Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 115-124 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 6 (2016) pp. 115-124 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.506.014 Association and Variation of Endophytic Fungi among Some Macrolichens in Central Western Ghats, Southern India K.S. Vinayaka1*, Y.L. Krishnamurthy2, Shivakumar Banakar3 and T. R. Prashith Kekuda4 1Department of Botany, Kumadvathi First Grade College, Shikaripura, Shivamogga-577427, Karnataka, India 2Department of Post Graduate Studies and Research inApplied Botany, Kuvempu University, Shankaraghatta, Shivamogga-577451, Karnataka, India 3Department of Post Graduate Studies and Research in Microbiology, Shri JJT University, Vidyanagari, Chudela, Jhunjhunu, Rajastan, India 4Department of Microbiology, Sahyadri Science College (Autonomous), Kuvempu University, Shivamogga-577203, Karnataka, India *Corresponding author ABSTRACT K eywo rd s Association of endolichenic fungi with eleven lichen species belongs to five Macrolichens, families distributed in Bhadra Wildlife Sanctuary, South India were investigated Endolichenic during May, 2013 to January, 2014. A total of 30 endolichenic taxa from 486 fungi, segments were isolated. The colonization rate ranged from 61.5% to 90.6 % among Western Ghats, lichen host species. Chaetomium globosum was occurred in eight, among 11 Chaetomium , species of lichen, it was the dominant species of endolichenic fungi in thalli of Parmotrema . lichens screened. The Ramalina pacifica and Parmotrema tinctorum showed maximum colonization by the endolichenic fungi. Out of 30 species of fungi 24 Article Info were sporulated and they were identified up to genus/species level. Chaetomium globosum, Fusarium spp., Xylaria sp. were isolated from thalli of 8, 5, 6 lichen Accepted: species respectively. The Shannon index was 0.18-0.26 and Simpson species 10 May 2016 Available Online: abundance was 0.004-0.015. Results of this study revealed that fungi were 10 June 2016 associated within lichen thalli other than symbiotic fungi and their compositions were varied with lichen species. Introduction Microbial endophytes have been found in (Hawksworth, 1988) and mosses (Dreyfuss, the leaves, bark and xylem of higher plants 1986). Endolichenic fungi also reported as (Schulz et al., 1993; Arnold and Lutzoni, parasymbionts in lichens (Petrini et al., 2007; Murali et al., 2007; Krishnamurthy et 1990). A number of fungi grow and produce al., 2008) including grasses (White and fruiting bodies on the surface of lichen thalli Cole, 1985; Krishnamurthy and Hemalatha, with identical fructifications. The hyphae of 2003) also in lower forms like algae these fungi penetrate the lichen thalli and 115 Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 115-124 form intimate union with cells of the algal from the internal tissues of macrolichens symbiont (Ahmadjian, 1967). There are which commonly occur in forests of Bhadra about 300 genera and 1000 species of Wildlife Sanctuary, Southern India. obligatory lichenicolous fungi known which form parasitic, saprophytic, parasymbiotic Materials and Methods and obligatory symbiotic mode of biological relationships on lichen species. Endolichenic Sampling of Lichens fungi are fungi that live within their host plants causing any noticeable symptoms of A total of eleven lichen specimens were disease (Hawksworth and Hill, 1984). These collected from different habitats of Bhadra are distinct from lichen mycobionts or any wildlife sanctuary, (75015' and 75050' E to other previously recognized fungal 1325' and 1350' N) Karnataka, Southern associates of lichens. They represent the India (Table 1). Apparently healthy looking same major lineages of Ascomycota as do lichen thalli were brought to the laboratory, fungal endophytes, largely parallel the high in paper bags stored in refrigerator and diversity of endophytes from the arctic to processed within 24 hours. The lichen the tropics (Arnold et al., 2009). For about specimens were identified by using the 32 taxa of endophytic fungi with 14 manual (Awasthi, 1988). These lichen ascomycetes, 16 hypomycetes, each of samples are stored in the herbarium of coelomycete and yeast were reported (Wen- Department of Applied Botany, Kuvempu Chao et al., 2007). Scopyleriopsis was University, Karnataka, India. distributed in all 6 species of macrolichens. About 70 cultures of black fungi belong to Isolation of Endolichenic Fungi from Chaetothriomycetidae and Dothideomyce- Lichen Thalli tidae from 14 different species of lichen thalli, they showed that some lineages of Surface sterilization of lichen thalli was black fungi can opportunistically grow on performed using the modified method (Guo lichens (Harutyunyan et al., 