Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.     

Analysis of genetic structure in soil populations of Rhizobium leguminosarum recovered from the USA and the UK

Although many studies have shown that animal-associated bacterial species exhibit linkage disequilibrium at chromosomal loci, recent studies indicate that both animal-associated and soil-borne bacterial species can display a nonclonal genetic structure in which alleles at chromosomal loci are in linkage equilibrium. To examine the situation in soil-borne species further, we compared genetic structure in two soil populations of Rhizobium leguminosarum bv. trifolii and two populations of R. leguminosarum bv. viciae from two sites in Oregon, with genetic structure in R. leguminosarum bv. viciae populations recovered from peas grown at a site in Washington, USA, and at a site in Norfolk, UK. A total of 234 chromosomal types (ET) were identified among 682 strains analysed for allelic variation at 13 enzyme-encoding chromosomal loci by multilocus enzyme electrophoresis (MLEE). Chi-square tests for heterogeneity of allele frequencies showed that the populations were not genetically uniform. A comparison of the genetic diversity within combined and individual populations confirmed that the Washington population was the primary cause of genetic differentiation between the populations. Each individual population exhibited linkage disequilibrium, with the magnitude of the disequilibrium being greatest in the Washington population and least in the UK population of R. leguminosarum bv. viciae. Linkage disequilibrium in the UK population was created between two clusters of 9 and 23 ETs, which, individually, were in linkage equilibrium. Strong linkage disequilibrium between the two major clusters of 8 and 12 ETs in the Washington population was caused by the low genetic diversity of the ETs within each cluster relative to the inter-cluster genetic distance. Because neither the magnitude of genetic diversity nor of linkage disequilibrium increased as hierarchical combinations of the six local populations were analysed, we conclude that the populations have not been isolated from each other for sufficient time, nor have they been exposed to enough selective pressure to develop unique multilocus genetic structure.     

Genetic Structure of Rhizobium leguminosarum biovar trifolii and viciae Populations Found in Two Oregon Soils under Different Plant Communities

An investigation was carried out to determine the genetic structure in soil populations of Rhizobium leguminosarum bv. trifolii and viciae at each of two Oregon sites (A and C) that were 1 km apart. Although the soils were similar, the plant communities were quite different because grazing by domestic animals had been allowed (site A) or prevented (site C). Analysis of allelic variation at 13 enzyme-encoding loci by multilocus enzyme electrophoresis delineated 202 chromosomal types (ETs) among a total of 456 isolates representing two populations of R. leguminosarum bv. trifolii (AT and CT) and two populations of R. leguminosarum bv. viciae (AV and CV). Regardless of their site of origin or biovar affiliation, isolates of the same ET were confirmed to be more closely related to each other than to isolates of other ETs by repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences and the PCR technique. Despite the wide range in densities of the Rhizobium populations (<10² to >10⁵/g of soil), their overall genetic diversities were similar (mean genetic diversity, 0.45 to 0.51), indicating that low-density populations of soil-borne bacterial species are not necessarily of little genetic diversity. Linkage disequilibrium analysis revealed significant multilocus structure (nonrandom associations of alleles) within each of the four populations. From a combination of cluster and linkage disequilibrium analyses, a total of eight distinct groups of ETs were defined in the four populations. Two groups (I and III) contributed significant numbers of ETs and isolates to each population. The two populations of R. leguminosarum bv. viciae (AV and CV) exhibited similar genetic structures despite existing at different densities, in different plant communities, and in the presence (CV) or absence (AV) of their local Vicia hosts. In contrast, total linkage disequilibrium was partitioned differently in two biovar populations occupying the same soil (AV and AT), with disequilibrium in the latter being due entirely to the presence of group V.     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

Elevated atmospheric CO2 alters microbial population structure in a pasture ecosystem

