Rapid In Situ Evolution of Nodulating Strains for Biserrula pelecinus L. through Lateral Transfer of a Symbiosis Island from the Original Mesorhizobial Inoculant

Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA and nifH from an inoculant to soil bacteria. Transfer of these chromosomal genes and the presence of an identical integrase gene adjacent to a Phe tRNA gene in both the inoculant and recipients indicate that there was lateral transfer of a symbiosis island.     

Methods To Alter the Recovery and Nodule Location of Bradyrhizobium japonicum Inoculant Strains on Field-Grown Soybeans

Three strains of Bradyrhizobium japonicum, I17, 110, and 61A76, were evaluated for their ability to form nodules on field-grown soybeans in soil with a highly competitive indigenous B. japonicum population. The predominant indigenous strain, 0336, in the field site used was unlike the more common isolates from Midwestern soils which belong to the 123 or 138 serogroups. This strain persisted in the soil for at least 30 years without any soybean crops. The three inoculant strains differed in their ability to compete with indigenous strains for nodule formation. Four different inoculation treatments were tested in three adjacent fields. When the amount of inoculum was increased, a higher proportion of nodules contained the inoculant strain. The most competitive inoculant strain was I17, a recent field isolate. Strain 61A76 was better than 110. There was no difference in recovery of the inoculant strains on the Hodgson or Corsoy soybean cultivars, nor was there a difference in recovery of the inoculant strains during the growing season. The vertical distribution of nodules containing the inoculant strains was affected by the method of adding the inoculant to the soil. Inoculant added to the seed furrow produced nodules mainly in the top region of the soybean root. Inoculant tilled into the soil produced nodules primarily in the bottom part of the root. The nodules that were produced in the bottom part of the root are younger and may contribute significant amounts of fixed nitrogen to the soybean during seed formation.     

Improvement of Rhizobium Inoculants

A practical approach was used to develop a Rhizobium (Bradyrhizobium) japonicum inoculant that increases soybean (Glycine max (L.) Merr.) yield in fields with indigenous Rhizobium populations, which typically outcompete strains present in existing commercial inoculants and therefore decrease the value of inoculant use. Field tests managed by several universities in the Mississippi delta region averaged a 169-kg/ha (P < 0.01) grain yield increase. The inoculant contains a mixture of mutants selected for increased nitrogen fixation ability. These mutants were derived from indigenous wild-type strains that are capable of high-level occupancy of nodules in soybean fields in the Mississippi delta region. To ensure microbiological purity, the inoculant is fermented directly in the point-of-use container with a vermiculite carrier (L. Graham-Weiss, M. L. Bennett, and A. S. Paau, Appl. Environ. Microbiol. 53:2138-2140, 1987). It should be possible to use this approach to produce more effective Rhizobium inoculants for any legume in any geographical area.     

Bradyrhizobium japonicum Inoculant Mobility, Nodule Occupancy, and Acetylene Reduction in the Soybean Root System

In the American Midwest, superior inoculant rhizobia applied to soybeans usually occupy only 5 to 20% of nodules, and response to inoculation is the exception rather than the rule. Attempts to overcome this problem have met with limited success. We evaluated the ability of Bradyrhizobium japonicum, supplied as a seed coat inoculant, to stay abreast of the infectible region of the developing soybean root system. The rhizoplane population of the inoculant strain declined with distance from site of placement, the decrease being more pronounced on lateral than on taproots. This decline was paralleled by a decrease in inoculant-strain nodule occupancy. Inoculant bradyrhizobia contributed little to nodulation of lateral roots, which at pod-fill accounted for more than 50% of nodule number and mass, and were major contributors to acetylene reduction activity. From these data, it appears that inoculant bradyrhizobia are competitive with indigenous soil strains at the point of placement in the soil but have limited mobility and so are incapable of sustaining high populations throughout the developing root system. The result is low nodule occupancy by the inoculant strain in the tapand lateral roots. Future studies should address aspects of inoculant placement and establishment.     

Identification of Bradyrhizobium japonicum Nodule Isolates from Wisconsin Soybean Farms

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a more discriminating method than serotyping for identifying strains of Bradyrhizobium japonicum. Analysis of 543 nodule isolates from southeastern Wisconsin soybean farms revealed that none of the isolates were formed by any of the inoculant strains supplied by either of two inoculant companies. Twenty-nine indigenous strains and six inoculant strains were identified. Strain 61A76, the most competitive indigenous strain, formed 21% of the nodules. Indigenous strains 3030, 3058, 0336, and 3052 formed 15, 11, 9, and 9% of the nodules, respectively. These predominant strains were not associated with a particular soybean cultivar, soil type, or farm location.     

