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Induction of toluene degradation and growth promotion in corn and wheat by horizontal gene transfer within endophytic bacteria
Soil Biology & Biochemistry 42 (2010) 1051e1057
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Induction of toluene degradation and growth promotion in corn and wheat by horizontal gene transfer within endophytic bacteria Yujing Wang, Hui Li, Wei Zhao, Xiaoli He, Jun Chen, Xiaolu Geng, Ming Xiao* College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2009 Received in revised form 26 February 2010 Accepted 2 March 2010 Available online 17 March 2010
Some experiments involving important crop plants, corn and wheat, were carried out to characterize the agronomic and environmental application of Burkholderia cepacia strain FX2 able to degrade toluene and containing plasmids with the gene encoding for catechol 2, 3-dioxygenase (C23O), a key enzyme in the degradation pathway of monocyclic aromatic compounds. The inoculation of corn and wheat with FX2 led to the promotion of plant growth and reduction in evapotranspiration of toluene into the air. Endophytic bacteria able to grow on toluene as the only source of carbon and containing a C23O gene were found in the plants inoculated with FX2 but not in their non-inoculated controls. Compared to control plants, a greater number of toluene-degrading, phosphate-solubilizing and siderophoreproducing endophytes were found in inoculated plants. Furthermore, a direct correlation occurred between plant biomass responses and the magnitude of C23O-containing endophytes. Phylogenetic tree comparison, plasmid analysis and filter mating assays showed that the C23O gene was transferred horizontally from FX2 to the natural endophytic bacteria of corn and wheat. Horizontal gene transfer among endophytic bacteria might contribute to pollutant degradation, growth promotion and potential for disease suppression in corn and wheat. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Burkholderia cepacia Horizontal gene transfer Catechol 2, 3-dioxygenase
1. Introduction Certain endophytic bacteria are able to promote phytoremediation of highly volatile organic pollutants, such as phenol and toluene which cannot be completely degraded by the plant thus resulting in phytotoxicity or volatilization of chemicals through the leaves (van der Lelie et al., 2001; Doty, 2008; Ryan et al., 2008; Weyens et al., 2009a). Some endophytic bacteria are capable of using certain pollutants as a nutrient source and are therefore able to degrade these target pollutants (Siciliano et al., 2001; Barac et al., 2004; Kuiper et al., 2004; Wang et al., 2007). The genetic information required for the efficient degradation of a pollutant is a predetermined feature of pollutant-degrading endophytic bacteria and is located on mobile genetic elements such as plasmids or bacterial chromosomes (Shields et al., 1995; Barac et al., 2004; Kuiper et al., 2004; Jussila et al., 2007). Horizontal transfer of this genetic information within the microbial population is a major mechanism by which microorganisms acquire new metabolic traits and rapidly adapt to new environmental stresses (Eltis and Bolin, 1996; Dong
* Corresponding author. Biology Department, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China. Tel.: þ86 21 64321022; fax: þ86 21 65642468. E-mail address: [email protected] (M. Xiao). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.03.002
et al., 1998; Mars et al., 1999; van der Lelie et al., 2005). Recent research reports have shown that endophytic bacteria equipped with a toluene-degradation pathway are able to reduce pollutant phytotoxicity and improve phytoremediation of these pollutants (Barac et al., 2004; Newman and Reynolds, 2005; Taghavi et al., 2005; van der Lelie et al., 2005). Other attractive properties of endophytic bacteria which make them suitable for agronomic applications include the induction of systemic resistance in plants against soil-borne pathogenic microorganisms, suppression of plant-pathogenic fungi, and promotion of plant growth (Weyens et al., 2009b). The endophytic bacteria, Bacillus strain EPB22, is able to enhance the growth of banana plants, and reduce the damage caused by banana bunchy top virus (Kavino et al., 2007). Individual bacterial isolates or mixtures of endophytic bacteria can induce growth promotion and suppression of wilt disease in oilseed rape and tomato (Nejad and Johnson, 2000). It has been reported that many of the cultivated endophytic bacteria, belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium, isolated from inside the roots and stems of sugarcane plants, were shown to produce the plant growth hormone indoleacetic acid. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin (Mendes et al., 2007). Some potato-associated endophytes have been found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds (Sessitsch et al., 2004). Endophytic bacterial
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Enterobacter sp. strain 638 exhibits a beneficial effect on growth and development of poplar trees (Taghavi et al., 2009). In our previous work, several strains of endophytic bacteria (FX1FX5), classified as Pseudomonas fluorescens and Burkholderia cepacia, were isolated from a corn plant (Zea mays), and found to degrade phenol. The strains were found to possess the plasmids loaded with a gene encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the degradation pathway of monocyclic aromatic compounds. Horizontal transfer of this gene has been found to occur between the bacteria living within a plant and its rhizosphere (Wang et al., 2007). In addition, strain FX2 was found to degrade toluene. However, characterization of the agronomic and environmental applications of these endophytic bacteria, especially to important crop plants, has not been carried out to date. In this paper we describe experiments that have been carried out on the potential applications of B. cepacia strain FX2 to important crop plants, corn and wheat. It was observed that this endophytic strain induced environmental remediation and the promotion plant growth, and even the potential for disease suppression, which might be a possible result of horizontal transfer of the C23O gene among those endophytic bacteria in corn and wheat.
transferred into airtight glass jars with the half-strength sterile Hoagland’s solution containing 100 mg l1 toluene. In order to avoid gas exchange between the upper and lower areas of the jars, the latter were divided into upper and lower sections with glass plates which contained holes through which the roots of the plants were able to grow and make contact with the Hoagland’s solution in the lower section of the jar. The glass jars were equipped with an injection port through which synthetic air was introduced. Activated charcoal tubes with a link to the headspace in the opening of the upper part of the glass jars were used for collecting the toluene that had volatilized from the plants. The glass jars containing the plants were placed in a growth chamber at a constant temperature of 22 C and a cycle of 14 h of light (150 mmol m2 s1) and 10 h of darkness. The toluene concentrations in the activated charcoal tubes were determined by GCeMS for a period of 120 h after the start of the experiment. The amount of toluene volatilized from those plants was calculated per unit of leaf surface area. The data are the means of 5 replicates and the error bars indicate the standard deviation from the mean. Two-way analysis of variance (ANOVA) was used to determine statistical significance (P < 0.05).
2. Materials and methods
2.3. Isolation and characterization of endophytic bacteria
2.1. Biomass for plant under greenhouse conditions and in field trials
Isolation of endophytic bacteria was performed as described previously (Wang et al., 2007). In brief, soil was removed from the roots under running tap water. The tissue were rinsed with deionized water, washed with sterile distilled water, and drained. The tissue was sterilized using 0.2% HgCl2 for 30 s, washed thoroughly with distilled water, cut into small pieces and homogenized in sterile distilled water. The cultures used to screen for toluene-degrading bacteria were grown in Stanier’s minimal medium (MSB) (Stanier et al.,1966) supplemented with toluene in the vapor phase, which was introduced into the headspace through a foam plug in the opening of the flask. Mineral phosphate solubilization was assayed on TY medium supplemented with 5 g l1 of Ca3(PO4)2. Aliquots of fresh culture were spread onto plates, and incubated at 28 C for 48 h. A clear zone around the colonies indicated solubilization. Siderophore production was detected by the formation of a bright zone with a yellowish fluorescence surrounding bacterial colonies on chrome azurol S (CAS) agar (Schwyn and Neilands, 1987) after 48 h of incubation at 28 C.
