Rhizobial Populations Research Articles

Antibiotics Resistance in Rhizobium: Type, Process, Mechanism and Benefit for Agriculture.

The use of high-quality rhizobial inoculants on agricultural legumes has contributed substantially to the N economy of farming systems through inputs from biological nitrogen fixation (BNF). Large populations of symbiotically effective rhizobia should be available in the rhizosphere for symbiotic BNF with host plants. The rhizobial populations should also be able to compete and infect host plants. However, the rhizosphere comprises large populations of different microorganisms. Some of these microorganisms naturally produce antibiotics which are lethal to susceptible rhizobial populations in the soil. Therefore, intrinsic resistance to antibiotics is a desirable trait for the rhizobial population. It increases the rhizobia's chances of growth, multiplication and persistence in the soil. With a large population of rhizobia in the soil, infectivity of host plants and the subsequent BNF efficiency can be guaranteed. This review, therefore, puts together findings by various researchers on antibiotic resistance in bacteria with the main emphasis on rhizobia. It describes the different modes of action of different antibiotics, the types of antibiotic resistance exhibited by rhizobia, the mechanisms of acquisition of antibiotic resistance in rhizobia and the levels of tolerance of different rhizobial species to different antibiotics.

Isolation and Molecular Characterization of Elite Indigenous <i>Rhizobia</i> Nodulating <i>Phaseolus</i> bean (<i>Phaseolus vulgaris</i> L.)

Nowadays application of biological nitrogen fixation (BNF) through rhizobia inoculums is highly promoted as a solution to solve the problem of poor soil fertility in areas where legumes are cultivated. This is due to the fact that, rhizobia enhance nitrogen fixation, induce disease resistance, reduce heavy metal in the soil, facilitate bioavailabity of iron in soil and is environmental friendly. To get rhizobia strains which are suitable for inoculants production, isolation and molecular characterization of elite rhizobia are highly needed. Molecular characterization acts as a spark plug for discovery of many microbes including Rhizobia. Multi Locus Sequence Analysis (MLSA), 16S rRNA gene sequence analysis, DNA-DNA hybridization and SDS-PAGE analysis of the whole-cell proteins are the molecular techniques mostly used in characterizing rhizobia. But before deciding to use or not to use rhizobia inoculants in certain areas, knowing the population size of indigenous rhizobia found in that area is very important, because this is a major factor which determines inoculums responses as well gives clues on which areas need or do not need inoculation. The Most Probable Number (MPN) method is mostly used in enumerating rhizobia population of the soil. Given that, in most of the developing countries, including Tanzania, Biological Nitrogen Fixation (BNF) technology is not fully flourished; more efforts in isolation, molecular characterization of elite rhizobia and estimation of indigenous rhizobia population in various areas are required.

Influence of chemical and biological properties of field grown pea and lentil rhizospheres on nodulation status and resident rhizobial population in acidic soils of Assam, India

The influence of rhizospheric properties on nodulation status and resident rhizobial population of pea and lentil grown at twenty three sites of Assam were assessed. Besides the plant infectivity of rhizobia isolated from pea and lentil nodules were tested for symbiotic effectiveness. The survey established significant variation of nodule number ((35.-223.plant)), nodule dry weight (6.6 to 271. mgplant) and most probable number count ( to 147g soil) across the twenty three diverse sites. Rhizospheric properties <italic>viz</italic> pH, organic carbon, available P<sub>2</sub>O<sub>5</sub> and K<sub>2</sub>O could establish significant correlation with nodule number (r=0.892*, 0.771*, 0.753* and 0.669*) and most probable number(r=0.784*, 0.580*, 0.615* and 0.649*) counts, correspondingly. Microbial biomass carbon showed significant correlation with nodule number(r=0.672*). Dehydrogenase and phosphomonoesterase activities too showed significant correlation with nodule number(r=0.695* and 0.634*) and most probable number (r=.575* and 0.534*) count, respectively. The symbiotic effectiveness of isolated rhizobia demonstrated significant variation of nodule score (1.33-3.67). Only 6 isolates could attain nodule score of ≥3.0, irrespective of resident rhizobial population.

