCTTGTTTTATTGTATCTTCTGAGCGTC
TTATTTTAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCAT
CGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCA
GTGAATCATCGAATCTTTGAACGCACATTGCGCCCATCAGTATTCTGGTG
GGCATGCCTGTTCGAGCGTCATTTCAACCCTTAAGCCTAGCTTAGTGTTG
GGAATCGACCGTAGGGTCGTTCCTTAAAGACAGTGGCGGAGCGGCAGTGG
TCCTCTGAGCGTAGTAAATTTATTTCTCGCTTTTGTCAGGCCCTGTCCTC
CCGCCATAAAACCCCCAATTTTTTAGTGGTTGACCTCGGATCAGGTAGGA
ATACCCGCTGAACTTAAGCATATCATAAGCCGGGAGGAAAAATAATTAGG
GAGTAATACAAC
18S rRNA reverse sequence
AAACTCGGCAATCCTACCTGATCCGAGGTCACCACTAAAAAAATTGGGGG
TTTTATGGCGGGAGGACAGGGCCTGACAAAAGCGAGAAATAAATTTACTA
CGCTCAGAGGACCACTGCCGCTCCGCCACTGTCTTTAAGGAACGACCCTA
CGGTCGATTCCCAACACTAAGCTAGGCTTAAGGGTTGAAATGACGCTCGA
ACAGGCATGCCCACCAGAATACTGATGGGCGCAATGTGCGTTCAAAGATT
CGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGC
GTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTAACT
TATTAAAATAAGACGCTCAGAAGATACAATAAAACAAGAGTTTCGTTTCC
ACCGGCGGGCCGCCGCAGGGTAACGCTACAGGGTAGCGCTACAGGGTAAG
CTACAGGGTAGCTGTCGCCCGCAGCGCAACCCGGCAGCCCCGTACGGAGC
TTACGCCGAGGCATAACTGAGTAAAGTTGGCAAATGGTGTGGGAGTTGTA
TAACTCTGTAATGATCCCTCCGCAGTCCCCCCCTTCCGG
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Fig. 4.1 Phylogenetic tree of the endophytic fungus, Arthrinium phaeospermum
inferred by neighbor-joining analysis of 18S rDNA sequences.
The rRNA sequence is deposited in NCBI database and the accession number given is
KM668704. Relationship of the selected endophytic isolate with other similar fungi has
been analysed based on 18S r DNA sequences. The selected endophytic fungal strain
were shown by species names or our university collection number followed by
Genbank accession numbers. The tree was constructed by using phylogeny.fr online
phylogeny programs. The numbers at the nodes indicate levels of boot strap support
based on neighbor joining analysis of sequence data. The rRNA sequences up on
BLAST analysis for alignment and comparison with NCBI database, OU E 38
(KM668704) is showing close similarity (99%) with Arthrinium phaeospermum (Fig.
4.1; Table 4.1). Therefore it has been identified as A. phaeospermum. These results
justified our identification using morphological characters.
OU E 203
The selected endophytic fungus OU E 203 was isolated from Pupalia lappacea and
identified on the basis of morphological features as Mycelia sterilia (OU E 203). The
identity of the endophyte was further subjected to molecular characterization by
analyzing 18S rRNA.
