Ann Microbiol (2015) 65:785–797 DOI 10.1007/s13213-014-0919-7 ORIGINAL ARTICLE Molecular profiling of fungal assemblages in the healthy and infected roots of Decalepis arayalpathra (J. Joseph & V. Chandras) Venter, an endemic and endangered ethnomedicinal plant from Western Ghats, India Kandasamy Premalatha & Sivaraman Gokul & Amit Kumar & Priyanka Mishra & Pooja Mishra & Kaliamoorthy Ravikumar & Alok Kalra Received: 1 October 2013 /Accepted: 13 May 2014 /Published online: 27 May 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Decalepis arayalpathra, an endangered, endemic typical clearly visible symptoms, such as a severe brown ethnomedicinal plant from southern Western Ghats, India, is discolouration on the taproot. Molecular profiling of all the targeted for its aromatic and medicinal properties. This study pure fungal isolates, viz., Trichoderma, Penicillium, aimed at to identify fungal endophyte populations associated Aspergillus, Fusarium and Mucor, revealed high sequence with healthy and diseased roots of this perennial shrub. similarities (≥ 98 %) to corresponding reference sequences. Healthy and rotted root samples of D. arayalpathra were Sequencing of Trichoderma pure cultures isolated from collected, fungal endophytes assemblages were identified healthy and diseased roots revealed sequence similarities to both by culture-dependent and culture-independent ap- Trichoderma harzianum, T. hamatum, T. koningiopsis, proaches, further sequenced and the retrieved sequences were T. asperellum, T. pubescens and Hypocrea sp. This confirms analysed with the reference sequences in GenBank to know the morphological examinations, as Hypocrea is the their phylogenetic relationships. Analysis of the ITS rDNA teleomorph stage of Trichoderma sp. This study signifies the region generated 24 different Ascomycota and three Basidio- first work pertaining to the taxonomy of the fungal endophytic mycota taxa. Trichoderma sp. was most abundant in healthy community of D. arayalpathra, and the results reported in this and diseased root samples, while Penicillium and Aspergillus work may help to ascertain the cause of root rot disease often were confined to healthy roots. Furthermore, Fusarium solani, perceived in D. arayalpathra. Also, it could be useful to Fusarium oxysporum and Mucor velutinosus were found to be identify the promising endophytic communities against the the most frequent fungi identified from the rotted root sam- root rot diseases occurring in D. arayalpathra. ples, thus substantiated to be the cause for D. arayalpathra decline in the wild. Interestingly, the strains assigned to Keywords Decalepis arayalpathra . Endophytes . Root rot . Fusarium sp. were isolated from diseased roots showing rDNA . Phylogenetic analysis * : : K. Premalatha ( ) P. Mishra A. Kalra Introduction Department of Microbial Technology and Entomology, CSIR– Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, India Decalepis arayalpathra (J. Joseph & V. Chandras) Venter, e-mail: [email protected] (Syn. Janakia arayalpathra J. Joseph & V. Chandras) belongs to the family Apocynaceae and is a narrow endemic species A. Kumar : P. Mishra Department of Plant Biology and Systematics, CSIR–Central confined to the Southern Western Ghats of Tamil Nadu and Institute of Medicinal and Aromatic Plants Research Centre, Kerala (Joseph and Chandrasekaran 1978;Venterand Bangalore 560 065, India Verhoeven 1997; Prabakaran et al. 2001; Prabakaran and Ravikumar 2003). Kallarasan and Amirthapala are the ver- S. Gokul : K. Ravikumar School for Conservation of Natural Resources, Repository for nacular names of this plant in Tamil and Malayalam, respec- Medicinal Resources I-AIM (FRLHT), Bangalore 560 106, India tively. It is a deciduous perennial shrub that grows at 45–90° 786 Ann Microbiol (2015) 65:785–797 on vertical rocky slopes among the grasslands, surrounded by roots, using both culture and culture-independent evergreen and shola forests. The plant grows in clumps with- (metagenomic) approaches. out any firm holding and is exposed to heavy wind velocity, high temperature and moderately good rainfall almost throughout the year. The only holding for the rootstocks of Materials and methods these plants is grasses with a meager amount of sedimentary black soil. Sampling site and source of material The moniliferous tuberous root of this plant is highly aromatic and is used for peptic ulcer, cancer-like afflictions, About 50 g of tubers of three different plants—Healthy Root for stomachache, as a rejuvenating tonic, and for preparing (HR) from healthy plants, Rotted Root (RRI) from completely pickles by the Kani tribes of southern Western Ghats dead plants and Rotted Root (RRII) from plants with partial (Pushpangadan et al. 