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Endophytic Bacteria Associated with Growing Shoot Tips of Banana (Musa sp.) cv. Grand Naine and the Affinity of Endophytes to the Host

  • Plant Microbe Interactions
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Abstract

A cultivation-based assessment of endophytic bacteria present in deep-seated shoot tips of banana suckers was made with a view to generate information on the associated organisms, potential endophytic contaminants in tissue-cultured bananas and to assess if the endophytes shared a beneficial relationship with the host. Plating the tissue homogenate from the central core of suckers showed colony growth on nutrient agar from just 75% and 42% of the 12 stocks during May and November, respectively (average 58%; 6 × 103 colony-forming units per gram), yielding diverse organisms belonging to firmicutes (Bacillus, Brevibacillus, Paenibacillus, Virgibacillus, Staphylococcus spp.), actinobacteria (Cellulomonas, Micrococcus, Corynebacterium, Kocuria spp.), α-proteobacteria (Paracoccus sp.), and γ-proteobacteria (Pseudomonas, Acinetobacter spp.). Each shoot tip showed one to three different organisms and no specific organism appeared common to different sucker tips. Tissue homogenate from shoot tips including the ones that did not yield culturable bacteria displayed abundant bacterial cells during microscopic examination suggesting that a high proportion of cells were in viable-but-nonculturable state, or their cultivation requirements were not met. Direct application of cultivation-independent approach to study endophytic bacterial community using bacterial 16S ribosomal RNA universal primers resulted in high interference from chloroplast and mitochondrial genome sequences. Dislodging the bacterial cells from shoot tips that did not show cultivable bacteria and incubating the tissue crush in dilute-nutrient broth led to the activation of four organisms (Klebsiella, Agrobacterium, Pseudacidovorax spp., and an unidentified isolate). The endophytic organisms in general showed better growth at 30–37 °C compared with 25 °C, and the growth of endophytes as well as pathogenic Erwinia carotovora were promoted with the supply of host tissue extract (HTE) while that of the isolates from nonplant sources were inhibited or unaffected by HTE, suggesting an affinity or dependence of the endophytes on the host and the prospect of an HTE-based assay for discriminating the nonendophytes from endophytes.

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References

  1. Alexander E, Pham D, Steck TR (1999) The viable-but-nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum. Appl Environ Microbiol 65:3754–3756

    PubMed  CAS  Google Scholar 

  2. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    PubMed  CAS  Google Scholar 

  3. Araújo WI, Marcon J, Maccheroni W Jr, van Elsas JD, van Vuurde JWL, Azevedo JL (2002) Diversity of endophytic bacteria populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68:4906–4914

    Article  PubMed  CAS  Google Scholar 

  4. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (2005) Current protocols in molecular biology, vol 1. Wiley, New York, pp 2.4.1–2.4.2

    Google Scholar 

  5. Azevedo JL, Maccheroni W Jr, Pereira JO, Araújo WL (2000) Endophytic microorganisms: a review on insect control and recent advances on tropical plants. Electronic J Biotech 3:40–65 http://bioline.utsc.utoronto.ca/archive/00000242/

    Google Scholar 

  6. Bacon CW, Glenn AE, Hinton DM (2002) Isolation, in planta detection and culture of endophytic bacteria and fungi. In: Hurst CJ, Crawford RL, McInerney MJ, Knudsen GR, Stetzenbach LD (eds) Manual of environmental microbiology, 2nd edn. ASM, Washington, DC, pp 543–553

    Google Scholar 

  7. Bacon CW, Hinton DM (2006) Bacterial endophytes: the endophytic niche, its occupants and its utility. In: Gnanamanickam SS (ed) Plant associated bacteria. Springer, New Delhi, pp 155–194

    Chapter  Google Scholar 

  8. Barac T, Taghavi S, Borremans B, Provoost A, Oeyen L, Colpaert JV, Vangromsveld J, van der Lelie D (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nat Biotech 22:583–588

    Article  CAS  Google Scholar 

  9. Bell CR, Dickie GA, Harvey WLG, Chan JWYF (1995) Endophytic bacteria in grapevine. Can J Microbiol 41:46–53

    Article  CAS  Google Scholar 

  10. Chelius MK, Triplett EW (2001) The diversity of archaea and bacteria in association with the roots of Zea mays L. Microb Ecol 41:252–263

    PubMed  CAS  Google Scholar 

  11. Compant S, Duffy B, Nowak J, Clément C, Ait Barka E (2005) Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl Environ Microbiol 71:4951–4959

