Molecular markers to study competition and diversity of Rhizobium

The research described in this thesis was directed to the development of molecular identification and detection techniques for studying the ecology of Rhizobium, a nitrogen- fixing bacterium of agricultural importance. Competition of inoculant strains with indigenous microbes is a serious problem in agricultural practice and was therefore addressed in this work using the developed tools. Furthermore, various molecular techniques have been applied to analyse rhizobial populations nodulating common bean and a new species was characterized.In this chapter the results obtained are summarized and potential future applications are discussed.Development of gusA - and celB -minitransposons and their use in rhizobial competition studiesThe use of marker genes in rhizobial competition studies is reviewed in Chapter 1. Specific attention is given to the gusA gene, encoding β-glucuronidase (GUS). This gene is a highly suitable marker for studying plant-microbe interactions due to the absence of GUS activity in plants and in most bacteria that are of relevance in agriculture. In Chapter 2 the construction of several GUS transposons containing the marker gene in combination with different regulation systems to be used for ecological and genetic studies is described. The minitransposon mTn5SS gusA 20 contains the aph promoter which was demonstrated to be expressed in a wide variety of Gram-negative bacteria (de Bruijn and Lupski, 1984) and its use in Rhizobium resulted in high-level constitutive expression. Transposon mTn5SS gusA 21 is similar to mTn5SS gusA 20, except that it contains a unique site for Spe I, a rare-cutting enzyme in bacteria with high G+C contents such as rhizobia (Sobral et al., 1991). The use of mTn5SS gusA 2I therefore may be instrumental for the genetic mapping of insertions. The tac promoter was used to drive the expression of the gusA gene in the transposon mTn5SS gusA 11 resulting in high GUS activity in the free-living state. The transposons with constitutive gusA expression are optimal for studying rhizosphere colonization and for studying nodule occupancy in young plants. In order to reduce any metabolic load due to GUS production, the mTn5SS gusA 10 transposon with regulated gusA expression was constructed. It contains the tac promoter in combination with the lacl q repressor gene and gusA gene expression is repressed until an inducer, such as IPTG, is added. This regulation should avoid possible effects on the ecological fitness, Two transposons carrying symbiotically activated gusA genes, mTn5SS gusA 30 and mTn5SS gusA 31, were made by using the nifH promoter of a Rhizobium and a Bradyrhizobium strain, respectively. The nifH gene encodes the Fecomponent of nitrogenase and is only expressed in symbiotic or microaerobic conditions (Fischer, 1994). These constructs are recommended for longer-term nodule occupancy experiments. Furthermore, a promoter-less GUS transposon,mTn5SS gusA 40, is described that should be of use for molecular genetic studies as well as for screening bacteria for their response to specific environmental conditions or signals. The developed transposons carry a gene conferring resistance tospectinomycin and streptomycin that proved to be an appropriate marker for many strains. Nevertheless, few strains with an endogenous resistance exist. Therefore, the development of additional transposons conferring an alternative resistance would be advantageous. This may be realized by inserting the developed GUS expression cassettes into minitransposons: containing other antibiotic (de Lorenzo et al., 1990) or natural resistance markers (Herrero et al., 1990). Chapter 2 also addresses also theapplication of the different transposons in studies on root colonization and nodule occupancy, while various GUS assays are described in detail.Potential effects on the fitness of a strain due to insertion of a GUS transposon were evaluated in Chapter 3. Only few data exist on the impact of foreign genes on the fitness of an organism (Doyle et al., 1995) and before using any marker system for ecological studies its ecological effects have to be studied rigorously. In the case of Rhizobium, it is essential that the nodulation behaviour and competitive ability are maintained. The competitive abilities, nodulation characteristics, and growth rates of five independent derivatives of R. tropici strain CIAT899 marked with the gusA gene on minitransposon mTn5SS gusA 10 were determined relative to the parent strain. Insertion of mTn5SS gusA 10 did not affect the nodulation or nitrogen fixation efficiency of the wild-type strain. Nevertheless, the competitiveness index of the different gusA derivatives relative to the parental strain CIAT899 varied between isolates. One isolate was less competitive than the wild-type strain in three independent experiments, while the other isolates proved to be either equally competitive or more competitive. The utilization of this methodology to assess competitivity resulted in highly significant calculations as all the nodules on each plant were analysed for nodule occupancy. The results showed that the insertion of mTn5SS gusA 10 may have an impact on the ecological behaviour of a strain, but derivatives indistinguishable from the parent strain can be obtained. A primary selection of marked strains is recommended, which may be achieved by