Diversity of Fungal Endophytes in Cymbopogon Caesius

Diversity of Fungal Endophytes in Cymbopogon Caesius Diversity of Fungal Endophytes in Cymbopogon caesius (Hook. Arn.) Stapf. of Kemmannugundi Regions of Karnataka, India Avinash K.S. and Y.L. Krishnamurthy* Abstract: Cymbopogon grass produces many bioactive compounds and used for many medicinal purposes. In this study healthy leaf, stem and root segments were cultured on media like Potato dextrose agar and Malt extract agar medium and pure cultured. Endophytic fungi were isolated from 1200 samples of Cymbopogon caesius grass collected from Kemmannugundi regions of Karnataka. Overall 56% of colonisation rate from surface sterilised tissues were recorded. About 30 different fungal species were isolated and 12 were identified and 18 were grouped as morph taxa. Less isolates were recovered from leaf segments and more isolates were recovered from root segments. Curvularia sp. and Fusarium sp. were frequently isolated endophytes with the high colonisation rate. Key Words: Endophytes, Cymbopogon caesius, Grass, Curvularia, Fusarium. 1. Introduction Endophytes are microorganisms which live inside the host without causing any negative effect by their presence. The plant and endophytic fungi show symbiotic association. Plant protects and feeds the endophyte which in return produces bioactive secondary metabolites to enhance the growth and competitiveness of the host in nature (Carrol, 1988 and Hong Lu et al., 2000). Endophytes are widely investigated for their bioactive metabolites and have proven to produce potential compounds which are applicable in medicine (Clovis and Ewald, 2012). The compounds usually isolated from medicinal plants may not be plant metabolites they may be from fungal endophytes which reside in the host plant. Endophytes of tropical plants are among the groups of fungi that have been studied to arrive at the predicted figure of 1.5 million (Hawksworth, 1991; Subramanian et al 2003) Cymbopogon species are traditional medicinal grasses well recognised for its aromatic oil. Cymbopogon caesius grass was wide spread in areas of Kemmannugundi of Chikmagalur Dist, Karnataka. Despite the wide distribution of Cymbopogon grass along the central Western Ghats of Karnataka only limited work has been done for their association with fungal endophytes. Even though the oil extracted from the grass has been used in medicine currently there is no enough information regarding the diversity of the fungal endophytes with the species. We have selected Cymbopogon caesius (Hook. Arn.) Stapf for the study of diversity of the fungal endophytes. Fungi were isolated from leaves, stem and root followed by the surface sterilisation process. The objectives of the study were to isolate and identify the fungal endophytes from different parts of Cymbopogon caeceous grass of Kemmannugundi regions of Karnataka. 2. Meterials and Methods 2.1 Sampling: C. caeseus (root, stem and leaf samples) was collected from Kemmannugundi (13 º2826 N 75 º4450 E 4275ft) Chikmagalur District. Roots were collected with a ball of soil so that to avoid damage to the roots. Twenty healthy looking culms of grass were collected and transported in a clean polythene bag in closed condition and processed within 24 hour of collection. 2.2 Isolation of fungi The collected samples were washed thoroughly by running tap water to clean soil from roots and were cut into small pieces. The cut segments were surface sterilised using 70% ethanol followed by immersion in 3% Sodium hypochlorite for 4 minutes and again washed twice with distilled water (Maheshwari and Rajagopal, 2013). The efficacy of the surface sterilisation was confirmed by pressing the sterilised segments onto the surface of the medium, the surface sterilised small pieces were cut into .03-.05cm segments (Chuyang et al., 2001). Four hundred segments were inoculated into potato dextrose agar medium which is supplemented with 100mg of Amoxillin to inhibit the growth of bacteria. The inoculated petri plates were wrapped with petriseal and incubated at 27  ±1 degree centigrade. The plates were observed daily after 3 days of inoculation up to one month the emerged fungal endophytes were transferred to new petri plates containing PDA Medium. 