The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

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.     

Lime pelleting inoculated serradella (Ornithopus spp.) increases nodulation and yield

Lime pelleting of the inoculated seed is recommended for most pasture legume species to improve survival of the rhizobia on the seed and to counter deleterious effects of soil or fertiliser acidity on rhizobial numbers. Except for New South Wales, lime pelleting is specifically not recommended for serradella (Ornithopus spp.). Our objectives were to evaluate effects of lime pelleting on bradyrhizobial numbers on seed, and nodulation and growth of the serradella plants. Three experiments are reported at two acid-soil sites in northern New South Wales involving four cultivars of yellow serradella (Ornithopus compressus) and Bradyrhizobium sp. (Lupinus) strains WSM471 (current inoculant strain) and WU425 and WSM480. Lime pelleting increased bradyrhizobial numbers on seed, 24 h after inoculation, by an average of 90%. Similarly, lime pelleting increased nodulation and shoot dry matter of the inoculated plants by an average of 57 and 28%, respectively. The three strains were similar in effects on plant growth. Relative values for shoot dry weight, averaged over sites, were 100 for WSM471 and 98 for both WU425 and WSM480. Our results confirmed previous research that lime pelleting inoculated serradella seed was not deleterious to survival of the bradyrhizobial inoculum, and showed that it could result in enhanced symbiotic activity of the inoculum in some instances. We recommend lime pelleting of serradella and that WSM471 remain the inoculant strain.     

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.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

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.     

Host-rhizobia interaction for effective inoculation: evaluation of the potential use of the ureide assay to monitor the symbiotic performance of tepary bean (Phaseolus acutifolius A. Gray)

Domesticated and wild-type tepary beans (Phaseolus acutifolius A. Gray) were grown with or without inoculation with rhizobia in pots under bacteriologically controlled conditions in a temperature-controlled glasshouse. Seeds were inoculated with a mixture of seven strains isolated from nodules collected from domesticated field-grown tepary bean in Arizona, USA, or with a commercial inoculant strain for Phaseolus vulgaris (CC511). Different degrees of plant reliance upon N₂ fixation for growth were generated by supplying the inoculated plants throughout growth with nutrients containing a range of concentrations of ¹⁵N-labeled NO₃ (0, 1, 2, 5 or 10 mM). An uninoculated treatment that received 10 mM ¹⁵N-labeled NO₃ was included to provide data for plants solely dependent upon NO₃ for growth. Six weeks after sowing, shoots were harvested for dry matter determination and subsequent ¹⁵N analysis, root-bleeding xylem sap was collected, and nodulation assessed. With regard to shoot biomass production, domesticated lines were more responsive to inoculation, but less responsive to applied N than wild types. All inoculated plants were nodulated, but the field isolates from tepary bean were more effective in N₂ fixation than strain CC511. It was concluded that tepary bean requires a specific inoculant to benefit from fixation of atmospheric N₂. Xylem sap samples were analysed for ureides (allantoin and allantoic acid), amino acid content (α-amino-N), and NO₃ concentration. The amount of ureide-N present in xylem sap was expressed as a percentage of total solute N, described as the relative abundance of ureide-N (RUN), for each N treatment and was compared to the proportion of plant N derived from N₂ fixation (%Ndfa) calculated using a ¹⁵N dilution technique. The RUN values ranged from 8% for saps collected from uninoculated plants provided with 10 mM NO₃ in the nutrient solution (%Ndfa=0) to 86-91% for nodulated plants grown in the absence of externally supplied NO₃ (%Ndfa=100). These data indicated that ureides were the principal product of N₂ fixation exported from the nodules to the shoot in xylem sap. Since RUN values were closely related to %Ndfa, it was proposed that N-solute analysis of xylem sap could provide a valuable analytical tool to monitor the symbiotic performance of tepary bean.     

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.     