Rhizobial counts in peat inoculants vary amongst legume inoculant groups at manufacture and with storage: implications for quality standards

Background and aims

Inoculation of legumes at sowing with rhizobia has arguably been one of the most cost-effective practices in modern agriculture. Critical aspects of inoculant quality are rhizobial counts at manufacture/registration and shelf (product) life.

Methods

In order to re-evaluate the Australian standards for peat-based inoculants, we assessed numbers of rhizobia (rhizobial counts) and presence of contaminants in 1,234 individual packets of peat–based inoculants from 13 different inoculant groups that were either freshly manufactured or had been stored at 4 °C for up to 38 months to determine (a) rates of decline of rhizobial populations, and (b) effects of presence of contaminants on rhizobial populations. We also assessed effects of inoculant age on survival of the rhizobia during and immediately after inoculation of polyethylene beads.

Results

Rhizobial populations in the peat inoculants at manufacture and decline rates varied substantially amongst the 13 inoculant groups. The most stable were Sinorhizobium, Bradyrhizobium and Mesorhizobium with Rhizobium, particularly R. leguminosarum bv. trifolii the least stable. The presence of contaminants at the 10^?6 level of dilution, i.e. >log 6.7 g^?1 peat, reduced rhizobial numbers in the stored inoculants by an average of 37 %. Survival on beads following inoculation improved 2–3 fold with increasing age of inoculant.

Conclusions

We concluded that the Australian standards for peat-based rhizobial inoculants should be reassessed to account for the large differences amongst the groups in counts at manufacture and survival rates during storage. Key recommendations are to increase expiry counts from log 8.0 to log 8.7 rhizobia g^?1 peat and to have four levels of inoculant shelf life ranging from 12 months to 3 years.     

Effectiveness of Rhizobium Strains Used in Inoculants after Their Introduction into Soil

Rhizobium strains used in inoculants for Trifolium spp., Medicago spp., Glycine max, and Lotus pedunculatus were isolated from nodules of these legumes grown in soils into which the rhizobia had been introduced 4 to 8 years before. Isolations were made from a total of 420 nodules. Nodule occupancy by the inoculant strains varied from 17.7% for a soybean strain to 100% in the case of L. pedunculatus whose specific rhizobia did not occur in the soils studied. In general, inoculant strains isolated from nodules did not differ in effectiveness from cultures of the same strains concurrently maintained in lyophilized form. The average effectiveness of all of the isolates (identified and unidentified) from a legume was 7.1 to 73.3% higher than that of the unidentified isolates alone, demonstrating the prolonged effect that a single-seed inoculation has on the rhizobial population in a soil which had not been planted with legumes before. Relatively weak recovery of a Rhizobium japonicum strain introduced into soil 4 years after soybean seed inoculated with a different strain had been planted in the same soil confirmed the advantage of a resident population over an introduced inoculant strain.     

Structure and Dynamics of Experimentally Introduced and Naturally Occurring Laccaria sp. Discrete Genotypes in a Douglas Fir Plantation

Ectomycorrhizal fungi have been introduced in forest nurseries to improve seedling growth. Outplanting of inoculated seedlings to forest plantations raises the questions about inoculant persistence and its effects on indigenous fungal populations. We previously showed (M.-A. Selosse et al. Mol. Ecol. 7:561–573, 1998) that the American strain Laccaria bicolor S238N persisted 10 years after outplanting in a French Douglas fir plantation, without introgression or selfing and without fruiting on uninoculated adjacent plots. In the present study, the relevance of those results to sympatric strains was assessed for another part of the plantation, planted in 1985 with seedlings inoculated with the French strain L. bicolor 81306 or left uninoculated. About 720 Laccaria sp. sporophores, collected from 1994 to 1997, were typed by using randomly amplified polymorphic DNA markers and PCR amplification of the mitochondrial and nuclear ribosomal DNAs. All plots were colonized by small spontaneous discrete genotypes (genets). The inoculant strain 81306 abundantly fruited beneath inoculated trees, with possible introgression in indigenous Laccaria populations but without selfing. In contrast to our previous survey of L. bicolor S238N, L. bicolor 81306 colonized a plot of uninoculated trees. Meiotic segregation analysis verified that the invading genet was strain 81306 (P < 0.00058), implying a vegetative growth of 1.1 m · year⁻¹. This plot was also invaded in 1998 by strain S238N used to inoculate other trees of the plantation. Five other uninoculated plots were free of these inoculant strains. The fate of inoculant strains thus depends less on their geographic origin than on unknown local factors.     