An increase in concentration of atmospheric CO₂ is one major factor influencing global climate change. Among the consequences of such an increase is the stimulation of plant growth and productivity. Below‐ground microbial processes are also likely to be affected indirectly by rising atmospheric CO₂ levels, through increased root growth and rhizodeposition rates. Because changes in microbial community composition might have an impact on symbiotic interactions with plants, the response of root nodule symbionts to elevated atmospheric CO₂ was investigated. In this study we determined the genetic structure of 120 Rhizobium leguminosarum bv. trifolii isolates from white clover plants exposed to ambient (350 μmol mol^?1) or elevated (600 μmol mol^?1) atmospheric CO₂ concentrations in the Swiss FACE (Free‐Air‐Carbon‐Dioxide‐Enrichment) facility. Polymerase Chain Reaction (PCR) fingerprinting of genomic DNA showed that the isolates from plants grown under elevated CO₂ were genetically different from those isolates obtained from plants grown under ambient conditions. Moreover, there was a 17% increase in nodule occupancy under conditions of elevated atmospheric CO₂ when strains of R. leguminosarum bv. trifolii isolated from plots exposed to CO₂ enrichment were evaluated for their ability to compete for nodulation with those strains isolated from ambient conditions. These results indicate that a shift in the community composition of R. leguminosarum bv. trifolii occurred as a result of an increased atmospheric CO₂ concentration, and that elevated atmospheric CO₂ affects the competitive ability of root nodule symbionts, most likely leading to a selection of these particular strains to nodulate white clover.     

Reticulated and epidemic population genetic structure of Rhizobium etli biovar phaseoli in a traditionally managed locality in Mexico

We conducted a multilocus enzyme electrophoresis (MLEE) study to assess the genetic structure of the nitrogen-fixing bacteria Rhizobium etli bv. phaseoli . We analysed the genetic variation at 10 enzyme-encoding chromosomal loci of 482 isolates from root nodules of Phaseolus vulgaris and P. coccineus bean plants. The isolates were obtained from six traditionally managed agricultural plots in two localities in the State of Puebla, in Central Mexico. The total mean genetic diversity ( H _E) for the six plots was 0.531. Among the 482 isolates collected, 126 distinctive multilocus genotypes (electrophoretic types [Ets]) were obtained, and approximately half of the isolates are represented by five widespread ETs. A significant degree of genetic differentiation among the six plots ( G _ST = 0.072) and between the two localities ( G _ST = 0.022) was detected. The main part of the observed variability (70%) was found among the isolates within the plants. The cluster analysis revealed two deeply diverging lineages, separated at a genetic distance of 0.7. When a multilocus linkage disequilibrium analysis was performed at different hierarchical levels, we found significant linkage disequilibrium, but when the analysis was performed for the genotypes within the two diverging lineages, we found evidence of recombination. We propose for R. etli bv. phaseoli a reticulated and epidemic genetic structure, in which few genotypes increase in frequency to produce numerically dominant clones, and genetic exchange occurs mainly among genotypes within each lineage.     

Genotypic and Phenotypic Comparisons of Chromosomal Types within an Indigenous Soil Population of Rhizobium leguminosarum bv. trifolii

The relative genetic similarities of 200 isolates of Rhizobium leguminosarum bv. trifolii recovered from an Oregon soil were determined at 13 enzyme loci by multilocus enzyme electrophoresis (MLEE). These isolates represented 13 antigenically distinct serotypes recovered from nodules formed on various clover species. The MLEE-derived levels of relatedness among isolates of R. leguminosarum bv. trifolii were found to be in good agreement with the levels of relatedness established by using repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the PCR technique and with levels of relatedness from previously published DNA reassociation studies. BIOLOG substrate utilization patterns showed that isolates within an electrophoretic type (ET) were phenotypically more similar to each other than to isolates of other ETs. The soil isolates were represented by 53 ETs which could be clustered into seven groups (groups B, E, G, H1, H2, I, and J). Evidence for multilocus structure within the population was obtained, and group B was identified as the primary creator of the disequilibrium. Of 75 isolates belonging to the nodule-dominant serotype AS6 complex, 72 were found in group B. Isolates WS2-01 and WS2-02 representing nodule-dominant serotypes recovered from subclover grown at another Oregon site were also found in group B. Isolates representing the most numerous ETs in group B (ETs 2 and 3) were either suboptimally effective or completely ineffective at fixing nitrogen on six different clover species. Another four groups of isolates (groups A, C, D, and F) were identified when 32 strains of diverse origins were analyzed by MLEE and incorporated into the cluster analysis. Group A was most dissimilar in comparisons with other groups and contained strain USDA 2124 (T24), which produces trifolitoxin and has unique symbiotic characteristics.     

