Molecular Biology Reports

, Volume 42, Issue 11, pp 1533–1543 | Cite as

Genetic diversity and antimicrobial activity of endophytic Myrothecium spp. isolated from Calophyllum apetalum and Garcinia morella

  • Karmakar Ruma
  • Kumar Sunil
  • Kukkundoor R. Kini
  • Harischandra Sripathy Prakash
Original Paper


Calophyllum apetalum and Garcinia morella, medicinal plants are endemic to Western Ghats, Karnataka, India. Sixteen Myrothecium isolates were obtained from the tissues of bark and twigs of these plants. The purpose of this study was to explore the antimicrobial activity and genetic variability of the endophytic Myrothecium isolates. The antimicrobial activity as well as the genetic diversity of endophytic Myrothecium species was investigated through RAPD, ISSR and ITS sequence analysis. Myrothecium isolates were genotypically compared by RAPD and ISSR techniques, 510 and 189 reproducible polymorphic bands were obtained using 20 RAPD and ten ISSR primers respectively. The isolates grouped into four main clades and subgroups using unweighted pair group method with arithmetic mean cluster analysis. rDNA ITS sequence analysis presented better resolution for characterising the isolates of Myrothecium spp. The clustering patterns of the isolates were almost similar when compared with RAPD and ISSR dendograms. The results signify that RAPD, ISSR and ITS analysis can be employed to distinguish the genetic diversity of the Myrothecium species. The endophytic and pathogenic strains were compared by maximum parsimony, maximum likelihood and neighbour joining methods. One isolate (JX862206) amongst the 16 Myrothecium isolates exhibited potent antibacterial and as well as anti-Candida activity.


Myrothecium RAPD ISSR ITS Phylogenetic analysis Maximum parsimony 



The authors acknowledge the support under Institution of Excellence Program of University of Mysore awarded by Ministry of Human Resource Development and UGC, Government of India.

Conflict of interest

There is no conflict of interest.

Supplementary material

11033_2015_3884_MOESM1_ESM.doc (6.3 mb)
Table S1. List of ISSR primers along with the corresponding annealing temperatures used in the study Table S2. The details of Myrothecium spp. sequences retrieved from GenBank Fig S1. Colony morphology and conidial characters of endophytic Myrothecium spp. Fig. S2. Agarose gel showing RAPD products of Myrothecium isolates obtained by amplifying 50 ng of DNA using the Primer OPM-02 (2a), OPM-04 (2b), OPM-07 (2c), OPM-12 (2d), OPM-19 (2e) and OPM-20 (2f). Lane numbers 1 to 16 correspond to the isolate numbers in Table 1; M: 1 kb ladder Fig. S3. Agarose gel showing ISSR products of Myrothecium isolates obtained by amplifying 50 ng of DNA using the Primer ISSR-03 (3a), ISSR-05 (3b), ISSR-07 (3c) and ISSR-10 (3d). Lane numbers 1 to 16 correspond to the isolate numbers in Table 1; M: 1 kb ladder Fig. S4. Agarose gel showing ITS products of Myrothecium isolates obtained by amplifying 50 ng of DNA using the primers ITS1 and ITS4. Lane numbers 1 to 16 corresponds to the isolate numbers in Table 1; M: 100 bp ladder Fig. S5. Phylogenetic analysis of ITS sequences of Myrothecium spp. The tree shown was derived by ClustalW analysis of 130 isolates of Myrothecium spp from different origins obtained from GenBank. The 16 Myrothecium isolates used in the study has been marked using a circle Fig. S7. Phylogenetic analysis using neighbour joining method of the ITS sequences of Myrothecium spp. The tree shown was derived by MEGA5 analysis of 130 isolates of Myrothecium spp from different origins obtained from GenBank. The 16 Myrothecium isolates used in the study has been marked using a circle. (E) = Endophyte; (P) = Pathogen; (Ma) = Marine; (O) = Others Fig. S8. Antibacterial activity of crude extract of Myrothecium isolate M1-CA-102. a: Klebsiella pneumonia; b: Staphylococcus aureus; c: E. coli; d: Salmonella typhi; e: Shigella sp.; f: Bacillus subtilis; g: Control


