Plant Cell Tiss Organ Cult (2008) 95:13–19 DOI 10.1007/s11240-008-9408-7
ORIGINAL PAPER
Identification and control of bacterial contaminants from Ilex dumosa nodal segments culture in a temporal immersion bioreactor system using 16S rDNA analysis Claudia Luna Æ Mo´nica Collavino Æ Luis Mroginski Æ Pedro Sansberro
Received: 25 February 2008 / Accepted: 31 May 2008 / Published online: 14 June 2008 Ó Springer Science+Business Media B.V. 2008
Abstract Endogenous bacterial contaminants isolated from infected cultures of Ilex dumosa nodal segments were identified as Stenotrophomonas maltophilia and Achromobacter sp. using 16S rDNA analysis. A range of antibiotics with different mechanism of actions and the commercial biocide PPMTM were tested for their capacity to repress the growth of Gram negative bacteria grown in liquid medium during the establishment phase of temporal immersion systems. The best results were obtained with the addition of 0.5 mg ml-1 cefotaxime to the culture media obtaining 100% of uncontaminated cultures without suppress of shoot growth. Keywords Micropropagation of woody plants Contamination RITAÒ 16S rDNA analysis Cefotaxime
Abbreviations Ap Ampicilline Cf Cefotaxime Cm Chloramphenicol Gm Gentamycin
C. Luna M. Collavino L. Mroginski P. Sansberro (&) Facultad de Ciencias Agrarias (UNNE), Instituto de Bota´nica del Nordeste, Sgto. Cabral 2131, CC: 209, W3402BKG Corrientes, Argentina e-mail:
[email protected]
Km Sm PPMTM
Kanamycin Streptomycin Plant preservative mixtureTM
Introduction Ilex dumosa var. dumosa R. from the family of holly plants, Aquifoliaceae, is a native South American evergreen shrub of which leaves are used to make an infusion similar to yerba mate (I. paraguariensis) tea. The literature reports that the infusion from yerba mate has several health benefits as hypocholesterolemic, hepatoprotective, central nervous system stimulant, diuretic and antioxidant. It also has benefits to the cardiovascular system and is a protector of DNA oxidation (Filip and Ferraro 2003; Schinella et al. 2005). Numerous active phytochemicals have been identified in mate tea that may be responsible for its health benefits, principally, polyphenols, xanthines, purine alkaloids, flavonoids, amino acids and minerals (Heck and Mejia 2007 and literature cited here in). In contrast, the I. dumosa leaves have less caffeine (xanthine) and saponins contents than I. paraguariensis keeping the other components and pharmacological properties (Choi et al. 2005; Filip et al. 1999). This fact and the advantages of I. dumosa respect to same agronomic characteristics arose with the interest of industries and breeders for its economical development. At present, seedlings and macrocuttings are used to producing commercial planting.
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Micropropagation is widely recommended as a biotechnological tool for the multiplication of select plants. To date, there have been three in vitro studies on I. dumosa, including zygotic embryo culture for recovering plants from rudimentary embryos (Sansberro et al. 2001a) and plant regeneration from nodal segment containing single axillary buds (Luna et al. 2003; Sansberro et al. 2001b) using a semi-solid method. However, such systems have been moderately efficient in term of multiplication rate, requiring a long period of culture due to a reduced growth rate, and hindered the automation. In this way, temporary immersion bioreactor system such as RITAÒ should be an important alternative to micropropagate Ilex species due to its numerous advantages compared to the semi-solid methods. Temporary immersion system combined the advantages of gelled and liquid medium, in particular having intermittent availability of nutrients, but still allowing the plants to grow in an air space (McAlister et al. 2005). In the last years, reports in the literature have shown that this technique improved the multiplication rate of many woody species including Eucalyptus, Coffea, Hevea, and Malus (Etienne-Barry et al. 1999; McAlister et al. 2005; Zhu et al. 2005) and at the same time, reduced the labor costs (Etienne et al. 1997). Additionally, the plants obtained have been found to be more suitable for acclimatization and development for autotrophy (Aitken-Christie et al. 1995). Although semi-solid cultures have been established without difficulty from nodal segments explants of I. dumosa following surface sterilization with hypochlorite aqueous solutions (Luna et al. 2003), the use of liquid culture system requires strict control of pathogens during the establishment phase of micropropagation. The preliminary results obtained with I. dumosa nodal segments cultures in a RITAÒ system showed a hazard contamination due to the fast proliferation of endogenous bacteria into the liquid medium killing the explants by nutrient competitions (data not published). The aim of this study was to identify the endogenous contaminants by means of 16S rDNA analyses and explore the feasibility of using antibiotics and biocides as effective treatments for contaminated nodal segmentcultures of Ilex dumosa grown in a temporary immersion bioreactor system. The experiments were undertaken on visibly bacterial contaminated cultures to test its effectiveness at controlling contamination.
