Bangladesh J. Plant Taxon. 29(1): 129-136, 2022 (June) DOI: https://doi.org/10.3329/bjpt.v29i1.60453 © 2022 Bangladesh Association of Plant Taxonomists FIRST REPORT OF THE ECTOMYCORRHIZAL STATUS OF CLAVARIADELPHUS PAKISTANICUS HANIF & KHALID BASED ON MORPHOTYPING AND MOLECULAR EVIDENCE MUHAMMAD HANIF*, BUSHRA ARSHAD, SAMINA SARWAR1 AND NOUSHEEN YOUSAF Department of Botany, GC University, Lahore, Pakistan Keywords: Aphyllophorales; Basidiomycetes; Khanspur; Morphotypes; nrDNA; Symbiosis. Abstract The ectomycorrhizae of a newly described club fungus Clavariadelphus pakistanicus Hanif & Khalid were collected from Ayubia, Khyber Pakhtunkhwa, Pakistan and described morpho-anatomically. Its Pinus wallichiana associated ectomycorrhizae have been characterized by dichotomously branched, reddish brown color of mature and dark brown to blackish young ectomycorrhizal tips with frequent unbranched and septate emanating hyphae. During molecular and phylogenetic analyses, these mycobionts showed maximum similarity and were clustered with basidiocarps sequences of C. pakistanicus. Hence it was confirmed that these ectomycorrhizae belong to C. pakistanicus and being first time reported from Pakistan. Introduction Clavariadelphus Donk is widely distributed in the temperate forests and has 20 species worldwide (Methven, 1990; Kirk et al., 2008; Hanif et al., 2014). Clavariadelphus is generally considered to be an element in the biota of the Northern Coniferous Forest and several species in North America are confined to mixed deciduous coniferous forests (Methven, 1990). Little is known about the nutritional status of the Clavariadelphus species. Pines are generally considered as ideal hosts for many of the ectomycorrhizal (ECM) fungi (Hanif et al., 2012; Hanif, 2012). There are many reports that indicate the ectomycorrhizal nature of pines. Some examples from literature are: Visser (1995) determined Coltricia perrinis, Thelephora spp., Suillus brevipes, Cenococcum geophillum, Cortinarius spp., Lactarius spp., Russula spp., Tricholoma spp. as ECM fungi with Pinus banksiana. Guo et al. (2020) reported 104 ectomycorrhizal operational taxonomic units (OTUs) from Pinus sylvestris roots. Zhao et al. (2020) claimed to isolate 805 OTUs from the same host. Hilszczańska et al. (2011) reported ectomycorrhizal symbiosis in P. sylvestris with Suillus luteus, Thelophora terrestris, Tomentella spp., Dermocybe palustris and Dermocybe spp. Margit et al. (2010) reported the different mycobionts (Amphinema byssoides, Wilcoxina sp. Flexipes, Suillus ploranus and Tomentella) in different host plants including P. cembra. Hawley et al. (2008) reported the ECM of Phialocephala tortnii and Hymenocyphus ericae with Pinus patula. Chung et al. (2003) reported many ectomycorrhizal fungi with P. densiflora and P. rigita. Koizumi and Nara (2019) reported 154 ECM fungal species from the root tips of P. pumila. Niazi et al. (2010) reported the ECM of Cantharellus cibarius with P. wallichiana from the Himalayan Temperate Forest of Pakistan. Tyub et al. (2018) reported 33 fungal taxa associated with P. wallichiana out of which 23 were ectomycorrhizal and rest were non-mycorrhizal. Murata et al. (2017) reported 42 putative ECM *Corresponding author, Email: dr.mhanif@gcu.edu.pk 1Department of Botany, Lahore College for Women University, Lahore, Pakistan. https://doi.org/10.3329/bjpt.v29i1.60453 mailto:dr.mhanif@gcu.edu.pk 130 HANIF et al. fungi in association with P. amamiana. Clavariadelphus species are reported as mycorrhizal with diverse hosts i.e., Clavariadelphus americanus is reported as mycorrhizal with oaks and pines (Corner, 1950; Methven, 1989, 1990; Kuo, 2007), C. occidentalis with conifers (Methven, 1989). Mostly ectomycorrhizal mushrooms including Clavariadelphus are widely distributed in the moist temperate forests of the World. Pakistan also has diversity rich hotspot areas in Himalayan moist temperate region and many mushrooms along with their mycobionts have been documented from this region (Niazi, 2008; Hanif, 2012; Sarwar, 2012; Ilyas, 2013; Jabeen, 2016) but unfortunately very little is known about complete picture of diversity of these fungi. Previously, Niazi (2008) described the ECM of Clavariadelphus truncatus with P. wallichiana. Some club fungi fruiting bodies have been reported by Hanif (2012) but their ectomycorrhizae are not well reported from Pakistan. In the present investigation, fruiting body of the C. pakistanicus and its ECM is illustrated and described morpho-anatomically and phylogenetically from the roots P. wallichiana. It is the first report of the mycorrhizal status of C. pakistanicus from Pakistan. Materials and Methods Isolation and clearing of ectomycorrhizae The sampling was carried out during the rainy season (July–August) from the coniferous forests of Pakistan located at an elevation of around 2200 m.a.s.l. Ectomycorrhizae of Clavariadelphus