76
The forward and reverse sequences of O_U_E203 are:
18S rRNA forward sequence
AAAAACTTTAATCATGGTGCGGCGCGGCCCCCGAGGAGCGGAACAATCCT
TGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGC
CATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGC
CGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGGAA
CAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTGGCATCGA
TGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTG
AATCATCGAATCTTTGAACGCACATTGCGCCCCTCGGTATTCCGTGGGGC
ATGCCTGTTCGAGCGTCATCTACACCCTCAAGCTCTGCTTGGTGTTGGGC
GTCTGTCCCGCCTCCGCGCGTGGACTCGCCCCAAATTCATTGGCAGCGGT
CCTTGCCTCCTCTCGCGCAGCACATTGCGCTTCTCGAGGGGCTACGGCTC
GCGTCCAACAAGCACATTTACCGTCTTTGACCTCGGATCAGGTAGGGATA
CCCGCTGAACTTAAGCATATCAATAAGCCCGGAGGAAAGATCATTAACCT
TTCAAATCAGGGTGCGGCGCGGCCCCGAGGAGCGGAAACAATCCTTGGGG
AGGTATGCGGGGGGCTTCGAGCCCCCATTTACGCAGCACGACTGCCATCC
TTACTTTACGAGCACCTTCTGATTCTCCTCGGCGGGGCAACCTGCCGTTG
GAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTTCGGGAACAAA
CAATCGTTACAACTTTCAACAATGGGATCTCTTGGGCTCTGGCATCGATG
AAAGAACGCAGCGAAAATGCGAATAAGGTAGTTGTGGAATTTGCAAAAAA
TTCAGTTGAAATCCATCGAAATCTTTTGAAACCCATTGGCCCTCGGTTAT
TTCGTGGGGGGCATGCCGGTTTCAAGGGTCATCTAACCCAAGCTGGCTTG
GGGGGTTTTGGGGGGTTTTTTCCCCCCCCCGGGGGGAATCCCCCAAATTC
ATTGGGGGGCGGGTCCTTTGCTTCCTCCTCGCGGCAACAACAATTTTGCG
TTTTTCCTTTCGGGGGGAGAGAAGCGTCGGGCCGCCGCACCAAAAAAAAA
AATAAATTTTTATCCTTCTTGTTATTTCCATGAAGAAAGAAGGGGGGGGG
GGGGCCCGCCACCCCCCATTATAAATTTAATTAAAAAAAGCGCAGGAAAG
GAAGAAAAAAAAAGAAAAAAAAAAAAGAAGGGGAAAGTAAAGGGACACCG
AACCAACCGAGCCAAGGTACAGTCAATTCCCATTTCATACCAATTCTGGC
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CGCTTGATTGCCTCCGATTAACCGTAAACTGGGCATTGAGCCGTCGATTG
GCACTGGACTTGGAATTTGATTAAGGCCCTGCCCAAT
18S rRNA reverse sequence
TCGGGGATCCCTACCTGATCCGAGGTCAAGACGGTAAATGTGCTTGTTGG
ACGCGAGCCGTAGCCCCTCGAGAAGCGCAATGTGCTGCGCGAGAGGAGGC
AAGGACCGCTGCCAATGAATTTGGGGCGAGTCCACGCGCGGAGGCGGGAC
AGACGCCCAACACCAAGCAGAGCTTGAGGGTGTAGATGACGCTCGAACAG
GCATGCCCCACGGAATACCGAGGGGCGCAATGTGCGTTCAAAGATTCGAT
GATTCACTGAATTCTGCAATTCACACTACTTATCGCATTTCGCTGCGTTC
TTCATCGATGCCAGAGCCAAGAGATCCATTGTTGAAAGTTGTAACGATTG
TTTGTTCCGGAACAGGTAATGCTAGATGCAAAAAGAGTTTATTCGGTTCC
AACGGCAGGTTGCCCCGCCGAGGGAGAACAGAAGGTGCTCGTAAAGTAAG
GATGGCAGTCGTGCGTGCGTAAATGGGGGGCTCGAAGCCCCCGCATACCT
CCCAAGGATTGTTTCCGCTCCTCGGGGGCCGCGCCGCACCCTGATTTGAA
AGGTTAATGATCCTTCCGCAGCCCCCCCCCCTCGGAAAAGG
Fig.4.2 Phylogenetic tree of the endophytic fungus OU E 203, Leptosphaerulina
chartarum inferred by neighbor-joining analysis of 18S rDNA sequences.
