Abstract
Ex situ conservation of vegetatively propagated crops by tissue culture is sometimes hampered by covert endophytic bacteria which become visible after repeated subculture over prolonged periods. In the present study, we identified bacterial contaminants and devised a strategy for their elimination from 20 in vitro conserved accessions of taro [Colocasia esculenta (L.) Schott] held in the In Vitro Gene Bank (IVGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR). Visually, the cultures were exhibiting white to dark brown exudation in tissue culture growth medium and undergoing slow degeneration as recorded by wilting and necrosis of leaves and shoots. On culturing the macerated pieces of shoots, corms, and leaf petioles from the contaminated taro cultures on bacterial indexing medium, six distinct bacterial colonies were observed. The identity of these bacteria was established through 16S rDNA sequencing as gram-negative strains (2) of Ralstonia spp. and gram-positive strains (4) of Paenibacillus spp. Thereafter, two strategies were adopted for elimination of contaminants from the cultures: (i) use of antibiotic supplemented media and (ii) hardening of micro-corms in field and re-establishment under in vitro conditions. Antibiotic supplemented media was not effective in eliminating contamination as reappearance of bacterial colonies was observed after subculturing in antibiotic-free growth medium. In contrast, re-establishment in vitro from hardened infected plants resulted in an average of 6.03% bacteria-free cultures in 14 accessions. The findings indicate that endophytic bacteria associated with the host (taro) plants under tissue culture conditions may efficiently be mitigated through periodic transfer of the tissue culture derived corms to ex vitro conditions and re-introduction of the bacteria-free shoot tip explants in vitro.
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References
Abd Alamer I, Tomah A, Li B, Zhang J (2020) Isolation, identification and characterization of rhizobacteria strains for biological control of bacterial wilt (Ralstonia solanacearum) of eggplant in China. Agriculture 10. https://doi.org/10.3390/agriculture10020037
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. https://doi.org/10.1128/aem.65.8.3754-3756.1999
Beatty PH, Jensen SE (2002) Paenibacillus polymyxa produces fusaricidin-type antifungal antibiotics active against Leptosphaeria maculans, the causative agent of blackleg disease of canola. Can J Microbiol 48:159–169. https://doi.org/10.1139/w02-002
Bent E, Chanway CP (2002) Potential for misidentification of a spore-forming Paenibacillus polymyxa isolate as an endophyte by using culture-based methods. Appl Environ Microbiol 68:4650–4652. https://doi.org/10.1128/AEM.68.9.4650-4652.2002
Bottini R, Cassán F, Piccoli P (2004) Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase. Appl Microbiol Biotechnol 65:497–503. https://doi.org/10.1007/s00253-004-1696-1
Boucher C, Genin S, Arlat M (2001) Current concepts of pathogenicity in plant pathogenic bacteria. Comptes Rendus l’Académie des Sci - Ser III - Sci la Vie 324:915–922. https://doi.org/10.1016/s0764-4469(01)01375-0
Brunner I, Echegaray A, Rubluo A (1995) Isolation and characterization of bacterial contaminants from Dieffenbachia amoena Bull, Anthurium andreanum Linden and Spathiphyllum sp. Schoot cultured in vitro. Sci Hortic (Amsterdam) 62:103–111. https://doi.org/10.1016/0304-4238(94)00743-Y
Buddenhagen I, Kelman A (1964) Biological and physiological aspects of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 2:203–230
Castellano-Hinojosa A, Pérez-Tapia V, Bedmar E, Santillana N (2018) Purple corn-associated rhizobacteria with potential for plant growth promotion. J Appl Microbiol 124:1254–1264
Compant S, Duffy B, Nowak J, Clément C, Barka EA (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. https://doi.org/10.1128/AEM.71.9.4951
Denny T (2006) Plant pathogenic Ralstonia species. In: Gnanamanickam S (ed) Plant-associated bacteria. Springer Publishing, Dordrecht, pp 573–644
FAO (2014) www.fao.org 2014 as accessed on Feb. 2014
Fegan M, Prior P (2005) How complex is the “Ralstonia solanacearum species complex”. In: Allen C, Prior P, Hayward A (eds) Bacterial wilt disease and the Ralstonia solanacearum species complex. APS Press, St. Paul, p 449
Fullagar R, Field J, Denham T, Lentfer C (2006) Early and mid Holocene tool-use and processing of taro (Colocasia esculenta), yam (Dioscorea sp.) and other plants at Kuk Swamp in the highlands of Papua New Guinea. J Archaeol Sci 33:595–614
