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Department of Biotechnology, Thapar Institute of Engineering & Technology, Patiala 147 001, India
K. Natarajan
G. Senthilarasu
Centre of Advanced study in Botany, University of Madras, Guindy Campus, Chennai 600 025, India
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ABSTRACT |
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Pisolithus is cosmopolitan in both tropical and temperate regions and forms ectomycorrhizal associations with a wide range of woody plants. Pisolithus indicus, a new species associated with Vateria indica (Dipterocarpaceae) is reported in this study from a dipterocarp native forest in the Western Ghats in India, using both morphological and molecular tools. The length of ITS1 and ITS2 regions of the present collection differed with other sequences of Pisolithus available in the databases. Phylogenetic analysis indicates that this species did not show significant homology with existing Pisolithus sequences reported previously and formed a separate branch linking with another Pisolithus isolate from dipterocarps. Molecular and morphological evidence showed that P. indicus is a new species associated with dipterocarps in India.
Key words: ITS rDNA, PCR, phylogeny, Pisolithus, Pisolithus indicus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) including members of the Pinaceae, Myrtaceae, Fagaceae, Mimosaceae, Dipterocarpaceae and Cistaceae. Although considerable heterogeneity exists in terms of basidiome, spore and isolate culture morphology, taxa within the genus Pisolithus have been regarded widely as conspecific and grouped as Pisolithus tinctorius (Pers.) Coker and Couch (= P. arhizus [Scop.] Ranschert) (Chambers and Cairney 1999). The confusion in Pisolithus systematics results from the species being poorly described, leading to many different species being placed in synonymy, or that the epithet "tinctorius" was extensively used for more than two different species. Recent molecular analysis have revealed considerable genetic variation within the species suggesting that P. tinctorius group worldwide comprises a complex of several species (Martin et al 1998, Anderson et al 2001, Martin et al 2002, Kanchanaprayudh et al 2003a, Moyersoen et al 2003, Singla et al 2004), which cannot be separated by morphological studies. In India Pisolithus populations are widespread, although they are found mostly in Eucalyptus plantations and were identified as P. albus (Cooke & Mass.) M.J. Priest, nom. prov., based on the ITS sequence analysis (Singla et al 2004). During our studies on the biodiversity of ectomycorrhizal fungi associated with members of the Dipterocarpaceae, we have collected a species of Pisolithus, which cannot be placed in any known species by morphological and molecular studies. We describe this as a new species. |
MATERIALS AND METHODS |
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. The ranges of spore sizes as well as average spore sizes are given in the form of (a)b–c(d). The range b–c includes the measured values taken by the maximum number of spores observed and extreme values are given in parenthesis. Arithmetic mean and standard deviations are provided, followed by spore quotient. Spore quotient (Q value) was obtained by mean length divided by mean width ratio of a spore in profile view. The specimens are deposited at the Herbarium of Madras University Botany Laboratory (Herb. MUBL No. 3156).
DNA extraction and ITS PCR.— –
For molecular characterization, the genomic DNA was extracted from the dried fruit body by placing it in liquid nitrogen and grinding into a fine powder with a mortar and pestle. Genomic DNA was extracted by the method of van Kan et al (1991). The ITS region of the rDNA was amplified by PCR with the primers ITS1 and ITS4 as described by White et al (1990). The 50 µL reaction mixture for PCR amplification contained: 10 ng DNA, 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTPs, 0.5 µM of each primer and 2.5 units of Taq Polymerase (Amersham Pharmacia, USA). Amplifications were performed in a thermal cycler (Perkin Elmer, USA) with an initial denaturation step of 94 C for 3 min followed by 37 cycles of 94 C for 2 min, 50 C for 1 min and 72 C for 2 min and a final extension of 72 C for 8 min. Controls containing no DNA template were included in every step of amplification to test for the presence of contamination of reagents and reaction buffer.
ITS sequencing and analysis.— –
The ITS-PCR product was run through 1.5% agarose gels in 0.5x TBE buffer, and the target band was excised. DNA from excised band was purified with QIAquick columns (QIAGEN, Valencia, California) and subcloned in pGEM-T easy vector system (Promega). Plasmid DNA was extracted from different clones and amplified with ITS1 and ITS4 primers. The amplified products of different clones were subjected for restriction fragment length polymorphism (RFLP) analysis to see the variation in the ITS region. The ITS product from three randomly selected clones were sequenced with Applied Biosystems automatic sequencer. Sequencing reactions were performed with the primers T7 and SP6 (Promega). These sequences were compared, and because all sequences showed similarity, one of the ITS sequences of Pisolithus (MSR-2) was used for further analysis. This sequence was compared with existing databases with BLAST (www.ncbi.nlm.nih.gov/blast). The sequences were aligned with some Pisolithus ITS sequences obtained from GenBank DNA database. They were chosen to cover 11 phylogenetic sequences identified by Martin et al (2002) along with the three other sequences of Pisolithus obtained from dipterocarprs reported from Thailand (Kanchanaprayudh et al 2003). The alignment was carried out with the program MultAlin (http://www.toulouse.inra.fr/multain.html). The resulting multiple alignments was optimized visually. Only unambiguous alignments were used in the phylogenetic analysis. ITS sequences of Paxillus involutus (AF167700
[GenBank]
) and Suillus luteus (L54110
[GenBank]
) were used as outgroup taxa. Phylogenetic analysis was performed by a neighbor joining (NJ) method using Kimura 2-parameter distances with MEGA (Kumar et al 2004). For analysis 1050 bootstrap replicates were performed to assess the statistical support for the tree.
