Isolation and Symbiotic Characteristics of Two Tn5-Derived Phage-Resistant <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> Strains that Nodulate Soybean

Using transponson Tn5 mutagenesis, two transconjugants of Bradyrhizobium japonicum with the properties of both phage resistance and ability to induce nodulation were isolated at the frequency of 0.02%. These transconjugants were tested for their symbiotic performance on soybean cv. JS335 under greenhouse and field conditions. Both phage-resistant mutants induced nodules (nod (+)), but the transconjugant B. japonicum E13 was ineffective in nitrogen fixation (fix (-)). Rhizobiophage presence in the inoculum of phage-resistant mutants did not influence the symbiotic effectiveness. The mixture of wild strain and phage in the inoculum caused reduced symbiotic performance under controlled conditions, while under a field environment phage (100 and 500 mul of approximately 10(8) particles ml(-1)) presence did not have any recognizable effect on increased nodule dry weight, nitrogenase activity, or foliar N(2) content. On the basis of restriction fragment length polymorphism analysis, phage-sensitive, less effective, homologous bradyrhizobia belonging to B. japonicum were detected in root nodules of both inoculated and uninoculated plants. Inoculation of a higher concentration of phage in the inoculum significantly reduced the symbiotic performance, while the lower concentration of phage did not show any effect on phage-susceptible, less effective, homologous bradyrhizobia or, thus, symbiotic efficiency under field conditions. The phage-resistant mutant B. japonicum A49 showed effective symbiosis as efficient as that of the wild strain. Inoculation of phage-resistant mutants with lytic phage may reduce the occupancy of phage-susceptible, ineffective/less effective/mediocre homologous bradyrhizobia strains under natural complex soil conditions.     

Biological characteristics of plasmids of <Emphasis Type="Italic">Mesorhizobium huakuii</Emphasis> HN3015 from <Emphasis Type="Italic">Astragalus sinicus</Emphasis>

A Mesorhizobium huakuii strain HN3015 was isolated from Astragalus sinicus in a rice-growing field of Southern China. Strain HN3015 contained three large plasmids. The three indigenous plasmids, named as pMhHN3015a, pMhHN3015b and pMhHN3015c of M. huakuii HN3015, were, respectively, cured by Tn5-sacB insertion. The mutant strain HN3015-1 cured with its largest plasmid pMhHN3015c formed only white null nodules. Mutant HN3015-3 cured with its smallest plasmid pMhHN3015a could form pink effective nodules. However, mutant HN3015-2 cured of the second largest plasmid pMhHN3015b lost nodulation ability. Furthermore, curing of pMhHN3015a had enhanced competitive nodulation ability and symbiotic efficiency of HN3015-3. The results from acidity tolerance assays indicated that the three plasmids in M. huakuii HN3015 had a positive control effect on acidity tolerance of HN3015, and all indigenous plasmids of M. huakuii HN3015 had a negative control effect on the alkali tolerance capacity of HN3015. Surprisingly, all plasmids in M. huakuii HN3015 had also a negative control effect on its growth rate. The results showed an interactive and functional complexity of plasmids in strain HN3015.     

Genetic variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains nodulating soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill]

Brazil has succeeded in sustaining production of soybean [Glycine max (L.) Merrill] by relying mainly on symbiotic N₂ fixation, thanks to the selection and use in inoculants of very effective strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is desirable that rhizobial strains used in inoculants have stable genetic and physiological traits, but experience confirms that rhizobial strains nodulating soybean often lose competitiveness in the field. In this study, soybean cultivar BR 16 was single-inoculated with four B. japonicum strains (CIAT 88, CIAT 89, CIAT 104 and CIAT 105) under aseptic conditions. Forty colonies were isolated from nodules produced by each strain. The progenitor strains, the isolates and four other commercially recommended strains were applied separately to the same cultivar under controlled greenhouse conditions. We observed significant variability in nodulation, shoot dry weight, shoot total N, nodule efficiency (total N mass over nodule mass) and BOX-PCR fingerprinting profiles between variant and progenitor strains. Some variant strains resulted in significantly larger responses in terms of shoot total N, dry weight and nodule efficiency, when compared to their progenitor strain. These results highlight the need for intermittent evaluation of stock bacterial cultures to guarantee effective symbiosis after inoculation. Most importantly, it indicates that it is possible to improve symbiotic effectiveness by screening rhizobial strains for higher N₂ fixation capacity within the natural variability that can be found within each progenitor strain.     

