Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

Nodulation of Trifolium subterraneum by introduced rhizobia in competition with naturalized strains

The ability of 4 strains of Rhizobium trifolii to compete with naturalized strains in nodulating Trifolium subterruneum cv. Mt Barker and cv. Woogenellup was assessed at 5 sites in New South Wales. The populations of naturalized rhizobia at these sites ranged from 4 × 10⁶ rhizobia/g to one where no rhizobia were detected. The introduced strains were inoculated singly or as mixed strain inocula onto seed of the host at 2 × 10⁶ rhizobia/seed. There were marked differences in competitive ability between the strains but these differences were modified by the host cultivar and the site.At the R. trifolii-free site the inoculum strain formed 100% of the nodules in the 1st yr; by the second year serologically unrelated strains had invaded the plots and these formed almost all of the nodules in the 3rd yr. At the site where competition was greatest (4 × 10⁶ naturalized rhizobia/g), there were no differences in the competitive abilities of the strains in the first year but at all other sites WU95 was superior whether used as a single strain or in a mixed strain inoculum. In these sites also the proportion of nodules formed by the inoculum strains declined markedly by the 2nd yr.     

Selection of Rhizobium strains on their competitive ability for nodulation

The shoot dry weight of alfalfa inoculated with an effective strain of Rhizobium meliloti mixed with an ineffective strain in different ratios was found to be directly proportional to the log of the number of effective nodules. Consequently the comparison of the shoot dry weight of plants inoculated with a mixture of effective and ineffective strains with the shoot dry weight of plants inoculated with the effective strain should allow the estimation of the relative competitiveness of the effective strains. To check this. the competitiveness of 14 antibiotic-resistant strains of R. leguminosarum was evaluated in this way and compared with the ability of the strains to form nodules when inoculated to seeds of Vicia faba planted in a soil containing indigenous R. leguminosarum. The percentage of recovery of the inoculum strains in the nodules of field-grown fababeans was positively correlated with the competitiveness of the strains as estimated by the greenhouse test. This simple way of evaluating the nodulating competitiveness of strains of rhizobia being indicative of their competitive behaviour with indigenous rhizobia in the field could therefore be useful for screening a large number of strains for competitiveness.     

Burr medic (Medicago polymorpha L.) selections for improved N2 fixation with naturalised soil rhizobia

Burr medic (Medicago polymorpha L.) is an annual pasture legume that is widely distributed in southern Australian farming systems. Burr medic is nodulated by rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae) that reside in many Australian soils, but the symbioses that develop are often sub-optimal in their rate of N₂ fixation. We attempted to identify burr medic lines, which are able to form effective symbioses with the naturalised Sinorhizobium in Australian field soils, as potential parents for a breeding program. There were three glasshouse experiments. Initially, 222 lines (including the M. polymorpha cvv. Santiago, Serena and Circle Valley) were inoculated with extracts of two soils that had been collected near Waikerie (soil S109) and Lochiel (soil S142) in South Australia. These soils were used because they contained numerically large communities of naturalised Sinorhizobium spp. that produced sub-optimal rates of N₂ fixation with cv. Santiago. None of the 222 lines of burr medic were able to form an effective symbiosis with the rhizobia from soil S109. However, when nodulated by the rhizobia from soil S142, some lines (e.g. SA8194) formed a very effective symbiosis, producing up to double the shoot dry matter (DM) of Santiago and eight times the DM of uninoculated plants. Seven promising lines were selected for further testing (with extracts of nine soils). Subsequently, two lines (SA20056 and SA8194) were selected and their symbiotic performance compared with that of Santiago, using extracts from 28 soils. While soil treatment had a major effect on mean shoot DM (soil N103=120 mg, soil N105=17 mg), the three medic lines performed similarly. Santiago, SA20056 and SA8914 all formed ineffective symbioses with the rhizobia in at least half of the 28 soils, even though >95% of the plants were nodulated. These experiments confirm that ineffective symbioses are common between burr medics and the rhizobia that have become naturalised in many Australian soils. Although some lines of burr medic were identified that were able to form more effective symbioses with the rhizobia in individual soils, none were able to form effective symbioses with a wide range of soil rhizobia. If a plant breeding approach is to be used to improve symbiotic performance of burr medic we propose that its hybridisation with other medic species, that have less specific rhizobial needs, will be required.     