Genotyping by sequencing suggests overwintering of Peronospora destructor in southwestern Québec,Canada

Several Peronospora species are carried by wind over short and long distances, from warmer climates where they survive on living plants to cooler climates. In eastern Canada, this annual flow of sporangia was thought to be the main source of Peronospora destructor responsible for onion downy mildew. However, the results of a recent study showed that the increasing frequency of onion downy mildew epidemics in eastern Canada is associated with warmer autumns, milder winters, and previous year disease severity, suggesting overwintering of the inoculum in an area where the pathogen is not known to be endogenous. In this study, genotyping by sequencing was used to investigate the population structure of P. destructor at the landscape scale. The study focused on a particular region of southwestern Québec—Les Jardins de Napierville—to determine if the populations were clonal and regionally differentiated. The data were characterized by a high level of linkage disequilibrium, characteristic of clonal organisms. Consequently, the null hypothesis of random mating was rejected when tested on predefined or nonpredefined populations, indicating that linkage disequilibrium was not a function of population structure and suggesting a mixed reproduction mode. Discriminant analysis of principal components performed with predefined population assignment allowed grouping P. destructor isolates by geographical regions, while analysis of molecular variance confirmed that this genetic differentiation was significant at the regional level. Without using a priori population assignment, isolates were clustered into four genetic clusters. These results represent a baseline estimate of the genetic diversity and population structure of P. destructor.     

Microsatellite markers for Sclerotinia subarctica nom. prov., a new vegetable pathogen of the High North

Eight polymorphic microsatellite loci were isolated from the ascomycete fungus Sclerotinia subarctica nom. prov. In Alaska, this pathogen causes white mold vegetable diseases sympatrically with the cosmopolitan and closely related Sclerotinia sclerotiorum. Eighteen alleles were observed across the 41 isolates tested and ranged from two to three alleles per locus. Together, the alleles from the eight polymorphic loci yielded only four haploid multilocus genotypes and exhibited significant linkage disequilibrium, reflecting extreme selfing and clonal vegetative reproduction.     

Genetic variation and recombination in Penicillium miczynskii and Eupenicillium species

Reproduction in the genus Penicillium is thought to be completely asexual. Sexual reproduction, as occurs in the related genus Eupenicillium, is thought to provide evolutionary benefits because it allows for new combinations of alleles and therefore increases the amount of variation within the species. This hypothesis was tested using inter-simple sequence repeats (ISSRs) to assess the amount of intraspecific and intra-population variation within Penicillium miczynskii and the closely related Eupenicillium shearii. The data for both genera were also used to test for clonal reproduction against the null hypothesis of panmixis, using measures of genotypic diversity, linkage disequilibrium and phylogenetic tree length. The ISSR fingerprints indicated that the 70 Eupenicillium strains actually included two distinct species, Eupenicillium shearii and Eupenicillium tropicum sp. nov., each represented by populations in both Costa Rica and India. While none of the species or populations were found to be randomly recombining, the relative strength of the clonal component differed among the species. Penicillium miczynskii had the smallest clonal component, with the highest genotypic diversity, lowest Index of Association, 40 % of alleles non-randomly associated, and phylogenetic tree length closer to that of recombined data sets than to the minimum possible. Eupenicillium tropicum showed nearly complete clonal reproduction with the lowest genotypic diversity and 100 % of alleles non-randomly associated in both populations. On the other hand, it also had the greatest amount of intraspecific variation, with as little as 38 % similarity among strains. The results indicate that Penicilliumspecies may, on rare occasion, genetically recombine; the regular occurrence of meiosis in the life cycle of Eupenicilliumspecies does not facilitate recombination; and the greatest amount of genetic variation was not associated with recombination, but with clonal propagation.     

