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Additions to the Ectomycorrhizae Associated with Himalayan Cedar (Cedrus deodara) using rDNA-ITS.

Byline: MUHAMMAD HANIF, ABDUL NASIR KHALID AND SAMINA SARWER

ABSTRACT

Himalayan cedar (Cedrus deoadara Roxb. Loud.), a member of Pinaceae, is an ever green tree of Himalayan origin and distributed from western Himalayan in Eastern Afghanistan, Pakistan, India and Western Nepal. Despite its usage in manufacturing of building material and furniture, it is an ectomycorrhizal host. In the present study, its four ectomycorrhizal fungi are characterized and identified using morpho-anatomic and molecular methods targeting its rDNA. The morphotypes of Peziza sp. MHSUC-01, Russula livescens and three species of Tomentella (Tomentella sp. 2ENA19_11, Tomentella sp. ENA35_13 and Tomentella sp. 2ENA35_13) are described first time from Pakistan. (c) 2012 Friends Science Publishers

Key Words: Himalayan moist temperate forests; rDNA; Mantle; PCR; Convergent

INTRODUCTION

Cedrus deodara (Roxb.) Loud. (Deodar cedar or Himalayan cedar), a member of Pinaceae, is native to Western Himalayas in Eastern Afghanistan, North Pakistan, North Central India, south western-most Tibat and Wesetrn Nipal. The trees are found to flourish at 1500-3200 m.a.s.l (Farjon, 1990). It is third most frequent (10%) after Abies pindrow Royle (60%) and Picea simthiana (15%) in Pakistan's part of Himalayan forests (Champion et al., 1968). Ahmed et al. (2011) recorded C. deodara from 23 localities from Hindukush and Himalayan Moist Temperate forests of Pakistan. This extensive survey emphasizes its importance in ecosystem. This association of fungi, especially macrofungi, is of vital importance for tree growth and survival. This association is present in 85% of trees of world (Kirk et al., 2008).

Himalayan cedar has long been used in Eastern medicines and its wood extract is carminative, diaphoretic and useful in fever, flatulence, pulmonary and urinary disorders (Baquar, 1989). Several alkaloids have been reported (Zhang et al., 1990; Digrak et al., 1999; Shinde et al., 1999a, b; Rawat et al., 2000; Wolff et al., 2001; Dimri and Sharma, 2004b) and extracted from different parts of C. deodara tree, which are of very useful medicinal importance.

The ectomycorrhizal symbioses have a different host range allowing the formation of ectomycorrhiza on a limited set of trees and shrubs. In temperate and boreal forests, up to 95% of the short roots form ectomycorrhizae (Smith and Read, 2008). Ectomycorrhizae have a beneficial impact on plant growth in natural and agroforestry ecosystems.

Fundamental to the success of these symbioses is the switch of nutrients between the symbionts. In addition, the establishment of the symbiosis is required for the completion of the fungal life cycle. Himalayan cedar is also an excellent photobiont that makes ectomycorrhizal association with several groups of fungi. A few species have been reported forming ectomycorrhizae with C. deodara only from India (Singh and Lakhanpal, 2000; Deepika et al., 2011; Saini and Singh, 2011) and from Pakistan (Niazi et al., 2006, 2008). All these reports were based on cultural studies and morpho-anatomic characterization.

To understand the functioning of forest ecosystem, it is very important to characterize and identify the ectomycorrhizal fungi (Brand, 1992). Ectomycorrhizae are being identified using color, shape, macroscopic (ramification and presence of rhizomorphs) and microscopic (mantle organization and features of cystidia) characters (Agerer, 2002). But these features are not supportive to identify all the fungal species associated with roots, especially the mycorrhizae of related species. These often have similar morphological characters and are confused. The ectomycorrhizae formed by Tuber spp. (T. maculatum and T. melanosporum) morphologically appears to be similar to T. indicum (Comandini and Pacioni, 1997; Zambonelli et al., 1997, 1999). To resolve the problem encountered, DNA based methods have been introduced for correct identification of ectomycorrhizal fungi (Lanfranco et al., 1998; Horton and Bruns, 2001; Landweert et al., 2003).

These methods are based on targeting ribosomal DNA regions and preferred, because of th ir specificity and sensitivity. For this purpose, species specific primers have been designed for precise and accurate identification. Buncci et al. (2011) designed species specific primers to identify the Tuber macrosporum.

In this study, focused on exploration of ectomycorrhizal morphotypes in Pakistan's Himalayan Moist Temperate forests, C. deodara morphotypes were sampled to measure the biodiversity of associated ectomycorrhizal fungi and indentify them using morpho- anatomic features and its rDNA genes. We used fungal specific primer (ITS1F) and universal primer (ITS4) to amplify its rDNA to confirm our morpho-anatomic identification.

