1887

Access Microbiology open research platform logo

Volume 3, Issue 9

Phylogenetic diversity and activity screening of cultivable Actinobacteria isolated from marine sponges and associated environments from the western coast of India Open Access

Abstract

The phylogenetic diversity of cultivable actinobacteria isolated from sponges ( spp.) and associated intertidal zone environments along the northern parts of the western coast of India were studied using 16S rRNA gene sequences. A subset of randomly selected actinobacterial cultures were screened for three activities, namely predatory behaviour, antibacterial activity and enzyme inhibition. We recovered 237 isolates from the phylum Actinobacteria belonging to 19 families and 28 genera, which could be attributed to 95 putative species using maximum-likelihood partition and 100 putative species using Bayesian partition in Poisson tree processes. Although the trends in the discovery of actinobacterial genera isolated from sponges were consistent with previous studies from different study areas, we provide the first report of nine actinobacterial species from sponges. We observed widespread non-obligate epibiotic predatory behaviour in eight actinobacterial genera and we provide the first report of predatory activity in , , , , and . Sponge-associated actinobacteria showed significantly more predatory behaviour than environmental isolates. While antibacterial activity by actinobacterial isolates mainly affected Gram-positive target bacteria with little or no effect on Gram-negative bacteria, predation targeted both Gram-positive and Gram-negative prey with equal propensity. Actinobacterial isolates from both sponges and associated environments produced inhibitors of serine proteases and angiotensin-converting enzyme. Predatory behaviour was strongly associated with inhibition of trypsin and chymotrypsin. Our study suggests that the sponges and associated environments of the western coast of India are rich in actinobacterial diversity, with widespread predatory activity, antibacterial activity and production of enzyme inhibitors. Understanding the diversity and associations among various actinobacterial activities – with each other and the source of isolation – can provide new insights into marine microbial ecology and provide opportunities to isolate novel therapeutic agents.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antibiotic production , bacterial predation , enzyme inhibition , molecular phylogeny and secondary metabolites
Funding
This study was supported by the:
  • Maharashtra Gene Bank Programme, Rajiv Gandhi Science and Technology Commission, Government of Maharashtra, India (Award RGSTC/File-2007/DPP-054/CR-28)
    • Principle Award Recipient: NotApplicable
© 2021 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/acmi/10.1099/acmi.0.000242
2021-09-21
2024-04-26
Loading full text...

Full text loading...

/deliver/fulltext/acmi/3/9/acmi000242.html?itemId=/content/journal/acmi/10.1099/acmi.0.000242&mimeType=html&fmt=ahah