Characterization of the papilionoid–Burkholderia interaction in the Fynbos biome: The diversity and distribution of beta-rhizobia nodulating Podalyria calyptrata (Fabaceae, Podalyrieae)

The South African Fynbos soils are renowned for nitrogen-fixing Burkholderia associated with diverse papilionoid legumes of the tribes Crotalarieae, Hypocalypteae, Indigofereae, Phaseoleae and Podalyrieae. However, despite numerous rhizobial studies in the region, the symbiotic diversity of Burkholderia has not been investigated in relation to a specific host legume and its geographical provenance. This study analyzed the diversity of nodulating strains of Burkholderia from the legume species Podalyria calyptrata. Diverse lineages were detected that proved to be closely related to Burkholderia taxa, originating from hosts in other legume tribes. By analyzing the genetic variation of chromosomal (recA) and nodulation (nodA) sequence data in relation to the sampling sites we assessed the geographical distribution patterns of the P. calyptrata symbionts. Although we found a degree of genetically differentiated rhizobial populations, a correlation between genetic (recA and nodA) and geographic distances among populations was not observed, suggesting high rates of dispersal and rhizobial colonization within Fynbos soils.

Indigenous rhizobia population influences the effectiveness of Rhizobium inoculation and need of inorganic N for common bean (Phaseolus vulgaris L.) production in eastern Ethiopia

Supplement with inorganic N application is essential to improve the common bean production in sub-Saharan Africa. However, the influence of indigenous rhizobial population on the inorganic N requirement with Rhizobium inoculation to secure sustainable way of common bean production system is not well known. The effect of different rates of N application either alone or in combination with Rhizobium inoculation on the nodulation, yield and yield traits of common bean cultivated in soils with different rhizobial population were conducted. Twelve treatments were produced by factorially combined six levels of N fertilizer (0, 20, 40, 60, 80 and 100 kg N ha−1) and two Rhizobium inoculation treatments (inoculated and uninoculated). The treatments were laid out in randomized completely block design and all treatments were replicated three times. Regardless of soil types, nodule number and nodule dry weight decreased with increasing rates of N application. 20 kg N ha−1 both alone and in combination with Rhizobium inoculation resulted in the largest nodulation in all soil types. The largest nodulation were induced in soil with large rhizobial population. Rhizobium inoculation significantly (P < 0.05) improved yield and yield traits of common bean. Moreover, our result revealed that the largest values of investigated traits were observed in inoculated treatment, as compared to the corresponding N rates of uninoculated treatments. The 20, 100 and 40 kg N ha−1 treatments resulted in significantly greater plant total tissue N at soil types with small, medium and large rhizobial population, respectively, as compared to unfertilized control. The highest total biomass yield (TBY) and grain yield (GY) at soil types with small and medium rhizobial population were obtained by the 100 kg N ha−1 treatment in combination with Rhizobium inoculation, while 20 and 40 kg N ha−1 applications produced the greatest TBY and GY, respectively, in soil with large rhizobial population. These results indicate that N requirement is varied based on rhizobial population and effectiveness of native rhizobia in N2 fixation.

Evolution of host-beneficial traits in nitrogen-fixing bacteria: Modeling and construction of systems for interspecies altruism

The literature and our own data on N2-fixing bacteria forming symbioses with plants and providing convenient models to study the evolution of interspecies (microsymbionts --> hosts) altruism are considered in the review. It is presented as a deeply reorganized intraspecies altruism implemented in the clonal population of rhizobia (bacteroids --> undifferentiated bacteria) under the control of kin selection induced by plant hosts. The analysis of this model suggests that it is possible to engineer practically valuable rhizobial strains in which high N2-fixing activity is combined with decreased survival outside of plants.

Effect of plant population and N fertilizer on the growth and yield of bambara groundnut (&lt;i&gt;Vigna subterranean&lt;/i&gt; (L.) Verdc.)

