Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations

The size of the background rhizobial population can often determine the success of field nodulation and persistence of inoculant rhizobia. Field experiments were conducted to determine the nodulation response of annual medics (Medicago spp.) in a pasture-wheat-pasture rotation when grown in soils of contrasting pH and rhizobial populations. Medicago truncatula Gaertn. and M. polymorpha L. were inoculated with one of three different strains of Sinorhizobium medicae (WSM540, WSM688) or S. meliloti (NA39) or left uninoculated and sown in two fields of pH (CaCl₂) 5.9 and 7.2 of differing soil rhizobial backgrounds (11 and 7.1 × 10⁴ cells/g soil, respectively). Nodulation was assessed in years 1 and 3 of the rotation. At the site with a small rhizobial background, M. polymorpha nodulated poorly when inoculated with the acid-sensitive strain NA39 but nodulated well when inoculated with acid-tolerant strains WSM688 and WSM540. M. truncatula had a similar extent of nodulation with each of the rhizobial inoculants. At the site with a large rhizobial background all treatments had greater than 85% of plants nodulated. Nodule occupancies, assessed by PCR, provided further insight: at the site with a small rhizobial background both medic species successfully nodulated with the acid-tolerant strains WSM540 and WSM688 and these strains persisted to year 3. However, at the site with large rhizobial background, only one strain, WSM688, was identified from M. truncatula nodules in year 3. This study highlights the importance of edaphic constraints and plant–rhizobia interactions to the successful development of nodulation in a field environment.     

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.     

The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

For optimum production, the use of commercial rhizobial inoculant on pea (Pisum sativum L.) at seeding is necessary in the absence of compatible rhizobial strains or when rhizobial soil populations are low or symbiotically ineffective. Multiple site experiments were conducted to characterize the abundance and effectiveness of resident populations of Rhizobium leguminosarum bv. viciae (Rlv) in eastern Canadian prairie soils. A survey of 20 sites across a broad geographical range of southern Manitoba was carried out in 1998 and was followed by more intensive study of five of the sites in 1999 and 2000. Appreciable nodulation of uninoculated pea was observed at all sites which had previously grown inoculated pea. However, uninoculated pea grown at two sites, which had not previously grown pea, had negligible nodulation. Likewise, wild Lathyrus sp. and Vicia sp. plants collected from uncultivated areas adjacent to agricultural sites were poorly nodulated. In the more intensively studied sites, there was a tendency towards higher nodulation in pea plants receiving commercial inoculant containing Rlv strain PBC108 across all site-years (e.g., 4.7% in nodulation and 22% in nodule mass), but the effect was significant at only 2 of 10 site-years. Despite a relatively high range of soil pH (6-8), regression analysis indicated that decreasing soil pH resulted in lower nodulation rates. Likewise, electrical conductivity (EC) was correlated to nodulation levels, however the effect of EC was likely more indicative of the influence of soil texture and organic matter than salinity. As with nodulation, commercial inoculation tended to increase above-ground dry matter (DM) and fixed-N (estimated by the difference method) at the early pod-filling stage, but again the effects were significant at only 2 of 10 site-years. Specifically, above-ground DM and fixed-N levels were up to 29 and 51% greater, respectively, in inoculated compared to non-inoculated treatments at these sites. Addition of N-fertilizer at a rate of 100 kg N ha⁻¹ decreased nodulation at almost all site-years (by as much as 70% at one site), but rarely resulted in increases in above-ground DM compared to inoculated plots. The study indicates for the first time that populations of infective, and generally effective strains of Rlv occur broadly in agricultural soils across the eastern Canadian prairie, but that there is a tendency for increased symbiotic efficiency with the use of commercial inoculant.     