References

  1. Ward AC, Bora N. Diversity and biogeography of marine actinobacteria. Curr Opin Microbiol 2006; 9:279–286 [View Article] [PubMed]
     [Google Scholar]
  2. Taylor MW, Radax R, Steger D, Wagner M. Sponge-associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev 2007; 71:295–347 [View Article] [PubMed]
     [Google Scholar]
  3. Lam KS. Discovery of novel metabolites from marine actinomycetes. Curr Opin Microbiol 2006; 9:245–251 [View Article] [PubMed]
     [Google Scholar]
  4. Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C et al. Taxonomy, physiology, and natural products of Actinobacteria. Microbiol Mol Biol Rev 2016; 80:1–43 [View Article] [PubMed]
     [Google Scholar]
  5. Tan LTH, Ser HL, Yin WF, Chan KG, Lee LH et al. Investigation of antioxidative and anticancer potentials of Streptomyces sp. MUM256 isolated from Malaysia mangrove soil. Front Microbiol 2015; 6:1316
     [Google Scholar]
  6. Braesel J, Lee JH, Arnould B, Murphy BT, Eustáquio AS. Diazaquinomycin biosynthetic gene clusters from marine and freshwater actinomycetes. J Nat Prod 2019; 82:937–946 [View Article] [PubMed]
     [Google Scholar]
  7. Mincer TJ, Jensen PR, Kauffman CA, Fenical W. Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments. Appl Environ Microbiol 2002; 68:5005–5011 [View Article] [PubMed]
     [Google Scholar]
  8. Kokare CR, Mahadik KR, Kadam SS. Isolation of bioactive marine actinomycetes from sediments isolated from Goa and Maharashtra coastlines (west coast of India. Indian J Mar Sci 2004; 33:248–256
     [Google Scholar]
  9. Jose PA, Jebakumar SRD. Unexplored hypersaline habitats are sources of novel actinomycetes. Front Microbiol 2014; 5:242 [View Article] [PubMed]
     [Google Scholar]
  10. Pathom-Aree W, Stach JE, Ward AC, Horikoshi K, Bull AT et al. Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench. Extremophiles 2006; 10:181–189 [View Article] [PubMed]
     [Google Scholar]
  11. Mohammadipanah F, Wink J. Actinobacteria from arid and desert habitats: diversity and biological activity. Front Microbiol 2016; 6:1541 [View Article] [PubMed]
     [Google Scholar]
  12. Shivlata L, Tulasi S. Thermophilic and alkaliphilic Actinobacteria: biology and potential applications. Front Microbiol 2015; 6:1014 [View Article]
     [Google Scholar]
  13. Riquelme C, Hathaway JJM, Enes Dapkevicius MLN, Miller AZ, Kooser A et al. Actinobacterial diversity in volcanic caves and associated geomicrobiological interactions. Front Microbiol 2015; 6:1342 [View Article]
     [Google Scholar]
  14. Yang J, Li X, Huang L, Jiang H. Actinobacterial diversity in the sediments of five cold springs on the Qinghai-Tibet plateau. Front Microbiol 2015; 6:1345 [View Article] [PubMed]
     [Google Scholar]
  15. Book AJ, Lewin GR, McDonald BR, Takasuka TE, Wendt-Pienkowski E et al. Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression. PLoS Biol 2016; 14:e1002475 [View Article] [PubMed]
     [Google Scholar]
  16. Qin Z, Munnoch JT, Devine R, Holmes NA, Seipke RF et al. Formicamycins, antibacterial polyketides produced by Streptomyces formicae isolated from African Tetraponera plant-ants. Chem Sci 2017; 8:3218–3227 [View Article] [PubMed]
     [Google Scholar]
  17. Li Z, Sun W, Zhang F, He L, Loganathan K. Actinomycetes from the South China Sea sponges: isolation, diversity and potential for aromatic polyketides discovery. Front Microbiol 2015; 6:1048
     [Google Scholar]
  18. Mahmoud HM, Kalendar AA. Coral-associated Actinobacteria: diversity, abundance, and biotechnological potentials. Front Microbiol 2016; 7:204 [View Article] [PubMed]