Characterization of surface motility in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>: regulation and role in symbiosis

Sinorhizobium meliloti can exhibit diverse modes of surface translocation whose manifestation depends on the strain. The mechanisms involved and the role played by the different modes of surface motility in the establishment of symbiosis are largely unknown. In this work, we have characterized the surface motility shown by two S. meliloti reference strains (Rm1021 and GR4) under more permissive conditions for surface spreading and analyzed the symbiotic properties of two flagella-less S. meliloti mutants with different behavior on surfaces. The use of Noble agar in semisolid minimal medium induces surface motility in GR4, a strain described so far as non-motile on surfaces. The motility exhibited by GR4 is swarming as revealed by the non-motile phenotype of the flagella-less flaAB mutant. Intriguingly, a flgK mutation which also abolishes flagella production, triggers surface translocation in GR4 through an as yet unknown mechanism. In contrast to GR4, Rm1021 moves over surfaces using mostly a flagella-independent motility which is highly reliant on siderophore rhizobactin 1021 production. Surprisingly, this motility is absent in a flagella-less flgE mutant. In addition, we found that fadD loss-of-function, known to promote surface motility in S. meliloti, exerts different effects on the two reference strains: while fadD inactivation promotes a flagella-independent type of motility in GR4, the same mutation interferes with the surface translocation exhibited by the Rm1021 flaAB mutant. The symbiotic phenotypes shown by GR4flaAB and GR4flgK, non-flagellated mutants with opposite surface motility behavior, demonstrate that flagella-dependent motility positively influences competitiveness for nodule occupation, but is not crucial for optimal infectivity.     

Effects of temperature on competition and relative dominance of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">Bradyrhizobium elkanii</Emphasis> in the process of soybean nodulation

Background and aims

Bradyrhizobium japonicum and Bradyrhizobium elkanii dominated soybean nodules in temperate and subtropical regions in Nepal, respectively, in our previous study. The aims of this study were to reveal the effects of temperature on the nodulation dominancy of B. japonicum and B. elkanii and to clarify the relationship between the effects of temperature and the climate-dependent distribution of Bradyrhizobium species.

Methods

A laboratory competition experiment was conducted between B. japonicum and B. elkanii strains isolated from the same temperate location in Nepal. A mixture of each strain was inoculated into sterilized vermiculite with or without soybean seeds, and inoculated samples were incubated at 33/27 (day/night) and 23/17 °C. Relative populations in the non-rhizosphere, rhizosphere, and nodules were determined by competitive PCR using specific primers for each strain at 0, 1, 2, and 4 weeks after inoculation.

Results

Both separately inoculated B. japonicum and B. elkanii strains formed nodules at both temperatures. Under competitive conditions, B. japonicum strains dominated at low temperature; however, at high temperature, both strains achieved co-nodulation in 1 week, with B. elkanii dominating after 2 weeks. The relative populations of both strains were similar in the non-rhizosphere and rhizosphere at low temperature, but B. elkanii strains dominated in these regions at high temperature.

Conclusions

The domination of B. japonicum strains in nodules at the low temperature appeared to be due to preferential infection, while the domination of B. elkanii strains at high temperature appeared to be due to the higher population of B. elkanii in the non-rhizosphere and rhizosphere, in addition to its domination in nodules after co-nodulation. The effects of temperature on the competition between B. japonicum and B. elkanii strains were remarkable and corresponded with the distribution of bradyrhizobial species in Nepal.