A multi-site field evaluation of granular inoculants for legume nodulation

The most common method of inoculating legume crops in Australia is the application of peat slurry inoculant to seed. The recent introduction of granular (solid) formulations of inoculants into the Australian market has provided the potential to apply rhizobia with greater ease, but their efficacy has not been independently evaluated. Here, we compare the efficacy of a range of experimental and commercially-available granular inoculants on chickpea, faba bean, lentil, lupin and pea crops in comparison with un-inoculated treatments, and with conventional seed-applied peat slurry inoculants. Thirty-seven field experiments were established in Victoria, South Australia and southern New South Wales over five years. Peat slurry inoculants provided effective nodulation of all legumes. Granular inoculants varied markedly in their ability to improve grain legume nodulation. The size of response depended inversely on background nodulation from soil rhizobial populations. At sites with median background nodulation, peat granules and attapulgite clay granules placed with seed resulted in nodulation similar to peat-slurry-based inoculation, but treatments with bentonite clay granules did not increase nodule numbers much above those in un-inoculated treatments. The generally lower numbers of rhizobia g⁻¹ in the bentonite granules, translated to lower rhizobia application rate to the soil. However, differences in number of rhizobia g⁻¹ granule did not fully explain the nodulation differences between granules. Granule moisture content and granule particle size differed markedly between granule types but their influence on nodulation was not tested. Grain yields did not differ between attapulgite granules placed with seed, peat granules and peat slurry inoculants (all well-nodulated treatments), but were lower with bentonite granule inoculants. Yield differences within sites were related to nodulation and the differences between treatments attenuated as background nodulation increased. Overall, these studies demonstrate that certain granule types have the potential to be used in Australia with grain legumes, particularly in circumstances when seed-applied inoculants are problematic, such as where seed fungicides or insecticides need to be applied. However, granular inoculant formulations differ substantially in their potential to produce nodules on a range of grain legumes.     

Strain of Rhizobium lupini determines natural abundance of 15N in root nodules of Lupinus spp

Discrepancies between French and Australian results in measurement of the natural enrichment of ¹⁵N in nodules of Lupinus spp were due to the strains of Rhizobium lupini used. The Australian inoculant strain, WU425, produced nodules enriched in ¹⁵N above values for the entire plants, on all lines of L. luteus and L. albus tested. The total nitrogen and the natural abundance of ¹⁵N in nodules produced by the French strain, LL13, were much lower. In tests in Australia and in France with L. luteus inoculated with 15 other strains of R. lupini from diverse origins, 9 produced nodules with elevated ¹⁵N abundance, but of these 8 were less than with strain WU425. Most strains producing ¹⁵N-enriched nodules were slower-growing on yeast extract-mannitol agar; most faster-growing strains produced little or no nodular ¹⁵N enrichment. For strains with ¹⁵N-enriched nodules, enrichment increased with increasing nodule N·plant⁻¹, and shoot nitrogen tended to be depleted in ¹⁵N. There was no relationship between symbiotic effectiveness of strains (mg N₂ fixed·plant⁻¹) and ¹⁵N enrichment of nodules.     

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.     

A comparison of competitiveness and persistence amongst five strains of Rhizobium tripolii

Five strains of Rhizohium trifolii were used to inoculate Trifolium subterraneum cv. Woogenellup sown into two soils with naturally-occurring populations of R. trifolii. In the 1st year all inoculant strains used singly were present at high frequency in the sampled nodule populations from the inoculated plots. Where an inoculant containing a mixture of equal parts of the 5 strains was used. one strain (WU95) predominated at both sites.The persistence of the strains was followed for a further 3 years at one site. Three of the strains WU95, CC2480a and WU290, were maintained at a high frequency (>75% of nodules sampled) for the entire period, but the other two strains showed poor persistence in this environment. Highly effective strains of rhizobia, not identifiable as inoculant strains, nor present in the nodule population at the first sampling, appeared during the course of the study. One strain. WU290, showed a high degree of variation in symbiotic effectiveness between single colony isolates from the stock culture and also between field isolates that were serologically identical with this strain.     

Rhizobium inoculation of Calopogonium caeruleum

The shade-tolerant cover legume Calopogonium caeruleum is promiscuous in its nodulating habits. In sand culture, symbiotic effectiveness of the strains tested was variable; 6 strains of rhizobia markedly improved shoot yields and 20 increased shoot N content. In pot experiments using cultivated and non-cultivated soils, inoculation gave no significant increase in shoot yields. When grown under rubber in plantation conditions at four localities, shoot dry matter yields, N content and nodulation also were not different from uninoculated plants when sampled for up to 2 yr after planting. This occurred despite the low numbers (< 10 g^?1 soil) of native rhizobia at some sites and an appreciable establishment (> 70% recovery in nodules) by the inoculant strains.     