Responses of bambara groundnut (Vigna subterranea (L.) Verdc) to 3 levels of fertilizer N (0, 50, and 100 kg N/ha) and seven plant populations (55555, 63492, 74074, 88888, 111111, 148148 and 222222 plants/ha) were studied under field conditions in Nsukka, Nigeria. The experimental design was a randomized complete block and there were three replications. Seed yield progressively increased with increasing plant population, being significantly highest at 222,222 plants/ha. The application of 100 kg N/ha increased the total dry matter per plant compared with no application or with 50 kg N/ha, although seed yield benefits were not readily apparent. Nodulation was not statistically influenced by N treatment although zero or- low N application showed a slight increase in nodulation, suggesting a lack of adequate population of the effective rhizobium in the location. Innoculation of bambara groundnut seeds with effective rhizobium strain before planting becomes necessary for good nodulation and production of the crop under Nsukka agro-ecological conditions.Key words: Vigna subterranea, nodulation, N-fertilizer, plant population, seed yield, drymatter yield.

&lt;b&gt;Rhizobial diversity from stem and root nodules of &lt;i&gt;Discolobium&lt;/i&gt; and &lt;i&gt;Aeschynomene&lt;/i&gt;

Among the genera that exhibit stem nodulation, Aeschynomene and Discolobium are found in the Pantanal Mato-grossense (wetlands in the state of Mato Grosso, Brazil). Isolates obtained from the stem and root nodules of D. pulchellum, D. psoraleaefolium, D. leptophyllum and A. fluminensis were collected from various locations in the Pantanal de Pocone sub-region and phenotypically characterized and genotyped by restriction analysis of the 16S rDNA gene. Of the 282 isolates obtained from the stem and root nodules, 84.3% alkalized YMA media and 74.1% showed slow growth. No differences in either the phenotype or the genotype among the rhizobial populations isolated from the root or stem nodules of the species Discolobium and A. fluminensis were observed. Among the isolates obtained from D. pulchellum, there was a group that was not similar to any of the reference strains used, and most of the isolates analyzed by PCR-RFLP were similar to the genus Bradyrhizobium.

Predominant populations of indigenous soybean-nodulating Bradyrhizobium japonicum strains obtained from organic farming systems in Minnesota.

Bradyrhizobium from organic fields in Minnesota were isolated and genotyped to assess diversity of soybean-bradyrhizobia in organic farming systems that can be used to improve soybean productivity. Soil samples were collected from 25 organic fields in Minnesota during May to July 2012. Soybean (cv. Lambert) was used as a host to trap indigenous bradyrhizobia in each sample. Genetic diversity of Bradyrhizobium strains (n=733) was determined using the horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (HFERP) DNA fingerprinting technique and the soybean-bradyrhizobia were classified into 79 different genotypes. Of these, 15 dominant genotypes were found and were highly similar (>92% fingerprint similarity) to serotypes USDA 127 (40.4%), USDA 4 (31.8%) and USDA 123 (15.5%), which were the three main populations of soybean-bradyrhizobia in organic fields. Bradyrhizobium japonicum serogroup USDA 4 strains were found to make up a previously unrecognized, predominant rhizobial population in the organic farming soils examined. The relative abundance of strain USDA 4 was negatively correlated with that of USDA 127 and this relationship may be influenced by the levels of NO3 -N and other soil edaphic factors. The local community of bradyrhizobia can be affected by applying inoculant bacteria to organic fields. Based on these results, soybean production in organic farms may be improved by displacing strains similar to USDA 4 with those better at nitrogen fixation and competitive ability than indigenous strains.

