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Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures

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Abstract

Endophytic bacterial contamination is a major constraint to the establishment and maintenance of aseptic Aglaonema cultures. The objectives of the present study included the identification of endophytic bacterial contaminants from micropropagated Aglaonema cultures and the investigation of effective antibiotic treatment for their control. Bacterial contaminants isolated from 181 infected stem nodal explants of six Aglaonema cultivars were identified following the amplification of 16S ribosomal RNA gene and partial sequence analysis. A total of thirteen different bacterial species were identified and these were found to be mostly associated with soil and water. The bacteria were subsequently subjected to antimicrobial susceptibility and minimum inhibitory concentration tests. Three antibiotics, including gentamicin, tetracycline and chloramphenicol, were selected for their effectiveness at low concentrations of 4–32 mg l−1 to inhibit bacterial growth in most of the bacterial species found in the present study. The incorporation of these antibiotics into the culture medium was found to effectively reduce the incidence of bacterial contamination in three of the four Aglaonema cultivars tested. Therefore, sanitation of the irrigation water and growth substrate while raising the stock plants, as well as the appropriate use of antibiotics during the in vitro culture stage will be important factors governing the success of Aglaonema micropropagation in the future.

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References

  • Attree SM, Sheffield E (1986) An evaluation of ficoll density gradient centrifugation as a method for eliminating microbial contamination and purifying plant protoplasts. Plant Cell Rep 5:288–291

    Article  Google Scholar 

  • 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 Press, Washington, pp 542–553

    Google Scholar 

  • Bauer RW, Kirby MDK, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by standard single disc diffusion method. Am J Clin Pathol 45:493–496

    PubMed  CAS  Google Scholar 

  • Bosshard PP, Abels S, Zbinden R, Bottger EC, Altwegg M (2003) Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-Month Evaluation). J Clin Microbiol 41:4134–4140

    Article  PubMed  CAS  Google Scholar 

  • Brunner I, Echegaray A, Rubluo A (1995) Isolation and characterization of bacterial contaminants from Dieffenbachia amoena Bull, Anthurium andreanum Linden and Spathiphyllum sp. shoot cultured in vitro. Sci Hortic 62:103–111

    Article  Google Scholar 

  • Buckley PM, DeWilde TN, Reed BM (1995) Characterization and identification of bacteria isolated from micropropagated mint plants. In Vitro Cell Dev Biol-Plant 31P:58–64

    Google Scholar 

  • Cerritos R, Vinuesa P, Eguiarte LE, Herrera-Estrella L, Alcaraz-Peraza LD, Arvizu-Gómez JL, Olmedo G, Ramirez E, Siefert JL, Souza V (2008) Bacillus coahuilensis sp. nov., a moderately halophilic species from a desiccaion lagoon in the Cuatro Ciénegas Valley in Coahuila, Mexico. Int J Syst Evol Microbiol 58:919–923

    Article  PubMed  CAS  Google Scholar 

  • Chen WL, Yeh DM (2007) Elimination of in vitro contamination, shoot multiplication, and ex vitro rooting of Aglaonema. HortScience 42:629–632

    CAS  Google Scholar 

  • Chen J, Henny RJ, McConnell DB (2002) Development of new foliage plant cultivars. In: Janick J, Whipkey A (eds) Trends in new crops and new uses. Timber Press, Portland, pp 466–472

    Google Scholar 

  • Clinical and Laboratory Standards Institute (2007) Disk diffusion supplemental tables. Document M100-S17. CLSI, Wayne

  • Dias SCF, Costa FEC, Andreote FD, Lacava PT, Teixeira MA, Assumpçao LC, Araújo WL, Azevedo JL, Melo IS (2009) Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion. World J Microbiol Biotechnol 25:189–195

    Article  CAS  Google Scholar 

  • Duncan DB (1955) Multiple range and multiple F-tests. Biometrics 11:1–42

    Google Scholar 

  • Falkiner FR (1988) Strategy for the selection of antibiotics for use against common bacterial pathogens and endophytes of plants. Acta Hortic 225:53–56

