Abstract
In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.
The genus Burkholderia is an important component of the soil microbial community (18). For instance, Burkholderia cepacia was first described as the causative agent of onion soft rot (11), but several strains of this species are not phytopathogenic and play an important role in promoting plant health (5). Moreover, many species belonging to the genus Burkholderia have the ability to produce compounds with antimicrobial activity (13, 20, 28, 30) and thus can be used as biocontrol agents with activity against phytopathogens. In addition, other Burkholderia strains have been shown to be plant-growth-promoting rhizobacteria (42), and introduction of Burkholderia species in crops such as maize and sorghum has resulted in increases in both root and shoot dry weights (4, 14). The mechanisms involved in plant growth promotion may range from production of phytohormones to fixation of atmospheric nitrogen, as shown for Burkholderia vietnamiensis (42). Estrada-De Los Santos et al. (21) recently showed that nitrogen fixation is a common property in the genus Burkholderia, after they isolated new diazotrophic Burkholderia species which were phylogenetically unrelated to B. vietnamiensis from coffee and maize plants. Furthermore, nonculturable bacteria belonging to the genus Burkholderia have been found as endosymbionts of arbuscular mycorrhizal fungi (6), and genes involved in nitrogen fixation have been shown to be active at least during the germination of spores (33). The endosymbionts were detected mainly in members of the family Gigasporaceae and were present as homogeneous populations throughout the fungal life cycle (7). In addition to all these features, the great nutritional versatility of the genus Burkholderia, reflected in its ability to use a wide range of organic compounds as carbon sources (24), certainly contributes to its capacity to successfully compete for root exudates and thus to efficiently colonize habitats such as the plant root. This nutritional versatility has also led to the use of Burkholderia strains for biodegradation of environmental pollutants (22).
Concomitant with the use of members of the genus Burkholderia, there is increasing concern about the risk of using this group of bacteria in processes such as biological control and bioremediation (12) since some species are important pathogens in cystic fibrosis patients (25, 43).
The list of species belonging to the genus Burkholderia has changed several times since 1992, when Yabuuchi et al. (47) proposed that seven former Pseudomonas species belonging to so-called rRNA group II should be grouped in this new genus, based on the results of a polyphasic taxonomic study. Now, the genus Burkholderia comprises 21 species (1, 8, 43, 46, 48). Moreover, several strains previously identified as B. cepacia were grouped in the so-called B. cepacia complex, which comprises at least six genomic species or genomovars (16).
The microbial community in soil is inherently complex, and assessments performed with such a complex population do not always reveal its specific components. Moreover, cultivation-based methods are limited because they do not assess the nonculturable fraction of the soil microbiota (44). Hence, an analysis of distinct phylogenetic groups of bacteria on the basis of soil DNA is required, because such an analysis reduces the complexity and thereby facilitates assessment of the subgroups that contribute to the total diversity (35). This can be achieved by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments, a technique that has been widely used to assess the diversity of various phylogenetic groups (34).
Burkholderia spp. have been identified by techniques such as DNA-DNA hybridization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amplified fragment length polymorphism fingerprinting, and PCR performed with primers with different degrees of specificity (3, 17, 43). In addition, assessment of Burkholderia species in environmental samples has been based mainly on analyses of the B. cepacia complex in which restriction fragment length polymorphism analyses of the recA gene or 16S rDNA have been used (5, 18, 23). However, none of these methods, including the PCR-based approaches, can be used to directly evaluate the diversity of the genus Burkholderia in natural settings.
The main goal of this work was to develop a method, based on PCR-DGGE, that allows direct analysis of the diversity of Burkholderia species in environmental samples. To achieve this goal, primers specific for the genus Burkholderia were developed based on the 16S rRNA gene. The PCR system was first evaluated for specificity and sensitivity by using DNAs isolated from Burkholderia and non-Burkholderia species. After optimization of the method, the PCR-DGGE system specific for Burkholderia was used to assess the diversity of this genus in soil samples.