  1. 1.
    Frohlich J, Hyde KD (1999) Biodiversity of palm fungi in the tropics: are the global fungal biodiversity estimates realistic? Biodivers Conserv 8:977–1004CrossRefGoogle Scholar
  2. 2.
    Hyde KD, Soytong K (2008) The fungal endophyte dilemma. Fungal Divers 33:163–173Google Scholar
  3. 3.
    Lin X, Huang Y, Zheng Z, Su W, Quiang X, Shen Y (2010) Endophytes from the pharmaceutical plant, Annona squamosa: isolation, bioactivity, identification and diversity of its polyketide synthase gene. Fungal Divers 43:41–51CrossRefGoogle Scholar
  4. 4.
    Azevedo JL Jr, Maccheroni W, Pereira JO, Araujo WL (2000) Endophytic microorganisms: a review on insect control and recent advances on tropical plants. Electron J Biotechnol 3:41–65CrossRefGoogle Scholar
  5. 5.
    Hyde KD, Bussaban B, Paulus B, Crous PW, Lee S, Mckenzie EHC, Photita W, Lumyong S (2007) Diversity of saprobic microfungi. Biodivers Conserv 16:17–35CrossRefGoogle Scholar
  6. 6.
    Wang X, Yang R, Feng S, Hou X, Zhang Y et al (2012) Genetic variation in Rheum palmatum and Rheum tanguticum (Polygonaceae), two medicinally and endemic species in China using ISSR markers. PLoS One 7(12):e51667PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Chen J, Zhang L-C, Xing Y-M, Wang Y-Q, Xing X-K et al (2013) Diversity and taxonomy of endophytic Xylariaceous fungi from medicinal plants of Dendrobium (Orchidaceae). PLoS One 8(3):e58268PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Liu JY, Huang LL, Ye YH, Zou WX, Guo ZJ, Tan RX (2001) Antifungal and new metabolites of Myrothecium sp. Z16, a fungus associated with white croaker Argyrosomus argentatus. J Appl Microbiol 100:195–202CrossRefGoogle Scholar
  9. 9.
    Amagata T, Rath C, Rigot JF, Tarlov N, Tenney K, Valeriote FA, Crews P (2003) Structures and cytotoxic properties of trichoverroids and their macrolide analogues produced by saltwater culture of Myrothecium verrucaria. J Med Chem 46:4342–4350CrossRefPubMedGoogle Scholar
  10. 10.
    Isaka M, Punya J, Lertwerawat Y, Tanticharoen M, Thebtaranonth Y (1999) Antimalarial activity of macrocyclic trichothecenes isolated from the fungus Myrothecium verrucaria. J Nat Prod 62:329–331CrossRefPubMedGoogle Scholar
  11. 11.
    Leslie JF, Zeller KA, Lamprecht SC, Rheeder JP, Marasas WFO (2005) Toxicity, pathogenicity and genetic differentiation of five species of Fusarium from Sorghum and Millet. Phytopathology 95:275–283CrossRefPubMedGoogle Scholar
  12. 12.
    Magnani RF, Rodrigues-Fo E, Daolio C, Ferreira AG, de Souzab AQL (2003) Three highly oxygenated caryophyllene sesquiterpenes from Pestalotiopsis sp., a fungus isolated from bark of Pinus taeda. Zeitschrift für Naturforschung 58c:319–324Google Scholar
  13. 13.
    Albrectsen BR, Bjorken L, Varad A, Hagner A, Wedin M, Karlsson J, Jansson S (2010) Endophytic fungi in European aspen (Populus tremula) leaves-diversity, detection, and a suggested correlation with herbivory resistance. Fungal Divers 41:17–28CrossRefGoogle Scholar
  14. 14.
    Sun X, Guo L-D (2012) Endophytic fungal diversity: review of traditional and molecular techniques. Mycology 3(1):65–76Google Scholar
  15. 15.
    Begerow D, Nilsson H, Unterseher M, Maier W (2010) Current state and perspectives of fungal DNA barcoding and rapid identification procedures. Appl Microbiol Biotechnol 87:99–108CrossRefPubMedGoogle Scholar
  16. 16.
    Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Tejesvi MV, Kini KR, Prakash HS, Subbiah V, Shetty HS (2007) Genetic diversity and antifungal activity of species of Pestalotiopsis isolated as endophytes from medicinal plants. Fungal Divers 24:37–54Google Scholar
  18. 18.
    Jana T, Sharma TR, Prasad RD, Arora DK (2003) Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique. Microbiol Res 158:249–257CrossRefPubMedGoogle Scholar
  19. 19.
    Meyer W, Koch A, Niemann C, Beyermann B, Epplen JT, Borner T (1993) Differentiation of species and strains among filamentous fungi by DNA fingerprinting. Curr Genet 19:239–242CrossRefGoogle Scholar
  20. 20.
    Burgess T, Wingfield VD, Wingfield MJ (2001) Comparison of genotypic diversity in native and introduced populations of Sphaeropsis sapinea isolated from Pinus radiate. Mycol Res 105:1331–1339CrossRefGoogle Scholar
  21. 21.
