BIODIVERSITY OF ENDOPHYTIC FUNGI IN CHEILANTHES TENUIFOLIA (PTERIDACEAE) THROUGH NUCLEAR rDNA ITS ANALYSIS

Thushara Susan Sabu, Linu Mathew* School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala *corresponding author Email id: linumathew@mgu.ac.in, Contact number: 9447505690 INTRODUCTION Endophytes are microbes that colonize the living internal tissues of plants without causing any immediate negative effects. Endophytes provide protection to their host from insect, pest and herbivore and help their host to adapt in different stress conditions. Cheilanthes tenuifolia is a prominent species of the family Pteridaceae . Pteridophytes being an important member of the biodiversity, the molecular aspects of the endophytes are to be studied in detail.

OBJECTIVES
1. To isolate the endophytic fungi associated with Cheilanthes tenuifolia 2.To characterize the endophytic fungi associated with Cheilanthes tenuifolia

MATERIALS AND METHODS

STUDY SITE AND SAMPLING STRATEGY The study was conducted during the monsoon season of 2012 at different parts of Kerala. The monsoon season is found to be the major growth period of ptreidophytes. Samples were collected from different parts of Kozhikode, Thrissur, Ernakulam, Kottayam and Kollam districts. A nested design (Ghazis & Chaveri, 2009) was used to survey the fungal endophytic diversity from pteridophyte. Three sites were selected within the sampling area, which were 1-5km apart from each other. From each site, five samples of each species were randomly chosen for endophyte collection. The plant materials were brought to the laboratory in sterile bags and processed within hours.

SURFACE STERILIZATION AND INCUBATION Isolation of endophytic fungi was done according to the method described by Petrini (1986). The plant samples were rinsed gently in running water to remove dust and debris. Leaf, stem, root and rhizoid samples were cut into small pieces and disinfected with 75% ethanol for 1 min followed by immersion in 0.1% Hg2Cl2 for 1 min and then washed thoroughly with sterile distilled water so that even a minute trace of mercury is being washed out. The surface sterilized segments were then blotted- dry on sterile blotting paper. The isolation of the fungus from leaves, stem, roots and rhizoids were made separately. About 4-5 segments were placed on Potato Dextrose Agar (PDA) supplemented with streptomycin sulphate (300mg/l) . Imprints of the inoculums were taken on PDA plates to check the efficacy of surface sterilization which were used as controls. The dishes were sealed with parafilm and incubated at 27 2 C for 7-10 days. Most of the fungal growth were initiated on the 4th or 5th day of inoculation. The fungi that grew out from the segments were periodically isolated and identified microscopically. The stain used for microscopic analysis was lactophenol cotton blue and the conidial morphology were compared using fungal atlas.The colonization frequency was calculated as described by Suryanarayanan et al (2003). Colonization frequency (% CF) = ( Ncol / Nt ) 100 ; Ncol = Number of segments colonized by each fungus and Nt = Total number of segments studied. MOLECULAR CHARACTERIZATION OF THE ENDOPHYTES The fungal isolates were inoculated into PD broth and incubated at 25 C in an orbital shaker at 100rpm for 5 days prior to DNA isolation. The resulting mycelia was filtered and dried on filter paper with three washes in sterile distilled water. The mycelium was then freeze-dried before being ground in a pestle and mortar containing liquid nitrogen. 0.01 gm of mycelia was weighed and ground well. 75l of the extraction buffer was added. The ground mycelia was incubated at 65C for 1hr and centrifuged at 13,000rpm for 10 min. To the supernatant, equal volume of chloroform: isoamylalcohol is added and centrifuged again at 13,000rpm for 10 min. To the aqueous layer 1/10 volume of sodium acetate and 2/3rd volume of isopropanol is added. Repeated the centrifugation and the pellet is washed with 70% ethanol by centrifuging. The resulting pellet was dried and dissolved in TE buffer (50l). The DNA samples were labelled and stored at -20C. The DNA sample was run on 0.8% agarose gels to check the bands prior to PCR amplification. The DNA samples were labelled and stored at -20C. Quality and quantity estimated by