The rRNA sequences up on BLAST analysis for alignment and comparison with NCBI
database, the isolate OU E 203 showed close relationship with Leptosphaerulina
chartarum and other uncultured fungal clones. The rRNA sequence is deposited in
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NCBI database and the accession number given is KM668707. Relationship of the
selected endophytic isolate with other similar fungi has been analysed based on 18S
rDNA sequences. The selected endophytic fungal strains were shown by species names
or our university collection number followed by Genbank accession numbers. The tree
was constructed by using phylogeny.fr online phylogeny programs. The numbers at the
nodes indicate levels of boot strap support based on neighbor joining analysis of
sequence data. OU E 203 (KM668707) is showing close similarity (99%) with
Leptosphaerulina chartarum (Fig 4.2; Table 4.1). Therefore the sterile mycelial fungus
has been identified as Leptosphaerulina chartarum.
OU E 211
The selected endophytic fungus OU E 211 was isolated from Terminalia bellerica and
identified on the basis of morphological features as Acremonium sp. (OU E 211). The
identity of the endophyte was further subjected to molecular characterization by
analyzing 18S rRNA.
The forward and reverse sequences of O_U_E 211 are as follows
18S rRNA forward sequence
AGGGGAAACGAAGTGTAAACTCCCAACCATTGTGTACCTACCACTGTTGC
TTCGGCGGCCTCGCCCCGGGCGCGTTCGCGCGGCCCGGACCCAGGCGTCC
GCCGGAGGCTCCAAACTCTTGTCTTTTAGTGTATTTCTGAGTGGCATAAG
CAAATAAATCAAAACTTTCAGCAACGGATCTCTTGGTTCTGGCATCGATG
AAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAA
TCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCAT
GCCTGTCTGAGCGTCATTTCAACCCTCAGGACCCGTTCGCGGGACCTGGC
GTTGGGGATCAGCCTGCCCCTGGCGGCGGCTGGCCCTGAAATCCAGTGGC
GGTTCCCTCGCGAACTCCTCCGTGCAGTAATTAAACCTCTCGCGGCAGGA
TAGCGGTTGAACCACGCCGTTAAACCCCCCACTTCTCAAGGTTGACCTCA
GATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAAAGCCGGGAGGA
AACCAAGACCGAGGGCACA
79
18S rRNA reverse sequence
AAATGGGGATTCTACCTGATCTGAGGTCACCTTGAGAAGTGGGGGGTTTA
ACGGCGTGGTTCAACCGCTATCCTGCCGCGAGAGGTTTAATTACTGCACG
GAGGAGTTCGCGAGGGAACCGCCACTGGATTTCAGGGCCAGCCGCCGCCA
GGGGCAGGCTGATCCCCAACGCCAGGTCCCGCGAACGGGTCCTGAGGGTT
GAAATGACGCTCAGACAGGCATGCCCGCCAGAATACTGGCGGGCGCAATG
TGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTA
TCGCATTTTGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGC
TGAAAGTTTTGATTTATTTGCTTATGCCACTCAGAAATACACTAAAAGAC
AAGAGTTTGGAGCCTCCGGCGGACGCCTGGGTCCGGGCCGCGCGAACGCG
CCCGGGGCGAGGCCGCCGAAGCAACAGTGGTAGGTTCACAATGGTTTGGG
AGTTTTTACACTCGGTAATGATCCCTCCGCAGTCCCCCCTTACGGGAAAA
Fig 4.3 Phylogenetic tree of the endophytic fungus OU E 211, Acremonium
sclerotigenum inferred by neighbor-joining analysis of 18S rDNA sequences.
The rRNA sequences up on BLAST analysis for alignment and comparison with NCBI
database, the rRNA sequence is deposited in NCBI database and the accession number
given is KM668706. Relationship of the selected endophytic isolates with other similar
fungi has been analysed based on 18S r DNA sequences. The selected endophytic
fungal strains were shown by species names or our university collection number
followed by Genbank accession numbers. The tree was constructed by using
phylogeny.fr online phylogeny programs. The numbers at the nodes indicate levels of
80
boot strap support based on neighbor joining analysis of sequence data. OU E 211
(KM668706) is showing close similarity (99%) with Acremonium sclerotigenum (Fig.
4.3; Table 4.1). Therefore it has been identified as Acremonium sclerotigenum. These
results justified our identification using morphological characters.