Spectral studies of Amaranthus tristis Linn. in Bioremediated Silk dyeing effluent with mixed biofertilizer inoculants

Microbial degradation as a treatment, with the combination of mixed inoculants of the Biofertilizer of Pseudomonas sp., Azospirillium sp. and Rhizobium sp., was employed for the remediation of Silk dyeing effluent. Remediating studies was undertaken to assess the feasibility of the mixed biofertilizer inoculant source for degradation of the Azodye effluent from the Silk dyeing Industry. The Green leafy vegetable (GLV), Amaranthus tristis Linn used as investigational prototypical plant species is selected for examining the phytochemicals, functional groups and its compounds grown in the effluent and biotreated environment and compared. The laboratory scale investigation showed that leaves, stem and root of the Amaranthus tristis Linn was qualitatively analysed for 20 phytochemicals which was grown in the different treatments of raw effluent and the biotreated effluent and the results showed the phytochemicals on the effluent’s influence reduced from strong positive to trace amounts while recovered on the biotreated environment. The FTIR analysis of the GLV grown in effluent and biotreated environments on comparison resulted in the functional group Alkene rescued in the biotreated effluent environment compared to the effluent contaminated area. The HPLC analysis of methanolic extracts of A. tristis grown in fresh water has 6 peaks of retention time of 2.6, 3, 3.9, 4, 4.2, and 4.6 RT whereas GLV effluent had only one peak of retention time of 4.1 RT. In the GLV from biotreated environment have 4 peaks were found with the maximum percentage area of 95.2% which proves that the compounds are rescued in the biotreated environment and few active compounds were confirmed in GCMS analysis. The Soil analysis results also indicate that the biotreatment of mixed inoculant of biofertilizers in the biotreated soil had influence resulting in improved levels of Ca, N, P and K with 114, 213, 10.5, 268 kg/ha respectively in the mixed inoculant biotreated soil. Similarly the micronutrients suchas Fe, Mn, Cu and Zn ranges to 4.1, 20.22, 2.13, 1.13 ppm respectively in the mixed inoculant biotreated soil within the optimal range. The study revealed that mixed biofertilizer inoculant has the recovery effect on the Silk dyeing (Azodyes) effluents effective reducing the pollutant capacity thereby meeting the discharged standards.     

Dilution of Liquid Rhizobium Cultures To Increase Production Capacity of Inoculant Plants

Experiments were undertaken to test whether peat-based legume seed inoculants, which are prepared with liquid cultures that have been deliberately diluted, can attain and sustain acceptable numbers of viable rhizobia. Liquid cultures of Rhizobium japonicum and Rhizobium phaseoli were diluted to give 10⁸, 10⁷, or 10⁶ cells per ml, using either deionized water, quarter-strength yeast-mannitol broth, yeast-sucrose broth, or yeast-water. The variously diluted cultures were incorporated into gamma-irradiated peat, and the numbers of viable rhizobia were determined at intervals. In all of the inoculant formulations, the numbers of rhizobia reached similarly high ceiling values by 1 week after incorporation, irrespective not only of the number of cells added initially but also of the nature of the diluent. During week 1 of growth, similar multiplication patterns of the diluted liquid cultures were observed in two different peats. Numbers of rhizobia surviving in the various inoculant formulations were not markedly different after 6 months of storage at 28°C. The method of inoculant preparation did not affect the nitrogen fixation effectiveness of the Rhizobium strains.     