Seeds of corn (Z. mays) and wheat (Triticum durum) were surface sterilized with 0.1% HgCl2 for 5 min and then washed thoroughly with distilled water. Seedlings were placed with their roots in the Hoagland’s solution containing strain FX2 at a final concentration of 108 CFU/ml for 96 h (Hoagland and Arnon, 1950). The Hoagland’s solution with strain FX2 was supplemented with 200 ml of LB medium per liter. The control plants were treated with the Hoagland’s solution without strain FX2. The seedlings were then transferred into the pots containing sandy soil and either maintained in a greenhouse, or used in field trials. In the greenhouse studies, earthenware pots, 20 cm in height and 20 cm in diameter were used in the experiment, and 4 corn seedlings or 6 wheat seedlings were grown in each pot containing 5 kg of soil. Six pots per treatment were used. The treated seedlings and the control seedlings were watered for 5, 10 or 15 days with half-strength Hoagland’s solution containing 0 or 400 mg toluene liter1 (one time everyday). Field trials were conducted in a field with a history of toluene pollution (5.238 mg of toluene per kg of soil). The soil was finely pulverized clayeloam with a pH of 7.5. Plots of 2 1 m in size were prepared. Each plot contains 8 rows, and 8 plants were grown in per row, so, 64 plants were included in per plot. The seedlings treated with strain FX2 and their controls were planted in rows (10 cm apart) and watered for 10, 15 or 20 days with half-strength Hoagland’s solution containing 0 or 400 mg toluene liter1 (one time everyday). At the indicated time points, the seedlings were harvested, and the growth parameter, plant biomass (in gram), was determined (fresh weight) and designated Wt. The plant biomass was determined (fresh weight) before the addition of toluene and designated Wo. The value of (WtWo)/Wo was then calculated. The data are the means of 5 replicates and the error bars indicate the standard deviation from the mean. Two-way analysis of variance (ANOVA) was used to determine statistical significance (P < 0.05).
2.4. Plasmid analysis, 16S rDNA amplification, clone of C23O-encoding sequence, southern hybridization For each strain studied, a single colony was picked from a fresh culture, and resuspended in 50 ml sterile deionized water. The genomic DNA was isolated according to standard method (Sambrook et al., 1989). If needed, DNA solution was re-extracted with phenol, and chloroform, precipitated with isopropanol, and washed twice in ethanol. Plasmid extraction was conducted according to standard method (Sambrook et al., 1989). The plasmids were subject to HindIII digestion, and separated on a 0.7% agarose gel. PCR amplification of 16S rDNA based on the genomic DNA was performed with the universal primer pair: 27f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492r (50 TACCTTGTTACGACTT-30 ). (Weissburg et al., 1991; Polz and Cavanaugh 1998; Martin-Laurent et al., 2001). PCR product was purified using the gel extraction kit (Pharmacia), sequenced through dideoxynucleotide sequencing. The PCR primers for clone of the C23O gene were designed based on the previous reports (Mars et al., 1999):
2.2. Amount of toluene volatilized from the corn and wheat Amount of toluene volatilized from the corn and wheat plants was determined essentially as previously described with the slight modifications (Barac et al., 2004). The corn and wheat seedlings inoculated with strain FX2 together with their controls were
P1, 50 -GCTGCTCCATGGGTATTATGAGAATTGGC-30 ; P2, 50 -GACGTCGGATCCTCATCATGTGTACACGGTG-30 A PCR product was ligated in the pGEM-T vector and was transformed into the cell of E. coli DH5a. Selection of transformants
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was performed as described previously (Sambrook et al., 1989). The inserted regions of all clones were sequenced through dideoxynucleotide sequencing. A digoxigenin-labeled DNA fragment was used as the probe for Southern hybridization to detect the C23O gene. The probe was generated from the PCR amplicon generated from the primers detailed above according to the instructions of the manufacturer of the kit (Boehringer, Mannheim, germany). The plasmids were isolated and transferred to nitrocellulose membranes. Southern hybridization was performed according to standard protocols (Sambrook et al., 1989). 2.5. Sequence alignment and phylogenetic analysis Complete alignment of the 16S rDNA or the C23O genes was performed using the CLUSTAL software. Phylogenetic analysis of the above sequences was preformed by using the programs DNADIST, FITCH and DRAWTREE in PHYLIP (Fitch and Margoliash, 1967; Kimura, 1980). First, sequence evolutionary distances were determined by using the program DNADIST, and then, converted to dendrograms by the least-squares distance algorithm in the program FITCH. Input order was randomized, and 20 input orders were examined. Output was converted into unrooted dendrograms with the program DRAWTREE. 2.6. Filter mating assays Filter mating assays was performed as described previously (Stuart-Keil et al., 1998; Wang et al., 2007). To obtain recipient strains for mating, the toluene positive strains were grown in LB broth and subcultured everyday until such time as toluene-negative and plasmidless colonies were obtained. The toluene-negative and plasmidless strains were streaked onto the LB agar plates containing rifampin. The toluene-negative, plasmidless and antibiotic-resistant recipient strains were obtained. About 3 109 recipient cells and 1 109 donor cells were harvested from LB broth, added to the same tube and in combination placed on a sterile filter (0.22-mm pore) on the LB agar plate. The conjugation plates were incubated at 30 C for 12 h. Transconjugants were spread onto selective medium. Recipient and donor inocula alone served as negative control. Transfer frequencies were measured by dividing the number of transconjugants by the viable counts of donor cells in the mating mixture at the end of filter mating assays. Average values were obtained from three mating experiments for each donorerecipient pair. 3. Results 3.1. Growth promotion and evapotranspiration reduction To characterize the application of B. cepacia strain FX2 to important crop plants, the seedlings of corn (Z. mays) and wheat (T. durum) were inoculated with half-strength Hoagland’s solution containing strain FX2 at a final concentration of 108 CFU/ml, and transferred into the pots containing sandy soil, and used for greenhouse experiments. As shown in Fig. 1A, the plants inoculated with strain FX2 produced more biomass than their controls, even in the absence of toluene. The plants exposed to toluene exhibited a reduction in biomass. Similar results were observed when the seedlings of corn and wheat inoculated with strain FX2 were planted in a field with a history of toluene pollution (Fig. 1B). Taken together, these data suggest that the growth promotion and reduction in phytotoxicity observed was a result of the inoculation of the plants with strain FX2.
Fig. 1. Plant biomass for corn and wheat in the presence or absence of toluene (Tol) under greenhouse conditions (A) and in field trials (B). The treated seedlings with strain FX2 and the control seedlings of corn (Zea mays) and wheat (Triticum durum) were watered for 15 days (greenhouse studies) or 20 days (field trials) with halfstrength Hoagland’s solution containing 0 or 400 mg toluene l1. At the indicated time points, the seedlings were harvested, plant biomass (in gram) was determined and designated Wt. The plant biomass was determined before the addition of toluene and designated Wo. The value of (WtWo)/Wo was then calculated. (C) The amount of toluene volatilized from the corn and wheat. The amount of toluene volatilized from plants was determined at 120 h after the start of the experiment.