OU E 208
The selected endophytic fungus OU E 208 was isolated from Emblica sp. and identified
on the basis of morphological features as Mycelia sterilia. (OU E 208). The identity of
the endophyte was further subjected to molecular characterization by analyzing 18S
rRNA sequence.
The forward and reverse sequences of O_U_E 208 is as follows
18S rRNA forward sequence
GGGGAATGTCTGGACCGCCTCGGCGCACCCAGAAACCCTTTGTGAACTTA
TACCTTTCTGTTGCCTCGGCGCAGGCCGGCCTCTTAGCTGAGGCCCCCCG
GAGACGGGGAGCAGCCCGCCGGCGGCCAACCAAACTCTTGTTTCTACAGT
GGATCTCTGAGTAAAAAACATAAATGAATCAAAACTTTCAACAACGGATC
TCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGT
GAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCC
TCTGGTATTCCGGAGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAG
CCTGGCTTGGTGTTGGGGCACTGCTCCGAGAGGAGCAGGCCCTGAAATCT
AGTGGCGAGCTCGCTAGGACCCCGAGCGTAGTAGTTATATCTCGTTCTGG
AAGGCCCTGGCGGTGCCCTGCCGTTAAACCCCCAACTTCTGAAATTTTGA
CCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGGCG
GGAGGAAAGTCAATTGTT
18S rRNA reverse sequence
AACTGGGTATTCTACCTGATCCGAGGTCAAATTTCAGAAGTTGGGGGTTT
AACGGCAGGGCACCGCCAGGGCCTTCCAGAACGAGATATAACTACTACGC
TCGGGGTCCTAGCGAGCTCGCCACTAGATTTCAGGGCCTGCTCCTCTCGG
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AGCAGTGCCCCAACACCAAGCCAGGCTTGAGGGTTGAAATGACGCTCGAA
CAGGCATGCCCTCCGGAATACCAGAGGGCGCAATGTGCGTTCAAAGATTC
GATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCG
TTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTTTGATTC
ATTTATGTTTTTTACTCAGAGATCCACTGTAGAAACAAGAGTTTGGTTGG
CCGCCGGCGGGCTGCTCCCCGTCTCCGGGGGGCCTCAGCTAAGAGGCCGG
CCTGCGCCGAGGCAACAGAAAGGTATAAGTTCACAAAGGGTTTCTGGGTG
CGCCGAGGCGCGTTCCAGCAATGATCCCTCCGCAGTACCCCCTTT
Fig. 4.4 Phylogenetic tree of the endophytic fungus OU E 208, Phomopsis inferred
by neighbor-joining analysis of 18S rDNA sequences.
The rRNA sequences up on BLAST analysis for alignment and comparison with NCBI
database, a close similarity was evident from the phylogenetic tree with the species of
Phomopsis (Fig. 4.4). The rRNA sequence was deposited in NCBI database and the
accession number given is KM668705. Relationship of the selected endophytic isolates
with other similar fungi has been analysed based on 18S r DNA sequences. The
selected endophytic fungal strains were shown by species names or our university
collection number followed by Genbank accession numbers. The tree was constructed
by using phylogeny.fr online phylogeny programs. The numbers at the nodes indicate
levels of boot strap support based on neighbor joining analysis of sequence data. OU E
208 (KM668705.) is showing close similarity (99%) with Phomopsis sp. (Fig. 4.4;
Table 4.1). Therefore, it has been identified as Phomopsis sp.
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Table 4.1 Molecular identification of endophytic fungi as revealed by BLAST
analysis of 18S rRNA sequences (NCBI -National Center for Biotechnology
Information data base)
S.no
Endophyte
isolate code
Plant
name
Query
Identity
Evalue
Most probable
identity
Accession
number
cover
1
OU E 38
A.
paniculata
94%
99%
0
Arthrinium
phaeospermum
KM668704
2
OU E 208
Emblica
sp.
96%
99%
0
Phomopsis sp.
KM668705
3
OU E 211
T. bellerica
95%
99%
0
Acremonium sp.