    Google Scholar 

  • Fisse L, Batalle A, Pera J (1987) Endogenous bacteria elimination in ornamental plants. Acta Hortic 212:87–90

    Google Scholar 

  • Grisham MP, Pan Y-B, Richard EP Jr (2007) Early detection of Leifsonia xyli subsp. xyli in sugarcane leaves by real-time polymerase chain reaction. Plant Dis 91:430–434

    Article  CAS  Google Scholar 

  • Gunson HE, Spencer-Phillips PTN (1994) Latent bacterial infections: epiphytes and endophytes as contaminants of micropropagated plants. In: Nicholas JR (ed) Physiology, growth and development of plants in culture. Kluwer Academic Publishers, Dordrecht, pp 379–396

    Chapter  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Herman EB (2004) Recent advances in plant tissue culture VIII. Microbial contaminants in plant tissue cultures: solutions and opportunities 1996–2003. Agritech Consultants Inc, Shrub Oak

    Google Scholar 

  • Hirano SS, Upper CD (1990) Population biology and epidemiology of Pseudomonas syringae. Annu Rev Phytopathol 28:155–177

    Article  Google Scholar 

  • Horsch RB, King J (1983) A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium spp. from cultures of Datura innoxia (Mill.). Plant Cell Tissue Organ Cult 2:21–28

    Article  Google Scholar 

  • Hung PQ, Kumar SM, Govindsamy V, Annapurna K (2007) Isolation and characterization of endophytic bacteria from wild and cultivated soybean varieties. Biol Fertil Soils 44:155–162

    Article  Google Scholar 

  • Kneifel W, Leonhardt W (1992) Testing of different antibiotics against gram positive and gram negative bacteria isolated from plant tissue cultures. Plant Cell Tissue Organ Cult 29:139–144

    Article  CAS  Google Scholar 

  • Kritzinger EM, Vuuren RJV, Woodward B, Rong IH, Spreeth MH, Slabbert MM (1998) Elimination of external and internal contaminants in rhizomes of Zantedeschia aethiopica with commercial fungicides and antibiotics. Plant Cell Tissue Organ Cult 52:61–65

    Article  CAS  Google Scholar 

  • Kunisaki JT (1980) In vitro propagation of Anthurium andreanum Lind. HortScience 15:508–509

    Google Scholar 

  • Lata H, Li XC, Silva B, Moraes RM, Halda-Alija L (2006) Identification of IAA-producing endophytic bacteria from micropropagated Echinaceae plants using 16S rRNA sequencing. Plant Cell Tissue Organ Cult 85:353–359

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Leifert C, Waites WM (1992) Bacterial growth in plant tissue culture media. J Appl Bacteriol 72:460–466

    Article  Google Scholar 

  • Leifert C, Ritchie JY, Waites WM (1991) Contaminants of plant tissue and cell cultures. World J Microbiol Biotechnol 7:452–469

    Article  Google Scholar 

  • Leifert C, Camotta H, Waites WM (1992) Effect of combinations of antibiotics on micropropagated Clematis, Delphinium, Hosta, Iris and Photinia. Plant Cell Tissue Organ Cult 29:153–160

    Article  CAS  Google Scholar 

  • Leifert C, Morris CE, Waites WM (1994) Ecology of microbial saprophytes and pathogens in tissue culture and field-grown plants: reasons for contamination problems in vitro. Crit Rev Plant Sci 13:139–183

    Google Scholar 

  • Liu T-H, Hsu N-W, Wu R-Y (2005) Control of leaf-tip necrosis of micropropagated ornamental statice by elimination of endophytic bacteria. In Vitro Cell Develop Biol Plant 41:546–549

    Article  Google Scholar 

  • Lodewyckx C, Vangronsveld J, Porteous Moore F, Moore ERB, Taghavi S, van der Lelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606

    Article  Google Scholar 

  • 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 Tissue Organ Cult 100:189–197