Response of field-grown bean (Phaseolus vulgaris L.) to Rhizobium inoculation and nitrogen fertilization in two Cerrados soils

 Most soils sown with field beans (Phaseolus vulgaris L.) contain indigenous rhizobia which might interfere with the establishment of inoculated strains. As a consequence, the benefits of bean inoculation are usually questioned, and the use of N fertilizer is gradually becoming a common practice. The present study had the objective of evaluating the effectiveness of inoculation and N fertilization in field soil with (site 1) and without (site 2) a previous bean-cropping history. At site 1, which had a rhizobial population of 7×10² cells g^–1 soil, inoculation had no effect on nodulation or yield, whereas at site 2 (<10 cells g^–1 soil) inoculation increased nodulation, nodule occupancy by the inoculated strain and grain yield. N fertilizer decreased nodulation at both sites, but increased grain yield at site 1 but not at site 2, indicating that the response to inoculation and N fertilization depends on the cropping history. When bean was cultivated for the first time, indigenous populations of rhizobia were low and high yields were accomplished solely with seed inoculation, with no further response to N fertilizer. In contrast, previous cultivation of bean increases soil rhizobia, preventing nodule formation by inoculated strains, and N fertilizer may be necessary for maximum yields. A significant interaction effect between N fertilizer and inoculation was detected for serogroup distribution only at site 2, with N fertilizer decreasing nodule occupancy by the inoculated strain and increasing the occurrence of indigenous strains. Consequently, although no benefits were obtained by the combination of inoculation and N fertilizer, this practice may be feasible with the selection of appropriate N-tolerant strains from the indigenous rhizobial population. Received: 26 May 1999     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.     

Effect of strains of Rhizobium trifolii on the establishment of oversown white clover (Trifolium repens)

Strains of Rhizobium trifolii incorporated into commercial peat inoculants were compared for their effect on the establishment and growth of oversown white clover (Trifolium repens) on soils devoid of infective rhizobia.There were marked differences in numbers of seedlings establishing and clover dry matter production per hectare with the various strains. However, when adjusted to a constant number of established seedlings, dry matter production from all strains, apart from one strain at one site, were similar indicating that the strains did not appear to influence the growth of individual clover plants.The marked differences in establishment of clover inoculated with the various strains could not be accounted for by differences in the number of rhizobia in the peat inoculant.Selecting strains of rhizobia for ability to increase establishment is considered important where clover is oversown onto soils devoid of rhizobia.     

Screening clover and Lotus rhizobia for tolerance of acidity and aluminium

Clover rhizobia (55 strains) were screened for tolerance of acidity and Al, using the technique of Keyser and Munns (1979). Assessment of visible turbidity after 14 days indicated three strains tolerant of pH 4.5 (although growth rate was reduced), 25 strains tolerant of 5μm Al and no strains tolerant of 50 μ m Al at pH 5.5.50 μmAl caused a decrease in the numbers of acid-tolerant strains at pH 4.5. Tolerance of acidity or Al was not associated with the pH or Al status of the soil from which a strain was isolated.Screening of eight strains of clover rhizobia and nine strains of Lotus rhizobia using turbidity assessment and viable counts indicated seven strains of clover rhizobia with different degrees of tolerance of 20 μm Al but none tolerant of 50 μm Al at pH 5.5. All Lotus rhizobia (both slow- and fast-growers) were tolerant of 20 and 50 μm Al at pH 5.5, with 50 μm Al causing a reduction in growth rate.Subculturing of strains in non-stressed and stressed media had no effect on the response to 50 μmAl at pH 5.5.     

Rhizobia in tropical legumes—IX. pot and field trials with inoculants for Psophocarpus tetragonolobus (L.) DC.