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RESULTS |
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In solo, sub Vateria indica ad Uppangala, Karnataka, India, conservatus in Herbarium Madras University Botany Laboratory. Holotypus Her. MUBL No. 3156.
Basidiome composed of peridium and stipe (FIG. 1A
). Peridium brownish grey (6F8), surface smooth at first, then cracking giving it a somewhat rugose aspect, finely cracked especially at the attachment portion of stipe and peridium. Peridium 10 cm diam., globose to subglobose. Stipe 7.5 x 3 cm, surface concolorous with the pileus, finely cracked, rugose squamulose, cylindric, equal, solid, arising from an aguglutinated mycelial base, with whitish rhizomorphs. Gleba greyish (7E1), powdery mass formed by the breakdown of rounded, smooth peridioles. Peridioles thin-walled, soon breaking down into powdery, fluffy mass. Constituent hyphae thin and thick-walled, ~ 7 µm diam., varying from hyaline to brown. Basidiospores (FIG. 1B, C) globose to subglobose, spiny, (7)11.5–19.5(21) x (7)11–17.5(15.6 ± 2 x 14.5 ± 1.7) µm Q = 1.07, including spines; spines ~ 2.5 µm long, interconnected to form a complete reticulum, grayish brown in alkaline solution, becoming rust brown in Melzer’s reagent. Basidia not observed. Cystidia absent. Clamp connections present. Terrestrial, under Vateria indica, native forest in Up-pangala, Karnataka, India, Jun 2001.
Molecular characterization.— –
The rDNA ITS region of the Pisolithus isolate (MSR2) was amplified with conserved fungal primers ITS1 and ITS4. The size of the ITS fragment was approximately 650 bp, which includes ITS1, 5.8S and ITS2 regions. The ITS region was sequenced and the sequence data was submitted to GenBank nucleotide database (accession number AY756113
[GenBank]
). The sequence was compared with the published Pisolithus sequences available in the NCBI database. Alignment of the sequence with the existing sequences of Pisolithus revealed variability in the ITS-1 and ITS-2 regions, and little or no variability in the 5.8S ribosomal gene (TABLE I). The length of ITS-1 and ITS-2 of the other isolates used to construct the phylogenetic tree (TABLE I) differed with the present isolate.
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). All sequences in the dendrogram were divided into three lineages (A, B and C) with high bootstrap values as shown by Martin et al (2002) and Kanchanaprayudh et al (2003a). Sequences obtained from the database generated the same phylogenetic dendrogram as described by Martin et al (2002) and Kanchanaprayudh et al (2003a). These lineages comprised 13 major phylogenetic species. Lineage C comprised only P. aurantioscabrosus Wat., which formed a basal clade. The present isolate did not show any significant homology with other isolates reported previously. It was grouped as a separate branch in lineage A along with three sequences of dipterocarp Pisolithus isolates (P. abditus nov. sp. Kanchanaprayudh, Sihan., Hogetsu & Watling). Pisolithus tinctorius and P. marmarotus (Berk.) Priest, nom. prov., also grouped in lineage A. Lineage B comprised P. albus and P. microcarpus (Cke & Mass.) G. Cunn. (FIG. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and P. adbitus collected under Dipterocarpus alatus in Thailand (Kanchanaprayudh et al 2003b). The present collection is the third species, which we are describing from under dipterocarp trees. It differs from both the above-mentioned species in having larger, heavily ornamented spores with reticulations on the surface, and in the basidiome color, size and presence of clamp connections.