Influence of Location, Host Cultivar, and Inoculation on the Composition of Naturalized Populations of Rhizobium meliloti in Medicago sativa Nodules

A phage typing system was used to evaluate the composition of indigenous populations of Rhizobium meliloti inhabiting nodules of Medicago sativa cultivars grown with and without inoculation at two field sites during 1983 and 1984. Soil at both locations contained established populations of R. meliloti at planting. Analysis of 1,920 nodule isolates revealed 55 unique phage types of indigenous R. meliloti at one site and 65 indigenous types at the other location. The distributions of phage types differed markedly between locations. At one site, the nodule population was dominated by two phage types; seven others occurred consistently but at lower frequency, and the remainder were encountered infrequently. No indigenous types predominated at the other location, although nine occurred more frequently than the remaining types. Indigenous R. meliloti predominated in nodules from inoculated plots at both sites, with inoculant recovery varying between 10 and 38% in each of two years. The frequency of occurrence of particular phage types at one location was significantly influenced by both M. sativa cultivar and inoculation. At this location, the interaction of cultivar and inoculation on the incidence of phage types suggests that the presence of an inoculant strain differentially affected nodule occupancy of M. sativa cultivars by members of the indigenous R. meliloti population. At both sites, the frequency of specific phage types differed between years. The data emphasize the importance of understanding the ecology and characteristics of indigenous Rhizobium populations as a prerequisite for elucidating problems of inoculant establishment and persistence in competitive situations.     

Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism

The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected.     

Competitive Abilities of Rhizobium meliloti Strains Considered to Have Potential as Inoculants

Twenty four strains of Rhizobium meliloti considered to have potential for inoculant production were grouped in pairs and tested for their ability to compete for nodulation on Medicago sativa, Medicago truncatula, and Medicago littoralis. At the outset, each pair of strains, which consisted of a wild type and a selected streptomycin-resistant mutant of another strain, was tested in an autoclaved soil. Six strain pairs, each consisting of a good and a poor competitor, reacted consistently when tested in each of five other autoclaved soils; eight pairs consisting of strains with comparable competitive abilities varied in their reactions in some of the soils, or even in the same soil when retested. An effect of soil pH on competitive ability was observed with some of these strains. Not all of the strains identified as good competitors on one or more of the Medicago spp. in the autoclaved soils were able to nodulate these plants satisfactorily in a field soil containing an established population of R. meliloti. Strain RF24, which seemed to be the best competitor on each of the three Medicago spp., grouped among the less effective strains on two of the legumes. Two strains of R. meliloti frequently used for inoculant production differed markedly with regard to competitive ability; this places some doubt on the relevancy of singling out competitive ability for special attention when selecting a strain for inoculant production.     

Influence of commercial inoculation with Glomus intraradices on the structure and functioning of an AM fungal community from an agricultural site

The use of commercial arbuscular mycorrhizal (AM) inoculants is growing. However, we know little about how resident AM communities respond to inoculations under different soil management conditions. The objective of this study was to simulate the application of a commercial AM fungal inoculant of Glomus intraradices to soil to determine whether the structure and functioning of that soil’s resident AM community would be affected. The effects of inoculation were investigated over time under disturbed or undisturbed soil conditions. We predicted that the introduction of an infective AM fungus, such as G. intraradices, would have greater consequences in disturbed soil. Using a combination of molecular (terminal restriction length polymorphism analysis based on the large subunit of the rRNA gene) and classical methods (AM fungal root colonization and P nutrition) we found that, contrary to our prediction, adding inoculant to soil containing a resident AM fungal community does not necessarily have an impact on the structure of that community either under disturbed or undisturbed conditions. However, we found evidence of positive effects of inoculation on plant nutrition under disturbed conditions, suggesting that the inoculant interacted, directly or indirectly, with the resident AM fungi. The inoculant significantly improved the P content of the host but only in presence of the resident AM fungal community. In contrast to inoculation, soil disturbance had a significant negative impact on species richness of AM fungi and influenced the AM fungal community composition as well as its functioning. Thus, we conclude that soil disturbance may under certain conditions have greater consequences for the structure of resident AM fungal communities in agricultural soils than commercial AM fungal inoculations with G. intraradices.     

Rhizobium Strain Identification and Quantification in Commercial Inoculants by Immunoblot Analysis

An immunoblot procedure for the strain-specific quantitative analysis of commercial Rhizobium inoculants was developed. The technique greatly reduced the time required for inoculant analysis. Correlation between immunoblot analysis and traditional plant nodule grow-out most-probable-number techniques was r = 0.90 for 16 commercial alfalfa inoculants tested.     