AGE (0.8%).ITS fragment was amplified by PCR from fungal genomic DNA using ITS - PCR universal primers ITS1:5 TCCGTAGGTGAACCTGCGG-3 and ITS4:5TCCTCCGCTTATTGATATGC-3. PCR(AGILENT SURECYCLER8800) was carried out in a final reaction volume of 25ul in 200 ul capacity thin wall PCR tube which contained Deionized water17.1, Taq buffer(10x) 2.5ul,MgCl2(25mM) 0.6ul, Primer forward ITS1 (10pM/ul) 0.5ul, Primer reverse ITS 4 (10pm/ul) 0.5ul, dNTPmix (2.5mM ) 2ul, Taq (3u/ul) 0.8ul Template DNA (25ug/ul) 1ul. .A negative DNA control was also kept. The PCR programme was designed for 35 cycles 5 min Initial denaturation 94oC followed by Denaturation 94oc for 30Sec, 30Sec primer annealing at 55oc,1 min extension at 72 oc and final extension 72oC for 10min. The amplified product was visualised in 1.5% AGE gel with 100bp ladder and it was were purified and subjected to sequencing. Sequencing of amplicon with forward and reverse primers was carried out in ABI 37301 cycle sequencer. PHYLOGENETIC ANALYSES Sequences were compared with ITS sequence data from strains available at the public databases GENBANK (http://www.ncbi.nlm.nih.gov) by using the BLAST N sequence match routines. The sequences were multiple aligned using the CLUSTAL W 1.6 (Thompson et al 1994) program and adjusted manually to maximize alignment using BioEdit 7.0.0 (Tom Hall 2006) The phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 (Tamura et al 2011)

RESULTS
The plant materials were collected from different parts of Kerala. Cheilanthes tenuifolia were the selected as explant for endophytic fungi isolation (fig: 1). The endophytic fungi were isolated from leaf, stem, root and rhizome segments. No fungus were isolated from root segments. The plant parts served as a good substrate for both ascomycota and basidiomycota. Fig 1: Cheilanthes tenuifolia

Cheilanthes tenuifolia Kingdom: Phylum: Class: Order: Family: Genus: Species: Plantae Pteridophyta Pteridopsida Polypodiales Pteridaceae Cheilanthes C.tenuifolia

Endophytes from Cheilanthes tenuifolia A total of three fungal isolates were isolated from the healthy leaf, stem and rhizome of Cheilanthes tenuifolia (fig 3,4,5) . There was a single isolate from the leaf which was Fusarium sp (CT- 1). From the stem, the endophyte isolated was Colletotrichum gloeosporoides (CT-2) and rhizome was Lasiodiplodia theobromae (CT-3). Based on the microscopic observation of the mycelia and conidia, the morphological features were analyzed. Using the sequences of the ribosomal DNA internal transcriber spacer (ITS) regions, the major endophytes were Fusarium oxysporum, Colletotrichum gloeosporoides and Lasiodiplodia theobromae(fig 11). Colletotrichum gloeosporoides Kingdom: Phylum: Fungi Ascomycota Fusarium oxysporum Fungi Ascomycota Pezizomycotina Sordariomycetes Hypocreales Nectriaceae

Subphyllum: Pezizomycotina Class: Order: Family: Sordariomycetes Glomerellales Glomerellaceae

Genus:

Colletotrichum

Fusarium

Hyphal growth in potato dextrose agar was found to be rapid with greenish grey or pinkish to salmon patches, powdery to velutine, profusely sporulated, and with abundant production of conidiomata; the reverse was grayish. The conidia were straight, cylindrical to slightly clavate. Fusarium showed rapid growth in PDA plates. Colonies are initially white, becoming tinged with salmon and lavender at maturity. Lavender to purple reverse. Lasiodiplodia theobromae Kingdom: Phylum: Class: Order: Family: Genus: Fungi Ascomycota Dothediomycetes Botryosphaeriales Botrosphaeriacea Lasiodiplodia

The Lasiodiplodia colonies were greyish sepia to mouse grey to black fluffy with aerial mycelium. FIG 2: EMERGENCE OF ENDOPHYTES FROM LEAF, STEM AND RHIZOME OF CHEILANTHES TENUIFOLIA

Microscopical identification of the endophytes

Fig 3:Colletotrichum gloeosporoides

Fig 4: Fusarium oxysporum

Fig 5: Lasiodiplodia theobromae

Molecular identification of the endophytes The DNA was isolated and amplified using ITS (fig 13). The amplified product was sequenced and then submitted to BLAST which revealed the near similar sequences of related species. The sequences which showed maximum identity were selected and submitted to BioEdit 7.0.0, aligned using CLUSTAL W and edited manually. A dendrogram was constructed using MEGA 5.05. Cheilanthes tenuifolia As the phylogenic tree(Fig 8) states the isolate CT-1 formed a clade with other members being Fusarium oxysporum with a boot strap support of 100 showing CT-1 may be a member of Genus Fusarium.. The isolate CT-2 formed a clade with other members being strain of Colletotrichum gloeosporoides with 100% bootstrap support showing CT-2 may be a member of Colletotrichum.The isolate CT-3 also formed a clade with members of Lasiodiplodia theobromae with 100% bootstrap

support showing that the strain might be of the Genus Lasiodiplodia.From the distance matrix of Cheilanthes tenuifolia (M:1), the pair wise distance of CT-1 to Fusarium oxysporum, CT-2 to Colletotrichum gloeosporoides and CL-3 to Lasiodiplodia theobromae is 0.000. Thus it can be assumed that the isolates CT-1, CT-2 & CT-3 are Fusarium oxysporum, Colletotrichum gloeosporoides and