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. https://doi.org/10.1016/S0168-6496(99)00056-2
Grady EN, MacDonald J, Liu L, Richman A, Yuan ZC (2016) Current knowledge and perspectives of Paenibacillus: a review. Microb Cell Fact 15:203. https://doi.org/10.1186/s12934-016-0603-7
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. https://doi.org/10.1128/AEM.67.9.3866
Hallman J (2001) Plant interactions with endophytic bacteria. In: Jeger MJ, Spence NJ (eds) Biotic interactions in plant-pathogen associations. CABI Publishing, Wallingford, pp 87–119
Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914
Hayward A (1991) Bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 29:65–87. https://doi.org/10.1146/annurev.py.29.090191.000433
Holland MA, Polacco JC (1994) PPFMs and other covert contaminants: is there more to plant physiology than just plant ? Annu Rev Plant Physiol Plant Mol Biol 45:197–209
Kelman A (1953) The bacterial wilt caused by Pseudomonas solanacearum. In: A literature review and bibliography. Raleigh: North Carolina State College
Kelman A (1954) The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693–695
Kulkarni AA, Kelkar SM, Watve MG, Krishnamurthy KV (2007) Characterization and control of endophytic bacterial contaminants in in vitro cultures of Piper spp., Taxus baccata subsp. wallichiana, and Withania somnifera. Can J Microbiol 53:63–74. https://doi.org/10.1139/W06-106
Lebot V, Tuia V, Ivancic A, Jackson GV, Saborio F, Reyes G, Rodriguez S, Robin G, Traore R, Aboagye L, Onyeka J (2017) Adapting clonally propagated crops to climatic changes: a global approach for taro (Colocasia esculenta (L.) Schott). Genet Resour Crop Evol 65:591–606. https://doi.org/10.1007/s10722-017-0557-6
Leifert C, Cassells A (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol - Plant 37:133-138
Leifert C, Ritchie JY, Waites WM (1991) Contaminants of plant-tissue and cell cultures. World J Microbiol Biotechnol 7:452–469. https://doi.org/10.1007/BF00303371
Leifert C, Woodward S (1998) Laboratory contamination management: the requirement for microbiological quality assurance. Plant Cell Tiss Org Cult 52:83–88
Li B, Yu R, Tang Q, Su T, Chen X, Zhu B, Wang Y, Xie G, Sun G (2011) Biofilm formation ability of Paenibacillus polymyxa and Paenibacillus macerans and their inhibitory effect against tomato bacterial wilt. African J Microbiol Res 5:4260–4266. https://doi.org/10.5897/ajmr10.549
Mano H, Morisaki H (2008) Endophytic bacteria in the rice plant. Microbes Environ 23:109–117. https://doi.org/10.1264/jsme2.23.109
Matthews PJ (2004) Genetic diversity in taro, and the preservation of culinary knowledge. Ethnobot Res Appl 2:55–71
Matthews PJ, Lockhart PJ, Ahmed I (2017) Phylogeography, ethnobotany, and linguistics: Issues arising from research on the natural and cultural history of taro Colocasia esculenta (L.) Schott. Man in India 97:353–380
Misra P, Gupta N, Toppo DD, Pandey V, Mishra MK, Tuli R (2010) Establishment of long-term proliferating shoot cultures of elite Jatropha curcas L. by controlling endophytic bacterial contamination. Plant Cell Tiss Org Cult 100:189–197. https://doi.org/10.1007/s11240-009-9636-5
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
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. https://doi.org/10.1128/AEM.72.5.3482-3488.2006
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. https://doi.org/10.1007/s11627-007-9061-6
Poussier S, Thoquet P, Trigalet-Demery D, Barthet S, Meyer D, Arlat M, Trigalet A (2003) Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene. Mol Microbiol 49:991–1003. https://doi.org/10.1046/j.1365-2958.2003.03605.x
Rajamanickam S, Karthikeyan G, Kavino M, Manoranjitham SK (2018) Biohardening of micropropagated banana using endophytic bacteria to induce plant growth promotion and restrain rhizome rot disease caused by Pectobacterium carotovorum subsp. carotovorum. Sci Hortic (Amsterdam) 231:179–187. https://doi.org/10.1016/j.scienta.2017.12.037
Reed BM, Buckley PM, Dewilde TN (1995) Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cell Dev Biol Plant 31:53–57
Reed BM, Tanprasert P (1995) Detection and control of bacterial contaminants of plant tissue cultures. A review of recent literature. Plant Tiss Cult Biotechnol 1:137–142
Roszak DB, Colwell RR (1987) Survival strategies of bacteria in the natural environment. Syst Res 4:111–117. https://doi.org/10.1002/sres.3850040205
Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett 278:1–9. https://doi.org/10.1111/j.1574-6968.2007.00918.x
