Inoculation with indigenous <Emphasis Type="Italic">rhizobium</Emphasis> strains increases yields of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) in northern Spain,although its efficiency is affected by the tillage system

Common bean (Phaseolus vulgaris L.) crops hold the potential to obtain higher yields by enhancing their biological nitrogen fixation (BNF) with Rhizobium. However in contrast to other legumes, common bean has shown a lack of positive response to inoculation with Rhizobium in many cases. This has led to a limited use of rhizobial inoculants in this crop, especially in Europe. The adaptation of bacterial strains to the rhizosphere is a key factor in the success of any inoculant, especially in a promiscuous legume such as common bean. This research aimed at increasing common bean yields via inoculation with effective indigenous Rhizobium leguminosarum strains. Three highly effective strains (LCS0306, LBM1123 and ZBM1008) which were selected according to their effectiveness at BNF in hydroponic conditions were separately inoculated onto common bean in a field experiment. The experiment was carried out under three environments and three tillage systems: conventional-tillage (CONVT), no-tillage (NT) and a cover-crop (CC). The grain yield observed with seed inoculation was significantly higher than the yield obtained with uninoculated seed under CONVT and CC. However, under NT inoculation had no effect. Furthermore, under CONVT and CC, inoculation with R. leguminosarum LCS0306 produced even higher yields than those obtained in nitrogen-fertilised or control plots. This is the first attempt to explain the inoculation performance of common bean under different tillage systems in Europe. A gene–based hypothesis has been used to explain the effectiveness of indigenous common bean rhizobia as nitrogen fixers in this crop.     

Serologic characteristics of certain root-nodule bacteria of legumes

Rhizobium trifolii organisms associated with eleven species ofTrifolium were isolated and studied by means of antigen-antibody agglutination and immune diffusion techniques. Ten serologic types were differentiated from the eleven species of nativeTrifolium. Certain serologic types of rhizobia were widely distributed among the native species ofTrifolium, whereas other rhizobial types were found only on one or two species. The distribution pattern appears to be independent of the proportion of each type present in the soil population. This may indicate specificity of selection between a host legume and serologic types of rhizobia.     

Biochemical evidence bearing on the domestication of<Emphasis Type="Italic">Phaseolus</Emphasis> (Fabaceae) beans

The genusPhaseolus (Fabaceae) consists of some 50 species, all of which are distributed in the Americas. Four of these contain cultigens.P. vulgaris (common bean),P. lunatus (lima bean),P. acutifolius (tepary bean),P. coccineus subsp.coccineus (runner bean); andP. coccineus subsp.polyanthus (no English vernacular name). Biochemical markers—phaseolin seed storage protein and isozymes—have provided new evidence on the organization of the first three species. Domestication has possibly caused a strong reduction in genetic diversity inP. vulgaris andP. acutifolius. BothP. vulgaris andP. lunatus cultivars result from at least two independent domestications, in Mesoamerica and in the Andes. These two species consist of two gene pools, each of which includes wild ancestors and their respective cultivated descendants. Our findings suggest the need for additional emphasis on genetic conservation of wild ancestors and their use in breeding programs and for a comparison of inter-gene pool vs. intra-gene pool crosses in breeding programs.     

Divergent Evolution of Symbiotic Bacteria: Rhizobia of the Relic Legume <Emphasis Type="Italic">Vavilovia formosa</Emphasis> Form an Isolated Group within <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">viciae</Emphasis>

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.     

Recent changes to the classification of symbiotic,nitrogen-fixing,legume-associating bacteria: a review

The Rhizobia are collectively comprised of gram negative soil bacteria that have the ability to form symbiotic nitrogen-fixing root and/or stem nodules in association with leguminous plants. The taxonomy of these bacteria is continually in a state of flux, in large part due to rapid development of refined molecular biology techniques. The isolation and characterization of new, and often different, legumes-nodulating bacteria on a variety of plant hosts has resulted in the naming of many new rhizobial species. Here we update the taxonomy of the legume-nodulating bacteria and describe newly identified rhizobia capable of nodulating edible legumes and legume trees. In 1990, there was only one bacterial species that was known to nodulate common bean worldwide (Rhizobium leguminosarum sv. phaseoli), one species that nodulated faba bean (Rhizobium leguminosarum sv. viciae), and two species that nodulated soybean (Bradyrhizobium japonicum and Rhizobium fredii). Today, nearly 14, 11, 6, 5, 5, 4, 3 and 2 species have been defined that are capable of nodulating common bean, soybean, cowpea, chickpea, peanut, lentils, faba bean and pea, respectively. The recent use of whole genome based taxonomy (genomotaxonomy) will surely change how we define this important group of bacteria. The identification of several rhizobial species that are able to nodulate and fix nitrogen with edible legumes may enhance the production of these crops and can compensate for worldwide deficiencies in human nutritional needs in the future.     