pakistanicus associated with Pinus wallichiana were sampled by tracing the rhizomorphs extending from the base of fruiting bodies towards plant roots. Soil blocks with roots were dug and packed in polythene bags and brought in laboratory for further analyses. The sampled roots were cleaned with running tap water and mycorrhizal roots were separated from non-mycorrhizal roots. The unramified ends of ectomycorrhizal morphotypes were cut in such a way to retain the particular system. These were then preserved in 2% CTAB buffer. The ectomycorrhizal systems were studied morpho-anatomically with the help of stereo microscope (for morphological studies) and compound microscope (for anatomical studies). Morphological studies The ectomycorrhizal system was studied under stereo microscope for length of mycorrhizal system, length of unramified ends, diameter of unramified ends, diameter of axis, branching system, shape of unramified ends, distinct features of mantle surface and the color of system following Agerer (1991, 1987–2002) Anatomical description Mantle was peeled off under stereo microscope in one drop of lactic acid and observed under compound microscope to study anatomical features of mantle surface like hyphal arrangement, shape and size of cells and dimensions of hyphal cells and drawn with Camera Lucida. Molecular characterization and phylogenetic analysis DNA was extracted from ECM root tips by following a modified CTAB method (Gardes and Bruns, 1996). Primer pairs ITS1F/ITS4 (White et al., 1990) for the ITS region were used for PCR and Sanger sequencing. All PCR products were evaluated for successful amplification using SYBR Green and 1.5% agarose gels with TAE buffer for gel electrophoresis. Amplicons were prepared for sequencing via enzymatic purification using exonuclease I and shrimp alkaline phosphatase enzymes (Werle et al., 1994). Purified products were sequenced through MacroGen Company (Seoul, South Korea). Sequence chromatograms were trimmed, edited, and assembled using Sequencher4.1 (Gene Codes, Ann Arbor, MI). Consensus sequences were analyzed using BLAST searches at NCBI (http://www.ncbi.nlm.nih.gov/). The most similar sequences for ITS http://www.ncbi.nlm.nih.gov/). FIRST REPORT OF THE ECTOMYCORRHIZAL STATUS OF C. PAKISTANICUS 131 region were retrieved from GenBank. These ITS sequences were then aligned using MUSCLE Alignment Tool to generate alignments (Edgar, 2004). MEGA5 software was used for phylogenetic analysis with maximum likelihood criterion by following algorithm and Jukes and Cantor (1969) model of sequences evolution (Tamura et al., 2011). One thousand bootstrap iterations were performed with rapid bootstrapping significant support was considered to be ≥70%. All phylogenetic analyses were performed on the CIPRES Portal v. 3.1. (Miller et al., 2010). Results and Discussion Morpho-anatomical characterization of ectomycorrhizal system of Clavariadelphus pakistanicus Ectomycorrhizal system dichotomous, main axis 44.5mm long, axis 0.5mm in diameter. unramified ends bent, 1mm long and 0.5mm in diameter, younger unramified ends reddish brown, older ends blackish brown. Texture of the system was smooth, host tissues not visible under the sheath; mantle surface smooth or cottony; Rhizomorphs absent. Emanating hyphae were common, concentrated around the sides of unramified ends, honey brown in colour (Fig. 1A-D). Fig. 1. Ectomycorrhizae of Clavariadelphus pakistanicus, A. ECM (habit) showing important morphological features; B. Pseudoparenchymatous (type M) outer mantle; C. Pseudoparenchymatous (type M) inner mantle; D. Emanating hyphae. Scale Bar: for A= 0.7cm; B = 0.41μm; C = 0.52 μm; D = 0.29 μm. 132 HANIF et al. Outer mantle layer pseudoparanchymatous, cells 6.24 μm in length and 1.35μm wide, matrix material pale yellow, densely packed round lobed cells, epidermoid cells of pale yellow colour, hyphae compactly packed and forked. Inner mantle layer also pseudoparanchymatous, gelatinous matrix material visible, cells 7.9μm long and 1.2μm wide, cells were same in size as the outer mantle, cells contents not clear (Fig. 1 (A-D). Rhizomorphs absent, emanating hyphae common, clamps absent, septate, unbranched, cylindrical hyphae, not constricted at the septa, cell content clear, cells 0.58μm width and 38.6μm long, cells thick walled. Molecular identification and Phylogenetic analysis The morpho-anatomic identification of ectomycorrhizae was supported by rDNA-ITS sequence based molecular identification. Sequences originated from the ITS region were used as a reference to BLAST against GenBank data. All sequences showed maximum similarity (100%) with Clavariadelphus pakistanicus sequences of sporocarp (HQ379937). Similar sequences were retrieved from GenBank and aligned with Pakistani ectomycorrhizal sequences reported during this study. Final data set for phylogenetic tree included 20 sequences. Tree was