MATERIALS AND METHODS

Himalayn cedar (Cedrus deodara) roots were sampled from Ayubia National Park, KPK. Sampled roots were voucher and wrapped in polythene bags. Morphotypes of C. deodara were manually selected after removing the soil particles from the morphotypes and kept in 2% CTAB buffer for DNA extraction and in distilled autoclaved H2O for morpho-anatomic characterization. The selected morphotypes were characterized morpho-anatomically following Agerer (2002) and were deposited in Herbarium of Botany Department (LAH), University of the Punjab, Lahore. Morphological and anatomical characterization of ectomycorrhizal system was carried out under stereo and compound microscopes, photographed and illustrations were made with the help of Camera Lucida.

For molecular characterization, DNA was extracted from the selected morphotypes using modified CTAB method given by Bruns (1995). Amplification of the extracted DNA was performed using fungal specific and universal primers (ITS1F, ITS1 and ITS4). The hot-start enzyme JumpStart (Sigma, St Louis, MO, USA) was used to catalyse the PCR with 2 min at 94degC, followed by 30 cycles of 30 s at 94degC, 30 s at 53degC, 40 s + 5 s per cycle at 72degC, and finishing with 5 min at 72degC. The PCR products were purified with QIA quick (Qiagen Inc., Valencia, CA, USA), sequenced bi-directionally using the reverse and forward primers and Big Dye Terminator Cycle Sequenceing V3.1 on an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA) and edited in sequencher 4.5 (Gene Codes, Ann Arbor, MI, USA) in Jodrell Laboratory, Royal Botanical Gardens, Kew, UK. DNA sequences were submitted to BLAST and used to query the nucleotide collection using default settings.

The divergence in rDNA-ITS was measured by comparing sequence pairs reconstructed by using MegAlign (DNASTAR). DNA sequences obtained from Cedrus deodara morphotypes were submitted in GenBank. These sequences were manually edited using MacClade 4.08 and Bioedit (version 7.0.9).

RESULTS

Peziza sp. ENASUC_4.11 Gen Bank Accession No. JN836754 Morphological Description: Ectomycorrhizal system this is highly dichotomous to coralloid. 5-7 mm long, axis 0.7-1.2 mm in diam. Unramified ends rounded to bent, bifurcate, 2-4 mm long and 1-1.5 mm in diam., younger tips creamy white, older tips brown to black. Texture of system was finely grainy, host tissue not visible under the sheath; Rhizomorphs absent, Emanating hyphae absent (Fig. 1a). Anatomical characteristics of mantle in plan views: Mantle parenchymatous in all layers. Outer mantle layers parenchymatous with irregular shape of cells, light yellowish, no cell contents visible, cells 14-15 um in length, 10-14 um in width (Fig. 1b). Inner mantle layer parenchymatous, cells colorless to light yellowish, cells irregular in shape, no matrix material observed, cells 13-15 um in length, 11-14 um in width (Fig. 1c).

Molecular Description

Sequence data evidence: rDNA sequence of Peziza sp. ENASUC_4.11 was BLAST searched. This species shared 91% identity with P. succosella (DQ200841.1) and 89% with P. cf. succosa (EU819417.1) and identified as Peziza sp. ENASUC_4.11 (Table I). The genus Peziza (Pezizales) has first time been reported as ectomycorrhizal with C. deodara.

Tomentella spp. Gen Bank Accession Nos. JN836750, JN836751, JN836752

Morphological Description: Mycorrhizal system the system is dichotomous, 2.5 mm long, main axis less than 1 mm wide, unramified tips straight, length of tips more than 1 mm and about 0.5 mm wide, colour of young and older tips dark brown to black, apices yellowish brown, texture rough, no visibility of host tissue through mantle. Rhizomorphs absent. Emanating hyphae frequent, restricted at tips, straight, blackish brown (Fig. 2a).

Anatomical characteristics of mantle in plan views: Outer mantle layer parenchymatous, cells 11-12 um in length and 11.5 um wide, no matrix material, no ornamation of cells, cell contents clear, brownish cells, cells rounded to irregular shape (Fig. 2b). Inner mantle layer also parenchymatous, no matrix material visible, cells 6.5-7 um long and about 6 um wide, cells smaller than outer mantle, cell contents clear (Fig. 2c).