Influence of pH and calcium on the growth,polysaccharide production and symbiotic association of<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> SEMIA 116 with alfalfa roots

The Sinorhizobium -legume interaction is sensitive to a number of environmental factors, soil acidity being one of the most important. In the typic Hapludoll soil of the central-southern region of Córdoba (Argentine) it was found that the nodulation of alfalfa ( Medicago sativaL.) roots was impaired with a reduction in shoot dry matter under conditions of soil acidity (pH 5.5) Our results showed that the addition of lime as dolomitic limestone at a concentration of 1 t ha ^-1to acid soil caused a significant increase in the nodulation of alfalfa roots inoculated with the strain Sinorhizobium meliloti SEMIA 116 (recommended inoculum for alfalfa) in the greenhouse experiments. The success of the lime treatment may be related not only to an increase in the pH values but also to an increase in the Ca concentration, improving the growth of S. meliloti and its nodulation ability under acidic conditions. In this study, we also demonstrated an increment in the bacterial growth rate as well as in the production of exopolysaccharides and lipopolysaccharides under low pH (5.5) and a high Ca concentration (5 mM) in the culture medium.     

Competitiveness and persistence of introduced rhizobia on oversown clover: Influence of strain,inoculation rate and lime pelleting

Five strains of R. trifolii were evaluated, at two inoculation levels in the presence or absence of lime pelleting, for their ability to compete and persist in a tussock grassland soil containing a naturalized population of rhizobia. Effects on the growth of the oversown white clover (Trifolium repens) were also investigated.Strains showed marked differences in their ability to form nodules on the host in competition with the naturalized population of rhizobia and also differed in their persistence over 15 months. The most competitive and persistent strain was PDDCC 2163 followed by 2153, 2666 and 2668 and the least competitive was 4144. Lime pelleting or increasing the rate of inoculation increased the competitive ability of strains. Strains that were highly competitive increased clover dry matter and N uptake.     

Evaluation of inoculation procedures for Calliandra calothyrsus Meisn. grown in tree nurseries

A series of inoculation experiments was conducted in glasshouses in Senegal and Kenya to evaluate inoculation procedures designed to optimise nodulation and N₂ fixation of Calliandra calothyrsus Meisn. seedlings. Nodulation and plant growth were used as indices of inoculation success. In an experiment carried out in sterile peat/vermiculite mixture, it was established that inoculation of C. calothyrsus with an effective rhizobial strain at the low rate of 1᎒² rhizobia per seedling was satisfactory for nodulation and growth, but further response occurred at rates of up to 1᎒⁹. A second experiment in (unsterilised) Sangalkam soil (Senegal) containing indigenous rhizobia demonstrated that the most successful form of inoculation was liquid inoculant applied around the root collar immediately after transplanting. This method was more successful than seed inoculation or application of alginate bead inoculant. A third experiment was conducted using filtermud inoculant in Leonard jars and unsterilised Muguga nursery soil from Kenya, containing a large population of indigenous rhizobia. Application of liquid inoculant to seedlings was better than seed inoculation. On the basis of our study, we recommend that C. calothyrsus seedlings raised in the nursery should be inoculated with a liquid inoculant immediately or soon after germination.     

NODULATION,NITROGENASE ACTIVITY,AND DRY WEIGHT OF CHICKPEA AND PIGEON PEA CULTIVARS USING DIFFERENT BRADYRHIZOBIUM STRAINS

Chickpea [Cicer arietinum (L.)] and pigeon pea [Cajanus cajan (L.) Millsp.] were grown outside in large clay pots from 1992 to 1995 in Edmond, Oklahoma. Plants were studied to evaluate nodulation, nitrogenase activity, and shoot dry weight (DW) of ‘ICCV-2’ and ‘Sarah’ chickpea inoculated with multistrain, TAL 1148, and TAL 480 Bradyrhizobium, as well as ‘Georgia-1’ and ‘ICPL-87’, pigeon pea inoculated with multistrain TAL 1127, and TAL 1132 Bradyrhizobium. Following wheat [Triticum aestivum (L.) emend. Thell.] harvests in the spring, legumes were planted in the summer and harvested at three successive dates during the following months. Leaves and stems from remaining plants were incorporated into the soil after the last harvest. Across year, chickpea measurements were sensitive to temperature and precipitation whereas pigeon pea measurements were sensitive to length of growing season as well as climate. Pigeon pea consistently demonstrated higher nitrogen-fixing capacity and shoot DW compared with chickpea. Nodule and shoot DW of both species increased with plant age whereas nodule count and nitrogenase activity generally increased with plant age and leveled off or decreased at flowering. Sarah chickpea demonstrated higher nodule count and nodule DW than ICCV-2, as did the Georgia-1 pigeon pea compared with ICPL-87. Shoot DW of Georgia-1 pigeon pea was generally higher than that of ICPL-87. Multistrain inoculum improved nodulation and shoot DW of chickpea, and TAL 1127 improved nodulation of pigeon pea compared with other treatments. These results indicate that specific chickpea and pigeon pea cultivars, along with appropriate Bradyrhizobium strains, may improve nitrogen fixation and DW of these species.     

