Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population. S. Tabacchioni, L. Chiarini, A. Bevivino, ...
MICROBIAL ECOLOGY Microb Ecol (2000) 40:169–176 DOI: 10.1007/s002480000015 © 2000 Springer-Verlag New York Inc.
Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population S. Tabacchioni, L. Chiarini, A. Bevivino, C. Cantale, C. Dalmastri Dipartimento Innovazione, Divisione Biotecnologie e Agricoltura, ENEA (Ente Nazionale per le Nuove Tecnologie, l’Energia e l’Ambiente) C.R. Casaccia,, 00060 Rome, Italy Received: 29 April 1999; Accepted: 27 January 2000; Online Publication: 28 August 2000
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The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT. From each medium, 50 randomly chosen colonies were isolated. On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B. cepacia species, whereas among PCAT isolates only 74% were assigned to the B. cepacia species. Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique. The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns. Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001). The results indicate that the two different isolation media select for B. cepacia populations with a different degree of genetic diversity. Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium.
Introduction Understanding the genetic diversity of a bacterial species in nature is shaped by the need to isolate pure cultures of microorganisms. Since cultivable bacteria are necessary for genetic studies and these represent only 0.1–20% of the bac-
Correspondence to: S. Tabacchioni; Fax: +39 06 30484808; E-mail: microbi@ casaccia.enea.it
teria present in microbial communities, it is likely that only a small portion of genetic diversity of a microbial species has been documented [1]. Even within the cultivable fraction of bacteria, obtaining microorganisms that are representative of the diversity existing in nature can be difficult as a result of the inherent bias due to culture media. Numerous selective and nonselective media have been used for enumerating and isolating soil and rhizosphere microorganisms. Previous work suggests that the choice of the
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medium has great impact on both the total count and the relative abundance of various taxonomic groups of microorganisms [3, 24]. Sørheim et al. [29] found that the populations of soil bacteria isolated on three different media were significantly different from each other. A recent paper by Caballero-Mellado et al. [5] hypothesizes that the limited genetic diversity observed in a population of Acetobacter diazotrophicus, recovered from different host species collected in widely separated regions of the world, could be explained, among other factors, by the high selectivity of the medium used for isolation. The present work aimed to investigate the bias associated with the choice of the medium used to isolate a natural population of Burkholderia cepacia from the rhizosphere of Zea mays. This report is a part of a greater study on biodiversity of natural populations of B. cepacia isolated from maize rhizosphere. B. cepacia is a particularly useful bacterial species to study the genetic polymorphism of microbial populations in a highly fluctuating environment such as the rhizosphere because of its ecological and genomic properties. In fact, B. cepacia has an unusual genomic organization characterized by the presence of multiple chromosomes regulated by separate control systems and by an extensive array of insertion sequences, which play a fundamental role in the ability to adapt to different environments by genetic transfer and mutations [23]. Since B. coppica is widespread in different environments, being reported both as plant growth promoting rhizobacterium [2, 8, 16, 19] and as an opportunistic human pathogen [18, 21, 22], studies regarding genetic diversity are important to assess the differences among populations isolated from different sources and to evaluate the risk associated with releasing these bacteria into the environment. Several studies regarding the structure and genetic diversity of B. cepacia populations isolated from different environments have been published [25, 34, 35], and we found that the genetic diversity of B. cepacia populations associated with maize roots was affected by such factors as plant development, soil type, maize cultivar, and localization on the root system [6, 7, 9]. Different selective media have been developed for the isolation of these bacteria from terrestrial, aquatic, and clinical environments [4, 15, 17, 36]. In particular, TB-T [15] and PCAT [4] media have been employed for the isolation of strains used for plant-growth promotion, biocontrol, and biodiversity studies. The selectivity of these media is due to the presence of specific nitrogen and carbon sources such as tryptamine and azelaic acid in PCAT, and antimicrobial agents such as trypan blue and tetracycline in TB-T. Based
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on these differences in selective properties, these media were chosen to assess the differences that choice of medium might impose on the evaluation of the diversity of a natural population of B. cepacia recovered from the environment. This report describes the genetic diversity of a natural population of B. cepacia recovered from the rhizosphere of maize on both PCAT and TB-T media. Strains isolated from maize roots were first assigned to the B. cepacia species based on the analysis of restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA) [7, 9, 13, 31]. Genetic diversity among strains assigned to the B. cepacia species was then checked by genomic fingerprints obtained using the Random Amplified Polymorphic DNA (RAPD) technique [32]. This PCR-based technique has been successfully applied to the study of genetic variation of bacterial populations isolated from different natural environments [6, 7, 9, 11, 14, 26, 30]. Data obtained by random amplification of genomic DNA were further investigated by the analysis of the molecular variance (AMOVA) method [10].