In order to investigate the evapotranspiration of this pollutant into the air, the amount of toluene volatilized from the aerial parts of these plants was determined using a method similar to that documented in a previous report (Barac et al., 2004; Taghavi et al., 2005). The seedlings of the corn and wheat inoculated with strain FX2 and their controls were transferred into airtight glass jars containing half-strength sterile Hoagland’s solution and toluene. After 120 h the amount of toluene volatilized from these plants was measured. Results showed that the plants inoculated with FX2 released much less toluene than the control plants, strongly suggesting that strain FX2 triggered the degradation of toluene within the plants (Fig. 1C). 3.2. Recovery of endophytic bacteria The numbers and species of endophytic bacteria were determined in the plants watered for 15 days (greenhouse studies) or
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Table 1 Recovery of bacteria from the plants inoculated with FX2 and their controls. Corn (Zea mays)
No. of cells on toluenea No. of cells with C23Ob Species with C23Oc B. cepacia B. brasilensis P. fluorescens P. migulae P. putida P. oryzihabitans No. of cells on Ca3(PO4)2a Species on Ca3(PO4)2c B. cepacia P. fluorescens P. putida No. of cells on CASa Species on CASc B. cepacia P. fluorescens P. putida P. oryzihabitans
Wheat (Triticum durum)
Greenhouse studies
Field trials
Greenhouse studies
Field trials
Incoculated
Controls
Incoculated
Controls
Incoculated
Controls
Incoculated
Controls
3.4 107 2.5 107
0 0
2.4 107 2.1 107
0 0
4.8 106 3.7 106
0 0
3.6 106 2.4 106
0 0
þ þ þ þ þ þ 9.7 106
7.6 103
þ þ þ þ þ þ 8.6 106
2.6 104
þ þ þ þ þ þ 6.2 105
5.3 103
þ þ þ þ þ þ 5.2 105
2.6 104
þ þ þ 2.4 107
þ 5.3 104
þ þ þ 1.6 107
þ þ 3.1 104
þ þ 3.9 106
þ 9.8 103
þ þ þ 2.6 106
þ 9.6 104
þ þ þ þ
þ þ
þ þ þ þ
þ þ
þ þ þ þ
þ þ þ
þ þ
The numbers and species of endophytic bacteria were determined in the plants watered for 15 days (greenhouse studies) or 20 days (field trials) with half-strength Hoagland’s solution containing 400 mg toluene liter-1. a Numbers of the cultivable endophytic bacteria grown on toluene, or Ca3(PO4)2, or CAS, from per gram (fresh weight) of plant material. The data are expressed as the means from five independent experiments with similar results. b Numbers of the cultivable endophytic bacteria containing the C23O gene per gram (fresh weight) of plant material. The data are expressed as the means from five independent experiments with similar results. c Species containing a C23O gene, or capable of phosphate solubilization, or siderophore production.
20 days (field trials) with half-strength Hoagland’s solution containing 400 mg toluene liter1. Cultivable endophytic bacteria able to grow on toluene as the only source of carbon and containing a C23O gene, were found in the plants inoculated with strain FX2 but not in their non-inoculated controls. Six different strains (B1eB6) were isolated from these endophytic bacteria carrying the C23O gene. The morphological and biochemical properties and 16S rDNA sequences of the six strains were nearly identical to those of B. cepacia, Burkholderia brasilensis, P. fluorescens, Pseudomonas migulae, Pseudomonas putida and Pseudomonas oryzihabitans, respectively (Table 1 and results not shown). A greater number of phosphate-solubilizing and siderophore-producing endophytic bacteria were found in the corn and wheat inoculated with strain FX2 than in their controls (Table 1). To examine the correlation between plant biomass responses and the magnitude of C23O-containing endophytic bacteria, we determined the numbers of C23O-containing endophytic bacteria and the plant biomass at indicated time points. A direct correlation was observed in greenhouse experiments. It was found that the increase in the magnitude of C23O-containing endophytic bacteria correlated well with plant biomass. Although plant biomass increased with time in the uninoculated control plants, increased levels of plant biomass in inoculated groups were higher than those in control groups (Table 2). A similar correlation between plant
biomass responses and the magnitude of C23O-containing endophytic bacteria was also found in field trials (Table 3). 3.3. HGT among endophytic bacteria The fact that C23O-containing endophytes were found in inoculated groups and not in control groups suggests that horizontal transfer of C23O gene occurred among endophytic communities of the plants and the C23O gene was originated from strain FX2. Comparison of phylogenetic trees based on patristic distances and sequence alignment illustrated that the C23O genes isolated from the six different strains were phylogenetically more closely related than the 16S rDNAs from their hosts (Fig. 2A, B). Generally, horizontal gene transfer (HGT) would be indicated if the lineage of the target gene is phylogenetically more closely related than that of the hosts. The phylogeny of 16S rDNA is putatively considered representative of the phylogeny of the bacterium possessing it. Therefore, the C23O gene might have been transferred horizontally among the natural endophytic bacteria of corn and wheat. To obtain more evidence for HGT, the cells of the above six strains and the FX2 strain were lysed, and plasmids were isolated. It was found that each of the six C23O positive strains isolated from inoculated plants possessed a large plasmid, which had similar HindIII restriction patterns (Fig. 3A, B). Strains B1 and FX2
Table 2 A correlation between plant biomass and the magnitude of bacteria with C23O (in greenhouse experiments). 5 days
Corn Wheat
inoculated controls inoculated controls
10 days
15 days
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
1.1 0.8 0.5 0.5
3.4 104 0 6.4 103 0
2.6 1.1 1.3 1.1
1.8 106 0 2.4 105 0
4.2 1.5 2.1 1.6
2.4 107 0 3.7 106 0
The data are expressed as the means from five independent experiments with similar results.
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Table 3 A correlation between plant biomass and the magnitude of bacteria with C23O (in field trials). 10 days
Corn Wheat
Inoculated Controls Inoculated Controls
15 days
20 days
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
1.2 0.8 0.5 0.5
3.4 105 0 7.2 104 0
2.8 1.2 1.4 1.1
1.7 107 0 1.1 106 0
3.2 1.7 1.9 1.2
2.7 107 0 3.2 106 0
The data are expressed as the means from five independent experiments with similar results.
were identical with morphological and biochemical properties, 16S rDNA and C23O gene sequences and plasmid profiles. Therefore, it is likely that strain B1 is the FX2 strain re-isolated from the inoculated plants. Furthermore, filter mating assays were performed among those C23O-containing endophytes. First, filter mating assays were carried out between different cells of FX2. The filter matings, with the cured antibiotic-resistant and plasmidless FX2 strain as the recipient and its parent FX2 strain as the donor, were done on a selective medium with toluene as a single carbon source. The transconjugants that grew on toluene were obtained and transfer frequencies were calculated. Plasmid and hybridization profile analysis showed that a large plasmid and a C23Oencoding sequence on the plamid were detected in transconjugant colonies, as in donor colonies (Fig. 4A, B). Transfer of a C23O gene between strain FX2 cells occurred at a mean frequency of 9.84 101 per donor (Table 4). When FX2 served as the donor and the rifampin-resistant B3 strain as the recipient, a large plasmid and a C23O-encoding sequence on the plamid were also detected in transconjugant colonies (Fig. 4). Evidently, transfer frequency between strains FX2 and P. fluorescens strain B3 was lower than that between different cells of strain FX2 (Table 4). Other filter mating assays results were shown in Table 4. Taken together, it was proved that the plasmid containing the C23O gene is able to transferred between the FX2 cells, from FX2 to other endophytic bacteria, between the six endophytes each other. Therefore, the C23O gene was transferred horizontally from FX2 to the natural endophytic communities of corn and wheat.
Fig. 2. Phylogenetic trees of 16S rDNA (A) and C23O genes (B) of B. cepacia (B1), B. brasilensis (B2), P. fluorescens (B3), P. migulae (B4), P. putida (B5) and P. oryzihabitans (B6).