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand-vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.     

Development of populations of rhizobium trifolii and nodulation of subterranean clover following the cropping phase in crop-pasture rotations in southeastern Australia

Soil samples taken from 28 sites following varying periods of cropping in a crop-pasture rotation contained very low populations of Rhizobium trifolii. Populations were less than 10³g^?1R. trifolii of soil for 89% of the sites and were significantly correlated with soil pH. Application of lime resulted in a build-up of R. trifolii in the absence of the host legume, subterranean clover, but when inoculated clover seed was sown the populations built up to satisfactory levels after the first season's growth, regardless of soil pH.The number of nodules per plant was increased by the application of lime, but the plants growing in unlimed soil had fewer, larger nodules. The increase in nodulation with lime on these low-calcium acid soils persisted to the third growing season.     

Symbiotic relationships and root nodule ultrastructure of the pasture legume Biserrula pelecinus L.—a new legume in agriculture

Biserrula pelecinus is a pasture legume species new to Australian agriculture. The potential N benefit from B. pelecinus pastures in agricultural systems may not be realised if its symbiotic interactions with Mesorhizobium spp. are not well understood. This study evaluated the symbiotic interactions of four strains of Biserrula root-nodule bacteria (WSM1271, WSM1283, WSM1284, WSM1497) with four genotypes of B. pelecinus (cv. Casbah, 93GRC4, 93ITA33, IFBI1) and with a range of related legumes, including species known to be nodulated by strains of Mesorhizobium loti and other Mesorhizobium spp. Structures of root nodules were studied using light and electron microscopy enabling the ultrastructure of effective and ineffective nodules to be compared. B. pelecinus always formed typical indeterminate, finger-like nodules. The number of bacteroids inside symbiosomes varied between host×strain combinations, however, nodules formed by ineffective associations had well developed peribacteroid membranes and abundant bacteroids. Considerable variation was found in N₂-fixing effectiveness of strains isolated from B. pelecinus on the four B. pelecinus genotypes. Strains WSM1271, WSM1284 and WSM1497 nodulated Astragalus membranaceus, only strains WSM1284 and WSM1497 nodulated Astragalus adsurgens. Strain WSM1284 also nodulated Dorycnium rectum, Dorycnium hirsutum, Glycyrrhiza uralensis, Leucaena leucocephala, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus and Ornithopus sativus. The four strains from B. pelecinus did not nodulate Amorpha fruticosa, Astragalus sinicus, Cicer arietinum, Hedysarum spinosissimum, Lotus parviflorus, Macroptilium atropurpureum or Trifolium lupinaster. M. loti strain SU343 nodulated all four genotypes of B. pelecinus. However, M. loti strain CC829 only nodulated B. pelecinus genotypes 93ITA33 and IFBI1 and the nodules were ineffective. The root nodule isolates from H. spinosissimum (E13 and H4) nodulated B. pelecinus cv. Casbah whereas the commercial inoculant strain for Cicer (CC1192) could not nodulate any genotype of B. pelecinus. These results indicate that strains WSM1271, WSM1283 and WSM1497 isolated originally from B. pelecinus have a specific host range while strain WSM1284 is promiscuous in its capacity to nodulate with a broad range of related species. As B. pelecinus can be nodulated by Mesorhizobium spp. from other agricultural legumes, particularly Lotus, there is an opportunity to utilise this trait in cultivar development.     

Effect of inoculum rate on the performance of chickpea (Cicer arietinum L.) Rhizobium strains in the field

Summary The influence of three inoculum rates on the performance of three chickpea (Cicer arietinum L.) Rhizobium strains was examined in the field on a Mollisol soil. Increasing amounts of inoculum improved the performance of the strains. A normal dose (10⁴ cells per seed) applied at different intervals gave non-significant increases in nodulation, nitrogenase activity (acetylene reduction assay), nitrogen uptake and grain yield. A ten-fold increase in inoculum increased nodule number, shoot dry weight, nitrogenase activity (ARA) and grain yield, but increases over the control were significant only for nodule dry weight and nitrogen uptake by shoot and grain. The highest level of inoculum (100 × normal) significantly increased nodule dry weight, grain yield, total nitrogenase activity (ARA) and nitrogen uptake by shoot and grain. Strain TAL 620 was more effective than the other two. Combined nitrogen (60 kg N ha^–1) suppressed nodulation and nitrogenase activity (ARA).Research paper No. 4345 from the Experiment Station, G. B. P. U. A. & T., Pantnagar, Nainital, U. P.     