2.3 Identification of Endophytic fungi For the identification of endophytic fungi, slides were prepared from pure cultures and were stained with Lacto phenol cotton blue stain and observed under Karl Zeiss Primo star microscope. Morphological Characteristics such as growth pattern colour of colony, mycelium texture, spore production type and characters of the spore (Barnet Hunter, Ellis, Subramanian). 3. Statistical analysis The colonisation rate (CR) was determined by total number of segments yielding endophytes divided by the total number of segments inoculated (Petrini et al., 1982). Number of segments yielding endophytes Total number of segments inoculated 4. Results A total of 959 isolates of endophytes were recovered out of 1200 segments inoculated. Total 400 segments each of root, stem and leaf segments were inoculated for the isolation of endophytes. Most frequently found endophytes were Fusarium oxysporum (11.46%), Aspergillus clavatus (8.7%), and Curvularia spp. (7.78%) (Table 1). Table 1: Colonisation Frequency of fungal endophytes of C. caesius. Sl. No. Endophyte Name Colonisation frequency (%) Root Stem Leaf Total Alternaria alternata 4.1 1.09 1.73 Aspergillus clavatus 15.3 6.0 4.8 8.7 Bipolaris Sp. 2.00 0.66 Cephalosporium sp. 0.8 1.9 0.9 Cladosporium sp. 0.6 0.2 0.26 Curvularia andropogonis 16.0 5.2 1.7 7.63 Curvularia lunata 12.2 7.9 3.7 7.93 Fusarium oxysporum 16.1 11.9 6.4 11.46 Hansfordia ovalispora 0.9 0.3 Oidiodendron sp. 1.6 0.9 0.83 Trichoderma sp. 0.4 1.3 0.56 Wardomyces anomala 0.6 0.2 Unidentified Morphotypes 16.4 14.3 14.6 15.1 Table 2: Infection frequency and isolation rate of fungal endophytes of C. caesius. C. caesius plant tissues Leaves Stem Root Total Number of Samples 400 400 400 1200 Number of isolates recovered 126 198 348 672 Colonisation rate 31.5% 49.5% 87% 56% Number of Morph taxa 4 5 9 18 More isolates of endophytes (87%) were recovered from the root segments of the Cymbopogon grass compared to stem (49.49%) and leaf segments (56%). 18 unidentified fungi were numbered and stored as morph taxa. 5. Discussion The Cymbopogon grass is the very popular for its aromatic oil. The purpose of the study was to document endophytic diversity of Cymbopogon grass of the study area which was widely distributed in the area. The endophytic fungi were cultivated on artificial medium (PDA) as pure culture. Some of the fungi exhibited characteristic colony characters and microscopic characters which were helpful in identifying them whereas some of the fungus was produced good mycelia but did not show any sporulation were kept as morph types. All the isolated endophytes belonging to 11 different genera. The endophytes were more resided in root. The endophytes may not show any host specificity as they were recovered from different groups of plants (Petrini 1986). In the present investigation leaves, stems and roots of C. caesius were used for isolation of endophytic fungi. The fungal colonisation was higher in roots as compare to stem and leaves. Higher colonisation of endophytes in leaf and stem tissues, as compared to roots, was reported by Siegel and Latch (1991) and Clay and Schardle (2002) Ajay et al (2012) in grasses and in the study of medicinal plant species leaves colonised greater number of endophytes compared to stem and bark (Raviraja 2005). About 11 genera of fungal species were isolated which indicates the various fungal populations in the grass. The variation in the colonisation rate depends on the host habitat, Soil environment (Shankar Naik et al, 2014). In the study the CR is varied in different parts of the plant. The more number of fungal endophytes isolated from root which similar to the results of Sita et, al., 2011 where thy obtained almost three times more endophytes in roots compared to shoot tissues. Fungi have been widely investigated as a source of bioactive compounds. 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