Lasiodiplodia theobromae respectively

Fig 6:GEL PHOTOGRAPH OF THE AMPLICON

FIG7: ITS PHYLOGENY OF ENDOPHYTES ISOLATES FROM CHEILANTHES TENUIFOLIA BY N-J ANALYSIS

gi|348161199|gb|JN624908.1| Fusarium sp. Sa/1/2 gi|348161197|gb|JN624906.1| Fusarium oxysporum strain Pc/12/1 gi|348161192|gb|JN624901.1| Fusarium oxysporum strain Ks/9/1 gi|348161179|gb|JN624888.1| Fusarium oxysporum strain Af/8/2 gi|400538413|emb|HE974453.1| Fusarium oxysporum CT-1 gi|333123084|gb|JF710554.1| Colletotrichum gloeosporioides gi|260751204|gb|GQ924904.1| Glomerella cingulata strain CMU15 gi|260751206|gb|GQ924906.1| Glomerella cingulata strain CMU17 CT-2 gi|333499500|gb|JF766989.1| Lasiodiplodia theobromae gi|390433018|gb|JX104215.1| Lasiodiplodia pseudotheobromae gi|377810541|gb|HM346873.2| Lasiodiplodia theobromae gi|377810536|gb|GU228527.2| Lasiodiplodia theobromae CT-3

Table 1: FREQUENCY OF COLONIZATION OF ENDOPHYTIC FUNGI ISOLATED FROM TENUIFOLIA SL. NO FUNGAL ISOLATE LEAF STEM ROOT RHIZOME % FREQUENCY OF COLONIZATION

CHEILANTHES

1.

C. gloeo sporoides

100

2.

Fusarium oxysporum

100

3.

Lasiodiplodia theobromae

100

Fig8 : Graphical representation of the colonization frequency of endophytes in C.tenuifolia

120 100 80 60 40 20 0 leaf stem root rhizoid

C.gloeosporoides F.oxysporum L.theobromae

DISCUSSION Endophytic microorganisms are found virtually in every plant on earth. Endophytic microorganisms, a group of organisms relatively unstudied, represent an important genetic resource for biotechnology, and had been target of ecological and taxonomic studies. Despite the

environmental and biotechnological importance of fungi, most of these organisms remain uncharacterized and there is no considerable knowledge about evolutionary relationship in many taxa (Sette et al 2006). In addition, microbial characterization becomes an important aspect of biotechnological application, in order to avoid problems associated with the use and release of potential plant and/or animal pathogens in the environment (Sette et al. 2006). Development of molecular techniques has been opening new perspectives for the taxonomic characterization and relationship inference of complex group of organisms. In the present study, the use of ITS sequencing and phylogenetic analyses identified 3 strains of endophytic filamentous fungi at genus level from C.tenuifolia. The most common endophytes from C.tenuifolia, rhizome was Lasiodiplodia theobromae (CT3), from leaf was Fusarium sp. (CT-1) and from stem was Colletotrichum gloeosporoides (CT-2). Lasiodiplodia theobromae is a plant pathogen with a very wide host range. L.theobromae is an anamorph of Botryosphaeria rhodium. It is found to cause cankers in grapevines( Burmo et al 2008) mango (Khanzada et al 2004) etc. It has also been isolated as an endophyte from Piper hispidum (Orlandelli et al 2012).This endophytic fungi is also found to produce taxol in Morinda citrifolia leaves (Pandi et al 2011). C.gloeosporioides is a common endophyte in Theobroma cacao, Amomum siamense, Coffea Arabica, Musa acuminate, Anacardium occidentale, Citrus spp., Eutrepe oleracea, Glycine max, Guarea Guidonia, Ilex paraguariensis, Malus domestica, Himatanthus sucuuba, Palicourea longiflora, Strychnos cogens, Spondias mombin and other species(Arnold et al 2003, Bussban et al 2001, Photita et al 2004, Gamboa & Bayman 2001, Lumyong et al 2002, Peixoto Neto et al 2002 ,Camatti-Sartori et al 2005, Rubini et al 2005, Arnold 2007,Huang et al 2008 ). This endophyte is also found to produce an anti cancer drug, Taxol in the leaves of Justicia gendarussa (Gangadevi & Muthumary 2008). Fusarium verticillioides (= F. moniliforme) is often found in maize seeds, constituting an important source of inoculum in the field. Fusarium spp., associated with symptomatic and asymptomatic plants, may be a primary causal agent of disease, a secondary invader or an endophyte.(Pamphville et al 2002) The rDNA internal transcribed spacer region sequences are now used in the identification and detection of fungi (Pryor and Gilbertson 2000, Anderson et al 2001, Guo et al 2001). Result from the BLAST N revealed that the 3 endophytes belonged to the Ascomycetes group; Genera Colletotrichum , Genera Lasiodiplodia and Genera Fusarium.