Calcium concentrations of soil affect suppressiveness against Aphanomyces root rot of pea

The potential for field soils to cause Aphanomyces root rot of pea (Pisum sativum) was estimated for a large number of samples from commercial pea fields over a period of 5 years, using a greenhouse bioassay. The aim of the research project was to gain a mechanistic understanding of soil suppressiveness to the disease. Regression analysis showed that of the measured soil variables (Ca, Mg, K, P, pH), soil Ca concentrations had the strongest (negative) correlation with disease prevalence, and also a significant negative correlation with disease severity in samples with confirmed presence of the disease. Greenhouse bioassays using a set of non-infested soils inoculated with artificially produced oospore inoculum of the casual organism Aphanomyces euteiches, showed a similar negative correlation between soil Ca content and disease severity. Disease severity was not consistently affected by soil sterilisation, but was lowered by the addition of two different Ca salts. In contrast, addition of sodium bicarbonate to two soils lowered the content of water-soluble Ca in the soils and increased disease severity. Studies of cultures of A. euteiches exposed to varying Ca concentrations in vitro showed that zoospore production was inhibited at submillimolar concentrations, while mycelial growth was stimulated or unaffected. We conclude that free Ca is a major variable controlling the degree of soil suppressiveness against A. euteiches, and that inhibition of zoospore production from oospores is a possible mechanism.     

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.     

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.     

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.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.     

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.     

Interaction between the soil yeast Rhodotorula mucilaginosa and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea

The effect of the soil yeast, Rhodotorula mucilaginosa LBA, on Glomus mosseae (BEG n°12) and Gigaspora rosea (BEG n°9) was studied in vitro and in greenhouse trials. Hyphal length of G. mosseae and G. rosea spores increased significantly in the presence of R. mucilaginosa. Exudates from R. mucilaginosa stimulated hyphal growth of G. mosseae and G. rosea spores. Increase in hyphal length of G. mosseae coincided with an increase in R. mucilaginosa exudates. No stimulation of G. rosea hyphal growth was detected when 0.3 and 0.5 ml per petri dish of yeast exudates was applied. Percentage root length colonization by G. mosseae in soybean (Glycine max L. Merill) and by G. rosea in red clover (Trifolium pratense L. cv. Huia) was increased only when the soil yeast was inoculated before G. mosseae or G. rosea was introduced. Beneficial effects of R. mucilaginosa on arbuscular mycorrhizal (AM) colonization were found when the soil yeast was inoculated either as a thin agar slice or as a volume of 5 and 10 ml of an aqueous solution. R. mucilaginosa exudates (20 ml per pots) applied to soil increased significantly the percentage of AM colonization of soybean and red clover.     

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.     

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.     

Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

The potential use of the legume-rhizobium symbiosis for the remediation of arsenic contaminated sites

The sustainable remediation of arsenic (As) contaminated sites requires an understanding of how As alters the biogeochemical processes in soil. Leguminous species are often used in the remediation of contaminated sites because of their capacity to fix nitrogen and enhance site fertility. While excess As is known to reduce the formation of root nodules in legumes, currently, little is known about how the legume-rhizobium symbiosis is affected by high As concentrations. Soybean (Glycine max) cv. Curringa and its rhizobial symbiont, Bradyrhizobium japonicum strain CB1809, were studied in dilute solution culture at As concentrations of 0, 1, 5 and 10 μM. As the As concentration of the nutrient solution increased, greater time was required for inoculated plants to produce root nodules (P=0.001) and the number of root nodules per plant at harvest decreased (P=0.007). Inspection of the soybean roots showed the number of root hairs decreased as the As concentration in the solution increased. The dry weight of soybean roots and shoots decreased significantly as the As concentration of the nutrient solution increased (P<0.05). Inoculated plants had significantly larger dry weights than noninoculated plants (P<0.05) including a 38% greater biomass for inoculated vs. noninoculated plants in the 10 μM As treatment. The increased biomass in inoculated plants could not be explained by improved N nutrition nor decreased As absorption and it is hypothesised that B. japonicum stimulated the growth of soybean via the production of growth-promoting hormones. This is the first reported evidence of rhizobial bacteria promoting the growth of plants at elevated concentrations of a heavy metal via a mechanism other than improved nitrogen nutrition. The potential use of rhizobia as growth-promoting bacteria for the remediation of heavy metal contaminated sites is an exciting new area of research.     

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.