     

Effects of the <Emphasis Type="Italic">purL</Emphasis> Gene Expression Level on the Competitive Nodulation Ability of <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis>

Purine pathway in Rhizobium is important during the nodulation processes. The purL gene in Sinorhizobium fredii (S. fredii) has been identified to be required for the whole establishment of a nitrogen-fixing nodule. To get a better understanding of the purL gene’s impacts on Rhizobium–plant interaction, the competitive nodulation abilities of S. fredii containing different purL expression plasmids were studied. Several kinds of coinoculations were performed, including using different bacterial concentration ratios, with or without the supplementation of purine source in the plant nutrient solution, and the delayed coinoculation tests. The results indicated that the competitive nodule occupancy of S. fredii was affected significantly by the purL expression level during the early nodulation periods. The mutant strain containing no purL expression could not elicit competitive nodules both in the presence and absence of purine source. A positive linear correlation within certain limits was observed between strain’s competitive nodule occupancy and purL gene expression level. All these results suggested that the purL gene played a role in the competitive nodulation of S. fredii.     

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.     

Cowpea Rhizobia Producing Dark Nodules: Use in Competition Studies

During a program of screening rhizobia from West Africa, it was found that some strains produced nodules of unusually dark appearance on cowpeas, but not on peanuts, soybeans, pigeon peas, or mung beans. The dark pigmentation was in the bacteroid zone, was not correlated with nodule effectiveness, and was additional to the leghemoglobin pigment. Only rhizobial strains with a nongummy (“dry”) colony morphology produced dark nodules. Visually distinguishable pink and dark nodules formed on the same root when a mixture of pink and dark strains was applied as inoculum. The dark-nodule phenotype was therefore appraised as a marker and found to be useful for studying nodulation competition with strains of the orthodox pink-nodule type. The competitiveness of 10 pink-nodule strains was examined relative to a black-nodule strain, IRc 256; a range of competitiveness was obtained of less competitive than, equally competitive to, or more competitive than IRc 256. Patterns of primary (early) nodulation were generally the same as patterns of secondary (later) nodulation. Mixed infections by dark and pink strains produced piebald nodules, the frequency of occurrence of which was much greater among primary than among secondary nodules.     

<Emphasis Type="Italic">tolC</Emphasis> mutant of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> strain CXM1-188 fails to establish effective symbiosis with alfalfa

The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5 mutagenesis in the effective strain CXM1-188. The mutant did not produce fluorescent halos in UV light on the LB medium containing Calcofluor white, which suggests that modification occurred in the production of exopolysaccharide EPS1. Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS media, and this was most likely connected with the absence of the second exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula white round Fix^? nodules or nodules of irregular shape. These nodules possessed the structure usually described for nodules of EPS1 mutants. According to the data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63 contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome. This gene encodes the protein sharing homology with the TolC protein, a component of a type I secretion system responsible for the export of protein toxins and proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to be exported by the type I secretion system, was tested in cultural supernatants of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was ascertained that secretory proteins ExsH and ExpE1 are absent in the culture medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved in the excretion of proteins ExsH and ExpE1.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

Influence of soil fertility on the rhizobial competitiveness for nodulation of <Emphasis Type="Italic">Acacia senegal</Emphasis> and <Emphasis Type="Italic">Acacia nilotica</Emphasis> provenances in nursery and field conditions

Within the framework of our study, we assessed the nodule occupancy of a mixture of various strains of rhizobia to inoculate several provenances of Acacia senegal and Acacia nilotica. The first part of the experiment was carried out under greenhouse conditions where the plants were cultivated in polyvinyl chloride tubes containing an unsterilized Sangalkam soil low in organic matter and nitrogen. The results showed that 4 and 8 months after sowing, rhizobial strains CIRADF 306 and CIRADF 300 were mainly present in nodules of A. nilotica and A. senegal, respectively. After transferring the seedlings to the more fertile soil in Bel Air field station, the molecular analysis of the nodules showed that strain CIRADF 306 was absent from the nodules of A. nilotica, whereas strain CIRADF 305 which occurred only at low nodule occupancy in the nursery, predominated in the field conditions. On the other hand, strain CIRADF 300 occurred in the majority of the nodules from the various provenances of A. senegal. These results demonstrated actual interaction between inoculated rhizobial strains, soil type and host plant genotype in terms of competitiveness, nodulation and symbiotic nitrogen fixation.     