Antigenically identifiable inoculants for Psophocarpus tetragonolobus were evaluated in three non-sterile soils contained in pots (sandy-clay, Renggam series; a loamy-sand, Sungei Buloh series; silty-clay, Munchong series). Most-probable-numbers of indigenous rhizobia ranged from 4 (Renggam series) to 13 (Munchong series) g^?1. Only two (RRIM 56 and 968) of the eight rhizobia tested formed > 50% of the nodules in all soils. Recovery of two strains (RRIM 968 and UMKL 12) was significantly poorer from the Munchong series soil which had the most indigenous rhizobia and the highest silt plus clay content. In a field trial using a Sungei Buloh series soil containing 700 rhizobia g^?1 capable of nodulating P. tetragonolobus, none of the applied strains formed > 18% of the nodules; two formed no nodules. There were no significant increases in plant yield in response to inoculation in the field trial and in two soils in the pot trials. In Sungei Buloh series soil, RRIM 56 formed 90% of the nodules when the indigenous rhizobia were 5 cells g^?, and 14% when the population was 700 g^?1. This raises the question of the need to inoculate seed sown into soils with high indigenous rhizobial populations, but there was some indication of increasing representation of inoculant strains in nodules with time.     

Evaluation of symbiotic effectiveness and size of resident Rhizobium leguminosarum var. viciae nodulating lentil (Lens culinaris medic) in some Ethiopian soils

This study was initiated to isolate, characterize and select symbiotically effective rhizobia nodulating lentil (Lens culinaris medic) and to enumerate indigenous rhizobia nodulating lentil in some Ethiopian soils. More than 84 nodule and soil samples were collected. In sand culture, only 62 isolates were authenticated as rhizobia nodulating lentil. Analyses of variance indicated that most of the parameters measured were significantly (p < 0.05) improved by inoculation, with the exception of root length. Inoculation increased shoot length, shoot dry weight and plant total nitrogen by 82.3, 196 and 452%, respectively, over negative control (without inoculation and N fertilization). The tested isolates were found to be very effective (20.9%) and effective (77.4%), with only one ineffective isolate. Indigenous rhizobia in the investigated soils ranged from 30 to 5.8 × 10³ cell g^?1 dry soil. A pot experiment with selected rhizobia and nitrogen fertilizer on Chefedonsa and Debrezeit soils did not show any significant difference in shoot dry weight at p < 0.05. From the study, it was observed that most Ethiopian soils were inhabited by a moderate to high number of indigenous rhizobia and rhizobia inoculation did not improve lentil productivity in the investigated soils.     

Diversity in the nutritional requirements of strains of various Rhizobium species

An examination of 85 strains of bacteria from five species of rhizobia (Rhizobium sp., R. japonicum, R. lupini, R. meliloti and R. trifolii), using a new semi-quantitative assay procedure, disclosed wide diversity among the strains in their requirement for, and response to, vitamins, carbon sources, and nitrogen sources. Approximately half of the strains in the first four species grew as well without vitamins as they did when supplied with a vitamin mixture or with yeast extract, but the other strains showed considerable variation in their requirements. Some strains were inhibited by yeast extract, or showed best growth in basic media supplemented with only one vitamin. The strains within the species differed widely in their utilization of gluconate, mannitol and arabinose as C-sources; there was less diversity in their use of glutamine, histidine, NH₄⁺-N and NO₃^?-N as N-sources. The significance of these observations in the culture of rhizobia in the laboratory, in their ecological adaptation to particular environments, and in their ability to form an effective symbiosis with particular host legumes, is discussed.     

N natural abundance of biologically fixed N2 in soybean is controlled more by the Bradyrhizobium strain than by the variety of the host plant

The ¹⁵N natural abundance technique is one of those most easily applied ‘on farm’ to evaluate the contribution of biological N₂ fixation (BNF) to legume crops. When proportional BNF inputs are high, the accuracy of this technique is highly dependent on an accurate estimate of the ¹⁵N abundance of the N derived from N₂ fixation (the ‘B’ value). The objective of this study was to determine the influence of soybean variety on ‘B’ value. Plants of five soybean varieties were inoculated separately with two Bradyrhizobium strains (one Bradyrhizobium japonicum and one Bradyrhizobium elkanii) grown in pots of soil virtually free of bradyrhizobia capable of nodulating soybean. The proportion of N derived from BNF (%Ndfa) was estimated in separate pots where a small quantity of enriched ¹⁵N ammonium sulphate was added. The %Ndfa was then used with the ¹⁵N natural abundance data of the nodulated soybean and non-N₂-fixing reference plants, to determine the ‘B’ value for each soybean variety/Bradyrhizobium association. The varieties nodulated by the B. japonicum strain showed significantly greater N content and %Ndfa than those nodulated by the B. elkanii strain, and in all cases the ‘B’ value of the shoot tissue (‘B_s’) was higher. The differences in ‘B_s’ values between varieties nodulated by the same Bradyrhizobium strain were insignificant, indicating that this parameter is influenced much more by the Bradyrhizobium strain than by the variety of the host plant.     

