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.     

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.     

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.     

Bradyrhizobium technology: a promising substitute for chemical nitrogen fertilizer in Bangladesh agriculture

Nitrogen is the most limiting element in Bangladesh soils and urea is the fertilizer commonly used for supplying it. Bradyrhizobium/Rhizobium inoculant was tried as a source of N nutrition for grain legumes in a number of field experiments. The inoculants markedly increased nodule number, nodule mass, shoot weight and yield of the crops compared to uninoculated control and urea-N treatments. For soybean (Glycine max), inoculation increased yield 113 percent over the control and 49 percent over the urea treament. For groundnut (Arachis hypogaea), the increases were 36 and 11 percent; for lentil (Lens culinaris), 30 and 13 percent; and for mungbean (Vigna radiata), 47 and 7 percent. The local inoculant strains were suitable for dependable inoculant production. The inoculant technology can be used as a promising and cheap substitute of urea for growing food legume crops in Bangladesh.     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

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.     

Genetic diversity of indigenous Bradyrhizobium nodulating promiscuous soybean [Glycine max (L) Merr.] varieties in Kenya: Impact of phosphorus and lime fertilization in two contrasting sites

While soybean is an exotic crop introduced in Kenya early last century, promiscuous (TGx) varieties which nodulate with indigenous rhizobia have only recently been introduced. Since farmers in Kenya generally cannot afford or access fertilizer or inoculants, the identification of effective indigenous Bradyrhizobium strains which nodulate promiscuous soybean could be useful in the development of inoculant strains. Genetic diversity and phylogeny of indigenous Bradyrhizobium strains nodulating seven introduced promiscuous soybean varieties grown in two different sites in Kenya was assayed using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 16S-23S rDNA intergenic spacer region and 16S rRNA gene sequencing. PCR-RFLP analysis directly applied on 289 nodules using Msp I distinguished 18 intergenic spacer groups (IGS) I–XVIII. Predominant IGS groups were I, III, II, IV and VI which constituted 43.9%, 24.6%, 8.3% 7.6% and 6.9% respectively of all the analyzed nodules from the two sites while IGS group VII, IX, X, XI, XII, XIV, XVI, XVII, XVIII each constituted 1% or less. The IGS groups were specific to sites and treatments but not varieties. Phylogenetic analysis of the 16S rRNA gene sequences showed that all indigenous strains belong to the genus Bradyrhizobium. Bradyrhizobium elkanii, Bradyrhizobium spp and Bradyrhizobium japonicum related strains were the most predominant and accounted for 37.9%, 34.5%, and 20.7% respectively while B. yuanmigense related accounted for 6.9% of all strains identified in the two combined sites. The diversity identified in Bradyrhizobium populations in the two sites represent a valuable genetic resource that has potential utility for the selection of more competitive and effective strains to improve biological nitrogen fixation and thus increase soybean yields at low cost.     

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.     

Enhancement of rice production using endophytic strains of Rhizobium leguminosarum bv. trifolii in extensive field inoculation trials within the Egypt Nile delta

This study assessed the ability of biofertilizer inoculants containing Rhizobium leguminosarum bv. trifolii to enhance production of rice (Oryza sativa L.) under actual agricultural conditions in the Nile delta. Large-scale field experiments evaluated 5 rice varieties inoculated with 7 endophytic rhizobial strains during 5 growing seasons, including at sites ranked as the world’s highest in rice production. Inoculation with single strains or multi-strain consortia significantly increased grain yield in 19 of the 24 trials. By combining superior rhizobial inoculants with agricultural extension training, grain yield increased up to 47% in farmers’ fields, with an average increase of 19.5%. Data on rice straw production, harvest index and the agronomic fertilizer N-use efficiency also indicated positive agronomic benefits of rhizobial inoculation. These results establish the merit of deploying our biofertilization strategy using selected rhizobial strains to promote rice production capacity while reducing the need for additional chemical N-fertilizer inputs to maintain agricultural sustainability and acceptable production economy. Technology transfer of this important translational research can significantly help to alleviate hunger and meet the nutritional needs of many people in developing countries.     

Salt and pH tolerance of Rhizobia

Salt and pH tolerance ofRhizobium strains specific forSesbania cannabina Pers., sannhemp (Crotalaria juncea L.) and soybean [Glycine max (L) Merr.] have been studied. The salt effect appeared to be ion-specific. Chlorides were more toxic than sulphates of Na, K and Mg. MgSo₄ has a stimulatory effect at lower than 1.0%. 0.2–0.8% NaHCO₃ was lethal for all the strains.S. cannabina was most salt (3.0%) and alkali (pH 10.0) resistant. Both salt-resistant and salt-sensitive strains were present in the case of sannhemp and soybean. pH 10.0 was not inhibitory for any strain but the strains were sensitive to acidity (pH 3.5–4.0).     