constructed through maximum likelihood criterion and showed highest log likelihood (-2451.4566). Phylogram consisted of 2 major clades and a few independent leaves (Fig. 2). Ectomycorrhizae of C. pakistanicus clustered with its basidioma (mh99, mh126, mh129), the above ground partner with strong bootstrap percentage (99%). All sequences of C. pakistanicus nested within clade of species that were previously reported as ectomycorrhizal with various photobionts. Placement of all these species with C. pakistanicus indicates ectomycorrhizal status. Fig. 2. Phylogenetic position of of Clavariadelphus pakistanicus ectomycorrhizae from Pakistan with respect to other related spp. Tree inferred by maximum likelihood analysis based on rDNA sequences, including ITS region. The numbers against branches indicate the percentage (>50%) at which a given branch was supported in 1000 bootstrap replications. GenBank accession numbers are given at the end of species names.■ indicate species reported from Pakistan. FIRST REPORT OF THE ECTOMYCORRHIZAL STATUS OF C. PAKISTANICUS 133 Interaction between photobionts and mycobionts is the archetype of symbiosis or mutualism (Ågren et al., 2019). These mycorrhizal associations are beneficial to the plant. The identification of ectomycorrhizal morphotypes based on morphological criteria is difficult to disseminate species. Therefore, the use of molecular techniques is an effective alternative. During present work ectomycorrhizal morphotype of a novel club fungus Clavariadelphus pakistanicus (Hanif et al., 2014) has been reported from Pakistan growing with Pinus wallichiana. This is the first report of ectomycorrhizae of this fungus from the World. Previously ectomycorrhizal association of Clavariadelphus ligula (Quel.) Donk was found at Hurpora and Yusmarg with Cedrus deodara and Pinus wallichiana (Itoo and Reshi, 2014). Ectomycorrhizal morphotypes of C. pakistanicus were collected by tracing method and identified through its rDNA-ITS sequence following Landeweert et al. (2003) and characterized by morphotyping method (Agerer, 1991; Agerer, 1987–2002; Mello et al., 2006). Clasen et al. (2018) reported that molecular tools based on sequencing of rDNA-ITS could be effective in species characterization and phylogenetic analysis. Their ectomycorrhizae have dichotomous ectomycorrhizal systems with bent unramified ends (reddish brown when young and blackish brown when old). Literature showed that mostly Pinus associated ectomycorrhizae have dichotomous branching pattern (Agerer, 1987–2002). Rhizomorphs were not recorded although some rhizomorphs like structures were present but their morphology and anatomy not support them as rhizomorphs. Emanating hyphae were frequent. Mantle organization in both outer and inner view was pseudoparanchymatous. These basic structure features may resemble with Pinirhiza lactariosimilis associated Pinus sylvestris (Golldack et al., 1997) but P. lactariosimilis may have some structural differences as well. ECM of C. pakistanicus has large (4-4.5mm) ectomycorrhizal system than P. lactariosimilis (2.7mm). Both these morphotypes also differed in cell size, smaller in later (2.5µm) and larger in earlier (6.24μm). The ECM of C. pakistanicus was also compared with the ECM of other related species. Ectomycorrhizae of both C. trancatus (Niazi et al., 2010) and C. pakistanicus have dichotomous branching. The ramification pattern in C. pistillaris was reported as monopodial pinnate associated with Fagus sylvatica (Iosifidou and Raidl, 2006). Rhizomorphs absent in C. pakistanicus whereas present in C. trancatus and C. pistillaris (Iosifidou and Raidl, 2006; Niazi, 2008; Niazi et al., 2010). All these three species have emanating hyphae. Mantle organization was pelectenchymatous in C. trancatus (Niazi et al., 2010). There are very few reports about mycorrhizal status of the species in genus Clavariadelphus. C. americanus was reported to form ECM with oaks and pines (Corner, 1950; Methven, 1989, 1990), C. occidentalis and C. unicolor with pines (Corner, 1950; Smith et al., 1981; Methven, 1989, 1990). The ectomycorrhizae of genus Clavariadelphus were also reported with Quercus spp. and many other deciduous trees (Izzo et al., 2005; Iosifidou and Raidl, 2006; Smith et al., 2007; Morris et al., 2008). C. mucorantus and C. ligula were reported in association with Pseudotsuga menziesii (Smith et al., 2002). The ectomycorrhizae of this fungus may increase the nutrients supply to the host. Same is reported by Corrales et al. (2018). They suggested that soils with poor nutrients are abundantly dominated by ectomycorrhizal fungi that can optimize plant nutrition and may contribute to the maintenance of forests. Many different mycobionts are being reported with deciduous hosts (Aryal et al., 2020; Defrenne et al., 2019; Albuquerque-Martins et al., 2019). 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