    Article  PubMed  CAS  Google Scholar 

  12. Compant S, Kaplan H, Sessitsch A, Nowak J, Ait Barka E, Clément C (2008) Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues. FEMS Microbiol Ecol 63:84–93

    PubMed  CAS  Google Scholar 

  13. Conn VM, Franco CMM (2004) Analysis of endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA clones. Appl Environ Microbiol 70:1787–1794

    Article  PubMed  CAS  Google Scholar 

  14. Conn VM, Walker AR, Franco CMM (2008) Endophytic actinobacteria induce defense pathways in Arabidopsis thaliana. Mol Plant Microbe Interact 21:208–218

    Article  PubMed  CAS  Google Scholar 

  15. Dalal A, Urban C, Segal-Maurer S (2008) Endocarditis due to Corynebacterium amycolatum. J Med Microbiol 57:1299–1302

    Article  PubMed  Google Scholar 

  16. Doty SL (2008) Enhancing phytoremediation through the use of transgenics and endophytes. New Phytol 179:318–333

    Article  PubMed  CAS  Google Scholar 

  17. Garbeva P, van Overbeek LS, van Vuurde JWL, van Elsas JD (2001) Analysis of the endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments. Microb Ecol 41:369–383

    PubMed  CAS  Google Scholar 

  18. Ghezzi JI, Steck TR (1999) Induction of the viable but non-culturable condition in Xanthomonas campestris pv. campestris in liquid microcosms and sterile soil. FEMS Microbiol Ecol 30:203–208

    Article  PubMed  CAS  Google Scholar 

  19. Govindarajan M, Balandreau J, Kwon S-W, Weon H-Y, Lakshminarasimhan C (2008) Effects of the inoculation of Burkholderia vietnamiensis and related endophytic diazotrophic bacteria on grain yield of rice. Microb Ecol 55:21–37

    Article  PubMed  Google Scholar 

  20. Grey BE, Steck TR (2001) The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection. Appl Environ Microbiol 67:3866–3872

    Article  PubMed  CAS  Google Scholar 

  21. Guo S, Wang Y, Sun X, Tang K (2008) Bioactive natural products from endophytes: a review. Prikl Biokhim Mikrobiol 44:153–158

    PubMed  CAS  Google Scholar 

  22. Habiba U, Reza S, Saha ML, Khan MR, Hadiuzzaman S (2002) Endogenous bacterial contamination during in vitro culture of banana: identification and prevention. Plant Tiss Cult 12:117–124

    Google Scholar 

  23. Hallmann J (2001) Plant interactions with endophytic bacteria. In: Jeger MJ, Spence NJ (eds) Biotic interactions in plant–pathogen associations. CABI, Wallingford, pp 87–119

    Google Scholar 

  24. Hallmann J, Quadt-Hallmann A, Mahaffe WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914

    CAS  Google Scholar 

  25. Horry JP, Sharrock S, Frison E (2000) Banana research and development: an international perspective. In: Singh HP, Chadha KL (eds) Banana improvement, production and utilization. Association for the Improvement in Production and Utilization of Banana, Trichy, pp 1–5

    Google Scholar 

  26. Izumi H, Anderson IC, Kilham K, Moore ER (2008) Diversity of predominant bacteria in European deciduous and coniferous trees. Can J Microbiol 54:173–179

    Article  PubMed  CAS  Google Scholar 

  27. James EK, Olivares FL (1998) Infection and colonization of sugarcane and other graminaceous plants by endophytic diazotrophs. Crit Rev Plant Sci 17:77–199

    Article  Google Scholar 

  28. Kukkurainen S, Leino A, Vähämiko S, Kärkkäinen HR, Ahanen K, Sorvari S, Rugienius R, Toldi O (2005) Occurrence and location of endophytic bacteria in garden and wild strawberry. HortSci 40:348–352

    Google Scholar 

  29. Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol Plant 37:133–138

    Article  Google Scholar 

  30. Li J, Zhao GZ, Chen HH, Wang HB, Qin S, Zhu WY, Xu LH, Jiang CL, Li WJ (2008) Antitumour and antimicrobial activities of endophytic streptomycetes from pharmaceutical plants in rain forest. Lett Appl Microbiol 47:574–580

    Article  PubMed  CAS  Google Scholar 

  31. Lian J, Wang Z, Zhou S (2008) Response of endophytic bacterial communities in banana tissue culture plantlets to Fusarium wilt pathogen infection. J Gen Appl Microbiol 54:83–92

    Article  PubMed  CAS  Google Scholar 

  32. Mano H, Morisaki H (2008) Endophytic bacteria in the rice plant. Microbes Environ 23:109–117

    Article  Google Scholar 

  33. Martinez L, Caballero-Mellado J, Orozco J, Martinez-Romero E (2003) Diazotrophic bacteria associated with banana (Musa spp.). Plant Soil 257:35–47

