Isolation and Identification of Shoot-Tip Associated Endophytic Bacteria from Banana cv. Grand Naine and Testing for Antagonistic Activity against Fusarium oxysporum f. sp. cubense

Aparna Chandra Sekhar, Pious Thomas^*

Endophytic and Molecular Microbiology Laboratory, Division of Biotechnology, ICAR-Indian Institute of Horticultural Research (IIHR), Bangalore, India.

DOI: 10.4236/ajps.2015.67101                

Abstract

Endophytic bacteria colonizing the shoot-tips of banana cv. Grand Naine were isolated and tested for the antagonistic activity against the Panama wilt pathogen Fusarium oxysporum f. sp. cubense (Foc). Pre-isolation, the suckers were given extensive disinfection treatments and the homogenate from the excised shoot-tip portion was plated on nutrient agar (NA) and trypticase soy agar (TSA). This yielded altogether 47 isolates: 26 on NA and 21 on TSA, respectively, from the 10 suckers collected during August to February. The number of bacterial isolates obtained per sucker varied from one to 15 based on colony characteristics registering up to 10 distinct species per shoot-tip based on 16S rRNA sequence analysis. The 47 isolates belonged to 19 genera and 25 species under the phylogenetic classes of Actinobacteria, α- and γ-Proteobacteria and Firmicutes. Actinobacteria constituted the predominant phylum (55% isolates) with the constituent genera of Arthrobacter, Brevibacterium, Corynebacterium, Curtobacterium, Kocuria, Kytococcus, Micrococcus, Naumanella, Rothia and Tessaracoccus spp. and an unidentified isolate belonging to the family Frankiaceae. Proteobacteria constituted the second major phylum (Brevundimonas, Enterobacter, Klebsiella, Pseudomonas, Serratia and Sphingomonas spp.) followed by Firmicutes (Bacillus and Staphylococcus spp.). Antagonistic activity of the endophytes against Foc was tested through agar plate assays (pit and spot applications on fungal lawn) employing potato dextrose agar and NA. Endophytic Pseudomonas aeruginosa (isolate GNS.13.2a) which was associated with a single sucker showed significant growth inhibition effect on Foc while Klebsiella variicola (GNS.13.3a) and Enterobacter cloacae (GNS13.4a) exhibited moderate inhibition. The study brings out considerable sucker to sucker variation in the associated cultivable endophytic bacteria in “Grand Naine” banana and identifies a few bacterial endophytes with biocontrol potential against the devastating Foc pathogen.

Keywords

Bacterial Endophytes, Bacterial-Fungal Antagonism, Biological Control, Fusarium oxysporum f. sp. cubense, Musa sp., Plantains, Pseudomonas aeruginosa