Competitiveness of a <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. Strain in Soils Containing Indigenous Rhizobia

The success of rhizobial inoculation on plant roots is often limited by several factors, including environmental conditions, the number of infective cells applied, the presence of competing indigenous (native) rhizobia, and the inoculation method. Many approaches have been taken to solve the problem of inoculant competition by naturalized populations of compatible rhizobia present in soil, but so far without a satisfactory solution. We used antibiotic resistance and molecular profiles as tools to find a reliable and accurate method for competitiveness assay between introduced Bradyrhizobium sp. strains and indigenous rhizobia strains that nodulate peanut in Argentina. The positional advantage of rhizobia soil population for nodulation was assessed using a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed an increase in nodule number per plant and nodule occupancy for strains established in vermiculite. In field experiments, only 9% of total nodules were formed by bacteria inoculated by direct coating of seed, whereas 78% of nodules were formed by bacteria inoculated in the furrow at seeding. In each case, the other nodules were formed by indigenous strains or by both strains (inoculated and indigenous). These findings indicate a positional advantage of native rhizobia or in-furrow inoculated rhizobia for nodulation in peanut.     

Biochemical and molecular characterization of a rhizobitoxine-producing <Emphasis Type="Italic">Bradyrhizobium</Emphasis> from pigeon pea plants

Out of a total of 8 bacterial strains isolated from the root nodules of pigeon pea plants grown in arid region, five were identified as rhizobia based on biochemical test and confirmed by 16S rDNA sequencing. PCR based screening for the rtxA gene (involved in biosynthesis of rhizobitoxine) revealed that the gene was present in one strain identified biochemically and genetically as belonging to species Bradyrhizobium (BS KT-24). The strain was resistant to phosphomycin, nalidixic acid, kanamycin, gentamicin and neomycin but sensitive towards streptomycin and spectinomycin. Bioinformatic-tool-guided phylogenetic analysis of rtxA gene revealed its distinctiveness from other known rtxA genes (present in B. japonicum, B. elkanii and Xanthomonas oryzae). The rhizobitoxine producing strain BS KT-24 is considered to exhibit better survival and nodulation protection besides competitiveness for pigeon pea and other legumes grown under abiotic stress and, thus, be a candidate in practical aspect of rhizobitoxine production by rhizobium and its application as rhizobial inoculants.     

Possible reasons for tolerance to mercury of <Emphasis Type="Italic">Lupinus albus</Emphasis> cv. G1 inoculated with Hg-resistant and sensitive <Emphasis Type="Italic">Bradyrhizobium canariense</Emphasis> strains

Inoculation with a mercury (Hg)-resistant Bradyrhizobium canariense strain (L7AH) confers on Lupinus albus the ability to grow under high concentrations of Hg and to accumulate this heavy metal. To elucidate the mechanism/s implicated in the acquisition of this tolerance, lupins were inoculated with resistant (L7AH) and sensitive (L3) strains and fed with different Hg solutions (0–200 μM HgCl₂). Mercury application resulted in cellular alterations in leaves and nodules, depending on the strain inoculated. Mesophyll cell chloroplasts from L7AH-inoculated plants treated with Hg showed similar structure to those in control plants, while those of L3-inoculated plants treated with Hg showed a large increase in the number and size of starch granules. This resulted in a large increase in chloroplast and cell size which produced altered grana distribution with a totally disorganized thylakoid structure and clear signs of degradation. The preservation of the distribution and morphology of chloroplasts in L7AH-inoculated plants may be a reason why the photosynthetic efficiency remained unchanged even after treatment with 200 μM of Hg. Mercury exposure produced changes in L3-infected nodule ultrastructure, with evident signs of degradation, especially in bacteroids. However, only slight alterations of nodule morphology were noticed in L7AH-infected nodules. X-ray microanalysis showed that, while Hg was present in the nodules formed by L3, in both cortex and infected zone, in those formed by L7AH only low levels of Hg in the outermost layers of the cortex were detected. The exclusion of Hg from the infected zone together with the conservation of the symbiosome structure in nodules from L7AH-inoculated plants may explain the maintenance of nitrogenase activity.     