Stability of Markers Used for Identification of Two Rhizobium galegae Inoculant Strains after Five Years in the Field

The stability of identification markers was examined for two Rhizobium galegae inoculant strains after 5 years in the field. The two strains are genetically closely related, but differ in their lipopolysaccharides. Strain HAMBI 540 has lipopolysaccharide of the rough type, whereas that of strain HAMBI 1461 is of the smooth type. The properties that were examined for 10 field isolates of each inoculant type were symbiotic phenotype, phage type, intrinsic antibiotic resistance, maximum growth temperature, lipopolysaccharide and total soluble protein patterns, immunological properties, DNA restriction profiles, and DNA hybridization patterns, which were determined by using nifHDK and recA sequences as probes. Of these properties, all remained stable in soil, with the exception of some variation in intrinsic antibiotic resistance and the acquisition of an extra EcoRI restriction fragment by one of the isolates. Thus, both the rough and the smooth lipopolysaccharide phenotypes persisted equally well in soil.     

Competition among Strains of Rhizobium leguminosarum biovar trifolii and Use of a Diallel Analysis in Assessing Competition

Competition between indigenous Rhizobium leguminosarum biovar trifolii strains and inoculant strains or between mixtures of inoculant strains was assessed in field and growth-room studies. Strain effectiveness under competition was compared with strain performance in the absence of competition. Field inoculation trials were conducted at Elora, Ontario, Canada, with soil containing indigenous R. leguminosarum biovar trifolii. The indirect fluorescent-antibody technique was used for the identification of nodule occupants. Treatments consisted of 10 pure strains, a commercial peat inoculant containing a mixture of strains, and an uninoculated control. Inoculant strains occupied 17.5 to 85% of nodules and resulted in increased dry weight and nitrogen content, as compared with the uninoculated control. None of the strains was capable of completely overcoming resident rhizobia, which occupied, on average, 50% of the total nodules tested. In growth-room studies single commercial strains were mixed in all possible two-way combinations and assessed in a diallel mating design. Significant differences in plant dry weight of red clover were observed among strain combinations. Specific combining ability effects were significant at the 10% level, suggesting that the effectiveness of strain mixtures depended on the specific strain combinations. Strains possessing superior effectiveness and competitive abilities were identified by field and growth-room studies. No relationship was detected between strain effectiveness and competitive ability or between strain recovery and host cultivar. The concentration of indigenous populations was not considered to be a limiting factor in the recovery of introduced strains at this site.     

Trametes versicolor: Potential for atrazine bioremediation in calcareous clay soil,under low water availability conditions

This study examined the feasibility of Trametes versicolor to actively degrade atrazine (0.5 μg g^?1) in non-sterile calcareous clay soil (Algarve, Portugal) microcosms for up to 24 weeks (20 °C), under low water availability (soil water potentials of ?0.7 and ?2.8 MPa). Soil respiration, laccase activity, and atrazine quantification by high-performance liquid chromatography (HPLC) were assessed. Respiration was significantly (p < 0.05) enhanced in soil containing the inoculant, particularly in the presence of atrazine, indicating that it remained metabolically active throughout the study. Furthermore, up to 98% and 85% (at ?0.7 and ?2.8 MPa, respectively) of atrazine was degraded in soil containing both the atrazine and the inoculant, compared to 96% and 50% in soil containing atrazine only. The contribution of T. versicolor to atrazine degradation was only significant (p < 0.005) under the driest soil treatment conditions. The strategies used for enhancing colonisation and biodegradation capabilities of the inoculant, as well as the selection of sawdust as carrier, were thus effective. However, there were no differences (p > 0.05) in quantified laccase activity in soil containing the inoculant and the control. Overall, this study demonstrated that T. versicolor was a strong candidate for atrazine bioremediation in soil with low moisture and organic matter contents, such as that found in semi-arid and Mediterranean-like ecosystems.     

Rhizobium leguminosarum Biovar viciae Symbiotic Hydrogenase Activity and Processing Are Limited by the Level of Nickel in Agricultural Soils

Analysis of levels of hydrogenase processing and activity in Rhizobium leguminosarum biovar viciae bacteroids from pea (Pisum sativum) plants showed that the oxidation of nitrogenase-evolved hydrogen is limited by the availability of nickel in agricultural soils. This limitation was overcome by using an inoculant strain engineered for higher hydrogenase expression.     

Evidence of Horizontal Transfer of Symbiotic Genes from a Bradyrhizobium japonicum Inoculant Strain to Indigenous Diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah Soil

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N₂-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.