Genetic Characterization of Rhizobia Associated with Horse Gram [Macrotyloma uniflorum (Lam.) Verdc.] Based on RAPD and RFLP

Aim: To genetically characterize rhizobia collected from the root nodules of Horse gram plants grown in different soil samples by using the methods RAPD and RFLP. Place and Duration of Study: Root nodules were collected from the plants grown in soil samples collected from different regions in Andhra Pradesh and Telangana states, India. Methodology: These isolates were characterized by two RAPD primers RPO4 and RPO5 of 10 nucleotide length. The PCR amplicons were purified by Qiaquick® PCR purification kit and digested by three restriction endonucleases i.e., TaqI, HpaII and AluI. Results: The RAPD profiles and restriction analysis of 16S rRNA gene with the three Original Research Article Edulamudi et al.; BMRJ, 5(4): 339-350, 2015; Article no.BMRJ.2015.035 340 endonucleases showed high polymorphism. RAPD and RFLP analysis of these 32 horse gram rhizobia showed that these isolates fall into four major clusters. The first clusters of both dendrograms from RAPD and RFLP contained the majority of the rhizobial isolates. Conclusion: From this, it is clear that RAPD and RFLP may play an important role if applied, to know the genetic diversity of rhizobia. High genetic diversity was observed among the horse gram rhizobial population and very few of the bacteria were considered to be identical. It clearly shows that the horse gram rhizobia are phylogenetically distinct.

The Diversity of Pea Microsymbionts in Various Types of Soils and Their Effects on Plant Host Productivity.

The growth and yield of peas cultivated on eight different soils, as well as the diversity of pea microsymbionts derived from these soils were investigated in the present study. The experimental plot was composed of soils that were transferred from different parts of Poland more than a century ago. The soils were located in direct vicinity of each other in the experimental plot. All soils examined contained pea microsymbionts, which were suggested to belong to Rhizobium leguminosarum sv. viciae based on the nucleotide sequence of the partial 16S rRNA gene. PCR-RFLP analyses of the 16S-23S rRNA gene ITS region and nodD alleles revealed the presence of numerous and diversified groups of pea microsymbionts and some similarities between the tested populations, which may have been the result of the spread or displacement of strains. However, most populations retained their own genetic distinction, which may have been related to the type of soil. Most of the tested populations comprised low-effective strains for the promotion of pea growth. No relationships were found between the characteristics of soil and symbiotic effectiveness of rhizobial populations; however, better seed yield was obtained for soil with medium biological productivity inhabited by high-effective rhizobial populations than for soil with high agricultural quality containing medium-quality pea microsymbionts, and these results showed the importance of symbiosis for plant hosts.

Does tree environment in agro-forestry ecosystems influence the population of N2 fixing soil bacteria?

The present study aimed to monitor the rhizobial population present in four “montado“ ecosystems from two different locations, Vaiamonte and Estremoz in the South of Portugal, with natural and sown pastures. The influence of tree canopy of Quercus suber was evaluated in relation to rhizobial population abundance, symbiotic effectiveness and genetic diversity. Results showed that the size of natural rhizobial population in the two older improved pastures analyzed, having more than 30 and 12 years, had the highest values outside the influence of cork tree canopy. However, under the canopy, the population size in these two improved pastures decreased. Also, rhizobial abundance in the youngest sown pastures (>5-years-old) had high values, either beneath the tree canopy or outside its influence. Concerning the effectiveness of symbiotic fixation, results revealed the existence of N2 fixing strains in the four pastures, the highest values being observed in the youngest improved pasture and under the tree canopy. A high genetic diversity of rhizobial population was also found in all pastures, especially outside the influence of the tree canopy.

Impact of the energy crop Jatropha curcas L. on the composition of rhizobial populations nodulating cowpea (Vigna unguiculata L.) and acacia (Acacia seyal L.)

Jatropha curcas, a Euphorbiaceae species that produces many toxicants, is increasingly planted as an agrofuel plant in Senegal. The purpose of this study was to determine whether soil priming induced by J. curcas monoculture could alter the rhizobial populations that nodulate cowpea and acacia, two locally widespread legumes. Soil samples were transferred into a greenhouse from three fields previously cultivated with Jatropha for 1, 2, and 15 years, and the two trap legumes were grown in them. Control soil samples were also taken from adjacent Jatropha-fallow plots. Both legumes tended to develop fewer but larger nodules when grown in Jatropha soils. Nearly all the nifH sequences amplified from nodule DNA were affiliated to the Bradyrhizobium genus. Only sequences from Acacia seyal nodules grown in the most recent Jatropha plantation were related to the Mesorhizobium genus, which was much a more conventional finding on A. seyal than the unexpected Bradyrhizobium genus. Apart from this particular case, only minor differences were found in the respective compositions of Jatropha soil versus control soil rhizobial populations. Lastly, the structure of these rhizobial populations was systematically imbalanced owing to the overwhelming dominance of a very small number of nifH genotypes, some of which were identical across soil types or even sites. Despite these weak and sparse effects on rhizobial diversity, future investigations should focus on the characterization of the nitrogen-fixing abilities of the predominant rhizobial strains.