     [Google Scholar]
  19. Trujillo ME, Riesco R, Benito P, Carro L. Endophytic actinobacteria and the interaction of Micromonospora and nitrogen fixing plants. Front Microbiol 2015; 6:1341 [View Article] [PubMed]
     [Google Scholar]
  20. Goodfellow M. Actinobacteria phyl. nov. Whitman W. eds In Bergey’s Manual of Systematics of Archaea and Bacteria New York: Wiley; 2015 https://dx.doi.org/10.1002/9781118960608.pbm00002
     [Google Scholar]
  21. Montalvo NF, Mohamed NM, Enticknap JJ, Hill RT. Novel actinobacteria from marine sponges. Antonie Van Leeuwenhoek 2005; 87:29–36 [View Article] [PubMed]
     [Google Scholar]
  22. Manivasagan P, Venkatesan J, Sivakumar K, Kim SK. Actinobacterial enzyme inhibitors–A review. Crit Rev Microbiol 2015; 41:261–272 [View Article] [PubMed]
     [Google Scholar]
  23. Olano C, Méndez C, Salas J. Antitumor compounds from marine actinomycetes. Mar Drugs 2009; 7:210–248 [View Article] [PubMed]
     [Google Scholar]
  24. Trischman JA, Tapiolas DM, Jensen PR, Dwight R, Fenical W et al. Salinamides A and B: anti-inflammatory depsipeptides from a marine streptomycete. J American Chem Soc 1994; 116:757–758 [View Article]
     [Google Scholar]
  25. Pimentel-Elardo SM, Kozytska S, Bugni TS, Ireland CM, Moll H et al. Anti-parasitic compounds from Streptomyces sp. strains isolated from Mediterranean sponges. Mar Drugs 2010; 8:373–380 [View Article] [PubMed]
     [Google Scholar]
  26. Cheng C, MacIntyre L, Abdelmohsen UR, Horn H, Polymenakou PN et al. Biodiversity, anti-trypanosomal activity screening, and metabolomic profiling of actinomycetes isolated from Mediterranean sponges. PLoS ONE 2015; 10:e0138528
     [Google Scholar]
  27. Gandhimathi R, Arunkumar M, Selvin J, Thangavelu T, Sivaramakrishnan S et al. Antimicrobial potential of sponge associated marine actinomycetes. J Mycol Med 2008; 18:16–22 [View Article]
     [Google Scholar]
  28. Abdelfattah MS, Elmallah MIY, Hawas UW, Abou El-Kassema LT, Eid MAG. Isolation and characterization of marine-derived actinomycetes with cytotoxic activity from the Red Sea coast. Asian Pacific Journal of Tropical Biomedicine 2016; 6:651–657 [View Article]
     [Google Scholar]
  29. Manivasagan P, Kang KH, Sivakumar K, Li-Chan EC, Oh HM et al. Marine actinobacteria: an important source of bioactive natural products. Environ Toxicol Pharmacol 2014; 38:172–188 [View Article]
     [Google Scholar]
  30. Imada C. Enzyme inhibitors and other bioactive compounds from marine actinomycetes. Antonie Van Leeuwenhoek 2005; 87:59–63 [View Article] [PubMed]
     [Google Scholar]
  31. Watve MG, Tickoo R, Jog MM, Bhole BD. How many antibiotics are produced by the genus Streptomyces?. Arch Microbiol 2001; 176:386–390 [View Article] [PubMed]
     [Google Scholar]
  32. Mahapatra GP, Raman S, Nayak S, Gouda S, Das G et al. Metagenomics approaches in discovery and development of new bioactive compounds from marine actinomycetes. Curr Microbiol 2020; 77:645–656 [View Article] [PubMed]
     [Google Scholar]
  33. Subramani R, Sipkema D. Marine rare actinomycetes: A promising source of structurally diverse and unique novel natural products. Marine Drugs 2019; 17:249 [View Article]
     [Google Scholar]
  34. Kumbhar C, Watve M. Why antibiotics: A comparative evaluation of different hypotheses for the natural role of antibiotics and an evolutionary synthesis. Nat Sci 2013; 5:26–40
     [Google Scholar]
  35. Vetter YA, Deming JW, Jumars PA, Krieger-Brockett BB. A predictive model of bacterial foraging by means of freely released extracellular enzymes. Microbial Ecology 1998; 36:75–92 [View Article]
     [Google Scholar]