    Article  CAS  Google Scholar 

  34. McInroy JA, Kloepper JW (1995) Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil 173:337–342

    Article  CAS  Google Scholar 

  35. Oliveira RP, Silveira DS, Silva SO (2001) Concentraçãa de BAP ea eficieêcia de micropropagação de bananeira tetraploide (Gropo AAAB). Sci Agricola 58:73–78

    Google Scholar 

  36. Ordax M, Marco-Noales E, López MM, Biosca EG (2006) Survival strategy of Erwinia amylovora against copper: induction of the viable-but-nonculturable state. Appl Environ Microbiol 72:3482–3488

    Article  PubMed  CAS  Google Scholar 

  37. Oszakir O (2007) Viable but non-culturable form of bacteria. Mikrobiyol Bul 41:477–484

    Google Scholar 

  38. Panicker B, Thomas P, Janakiram T, Venugopalan R, Narayanappa SB (2007) Influence of cytokinin levels on in vitro propagation of shy suckering chrysanthemum “Arka Swarna” and activation of endophytic bacteria. In Vitro Cell Dev Biol Plant 43:614–622

    Article  CAS  Google Scholar 

  39. Reiter B, Pfeifer U, Schwab H, Sessitsch A (2002) Response of endophytic bacterial communities in potato plants to infection with Erwinia carotovora subsp. atroseptica. Appl Environ Microbiol 68:2261–2268

    Article  PubMed  CAS  Google Scholar 

  40. Reiter B, Sessitsch A (2006) Bacterial endophytes of the wildflower Crocus albiflorus analyzed by characterization of isolates and by a cultivation-independent approach. Can J Microbiol 52:140–149

    Article  PubMed  CAS  Google Scholar 

  41. Roels S, Escalona M, Cejas I, Noceda C, Rodriguez R, Canal MJ, Sandoval J, Debergh P (2005) Optimization of plantain (Musa AAB) micropropagation by temporary immersion system. Plant Cell Tiss Org Cult 82:57–66

    Article  CAS  Google Scholar 

  42. Rosenblueth M, Martínez L, Silva J, Martínez-Romero E (2004) Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 27:27–35

    Article  PubMed  CAS  Google Scholar 

  43. Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interaction with hosts. Mol Plant Microbe Interact 8:827–837

    Article  CAS  Google Scholar 

  44. Roszak DB, Colwell RR (1987) Survival strategies of bacteria in the natural environment. Microbiol Rev 51:365–379

    PubMed  CAS  Google Scholar 

  45. Ryan R, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent development and applications. FEMS Microbiol Lett 278:1–9

    Article  PubMed  CAS  Google Scholar 

  46. Sessitsch A, Reiter B, Pfeifer U, Wilhelm E (2002) Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. FEMS Microbiol Ecol 39:23–32

    Article  PubMed  CAS  Google Scholar 

  47. Strosse H, Van den Houwe I, Panis B (2004) Banana cell and tissue culture—a review. In: Jain SM, Swennen R (eds) Banana improvement: cellular, molecular biology, and induced mutations. Science Publisher, New Hampshire (http://www.fao.org/docrep/007/ae216e/ae216e00.html)

    Google Scholar 

  48. Sturz Av, Christie BR, Matheson BG (1998) Association of bacterial endophyte populations from red clover and potato crops with potential for beneficial allelopathy. Can J Microbiol 44:162–167

    Article  CAS  Google Scholar 

  49. Sun L, Qiu F, Zhang X, Dai X, Dong X, Song W (2007) Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis. Microb Ecol 55:415–424

    Article  PubMed  CAS  Google Scholar 

  50. Sziderics AH, Rasche F, Trognitz F, Sessitsch A, Wilhelm E (2007) Bacterial endophytes contribute to abiotic stress adaptation in pepper plants (Capsicum annuum L.). Can J Microbiol 53:1195–1202

    Article  PubMed  CAS  Google Scholar 

  51. Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangronsveld J, van der Lelie D (2009) Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microbiol 75:748–757

    Article  PubMed  CAS  Google Scholar 

  52. Thomas P (2004) In vitro decline in plant cultures: detection of a legion of covert bacteria as the cause for degeneration of long-term micropropagated triploid watermelon cultures. Plant Cell Tiss Org Cult 77:173–179

    Article  Google Scholar 

  53. Thomas P (2004) A three-step screening procedure for detection of covert and endophytic bacteria in plant tissue cultures. Curr Sci 87:67–72

