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.     

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

Genetic diversity of symbiotic Bradyrhizobium elkanii populations recovered from inoculated and non-inoculated Acacia mangium field trials in Brazil

Acacia mangium is a legume tree native to Australasia. Since the eighties, it has been introduced into many tropical countries, especially in a context of industrial plantations. Many field trials have been set up to test the effects of controlled inoculation with selected symbiotic bacteria versus natural colonization with indigenous strains. In the introduction areas, A. mangium trees spontaneously nodulate with local and often ineffective bacteria. When inoculated, the persistence of inoculants and possible genetic recombination with local strains remain to be explored. The aim of this study was to describe the genetic diversity of bacteria spontaneously nodulating A. mangium in Brazil and to evaluate the persistence of selected strains used as inoculants. Three different sites, several hundred kilometers apart, were studied, with inoculated and non-inoculated plots in two of them. Seventy-nine strains were isolated from nodules and sequenced on three housekeeping genes (glnII, dnaK and recA) and one symbiotic gene (nodA). All but one of the strains belonged to the Bradyrhizobium elkanii species. A single case of housekeeping gene transfer was detected among the 79 strains, suggesting an extremely low rate of recombination within B. elkanii, whereas the nodulation gene nodA was found to be frequently transferred. The fate of the inoculant strains varied depending on the site, with a complete disappearance in one case, and persistence in another. We compared our results with the sister species Bradyrhizobium japonicum, both in terms of population genetics and inoculant strain destiny.     

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.     

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.     

Polyacrylamide-Entrapped Rhizobium as an Inoculant for Legumes

Pot experiments showed that Rhizobium japonicum cells entrapped in a polyacrylamide gel could be used as an inoculant for soybeans and compared favorably to laboratory-made peat base inoculant containing the same bacterial strain.     

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.     

Growth and Survival of Streptomycete Inoculants and Extent of Plasmid Transfer in Sterile and Nonsterile Soil

The growth and survival of strains of Streptomyces lividans and S. violaceolatus in sterile and nonsterile soil was investigated by using inoculated soil microcosms run as batch systems. It was evident that, after an initial short mycelial growth phase of 2 to 3 days, sporulation occurred and inoculants survived as spores. The transfer of a high-copy-number, self-transmissible plasmid, pIJ673, was detected by using intra- and interspecific crosses. The initial detection of transconjugants correlated with the development of the mycelial state of the inoculants (as confirmed by scanning electron microscopy) after 2 days of incubation. Subsequent spread of the plasmid was attributed to spread within existing mycelium followed by sporulation. In natural soil, inoculant numbers remained constant or declined, but plasmid transfer was readily detected.     

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.     

Impact of arbuscular mycorrhizal fungal inoculants on subsequent arbuscular mycorrhizal fungi colonization in pot-cultured field pea (Pisum sativum L.)

The use of commercial inoculants containing non-resident arbuscular mycorrhizal fungi (AMF) is an emerging technology in field crop production in Canada. The objective of this study was to assess the impact of AMF inoculants containing either a single species (Glomus irregulare) or mixed species (G. irregulare, Glomus mosseae, and Glomus clarum) on AMF root colonization and consequent plant growth parameters of field pea grown using pot cultures. Field pea was grown in both sterilized and non-sterile (i.e., natural) field-collected soil containing resident AMF and received three inoculation treatments: uninoculated control, G. irregulare only, and a mixture of AMF species of G. irregulare, G. mosseae, and G. clarum. After 42 days, the AMF community assembled in field pea roots was assessed by cloning and sequencing analysis on the LSU-ITS-SSU rDNA gene, together with a microscopic assessment of colonization, biomass production, nutrient uptake, and N₂ fixation. The identity of AMF inoculants had a significant effect on field pea performance. The mixed species AMF inoculant performed better than the single species G. irregulare alone by promoting mycorrhizal colonization, field pea biomass, N and P uptake, and N₂ fixation and did not result in a significant compositional change of the AMF community that subsequently assembled in field pea roots. In contrast, the single species G. irregulare inoculant did not significantly enhance field pea biomass, N and P uptake, and N₂ fixation, although a significant compositional change of the subsequent AMF community was observed. No significant interactions affecting these measurements were detected between the resident AMF and the introduced AMF inoculants. The observation that the mixed species AMF inoculant promoted plant growth parameters without necessarily affecting the subsequent AMF community may have important implications regarding the use of non-resident AMF inoculants in agricultural production.     

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.     

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.     

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.     

Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.     

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.     

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.     

Survival of endophytic bacteria in polymer-based inoculants and efficiency of their application to sugarcane

Background & aims

Studies have been conducted to evaluate maintenance of cell viability and stability, as well as to select cheap carriers to extend the shelf life of plant beneficial bacterial inoculants for agricultural crops. The purpose of this study was to evaluate the shelf life and the colonization efficiency of novel liquid and gel-based inoculant formulations for sugarcane. The different inoculant formulations were all composed of a mixture of five strains of diazotrophic bacteria (Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense and Burkholderia tropica), which are recognized as sugarcane growth promoters.

Methods

Different inoculant formulations containing as carrier the polymers carboxymethylcellulose (CMC) and corn starch (60/40 ratio) at five different concentrations (named PIC, for Polymeric Inoculant Carrier) were supplemented, or not, with 2?% MgO, an interfacial stabilizing agent. Bacterial survival in the different formulations during storage was evaluated under controlled conditions, and two experiments with mini-cuttings of sugarcane variety RB72454 were carried out under greenhouse conditions.

Results

Laboratory tests showed that in the formulation composed of 0.8?g of the polymeric mixture per 100?g of the final product (PIC 0.8), survival of G. diazotrophicus and A. amazonense was around 10⁹?CFU?mL^?1 after 120?days of storage, regardless of the supplementation with MgO. The other formulation (2.2?g of polymeric mixture, PIC 2.2) presented survival levels of 10⁸?CFU?mL^?1 for up to 60?days of storage for all the individual strains. In the greenhouse, sugarcane seedlings showed a positive growth response 50?days after inoculation when inoculated with the mixture of five bacteria, with and without PIC 2.2.

Conclusions

The polymer carriers described here allowed for the long-term survival of the five different bacterial strains tested. In addition, short-term experiments in the greenhouse showed that their application as part of an inoculant on sugarcane cuttings was at least as effective in terms of bacterial colonization and the promotion of plant growth as that of the bacterial mixture without carriers.     

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.     

An essential virulence protein of Agrobacterium tumefaciens,VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA

The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer.     

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.