Materials and Methods Isolation of Burkholderia cepacia Strains Zea mays plants (cv. Doge, KWS) were cultivated in a field located at Dragoni, Caserta, Italy. Maize had been cultivated for 8 years in this field. The soil composition was as follows: sand 58%, clay 27%, silt 15%, organic C 1.97% (wt/wt); the pH was 5.43. Two maize plants were harvested after 53 days of growth, i.e., at the stage of tassel appearance. Roots were excised from plants and loosely adhering soil was removed. Afterward, roots were weighed, blended, and then resuspended in phosphate buffered saline (PBS, FlowLaboratoires). Serial dilutions of these suspensions were plated on the selective media PCAT (2 g azelaic acid, 0.2 g tryptamine, 0.1 g MgSO4 ⭈ 7H2O, 4 g KH2PO4, 4 g K2HPO4, 0.02 g yeast extract, 15 g agar, in 1 L of distilled water, pH 5.5) and TB-T (2 g glucose, 1 g L-asparagine, 1 g NaHCO3, 0.5 g KH2PO4, 0.1 g MgSO4 ⭈ 7H2O, 0.05 g trypan blue, 0.02 g tetracycline, 15 g agar, in 1 L of distilled water, pH 5.5) to estimate and isolate microorganisms belonging to the B. cepacia species. Plates were incubated at 28°C for 4 days. In order to isolate B. cepacia strains for further genetic analysis, a total of 100 colonies (50 from each medium) were randomly picked up from the same dilution of root sample, i.e., 100-fold dilution from both PCAT and TB-T plates containing 50 to 100 colonies. The isolates were subjected to single colony isolation on PCAT or TB-T media and cryopreserved at -80°C in 10% glycerol for further analysis.
Amplification and Restriction Analysis of 16S rDNA (ARDRA) Amplification of 16S rDNAs were performed directly on 2 µl of cell lysates obtained from single colonies according to the procedure
171 described by Di Cello et al. [9]. Restriction analysis of the amplified 16S rDNA with the enzyme AluI enabled us to assign to the B. cepacia species all the isolates showing the same ARDRA pattern as that obtained from reference strain B. cepacia LMG11351 [7, 9].
Table 1. Data from isolation, identification, and RAPD fingerprinting of B. cepacia isolates recovered from maize roots
Growth of B. cepacia Isolates on PCAT, TB-T, and modified TB-T Media All putative B. cepacia strains isolated on PCAT were subsequently streaked on TB-T medium, whereas all those recovered on TB-T were subsequently streaked on PCAT medium. All strains were plated in triplicate and the plates incubated at 28°C for 72 h. B. cepacia strains isolated on PCAT were then plated in triplicate on different formulations of TB-T medium obtained by omitting and/ or adding specific components to the basal TB-T medium (see Table 3). Microbial growth was observed after 96 h incubation at 28°C.
Random Amplified Polymorphic DNA (RAPD) Analysis Amplification reactions of genomic DNAs were performed directly on the same cell lysate suspensions used to amplify the 16S rDNA, by using the 10-mer primer AP5 (5’-TCCCGCTGCG-3’) having an 80% GC content, as previously described [9]. The amplification patterns were manually analyzed.