2008). A et al., 2003). Healthy looking thalli of each recovery of endolichenic fungi from tissue lichen specimens were cleaned in tap water segments of lichen thalli showed that these to remove dirt and then thoroughly washed fungi not found on surface sterilized lichen under running tap water. These lichen thalli thalli (Arnold et al., 2009). The colonization were surface sterilized by consecutive of fungi was considered environmental immersion for one minute in 75% ethanol, 3 contaminants (Frankland, 1974). Fungi minute in 2% Sodium hypochlorite and 3 belong to soil and litter symbiosis or seconds in 75 % ethanol and washed several pathogenic also isolated from fruticose times with sterile distilled water. The thalli lichens which were symbionts or pathogens were surface dried with sterile paper and cut of higher plants and lichens inhabiting fungi into 1cm sized lichen segments and then (Petrini et al., 1990). Although there are plated on nutrient medium consisted of 2% several studies on endophytic malt extract, 0.4 yeast extract and 2% agar microorganisms of higher plants, very few to which 50mg/l chlortetracyline used as studies were carried out on the isolation of antibacterial antibiotic (Petrini et al., 1990). endolichenic fungi from the lichen thallus in The plates were incubated at 25ºC± 2 for 10- India (Suryanarayanan et al., 2005). 15 days under Philips fluorescent tube light Therefore, we have examined some lichen for 12/12 h day and night alternate light species for isolation of endolichenic fungi conditions. They were examined regularly 116 Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 115-124 and colonies developed were transferred according to the formula D’ = 1/∑ (pi) 2, immediately to new petri dishes with PDA where k is the total number of fungal species medium for identification. Sterilized tissue and pi is the proportion of individual that segments were pressed down on to the species i contributes to the total (Pielou, surface of PDA medium to check back the 1975). Simpson index was calculated to efficacy of surface sterilization procedure understand species diversity of endolichenic and to confirm endolichenic isolations only fungi occurred in lichen thalli. from internal tissues of lichen segments. Absence of growth of any fungi on the Results and Discussion medium confirmed that surface sterilization procedure was effective in removing the A total of 30 different endolichenic fungal surface fungi as reported in a previous study species were recovered from 11 species of (Schulz et al., 1993). Morphology of foliose and fruticose lichens (Table 2). subcultures on PDA were examined Twenty four species of fungi have produced periodically and identified when isolates fructifications in culture and have been were sporulated (Domsch and Gams, 1972; identified to genus or species level and rest Nagamani et al., 2006 ). The fungal cultures were categorized as sterile forms (Table 3). that failed sporulation even after two months The high colonization rate (61.5-90.6%) of of incubation were named as mycelia sterilia endolichenic fungi was observed among (Schulz et al., 1993). Pattern of fungal eleven lichen species in this study. association with lichens on nutrient rich and Maximum colonization rate was observed in poor media was also done in order to Parmotrema cristiferum (90.6%) and P. understand influence of different medium on reticulatum (90.3%) and moderate in fungal growth. The surface sterilized lichen Cladonia fruticulosa (61.5%). Among the segments were incubated on 3 such media 30 isolates, 24 species were identified in that viz., PDA (Dextrose-20g, Agar- 15g, potato most of them were belonging to ascomycota 200g/ 1000 ml pH-5.6±0.2), MYA (Malt and all other isolates categorized as Mycelia extract-10g, Yeast extract- 4g, Glucose-4g sterilia (6). A moderate to high per cent of and Agar 5g pH-7.5±0.2) and CZA endolichenic fungi occurred in this study (Sucrose-30g, NaNO3-3g, K2HPO4-1g, KCl- from eleven species of macrolichens. After a 0.5g, MgSO4.7H2O-0.01g, Agar-15g, thorough surface sterilization protocol, and distilled water 1000ml, pH 6.2±0.3). incubation, all the fungi expressed from the Incubation conditions maintained were as cut ends of the thalli. This is confirmed as mentioned earlier. sterilized thalli were pressed on to the medium to check back the possibility of Data Analysis contaminants on the surface of thallus to know effectiveness of surface sterilization. The Relative Frequency (RF %) of occurrence of endolichenic fungi was The fungal species associated with thalli of calculated as the total number of taxon lichen species such as Chaetomium divided by the total number of taxa obtained globosum (8 species of lichens), from lichen thalli