Anatomical characteristics of emanating elements: Rhizomorphs absent. Emanating hyphae frequent have no clamps, septate, branched, cell contents clear, cells width about 4 um and 25 um long, cells thick walled (Fig. 2d). Molecular Description: In the present investigation, genus Tomentella (Thelephoraceae) has also been reported as new mycobiont associated with roots of C. deodara. Three new isolates of Tomentella were described as ectomycorrhizal with C. deodara. rDNA from these three isolates when BLAST searched, they matched very close (92% and 93%) to Thelephoraceae sp. 'Taylor #2' (U83467.1) (Table I) and Thelephoraceae sp. P184 (FN669273.1) and second closest matched with Tomentella spp., (Tomentella fuscocinerea GU214810.1 and Tomentella sp., AJ534914.1). rDNA of these isolates were also found different from each other and showed divergence in ITS-rDNA ranging from 0.9-2.5 (Fig. 4a).

Table I: BLAST results of rDNA sequences from Pakistan along with closest matches and other parameters.

Matching results of each morphotype based on rDNA ITS sequences

Fungal species###Voucher No.###GenBank###Sequence Closest match in Origin###GenBank###Max###Query###E###Max.

###Accession No. length###GenBank###Country Accession No.###Score coverage Value Identity (%)

Tomentella sp.###ENA35_13###JN836750###605bp Thelephoraceae###Estonia FN669273.1###911###100%###0.0###93%

###sp. P184

Tomentella sp.###2ENA35_13###JN836751###816bp Thelephoraceae###Estonia FN669273.1###911###99%###0.0###93%

###sp. P184

Tomentellasp.###2ENAI9_11###JN836752###618bp Thelephoraceae###USA###U83467.1###887###99%###0.0###92%

###sp. `Taylor #2'

Russula livescens###ENA27.12###JN836753###585bp Russula livescens China###GU371297.1###1027###100%###0.0###98%

Peziza###sp. ENASUC 4.11###JN836754###420bp Peziza succosella USA###DQ200841.1###612###99%###0.0###91%

MHSUC 01

Table II: Tomentella spp. indicating the variations in molecular weight and G+C and A+T percentages

Fungal isolates###GenBank###Sequence Length (bp) Molecular Weight (Single stranded) in Daltons G+C Contents A+T Contents

###Accession No.

Tomentella sp. ENA3513###JN836750###605###183367###47.60%###52.40%

Tomentellasp.2ENA35###JN836751###618###187225###47.90%###52.1%

Tomentellasp.2ENA1911###JN836752###618###187172###47.57%###52.43

The G+C/A+T contents of these isolates showed slight variations (Fig. 3). When genetic characters of these isolates compared with each other, these were found to be different. Tomentella sp. 2ENA19_11, Tomentella sp. ENA35_13 and Tomentella sp. 2ENA35_13 shared 96.80-98.82% of genetic characters analyzed and showed 89.8% identity. Intra- specific variations in rDNA-ITS of these isolates observed in 671 base pairs rDNA sequence alignment length. There were total of 20 sites in the alignment of Tomentella spp. Sequences, which represented rDNA intra-specific variations in ITS and 5.8S gene of rDNA (Fig. 5) and hence treated as different isolates. But morphotypes of all these isolates possess similar morpho-anatomic features.

Thus, these seemed to be convergent morpho-anatomically and divergent phylogenetically.

Russula livescens (Batsch) Quelet Gen Bank Accession No. JN836753

Morphological Description: Ectomycorrhizal system dichotomously branched, system up to 7 mm long with 2 mm thick main axis, unramified tips 2-3 mm long and 0.3-0.5 mm thick, colour of system dark yellowish brown, younger tips dark honey brown, straight to bent slightly, surface of mycorrhizal system velvety, host tissue not visible under mantle surface.

Rhizomorphs absent. Emanating hyphae rare, branched, brownish (Fig. 4a). Anatomical characteristics of mantle in plan views: Mantle parenchymatous in all layers. Outer mantle layer is parenchymatous, cells elongated to irregular in shape, 17-18 um in length and 6-7 um in width, light yellowish, no cell contents, no septa and clamps (Fig. 4b). Inner mantle layer also parenchymatous, cells elongated to irregular in shape, 18.4 um long and 5.6 um in width, honey to light yellowish in color, no matrix material, no septa and clamps (Fig. 4c). Anatomical characteristics of emanating elements: Rhizomorphs absent.

Emanating hyphae branched, septa only at clamps, light yellowish, cells longer, thick walled, septa also thick, smooth, cells 3.5-4.3 um in diam., 66.5 um in length (Fig. 4d).