  36. Lee OO, Wong YH, Qian PY. Inter- and intraspecific variations of bacterial communities associated with marine sponges from San Juan Island, Washington. Appl Environ Microbiol 2009; 75:3513–3521 [View Article] [PubMed]
     [Google Scholar]
  37. Thomas TR, Kavlekar DP, LokaBharathi PA. Marine drugs from sponge-microbe association - a review. Mar Drugs 2010; 8:1417–1468 [View Article] [PubMed]
     [Google Scholar]
  38. Lee YK, Lee JH, Lee HK. Microbial symbiosis in marine sponges. J Microbiol 2001; 39:254–264
     [Google Scholar]
  39. Casida LE. Bacterial predators of Micrococcus luteus in soil. Appl Environ Microbiol 1980; 39:1035–1041 [View Article] [PubMed]
     [Google Scholar]
  40. Casida LE. Interaction of Agromyces ramosus with other bacteria in soil. Appl Environ Microbiol 1983; 46:881–888 [View Article] [PubMed]
     [Google Scholar]
  41. Kumbhar C, Mudliar P, Bhatia L, Kshirsagar A, Watve M. Widespread predatory abilities in the genus Streptomyces. Arch Microbiol 2014; 196:235–248 [View Article] [PubMed]
     [Google Scholar]
  42. Pund A, Holkar K, Watve M, Baig UI. Bacterial predator prey and the third beneficiary. Matters (Zur) 2020
     [Google Scholar]
  43. Harir M, Bendif H, Bellahcene M, Fortas Z, Pogni R. Streptomyces secondary metabolites. Enany S. eds In Basic Biology and Applications of Actinobacteria London: IntechOpen; 2018 pp 99–122 [View Article]
     [Google Scholar]
  44. Van der Meij A, Worsley SF, Hutchings MI, van Wezel GP. Chemical ecology of antibiotic production by actinomycetes. FEMS Microbiol Rev 2017; 41:392–416 [View Article] [PubMed]
     [Google Scholar]
  45. Singh HS. Marine protected areas in India. Indian J Marine Sci 2003; 32:226–233
     [Google Scholar]
  46. De K, Venkataraman K, Ingole B. Current status and scope of coral reef research in India: A bio-ecological perspective. Indian J Geo Marine Sci 2017; 46:647–662
     [Google Scholar]
  47. Kathiresan K. Mangrove forests of India. Curr Sci 2018; 114:976–981 [View Article]
     [Google Scholar]
  48. Mote S, Gupta V, De K, Nanajkar M, Damare SR et al. Bacterial diversity associated with a newly described bioeroding sponge, Cliona thomasi, from the coral reefs on the West Coast of India. Folia Microbiol 2020
     [Google Scholar]
  49. Watve M, Shejval V, Sonawane C, Rahalkar M, Matapurkar A et al. The “K” selected oligophilic bacteria: A key to uncultured diversity. Curr Sci 2000; 78:1535–1542
     [Google Scholar]
  50. ZoBell CE. Studies on marine bacteria. I. The cultural requirements of heterotrophic aerobes. J Mar Res 1941; 4:41–75
     [Google Scholar]
  51. Takizawa M, Colwell RR, Hill RT. Isolation and diversity of actinomycetes in the Chesapeake Bay. Appl Environ Microbiol 1993; 59:997–1002 [View Article] [PubMed]
     [Google Scholar]
  52. Jiang X, Ellabaan MMH, Charusanti P, Munck C, Blin K et al. Dissemination of antibiotic resistance genes from antibiotic producers to pathogens. Nature Comm 2017; 8:15784
     [Google Scholar]
  53. Heuer H, Krsek M, Baker P, Smalla K, Wellington EM. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl Environ Microbiol 1997; 63:3233–3241 [View Article] [PubMed]
     [Google Scholar]
  54. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999; 41:95–98
     [Google Scholar]
  55. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410 [View Article] [PubMed]
     [Google Scholar]
  56. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32:1792–1797 [View Article] [PubMed]
     [Google Scholar]
  57. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33:1870–1874 [View Article] [PubMed]
