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.     

Distribution and genetic diversity of rhizobia nodulating natural populations of Medicago truncatula in tunisian soils

A collection of 299 isolates of rhizobia nodulating Medicago truncatula was isolated from 10 Tunisian soils and was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR/RFLP) of 16S rRNA gene. Results showed that 227 and 72 isolates were assigned, respectively, to Sinorhizobium meliloti and Sinorhizobium medicae. In 9 out of 10 soils S. meliloti was detected, whereas S. medicae was recovered from only 5 out of 10 soils. The cross-nodulation of three populations of M. truncatula grown on Bulla Regia soil, which contained naturally the two Sinorhizobium species, showed that M. truncatula population collected from Amra site was selective to S. meliloti at least in soil conditions. Forty-eight isolates of each Sinorhizobium species trapped by M. truncatula populations collected from Bulla Regia, Soliman and Rhayet sites on Bulla Regia soil were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and showed a clear distinction between the two Sinorhizobium species and a higher diversity for S. meliloti.     

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.     

Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media

In acid soil, low pH, reduced availability of nutrients, and toxicity of Al and Mn limit plant growth and the survival and effectiveness of rhizobia. The symbiosis between legumes and rhizobia is particularly sensitive to acid soil stress. A pot experiment evaluated whether Bradyrhizobium japonicum strain growth on acidic agar media would predict ability to colonize the rhizosphere and form effective nodules in acidic soils. Three Indonesian strains of B. japonicum with similar effectiveness at neutral pH in sand culture but with different tolerance of acid soil stress factors in agar media, and an acid-tolerant commercial strain (CB1809) of comparable effectiveness, were tested in three acid soils using the Al tolerant soybean (Glycine max cv PI 416937). At 7 days after inoculation all strains had achieved large rhizosphere populations, but by day 14 the rhizosphere population of the acid-sensitive strain had decreased, while the more acid-tolerant strains increased. The acid-tolerant strains had significantly greater nodulation and symbiotic effectiveness than plants inoculated with the acid-sensitive strain. Laboratory prescreening of B. japonicum for acid, Al and Mn tolerance in acid media successfully identified strains which were symbiotically competent in low pH soils.     

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.     

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.     

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.     

Differential effects of soil disturbance and plant residue retention on function of arbuscular mycorrhizal (AM) symbiosis are not reflected in colonization of roots or hyphal development in soil

The effects of soil disturbance and residue retention on the functionality of the symbiosis between medic (Medicago truncatula L.) and arbuscular mycorrhizal fungi (AMF) were assessed in a two-stage experiment simulating a crop rotation of wheat (Triticum aestivum L.) followed by medic. Plants were inoculated or not with the AMF, Glomus intraradices and Gigaspora margarita, separately or together. The contribution of the arbuscular mycorrhizal (AM) pathway for P uptake was determined using ³²P-labeled soil in a small hyphal compartment accessible only to hyphae of AMF. In general AM colonization was not affected by soil disturbance or residue application and disturbance did not affect hyphal length densities (HLDs) in soil. At 4 weeks disturbance had a negative effect on growth and phosphorus (P) uptake of plants inoculated with G. margarita, but not G. intraradices. By 7 weeks disturbance reduced growth of plants inoculated with G. margarita or AMF mix and total P uptake in all inoculated plants. With the exception of plants inoculated with G. margarita in disturbed soil at 4 weeks, the AM pathway made a significant contribution to P uptake in all AM plants at both harvests. Inoculation with both AMF together eliminated the negative effects of disturbance on AM P uptake and growth, showing that a fungus insensitive to disturbance can compensate for loss of contribution of a sensitive one. Application of residue increased growth and total P uptake of plants but decreased ³²P in plants inoculated with the AMF mix in disturbed soil, compared with plants receiving no residue. The AMF responded differently to disturbance and G. intraradices, which was insensitive to disturbance, compensated for lack of contribution by the sensitive G. margarita when they were inoculated together. Colonization of roots and HLDs in soil were not good predictors of the outcomes of AM symbioses on plant growth, P uptake or P delivery via the AM pathway.     

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.     

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.     

Controlled ectomycorrhization of an exotic legume tree species Acacia holosericea affects the structure of root nodule bacteria community and their symbiotic effectiveness on Faidherbia albida, a native Sahelian Acacia

Many fast growing tree species have been introduced to promote biodiversity rehabilitation on degraded tropical lands. Although it has been shown that plant productivity and stability are dependent on the composition and functionalities of soil microbial communities, more particularly on the abundance and diversity of soil symbiotic micro-organisms (mycorrhizal fungi and rhizobia), the impact of tree introduction on soil microbiota has been scarcely studied. This research has been carried in a field plantation of Acacia holosericea (Australian Acacia species) inoculated or not with an ectomycorrhizal fungus isolate, Pisolithus albus IR100. After 7 year's plantation, the diversity and the symbiotic properties of Bradyrhizobia isolated from the plantation soil or from the surrounding area (Faidherbia albida (Del.) a. Chev. parkland) and able to nodulate F. albida, a native Sahelian Acacia species, have been studied. Results clearly showed that A. holosericea modified the structure of Bradyrhizobia populations and their effectiveness on F. albida growth. This negative effect was counterbalanced by the introduction of an ectomycorrhizal fungus, P. albus, on A. holosericea root systems.In conclusion, this study shows that exotic plant species can drastically affect genotypic and symbiotic effectiveness of native Bradyrhizobia populations that could limit the natural regeneration of endemic plant species such as F. albida. This effect could be counterbalanced by controlled ectomycorrhization with P. albus. These results have to be considered when exotic tree species are used in afforestation programs that target preservation of native plants and soil ecosystem rehabilitation.     