Molecular Description: Russula livescens is also reported as new mycobiont for Cedrus deodara. rDNA sequence of Russual ssp. from Pakistan when BLAST searched, it matched 98% with R. livescens (GU371297.1) (Table I) and a new record for Pakistan.

DISCUSSION

In the present study, we documented the ectomycorrhizal forming fungi from the roots of Himalayan cedar first time from Pakistan based on molecular methods. These fungi on the roots were described morpho- anatomically and identified using rDNA sequences from these molecular operational taxonomic units (MOTUs)/species.

We found this technique quite effective in the identification of fungi found symbiotically on roots of C. deodara. Our findings are in agreement with the study conducted by Iotti and Zambonelli (2006). They used the similar technique to identify Tuber ectomycorrhiza.

We report five ectomycorrhizal MOTUs/species associated with Himalayan Cedar first time from Pakistan viz; Peziza sp. MHSUC-01, Russula livescens, Tomentella sp. 2ENA19_11, Tomentella sp. ENA35_13 and Tomentella sp. 2ENA35_13. Genus Peziza has 104 species worldwide and is represented by eight species in Pakistan (Ahmad at al., 1997). Peziza sp. MHSUC-01 characterized and identified morpho-anatomically and using rDNA sequence data is found very close to P. succosella (DQ200841.1). To our knowledge, P. succosella is not reported as ectomycorrhizal. Another ally of Peziza sp. MHSUC-01 is P. succosa, which is reported as ectomycorrhizal by Tedersoo (2006). It has whitish cottony morphology, with pseudoparenchymatous mantle having spherical to rectangular, blunt-angled and thick walled cells in outer mantle and rectangular to polygonal thin walled cells in inner mantle (Tedersoo, 2006) as compared to Peziza sp. MHSUC-01. So, Peziza sp. MHSUC-01 is being treated as new MOTU/species.

With this addition, the number of Peziza spp., is raised to nine from Pakistan.

We reported three MOTUs/ species of Tomentella viz; Tomentella sp. 2ENA19_11, Tomentella sp. ENA35_13 and Tomentella sp. 2ENA35_13 as ectomycorrhizal. Genus Tomentella is also not reported as ectomycorrhizal from Pakistan. Previously, only seven species were reported by Ahmad et al. (1997). With the present report, this number has increased to ten. The morphotypes of these three ectomycorrhizal isolates has striking similarities in morpho-anatomic features (Fig. 2a-d) but differ from other related species of Tomentella. Raidl and Muller (1996) described T. ferrugenea, a close ally of Tomentella spp. from Pakistan, as ectomycorrhizal with Fagus sylvatica. It has monopodial pinnate or monopodial pyramidal ramification with plectenchymatous mantle organization (Raidl and Muller, 1996) in contrast with dichotomous branched ramification and parenchymatous mantle organization in all Tomentella isolates from Pakistan.

Species of Tomentella included in present study has intraspecific variations in their rDNA region. There are several insertions and deletions indicated in the sequenced and aligned data (Fig. 3). Intraspecific variations in their repeat regions were also observed by the relative measuring the G+C contents. Maoxian et al. (2005) studied the intraspecific variations in Pearl Oyster by measuring G+C contents. Another study conducted by Muthumeenakshi et al. (2001) showed the similar rDNA-ITS variations in Coniothyrium minitans (Ascomycota; Pleosporales) and its related species viz; C. sporulosum and C. cerealis. They differentiated C. minitans and C. cerealis on the basis of their G+C contents.

Himalayan Cedar has another mycobiont, Russula livescens, found symbiotically with its roots. R. livescens reported as ectomycorrhizal with Pinus yunnanensis (Xue- Dan, 2010) and Cistus sp. (Contu, 1984). Occurrence of R. livescens with C. deodara is also a new record. Previously, 23 species of Russula have been reported by Ahmad et al. (1997). There are some other reports (Khalid, 1998; Niazi, 2008) regarding the Russula from Pakistan, but not a single report about their ectomycorrhiza especially with C. deodara. R. livescens is reported as new record from Pakistan, raising their number to 24.

The present investigation emphasizes the use of rDNA based technology for the precise and accurate identification of ectomycorrhizal fungi. Ectomycorrhiza of C. deodara have been first time reported from Pakistan using molecular methods. This host tree is unexplored from Pakistan and comprehensive study could add the mycoflora associated with it and of Pakistan.

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Department of Botany, University of the Punjab, Lahore, Pakistan 1Corresponding author's e-mail: mhanif_r@hotmail.com
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Author:Hanif, Muhammad; Khalid, Abdul Nasir; Sarwer, Samina
Publication:International Journal of Agriculture and Biology
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Date:Feb 28, 2012
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