The taxonomic distribution of P. indicus from other species is confirmed further by molecular analysis. Phylogenetic analysis based on ITS sequences clearly separated the present collection from all the other species of Pisolithus reported previously. A phylogeny tree was constructed from the ITS sequences covering 11 phylogenetic species identified by Martin et al (2002) and the sequences of isolates of Pisolithus from dipterocarps identified by Kanchanaprayudh et al (2003a). Martin et al (2002) separated P. aurantioscabrosus from a major grouping divided into two strong branches, one (lineage B) containing four species surrounding the P. albus and P. microcarpus consortia and associated with eucalypts and acacias and a second (lineage A) containing the more familiar P. tinctorius and P. marmoratus consortia (six species), associated with a range of vascular plant hosts. Kanchanaprayudh et al (2003a) included P. abditus in the latter branch as an additional new species (species 12). Of interest, this isolate was linked with an unnamed African taxon associated with Afzelia, a caesalpinoid legume (Martin et al 2002), and of course Africa is the home of ancient ectomycorrhizal dipterocarp genus Marquesia, indicating a possible early link in these floristic components (Watling 1994). The present collection formed a separate branch linking with isolates of P. abditus from dipterocarps of Thailand described by Kanchanaprayudh et al (2003b). This isolate added another new species (species 13) to the phylogeny tree of Pisolithus described by Martin et al (2002), indicating that 13 major phylogentic species are present in the Pisolithus taxonomy. Martin et al (2002) showed that evolutionary lineages within Pisolithus are related to the biogeographical origin of the hosts. Several species identified in their analysis consisted of isolates limited to specific geographical regions, most of them confined to endemic plants such as Afzelia (species 1), Cistus (species 3), pines and oaks (species 4). In addition the results presented in this study suggest that regional floras and endemic plants could act as hosts of endemic species of Pisolithus as reported by Martin et al (2002). Pisolithus indicus, the new species identified in this study is in a branch of the tree with taxon associated with dipterocarps indicating that different dipterocarps arose as host species with different Pisolithoid fungi, which also has also been reported (Martin et al 2002, Kanchanaprayudh et al 2003b). Singla et al (2004) reported the occurrence of P. albus in India (grouped in lineage B), which is associated with eucalypts but not with any other tree species. The present isolate (P. indicus) is the second Pisolithus reported from India.
Because both morphological and molecular studies showed differences from other Pisolithus species reported previously, it was concluded that this is a new species and was identified as P. indicus, which form ectomycorrhizal association with dipterocarps of native forests in India. The host range of Pisolithus fungi has been considered to be relatively wide because Pisolithus species develop their basidiomes in association with many tree species (Marx 1977). As suggested by Kanchanaprayudh et al (2003a) the host range in Pisolithus species should be re-evaluated on the basis of molecular identification of species of the basidiomes and isolates in nature. Further studies are required to study the diversity of Pisolithus using greater sampling of taxa, different genetic loci, different methods, etc. to understand the complete taxonomy of this group of fungi.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Tel: 91-175-2393043. Fax: 91-175-2393738. E-mail: vasu70{at}yahoo.com
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Chambers SM, Cairney JWG. 1999. Pisolithus. In: Cairney JWG, Chambers SM, eds. Ectomycorrhizal fungi. Key genera in profile. Springer, Heidelberg. p 1–31.
Kanchanaprayudh J, Zhou Z, Yomyart S, Sihanonth P, Hogetsu T. 2003a. Molecular phylogeny of ectomycorrhizal Pisolithus fungi associated with pine, dipterocarp, and eucalyptus trees in Thailand. Mycoscience 44:287–294.[CrossRef]
———, ———, ———, ———, ———, Watling R. 2003b. A new species, Pisolithus abditus, an ectomycorrhizal fungus associated with dipterocarps in Thailand. Mycotaxon 88:463–467.
Kornerup A, Wanscher JH. 1978. Methuen Handbook of Colour. 3rd ed. Methuen & Co. Ltd. London. 243 p.
Kumar S, Tamura K, Nei M. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5: 150–163.
Moyersoen B, Beever RE, Martin F. 2003. Genetic diversity of Pisolithus in New Zealand indicates multiple long-distance dispersal from Australia. New Phytol 160:569–579.[CrossRef]
———, Delaruelle C, Ivory M. 1998. Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in low land Kenyan forests. New Phytol 139:341–352.[CrossRef]
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Marx DH. 1977. Tree host range and world distribution of the ectomycorrhizal fungus Pisolithus tinctorius. Can J Microbiol 23:217–223.[Medline]
Singla S, Reddy MS, Marmeisse R, Gay G. 2004. Genetic variability and taxonomic position of ectomycorrhizal fungus Pisolithus from India. Microbiol Res 159:203–210.[CrossRef][Medline]
van Kan JAL, van den Ackerveken GFJM, de Wit PJGM. 1991. Cloning and characterization of the cDNA of avirulence avr9 of the fungal pathogen Cladosporium fulvum, the casual agent of tomato leaf mold. Mol Plant Microbe Interact 4:52–59.[Medline]
Watling R. 1994. Ectomycorrhizal fungi in the Palaeotropics. Mycologia Heletica 2:129–138.
White T.J., Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ., eds. PCR Protocols: a guide to methods and applications. Academic Press, New York. 315–322.
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