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Materials and methods Plant material and source of explants Eight-year-old plants of Ilex dumosa var. dumosa R. grown in pots maintained under greenhouse conditions were used as a source of explants. The plants were kindly provided by INTA-EEA Cerro Azul (Misiones, Argentina), herbarium specimen CTES 321289. Explants (1.5–2 cm long stem segments containing one axillary bud) were collected from young nonlignified branches, surface sterilized in 70% ethanol for 1 min and 1.5% NaOCl with 0.1% TRITONÒ for 30 min, and washed with several rinses of sterile distilled water (Luna et al. 2003). Afterwards, the explants were cultured in 11 ml glass tubes containing 3 ml of quarter-strength MS salts and vitamins (MS), 3% sucrose with the pH adjusted to 5.8 prior the addition of agar (0.65% agar A-1296, SIGMAÒ Chem. Co.) and autoclaving (1.45 kg cm-2 for 20 min). Shoots from nodal segments were grown for at least 20 days in a growth room at 27 ± 2°C with 14 h photoperiod (116 lmol m-2 s-1 PPFD, from fluorescent lamps) before to subculture in bioreactors. Isolation and identification of the bacterial contaminant Bacterial colonies grown around the shoot were collected from infected medium during the establishment phase of micropropagation. Subsequently, an appropriate dilution was subcultured on Tryptone soya agar (TSA) and nutrient agar (NA) medium. Petri plates were incubated 7 days at 30°C in the dark. Two different groups, labeled as IdR and IdW, were distinguished on the basis of colony morphology and Gram reaction. Representative isolates from both groups were selected and further analyzed by 16S rDNA sequencing. The 16S rDNA gene was PCR amplified from genomic DNA isolated from pure bacterial colonies. The bacterial DNA extraction was performed using a ChelexÒ resin (Bio-Rad, Mu¨nchen, Germany). Briefly, 1 ml of an overnight culture of the bacterial cells was centrifuged for 2 min at 13,0009g. The pellet was re-suspended in 200 ll of NaCl (1 M), centrifuged and washed with 200 ll of distilled water. Finally, the cells were re-suspended in 150 ll of ChelexÒ 100
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(10% wt./vol) and incubated at 58°C for 10 min. The sample was briefly mixed and incubated at 99°C for 8 min. After a centrifugation for 5 min at 13,0009g, an aliquot (2 ll) of the supernatant containing DNA was directly used as template in the PCR reaction. The DNA was amplified with the universal primers rD1 (30 -AAGGAGGTGATCCAGCC-50 ) for positions 8-27 and fD1 (50 -AGAGTTTGATCCTGGCT CAG-30 ), for positions 1524–1540 of Escherichia coli strain K12 (Weisburg et al. 1991). The reactions were performed using the following conditions: one cycle of 95°C for 10 min, then 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, followed by one cycle of 72°C for 10 min. The resultant amplicons were cloned using pGEM-T easy (Promega), and plasmids containing the 16S rDNA inserts were purified using the Wizard miniprep DNA purification system. Sequencing of cloned DNA was undertaken by Macrogen Inc., Seoul, Korea. Nucleotide