Lima bean nodulates efficiently with <Emphasis Type="Italic">Bradyrhizobium</Emphasis> strains isolated from diverse legume species

Lima bean (Phaseolus lunatus L.) is an important legume species that establishes symbiosis with rhizobia, mainly of the Bradyrhizobium genus. The aim of this study was to evaluate the efficiency of rhizobia of the genus Bradyrhizobium in symbiosis with lima bean, in both Leonard jars and in pots with a Latossolo Amarelo distrófico (Oxisol). In the experiment in Leonard jars, 17 strains isolated from nodules of the three legume subfamilies, Papilionoideae (Vigna unguiculata, Pterocarpus sp., Macroptilium atropurpureum, Swartzia sp., and Glycine max), Mimosoideae (Inga sp.), and Caesalpinioideae (Campsiandra surinamensis) and two uninoculated controls, one with a low concentration (5.25 mg L^?1) and another with a high concentration (52.5 mg L^?1) of mineral nitrogen (N) were evaluated. The six strains that exhibited the highest efficiency in Leonard jars, isolated from nodules of Vigna unguiculata (UFLA 03–144, UFLA 03–84, and UFLA 03–150), Campsiandra surinamensis (INPA 104A), Inga sp. (INPA 54B), and Swartzia sp. (INPA 86A), were compared to two uninoculated controls, one without and another with 300 mg N dm^?3 (NH₄NO₃) applied to pots with samples of an Oxisol in the presence and absence of liming. In this experiment, liming did not affect nodulation and plant growth; the INPA 54B and INPA 86A strains stood out in terms of shoot dry matter production and provided increases of approximately 48% in shoot N accumulation compared to the native rhizobia populations. Our study is the first to indicate Bradyrhizobium strains isolated from the three legume subfamilies are able to promote lima bean growth via biological nitrogen fixation in soil conditions.     

Diversity and symbiotic divergence of endophytic and non-endophytic rhizobia of <Emphasis Type="Italic">Medicago sativa</Emphasis>

Knowledge of rhizobium diversity is helping to enable the utilization of rhizobial resources. To analyze the phenotypic and genetic diversity and the symbiotic divergence of rhizobia of Medicago sativa, 30 endophytic and non-endophytic isolates were collected from different parts of five alfalfa varieties in three geographic locations in Gansu, China. Numerical analyses based on 72 phenotypic properties and restriction fragment length polymorphism (RFLP) fingerprinting indicated the abundant phenotypic and genetic diversity of the tested strains. According to the phylogenetic analysis of 16S RNA, atpD, glnII, and recA gene sequences, Rhizobium and Ensifer were further classified into four different genotypes: Rhizobium radiobacter, Rhizobium sp., Rhizobium rosettiformans, and Ensifer meliloti. The differences in architecture and functioning of the rhizobial genomes and, to a lesser extent, environment diversification helped explain the diversity of tested strains. The tested strains exhibited similar symbiotic feature when inoculated onto M. sativa cvs. Gannong Nos. 3 and 9 and Qingshui plants for the clustering feature of their parameter values. An obvious symbiotic divergence of rhizobial strains was observed in M. sativa cvs. Longzhong and WL168HQ plants because of the scattered parameter values. Their symbiotic divergence differed according to alfalfa varieties, which indicated that the sensitivity of different alfalfa varieties to rhizobial strains may differ. Most of the tested strains exhibited plant growth-promoting traits including phosphate solubilization and production of indole-3-acetic acid (IAA) when colonizing plant tissues and soil.     