Sakiyama CCH, Paula EM, Pereira PC, Borges AC, Silva DO (2001) Characterization of pectin lyase produced by an endophytic strain isolated from coffee cherries. Lett Appl Microbiol 33:117–121. https://doi.org/10.1046/j.1472-765X.2001.00961.x
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Schell MA (2000) Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory network. Annu Rev Phytopathol 38:263–292
Smith EF (1896) A bacterial disease of the tomato, eggplant and Irish potato (Bacillus solanacearum n. sp.). In: 12th. Washington GPO, Bulletin (United States, Division of Vegetable Physiology and Pathology, pp 1–28
Smith EF (1920) The brown rot of Solanaceae. In: Bacterial diseases of plants. Saunders Company, USA, pp 177–201
Tanprasert P, Reed BM (1997) Determination of minimal bactericidal and effective antibiotic treatment concentrations for bacterial contaminants from micropropagated strawberries. In Vitro Cell Dev Biol - Plant 33:227–230. https://doi.org/10.1007/s11627-997-0027-5
Teng WL, Nicholson L (1997) Pulse treatments of penicillin-G and streptomycin minimise internal infections and have post-treatment effects on the morphogenesis of ginseng root culture. Plant Cell Rep 16:531–535. https://doi.org/10.1007/s002990050273
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 Tissue Organ Cult 77:173–179. https://doi.org/10.1023/B:TICU.0000016824.09108.c8
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. https://doi.org/10.1111/j.1365-2672.2006.02993.x
Thomas P (2007) Isolation and identification of five alcohol defying Bacillus spp. covertly associated with in vitro culture of seedless watermelon. Curr Sci 92:983–987
Thomas P (2011) Intense association of non-culturable endophytic bacteria with antibiotic-cleansed in vitro watermelon and their activation in degenerating cultures. Plant Cell Rep 30:2313–2325. https://doi.org/10.1007/s00299-011-1158-z
Thomas P, Kumari S (2010) Inconspicuous endophytic bacteria mimicking latex exudates in shoot-tip cultures of papaya. Sci Hortic (Amsterdam) 124:469–474. https://doi.org/10.1016/j.scienta.2010.02.013
Thomas P, Prakash GS (2004) Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 years in vitro. In Vitro Cell Dev Biol - Plant 40:603–607. https://doi.org/10.1079/IVP2004583
Thomas P, Swarna GK, Roy PK, Patil 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. https://doi.org/10.1007/s11240-008-9341-9
Timmusk S, Nicander B, Granhall U, Tillberg E (1999) Cytokinin production by Paenibacillus polymyxa. Soil Biol Biochem 31:1847–1852. https://doi.org/10.1016/S0038-0717(99)00113-3
Ulrich K, Stauber T, Ewald D (2008) Paenibacillus—a predominant endophytic bacterium colonising tissue cultures of woody plants. Plant Cell Tiss Org Cult 93:347–351. https://doi.org/10.1007/s11240-008-9367-z
Van den Houwe I, Gun J, Swennen R (1998) Bacterial contamination in Musa shoot tip cultures. Acta Hortic (490):485–492
Van Elsas JD, Kastelein P, De Vries PM, Van Overbeek LS (2001) Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water. Can J Microbiol 47:842–854. https://doi.org/10.1139/cjm-47-9-842
Viss PR, Brooks EM, Driver JA (1991) A simplified method for the control of bacterial contamination in woody plant tissue culture. In Vitro Cell Dev Biol - Plant 27:42–42. https://doi.org/10.1007/BF02632060
Wilson K (1987) Preparation of genomic DNA from bacteria. In: Current protocols in molecular biology. Wiley, New York, p 2.4.1–2.4.5
Acknowledgments
The authors gratefully acknowledge The Director, ICAR-NBPGR, New Delhi, for the facilities, Dr. P. Thomas (CEO & Director, Thomas Biotech & Cytobacts Centre for Biosciences, Bengaluru) for sharing his experience and publications, Dr. J.K. Ranjan (ICAR-IARI, New Delhi) for conducting statistical analysis and Dr. Pragya (ICAR-NBPGR, New Delhi) for providing valuable inputs in preparation of the manuscript.
Funding
This work was supported by the project “In vitro conservation of tuber crops with special reference to sweet potato, yams and taro” (project code: PGR/TCCU-BUR-DEL-01.01) funded by the Indian Council of Agricultural Research - National Bureau of Plant Genetic Resources (ICAR-NBPGR).
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Srivastava, V., Nerwal, D.K., Kandan, A. et al. Management of microbial contaminants in the In Vitro Gene Bank: a case study of taro [Colocasia esculenta (L.) Schott]. In Vitro Cell.Dev.Biol.-Plant 57, 152–163 (2021). https://doi.org/10.1007/s11627-020-10125-5
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DOI: https://doi.org/10.1007/s11627-020-10125-5