coinoculating the parent and the marked derivative in a one to one ratio and ensuring that the proportion of blue nodules does not differ significantly from the expected 50%.Furthermore, in Chapter 3, the detection of dual nodule occupancy is discussed. The appearance of partially stained nodules in mixed inoculum treatments but not in single strain treatments led to the conclusion that these were due to mixed infections. This was confirmed by nodule isolation and plating experiments. Partially stained nodules were also observed by Krishnan and Pueppke (1992) who reported that nodules were occupied by either a lacZ- marked or a non-marked R. fredii strain and X-gal was used for detection. However, the lacZ marker system has several disadvantages due to high background activity in plant and rhizobia whereas the gusA marker gene can be used to readily detect dual nodule occupancy on plant.A new marker gene system based on the celB gene is presented in Chapter 4. The celB gene has been isolated from the hyperthermophilic archaeon Pyrococcus juriosus and it encodes a thermostable and thermoactive β-glucosidase with a high β-galactosidase activity (Voorhorst et al., 1995). The latter enzyme activity can be used for the detection of rhizobia as endogenous background activity in plants as well as in bacteria can be easily eliminated by a heat treatment. Moreover, cheap histochemical substrates are available to determine β-galactosidase activity. The E. coli β-galactosidase gene, lacZ , has been used to monitor engineered soil bacteria (Drahos et al., 1986; Hartel et al., 1994) but was only found to be appropiate when used with Lac- bacteria. Transposons containing the ce1B gene were constructed in E. coli , based on the existing gusA transposons. The first transposon, mTn5SS celB 10 , contains the tac promoter which is regulated by the lacI q gene product and should reduce any metabolic stress to the marked strain due to marker gene activity. Transposon mTn5SS celB 31 carries celB expressed from a Bradyrhizobium nifH promoter and is active in nitrogen-fixing legume nodules. A third celB minitransposon, which contains the marker gene constitutively expressed is described elsewhere (Sessitsch et al., submitted). The celB marker gene system has several advantages in rhizobial competition studies over conventional techniques as the assay is simple to perform and the histochemical substrates are cheap. However, the greatest advantage is that gusA and celB marked strains can be localized simultaneously on a plant and a combined gusA/celB assay will enable studies of multi-strain rhizobial inocula competing with indigenous rhizobial populations. Although simultaneous detection of differently marked strains has been reported (Thompson et al., 1995; Bauchrowitz et al., 1996), the celB gene encoding the thermostable marker is better suited for double staining. In addition, a procedure has been described allowing detection of gusA and celB marked strains on plates (Sessitsch et al., submitted). In Chapter 4 the application of the celB marker gene is demonstrated for Rhizobium. However, because of the wide host range of the Tn5 based transposons and the portable expression signals this marking system is suitable for use in a variety of Gram-negative bacteria.The advantages of the different gusA and celB marker gene cassettes are discussed in Chapter 1, but also other reporter genes and their applications in studies on microbial ecology are presented. Moreover, the development of a GUS Gene Marking Kit is reported. This kit was made particularly for agronomists and microbiologists in developing countries who are not familiar with molecular techniques and who do not have the resources to establish this methodology in their laboratories. Meanwhile, a CelB Gene Marking Kit is also available that can be used either in combination with or instead of the GUS Gene Marking Kit (FAO/IAEA, 1992-1997).Ecology of rhizobia nodulating common beanIn the fields around the Seibersdorf laboratory common bean has not been cultivated during the last decades but is well nodulated. In earlier studies, common bean rhizobia populations in this soil have been found to be very competitive in nodulation (see Chapter 1). They were shown to outcompete R. tropici strain CIAT899 when inoculating Phaseolus vulgaris at an inoculation level of 10 5cells per seed. When increasing inoculation to 10 8cells per seed, 65% of the nodules were still occupied by the native strains. In Chapter 5, rhizobial populations isolated from common bean nodules grown in the Seibersdorf soil were characterized. Molecular methods targeting the whole genome such as PCR with repetitive primers were used, and specific chromosomal loci such as the 16S rRNA gene or the 16S- 23S rDNA intergenic spacer were analyzed. Plasmid profiles and Southern hybridization with a nifH probe gave information on symbiotic regions. In addition, the nodulation host range was determined. Two distinct groups were found, one of them was classified as R. etli according to the RFLP analysis of the 16S rRNA gene and because of the presence of three copies of the nifH gene. The members of the second group could not be assigned to any recognized common bean nodulating Rhizobium species, i.e. R. leguminosarum bv. phaseoli, R. etli and R. tropici, but showed high similarity to Rhizobium sp. (Phaseolus) strain R602sp isolated in France (Laguerre et al., 