A dry granular inoculant of Rhizobium for soil application

A dry granular inoculant of Rhizobium was prepared from sodium alginate and peralite. High numbers of two groundnut (Arachis hypogaea) Rhizobium strains, NC 92 and TAL 1000 used to prepare inoculants survived in dry granules beyond 180 days. The viable counts were 9.72 and 9.91 log₁₀ rhizobia g^-1 of dry granules for NC 92 and TAL 1000, respectively compared to 8.0 log₁₀ rhizobia g^-1 of peat inoculant for NC 92 at the end of six months storage. The granular inoculant was free from contaminants. In a pot culture experiment the granular inoculant applied to the soil gave similar results when seeds were dressed with a peat inoculant; nodulation and growth of groundnut were similar. The major advantage of this inoculant is that, it can be stored in a dry state without losing much viability.     

Two rhizobacterial strains,individually and in interactions with Rhizobium sp., enhance fusarial wilt control,growth, and yield in pigeon pea

A Pseudomonas aeruginosa strain, RRLJ 04, and a Bacillus cereus strain, BS 03, were tested both individually and in combination with a Rhizobium strain, RH 2, for their ability to enhance plant growth and nodulation in pigeon pea (Cajanus cajan L.) under gnotobiotic, greenhouse and field conditions. Both of the rhizobacterial strains exhibited a positive effect on growth in terms of shoot height, root length, fresh and dry weight, nodulation and yield over the non-treated control. Co-inoculation of seeds with these strains and Rhizobium RH 2 also reduced the number of wilted plants, when grown in soil infested with Fusarium udum. Gnotobiotic studies confirmed that the suppression of wilt disease was due to the presence of the respective PGPR strains. Seed bacterization with drug-marked mutants of RRLJ 04 and BS 03 confirmed their ability to colonize and multiply along the roots. The results suggest that co-inoculation of these strains with Rhizobium strain RH 2 can be further exploited for enhanced growth, nodulation and yield in addition to control of fusarial wilt in pigeon pea.     

Genetic diversity of rhizobia associated with root nodules of white lupin (Lupinus albus L.) in Tunisian calcareous soils

With a view to introducing white lupin (Lupinus albus L.) for cultivation in Tunisian calcareous soils, compatible indigenous rhizobia for nitrogen-fixing symbiosis were investigated and characterized. Two L. albus varieties, Mekna and Lumen, were used to trap rhizobia in soil samples collected from 56 sites with high active lime contents (0–49%). Nodulation occurred in only 15 soils. The local variety, Mekna, developed significantly more root nodules and had a trapping capacity in more soils than the imported variety Lumen. A phylogenetic analysis based on the partial 16S-23S ribosomal RNA internal transcribed spacer region (ITS) and multi-locus sequence analysis (MLSA) of three chromosomal housekeeping genes, recA, atpD and dnaK, showed that strains were affiliated to Agrobacterium, Rhizobium, and Neorhizobium, with large internal diversity, including separate lineages. Infectivity tests highlighted some nodulation specificity at the plant variety level, since the strains originating from Mekna could only nodulate this variety, while strains trapped in Lumen could nodulate both varieties. When inoculated, almost all strains resulted in a significant increase in plant shoot dry weight on L. albus. Although Agrobacterium sp. strains isolated from L. albus could nodulate and had a plant growth promoting effect, no nodA and nodC genes could be amplified. This is discussed together with the absence of bradyrhizobia and the general infrequency of L. albus–nodulating rhizobia in Tunisian soils. The adapted and efficient rhizobial strains reported here were promising candidates for inoculant development and represent a contribution towards successful cultivation of L. albus in Tunisia, especially the most promising Mekna variety.     

Influence of the Size of Indigenous Rhizobial Populations on Establishment and Symbiotic Performance of Introduced Rhizobia on Field-Grown Legumes

Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 × 10⁴ g of soil^-1 for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha^-1 in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P ≤ 0.05). On average, inoculation increased yield by 62%. Soybean (G. max) responded to inoculation most frequently, while cowpea (V. unguiculata) failed to respond in all trials. Inoculation responses in the other legumes were site dependent. The response to inoculation and the competitive success of inoculant rhizobia were inversely related to numbers of indigenous rhizobia. As few as 50 rhizobia g of soil^-1 eliminated inoculation response. When fewer than 10 indigenous rhizobia g of soil^-1 were present, economic yield was significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil^-1. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N₂-fixing crops indicates a potential for improving inoculation technology, the N₂ fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