    Article  Google Scholar 

  • Miyazaki J, Tan BH, Errington SG (2010) Eradication of endophytic bacteria via treatment for axillary buds of Petunia hybrida using Plant Preservative Mixture (PPM). Plant Cell Tissue Organ Cult 102:365–372

    Article  CAS  Google Scholar 

  • Mizra MS, Ahmad W, Latif F, Haurat J, Bally R, Normand P, Malik KA (2001) Isolation, partial characterization, and the effect of plant growth-promoting bacteria (PGPB) on micropropagated sugarcane in vitro. Plant Soil 237:47–54

    Article  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • NCCLS (1997) Performance Standards for Antimicrobial disk susceptibility tests: approved standard M2–A7. National Committee for Clinical Laboratory Standards, Wayne

    Google Scholar 

  • Nowak J, Asiedu SK, Bensalim S, Richards J, Stewart A, Smith C, Stevens D, Sturz AV (1998) From laboratory to applications: challenges and progress with in vitro dual cultures of potato and beneficial bacteria. Plant Cell Tissue Organ Cult 52:97–103

    Article  Google Scholar 

  • Nuebel U, Engelen B, Felske A, Snaidr J, Weishuber A, Amann RI (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643

    CAS  Google Scholar 

  • Reed BM, Buckley PM, DeWilde TN (1995) Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cell Develop Biol Plant 31:53–57

    Article  Google Scholar 

  • SAS Institute Inc. (1999) Version 8.02. SAS Institute, Cary

  • Seabrook JM, Farrell G (1993) City water can contaminate tissue culture stock plants. HortScience 28:628–629

    Google Scholar 

  • Shehata AM, Wannarat W, Skirvin RM, Norton MA (2010) The dual role of carbenicillin in shoot regeneration and somatic embryogenesis of horseradish (Armoracia rusticana) in vitro. Plant Cell Tissue Organ Cult 102:397–402

    Article  CAS  Google Scholar 

  • Shen H, Li Z, Han D, Yang F, Huang Q, Ran L (2010) Detection of indigenous endophytic bacteria in Eucalyptus urophylla in vitro conditions. Front Agric China 4:37–41

    Article  Google Scholar 

  • Sun L, Qiu F, Zhang X, Dai X, Dong X, Song W (2008) Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis. Microbiol Ecol 55:415–424

    Article  CAS  Google Scholar 

  • Thomas P (2004) In vitro decline in plant cultures: detection of a legion of covert bacteria as the cause for degenetation of long-term micropropagated troploid watermelon cultures. Plant Cell Tissue Organ Cult 77:173–179

    Article  Google Scholar 

  • 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 Develop Biol Plant 40:603–607

    Article  Google Scholar 

  • 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 

  • Thomas P, Swarna GK, Patil P, Rawal RD (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 Tissue Organ Cult 93:39–54

    Article  Google Scholar 

  • Thomas P, Goplakrishnan C, Krishnareddy M (2011) Soft rot inciting Pectobacterium carotovorum (syn. Erwinia carotovora) is unlikely to be transmitted as a latent pathogen in micropropagated banana. Plant Cell Tissue Organ Cult 105:423–429

    Article  Google Scholar 

  • Wackerow-Kouzova ND (2007) Ochrobactrum intermedium ANKI, a nitrogen-fixing bacterium able to decolorize azobenzene. Appl Biochem Microbiol 43:403–406

    Article  CAS  Google Scholar 

  • Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703

    PubMed  CAS  Google Scholar 

  • Yu Z, Morrison M (2004) Comparison of different hypervariabile regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturating gradient gel electrophoresis. Appl Environ Microbiol 70:4800–4806

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This research was financially supported by a grant from the Council of Agriculture, Taiwan (Contract No. 100AS-1.1.1-FD-Z2).

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Correspondence to Jong-Yi Fang.

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Fang, JY., Hsu, YR. Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures. Plant Cell Tiss Organ Cult 110, 53–62 (2012). https://doi.org/10.1007/s11240-012-0129-6

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  • DOI: https://doi.org/10.1007/s11240-012-0129-6

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