Yeast Mannitol Broth Research Articles

High throughput pH bioassay demonstrates pH adaptation of Rhizobium strains isolated from the nodules of Trifolium subterraneum and T. repens

The purpose of developing this high throughput assay was to determine whether there was evidence of pH adaptation in strains of rhizobia which nodulate subterranean clover (SC) and white clover (WC), and whether this was related to the pH of the soil of origin. pH is a first-order factor influencing the niche preferences of soil microorganisms and has been convincingly shown to be a key driver of soil bacterial communities. Naturalised strains of Rhizobium spp. that are pH-adapted may have the potential to better compete and/or persist in acidic or alkaline soils compared with introduced commercial strains. Three pilot studies were conducted to design the optimised bioassay. This bioassay tested the effect of pH-amended yeast mannitol broth (seven pH values from pH 4.5–9.0), across three time points, on the in vitro growth of 299 Rhizobium strains isolated from the nodules of SC and WC. The media pH where strains demonstrated fastest growth was related to the pH of the soil that strains were isolated from. However, the correlation between media pH and soil pH was strongly influenced by the growth of strains from alkaline soils (alkaline adaptation), especially in strains isolated from SC nodules.

Exploration of indigenous Trichoderma species for their use as biofertilizers, optimization of growth conditions and cultivation on cheap substrates

Members of the genus Trichoderma have substantially improved the agricultural economy through inhibiting the phytopathogens, assimilating nutrients and inducing defensive metabolism against the environmental stresses. The major aim of this study was to isolate indigenous Trichoderma that can be used as a potential biocontrol agent (BCA). Currently, we isolated 14 native fungal isolates from vermicompost, agricultural soils and infected substrates, and identified them as Trichoderma spp. based on their morphological characteristics. Isolates designated V1D, V1F, V3D, V3F, W1, W2, KAL, NAR, BIOC, AG, RD and NIM resembled to T. viride, while MUSH and RF resembled T. harzianum. The ability of these isolates to assimilate different forms of sugars, nitrogen source (N) and phosphates (PO4) were evaluated qualitatively. Isolates showed differing solubilization zones (cm) in agar plates containing cellulose (1.8-9.5 cm), amylose (0.1-2.1 cm), tri-calcium phosphate (0.1-0.17 cm) and di-calcium phosphate (0.33-0.53 cm), to substantiate their biofertilizer potentialities. Confrontation assay with dual culture technique against seven phytopathogens (i.e. Rhizoctonia solani, Aspergillus niger, Sclerotinia sclerotiorum, S. rolfsii, Fusarium solani, F. oxysporum and Botryodiplodia theobromae) revealed promising mycoparasitic activity. Three isolates (MUSH, BIOC and V3F) showed mycelial growth inhibition in the range of 33-77%, compared to the control plate (without isolates). With respect to isolate MUSH, a significantly higher (P< 0.05) dry biomass weight (g) was obtained at pH 7 (0.66 ± 0.05) and pH 6 (0.55 ± 0.05), than at pH 3, pH 4 and pH 5. Similarly, higher biomass significance (P< 0.001) was obtained in yeast mannitol broth (2.58 ± 0.11 g), compared to potato dextrose broth (PDB) and nutrient broth (NB). The production of spores by isolate MUSH was tested on four locally available solid substrates (i.e. corn stalk, rice husk, jackfruit molasses and sugarcane bagasse) through solid state fermentation (SSF). Production of conidia (cfu/g) was higher in corn stalk (72.6 ×108), followed by rice husk (68.4×108), jackfruit molasses (18.6×108) and sugarcane bagasse (12.4×108). High counts of conidia production on these substrates render MUSH isolate efficient to be used as a BCA by the farmers, to enhance their crop productivity.

Mesorhizobium norvegicum sp. nov., a rhizobium isolated from a Lotus corniculatus root nodule in Norway.

Strain 10.2.2T was isolated from a root nodule of a Lotus corniculatus plant growing near Skammestein (Norway). Phenotypic and chemotaxonomic characterization revealed that colonies grown on yeast-mannitol broth agar were circular, convex and slimy. Growth occurred at 28 °C in 0-1 % NaCl and in a pH range from above 4 to 10. Cells were resistant to kanamycin and phosphomycin. They could assimilate carbon sources such as l-lysine, d-mannose, d-mannitol, and l-alanine. Major fatty acids found in the organism were 11-methyl C18 : 1ω7c, C16 : 0, C18 : 1ω7c, C18 : 0 and C19 : 0 cyclo ω8c. Genome sequencing and characterization of the genome revealed its size to be 8.27 Mbp with a G+C content of 62.4 mol%. Phylogenetic analyses based on the 16S rRNA gene and housekeeping gene alignments placed this strain within the genus Mesorhizobium. Pairwise genome-wide average nucleotide identity values supported that strain 10.2.2T represents a new species, for which we propose the name Mesorhizobium norvegicum sp. nov. with the type strain 10.2.2T (=DSM 108834T=LMG 31153T).