A Selective Medium for the Isolation and Quantification of Bradyrhizobium japonicum and Bradyrhizobium elkanii Strains from Soils and Inoculants

The ecological examination of members of the family Rhizobiaceae has been hampered by the lack of a selective medium for isolation of root nodule bacteria from soil. A novel non-antibiotic-containing medium has been developed which allows selective isolation of Bradyrhizobium japonicum and B. elkanii strains from soil and inoculants. The medium, BJSM, is based on the resistance of B.japonicum and B. elkanii strains to more than 40 μg of the metals ions Zn²⁺ and Co²⁺ per ml. BJSM does not allow growth of Rhizobium sp. strains. We used BJSM to isolate bacteria from a Hubbard soil and from several commercially prepared soybean inoculants. Ninety-eight percent of the isolates obtained from Hubbard soil nodulated Glycine max cv. Kasota, and between 55 and 95% of the isolates from the commercial inoculants had the ability to nodulate soybeans. Numbers of bradyrhizobia obtained by using BJSM, strain-specific fluorescent antibodies, and the most-probable-number plant infection assay indicated that the three techniques were comparable in quantifying B. japonicum strains in soils and inoculants, although most-probable-number counts were generally 0.5 order of magnitude greater than those obtained by using BJSM. Results of our studies indicate that BJSM is useful for direct isolation and quantification of B. japonicum and B. elkanii from natural soils and inoculants. This medium may prove to be an important tool for autecological and enumeration studies of diverse populations of bradyrhizobia and as a quality control method for soybean inoculants.     

Inoculant Production with Diluted Liquid Cultures of Rhizobium spp. and Autoclaved Peat: Evaluation of Diluents, Rhizobium spp., Peats, Sterility Requirements, Storage, and Plant Effectiveness

Fully grown broth cultures of various fast- and slow-growing rhizobia were deliberately diluted with various diluents before their aseptic incorporation into autoclaved peat in polypropylene bags (aseptic method) or mixed with the peat autoclaved in trays (tray method). In a factorial experiment with the aseptic method, autoclaved and irradiated peat samples from five countries were used to prepare inoculants with water-diluted cultures of three Rhizobium spp. When distilled water was used as the diluent, the multiplication and survival of rhizobia in the peat was similar to that with diluents having a high nutrient status when the aseptic method was used. In the factorial experiment, the mean viable counts per gram of inoculant were log 9.23 (strain TAL 102) > log 8.92 (strain TAL 82) > log 7.89 (strain TAL 182) after 24 weeks of storage at 28°C. The peat from Argentina was the most superior for the three Rhizobium spp., with a mean viable count of log 9.0 per g at the end of the storage period. The quality of inoculants produced with diluted cultures was significantly (P = 0.05) better with irradiated than with autoclaved peat, as shown from the factorial experiment. With the tray method, rhizobia in cultures diluted 1,000-fold or less multiplied and stored satisfactorily in the presence of postinoculation contaminants, as determined by plate counts, membrane filter immunofluorescence, and plant infection procedures. All strains of rhizobia used in both the methods showed various degrees of population decline in the inoculants when stored at 28°C. Fast- and slow-growing rhizobia in matured inoculants produced by the two methods showed significant (P < 0.01) decline in viability when stored at 4°C, whereas the viability of some strains increased significantly (P < 0.01) at the same temperature. The plant effectiveness of inoculants produced with diluted cultures and autoclaved peat did not differ significantly from that of inoculants produced with undiluted cultures and gamma-irradiated peat.     

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.     

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.     

Competitiveness of Rhizobium trifolii Strains Associated with Red Clover (Trifolium pratense L.) in Mississippi Soils

Five strains of Rhizobium trifolii were evaluated in competition with indigenous populations in nodulating red clover (Trifolium pratense L.) cv. Kenland in two different soils in Mississippi. Double antibiotic resistance acquisition was used to measure the proportion of nodules occupied by the introduced mutant strains. In vertisol soil, strains RP113-7, 162BB1, LM1, and 162P17 were recovered in at least 94% of the assayed nodules, whereas TA1 was found in 83.8% of the nodules. At an ultisol location, significant differences were detected within the introduced rhizobia. Strain RP113-7 was recovered at very high rates (99.2% of the assayed nodules), whereas strains 162BB1, LM1, 162P17, and TA1 were all found in 84.9 to 96.0% of the nodules sampled. Forage yield and percent crude protein levels were lower with the less effective but competitive strain (TA1) at both locations. Results indicated that more effective strains of R. trifolii can increase red clover production and symbiotic nitrogen fixation under different environmental conditions in Mississippi.