Improved zinc tolerance in Eucalyptus globulus inoculated with Glomus deserticola and Trametes versicolor or Coriolopsis rigida

The potential of interactions between saprophytic and arbuscular mycorrhizal (AM) fungi to improve Eucalyptus globulus grown in soil contaminated with Zn were investigated. The presence of 100 mg kg ⁻¹ Zn decreased the shoot and root dry weight of E. globulus colonized with Glomus deserticola less than in plants not colonized with AM. Zn also decreased the extent of root length colonization by AM and the AM fungus metabolic activity, measured as succinate dehydrogenase (SDH) activity of the fungal mycelium inside the E. globulus root. The saprophytic fungi Trametes versicolor and Coriolopsis rigida increased the shoot dry weight and the tolerance of E. globulus to Zn when these plants were AM-colonized. Both saprophytic fungi increased the percentage of AM root length colonization and elevated G. deserticola SDH activity in the presence of all Zn concentrations applied to the soil. In the presence of 500 and 1000 mg kg⁻¹ Zn, there were higher metal concentrations in roots and shoots of AM than in non-AM plants; furthermore, both saprophytic fungi increased Zn uptake by E. globulus colonized by G. deserticola. The higher root to shoot metal ratio observed in mycorrhizal E. globulus plants indicates that G. deserticola enhanced Zn uptake and accumulation in the root system, playing a filtering/sequestering role in the presence of Zn. However, saprophytic fungi did not increase the root to shoot Zn ratio in mycorrhizal E. globulus plants. The effect of the saprophytic fungi on the tolerance and the accumulation of Zn in E. globulus was mediated by its effect on the colonization and metabolic activity of the AM fungi.     

Regulation of parasitism by host density: The Bdellovibrio-Rhizobium interrelationship

Rhizobium strains of the cowpea group did not lose viability readily when added to soil, but Bdellovibrio acting on these rhizobia were found in 32 of 90 soils examined. Bdellovibrio did not initiate replication in liquid media at low host densities, but it did multiply once the Rhizohium numbers increased through growth to about 10⁸ ml^?1. From about 10⁴ to 6 × 10⁵ ml^?1Rhizohium cells survived attack by the parasites in liquid media. In nutrient-free buffer, no significant increase in vibrio abundance was evident if the rhizobial frequency was low. whereas Rhizobium populations containing 6 × 10⁸ cells ml^?1 were lysed rapidly. Bdellovibrio did not multiply when introduced into sterile soil with small numbers of the host, but it replicated when the rhizobia were abundant because of the latter's use of soil organic matter for growth or because of the deliberate addition of 10⁸Rhizohium g^?1. Nevertheless, the host persisted in such vibrio-rich soil samples. The abundance of indigenous bdellovibrios increased appreciably in nonsterile soil if the rhizobia were introduced in large but not small numbers. It is suggested that a major reason for the lack of elimination of the host population in soil by its parasites is the need for a critical host cell frequency, large Rhizobium numbers being required for Btiellovibrio to initiate replication and low numbers of surviving hosts no longer being able to support the parasite.     