Clonal reproduction shapes evolution in the lizard malaria parasite Plasmodium floridense

The preponderant clonal evolution hypothesis (PCE) predicts that frequent clonal reproduction (sex between two clones) in many pathogens capable of sexual recombination results in strong linkage disequilibrium and the presence of discrete genetic subdivisions characterized by occasional gene flow. We expand on the PCE and predict that higher rates of clonal reproduction will result in: (1) morphologically cryptic species that exhibit (2) low within‐species variation and (3) recent between‐species divergence. We tested these predictions in the Caribbean lizard malaria parasite Plasmodium floridense using 63 single‐infection samples in lizards collected from across the parasite's range, and sequenced them at two mitochondrial, one apicoplast, and five nuclear genes. We identified 11 provisionally cryptic species within P. floridense, each of which exhibits low intraspecific variation and recent divergence times between species (some diverged approximately 110,000 years ago). Our results are consistent with the hypothesis that clonal reproduction can profoundly affect diversification of species capable of sexual recombination, and suggest that clonal reproduction may have led to a large number of unrecognized pathogen species. The factors that may influence the rates of clonal reproduction among pathogens are unclear, and we discuss how prevalence and virulence may relate to clonal reproduction.     

<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii rosR</Emphasis> is required for interaction with clover,biofilm formation and adaptation to the environment

Background  

Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.     

LOSS OF SEX IN CLONAL POPULATIONS OF A FLOWERING PLANT,DECODON VERTICILLATUS (LYTHRACEAE)

The loss of traits that no longer increase fitness is a pervasive feature of evolution, although detailed studies of the genetic, developmental, and evolutionary factors involved are few. Most perennial plants practice both sexual and clonal reproduction, and it has been hypothesized that populations with little sexual recruitment may lose the capacity for sexual reproduction by fixing mutations that disable one or more of the many processes involved in sex. The clonal, tristylous aquatic plant, Decodon verticillatus, exhibits marked geographical variation in sexual recruitment. Populations at the northern limit of the range are usually monomorphic for style length consist of single genotypes, and produce almost no seed, due, in part, to environmental conditions that inhibit pollination, fertilization, and seed maturation. Controlled crosses in a greenhouse provided evidence for greatly reduced sexual capacity in an exclusively clonal, monomorphic population. Plants from this infertile population produced only 3–18% as many seeds per pollination as fertile populations. Observations of pollen tube growth indicated that infertility is due to severe reductions in pollen tube numbers both early after pollination and later when pollen tubes were traversing the ovary, due primarily to the inability of pistils to support normal tube growth. A three-year greenhouse experiment comparing fertility, survival, and growth of F1 progenies produced from reciprocal crosses between plants from the infertile population and those from nearby fertile populations suggested that the genetic basis for infertility is simple and may involve a single recessive mutation. In addition, the results did not reveal any association between infertility and other aspects of survival and vegetative vigor. The infertile genotype was likely fixed in the population through founder effect rather than indirect selection resulting from antagonistic pleiotropy or direct selection of advantages associated with reduced investment in sexual reproduction. A broader comparison of sexual fertility in 15 clonal, monomorphic populations and five genotypically diverse, trimorphic populations under greenhouse conditions revealed substantial infertility in all but one monomorphic population. Populations varied somewhat in the stage at which infertility was expressed, however, pollen tube growth was impaired in all populations. These results provide strong support for the hypothesis that complex traits like sex are degraded by mutation when they no longer increase fitness.     