4. Discussion The use of bacteria to clean up environmental pollutants has gained momentum in past years although obstacles are present before they can be applied in field-scale projects (Berg et al., 2005; Newman and Reynolds. 2005; van der Lelie et al., 2005). In our previous work, B. cepacia strain FX2, a strain of endophytic bacterium able to degrade toluene and phenol, was isolated from a corn plant (Z. mays).The strain was found to possess a large plasmid encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the degradation pathway of monocyclic aromatic compounds (Wang et al., 2007). In this paper we describe experiments to investigate the potential applications of strain FX2 to important crop plants, corn and wheat. Greenhouse and field trials showed that plants produced more biomass when the FX2 strain was introduced in corn and wheat (Fig. 1A, B). Furthermore, we found in the evapotranspiration measurements that much less toluene was released from the plants inoculated by the FX2 (Fig. 1C). Those results suggested that the growth promotion and reduction in phytotoxicity observed was a result of the inoculation of the plants with strain FX2. Cultivable endophytic bacteria able to grow on toluene as the only source of carbon and containing a C23O gene were found in the plants inoculated with strain FX2 but not in their non-inoculated controls (Table 1). Many strains of these bacteria were often reported to be isolated from different plants, able to promote host plant growth, produce antifungal metabolite, degrade organic pollutants and possess C23O genes (Nejad and Johnson, 2000; Sessitsch et al., 2004; Taghavi et al., 2005, 2009; Kavino et al., 2007; Mendes et al., 2007; Wang et al., 2007). Furthermore, a greater number of phosphate-solubilizing and siderophoreproducing endophytic bacteria were found in the corn and wheat inoculated with strain FX2 than in their controls (Table 1). Phosphorus is one of the major essential macronutrients for biological growth and development and phosphate-solubilizing microorganisms may therefore play an important role in supplying phosphate to plants in a more environmentally-friendly and sustainable manner (Rodríguez et al., 2006; Khan et al., 2007). Production of siderophores by bacteria is important in the suppression of plant pathogens and induction of systemic resistance in plants (Compant et al., 2005). Our results suggested that growth promotion and disease suppression might be more effective in the plants inoculated with FX2. It was found that the increase in the number of C23O-containing endophytic bacteria was in agreement with plant biomass in greenhouse experiments and field trials (Table 2), indicating that a direct correlation occurred between plant biomass responses and the magnitude of C23O-containing endophytic bacteria. Comparison of phylogenetic trees illustrated that the C23O gene might have been transferred horizontally among these C23O-containing endophytic bacteria isolated from these corn and wheat plants inoculated by FX2. Indeed, these strains possessed a large plasmid and similar restriction enzyme-digested plasmid patterns
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Fig. 3. Plasmid analysis for endophytic bacterial strains B1eB6 and FX2 (lanes 1e7). The plasmids (A) isolated from endophytic bacterial strains and the HindIII-digested plasmids (B) were separated on a 0.7% agarose gel. Lane 8, molecular size markers.
(Fig. 3A, B). Filter mating assays further showed that the plasmid containing the C23O gene was able to be transferred among these isolated endophyte strains containing strain FX2 (Fig. 4A, B; Table 4). Therefore, the C23O gene has been transferred horizontally from FX2 to the natural endophytic communities of corn and wheat. The increasing number of C23O-containing endophytic bacteria, and of phosphate-solubilizing and siderophore-producing endophytic bacteria, in corn and wheat, might be a possible result of horizontal transfer of the C23O gene among those endophytic bacteria. We assume that horizontal transfer of the genetic determinants for the degradation of toluene from strain FX2 to the endophytic communities of the plants, might contribute to a reduction in phytotoxicity, thus creating microenvironments
which phosphate-solubilizing and siderophore-producing endophytic bacteria prefer to colonize. In summary, induction of toluene degradation and growth promotion and potential for disease suppression were found in the corn and wheat as a possible result of HGT within endophytic bacteria. This is very important for crop plants. Our results have revealed that horizontal transfer of genetic information required for the efficient degradation of a pollutant by the endophytic population might not only contribute to pollutant degradation, but also have other beneficial effects on plant, such as growth promotion and disease suppression. HGT, associated with an endophytic bacterium containing a transferable plasmid carrying the genetic information encoding the desired metabolic properties, is very
Fig. 4. Electrophoresis analysis of plasmid profiles (A) and Southern hybridization to plasmid profiles (B) for HGT between different cells of strain FX2 and between strains FX2 and B3. Lanes: 1 and 5, donor strain (the original parent FX2); 2 and 6, recipient strains (the toluene-negative, plasmidless and antibiotic-resistant FX2 and B3); 3 and 7, the transconjugant FX2 and B3.
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Table 4 Transfer frequencies among endophytic bacteriaa. Recipient strains
Donor strains B1(FX2)
B1(FX2) B2 B3 B4 B5 B6
9.84 1.98 2.49 1.80 3.20 9.33
101 101 101 101 101 102
B2 9.12 5.32 2.44 1.40 3.10 9.22
B3
102 101 101 101 101 102
2.52 2.93 9.02 2.90 2.41 8.82
B4
101 101 101 101 101 102
1.37 1.88 2.61 6.77 2.34 9.54
B5
101 101 101 101 101 102
3.00 3.45 2.90 2.71 6.42 1.99
B6
101 101 101 101 101 101
9.82 8.99 8.51 1.03 1.97 4.70
102 102 102 101 101 101
a Transfer frequencies were measured by dividing the number of transconjugants by the viable counts of donor cells in the mating mixture at the end of filter mating assays. Average values were obtained from three mating experiments for each donor-recipient pair. The names of different strains were referred to as Fig. 2.
important for agronomic and environmental applications in crop plants. Acknowledgement This work was supported by the National Natural Science Foundation of China (30670445, 30870492), the Shanghai Municipal Science and Technology Commission (07DZ12038, 08JC1416900), the Shanghai Municipal Education Commission (08ZZ66), the Shanghai Leading Academic Discipline Project (S30406) and the Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50401). References Barac, T., Taghavi, S., Borremans, B., Provoost, A., Oeyen, L., Colpaert, J.V., Vangronsveld, J., van der Lelie, D., 2004. Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nature Biotechnology 22, 583e588. Berg, G., Eberl, L., Hartmann, A., 2005. The rhizosphere as a reservoir for opportunistic human pathogenic bacteria. Environmental Microbiology 7, 1673e1685. Compant, S., Duffy, B., Nowak, J., Clément, C., Barka, E.A., 2005. Use of plant growthpromoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Applied and Environmental Microbiology 71, 4951e4959. Dong, Q.H., Springeal, D., Schoeters, D., Nuyts, G., Mergeay, M., Diels, L., 1998. Horizontal transfer of bacterial heavy metal resistance genes and its applications in activated sludge systems. Water Science and Technology 37, 465e468. Doty, S.L., 2008. Enhancing phytoremediation through the use of transgenics and endophytes. New Phytologist 179, 318e333. Eltis, L.D., Bolin, J.T., 1996. Evolutionary relationships among extradiol dioxygenases. Journal of Bacteriology 178, 5930e5937. Fitch, W.M., Margoliash, E., 1967. Construction of phylogenetic trees. Science 155, 279e284. Hoagland, D.R., Arnon, D.I., 1950. The Water Culture Method for Plants without Soil. Circular 374. University of California, Agriculture Experimental Station, Berkeley. Jussila, M.M., Zhai, J., Suominen, L., Lindstöm, K., 2007. TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo. Environmental Pollution 146, 510e524. Kavino, M., Harish, S., Kumar, N., Saravanakumar, D., Damodaran, T., Soorianathasundaram, K., Samiyappan, R., 2007. Rhizosphere and endophytic bacteria for induction of systemic resistance of banana plantlets against bunchy top virus. Soil Biology & Biochemistry 39, 1087e1098. Khan, M.S., Zaidi, A., Wani, P.A., 2007. Role of phosphate-solubilizing microorganisms in sustainable agriculture e a review. Agronomic Sustainable Development 27, 29e43. Kimura, M., 1980. A simple method for estimating evolutionary rate of basesubstitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111e120. Kuiper, I., Lagendijk, E.L., Bloemberg, G.V., Lugtenberg, B.J.J., 2004. Rhizoremediation: a beneficial plantemicrobe interaction. Molecular Plantemicrobe Interactions 17, 6e15. Mars, B.E., Kingma, J., Kaschabek, S.R., Reineke, W., Janssen, D.B., 1999. Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31. Journal of Bacteriology 181, 1309e1318. Martin-Laurent, F., Philippot, L., Hallet, S., Chaussod, R., Germon, J.C., Soulas, G., Catroux, G., 2001. DNA extraction from soils: old bias for new microbial diversity analysis methods. Applied and Environmental Microbiology 67, 2354e2359.