sequences were compared with NCBI GenBank entries and similarities were determined by using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/ BLAST). Partials 16S rDNA gene sequences retrieved in this study were deposited in the GeneBank data base under accession numbers EU442188 (Achromobacter sp.) and EU442189 (Stenotrophomonas maltophilia). Antibiotics treatment of plant material In order to inhibit the rise of contaminants three experiments were performed. The first experiment aimed to evaluate the effect of a short pretreatment to the explants with either antibiotics or commercial biocides before being transferred to bioreactors. Explants from semi-solid cultures infected with bacteria were cultured in 125 ml Erlenmeyer flasks (15 explants/flask) containing 20 ml of MS liquid medium with 1.5% sucrose, 20 lM 6-benzyladenine (BA), plus either the following antibiotics (in mg ml-1): Gm 100, Gm 80 + Km 25, Gm 80 + Cm 50, Cm 50 + Ap 200, Ap 200 + Gm 80 or the biocide PPMTM (Plant Cell Technology, Washington, DC) 0.05–0.1% v/v for 7 days (pre-treated) and transferred to the RITAÒ containers with 200 ml of the same basal medium but without antibiotics for 28 days. Based on experiment 1, the second experiment was executed to assess the effect of continuous exposure
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in the antibiotic mixtures. The explants were cultured directly in RITAsÒ and subjected to the identical basal medium (200 ml) and antibiotics combinations during the entire period (35 days). Founded on experiment 2, the third experiment was achieved to optimize type and doses of antibiotics. The infected explants were transferred to bioreactors (15 explants/recipient) containing 200 ml of basal medium with different combinations and concentrations (in mg ml-1) of Sm (200), Cm (50), Cf (0.25, 0.50, 1) and mixtures between Ap (200) + Cm (50), Sm (200) + Cf (0.50), Cf (0.25, 0.50, 1) + Ap (200) or Cm (50). Bioreactors containing the culture medium (pH: 5.8) were autoclaved at 1.45 kg cm-2 for 20 min. The antibiotics were dissolved in sterile deionised water (except for cloramphenicol which was suspended with an aliquot of ethanol) and sterilized by filtration (MilliporeÒ 0.22 lm pore size). For experiment 1, the Erlenmeyer flasks were subjected to 90 rpm in an orbital shaker. For the temporary immersion program, the explants were in contact with the medium during 1 min each 4 h. The cultures were incubated in a growth room at 27 ± 2°C with 14 h of photoperiod (180 lmol m-2 s-1 PPFD, from fluorescent lamps). Percentage of establishment as well as shoot proliferation and length of the regenerated shoots were recorded at the end of the experiments. Phytotoxicity of antibiotics was determined visually by checking for morphological changes, chlorosis and browning of the explants and/or the regenerated shoots. All experiments consisted of three replications for each treatment. Statistical analysis of data was performed with ANOVA analysis (GraphPad Software, San Diego, CA, USA) and the Tukey tests (P B 0.05) were used in order to compare differences among treatments.