1993). Isolates of this group also formed nodules on cowpea, Leucaena and Gliricidia. For a long time, R. leguminosarum bv. phaseoli was believed to be the only microsymbiont in Europe, but recently R. tropici and two new species have been found in French soils (Laguerre et al., 1993; Amarger, 1994). These studies and the results obtained in Chapter 5 indicate that strains originating in Mesoamerica could establish well in European soils. However, diversity was not high among the Austrian isolates due to the long absence of the host plant in this soil.The focus of Chapter 6 is on the taxonomy and phylogeny of the Austrian isolates showing high similarity to Rhizobium sp. R602sp. A Mexican common bean isolate, FL27, was included in this study since Laguerre et al. (1993) found the partial 16S rRNA gene sequence of R602sp to be identical to FL27. Sequence analysis of the 16S rRNA gene, determination of the copy number and heterogeneity of ribosomal genes, plasmid profiles and DNA-DNA hybridization resulted in valuable taxonomic information on these strains. Based on these results it was proposed that these strains belong to a new species that was named R. pueblae sp. nov., referring to the state Puebla, Mexico, where FL27 was isolated. The Mexican, French and Austrian isolates showed very similar 16S rDNA sequences with a maximum of two nucleotide substitutions. Comparison of the 16S rDNA sequences with those of other bacteria revealed highest similarity to R. leguminosarum strain IAM 12609, R. sp. OK50 and to R. etli. Although phylogenetic dendrograms always positioned R. pueblae sp. nov. strains in the vicinity of the above-mentioned species, the new species was found to belong to a lineage different from those of described Rhizobium species. The whole DNA relatedness among the European isolates was very high but showed lower levels with FL27, probably due to the presence of different plasmids. The DNA homology to other bean-nodulating species was very low. R. pueblae sp. nov. strains possess at least three copies of the 16S rRNA gene and the ribosomal gene organization is different to other species. Despite the high competitive ability of some strains in the Seibersdorf soil, little is known on the agronomic value of this species.Concluding remarksTo improve biological nitrogen fixation, adapted efficient nitrogen-fixing plant genotypes, effective rhizobial inoculants and appropiate agricultural management practices are needed. Consequently, plant breeders, microbiologists, soil scientists, agronomists as well as farmers have to cooperate in order to achieve this goal.The selection of superior sources of natural plant genetic variability and plant breeding in the presence of rhizobia instead of applying nitrogen fertilizers may lead to the identification of high-fixing lines. Other desirable traits such as disease resistance or stress tolerance could be transferred by appropiate breeding methods or by genetic engineering. Soils, in which legumes are cultivated, vary greatly and can be opposed to various environmental stresses such as low pH or high temperature. Efforts have been undertaken to develop appropiate plant genotypes whereas for a long time the stress tolerance of inoculant strains has not been considered. Inoculant strains have been recommended based on good symbiotic performance in a particular environmentbeen recommended based on good symbiotic performance in a particular environment while the soil status or the agroecological zone of the final application has not been taken into account. The vast genetic pool of natural soils containing not yet identified strains and species can provide a variety of inoculant strains that may show better performance in the field. A strong correlation between the indigenous population size and the nodule occupancy of the inoculant strain has been established (Thies et al., 1991). However, the effect of the diversity of indigenous rhizobia on competition has not been determined. Probably, different strategies are needed in order to outcompete highly dominant field isolates or to achieve successful competition of an inoculant strain with a variety of different indigenous strains that are present in low numbers. In addition, inoculation practices have to be developed that are convenient for the farmer and that allow distribution of the introduced strain into the entire rooting zone.It seems that the various aspects important for efficient nodulation and nitrogen fixation are presently not linked sufficiently. A more rational selection of efficient and competitive strains could be realized when a database existed containing data on soil properties, environmental conditions, rhizobial diversity and population size, as well as on the competitive ability and effectiveness of rhizobial strains in combination with particular plant genotypes. Furthermore, these data could be of use for the development of new strains or plants by genetic engineering.In this thesis, the development of new methods to assess rhizobial competition is presented. These marker gene-based techniques are appropiate for the large-scale screening of inoculant strains but can be also used for genetic analysis of a variety of Gram-negative bacteria. Molecular methods have facilitated the analysis of strains nodulating common bean and resulted in the description of a new Rhizobium species that includes strains with possible beneficial properties