Influence of soil type and indigenous pathogenic fungi on bean hypocotyl rot caused by Rhizoctonia solani AG4 HGI in Cuba

Calcisol, ferralsol and vertisol soils, representative of different bean production areas of Villa Clara province in Cuba, were selected to determine the impact of soil type on bean hypocotyl rot severity caused by Rhizoctonia solani AG4 HGI (isolate CuVC-Rs7). In inoculated autoclaved soil, hypocotyl rot was most severe in calcisol soil, followed by ferralsol soils and then vertisol soils. In inoculated natural soils, disease severity was lower in vertisol and calcisol soils and higher in ferralsol soil, indicating that biological factors are suppressing or stimulating the pathogenic efficiency of R. solani. Native binucleate Rhizoctonia AGF, Sclerotium rolfsii and R. solani AG 4 HGI were isolated from bean plants grown in natural calcisol, vertisol and ferralsol soils, respectively. Subsequent studies about the interaction between these fungi and R. solani indicated that they were involved in the variability of disease severity caused by R. solani. The addition of R. solani AG4 HGI (isolate CuVC-Rs7) into each autoclaved soil inoculated with binucleate Rhizoctonia or S. rolfsii resulted in a reduction of disease severity caused by this pathogen while in soils inoculated with native R. solani AG4 HGI, disease severity increased. Irrespective of fungal interactions, calcisol was always the most disease conducive soil and vertisol the most disease repressive soil. The mechanisms by which native pathogenic fungi could influence disease severity caused by R. solani are discussed.     

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.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.     

Quantification and bioavailability of scyllo-inositol hexakisphosphate in pasture soils

The recent identification of scyllo-inositol hexakisphosphate in alkaline soil extracts by solution ³¹P NMR spectroscopy allowed us to investigate this compound in soils by re-analyzing spectra from two previously published studies. Concentrations of scyllo-inositol hexakisphosphate in 29 temperate pasture soils from England and Wales ranged between 11 and 130 mg P kg⁻¹ soil and accounted for between 4 and 15% of the soil organic phosphorus. The ratio of scyllo-inositol hexakisphosphate to myo-inositol hexakisphosphate ranged between 0.29 and 0.79. In a 10 month pot experiment with six grassland soils from New Zealand, growth of pine seedlings (Pinus radiata D. Don) decreased scyllo-inositol hexakisphosphate concentrations by between 10 and 46%. Growth of ryegrass (Lolium perenne L.) decreased scyllo-inositol hexakisphosphate in three low-nutrient soils by 5-21%, but increased it in three other soils by 11-16%. We conclude that scyllo-inositol hexakisphosphate is an important component of soil organic phosphorus with potential ecological significance.     

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.     

New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

Soil texture and irrigation influence the transport and the development of Pasteuria penetrans, a bacterial parasite of root-knot nematodes

The transport of the spores of Pasteuria penetrans was studied in three contrasted textured soils (a sandy, a sandy-clay and a clay soils), cultivated with tomato, inoculated with juveniles of Meloidogyne javanica and watered with 25 or 150 mm day⁻¹. One month after inoculation of the nematodes, 53% of the spores inoculated were leached by water flow in the sandy soil but only 14% in the sandy-clay soil and 0.1% in the clay soil. No nematodes survived in the clay soil, while the population was multiplied both in the sandy and in the sandy-clay soils. But juveniles of M. javanica were more infected by P. penetrans in the sandy-clay soil than in the sandy soil. Comparing different combinations of bare soils containing 1.1-57% of clay showed that the best spore percolation and retention balance occurred in soils amended with 10-30% clay. However, the spore recoveries decreased when the soil was enriched with more than 30% clay. The role of clay particles on the extractability of spores and on their availability to attach to the nematode cuticle in the soil 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.     

Chickpea rhizobium populations: Survey of influence of season,soil depth and cropping pattern

Chickpea Rhizobium populations in soil samples from research stations and farmers' fields in different geographic regions of India ranged from <10 to > 10⁴ rhizobia g⁻¹ soil. Fields on research stations with a known history of chickpea cropping had more rhizobia (calc. 10³ to 10⁵ rhizobia g^1&#x0304; soil) than the majority of farmers' fields (calc. < 10 to 10³ rhizobia g⁻¹ soil). In the absence of chickpea in the cropping pattern, soils generally had < 10² rhizobia g^1&#x0304; and crops in such fields nodulated poorly. However, poor nodulation was also observed when populations of rhizobia were high, indicating that other factors were also important for nodulation. There was no obvious consistent correlation of Rhizobium population with pH, electrical conductivity and nitrate-nitrogen status of the soil.Rhizobium populations declined with soil depth and were highest (about 10⁴ rhizobia g⁻¹ soil) in the top 30 cm of the profile and lowest, but still present (calc. 10³–10³ rhizobia g'1 soil), at 90–120 cm—a depth where no nodules are found. Populations fluctuated most in the top 5 cm, being reduced during periods of high soil temperature in summer and recovering after rains. Rhizobium populations were at a maximum after chickpea but survived well under pigeonpea, groundnut and maize. When rice followed an inoculated chickpea crop, there was about a 100-fold decrease in the Rhizobium population.