Mendes, R., Pizzirani-Kleiner, A.A., Araujo, W.L., Raaijmakers, J.M., 2007. Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates. Applied and Environmental Microbiology 73, 7259e7267. Nejad, P., Johnson, P.A., 2000. Endophytic bacteria induce growth promotion and wilt disease suppression in oilseed rape and tomato. Biological Control 18, 208e215. Newman, L.A., Reynolds, C.M., 2005. Phytoremediation and bacteria: new uses of endophytic bacteria in plants. Trends in Biotechnology 23, 6e8. Polz, M.F., Cavanaugh, C.M., 1998. Bias in template-to-product ratios in multitemplate PCR. Applied and Environmental Microbiology 64, 3724e3730. Rodríguez, H., Fraga, R., Gonzalez, T., Bashan, Y., 2006. Genetics of phosphate solubilization and its potential applications for improving plant growthpromoting bacteria. Plant and Soil 287, 15e21. Ryan, R.P., Germaine, K., Franks, A., Ryan, D.J., Dowling, D.N., 2008. Bacterial endophytes: recent developments and applications. FEMS Microbiology Letters 278, 1e9. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning:a Laboratory Manual, second ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry 160, 47e56. Sessitsch, A., Reiter, B., Berg, G., 2004. Endophytic bacterial communities of fieldgrown potato plants and their plant-growth-promoting and antagonistic abilities. Canadian Journal of Microbiology 50, 239e249. Shields, M.S., Reagin, M.J., Gerger, R.R., Campbell, R., Somerville, C., 1995. TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas cepacia G4). Applied and Environmental Microbiology 61, 1352e1356. Siciliano, S.D., Fortin, N., Mihoc, A., Wisse, G., Labelle, S., Beaumier, D., Ouellette, D., Roy, R., Whyte, L.G., Banks, M.K., Schwab, P., Lee, K., Greer, C.W., 2001. Selection of specific endophytic bacterial genotypes by plants in response to soil contamination. Applied Microbiology and Biotechnology 67, 2469e2475. Stanier, R.Y., Palleroni, N.J., Doudoroff, M., 1966. The aerobic pseudomonads: a taxonomic study. Journal of General Microbiology 43, 159e271. Stuart-Keil, K.G., Hohnstock, A.M., Drees, K.P., Herrick, J.B., Madsen, E.L., 1998. Plasmids responsible for horizontal transfer of naphtalene catabolism genes between bacteria at a coal-contaminated site are homologous to pDTG1 from Pseudomonas putida NCIB 9816-4. Applied and Environmental Microbiology 64, 3633e3640. Taghavi, S., Barac, T., Greenberg, B., Borremans, B., Vangronsveld, J., van der Lelie, D., 2005. Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene. Applied Environmental Microbiology 71, 8500e8505. 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. Applied and Environmental Microbiology 75, 748e757. van der Lelie, D., Schwitzguebel, J.P., Glass, D.J., Vangronsveld, J., Baker, A., 2001. Assessing phytoremediation’s progress in the United States and Europe. Environmental Sceience & Technology 35, 446Ae452A. van der Lelie, D., Barac, T., Taghavi, S., Vangronsveld, J., 2005. Response to Newman: new uses of endophytic bacteria to improve phytoremediation. Trends Biotechnology 23, 8e9. Wang, Y., Xiao, M., Geng, X., Liu, J., Chen, J., 2007. Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere. Applied Microbiology and Biotechnology 77, 733e739. Weissburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.L., 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriol 173, 697e703. Weyens, N., van der Lelie, D., Taghavi, S., Vangronsveld, J., 2009a. Phytoremediation: planteendophyte partnerships take the challenge. Current Opinion in Biotechnology 20, 248e254. Weyens, N., van der Lelie, D., Taghavi, S., Newman, L., Vangronsveld, J., 2009b. Exploiting plantemicrobe partnerships for improving biomass production and remediation. available online. Trends in Biotechnology. doi:10.1016/j.tibtech. 2009.07.006.
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Induction of toluene degradation and growth promotion in corn and wheat by horizontal gene transfer within endophytic bacteria Yujing Wang, Hui Li, Wei Zhao, Xiaoli He, Jun Chen, Xiaolu Geng, Ming Xiao* College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2009 Received in revised form 26 February 2010 Accepted 2 March 2010 Available online 17 March 2010
Some experiments involving important crop plants, corn and wheat, were carried out to characterize the agronomic and environmental application of Burkholderia cepacia strain FX2 able to degrade toluene and containing plasmids with the gene encoding for catechol 2, 3-dioxygenase (C23O), a key enzyme in the degradation pathway of monocyclic aromatic compounds. The inoculation of corn and wheat with FX2 led to the promotion of plant growth and reduction in evapotranspiration of toluene into the air. Endophytic bacteria able to grow on toluene as the only source of carbon and containing a C23O gene were found in the plants inoculated with FX2 but not in their non-inoculated controls. Compared to control plants, a greater number of toluene-degrading, phosphate-solubilizing and siderophoreproducing endophytes were found in inoculated plants. Furthermore, a direct correlation occurred between plant biomass responses and the magnitude of C23O-containing endophytes. Phylogenetic tree comparison, plasmid analysis and filter mating assays showed that the C23O gene was transferred horizontally from FX2 to the natural endophytic bacteria of corn and wheat. Horizontal gene transfer among endophytic bacteria might contribute to pollutant degradation, growth promotion and potential for disease suppression in corn and wheat. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Burkholderia cepacia Horizontal gene transfer Catechol 2, 3-dioxygenase
1. Introduction Certain endophytic bacteria are able to promote phytoremediation of highly volatile organic pollutants, such as phenol and toluene which cannot be completely degraded by the plant thus resulting in phytotoxicity or volatilization of chemicals through the leaves (van der Lelie et al., 2001; Doty, 2008; Ryan et al., 2008; Weyens et al., 2009a). Some endophytic bacteria are capable of using certain pollutants as a nutrient source and are therefore able to degrade these target pollutants (Siciliano et al., 2001; Barac et al., 2004; Kuiper et al., 2004; Wang et al., 2007). The genetic information required for the efficient degradation of a pollutant is a predetermined feature of pollutant-degrading endophytic bacteria and is located on mobile genetic elements such as plasmids or bacterial chromosomes (Shields et al., 1995; Barac et al., 2004; Kuiper et al., 2004; Jussila et al., 2007). Horizontal transfer of this genetic information within the microbial population is a major mechanism by which microorganisms acquire new metabolic traits and rapidly adapt to new environmental stresses (Eltis and Bolin, 1996; Dong
* Corresponding author. Biology Department, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China. Tel.: þ86 21 64321022; fax: þ86 21 65642468. E-mail address: [email protected] (M. Xiao). 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.03.002
et al., 1998; Mars et al., 1999; van der Lelie et al., 2005). Recent research reports have shown that endophytic bacteria equipped with a toluene-degradation pathway are able to reduce pollutant phytotoxicity and improve phytoremediation of these pollutants (Barac et al., 2004; Newman and Reynolds, 2005; Taghavi et al., 2005; van der Lelie et al., 2005). Other attractive properties of endophytic bacteria which make them suitable for agronomic applications include the induction of systemic resistance in plants against soil-borne pathogenic microorganisms, suppression of plant-pathogenic fungi, and promotion of plant growth (Weyens et al., 2009b). The endophytic bacteria, Bacillus strain EPB22, is able to enhance the growth of banana plants, and reduce the damage caused by banana bunchy top virus (Kavino et al., 2007). Individual bacterial isolates or mixtures of endophytic bacteria can induce growth promotion and suppression of wilt disease in oilseed rape and tomato (Nejad and Johnson, 2000). It has been reported that many of the cultivated endophytic bacteria, belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium, isolated from inside the roots and stems of sugarcane plants, were shown to produce the plant growth hormone indoleacetic acid. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin (Mendes et al., 2007). Some potato-associated endophytes have been found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds (Sessitsch et al., 2004). Endophytic bacterial
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Enterobacter sp. strain 638 exhibits a beneficial effect on growth and development of poplar trees (Taghavi et al., 2009). In our previous work, several strains of endophytic bacteria (FX1FX5), classified as Pseudomonas fluorescens and Burkholderia cepacia, were isolated from a corn plant (Zea mays), and found to degrade phenol. The strains were found to possess the plasmids loaded with a gene encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the degradation pathway of monocyclic aromatic compounds. Horizontal transfer of this gene has been found to occur between the bacteria living within a plant and its rhizosphere (Wang et al., 2007). In addition, strain FX2 was found to degrade toluene. However, characterization of the agronomic and environmental applications of these endophytic bacteria, especially to important crop plants, has not been carried out to date. In this paper we describe experiments that have been carried out on the potential applications of B. cepacia strain FX2 to important crop plants, corn and wheat. It was observed that this endophytic strain induced environmental remediation and the promotion plant growth, and even the potential for disease suppression, which might be a possible result of horizontal transfer of the C23O gene among those endophytic bacteria in corn and wheat.