Results and discussion Bacterial isolate identification The Gram negative bacterium observed at the base of Ilex dumosa explants in the form of a red (IdR) or white (IdW) colony were isolated and identified using 16S rDNA sequence and BLAST algorithm. The sequence analysis of IdR strain displayed 99% identity with the
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16S rDNA sequences of Stenotrophomonas maltophilia strain 6B2-1 (GenBank accession no. AY445079.1) and S. maltophilia strain alfa-2 (AB180661.1). For IdW strain, the 16S rDNA sequences of Achromobacter xylosoxidans strain AU1011 (AF411020.1) and Achromobacter sp. NCW (EU220009.1) disclosed the highest level match (99%). These results suggest that strains IdR (EU442189) and IdW (EU442188) should be identified as Stenotrophomonas maltophilia and Achromobacter sp., respectively. Many endophytic bacteria have been identified from plant tissue cultures including species of Acinetobacter, Agrobacterium, Bacillus, Corynebacterium, Curtobacterium, Enterobacter, Erwinina, Flabobacterium, Lactobacillus, Micrococcus, Pseudomonas, Staphylococcus, Stenotrophomonas, and Xanthomonas (Isenegger et al. 2003; Lata et al. 2006; Tanprasert and Reed 1998). Stenotrophomonas maltophilia is found in soil and water (Crossman et al. 2008). Recently, we could identify and characterize its phosphate solubilizing activity in lateritic soils within the rhyzosphere of Ilex dumosa and I. paraguariensis plants grown in field conditions (unpublished data). Achromobacter sp. has been usually identified in soil and water (Adesina et al. 2007) and many beneficial functions have been reported, including stimulation of ionic transport to promote plant growth (Bertrand et al. 2000). Antibiotics treatment of plant material A range of antibiotics and commercial biocides with different mechanisms of action were tested for their Fig. 1 Percentage of bacterial contamination after treatment of explants with different antibiotics or biocides during either 7 (a) or 35 days (b). Note: Bars with the same letter are not significantly different by Tukey’s least significance range test (P \ 0.05)
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capacity to repress the growth of Gram-negative bacteria grown in liquid medium. Initial experiments showed that combination of 50 mg ml-1 chloramphenicol and 200 mg ml-1 ampicilline either as a pre-treatment for 7 days or constant exposure was highly effective against bacterial contamination (Fig. 1a–b). However, severe damage was evident in stem and leaves due to phytotoxicity. The addition of gentamycin alone or in combination with kanamycin, chloramphenicol or ampicilline retains the growth and development of shoots. Gentamycin belong to the group of aminoglycoside antibiotics. They bind to 30S ribosomal subunits in bacterial cells and inhibit olein synthesis and may also inhibit protein synthesis in chloroplasts and mitochondria in plant tissues, there for resulting in small and yellow leaves (Reed et al. 1998). Alternatively, PPMTM was shown to be highly effective only in constant exposure. Thirty-five-day treatment of explants infected with both bacterial isolates produced 67% bacteria free cultures (visibly clean) but the shoots showed an early defoliation and growth was severely inhibited by both concentrations tested. In fact, previous authors have reported sensitivity to PPMTM in seedlings of Arabidopsis thaliana, leaf explants of chrysanthemum, and bryophyte protonemata (George and Tripepi 2001; Paul et al. 2001; Rowntree 2006). A measurement of an appropriate effectiveness would be the antibacterial agent’s ability to control contamination without adversely affecting the vegetative growth of shoots. In this way, the use of cefotaxime at low doses was highly effective for controlling
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sprouted bud produced 2–4 elongated shoots with more than 7 cm in length (Fig. 3). This beneficial effect was earlier observed by Agrawal et al. (1998) in cultured cotton embryos. Cefotaxime is a cephalosporin antibiotic which works by weakening and rupturing the cell wall, killing the bacteria. It is extensively used in protocols of Agrobacterium-mediated transformation to arrest the bacterial proliferation (Gelvin 2000). Combinations of antibiotics are used against bacteria from plant tissue cultures in various species (Reed et al. 1998). However, our results showed that chloramphenicol alone or in combination with cefotaxime was less effective for killing bacteria than a single dose of the later. Concomitantly, nodal cuttings of Ilex dumosa showed the same antibiotic phytotoxicity to the combination of chloramphenicol and cefotaxime but severe damage was evident when the basal medium was supplemented with ampicilline plus either chloramphenicol (50 mg ml-1) or cefotaxime (0.5–1 mg ml-1).