1990). Biological activities including infections of D. arayalpathra—were collected from Kalakkad gastric antisecretory and anti-ulcer activities from the root Mudanthurai Tiger Reserve (KMTR) in Tirunelveli district have also been reported(Shine et al. 2007). and Kanyakumari Wildlife Sanctuary (KWS) in Decalepis arayalpathra is assessed as Critically Endan- Kannyakumari district of Tamil Nadu, India (Fig. 1). Geo- gered (B1, 2c, d)–Globally (CR-G) following International graphically, the area is located between 8°17’ to 8°42’N Union for Conservation of Nature (IUCN) criteria in the latitude and 77°06’ to 77°35’ E longitude with an altitude Conservation Assessment and Management Prioritization range between 550 and 1,500 MSL. The plant herbarium (CAMP) workshop by the Foundation of Revitalization of specimens from the samples collected are deposited at Local Health Tradition (FRLHT) and the Conservation Breed- FRLHT, Herbarium of FRLHT, Bangalore (Ravikumar and ing Specialist Group (CBSG) (Molur and Walker 1997; Gokul 114161; Gokul 113868) for future reference. Ravikumar and Ved 2000). The National Biodiversity Author- ity of India (NBA) declared this plant as a species of high Isolation of endophytic fungi conservation concern (Anonymous 2011). Furthermore, the natural regeneration as well as conventional propagation of Isolation of endophytic fungi was performed based on the this plant are overwhelmed by several factors like poor fruit procedures described by Xu et al. (2008). Root samples were set, seed germination and rooting on stem cuttings (Sudha and thoroughly washed in running tap water for 10 min before Seeni 2001). Rare plants growing in remote locations repre- disinfection. The samples of roots were surface sterilised by sent rich potential sources of novel microorganisms, which washing in 75 % ethanol for 1 min, sterile distilled water may have the ability to produce secondary metabolites of twice, and 0.05 g ml−1 sodium hypochlorite solution for medicinal and industrial interest (Rivera-Orduña et al. 2011). 3 min, followed by several rinses in sterile distilled water. Several studies reported that a high proportion of microorgan- Small pieces (0.5×0.5 cm) of surface sterilised roots were isms cannot be cultured on synthetic media at this time (Kent placed in Petri dishes containing Potato Dextrose Agar and Triplett 2002), whereas culture-independent methods, (PDA, HiMedia), prepared according to the manufacturer’s mostly supported by the development of PCR-based technol- instruction and incubated at 28 °C for 7–14 days. The antibi- ogies, better assist in identification and characterization of otics streptomycin sulphate and chloramphenicol were added, microorganisms directly from environmental samples (Filion each to a final concentration of 50 mg ml−1, to inhibit bacterial et al. 2004). However, a combination of culture-dependent contamination. When colonies appeared, they were and culture-independent approaches could afford more insight subcultured onto fresh plates containing the same medium into the evaluation of diversity (Jebaraj et al. 2010). and allowed to grow for 14 days before they were subjected Medicinal plants have been recognized as a repository of to morphological examination. To examine fungal structures, fungal endophytes with novel metabolites (Wiyakrutta et al. the culture was transferred into a drop of 0.01 % cotton blue in 2004). Studies on medicinal plants from the Western Ghats 60 % lactic acid on a microscope slide using a sterile needle, report the presence of a diverse community of endophytic and observed under a light microscope. fungi (Naik et al. 2008). There are practically no reports on the fungal endophytes associated with the endemic medicinal Characterization of fungi associated with D. arayalpathra plant D. arayalpathra of the Western Ghats. Hence, this study provides the first information on the fungal endophytes asso- Colony morphology ciated with this plant. As an initial step in the search of fungi capable of producing novel anticancer compounds, and since Fungal isolates were first grouped into morphotypes accord- there are no existing reports of fungal communities associated ing to colony morphology, and we referred to fungal genera with D. arayalpathra, the objective of this study is to profile when reproductive structures were present, using appropriate the main fungal taxa associated with healthy and infected taxonomic literature. For a further grouping of isolates, Ann Microbiol (2015) 65:785–797 787 Fig. 1 Habitat and sample collection sites of Decalepis arayalpathra in the Western Ghats, India. 1–Red fort estate Peak. 2–Ammankovil Mottai. 3– Kuthiraikatti Peak. 4–Flower and Fruit Twig. 5, 6–Habitat molecular identification of all