transferred into airtight glass jars with the half-strength sterile Hoagland’s solution containing 100 mg l1 toluene. In order to avoid gas exchange between the upper and lower areas of the jars, the latter were divided into upper and lower sections with glass plates which contained holes through which the roots of the plants were able to grow and make contact with the Hoagland’s solution in the lower section of the jar. The glass jars were equipped with an injection port through which synthetic air was introduced. Activated charcoal tubes with a link to the headspace in the opening of the upper part of the glass jars were used for collecting the toluene that had volatilized from the plants. The glass jars containing the plants were placed in a growth chamber at a constant temperature of 22 C and a cycle of 14 h of light (150 mmol m2 s1) and 10 h of darkness. The toluene concentrations in the activated charcoal tubes were determined by GCeMS for a period of 120 h after the start of the experiment. The amount of toluene volatilized from those plants was calculated per unit of leaf surface area. The data are the means of 5 replicates and the error bars indicate the standard deviation from the mean. Two-way analysis of variance (ANOVA) was used to determine statistical significance (P < 0.05).
2. Materials and methods
2.3. Isolation and characterization of endophytic bacteria
2.1. Biomass for plant under greenhouse conditions and in field trials
Isolation of endophytic bacteria was performed as described previously (Wang et al., 2007). In brief, soil was removed from the roots under running tap water. The tissue were rinsed with deionized water, washed with sterile distilled water, and drained. The tissue was sterilized using 0.2% HgCl2 for 30 s, washed thoroughly with distilled water, cut into small pieces and homogenized in sterile distilled water. The cultures used to screen for toluene-degrading bacteria were grown in Stanier’s minimal medium (MSB) (Stanier et al.,1966) supplemented with toluene in the vapor phase, which was introduced into the headspace through a foam plug in the opening of the flask. Mineral phosphate solubilization was assayed on TY medium supplemented with 5 g l1 of Ca3(PO4)2. Aliquots of fresh culture were spread onto plates, and incubated at 28 C for 48 h. A clear zone around the colonies indicated solubilization. Siderophore production was detected by the formation of a bright zone with a yellowish fluorescence surrounding bacterial colonies on chrome azurol S (CAS) agar (Schwyn and Neilands, 1987) after 48 h of incubation at 28 C.
Seeds of corn (Z. mays) and wheat (Triticum durum) were surface sterilized with 0.1% HgCl2 for 5 min and then washed thoroughly with distilled water. Seedlings were placed with their roots in the Hoagland’s solution containing strain FX2 at a final concentration of 108 CFU/ml for 96 h (Hoagland and Arnon, 1950). The Hoagland’s solution with strain FX2 was supplemented with 200 ml of LB medium per liter. The control plants were treated with the Hoagland’s solution without strain FX2. The seedlings were then transferred into the pots containing sandy soil and either maintained in a greenhouse, or used in field trials. In the greenhouse studies, earthenware pots, 20 cm in height and 20 cm in diameter were used in the experiment, and 4 corn seedlings or 6 wheat seedlings were grown in each pot containing 5 kg of soil. Six pots per treatment were used. The treated seedlings and the control seedlings were watered for 5, 10 or 15 days with half-strength Hoagland’s solution containing 0 or 400 mg toluene liter1 (one time everyday). Field trials were conducted in a field with a history of toluene pollution (5.238 mg of toluene per kg of soil). The soil was finely pulverized clayeloam with a pH of 7.5. Plots of 2 1 m in size were prepared. Each plot contains 8 rows, and 8 plants were grown in per row, so, 64 plants were included in per plot. The seedlings treated with strain FX2 and their controls were planted in rows (10 cm apart) and watered for 10, 15 or 20 days with half-strength Hoagland’s solution containing 0 or 400 mg toluene liter1 (one time everyday). At the indicated time points, the seedlings were harvested, and the growth parameter, plant biomass (in gram), was determined (fresh weight) and designated Wt. The plant biomass was determined (fresh weight) before the addition of toluene and designated Wo. The value of (WtWo)/Wo was then calculated. The data are the means of 5 replicates and the error bars indicate the standard deviation from the mean. Two-way analysis of variance (ANOVA) was used to determine statistical significance (P < 0.05).
2.4. Plasmid analysis, 16S rDNA amplification, clone of C23O-encoding sequence, southern hybridization For each strain studied, a single colony was picked from a fresh culture, and resuspended in 50 ml sterile deionized water. The genomic DNA was isolated according to standard method (Sambrook et al., 1989). If needed, DNA solution was re-extracted with phenol, and chloroform, precipitated with isopropanol, and washed twice in ethanol. Plasmid extraction was conducted according to standard method (Sambrook et al., 1989). The plasmids were subject to HindIII digestion, and separated on a 0.7% agarose gel. PCR amplification of 16S rDNA based on the genomic DNA was performed with the universal primer pair: 27f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492r (50 TACCTTGTTACGACTT-30 ). (Weissburg et al., 1991; Polz and Cavanaugh 1998; Martin-Laurent et al., 2001). PCR product was purified using the gel extraction kit (Pharmacia), sequenced through dideoxynucleotide sequencing. The PCR primers for clone of the C23O gene were designed based on the previous reports (Mars et al., 1999):
2.2. Amount of toluene volatilized from the corn and wheat Amount of toluene volatilized from the corn and wheat plants was determined essentially as previously described with the slight modifications (Barac et al., 2004). The corn and wheat seedlings inoculated with strain FX2 together with their controls were
P1, 50 -GCTGCTCCATGGGTATTATGAGAATTGGC-30 ; P2, 50 -GACGTCGGATCCTCATCATGTGTACACGGTG-30 A PCR product was ligated in the pGEM-T vector and was transformed into the cell of E. coli DH5a. Selection of transformants
Y. Wang et al. / Soil Biology & Biochemistry 42 (2010) 1051e1057
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was performed as described previously (Sambrook et al., 1989). The inserted regions of all clones were sequenced through dideoxynucleotide sequencing. A digoxigenin-labeled DNA fragment was used as the probe for Southern hybridization to detect the C23O gene. The probe was generated from the PCR amplicon generated from the primers detailed above according to the instructions of the manufacturer of the kit (Boehringer, Mannheim, germany). The plasmids were isolated and transferred to nitrocellulose membranes. Southern hybridization was performed according to standard protocols (Sambrook et al., 1989). 2.5. Sequence alignment and phylogenetic analysis Complete alignment of the 16S rDNA or the C23O genes was performed using the CLUSTAL software. Phylogenetic analysis of the above sequences was preformed by using the programs DNADIST, FITCH and DRAWTREE in PHYLIP (Fitch and Margoliash, 1967; Kimura, 1980). First, sequence evolutionary distances were determined by using the program DNADIST, and then, converted to dendrograms by the least-squares distance algorithm in the program FITCH. Input order was randomized, and 20 input orders were examined. Output was converted into unrooted dendrograms with the program DRAWTREE. 2.6. Filter mating assays Filter mating assays was performed as described previously (Stuart-Keil et al., 1998; Wang et al., 2007). To obtain recipient strains for mating, the toluene positive strains were grown in LB broth and subcultured everyday until such time as toluene-negative and plasmidless colonies were obtained. The toluene-negative and plasmidless strains were streaked onto the LB agar plates containing rifampin. The toluene-negative, plasmidless and antibiotic-resistant recipient strains were obtained. About 3 109 recipient cells and 1 109 donor cells were harvested from LB broth, added to the same tube and in combination placed on a sterile filter (0.22-mm pore) on the LB agar plate. The conjugation plates were incubated at 30 C for 12 h. Transconjugants were spread onto selective medium. Recipient and donor inocula alone served as negative control. Transfer frequencies were measured by dividing the number of transconjugants by the viable counts of donor cells in the mating mixture at the end of filter mating assays. Average values were obtained from three mating experiments for each donorerecipient pair. 3. Results 3.1. Growth promotion and evapotranspiration reduction To characterize the application of B. cepacia strain FX2 to important crop plants, the seedlings of corn (Z. mays) and wheat (T. durum) were inoculated with half-strength Hoagland’s solution containing strain FX2 at a final concentration of 108 CFU/ml, and transferred into the pots containing sandy soil, and used for greenhouse experiments. As shown in Fig. 1A, the plants inoculated with strain FX2 produced more biomass than their controls, even in the absence of toluene. The plants exposed to toluene exhibited a reduction in biomass. Similar results were observed when the seedlings of corn and wheat inoculated with strain FX2 were planted in a field with a history of toluene pollution (Fig. 1B). Taken together, these data suggest that the growth promotion and reduction in phytotoxicity observed was a result of the inoculation of the plants with strain FX2.