Statistics The presence or absence of RAPD markers (1 for the presence or 0 for the absence of each band in the gels) for each strain was used to calculate the genetic distance for each pair of strains. The Euclidean metric measurement (E) of Excoffier et al. [10], as defined by Huff et al. [20], was used as follows: E = e2xy = n(1 - 2nxy/2n), where 2nxy is the number of markers shared by two individuals, and n is the total number of polymorphic sites. The Analysis of Molecular Variance (AMOVA) procedure, based on 10,000 permutations, was applied to estimate the variance components for RAPD patterns, partitioning the variation among and within samplings. All analyses were processed using the Arlequin program kindly provided by L. Excoffier (University of Geneva), [28]. The Euclidean distances calculated between all possible combinations of strains taken in pairs were further analyzed using oneway ANOVA (StatView 512+, Brain Power Inc., CA, USA) to compare the level of internal variability between the two samples studied. To further investigate the genetic relationships among all the isolated B. cepacia strains, an analysis of RAPD data was performed using the programs FITCH (for applying the Fitch–Margoliash method), NEIGHBOR (for applying the Neighbor-joining method), and CONSENSE (for computing consensus trees) of the PHYLIP 3.5c software package [12]. More than 2,000 Neighbor and 30 Fitch bootstrap replications were performed to determine the statistical significance of the obtained branches. The Euclidean dis-
No. of B. cepacia isolated
No. of B. cepacia showing a RAPD pattern
Sample
PCAT
TB-T
PCAT
TB-T
PCAT
TB-T
Plant I Plant II
25 25
25 25
19 13
24 20
16 12a
20 18a
a
No. of haplotypes
One haplotype was found to be common to the two groups of isolates.
tance matrix was used as an input file. The obtained trees were then displayed using the TREEVIEW program [27].
Results Isolation and Identification of Bacterial Strains Recovered from the Maize Rhizosphere A total of 50 isolates were recovered on both PCAT and TB-T culture media (25 isolates from the root system of each plant). Each isolate was subjected to PCR amplification and restriction analysis of 16S rDNA (ARDRA). PCR amplification of 16S rDNA of each of the 100 isolates gave rise to an amplification fragment of about 1,450 bp. Data obtained by restriction analysis of amplified 16S rDNA with the enzyme AluI enabled us to assign all the 50 TB-T isolates to the B. cepacia species (Table 1). By contrast, six different ARDRA patterns were recognized among the 50 PACT isolates, with only 37 of the 50 isolates (Table 1) analyzed producing an ARDRA pattern typical of the B. cepacia group (data not shown). The strain B. cepacia LMG11351 (also named PHP7) was used to obtain a reference ARDRA pattern [9]. It was not possible to assign isolates showing the other five ARDRA patterns to a given bacterial species because of the lack of known reference strains. The numbers of B. cepacia recovered from the rhizosphere of each plant on PCAT and TB-T media are shown in Table 2. Values of colony forming units (CFU) per g (fresh weight) of root obtained on both the two different selective media were corrected based on the ARDRA results obtained for PCAT and TB-T isolates, respectively. Similar values of colonization were obtained, indicating that there are no differences in the total number of B. cepacia recovered on the two distinct selective media used. Growth of B. cepacia Isolates on PCAT, TB-T, and Modified TB-T Media All isolates recovered from the two different isolation media and assigned to the B. cepacia species by means of ARDRA
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Table 2. Enumeration of bacterial populations on PCAT and TB-T media CFU per g (fresh weight) of root
Table 3. Formulations of the different modified TB-T media and % of growth of B. cepacia strains isolated on PCAT Componentsa
% of isolates assigned to the B. cepacia speciesa
Sample
PCAT
TB-T
PCAT
TB-T
Plant I Plant II
2.0 × 106 1.9 × 105
9.6 × 105 2.2 × 105
76 72
100 100
a
The isolates recovered both from PCAT and TB-T medium (50 for each medium and 25 for each plant) were assigned by means of ARDRA analysis to the B. cepacia species.
analysis were cultured both on PCAT and TB-T media to confirm their ability to grow on these two different selective media reported to be specific for the B. cepacia species. Results showed that whereas all B. cepacia isolated on TB-T were able to grow on PCAT, only 64% of B. cepacia strains isolated on PCAT were able to grow on TB-T. Therefore, all B. cepacia isolated on PCAT were cultured on modified TB-T media (Table 3) in order to provide information on which of the components could limit their growth on this medium. The results indicated that all strains tested were able to grow on TB-T medium lacking tetracycline, whereas only 61 to 65% of the strains were able to grow on the other modified TB-T media (Table 3).