     [Google Scholar]
  58. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat Methods 2017; 14:587–589 [View Article] [PubMed]
     [Google Scholar]
  59. Schwarz G. Estimating the dimension of a model. Ann Stat 1978; 6:461–464 [View Article]
     [Google Scholar]
  60. Nei M, Kumar S. Molecular Evolution and Phylogenetics UK: Oxford University Press; 2000
     [Google Scholar]
  61. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Mol Biol Evol 2015; 32:268–274 [View Article] [PubMed]
     [Google Scholar]
  62. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: Improving the ultrafast bootstrap approximation. Mol Biol Evol 2018; 35:518–522 [View Article] [PubMed]
     [Google Scholar]
  63. Rambaut A. FigTree. ver 1.4.3; 2009 http://tree.bio.ed.ac.uk/software/%ef%ac%81gtree/
  64. Zhang J, Kapli P, Pavlidis P, Stamatakis A. A general species delimitation method with applications to phylogenetic placements. Bioinformatics 2013; 29:2869–2876 [View Article] [PubMed]
     [Google Scholar]
  65. Valli S, Suvathi SS, Aysha OS, Nirmala P, Vinoth KP et al. Antimicrobial potential of Actinomycetes species isolated from marine environment. Asian Pac J Trop Biomed 2012; 2:469–473 [View Article]
     [Google Scholar]
  66. Velho-Pereira S, Kamat NM. Antimicrobial screening of actinobacteria using a modified cross-streak method. Indian J Pharm Sci 2011; 73:223–228 [View Article] [PubMed]
     [Google Scholar]
  67. Cheung AL, Ying P, Fischetti VA. A method to detect proteinase activity using unprocessed X-ray films. Anal Biochem 1991; 193:20–23 [View Article] [PubMed]
     [Google Scholar]
  68. Tripathi VR, Kumar S, Garg SK. A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract. BMC Complement Altern Med 2011; 11:56 [View Article] [PubMed]
     [Google Scholar]
  69. Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol 1971; 20:1637–1648 [View Article] [PubMed]
     [Google Scholar]
  70. Ng KH, Lye HS, Easa AM, Liong MT. Growth characteristics and bioactivity of probiotics in tofu-based medium during storage. Ann Microbiol 2008; 58:477–487
     [Google Scholar]
  71. Hammer Ø, Harper DAT, Ryan PD. Past: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica 2001; 4:1–9
     [Google Scholar]
  72. Padgitt PJ, Moshier SE. Mycobacterium poriferae sp. nov., a scotochromogenic, rapidly growing species isolated from a marine sponge. Int J Syst Evol Microbiol 1987; 37:186–191
     [Google Scholar]
  73. Elfalah HW, Usup G, Ahmad A. Anti-microbial properties of secondary metabolites of marine Gordonia tearrae extract. J Agric Sci 2013; 5:94–101
     [Google Scholar]
  74. Santos JD, Vitorino I, De la Cruz M, Díaz C, Cautain B et al. Bioactivities and extract dereplication of Actinomycetales isolated from marine sponges. Front Microbiol 2019; 10:727 [View Article] [PubMed]
     [Google Scholar]
  75. Kiran GS, Sabarathnam B, Selvin J. Biofilm disruption potential of a glycolipid biosurfactant from marine Brevibacterium casei. FEMS Immunol Med Microbiol 2010; 59:432–438 [View Article] [PubMed]
     [Google Scholar]
  76. Palomo S, González I, de la Cruz M, Martín J, Tormo JR et al. Sponge-derived Kocuria and Micrococcus spp. as sources of the new thiazolyl peptide antibiotic kocurin. Mar Drugs 2013; 11:1071–1086 [View Article] [PubMed]
     [Google Scholar]
  77. Stach JEM, Maldonado LA, Masson DG, Ward AC, Goodfellow M et al. Statistical approaches for estimating actinobacterial diversity in marine sediments. Appl Environ Microbiol 2003; 69:6189–6200 [View Article] [PubMed]
     [Google Scholar]