Salt tolerance of rhizobial populations from contrasting environmental conditions: understanding the implications of climate change.

It is predicted that global climate change may alter environmental parameters such as rainfall distribution which in turn may alter the salinity of soils with unpredictable effects upon soil microbial populations. In the present work the tolerance to salinity of rhizobia, isolated from locations with contrasting climatic conditions, and the potential of strains to fix nitrogen symbiotically under saline conditions were investigated. Since plasmids may encode key genes related to growth and survival under environmental stress conditions, which will reflect on protein synthesis, both the plasmid and protein profiles were analyzed. A multivariate statistical approach related salt tolerance to the origin of the isolates, identifying rainfall and water availability as a possible factor explaining the differences in salt tolerance displayed by rhizobia isolates. The classification analysis allowed the subdivision of isolates in terms of salt tolerance into extremely sensitive (≤0.15%), sensitive (0.15-0.6%), moderately tolerant (0.9-1.5%), tolerant (2.1-3.6%) and extremely tolerant (≥5.4%). Taken all together it was shown that plasmids are involved in salt tolerance and that the impact of salinity on the protein profile and nitrogen fixation varied according to the salt tolerance of the strains, evidencing the susceptibility of rhizobial communities to changes in rainfall regimes.

Genetic diversity and evolution of Bradyrhizobium populations nodulating Erythrophleum fordii, an evergreen tree indigenous to the southern subtropical region of China.

The nodulation of Erythrophleum fordii has been recorded recently, but its microsymbionts have never been studied. To investigate the diversity and biogeography of rhizobia associated with this leguminous evergreen tree, root nodules were collected from the southern subtropical region of China. A total of 166 bacterial isolates were obtained from the nodules and characterized. In a PCR-based restriction fragment length polymorphism (RFLP) analysis of ribosomal intergenic sequences, the isolates were classified into 22 types within the genus Bradyrhizobium. Sequence analysis of 16S rRNA, ribosomal intergenic spacer (IGS), and the housekeeping genes recA and glnII classified the isolates into four groups: the Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi groups, comprising the dominant symbionts, Bradyrhizobium yuanmingense, and an unclassified group comprising the minor symbionts. The nodC and nifH phylogenetic trees defined five or six lineages among the isolates, which was largely consistent with the definition of genomic species. The phylogenetic results and evolutionary analysis demonstrated that mutation and vertical transmission of genes were the principal processes for the divergent evolution of Bradyrhizobium species associated with E. fordii, while lateral transfer and recombination of housekeeping and symbiotic genes were rare. The distribution of the dominant rhizobial populations was affected by soil pH and effective phosphorus. This is the first report to characterize E. fordii rhizobia.

Variations in infectivity of indigenous rhizobial isolates of some soils in the rainforest zone of Nigeria

Nodule formation in legumes is a process that starts with root infection by rhizobia. The present study assessed the population and infectivity of the indigenous rhizobial strains in rainforest soils of Nigeria. Soils were collected from three sites – Idi-Ayunre, Orile-Ilugun (OI) and the University of Ibadan Teaching and Research Farm (UITRF) – and analysed for physico-chemical properties and rhizobial population. Soybean varieties TGx1448-2E and TGx1456-2E and a cowpea variety IT89KD-288 were planted as trap crops on each of the soils, and rhizobia were isolated from their nodules. Infectivity assay was conducted using eight varieties of soybean and a cowpea variety. Most probable number estimate of the rhizobial population showed that the UITRF had significantly higher rhizobial population than the other two locations. OI and the UITRF soils planted with TGx1448-2E had significantly higher nodules and number of strains than other treatments. Among the 70 slow-grower strains isolated, only nine were infective. Three of the nine strains – IDC8, TRC2 and OISa-6e – nodulated at least seven of the eight soybean varieties used for infectivity test. Indigenous rhizobial infectivity of the studied locations was low, and cultivation of grain legume may require rhizobial inoculation for high productivity.

Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China

A total of 155 nodule isolates that originated from seven sites in Northwest China were characterized by PCR-RFLP of the 16S rRNA gene and sequence analysis of multiple core genes (16S rRNA, recA, atpD, and glnII) in order to investigate the diversity and biogeography of Glycine soja-nodulating rhizobia. Among the isolates, 80 were Ensifer fredii, 19 were Ensifer morelense, 49 were Rhizobium radiobacter, and 7 were putative novel Rhizobium species. The phylogenies of E. fredii and E. morelense isolates in a concatenate tree (assembly of all housekeeping genes) were generally consistent with those in individual gene trees. However, incongruence was found in the phylogenies of the different genes of Rhizobium isolates, indicating that lateral transfer or recombination possibly occurred in these gene loci. Despite their species identity, all the isolates in this study formed a single lineage related to E. fredii in nodAand nifH gene phylogenies, which also indicated that the symbiotic genes were laterally transferred between different species. Biogeographic patterns were found at the species and strain genomic type levels, as revealed by BOXA1R fingerprinting, demonstrating that the evolution of rhizobial populations in different geographic locations was related to soil types, altitude and spatial effects. This study is the first to report that E. morelense, R. radiobacter, and Rhizobium sp. are microsymbionts of G. soja, as well as showing that the diversity of G. soja rhizobia is enhanced and new rhizobia have evolved in Northwest China.

Genetic diversity of Rhizobium present in nodules of Phaseolus vulgaris L. cultivated in two soils of the central region in Chile

Although Phaseolus vulgaris L. is native from the Americas and is currently cultured in diverse areas, very little is known about the diversity of symbiotic nitrogen fixing Rhizobium (mycrosymbiont) in many of those cultures. Therefore, the aim of this study was to assess the genetic diversity of Rhizobium present in nodules of P. vulgaris in the central region of Chile. A method to extract DNA from surface-sterilized nodules was applied to two populations of the same seed variety grown in different fields. The 16S rRNA and nifH genes were amplified directly from the DNA extracted. DGGE analysis and clone libraries showed a restricted genetic diversity of the microsymbiotic populations that nodulate P. vulgaris. Both molecular markers revealed the presence of a microsymbiont closely related to Rhizobium etli in all the plants from the soils studied, indicating that the populations of Rhizobium sp. nodulating P. vulgaris in the central region of Chile displayed an extremely low genetic diversity. The level of genetic diversity in microsymbiont populations in plants grown in soils with different origin suggested that other factors rather than the indigenous soil rhizobial populations play a major role in the selection of the symbiotic partner in P. vulgaris.

Joint evolution of kin recognition and cooperation in spatially structured rhizobium populations.

In the face of costs, cooperative interactions maintained over evolutionary time present a central question in biology. What forces maintain this cooperation? Two potential ways to explain this problem are spatially structured environments (kin selection) and kin-recognition (directed benefits). In a two-locus population genetic model, we investigated the relative roles of spatial structure and kin recognition in the maintenance of cooperation among rhizobia within the rhizobia-legume mutualism. In the case where the cooperative and kin recognition loci are independently inherited, spatial structure alone maintains cooperation, while kin recognition decreases the equilibrium frequency of cooperators. In the case of co-inheritance, spatial structure remains a stronger force, but kin recognition can transiently increase the frequency of cooperators. Our results suggest that spatial structure can be a dominant force in maintaining cooperation in rhizobium populations, providing a mechanism for maintaining the mutualistic nodulation trait. Further, our model generates unique and testable predictions that could be evaluated empirically within the legume-rhizobium mutualism.

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

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. 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. 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. 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.