The IS1167 Insertion Sequence Is a Phylogenetically Informative Marker Among Isolates of Serotype 6B Streptococcus pneumoniae

The phylogenetic utility of the IS1167 insertion sequence was examined with restriction fragment length polymorphism (RFLP) analyses of a sample of 50, predominantly invasive, capsular serotype 6B Streptococcus pneumoniae isolates previously characterized by multilocus enzyme electrophoresis (MLEE). The strains represented a genetically diverse assemblage of 34 distinct clonotypes composed of 26 restriction fragment types and 23 multilocus enzyme types. All isolates carried the IS1167 insertion sequence, with an average of 9.5 copies. The cross-classification of isolates based on RFLP and MLEE typing schemes was 81% concordant. Phylogenetic analyses demonstrated a significant (P < 0.0001) association between strains of a given RFLP lineage with those of a given MLEE lineage. A significant correlation (P < 0.00004) was also found between the proportion of restriction fragments shared by any given pair of isolates and their genetic distances estimated from the MLEE data. Parity between the two genetic markers implied that the sampled isolates were in linkage disequilibrium. The existence of nonrandom associations among genetic loci was confirmed by Monte Carlo analyses of the MLEE data. These studies, thus, demonstrated that invasive pneumococcal isolates of a single capsule type recovered on a regional scale can retain a largely clonal population structure over a period of 8 years. The ability to detect linkage disequilibrium and generate relatively congruent dendrograms based on distance and parsimony methods suggested that the restriction fragment data were robust to phylogenetic analysis. Received: 20 May 1997 / Accepted: 20 November 1997     

VARIATION IN CONTRIBUTIONS OF ASEXUAL REPRODUCTION TO THE GENETIC STRUCTURE OF POPULATIONS OF THE SEA ANEMONE METRIDIUM SENILE

Northeastern North American populations of the sea anemone Metridium senile show marked differences in levels of genotypic diversity. Comparisons with expectations generated by computer simulation show that some populations are genotypically as diverse as expected for sexually reproducing populations with free recombination, whereas others are significantly less diverse than expected, despite efforts to avoid collecting clonemates. These reductions in diversity are not attributable to the Wahlund effect; they probably result from extensive clonal reproduction. Reduced genotypic diversity may be produced by low rates of recruitment of planktonic larvae, followed by asexual proliferation. The resulting founder effect may account for previously documented random allele-frequency variation between adjacent populations. It is presently uncertain whether the few genotypes found in some populations are particularly well-adapted to local conditions.     

Population structure of two beetle-associated yeasts: comparison of a New World asexual and an endemic Nearctic sexual species in the <Emphasis Type="Italic">Metschnikowia</Emphasis> clade

The genetic structure of two related yeast species, one sexual and one asexual, was compared using polymorphic DNA markers. Although both yeasts propagate by asexual budding of haploid cells, Metschnikowia borealis reproduces sexually when compatible strains come in contact. To what extent this has occurred in nature was not known. As Candida ipomoeae is a closely related, asexual species, the two yeasts provide an excellent model system to assess the role of sexual reproduction in a biogeographic context. Natural isolates of the two species were characterized using several polymorphic DNA markers. As predicted for an organism whose reproduction is strictly clonal, C. ipomoeae exhibited low haplotype diversity, high linkage disequilibrium, and high population differentiation. In contrast, M. borealis had unique haplotypes in most isolates, lower population differentiation, and little linkage disequilibrium, demonstrating that sexual recombination is prevalent. Geographic gradients were identified in both species, indicating that historical and climatic factors both play a role in shaping the populations. The spatial structure is also thought to be influenced by the ecology of the small floricolous beetles (family Nitidulidae) that vector the yeasts. For example, Hawaiian strains of C. ipomoeae show evidence of having undergone a genetic bottleneck, most likely when the vector was introduced to the islands. The two haplotypes found in Hawaii were nearly identical and were also found in North and Central America. M. borealis had a more continuous distribution where the genetic markers follow latitudinal and longitudinal gradients.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Sequence Analysis of Hypothetical Lysine Exporter Genes of Rhizobium leguminosarum bv. trifolii from Calamine Old Waste Heaps and Their Evolutionary History