CONCLUSION From the present study, what is evident is that indigenous ascomycetes have cryptically colonized the leaf, stem and rhizome. The ascomycetes (Stone et al 2004, Rubini et al 2005,Neubert et al 2006, Higgins et al 2007, Hoffman &) have been appeared predominant in the endophytic mycota. The resident endophytes could prevent initial colonization or displace the invading pathogen (Crozier et al 2006) and inorder to study the actual mechanism behind this displacement, the bioactive molecules produced by the endophytes are to be isolated and identified. Further studies are required to unveil the role played by the endophyte in the host development and survival in adverse conditions.

REFERENCES 1.Crozier , J., S., E., Thomas, M., C., Aime, H., C., Evans, K., A.,Holmes. 2006. Molecular characterization of fungal endophytic morphospecies isolated from stems and pods of Theobroma cacao. Plant Pathology. 55: 783-791. 2. L. D. Sette, M. R. Z. Passarini, C. Delarmelina, F. Salati, M. C. T. Duarte. 2006. Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants. World Journal of Microbiology and Biotechnology. ; 22(11):1185-1195 3. Jao Alencar Pamphville, Joao Lucio Azevedo. 2002. Molecular characterization of endophytic strains of Fusarium verticillioides (=Fusarium moniliforme) from maize (Zea mays. L). World Journal of Microbiology and Biotechnology.18 (5): 391-396.

M:1Distance matrix of pairwise distance between the isolates of Cheilanthes tenuifolia

gi|348161199|gb|JN624908.1|_Fusarium_sp. _Sa/1/2 gi|348161197|gb|JN624906.1|_Fusarium_ox ysporum_strain_Pc/12/1 gi|348161192|gb|JN624901.1|_Fusarium_ox ysporum_strain_Ks/9/1 gi|348161179|gb|JN624888.1|_Fusarium_ox ysporum_strain_Af/8/2 gi|400538413|emb|HE974453.1|_Fusarium_ oxysporum CT-1 gi|333123084|gb|JF710554.1|_Colletotrichu m_gloeosporioides gi|260751204|gb|GQ924904.1|_Glomerella_ cingulata_strain_CMU15 gi|260751206|gb|GQ924906.1|_Glomerella_ cingulata_strain_CMU17 CT-2 gi|333499500|gb|JF766989.1|_Lasiodiplodia _theobromae gi|390433018|gb|JX104215.1|_Lasiodiplodia _pseudotheobromae

0.0 00 0.0 00 0.0 00 0.0 00 0.0 00 0.2 17 0.2 17 0.2 17 0.2 17 0.3 18 0.3 18

0.0 00 0.0 00 0.0 00 0.0 00 0.2 17 0.2 17 0.2 17 0.2 17 0.3 18 0.3 18

0.0 00 0.0 00 0.0 00 0.2 17 0.2 17 0.2 17 0.2 17 0.3 18 0.3 18

0.0 00 0.0 00 0.2 17 0.2 17 0.2 17 0.2 17 0.3 18 0.3 18

0.0 00 0.2 17 0.2 17 0.2 17 0.2 17 0.3 18 0.3 18

0.2 17 0.2 17 0.2 17 0.2 17 0.3 18 0.3 18

0.0 00 0.0 00 0.0 00 0.3 13 0.3 13

0.0 00 0.0 00 0.3 13 0.3 13

0.0 00 0.3 13 0.3 13

0.3 13 0.3 13

0.0 00

gi|377810541|gb|HM346873.2|_Lasiodiplodia _theobromae gi|377810536|gb|GU228527.2|_Lasiodiplodia _theobromae CT-3

0.3 18 0.3 18 0.318

0.3 18 0.3 18 0.3 18

0.3 18 0.3 18 0.3 18

0.3 18 0.3 18 0.3 18

0.3 18 0.3 18 0.3 18

0.3 18 0.3 18 0.3 18

0.3 13 0.3 13 0.3 13

0.3 13 0.3 13 0.3 13

0.3 13 0.3 13 0.3 13

0.3 13 0.3 13 0.3 13

0.0 00 0.0 00 0.0 00

0.0 00 0.0 00 0.0 00

0.0 00 0.0 00

0.0 00