Isolation and Characterization of Dominant Fungi from Rhizospheric Soil of Saussurea obvallata (DC.) Edgew. (Brahma Kamal) of the Indian Himalayan Region

The Himalayan region is conferred by diverse wealth in term of flora and fauna. Among Himalayan plant biodiversity, Saussurea obvallata (common name Brahma Kamal) has an immense spiritual and medicinal significance. Owing to high demand, this herb is on the verge of extinction. Numerous studies have been carried out for preservation and in vitro regeneration of Saussurea obvallata. In view of above, this is the first study which aims to isolate and identify rhizospheric fungi associated with this plant. Soil samples were collected from three locations in Kedarnath valley and total 34 fungal isolates were isolated. Out of 34, three fungal isolates i.e. MaHaD1, MaHaD2 and MaHaD3 were selected for morphological and molecular characterization. The 16S rDNA sequencing identified MaHaD1, MaHaD2 and MaHaD3 as Phanerochaete chrysosporium, Trichoderma longibrachiatum and Aspergillus fumigatus respectively. The presence of these three dominant fungi in rhizospheric region seems to be results of climatic conditions and plant physiology. Results indicate that the presence of fungal microflora may play a vital role in survival and proliferation of Saussurea obvallata plant by providing tolerance and resistance to abiotic stress and fungal/nematode pathogens respectively.


INTRODUCTION
Saussurea obvallata (DC.) Edgew. (Brahma Kamal) is one of the valuable traditional herb of the Himalayan region belonging to the family Asteraceae 1 . Generally, it is found in different states of the Indian Himalayan region (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim and Arunanchal Pradesh) and other countries (Nepal, Bhutan, Myanmar, China and Pakistan) 2 with an altitudinal range from 3,000 -4,800 m above mean sea level 3,4,5 . The whole plant of Saussurea obvallata is used by the local people of the Himalaya, for traditional, cultural, and religious purposes 6,7 . The Conservation Assessment and Management Plan (CAMP) had categorized Saussurea obvallata as an endangered species. Several investigations have been carried out to study reproductive biology, genetic diversity, cultivation and propagation of S. obvallata in view of conservation and management of the endangered medicinal herb 3,6 ( Fig. 1). To the best of our knowledge, no study has been done to study rhizospheric microbes of Saussurea obvallata till now. As rhizospheric microflora plays an important role in germination, growth and survival of plant in respective niche. In order to know the role of rhizospheric microflora in growth of Saussurea obvallata, this study was conducted to isolate the fungal community of Saussurea obvallata rhizospheric region. In the present study, we revealed that the potential fungal community present in the rhizospheric soil of Saussurea obvallata.

Isolation of Fungus
The rhizospheric soil samples of Saussurea obvallata were collected from Madhu Ganga (MG), HathiParwat (HP) and MahaPanth (MP) in Kedarnath Valley (Fig. 1), Uttarakhand, India 8 . The samples were packed and sealed in plastic bags and immediately brought to the laboratory and stored in refrigerator till further use. The three soil samples were serially diluted and 100µl of dilution sample (10 0-5 ) was plated onto potato dextrose agar (PDA) (CDH, JO-0013, India) supplemented with antibiotic (50 mg L -1 of chloramphenicol) for the isolation of fungi and incubated for seven days at 20°C.

Morphology of Isolates
The fungi were identified based on the macroscopic (colony morphology, texture) and microscopic (fruiting bodies i.e. conidia and hyphae) culture characters. For microscopic identification fungal isolates were stained with lactophenol cotton blue stain and were observed at 40x and 100x. The classifications of the fungi were carried out following standard procedures. All the isolated fungi were named and stored in fungal broth containing 15% (v/v) glycerol at -20°C for further use.

DNA extraction, gene amplification and sequencing
Genomic DNA was isolated from the isolates viz. MaHaD1, MaHaD2 and MaHaD3 using the ultrapure DNA isolation kit.The identification of isolates carried out by internal spacer transcribed (ITS) region amplification and sequencing. The ITS region of rDNA was amplified by PCR using fungal universal primer pairs ITS4 and ITS5 9 . The sequencing PCR was set up with ABI BigDye®Terminatorv3.1 Sequence kit. The raw sequences were obtained from ABI 3100 automated DNA sequencer and aligned with publicly available sequences and analyzed for identification.

Deposition of fungus isolates in culture collection center
MaHaD1, MaHaD2 and MaHaD3 fungal isolates were deposited in the National Fungal Culture Collection of India (NFCCI) (http://nfcci. aripune.org/) for the accession no. and future research works.

RESULTS AND DISCUSSION Fungal Isolates
Total 34, 29 and 31 fungal isolates were isolated from rhizospheric soil samples of Saussurea obvallata collected from MG, HP and MP sites respectively. For further study one fungal isolate (MaHaD1, MaHaD2 and MaHaD3) from each collection site i.e. MG, MP and HP were selected on the basis of the best growth (Table 1).

Morphological identification
Morphological identification of MaHaD1, MaHaD2 and MaHaD3 isolates were carried out using of lactophenol cotton blue staining method (Fig. 2). The colony appearance and microscopic morphological details revealed by staining are summarized in Table 2.

DISCUSSION
In our study, isolation, morphological and molecular characterization of fungal isolates from rhizospheric region of Saussurea obvallata suggested occourence of Phanerochaete chrysosporium, Trichoderma longibrachiatum and A. fumigatus as dominant genera. These fungal genera have effective role in organic decomposition, bioremediation and sustainable development 12,13 . Aspergillus fumigatus is saprophytic, airborne opportunistic pathogen. The ubiquitous prevalence of Aspergillus fumigatus in comparison to other sp. of Aspergillus is due to tolerance to various stresses (freezing, low pH, dehydration) and growth on decaying vegetation and debris. Although Aspergillus fumigatus does not have any direct role in plant growth promotion, some studies reported vital role of Aspergillus fumigatus in C and N recycling 14,15 .
The Phanerochaete chrysosporium commonly known as white rot fungi 16 is well known for digestion of fragrant poisons, lignin and lipids and remediation of various pollutants such as PAH, heavy metals and dyes 17 . Phanerochaete chrysosporium is considered an uncommon organism for its capacity to depolymerize and change lignin, cellulose and hemicellulose, in comparison with different basidiomycetes 18 . Trichoderma longibrachiatum uses cellulases to digest cellulose from decaying plant biomass, and chitinases to digest the chitinous walls of other fungi and nematode which indicate role of Trichoderma as a biocontrol agent 19,20,21,22 . Moscatiello et al. 23 investigated that a mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by T.longibrachiatum, which may assume an imperative role in the beginning times of the plant-parasite association. Some Trichoderma sp. are also known to provide tolerance/resistance to abiotic stresses, disease in addition to better plant growth 24,22 . The results suggest roles played by these fungal isolates in survival of S. obvallata plants. The probable mechanism of which may be by protecting plants from pests, inducing systemic resistance to plants for abiotic stress and improve soil nutrition acquisition. Further study is required to confirm the mechanism and role demonstrated by rhizospheric microorganisms in survival and growth of S. obvallata.