Nodulation ability in different genotypes of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> by rhizobia from California agricultural soils

Phaseolus lunatus is the second economically most important species of the genus Phaseolus. It carries out N fixation through symbiosis with rhizobia. However, it is unclear whether P. lunatus can nodulate with native rhizobia from soils where this legume is not native or was not cultivated previously. Thus, this study assessed the ability of 14 geographically distant lima bean genotypes to nodulate with rhizobia from three California agricultural soils: without a history of legumes or P. lunatus cultivation, with a history of legumes as a cover crop, and with a history of P. lunatus cultivation. Nodulation only occurred on genotypes grown in the soil with a history of P. lunatus planting. The analysis of variance of nodulation traits showed that the genotype effect was highly significant in all the traits measured. Shoot biomass had a higher correlation with nodule size and nodule weight than with nodule number. In addition, shoot biomass and leaf N content were positively correlated with nodule coloration and with nodule position close to the main root of the plant. This study suggests that agricultural soils from California do not appear to have native rhizobia able to nodulate P. lunatus, which suggests the need to inoculate, at least initially, the seeds at planting in order to establish the population of rhizobia. Also, geographically distant lima bean genotypes have different responses to nodulating bacteria and it suggests that future studies to test these genotypes across different environments should be pursued.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.     

Diversity and symbiotic effectiveness of <Emphasis Type="Italic">Adesmia</Emphasis> spp. root nodule bacteria in central and southern Chile

Legumes in the genus Adesmia are wild species with forage and medicinal potential. Their nitrogen fixation efficiency depends on their association with soil bacteria known as rhizobia. The aim of this work was to assess the diversity and symbiotic effectiveness of root nodule bacteria from Adesmia boronioides, Adesmia emarginata and Adesmia tenella from different regions of Chile. Adesmia spp. nodules were collected from seven sites obtaining 47 isolates, which resulted in 19 distinct strains. The diversity of the strains was determined via partial sequencing of the dnaK, 16srRNA and nodA genes. The strains were authenticated as root nodule bacteria on their original host and assessed for symbiotic effectiveness on A. emarginata and A. tenella. The strains from Adesmia tenella clustered within the Mesorhizobium clade. Adesmia boronioides nodulated with Mesorhizobium sp., Rhizobium leguminosarum and Bradyrhizobium sp. The rhizobia from A. emarginata were identified as Burkholderia spp, which was symbiotically ineffective on this species and on A. tenella. Strains isolated from Adesmia emarginata nodules, but unable to induce nodulation, were identified as Labrys methylaminiphilus. Labrys strain AG-49 significantly increased root dry weight in A. emarginata. The nodA genes from Adesmia strains were unique and correlated to legume host. A. emarginata was effectively nodulated by Bradyrhizobium AG-64 and A. tenella by Mesorhizobium strains AG-51 and AG.52. It is concluded that Adesmia emarginata, A. tenella and A. boronioides are associated to diverse bacterial symbionts and selection of an effective inoculant is a key step to assist Adesmia spp. adaptation and restoration.     

Rhizobial diversity associated with the spontaneous legume <Emphasis Type="Italic">Genista saharae</Emphasis> in the northeastern Algerian Sahara

Genista saharae is an indigenous shrub legume that spontaneously grows in the northeastern Algerian Sahara. It is known for efficient dune fixation and soil preservation against desertification, due to its drought tolerance and its contribution to sustainable nitrogen resources implemented by biological N₂-fixation. In this study, the root nodule bacteria of G. saharae were investigated using phenotypic and phylogenetic characterization. A total of 57 rhizobial strains were isolated from nodules from several sites in the hyper-arid region of Metlili and Taibet (east Septentrional Sahara). They all nodulate G. saharae species but they differed in their symbiotic efficiency and effectiveness. The genetic diversity was assessed by sequencing three housekeeping genes (atpD, recA and 16S rRNA). The majority of isolates (81 %) belonged to the genus Ensifer (previously Sinorhizobium), represented mainly by the species Ensifer meliloti. The next most abundant genera were Neorhizobium (17 %) with 3 different species: N. alkalisoli, N. galegae and N. huautlense and Mesorhizobium (1.75 %) represented by the species M. camelthorni. Most of the isolated strains tolerated up to 4 % (w/v) NaCl and grew at 45 °C. This study is the first report on the characterization of G. saharae microsymbionts in the Algerian Sahara.     