Fig. 1. Plant biomass for corn and wheat in the presence or absence of toluene (Tol) under greenhouse conditions (A) and in field trials (B). The treated seedlings with strain FX2 and the control seedlings of corn (Zea mays) and wheat (Triticum durum) were watered for 15 days (greenhouse studies) or 20 days (field trials) with halfstrength Hoagland’s solution containing 0 or 400 mg toluene l1. At the indicated time points, the seedlings were harvested, plant biomass (in gram) was determined and designated Wt. The plant biomass was determined before the addition of toluene and designated Wo. The value of (WtWo)/Wo was then calculated. (C) The amount of toluene volatilized from the corn and wheat. The amount of toluene volatilized from plants was determined at 120 h after the start of the experiment.
In order to investigate the evapotranspiration of this pollutant into the air, the amount of toluene volatilized from the aerial parts of these plants was determined using a method similar to that documented in a previous report (Barac et al., 2004; Taghavi et al., 2005). The seedlings of the corn and wheat inoculated with strain FX2 and their controls were transferred into airtight glass jars containing half-strength sterile Hoagland’s solution and toluene. After 120 h the amount of toluene volatilized from these plants was measured. Results showed that the plants inoculated with FX2 released much less toluene than the control plants, strongly suggesting that strain FX2 triggered the degradation of toluene within the plants (Fig. 1C). 3.2. Recovery of endophytic bacteria The numbers and species of endophytic bacteria were determined in the plants watered for 15 days (greenhouse studies) or
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Table 1 Recovery of bacteria from the plants inoculated with FX2 and their controls. Corn (Zea mays)
No. of cells on toluenea No. of cells with C23Ob Species with C23Oc B. cepacia B. brasilensis P. fluorescens P. migulae P. putida P. oryzihabitans No. of cells on Ca3(PO4)2a Species on Ca3(PO4)2c B. cepacia P. fluorescens P. putida No. of cells on CASa Species on CASc B. cepacia P. fluorescens P. putida P. oryzihabitans
Wheat (Triticum durum)
Greenhouse studies
Field trials
Greenhouse studies
Field trials
Incoculated
Controls
Incoculated
Controls
Incoculated
Controls
Incoculated
Controls
3.4 107 2.5 107
0 0
2.4 107 2.1 107
0 0
4.8 106 3.7 106
0 0
3.6 106 2.4 106
0 0
þ þ þ þ þ þ 9.7 106
7.6 103
þ þ þ þ þ þ 8.6 106
2.6 104
þ þ þ þ þ þ 6.2 105
5.3 103
þ þ þ þ þ þ 5.2 105
2.6 104
þ þ þ 2.4 107
þ 5.3 104
þ þ þ 1.6 107
þ þ 3.1 104
þ þ 3.9 106
þ 9.8 103
þ þ þ 2.6 106
þ 9.6 104
þ þ þ þ
þ þ
þ þ þ þ
þ þ
þ þ þ þ
þ þ þ
þ þ
The numbers and species of endophytic bacteria were determined in the plants watered for 15 days (greenhouse studies) or 20 days (field trials) with half-strength Hoagland’s solution containing 400 mg toluene liter-1. a Numbers of the cultivable endophytic bacteria grown on toluene, or Ca3(PO4)2, or CAS, from per gram (fresh weight) of plant material. The data are expressed as the means from five independent experiments with similar results. b Numbers of the cultivable endophytic bacteria containing the C23O gene per gram (fresh weight) of plant material. The data are expressed as the means from five independent experiments with similar results. c Species containing a C23O gene, or capable of phosphate solubilization, or siderophore production.
20 days (field trials) with half-strength Hoagland’s solution containing 400 mg toluene liter1. Cultivable endophytic bacteria able to grow on toluene as the only source of carbon and containing a C23O gene, were found in the plants inoculated with strain FX2 but not in their non-inoculated controls. Six different strains (B1eB6) were isolated from these endophytic bacteria carrying the C23O gene. The morphological and biochemical properties and 16S rDNA sequences of the six strains were nearly identical to those of B. cepacia, Burkholderia brasilensis, P. fluorescens, Pseudomonas migulae, Pseudomonas putida and Pseudomonas oryzihabitans, respectively (Table 1 and results not shown). A greater number of phosphate-solubilizing and siderophore-producing endophytic bacteria were found in the corn and wheat inoculated with strain FX2 than in their controls (Table 1). To examine the correlation between plant biomass responses and the magnitude of C23O-containing endophytic bacteria, we determined the numbers of C23O-containing endophytic bacteria and the plant biomass at indicated time points. A direct correlation was observed in greenhouse experiments. It was found that the increase in the magnitude of C23O-containing endophytic bacteria correlated well with plant biomass. Although plant biomass increased with time in the uninoculated control plants, increased levels of plant biomass in inoculated groups were higher than those in control groups (Table 2). A similar correlation between plant
biomass responses and the magnitude of C23O-containing endophytic bacteria was also found in field trials (Table 3). 3.3. HGT among endophytic bacteria The fact that C23O-containing endophytes were found in inoculated groups and not in control groups suggests that horizontal transfer of C23O gene occurred among endophytic communities of the plants and the C23O gene was originated from strain FX2. Comparison of phylogenetic trees based on patristic distances and sequence alignment illustrated that the C23O genes isolated from the six different strains were phylogenetically more closely related than the 16S rDNAs from their hosts (Fig. 2A, B). Generally, horizontal gene transfer (HGT) would be indicated if the lineage of the target gene is phylogenetically more closely related than that of the hosts. The phylogeny of 16S rDNA is putatively considered representative of the phylogeny of the bacterium possessing it. Therefore, the C23O gene might have been transferred horizontally among the natural endophytic bacteria of corn and wheat. To obtain more evidence for HGT, the cells of the above six strains and the FX2 strain were lysed, and plasmids were isolated. It was found that each of the six C23O positive strains isolated from inoculated plants possessed a large plasmid, which had similar HindIII restriction patterns (Fig. 3A, B). Strains B1 and FX2
Table 2 A correlation between plant biomass and the magnitude of bacteria with C23O (in greenhouse experiments). 5 days
Corn Wheat
inoculated controls inoculated controls
10 days
15 days
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
1.1 0.8 0.5 0.5
3.4 104 0 6.4 103 0
2.6 1.1 1.3 1.1
1.8 106 0 2.4 105 0
4.2 1.5 2.1 1.6
2.4 107 0 3.7 106 0
The data are expressed as the means from five independent experiments with similar results.