Isotopic fractionation during N2 fixation by four tropical legumes

To quantify the contribution of biological nitrogen fixation (BNF) to legume crops using the ¹⁵N natural abundance technique, it is necessary to determine the ¹⁵N abundance of the N derived from BNF—the B value. In this study, we used a technique to determine B whereby both legume and non-N₂-fixing reference plants were grown under the same conditions in two similar soils, one artificially labelled with ¹⁵N, and the other not. The proportion of N derived from BNF (%Ndfa) was determined from the plants grown in the ¹⁵N-labelled soil and it was assumed that the %Ndfa values of the legumes grown in the two soils were the same, hence the B value of the legumes could be calculated. The legumes used were velvet bean (Mucuna pruriens), sunnhemp (Crotalaria juncea), groundnut (Arachis hypogaea) and soybean (Glycine max) inoculated, or not, with different strains of rhizobium. The values of %Ndfa were all over 89%, and all the legumes grown in unlabelled soil showed negative δ¹⁵N values even though the plant-available N in this soil was found to be approximately +6.0‰. The B values for the shoot tissue (B_s) were calculated and ranged from approximately −1.4‰ for inoculated sunnhemp and groundnut to −2.4 and −4.5‰ for soybean inoculated with Bradyrhizobium japonicum strain CPAC 7 and Bradyrhizobium elkanii strain 29W, respectively. The B (B_wp) values for the whole plants including roots, nodules and the original seed N were still significantly different between the soybean plants inoculated with CPAC 7 (−1.33‰) and 29W (−2.25‰). In a parallel experiment conducted in monoxenic culture using the same soybean variety and Bradyrhizobium strains, the plants accumulated less N from BNF and the values were less negative, but still significantly different for soybean inoculated with the two different Bradyrhizobium strains. The results suggest that the technique utilized in this study to determine B with legume plants grown in soil in the open air, yields B values that are more appropriate for use under field conditions.     

Five years of simulated atmospheric nitrogen deposition have only subtle effects on the fate of newly synthesized carbon in Calluna vulgaris and Eriophorum vaginatum

To understand the implications of atmospheric nitrogen deposition on carbon turnover in peatlands, we conducted a ¹³C pulse labeling experiment on Calluna vulgaris and Eriophorum vaginatum already receiving long-term (5 years) amendments of 56 kg N ha⁻¹ y⁻¹ as ammonium or nitrate. We examined shoot tissue retention, net ecosystem respiration returns of the ¹³C pulse, and soil porewater DOC content under the two species. ¹³C fixation in Eriophorum leaves was enhanced with nitrogen addition and doubled with nitrate supply. This newly fixed C appeared to be relocated below-ground faster with nitrogen fertilization as respiration returns were unaffected by N inputs. By contrast, increases in ¹³C fixation were not observed in Calluna. Instead, net ecosystem respiration rates over Calluna increased with N fertilization. There was no significant label incorporation into DOC, suggesting a conservative strategy of peatland vegetation regarding allocation of C through root exudation. Greater concentrations of total DOC were identified with nitrate addition in Calluna. Given the long-term nature of the experiment and the high N inputs, the overall impacts of nitrogen amendments on the fate of recently synthesized C in Eriophorum and Calluna in this ombrotrophic peatland were surprisingly more moderate than originally hypothesized. This may be due to N being effectively retained within the bryophyte layer, thus limiting, and delaying the onset of, below-ground effects.     

Rhizobia nodulating African Acacia spp. and Sesbania sesban trees in southern Ethiopian soils are metabolically and genomically diverse

The diversity of 110 rhizobial strains isolated from Acacia abyssinica, A. seyal, A. tortilis, Faidherbia albida, Sesbania sesban, Phaseolus vulgaris, and Vigna unguiculata grown in soils across diverse agro-ecological zones in southern Ethiopia was assessed using the Biolog™ system and amplified fragment length polymorphism (AFLP) fingerprinting technique. By cluster analysis of the metabolic and genomic fingerprints, the test strains were grouped into 13 Biolog and 11 AFLP clusters. Twenty-two strains in the Biolog method and 15 strains in the AFLP analysis were linked to eight and four reference species, respectively, out of the 28 included in the study. Most of the test strains (more than 80% of 110) were not related to any of the reference species by both methods. Forty-six test strains (42% of 110) were grouped into seven corresponding Biolog and AFLP clusters, suggesting that these groups represented the same strains, or in some cases clonal descendants of the same organisms. In contrast to the strains from S. sesban, isolates from Acacia spp. were represented in several Biolog and AFLP clusters indicating the promiscuous nature of the latter and widespread occurrence of compatible rhizobia in most of the soil sampling locations. The results showed that indigenous rhizobia nodulating native woody species in Ethiopian soils constituted metabolically and genomically diverse groups that are not linked to reference species.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.