Fig. 2 Percentage of bacterial contamination after treatment of explants with different antibiotics or biocides over time. Note: Bars with the same letter are not significantly different by Tukey’s least significance range test (P \ 0.05)
bacterial contamination from plant tissue cultures of Ilex dumosa (Fig. 2). The addition of 0.5 mg ml-1 cefotaxime to the culture medium produced 100% of uncontaminated cultures and improved the growth rate showing significant differences with the other treatments (Table 1), 27.3% of the explants manifested apical dominance rupture and near to 100% of the
Conclusions The control of bacterial contaminants is essential for successful micropropagation of Ilex dumosa using
Table 1 Shoot growth of Ilex dumosa explants subjected to a long exposure with antibiotics included in the culture media Antibioticsa
Range of phytotoxicityb
Sprouting and development of shoots Apical dominance rupture (%) 0
Shoots with more than 2 cm in length (%)
Shoot length (cm)
Cm 50
0.5
Cf 0.25
0
16.3 ± 1.9
d
64.3 ± 14
b
3.7 ± 0.2
bc
ab
85.2 ± 7.6
ab
2.4 ± 0.1
d
Cf 0.5
0
27.3 ± 3.1
a
98.8 ± 0.7
a
7.8 ± 0.2
a
Cf 1
0.5
3.3 ± 1.9
cd
87.8 ± 4.5
ab
3.4 ± 0.2
cd
Cm 50 + Cf 0.25
0.5
0
d
87.8 ± 1.6
ab
3.3 ± 0.4
cd
Cm 50 + Cf 0.5
0.5
7.1 ± 0.6
bcd
83.3 ± 4.8
ab
2.7 ± 0.2
cd
10.2 ± 1.1
bcd
87.5 ± 4.3
ab
2.9 ± 0.1
cd
Cm 50 + Cf 1
0.5
Ap 200 + Cm 50
1
Ap 200 + Cf 0.25
0.5
Ap 200 + Cf 0.5 Ap 200 + Cf 1
1 1
– 13.3 ± 1.9
– bc
– –
67 ± 4
– b
– –
Sm 200 + Cf 0.5
0.5
3.5 ± 3.5
cd
Sm 200
0.5
5.6 ± 5.6
bcd
4.3 ± 0.2
bc
– –
72 ± 6.9
ab
86.5 ± 3.7
ab
2.6 ± 0.04 2.89 ± 0.1
cd cd
a
Antibiotics tested (in mg ml-1): Ap, Ampicilline; Cf, Cefotaxime; Cm, Chloramphenicol; Sm, Streptomycin. Values are means ± SEM; n = 45. Means in each column followed by different letters are different by Tukey’s least significance range test at 5%
b
Phytotoxicity was scored on a semi-quantitative basis at the end of the experiment as 1, 0.5, and 0 for high, moderate, and nil phytotoxicity, respectively
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Fig. 3 Growth and development of Ilex dumosa explants culture in a temporal immersion system. (a) Vegetative growth after 35 days in MS plus sucrose 3%, BA 20 lM and cefotacime 0.5 g ml-1. (b) Apical dominance rupture and production of elongated shoots with more than 5 cm in length
temporary immersion reactors. This study was designed to test the efficacy of some antimicrobial agents to eliminate specific bacteria in nodal segments obtained from greenhouse-grown plants. The Gramnegative bacteria Stenotrophomonas maltophilia and Achromobacter sp. were identified using 16S rDNA. The use of a single antibiotic added to the culture medium during the multiplication phase in the bioreactor was the most effective treatment to control these endogenous contaminants. In fact, quarter-strength MS medium with 1.5% sucrose, 20 lM 6-benzyladenine plus 0.5 mg ml-1 cefotaxime produced 100% of cleaned cultures and promoted the growth and development of shoots. The use of other antibiotics, alone or in combinations of two, was less effective for killing bacteria and resulted in a range of phytotoxicity symptoms depending on the antibiotic used. Severe damage was evident when the basal medium included ampicilline plus either chloramphenicol or cefotaxime. Acknowledgements The authors are gratefully indebted to the supporting funding from ANPCyT (Grant no. 08-10849), CONICET, and Establecimiento Las Marı´as S.A. We extend our deep appreciation to Prof. Elsie O’Connor for English language checking. We thank anonymous reviewers for their critical comments.
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