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Mercado-Blanco, J. and Lugtenberg, B.J.J. (2014) Biotechnological Applications of Bacterial Endophytes. Current Biotechnology, 3, 60-75. http://dx.doi.org/10.2174/22115501113026660038
[2]	Podolich, O., Ardanov, P., Zaets, I., Pirttilä, A.M. and Kozyrovska, N. (2015) Reviving of the Endophytic Bacterial Community as a Putative Mechanism of Plant Resistance. Plant and Soil, 388, 367-377. http://dx.doi.org/10.1007/s11104-014-2235-1
[3]	Hardoim, P.R., van Overbeek, L.S. and Elsas, J.D.V. (2008) Properties of Bacterial Endophytes and Their Proposed Role in Plant Growth. Trends in Microbiology, 16, 463-471. http://dx.doi.org/10.1016/j.tim.2008.07.008
[4]	Compant, S., Duffy, B., Nowak, J., Clement, C. and Barka, E.A. (2005) Use of Plant Growth-Promoting Bacteria for Biocontrol of Plant Diseases: Principles, Mechanism of Action and Future Prospects. Applied and Environmental Microbiology, 71, 4951-4959. http://dx.doi.org/10.1128/AEM.71.9.4951-4959.2005
[5]	Azevedo, J.L., Maccheroni Jr., W., Pereira, J.O. and Luiz de Araújo, W. (2000) Endophytic Microorganisms: A Review on Insect Control and Recent Advances on Tropical Plants. Electronic Journal of Biotechnology, 3, 40-65. http://dx.doi.org/10.2225/vol3-issue1-fulltext-4
[6]	Hallmann, J., Quadt-Hallmann, A., Mahaffe, W.F. and Kloepper, J.W. (1997) Bacterial Endophytes in Agricultural Crops. Canadian Journal of Microbiology, 43, 895-914. http://dx.doi.org/10.1139/m97-131
[7]	Thomas, P., Swarna, G.K., Patil, P. and Rawal, R.D. (2008) Ubiquitous Presence of Normally Non-Cultivable Culturable Endophytic Bacteria in Field Shoot-Tips of Banana and Their Gradual Activation to Quiescent Cultivable Form in Tissue Cultures. Plant Cell Tissue and Organ Culture, 93, 39-54. http://dx.doi.org/10.1007/s11240-008-9340-x
[8]	Thomas, P. and Soly, T.A. (2009) Endophytic Bacteria Associated with Growing Shoot Tips of Banana (Musa sp.) cv. Grand Naine and the Affinity of Endophytes to the Host. Microbial Ecology, 58, 952-964. http://dx.doi.org/10.1007/s00248-009-9559-z
[9]	Thomas, P., Swarna, G.K., Roy, P.K. and Patil, P. (2008) Identification of Cultivable Culturable and Originally Non-Culturable Endophytic Bacteria Isolated from Shoot Tip Cultures of Banana cv. Grand Naine. Plant Cell Tissue and Organ Culture, 93, 55-63. http://dx.doi.org/10.1007/s11240-008-9341-9
[10]	Thomas, P. and Reddy, M.K. (2013) Microscopic Elucidation of Abundant Endophytic Bacteria Colonizing the Cell Wall—Plasma Membrane Peri-Space in the Shoot-Tip Tissue of Banana. AoB Plants, 5, Article ID: plt011. http://dx.doi.org/10.1093/aobpla/plt011
[11]	Thomas, P. and Sekhar, A.C. (2014) Live Cell Imaging Reveals Extensive Intracellular Cytoplasmic Colonization of Banana by Normally Non-Cultivable Endophytic Bacteria. AoB PLANTS, 6, Article ID: plu002. http://dx.doi.org/10.1093/aobpla/plu002
[12]	Stover, R.H. (1962) Fusarial Wilt (Panama Disease) of Bananas and Other Musa Species. Commonwealth Mycological Institute, Kew, England.
[13]	Ploetz, R.C., Thomas, J.E. and Slabaugh, W.R. (2003) Diseases of Banana and Plantains. In: Ploetz, R.C., Ed., Diseases of Tropical Fruit Crops, CABI Publishing, CABI International, Wallingford, Oxon, 73-134. http://dx.doi.org/10.1079/9780851993904.0073
[14]	Pérez-Vicente, L. and Dita, M.A. (2014) Fusarium Wilt of Banana or Panama Disease by Fusarium oxysporum f. sp. cubense: A Review on History, Symptoms, Biology, Epidemiology and Management. In: Pérez-Vicente, L., Dita, M.A. and Martínez-de la Parte, E., Eds., Technical Manual Prevention and Diagnostic of Fusarium Wilt (Panama Disease) of Banana Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4), FAO, Rome, 5-30.
[15]	Bancroft, J. (1876) Report of the Board Appointed to Enquire into the Cause of Disease Affecting Livestock and Plants. Votes and Proceedings, 3, 1011-1038.
[16]	Prasadji, J.K. and Smith, L. (2007) Fusarium Wilt of Banana: Cultivar Reaction, Race and Vegetative Compatibility Groups of Isolates of Fusarium oxysporum f. sp. cubense. In: Singh, H.P. and Uma, S., Eds., Banana: Technological Advancements, Association for the Improvement in Production and Utilization of Banana (AIPUB), C/o NRC for Banana, Trichy, 405-412.
[17]	Ploetz, R.C. (1990) Variability in Fusarium oxysporum f. sp. cubense. Canadian Journal of Botany, 68, 1357-1363. http://dx.doi.org/10.1139/b90-173
[18]	Sivamani, E. and Gnanamanickam, S.S. (1988) Biological Control of Fusarium oxysporum f. sp. cubense in Banana by Inoculation with Pseudomonas fluorescens. Plant and Soil, 107, 3-9. http://dx.doi.org/10.1007/BF02371537