Growth and nodulation in <Emphasis Type="Italic">Alnus sieboldiana</Emphasis> in response to <Emphasis Type="Italic">Frankia</Emphasis> inoculation and nitrogen treatments

Key message

We investigated a Frankia – Alnus sieboldiana symbiosis, including the minimum inoculum dose for constant nodulation, the period of time to nodulation after inoculation, and the effects of N on nodulation.

Abstract

Frankia is a nitrogen-fixing actinomycete that forms root nodules in some dicotyledonous plants, which are called actinorhizal. We studied nodule formation in Alnus sieboldiana, an actinorhizal plant, after inoculation with a Frankia isolate to establish techniques for Frankia inoculation and the cultivation of inoculated plants. Root nodules formed on seedlings of A. sieboldiana by 2 weeks after inoculation, and N₂ fixation measured by acetylene reduction activity started 3 weeks after inoculation. Nodulation was observed after inoculation with a Frankia isolate at 0.001 μL packed cell volume (pcv). The number of nodules formed on the seedlings inoculated with Frankia at more than 0.05 μL pcv was not significantly different. Nodule development and N₂ fixation were reduced when inoculated seedlings were treated weekly with 15 mM NH₄NO₃-N. In contrast, treatment with 3.75 or 0.9375 mM NH₄NO₃-N did not inhibit nodule development or N₂ fixation of inoculated seedlings by 15 weeks of N treatment.

     

Competition between introduced <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains and indigenous bradyrhizobia in Minnesota organic farming systems

Organic farmers recognize the importance of rhizobial associations with legume plants to help meet N fertility and plant productivity needs. A field experiment was done at three organic fields in Minnesota to assess the effect of indigenous Bradyrhizobium japonicum ORGS3 and ORGS5 and reference USDA 110 strains on the growth and yield performance of soybean. Soybean genotypes MN1505SP and Lambert inoculated with B. japonicum ORGS3 had significantly greater (P < 0.01) nodule numbers (42.1 ± 2.5), herbage N-contents (4.02 ± 0.01%), dry biomass (12.60 ± 1.45 g), and plant populations (117,890 ± 288.13 plant/acre) compared with the un-inoculated control. Grain yields were not affected by inoculation. Most nodules formed on non-inoculated Lambert (70%) and MN1505SP (53%) were occupied by strain ORGS5. The inoculant strains USDA110 and ORGS5 increased nodule occupancy by 10% on MN1505SP and Lambert. In contrast, strain ORGS3, and the combination of strains ORGS5 plus ORGS3, increased nodules occupancy on Lambert by 23 and 20%, respectively, compared with the control. The majority of nodules on Lambert (59%) and MN1505SP (52%) in the Farmington and Lamberton fields, respectively, were occupied by ORGS5. In contrast, 41 and 45% of nodules formed on Lambert and MN1505SP at Rosemount, respectively, were occupied by strain ORGS3. The lowest percentage of nodules formed on Lambert (4%) and MN1505SP (5%), in the Farmington field, were occupied by USDA110. These results showed that Bradyrhizobium strains ORGS3 and ORGS5 can be used to enhance N fixation and productivity of organically-grown soybeans grown in Minnesota fields.