Random Amplified Polymorphic DNA (RAPD) Analysis All the isolates assigned to the B. cepacia species were fingerprinted by the RAPD technique using the 10-mer primer AP5, obtaining a total of 32 amplified DNA for PCAT isolates and 44 for TB-T isolates, respectively (Table 1). A representative example of the amplification patterns obtained is shown in Fig. 1. The choice of this primer was based on previous experiments in which the amplification patterns obtained with AP5 exhibited a higher level of polymorphism than those obtained using other primers [9]. RAPD analyses were repeated three times and consistent results were obtained (data not shown). Amplification of the genomic DNA of B. cepacia strains gave rise to a total of 44 markers whose dimensions ranged from 216 to 1,661 bp. The RAPD patterns obtained were represented by the presence or absence of these 44 markers. The patterns obtained were compared with each other and a Euclidean distance matrix was constructed (data not shown). The matrix showed a high genetic variability within the population since 64 different haplotypes were found among the 76 strains analyzed. From the distance matrix E, we extracted AMOVA data showing that
Media
Tc
Tb
Ap
Tr
Aa
Glu
Ye
% of growth
TB-T TB-T (-Tc) TB-T (-Tb) TB-T (-Tb-Tc) TB-T (-Ap+Tr) TB-T (-Glu+Aa) TB-T (-Ap-Glu+ Tr+Aa) TB-T (+Ye)
+ + + +
+ + + +
+ + + + +
+ -
+
+ + + + + -
-
64 100 65 100 61 65
+ +
+ +
+
+ -
+ -
+
+
63 65
a
TC = 0.02 g/L tetracycline; Tb = 0.05 g/L trypan blue; Ap = 1.0 g/L asparagine; Tr = 0.2 g/L tryptamine; Aa = 2.0 g/L azelaic acid; Glu = 2.0 g/L glucose; Ye = 0.02 g/L yeast extract. The final pH of each medium was 5.5 ± 0.01.
most genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39 ± 3.95) were significantly higher (P < 0.0001) than those among TB-T isolates (9.36 ± 3.67). Genetic relationships among the 76 strains, calculated using the distance matrix E and the Fitch–Margoliash method, are presented as an unrooted tree (Fig. 2). The tree obtained has “bushlike” characteristics, as previously described for a bacterial population exhibiting frequent recombinations [33]. Moreover, different clusters can be recognized where PCAT isolates or TB-T isolates are predominant. An essentially identical tree was obtained when the Neighbor-joining method was used on the same data set (data not shown). Consensus trees were generated in which bootstrap values above 80% from over 2,000 Neighbor and 30 Fitch replications were obtained at the nodes, showing that the indicated branches were statistically significant (data not shown).
Discussion In this study, the bias related to the choice of isolation medium on the assessment of the genetic diversity of a natural B. cepacia population recovered from the rhizosphere of Z. mays was investigated. Two selective media, PCAT and TBT, were used to recover B. cepacia isolates from root samples. Our results show that the TB-T medium is more selective than the PCAT medium. In fact, on the basis of ARDRA analysis, all TB-T isolates were assigned to the B. cepacia
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Fig. 1. Electrophoretic patterns obtained by RAPD analysis of some B. cepacia strains isolated on PCAT (a) and TB-T (b) media with primer AP5. First lane (a) and last lane (b): 123-bp molecular size marker ladder.
species, whereas among PCAT isolates only 74% were assigned to this species. The percentage of B. cepacia isolates recovered on PCAT medium is consistent with the results of Di Cello et al. [9], which were obtained in a study on the biodiversity of a B. cepacia population isolated from maize roots at different plant growth stages. Hagedorn et al. [15]
defined the formulation of TB-T medium as highly selective based on observing the high efficiency of the recovery of B. cepacia strains from low dilutions of soil samples (101 to 103). The different levels of selectivity observed between PCAT and TB-T media may be attributed to their different chemi-
Fig. 2. Unrooted tree showing the genetic relationships among 76 B. cepacia isolates recovered from the rhizosphere of Zea mays on PCAT and TB-T media. Each isolate was named using the progressive number of isolation preceded by P or T for PCAT and TB-T medium, respectively. The tree is based on the Euclidean distance matrix. Clustering was performed using the Fitch– Margoliash method. Essentially identical trees were obtained when the Consense program was applied on more than 2,000 Neighbor and 30 Fitch bootstrap replications.