Promotion of Peanut Growth by Co-inoculation with Selected Strains of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Azospirillum</Emphasis>

The ability of inoculated rhizobial strains to increase root nodulation of host legumes often depends on their competitiveness with existing native soil strains. Results of studies to date on rhizobial inoculation for improvement of peanut (Arachis hypogaea L.) production in Argentina have been inconsistent and controversial. In many cases, nodulation and yield of peanut crops have been increased by inoculation of specific rhizobial strains. Native peanut-nodulating strains are generally present in soils of agricultural areas, but their growth-promoting effect is often lower than that of inoculated strains. Many species of the genus Bradyrhizobium interact in a host-specific manner with legume species and form nitrogen-fixing root nodules. Other free-living rhizobacteria such as species of the genus Azospirillum are facultatively capable of interacting with legume roots and promoting plant growth. We evaluated and compared the effects of various single inoculation and co-inoculation treatments on peanut growth parameters in greenhouse and field experiments. In the greenhouse studies, co-inoculation with various Bradyrhizobium strains (native 15A and PC34, and recommended peanut inoculant C145), and Azospirillum brasilense strain Az39 generally resulted in increases in the measured parameters. The growth-promoting effect of 15A was similar to or higher than that of C145. In the field studies, 15A-Az39 co-inoculation had a greater promoting effect on measured growth parameters than did C145-Az39 co-inoculation. Our findings indicate that careful selection of native rhizobacterial strains adapted to peanut soils is useful in strategies for growth promotion, and that 15A in particular is a promising candidate for future inoculant formulation.     

<Emphasis Type="Italic">Mesorhizobium jarvisii</Emphasis> sv. astragali as predominant microsymbiont for <Emphasis Type="Italic">Astragalus sinicus</Emphasis> L. in acidic soils,Xinyang, China

Background and aims

Common bean (Phaseolus vulgaris L.) nodulates with a wide range of rhizobia. Amongst these is Bradyrhizobium, which is inefficient but able to induce profuse nodulation on this crop. Based on this observation, we tested whether co-inoculating bradyrhizobia with a more standard common bean symbiont, Rhizobium tropici, could stimulate growth and nodulation of common bean, thus contributing to a more effective symbiosis.

Methods

Rhizobium tropici was co-inoculated with two Bradyrhizobium strains applied at three different doses (10⁴, 10⁶, and 10⁸ CFU seed^?1) under sterile conditions, and at a single dose (10⁸ CFU seed^?1) in non-sterile soil. Plant biomass, nodulation, and N accumulation in plant tissues were evaluated.

Results

Co-inoculated plants produced more nodules, and accumulated more shoot dry biomass and nitrogen than plants inoculated with R. tropici alone under gnotobiotic conditions. Significant responses were observed at the highest inoculum dose and a significant correlation between dose and shoot dry weight was observed. Co-inoculation increased biomass and N accumulation in non-sterile soil, although with a smaller magnitude.

Conclusions

Altogether, our findings suggest that the co-inoculation with bradyrhizobia contributed to an improved symbiotic interaction between R. tropici and common beans.

     

Rhizobia from Lanzarote,the Canary Islands,That Nodulate Phaseolus vulgaris Have Characteristics in Common with Sinorhizobium meliloti Isolates from Mainland Spain

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"DQ333891","term_id":"84872507","term_text":"DQ333891"}}DQ333891 and {"type":"entrez-nucleotide","attrs":{"text":"AF217267","term_id":"13810627","term_text":"AF217267"}}AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.A remarkable attribute of Phaseolus vulgaris (common bean) is its ability to nodulate with rhizobia from at least 20 different legume genera (summarized in reference 1). Of particular relevance is the report by Ishizawa (16), who described P. vulgaris nodulation ranging from doubtful to good by 14 strains recovered from Medicago sativa, Medicago denticulata, and Melilotus alba, while nodulation of the latter three legumes by four bean strains was negative.At the time of the host range experiments, such as those described by Ishizawa (16), rhizobial nomenclature depended on the legume host of origin; the taxonomy of the strains was based on cross-inoculation groups. Consequently, no information was available about the genetic relationships among the rhizobial strains that originated from the different host legume genera and formed nodules on P. vulgaris. Eventually, rhizobial nomenclature based on the cross-inoculation groups was abandoned because of the many unexplainable and incongruous nodulation data (44). The cross-inoculation groups consisted of different rhizobial species within the single genus Rhizobium. Eventually, rhizobial taxonomy was expanded to several different genera based on estimates of their phylogeny (38). Phylogenies of bean-nodulating rhizobia were estimated from variations in the 16S rRNA gene sequence (39), even though subsequently it became clear that this method is significantly limited by histories of genetic exchange and recombination (6, 40). Most reported phylogenies of rhizobia nodulating P. vulgaris have placed them in the genus Rhizobium (3, 39), but several surveys with isolates from North Africa and Spain have demonstrated that rhizobia in the genus Sinorhizobium also nodulate this legume species (12, 23, 24, 25, 41), supporting the nodulation data originally published by Ishizawa (16). The number of isolates described as originating from nodules of P. vulgaris in the genus Sinorhizobium is small, and for the most part, from the published evidence, it has been suggested that they are affiliated with Sinorhizobium fredii. However, nodules of P. vulgaris growing in a single Tunisian soil where beans are cultivated yielded four isolates that, according to the data, appeared to support an affiliation with Sinorhizobium meliloti rather than S. fredii (25). Whether these four cultures were of the same rhizobial genotype constituting a single example of S. meliloti isolated from P. vulgaris is unknown.P. vulgaris was introduced into Europe as a crop plant as early as the 16th century (31) but never became a very important part of agriculture in Lanzarote, one of the Canary Islands that lie in the Atlantic Ocean to the west of the North African coast. Since there is no record of any nodulation studies with P. vulgaris cultivated on Lanzarote Island, the first objective of this study was to examine bean plants that had grown in Lanzarote soil for nodulation. Considering that the diversity of rhizobia able to nodulate bean plants is extremely wide, the second objective was to characterize the isolates originating from the nodules of plants grown in Lanzarote soil.(Part of this work was presented as a poster at the First International Meeting on Microbial Phosphate Solubilization, Salamanca, Spain, July 2002.)     