    O’Brien HE, Parrent JL, Jackson JA, Jean Moncalvo J-M, Vilgalys R (2005) Fungal community analysis by large-scale sequencing of environmental samples. Appl Environ Microbiol 71:5544–5550PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Khare CP (2007) In: Khare CP (ed) Indian medicinal plant: an illustrated dictionary. Springer, New YorkGoogle Scholar
  23. 23.
    Ruma K, Shailasree S, Sampath KKK, Niranjana SR, Prakash HS (2012) Endophytic fungal assemblages in Calophyllum and Garcinia spp. of Clusiaceae family in Western Ghats, India. Curr Biotechnol 1:109–114CrossRefGoogle Scholar
  24. 24.
    Kang T-J, Yang M-S (2004) Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants. BMC Biotechnol 20:4–20Google Scholar
  25. 25.
    Joshi SD, Sanjay R, Baby UI, Mandal AKA (2009) Molecular characterization of Pestalotiopsis spp. associated with tea (Camellia sinensis) in southern India using RAPD and ISSR markers. Indian J Biotechnol 8:377–383Google Scholar
  26. 26.
    White TJ, Bruns TD, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, CaliforniaCrossRefGoogle Scholar
  27. 27.
    Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590PubMedCentralPubMedGoogle Scholar
  28. 28.
    Miller M (1997) Tools for population genetic analysis (TFPGA) 1.3: a windows program for the analysis of allozyme and molecular population genetic data. Computer software distributed by authorGoogle Scholar
  29. 29.
    Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526PubMedGoogle Scholar
  32. 32.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Hadacek F, Greger H (2000) Testing of antifungal natural products: methodologies, comparability of result and assay choice. Phytochem Anal 11:137–147CrossRefGoogle Scholar
  34. 34.
    Bacon CW, White JF (2000) Microbial endophytes. Marcel Deker Inc., New YorkGoogle Scholar
  35. 35.
    Williams JGK, Hanafey MK, Rafalski JA, Tingey SV (1993) Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol 218:704–740CrossRefPubMedGoogle Scholar
  36. 36.
    Tejesvi MV, Tamhankar SA, Kini KR, Rao VS, Prakash HS (2009) Phylogenetic analysis of endophytic Pestalotiopsis species from ethnopharmaceutically important medicinal trees. Fungal Divers 38:167–183Google Scholar
  37. 37.
    Glienke-Blanco C, Aguilar-Vildoso CI, Vieira MLC, Barroso PAV, Azevedo JL (2002) Genetic variability in the endophytic fungus Guignardia citricarpa isolated from citrus plants. Genet Mol Biol 25:251–255CrossRefGoogle Scholar
  38. 38.
    Rodrigues KF, Sieber TN, Grunig CR, Holdenrieder O (2004) Characterization of Guignardia mangiferae isolated from tropical plants based on morphology, ISSR-PCR amplifications and ITS1-5.8S-ITS2 sequences. Mycol Res 108:45–52CrossRefPubMedGoogle Scholar
  39. 39.
    Ratanacherdchai K, Wang HK, Lin FC, Soytong K (2009) ISSR for comparison of cross-inoculation potential of Colletotrichum capsici causing chilli anthracnose. Afr J Microbiol Res 4:76–83Google Scholar
  40. 40.
    Carlier J, Cabrita L, Leitao J, Sousa RM, Sousa AT (2011) ISSR and AFLP characterization of Phomopsis amygdali (Del.) Tuset & Portilla accessions. Acta Hortic (ISHS) 912:645–650CrossRefGoogle Scholar
  41. 41.
    Peay KG, Kennedy PG, Bruns TD (2008) Fungal community ecology: a hybrid beast with a molecular master. Bioscience 58:799–810CrossRefGoogle Scholar
  42. 42.
    Kim SC, Crawford DJ, Jansen RK (1996) Phylogenetic relationships among the genera of the subtribe Sonchinae (Asteraceae): evidence from ITS sequences. Syst Bot 21:417–432CrossRefGoogle Scholar
  43. 43.
    Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118CrossRefPubMedGoogle Scholar
  44. 44.
    Wei JG, Xu T, Guo LD, Liu AR, Zhang Y, Pan XH (2007) Endophytic Pestalotiopsis species associated with plants of Podocarpaceae, Theaceae and Taxaceae in southern China. Fungal Divers 24:55–74Google Scholar
  45. 45.
    Stierle A, Strobel G, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 260:214–216CrossRefPubMedGoogle Scholar
  46. 46.
    Boyle C, Götz M, Dammann-Tugend U, Schulz B (2001) Endophyte–host interactions III. Local vs. systemic colonisation. Symbiosis 31:259–281Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Karmakar Ruma
    • 1
  • Kumar Sunil
    • 1
  • Kukkundoor R. Kini
    • 1
  • Harischandra Sripathy Prakash
    • 1
  1. 1.Department of Studies in BiotechnologyUniversity of MysoreMysoreIndia

Personalised recommendations