First indications for the involvement of strigolactones on nodule formation in alfalfa (Medicago sativa)

Strigolactones have recently been suggested to be phytohormones that are present in all plants. Strigolactones are released by roots into the rhizosphere, stimulating the seed germination of parasitic plants such as Striga spp. and Orobanche spp. and play a crucial role in the interaction between plants and symbiotic arbuscular mycorrhizal fungi.By applying different concentrations of the synthetic strigolactone analogue GR24 to alfalfa (Medicago sativa) inoculated with Sinorhizobium meliloti we could show that in alfalfa nodulation is positively affected by the presence of the strigolactone analogue GR24. Moreover, we could show that this increased nodulation cannot be linked with a stimulatory effect of GR24 on the growth or the expression of nod genes of S. meliloti.Putative mechanisms operating in the plant in response to the addition of GR24 and leading to increased nodule formation by rhizobia are discussed.     

A proposal for re-evaluating the most probable number procedure for estimating numbers of Bradyrhizobium spp.

The reliability of the most probable number (MPN) method for estimating bradyrhizobial numbers was evaluated by comparison with the plate count procedure. MPN estimates increased with time of nodulation scoring after seedling inoculation through 6 weeks of incubation. Ratios of MPN to plate counts increased as the bradyrhizobial cell suspension concentration increased. The MPN method could not detect Bradyrhizobium japonicum numbers at concentrations of 10³ colony forming units (CFU) ml^-1 and below. A proposal for re-evaluating MPN estimates is discussed.     

The effect of acidity on the production of signal molecules by Medicago roots and their recognition by Sinorhizobium

The Medicago sativa-Sinorhizobium symbiosis is challenged by acidity, resulting in generally poor nodulation and production. Medicago murex, however, can nodulate and grow at low pH. The effect of low pH on signal exchange in the Sinorhizobium-Medicago symbiosis was studied to gain a greater understanding of the basis for poor nodulation of M. sativa compared to M. murex. Root exudates from M. sativa and M. murex grown in buffered nutrient solution at pH 4.5, 5.8 and 7.0, were collected to measure the expression of nodB induction in Sinorhizobium. A nodB-gusA fusion was constructed and inserted into Sinorhizobium medicae strains WSM419 (acid tolerant) and CC169 (acid sensitive). We identified greater induction by root exudates from both Medicago spp. collected at pH 4.5 than at pH 5.8 and 7.0, less induction by M. murex than M. sativa and less induction of WSM419 than CC169. The same major inducing compounds, 4′,7-dihydroxyflavanone (liquiritigenin), 4′,7-dihydroxyflavone, and 2′,4′,4-trihydroxychalcone (isoliquiritigenin), were identified in exudates of M. murex and M. sativa at all pH values, although in increasing amounts at lower pH. Poor nodulation of M. sativa relative to M. murex under acid conditions is not the consequence of decreased induction of Sinorhizobium nodB by chemical inducers present in the root exudates of both species at low pH.     

Bradyrhizobium sp. (Lupinus) in the winter rainfall region of South Africa

Widespread cultivation of lupin has resulted in the establishment of effective populations of Bradyrhizobium sp. (Lupinus) in the winter rainfall region of the Western Cape, South Africa. To determine whether inoculation increased yields of Lupinus angustifolius L., field trials were carried out at five sites in this region. Populations ranged from 380 rhizobia g^-1 in a moderately alkaline (pH 7.6) soil to >5,000 rhizobia g^-1 in four moderately acid (pH 5.5-5.8) soils. Soil isolates were generally similar to the inoculant strain WU425 in nitrogen-fixing effectiveness but several were significantly less effective. Average effectiveness of isolates from certain soils differed significantly. Although inoculation failed to appreciably increase nodule occupancy by WU425 in acid soils containing high populations of rhizobia, nodule occupancy was increased to 98% in the low population alkaline soil. The latter site was later abandoned because of disease. At the other sites, analysis of seed dry mass and protein content showed that yields were not significantly increased by either inoculation, nitrogen fertiliser (45 kg N ha^-1) or molybdenum applications. Analysis of genomic DNA by PCR fingerprinting showed that WU425 (isolated in Western Australia) and serologically related strains from other cultures clustered separately from the soil isolates. Isolates from the four acid soils were genomically diverse, whereas isolates from the alkaline soil formed a homogeneous cluster. Further investigation is required to determine the benefit of inoculation in alkaline soils of the winter rainfall region of the Western Cape.