<Emphasis Type="Italic">Rhizobium cellulosilyticum</Emphasis> as a co-inoculant enhances <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> grain yield under greenhouse conditions

The Rhizobium-legume symbiosis is a complex partnership with many factors, with initial bacterial colonization of the plant root surface and primary infection as key early stages. Two molecules are strongly involved in these processes: the structural carbohydrate cellulose and the enzyme cellulase, which breaks down the former and allows rhizobia to infect the roots. Here, we report the effect on common bean (Phaseolus vulgaris L.) after co-inoculation of the non-nodulating, cellulase-overproducing strain Rhizobium cellulosilyticum ALA10B2^T and the P. vulgaris-nodulating R. leguminosarum strain TPV08. In order to elucidate the effect of combined inoculation with both strains, we designed greenhouse assays, including single inoculation with strain TPV08, co-inoculation with both strains and an uninoculated treatment in non-sterile peat. Chemical fertilizers were not added. Chlorophyll content in the leaves was measured after the flowering stage by spectrophotometry and was considered to be indicative of the nutrient status of the plants. Nodule formation was observed on roots of the inoculated plants, while no nodulation was observed on roots of the uninoculated plants. The results indicate a synergistic effect between the two Rhizobium strains. Co-inoculated plants exhibited significant increases in seed yield and nitrogen content in comparison with the uninoculated control plants and with plants inoculated with a single strain. It is suggested that co-inoculation with strain ALA10B2^T greatly increased the efficiency of N fixation by strain TPV08.     

Genetic markers for search of rhizobia based on symbiotic genes

The possible application of rhizobial symbiotic genes as markers for the search and primary identification of rhizobia from temperate-zone legumes was studied. It was shown that conservative sym genes nifH and nifD could be used as markers for rapid search of rhizobia among the analyzed isolates, while more variable genes nifK and nodC could be used for their primary identification. Efficiency of the proposed method was shown in analysis of bacterial isolates obtained from Onobrychis arenaria and Astragalus cicer root nodules.     

Nectar trichome structure of aquatic bladderworts from the section <Emphasis Type="Italic">Utricularia</Emphasis> (Lentibulariaceae) with observation of flower visitors and pollinators

In Utricularia, the flower spur is a nectary and in this organ, nectar is produced and stored. This study aimed to examine the structure of the nectary trichomes in four Utricularia species (Utricularia vulgaris L., U. australis R.Br., U. bremii Heer and U. foliosa L.) from the generic section Utricularia. We have investigated whether species with different spur morphology had similar spur anatomy and nectary trichome structure. In Utricularia flowers, nectar is produced by spur capitate trichomes (sessile or stalked). Our results showed that regardless of the various spur morphology, trichomes have similar architecture and ultrastructure. Head cells of these trichomes are transfer cells with an eccrine nectar secretion. Examined species differed in the micromorphology of papillae in spurs. The fly Eristalis tenax was found to be a pollinator of U. vulgaris. Small Halictidae bees seem to be pollinators of U. foliosa.