    CAS  Google Scholar 

  54. Thomas P (2006) Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp as a covert contaminant in grape tissue cultures. J Appl Microbiol 101:764–774

    Article  PubMed  CAS  Google Scholar 

  55. Thomas P, Kumari S, Swarna GK, Gowda TKS (2007) Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host–endophyte interaction in vitro and in vivo. Can J Microbiol 53:380–390

    Article  PubMed  CAS  Google Scholar 

  56. Thomas P, Kumari S, Swarna GK, Prakash DP, Dinesh MR (2007) Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya. Plant Cell Rep 26:1491–1499

    Article  PubMed  CAS  Google Scholar 

  57. Thomas P, Swarna GK, Patil P (2008) Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures. Plant Cell Tiss Org Cult 93:39–54

    Article  Google Scholar 

  58. Thomas P, Swarna GK, Roy PK, Prakash P (2008) Identification of culturable and originally non-culturable endophytic bacteria isolated from shoot tip cultures of banana cv. Grand Naine. Plant Cell Tiss Org Cult 93:55–63

    Article  Google Scholar 

  59. Tian X, Cao L, Tan H, Han W, Chen M, Liu Y, Zhou S (2007) Diversity of cultivated and uncultivated actinobacterial endophytes in the stems and roots of rice. Microb Ecol 53:700–707

    Article  PubMed  Google Scholar 

  60. Tyler HL, Triplett EW (2008) Plants as a habitat for beneficial and/or human pathogenic bacteria. Ann Rev Phytopathol 46:53–73

    Article  CAS  Google Scholar 

  61. Ulrich K, Ulrich V, Ewald D (2008) Diversity of endophytic bacterial communities in poplar grown under field conditions. FEMS Microbiol Ecol 63:169–180

    Article  PubMed  CAS  Google Scholar 

  62. Veach LA, Pfaller MA, Barrett M, Koontz FP, Wenzel RP (1990) Vancomycin resistance in Staphylococcus haemolyticus causing colonization and bloodstream infection. J Clin Microbiol 28:2064–2068

    PubMed  CAS  Google Scholar 

  63. Vuong C, Otto M (2002) Staphylococcus epidermidis infections. Microbes Infections 4:481–489

    Article  Google Scholar 

  64. Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This study was supported by a grant from the Department of Biotechnology, Govt. of India, New Delhi, India under the project “Identification of covert endophytic microbes in plant tissue cultures and their management and control.” The supply of suckers of different banana cultivars by Dr. L. B. Naik/Dr. Saxena, laboratory assistance by G. K. Swarna, N. Shivrudriah, and B. Hanumanthraju (IIHR, Bangalore, India) are acknowledged. IIHR Contr. No. 55/2008

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  1. Division of Biotechnology, Indian Institute of Horticultural Research, Hessarghatta Lake, Bangalore, 560089, India

    Pious Thomas & Thyvalappil A. Soly

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Correspondence to Pious Thomas.

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Supply of live bacterial cultures or genetic material for research purpose is subject to their revival from glycerol stocks (as some of the organisms showed poor tolerance) and the requestor obtaining permission from the Indian Council for Agricultural Research (ICAR), New Delhi, 110001, India. All the cultures described in this study are in the process of deposition at the microbial culture collection, National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau Nath Bhanjan, Uttar Pradesh 275 101, India (www.nbaim.org).

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Supplementary Table 1

Growth of selected bacterial isolates from different phylogenetic groups in dilute-nutrient broth or broth supplemented with filter-sterilized host tissue extract (HTE) (RTF 42 kb)

Supplementary Fig. 1

Phylogenic tree based on 16S rRNA gene sequence of different endophytic bacterial isolates retrieved from banana shoot tips. Phylogenic relationships were estimated according to Jukes–Cantor model and the tree was constructed by the neighbor-joining method with 100–1,000 bootstrap resamplings. Genetic distances are indicated along the branch (PDF 150 kb)

Supplementary Fig. 2

Shoot-tip homogenate of banana under phase contrast (1000×) displaying plastids (P) and mitochondria (M) together with bacterial cells in the background (a), and filtered tissue homogenate (2.5 µm) displaying abundant bacterial cells after the sedimentation of larger particles (b) (GIF 132 kb)

High Resolution Image (TIFF 3.73 mb)

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Thomas, P., Soly, T.A. Endophytic Bacteria Associated with Growing Shoot Tips of Banana (Musa sp.) cv. Grand Naine and the Affinity of Endophytes to the Host. Microb Ecol 58, 952–964 (2009). https://doi.org/10.1007/s00248-009-9559-z

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