The genus Burkholderia is an important component of the soil microbial community (18). For instance, Burkholderia cepacia was first described as the causative agent of onion soft rot (11), but several strains of this species are not phytopathogenic and play an important role in promoting plant health (5). Moreover, many species belonging to the genus Burkholderia have the ability to produce compounds with antimicrobial activity (13, 20, 28, 30) and thus can be used as biocontrol agents with activity against phytopathogens. In addition, other Burkholderia strains have been shown to be plant-growth-promoting rhizobacteria (42), and introduction of Burkholderia species in crops such as maize and sorghum has resulted in increases in both root and shoot dry weights (4, 14). The mechanisms involved in plant growth promotion may range from production of phytohormones to fixation of atmospheric nitrogen, as shown for Burkholderia vietnamiensis (42). Estrada-De Los Santos et al. (21) recently showed that nitrogen fixation is a common property in the genus Burkholderia, after they isolated new diazotrophic Burkholderia species which were phylogenetically unrelated to B. vietnamiensis from coffee and maize plants. Furthermore, nonculturable bacteria belonging to the genus Burkholderia have been found as endosymbionts of arbuscular mycorrhizal fungi (6), and genes involved in nitrogen fixation have been shown to be active at least during the germination of spores (33). The endosymbionts were detected mainly in members of the family Gigasporaceae and were present as homogeneous populations throughout the fungal life cycle (7). In addition to all these features, the great nutritional versatility of the genus Burkholderia, reflected in its ability to use a wide range of organic compounds as carbon sources (24), certainly contributes to its capacity to successfully compete for root exudates and thus to efficiently colonize habitats such as the plant root. This nutritional versatility has also led to the use of Burkholderia strains for biodegradation of environmental pollutants (22).
Concomitant with the use of members of the genus Burkholderia, there is increasing concern about the risk of using this group of bacteria in processes such as biological control and bioremediation (12) since some species are important pathogens in cystic fibrosis patients (25, 43).
The list of species belonging to the genus Burkholderia has changed several times since 1992, when Yabuuchi et al. (47) proposed that seven former Pseudomonas species belonging to so-called rRNA group II should be grouped in this new genus, based on the results of a polyphasic taxonomic study. Now, the genus Burkholderia comprises 21 species (1, 8, 43, 46, 48). Moreover, several strains previously identified as B. cepacia were grouped in the so-called B. cepacia complex, which comprises at least six genomic species or genomovars (16).
The microbial community in soil is inherently complex, and assessments performed with such a complex population do not always reveal its specific components. Moreover, cultivation-based methods are limited because they do not assess the nonculturable fraction of the soil microbiota (44). Hence, an analysis of distinct phylogenetic groups of bacteria on the basis of soil DNA is required, because such an analysis reduces the complexity and thereby facilitates assessment of the subgroups that contribute to the total diversity (35). This can be achieved by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments, a technique that has been widely used to assess the diversity of various phylogenetic groups (34).
Burkholderia spp. have been identified by techniques such as DNA-DNA hybridization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amplified fragment length polymorphism fingerprinting, and PCR performed with primers with different degrees of specificity (3, 17, 43). In addition, assessment of Burkholderia species in environmental samples has been based mainly on analyses of the B. cepacia complex in which restriction fragment length polymorphism analyses of the recA gene or 16S rDNA have been used (5, 18, 23). However, none of these methods, including the PCR-based approaches, can be used to directly evaluate the diversity of the genus Burkholderia in natural settings.
The main goal of this work was to develop a method, based on PCR-DGGE, that allows direct analysis of the diversity of Burkholderia species in environmental samples. To achieve this goal, primers specific for the genus Burkholderia were developed based on the 16S rRNA gene. The PCR system was first evaluated for specificity and sensitivity by using DNAs isolated from Burkholderia and non-Burkholderia species. After optimization of the method, the PCR-DGGE system specific for Burkholderia was used to assess the diversity of this genus in soil samples.
| Original language | English |
|---|---|
| Pages (from-to) | 1595-1603 |
| Number of pages | 8 |
| Journal | Applied and environmental microbiology |
| Volume | 68 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Apr-2002 |
| Externally published | Yes |