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Table 3 A correlation between plant biomass and the magnitude of bacteria with C23O (in field trials). 10 days
Corn Wheat
Inoculated Controls Inoculated Controls
15 days
20 days
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
(WtWo)/Wo
No. of cells with C23O
1.2 0.8 0.5 0.5
3.4 105 0 7.2 104 0
2.8 1.2 1.4 1.1
1.7 107 0 1.1 106 0
3.2 1.7 1.9 1.2
2.7 107 0 3.2 106 0
The data are expressed as the means from five independent experiments with similar results.
were identical with morphological and biochemical properties, 16S rDNA and C23O gene sequences and plasmid profiles. Therefore, it is likely that strain B1 is the FX2 strain re-isolated from the inoculated plants. Furthermore, filter mating assays were performed among those C23O-containing endophytes. First, filter mating assays were carried out between different cells of FX2. The filter matings, with the cured antibiotic-resistant and plasmidless FX2 strain as the recipient and its parent FX2 strain as the donor, were done on a selective medium with toluene as a single carbon source. The transconjugants that grew on toluene were obtained and transfer frequencies were calculated. Plasmid and hybridization profile analysis showed that a large plasmid and a C23Oencoding sequence on the plamid were detected in transconjugant colonies, as in donor colonies (Fig. 4A, B). Transfer of a C23O gene between strain FX2 cells occurred at a mean frequency of 9.84 101 per donor (Table 4). When FX2 served as the donor and the rifampin-resistant B3 strain as the recipient, a large plasmid and a C23O-encoding sequence on the plamid were also detected in transconjugant colonies (Fig. 4). Evidently, transfer frequency between strains FX2 and P. fluorescens strain B3 was lower than that between different cells of strain FX2 (Table 4). Other filter mating assays results were shown in Table 4. Taken together, it was proved that the plasmid containing the C23O gene is able to transferred between the FX2 cells, from FX2 to other endophytic bacteria, between the six endophytes each other. Therefore, the C23O gene was transferred horizontally from FX2 to the natural endophytic communities of corn and wheat.
Fig. 2. Phylogenetic trees of 16S rDNA (A) and C23O genes (B) of B. cepacia (B1), B. brasilensis (B2), P. fluorescens (B3), P. migulae (B4), P. putida (B5) and P. oryzihabitans (B6).
4. Discussion The use of bacteria to clean up environmental pollutants has gained momentum in past years although obstacles are present before they can be applied in field-scale projects (Berg et al., 2005; Newman and Reynolds. 2005; van der Lelie et al., 2005). In our previous work, B. cepacia strain FX2, a strain of endophytic bacterium able to degrade toluene and phenol, was isolated from a corn plant (Z. mays).The strain was found to possess a large plasmid encoding the catechol 2, 3-dioxygenase (C23O), a key enzyme in the degradation pathway of monocyclic aromatic compounds (Wang et al., 2007). In this paper we describe experiments to investigate the potential applications of strain FX2 to important crop plants, corn and wheat. Greenhouse and field trials showed that plants produced more biomass when the FX2 strain was introduced in corn and wheat (Fig. 1A, B). Furthermore, we found in the evapotranspiration measurements that much less toluene was released from the plants inoculated by the FX2 (Fig. 1C). Those results suggested that the growth promotion and reduction in phytotoxicity observed was a result of the inoculation of the plants with strain FX2. Cultivable endophytic bacteria able to grow on toluene as the only source of carbon and containing a C23O gene were found in the plants inoculated with strain FX2 but not in their non-inoculated controls (Table 1). Many strains of these bacteria were often reported to be isolated from different plants, able to promote host plant growth, produce antifungal metabolite, degrade organic pollutants and possess C23O genes (Nejad and Johnson, 2000; Sessitsch et al., 2004; Taghavi et al., 2005, 2009; Kavino et al., 2007; Mendes et al., 2007; Wang et al., 2007). Furthermore, a greater number of phosphate-solubilizing and siderophoreproducing endophytic bacteria were found in the corn and wheat inoculated with strain FX2 than in their controls (Table 1). Phosphorus is one of the major essential macronutrients for biological growth and development and phosphate-solubilizing microorganisms may therefore play an important role in supplying phosphate to plants in a more environmentally-friendly and sustainable manner (Rodríguez et al., 2006; Khan et al., 2007). Production of siderophores by bacteria is important in the suppression of plant pathogens and induction of systemic resistance in plants (Compant et al., 2005). Our results suggested that growth promotion and disease suppression might be more effective in the plants inoculated with FX2. It was found that the increase in the number of C23O-containing endophytic bacteria was in agreement with plant biomass in greenhouse experiments and field trials (Table 2), indicating that a direct correlation occurred between plant biomass responses and the magnitude of C23O-containing endophytic bacteria. Comparison of phylogenetic trees illustrated that the C23O gene might have been transferred horizontally among these C23O-containing endophytic bacteria isolated from these corn and wheat plants inoculated by FX2. Indeed, these strains possessed a large plasmid and similar restriction enzyme-digested plasmid patterns
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Y. Wang et al. / Soil Biology & Biochemistry 42 (2010) 1051e1057
Fig. 3. Plasmid analysis for endophytic bacterial strains B1eB6 and FX2 (lanes 1e7). The plasmids (A) isolated from endophytic bacterial strains and the HindIII-digested plasmids (B) were separated on a 0.7% agarose gel. Lane 8, molecular size markers.
(Fig. 3A, B). Filter mating assays further showed that the plasmid containing the C23O gene was able to be transferred among these isolated endophyte strains containing strain FX2 (Fig. 4A, B; Table 4). Therefore, the C23O gene has been transferred horizontally from FX2 to the natural endophytic communities of corn and wheat. The increasing number of C23O-containing endophytic bacteria, and of phosphate-solubilizing and siderophore-producing endophytic bacteria, in corn and wheat, might be a possible result of horizontal transfer of the C23O gene among those endophytic bacteria. We assume that horizontal transfer of the genetic determinants for the degradation of toluene from strain FX2 to the endophytic communities of the plants, might contribute to a reduction in phytotoxicity, thus creating microenvironments
which phosphate-solubilizing and siderophore-producing endophytic bacteria prefer to colonize. In summary, induction of toluene degradation and growth promotion and potential for disease suppression were found in the corn and wheat as a possible result of HGT within endophytic bacteria. This is very important for crop plants. Our results have revealed that horizontal transfer of genetic information required for the efficient degradation of a pollutant by the endophytic population might not only contribute to pollutant degradation, but also have other beneficial effects on plant, such as growth promotion and disease suppression. HGT, associated with an endophytic bacterium containing a transferable plasmid carrying the genetic information encoding the desired metabolic properties, is very
Fig. 4. Electrophoresis analysis of plasmid profiles (A) and Southern hybridization to plasmid profiles (B) for HGT between different cells of strain FX2 and between strains FX2 and B3. Lanes: 1 and 5, donor strain (the original parent FX2); 2 and 6, recipient strains (the toluene-negative, plasmidless and antibiotic-resistant FX2 and B3); 3 and 7, the transconjugant FX2 and B3.
Y. Wang et al. / Soil Biology & Biochemistry 42 (2010) 1051e1057
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Table 4 Transfer frequencies among endophytic bacteriaa. Recipient strains
Donor strains B1(FX2)
B1(FX2) B2 B3 B4 B5 B6
9.84 1.98 2.49 1.80 3.20 9.33
101 101 101 101 101 102
B2 9.12 5.32 2.44 1.40 3.10 9.22
B3
102 101 101 101 101 102
2.52 2.93 9.02 2.90 2.41 8.82
B4
101 101 101 101 101 102
1.37 1.88 2.61 6.77 2.34 9.54
B5
101 101 101 101 101 102
3.00 3.45 2.90 2.71 6.42 1.99
B6
101 101 101 101 101 101
9.82 8.99 8.51 1.03 1.97 4.70
102 102 102 101 101 101
a Transfer frequencies were measured by dividing the number of transconjugants by the viable counts of donor cells in the mating mixture at the end of filter mating assays. Average values were obtained from three mating experiments for each donor-recipient pair. The names of different strains were referred to as Fig. 2.
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