The aim of this study was to identify heavy metal detoxification system in Rhizobium leguminosarum bv. trifolii isolated from Trifolium repens inhabiting old (70–100 years) Zn–Pb waste heaps in Poland by PCR reaction with czcD1 and czcD2 primers. By sequence analysis, four different genotypes of obtained amplicons were identified among eight examined isolates. Their sequence similarity ranged 91–99 %. They indicated the highest sequence identity to the hypothetical lysine exporter gene of R. leguminosarum bv. trifolii WSM1325 (91–97 %) and 76–81 % sequence similarity to hypothetical lysine exporter genes of R. leguminosarum bv. trifolii WSM2304 and R. etli CFN42 and CIAT652. On phylogenetic tree of obtained amplicons, all four studied R. leguminosarum bv. trifolii genotypes formed common monophyletic cluster with R. leguminosarum bv. trifolii WSM1325 at 100 % bootstrap support showing that all four amplicons obtained in PCR with czcD1 and czcD2 primers are fragments of hypothetical lysine exporter gene (lysE). We also suggest that Lys efflux exporter may participate in heavy metal transport out of R. leguminosarum bv. trifolii cells.     

The mass selection reservoir and sugarcane selection

Summary A method is proposed which extends the mass reservoir technique to the breeding of clonally propagated crops. The first phase produces a diverse array of clones by sexual recombination. Then the selection phase is conducted in one genotypically heterogeneous population. Such a population is termed a mass selection reservoir (MSR). In each generation of agricultural bulk planting, competitive ability is supplemented with a regime of artificial selection among propagules for fixing the rate at which each component genotype is advanced.A MSR programme has been initiated in sugarcane in Fiji. An analysis of the variation in selection characters demonstrated significant clonal effects at the single stalk (propagule) level. Sugar concentration was particularly repeatable on this basis. After two generations of selection, the MSR's performance at the population level at least equalled that of the best current commercial clone, Ragnar. It is therefore likely to include superior isolates of one or more clones.Two possible artificial selection methods are compared. These arise from either a linear (L) or multiplicative (M) combination of the two major selection criteria, sugar concentration and stalk weight. Although the M series differs genotypically from the L series, there is little difference to date in their respective population performances.     

The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress

Aims

The aim of this study was investigation of the response of R. leguminosarum bv. trifolii wild-type and its two rosR and pssA mutant strains impaired in exopolysaccharide (EPS) synthesis to oxidative stress conditions caused by two prooxidants: a superoxide anion generator- menadione (MQ) and hydrogen peroxide (H₂O₂).

Methods

The levels of enzymatic (catalase, superoxide dismutase, pectinase and β-glucosidase) and non-enzymatic (superoxide anion generator, formaldehyde, phenolic compounds) biomarkers were monitored using biochemical methods in both the supernatants and rhizobial cells after treatment with 0.3?mM MQ and 1.5?mM H₂O₂. The viability of bacterial cells was estimated using fluorescent dyes and confocal laser scanning microscopy. In addition, the effect of prooxidants on symbiosis of the R. leguminosarum bv. trifolii strains with clover was established.

Results

The tested stress factors significantly changed enzymatic patterns of the rhizobial strains, and the wild-type strain proved to be more resistant to these prooxidants than both pssA and rosR mutants. Significantly higher activities of both catalase and superoxide dismutase have been detected in these mutants in comparison to the wild-type strain. H₂O₂ and MQ also increased the levels of pectinase and β-glucosidase activities in the tested strains. Moreover, pre-incubation of R. leguminosarum bv. trifolii strains with the prooxidants negatively affected the viability of bacterial cells and the number of nodules elicited on clover plants.

Conclusions

EPS produced in large amounts by R. leguminosarum bv. trifolii plays a significant protective role as a barrier against oxidative stress factors and during symbiotic interactions with clover plants.