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cal compositions. Hagedorn et al. [15] reported that the high selectivity of TB-T medium is based on the presence of the antimicrobial agents trypan blue and tetracycline. Our experiments, performed to determine if any particular compounds were critical for the growth of B. cepacia strains, revealed that all PCAT isolates were able to grow on TB-T medium only when tetracycline was omitted. This means that tetracycline could be selective not only for other bacterial species, but also within the same B. cepacia species. As PCAT and TB-T media differ in their level of selectivity for bacteria belonging to the B. cepacia species, it is of great importance to assess how this finding could influence further investigations on the genetic diversity of the isolated population. In fact, since PCAT appears to be less selective than TB-T medium not only at an interspecific but also at an intraspecific level, it may be recommended to be used when a great representativity of B. cepacia populations is required. To evaluate the bias due to the use of the two different isolation media on the assessment of genetic diversity of B. cepacia strains, RAPD analysis of all isolates assigned to this species was performed. RAPD fingerprinting revealed a high level of genetic diversity since among 76 strains analyzed, 64 distinct haplotypes were found. Similar percentages of haplotypes were found among isolates recovered from different plant samples on the two selective media. The RAPD variation observed between the two groups of isolates was analyzed by the AMOVA method. This method, successfully applied to biodiversity studies of bacterial populations isolated from different natural environments, is a very powerful tool for investigating genetic variation within intraspecific populations [9, 26]. The comparison between B. cepacia populations isolated from maize roots on two different selective media resulted in a significant (P < 0.001) value of variance component attributable to divergences between them (9.41%), indicating that the two isolation media select for B. cepacia populations having different levels of genetic diversity. Data derived from RAPD results were useful to construct a tree representing how the strains were genetically related. Although this tree does not imply phylogenetic relationships, it has considerable value for interpreting data, allowing us to define the genetic structure of the isolated population, and indicating that there are major differences between the strains isolated on the two media. The unrooted structure of the tree allowed us to consider the isolated strains as a freely recombinant population. These results are consistent with those previously obtained for populations adapted to living in soil and lotic environments where genetic recombinations seem to be highly frequent [7, 9, 34,
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35]. Since PCAT and TB-T isolates were generally grouped together in all trees, they indicate that there are major differences between the two media investigated. These results are statistically significant, as the consensus method applied among all isolates showed that the distribution of the clusters was conserved among all replicates so indicated by bootstrap values (data not shown). The higher mean genetic distance observed among PCAT isolates confirms that the B. cepacia strains recovered on this medium have a higher degree of genetic variability than those recovered on TB-T medium. Hagedorn et al. [15] suggested that the presence in PCAT medium of tryptamine and azelaic acid as sole nitrogen and carbon sources, respectively, could limit the diversity of B. cepacia biotypes that can be recovered because of the high specificity of these two compounds. On the basis of our results, we have demonstrated that although TB-T medium contains glucose as a carbon source, the degree of genetic diversity within this group of isolates is lower than that obtained using PCAT medium. We hypothesize that the high concentration of tetracycline in the TB-T medium, in combination with other specific compounds such as asparagine and trypan blue, represents a greater limiting factor than the presence of azelaic acid and tryptamine in PCAT medium, in terms of selectivity within the B. cepacia species. In conclusion, the results obtained indicate that the evaluation of the genetic diversity of a natural microbial population can be distorted by the use of a specific isolation medium. Thus, the choice of the isolation medium must be carefully evaluated before attempting isolation of microorganisms from environmental sources when genetic diversity has to be assessed.
Acknowledgments We thank Gabriella Seri for technical assistance and Drs. Armando and Maria Laura Desideri Gaveglio for providing plant samples. This work was partially supported by Consiglio Nazionale delle Ricerche (C.N.R.) (ECOMI 0639 n. 97.01533.CT06), and by Ministero delle Risorse Agricole, Alimentari e Forestali (MiRAAF)—Piano Nazionale Biotecnologie Vegetali—D. M. 125/7240/96.
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