  78. Satheeja SV, Jebakumar SR. Phylogenetic analysis and antimicrobial activities of Streptomyces isolates from mangrove sediment. J Basic Microbiol 2011; 51:71–79 [View Article] [PubMed]
     [Google Scholar]
  79. Yi H, Schumann P, Sohn K, Chun J. Serinicoccus marinus gen. nov., sp. nov., a novel actinomycete with L-ornithine and L-serine in the peptidoglycan. Int J Syst Evol Microbiol 2004; 54:1585–1589 [View Article] [PubMed]
     [Google Scholar]
  80. Shinde VL, Meena RM, Shenoy BD. Phylogenetic characterization of culturable bacteria and fungi associated with tarballs from Betul beach, Goa, India. Mar Pollut Bull 2018; 128:593–600 [View Article] [PubMed]
     [Google Scholar]
  81. Zhang H, Zhang W, Jin Y, Jin M, Yu X. A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species. Antonie van leeuwenhoek 2008; 93:241–248 [View Article] [PubMed]
     [Google Scholar]
  82. Bennur T, Kumar AR, Zinjarde S, Javdekar V. Nocardiopsis species: Incidence, ecological roles and adaptations. Microbiol Res 2015; 174:33–47 [View Article] [PubMed]
     [Google Scholar]
  83. Menezes CB, Bonugli-Santos RC, Miqueletto PB, Passarini MR, Silva CH et al. Microbial diversity associated with algae, ascidians and sponges from the north coast of São Paulo state. Microbiol Res 2010; 165:466–482 [View Article] [PubMed]
     [Google Scholar]
  84. Ramasamy D, Kokcha S, Lagier J-C, Nguyen T-T, Raoult D et al. Genome sequence and description of Aeromicrobium massiliense sp. nov. Stand Genomic Sci 2012; 7:246–257 [View Article] [PubMed]
     [Google Scholar]
  85. Nand K, Rao DV. Arthrobacter mysorens–a new species excreting L-glutamic acid. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg 1972; 127:324–331 [PubMed]
     [Google Scholar]
  86. Jurkevitch E. Predatory behaviors in bacteria-diversity and transitions. Microbe 2007; 2:67–73
     [Google Scholar]
  87. Casida LE. Minireview: Nonobligate bacterial predation of bacteria in soil. Microb Ecol 1988; 15:1–8 [View Article] [PubMed]
     [Google Scholar]
  88. Zeph LR, Casida LE. Gram-negative versus gram-positive (actinomycete) nonobligate bacterial predators of bacteria in soil. Appl Environ Microbiol 1986; 52:819–823 [View Article] [PubMed]
     [Google Scholar]
  89. Ibrahimi M, Korichi W, Hafidi M, Lemee L, Ouhdouch Y et al. Marine Actinobacteria: screening for predation leads to the discovery of potential new drugs against multidrug-resistant bacteria. Antibiotics 2020; 9:91 [View Article]
     [Google Scholar]
  90. Linares JF, Gustafsson I, Baquero F, Martinez JL. Antibiotics as intermicrobial signaling agents instead of weapons. Proc Natl Acad Sci U S A 2006; 103:19484–19489 [View Article] [PubMed]
     [Google Scholar]
  91. Shintre NA, Tamhane VA, Baig U, Pund A, Patwardhan R et al. Diversity of culturable actinobacteria producing protease inhibitors from the intertidal zones of Maharashtra, India. Curr Microbiol 2020; 77:3555–3564 [View Article] [PubMed]
     [Google Scholar]
  92. Pérez J, Moraleda-Muñoz A, Marcos-Torres FJ, Muñoz-Dorado J. Bacterial predation: 75 years and counting!. Environ Microbiol 2016; 18:766–779 [View Article] [PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.0.000242
Loading
Phylogenetic diversity and activity screening of cultivable Actinobacteria isolated from marine sponges and associated environments from the western coast of India
Access Microbiology 3, 000242 (2021); https://doi.org/10.1099/acmi.0.000242
/content/journal/acmi/10.1099/acmi.0.000242
/content/journal/acmi/10.1099/acmi.0.000242
Loading

Data & Media loading...

Supplements

Supplementary material 1

EXCEL

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error