[19]	Thangavelu, R., Kumar, K.M., Ganga Devi, P. and Mustaffa, M.M. (2011) Genetic Diversity of Fusarium oxysporum f. sp. cubense isolatees (Foc) of India by Inter Simple Sequence Repeats (ISSR) Analysis. Molecular Biotechnology, 51, 203-211. http://dx.doi.org/10.1007/s12033-011-9457-8
[20]	Sun, J.B., Peng, M., Wang, Y.G., Zhao, P.J. and Xia, Q.Y. (2011) Isolation and Characterization of Antagonistic Bacteria against Fusarium Wilt and Induction of Defense Related Enzymes in Banana. African Journal of Microbiology Research, 5, 509-515.
[21]	Ho, Y.N., Chiang, H.M., Chao, C.P., Su, C.C., Hsu, H.F., Guo, C., Hsieh, J.L. and Huang, C.C. (2015) In Planta Biocontrol of Soilborne Fusarium Wilt of Banana through a Plant Endophytic Bacterium, Burkholderia cenocepacia 869T2. Plant and Soil, 387, 295-306. http://dx.doi.org/10.1007/s11104-014-2297-0
[22]	Thomas, P., Sekhar, A.C. and Mujawar, M.M. (2012) Non-Recovery of Varying Proportions of Viable Bacteria during Spread-Plating Governed by the Extent of Spreader Usage and Proposal for an Alternate Spotting-Spreading Approach to Maximize the CFU. Journal of Applied Microbiology, 113, 339-350. http://dx.doi.org/10.1111/j.1365-2672.2012.05327.x
[23]	White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: Innis, M.A., Gelfand, D.H., Shinsky, J.J. and White, T.J., Eds., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., New York, 315-322. http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1
[24]	Martinez, L., Caballero-Mellado, J., Orozco, J. and Martinez-Romero, E. (2003) Diazotrophic Bacteria Associated with Banana (Musa spp.). Plant and Soil, 257, 35-47. http://dx.doi.org/10.1023/A:1026283311770
[25]	Souza, S.A., Xavier, A.A., Costa, M.R., Cardoso, A.M.S., Pereira, M.C.T. and Nietsche, S. (2013) Endophytic Bacterial Diversity in Banana “Prata Anã” (Musa spp.) Roots. Genetics and Molecular Biology, 36, 252-264. http://dx.doi.org/10.1590/S1415-47572013000200016
[26]	Rosenblueth, M. and Martinez-Romero, E. (2006) Bacterial Endophytes and Their Interaction with Hosts. Molecular Plant Microbe Interactions, 19, 827-837. http://dx.doi.org/10.1094/MPMI-19-0827
[27]	Ting, A.S.Y., Sariah, M., Jugah, K., Son, R. and Gurmit, S. (2008) Endophytic Microorganisms as Potential Growth Promoters of Banana. BioControl, 53, 541-553. http://dx.doi.org/10.1007/s10526-007-9093-1
[28]	Ting, A.S.Y., Meon, S., Kadir, J., Radu, S. and Singh, G. (2010) Induction of Host Defence Enzymes by the Endophytic Bacterium Serratia marcescens, in Banana Plantlets. International Journal of Pest Management, 56, 183-188. http://dx.doi.org/10.1080/09670870903324198
[29]	Chernin, L., Ismailov, Z., Haran, S. and Chet, I. (1995) Chitinolytic Enterobacter agglomerans Antagonistic to Fungal Plant Pathogens. Applied and Environmental Microbiology, 61, 1720-1726.
[30]	Crawford, D.L., Lynch, J.M., Whipps, J.M. and Ousley, M.A. (1993) Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen. Applied and Environmental Microbiology, 59, 3899-3905.
[31]	Sun, J.B., Peng, M., Wang, Y.G., Zhao, P.J. and Xia, Q.Y. (2011) Isolation and Characterization of Antagonistic Bacteria against Fusarium Wilt and Induction of Defense Related Enzymes in Banana. African Journal of Microbiology Research, 5, 509-515.
[32]	Ayyadurai, N., Ravindra Naik, P., Sreehari Rao, M., Sunish Kumar, R., Samrat, S.K., Manohar, M. and Sakthivel, N. (2006) Isolation and Characterization of a Novel Banana Rhizosphere Bacterium as Fungal Antagonist and Microbial Adjuvant in Micropropagation of Banana. Journal of Applied Microbiology, 100, 926-937. http://dx.doi.org/10.1111/j.1365-2672.2006.02863.x
[33]	Gupta, G., Panwar, J. and Jha, P.N. (2013) Natural Occurrence of Pseudomonas aeruginosa, a Dominant Cultivable Diazotrophic Endophytic Bacterium Colonizing Pennisetum glaucum (L.) R. Br. Applied Soil Ecology, 64, 252-261. http://dx.doi.org/10.1016/j.apsoil.2012.12.016
[34]	Kumar, A., Munder, A., Aravind, R., Eapen, S.J., Tummler, B. and Raaijmakers, J.M. (2013) Friend or Foe: Genetic and Functional Characterization of Plant Endophytic Pseudomonas aeruginosa. Environmental Microbiology, 15, 764-779. http://dx.doi.org/10.1111/1462-2920.12031
[35]	Pukatzki, S., Kessin, R.H. and Mekalanos, J.J. (2002) The Human Pathogen Pseudomonas aeruginosa Utilizes Conserved Virulence Pathways to Infect the Social Amoeba Dictyostelium discoideum. Proceedings of the National Academy of Sciences, 99, 3159-3164. http://dx.doi.org/10.1073/pnas.052704399