KM668706
4
OU E 203
Pupalia
lappacea
96%
99%
0
Leptosphaerulina
chartarum
KM668707
4.4 Discussion
Anamorphic fungi and mycelia sterlia were the commonly isolated and predominant
endophytic mycoflora of the medicinal plants. Identification of fungi relies heavily on
reproductive structures and non sporulating mycelial forms, though predominant and
common among endophytes, do not find a place in taxonomic hierarchy. In view of
this, many mycologists working particularly on endophytic fungi have adopted the
concept of ‘morphospecies’ for the non-sporulating forms. Indeed the morphospecies
concept was developed on the basis of similarity of colony surface textures, hyphal
pigments, exudation, growth rate etc. (Bills, 1996). But the question still remain as to
how good the cultural similarities and differences are at indicating species identity and
species status. Therefore, the taxonomic status of morphospecies based on cultural
characteristics is questionable (Arnold et al., 2000; Guo et al., 2003). Dictionary of
fungi (8th edition) defined the concept of morphospecies as the traditional apporoach
recognizing units that could be delimited on the basis of morphological characters, and
ideally by discontinuities in several such as distinct from biological ecological
phylogenitic and polythetic species (Hawksworth et al., 1995). Schulthess and Faeth
(1998) preferred another term, morphotype to describe non-sporulating fungal
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endophytes in the leaves of Arizona fescue (Festuca arizonica). Morphotype has been
defined as a ‘group of morphologically differentiated individuals of a species of
unknown or of no taxonomic significance’ (Hawksworth et al., 1995) and is used in
preference over ‘morphospecies’ in the present study as done by Lacap et al. (2003).
Sequence analysis of rDNA has provided an useful tool in determining the taxonomic
placement of not only non- sporulating fungi and also the anamorphic fungi. Guo et al.
(2000) have identified endophytic fungal species using the 5.8S and ITS region and
showed that unidentified morphotypes from Livistona chinensis belonging to
filamentous ascomycetes. Lacap et al. (2003) compared six randomly selected
morphotypes with nucleotide sequence similarities of the rDNA ITS region and the
5.8S gene using UPGMA cluster analysis and showed that a degree of certainty exists
in assigning morphotypes to genera, as strain within each morphotypes clearly grouped
together as taxonomic units and they stated that different morphotypes represent
distinct taxa. It is likely that morphologically similar sterile mycelial forms may belong
to different unrelated taxa (Guo et al., 2003). Guo et al. (2003) identified 18
morphotypes from Pinus to various taxonomic levels based on rDNA sequence
analysis.
In the present study, we have selected two unidentified non-spourlating fungal
endophytes isolated from Emblica and Pupalia lappacea and charecterised by rDNA
sequencing using ITS primers. They were identified as Leptosphaerulina chartarum
and Phomopsis sp. based on sequence similarities of 18S rDNA with NCBI data base.
Our results, though limited, suggest the utility and importance of molecular
characterization in identifying non-sporulating morphotypes of mycelia sterilia.
Non-sporulating morphotypes from Emblica sp. and Pupalia lappacea in present study
segregated into different genera based on rDNA sequence analysis suggesting that
morphological similarities of mycelial forms are not reliable in the absence of
sporulation. These results are also in conformity with the earlier findings (Guo et al.,
2003; Promputtha et al., 2005).
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Our results also have confirmed morphological identification of spourlating fungi such
as A. phaeospermum and Acremonium sclerotiginum. by molecular analysis of the 18S
rDNA sequences. It may be concluded from the present study that molecular methods
of fungal identification are reliable and may be the only way of identifying the sterile
forms of fungi in the absence of any sexual structures.
4.5 Conclusions
1.
Molecular analysis of 18S rDNA confirmed the morphological identification of
A. phaeospermum and Acremonium sclerotiginum., isolated from A. paniculata
and T. bellerica respectively.
2.
Molecular identification of non-spourlating morphotypes revealed that the
endophytes are Leptosphaerulina chartarum and Phomopsis sp.
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