Development of liquid rhizobial inoculants and pre-inoculation of alfalfa seeds

Application of liquid microbial inoculants on legume seeds is a sustainable agricultural practice that can improve plant nutrient uptake and increase crop productivity. Inoculants should provide long-term survival of rhizobia in the final product and after application, to legume seeds. Ten different medium formulations of microbial inoculants were examined (yeast mannitol broth with the addition of agar, sodium-alginate, calcium chloride, glycerol or ferric chloride and combinations thereof) for the survival of the efficient nitrogen-fixing rhizobium, Sinorhizobium (Ensifer) meliloti L3Si strain. The most suitable liquid inoculant for survival of L3Si during a storage time of 150 days was the medium formulation containing glycerol in combination with agar or sodium-alginate. Alfalfa seeds were pre-inoculated with four formulations (yeast mannitol broth (YMB), YMB with agar (1 g L-1), YMB with 1 or 5 g L-1 sodium-alginate) for up to three months. Seeds pre-inoculated and stored for one month produced successful alfalfa plants. The nitrogen content in alfalfa obtained from pre-inoculated seeds one month before sowing was adequate and ranged from 3.72-4.19%. Using S. meliloti-based liquid inoculants for alfalfa and application of the pre-inoculation technique can increase the quality of alfalfa crops and reduce cultivation cost.

Hairy root cultures of Salvia viridis L. for production of polyphenolic compounds

Salvia viridis is a containing bioactive phenols plant, which has been used in Turkish medicine. The aim of the study was to establish hairy root culture of the species in order to increase the production of the compounds with pharmacological activity. Transformed roots are promising biotechnological systems that produce great amounts of secondary metabolites; this is attributed to their fast growth and their genetic and biosynthetic stability. Stable hairy root lines of S. viridis were established from shoot explants using Agrobacterium rhizogenes strain A4. The highest frequency of transformation (45%) was achieved when the shoot was inoculated at the node or internode by bacteria cultivated on Yeast/Mannitol/Broth (YMB) medium supplemented with acetosyringone. The transformation of the root clones was confirmed by polymerase chain reaction using aux1, aux2, rolB and rolC primers. Of the five obtained transformed root clones, the highest biomass was achieved for clone K3 (13.63 ± 0.5 g/L after 5 weeks) grown in Woody Plant (WP) medium in darkness. The UPLC-PDA-ESI–MS analysis of hydromethanolic extracts of transformed roots of S. viridis revealed the presence of 10 compounds identified as caffeic acid derivatives. Quantitative analysis showed that rosmarinic acid (RA) was the predominant compound in all clones. The highest RA (35.8 mg/g dry weight) and total polyphenol (41.24 mg/g dry weight) content were evaluated in clone K3 cultured in WP medium. These values were 8-fold higher than those of the non-transformed roots, indicating that hairy root cultivation could be a promising technique for the production of valuable compounds from S. viridis.

Plant growth promotion by Bradyrhizobium japonicum under heavy metal stress

The increase in usage of heavy metals in different industrial activities causes their existence in effluents. Excessive concentrations of these heavy metals pollute soil and water. Heavy metals cause toxicities and other harmful effects not only in humans and animals but also in plants and soil microorganisms. Heavy metals disrupt many biochemical and physiological activities in bacteria, including growth, development, enzyme and hormone production. Indole acetic acid (IAA) is one of the most important hormones in plants, which is secreted by both bacteria and plants. The present study assessed the effects of Ni, Pb and Cu on the growth of Bradyrhizobium japonicum and IAA production.B. japonicum were grown in yeast mannitol broth in a rotary shaker at 100rpm for 18h for each variant. Ni, Pb and Cu with concentrations ranging from 2.5–40mg/L were added to B. japonicum cultures and turbidity was measured at 600nm. Effects of Ni, Pb and Cu on IAA production were determined by measuring the IAA content with Salkowski method. The metal uptake by B. japonicum was determined in the range of 5–50mg/L. A lettuce seedling assay was conducted to observe the effect of B. japonicum on plant growth under heavy metal stress. Lettuce seedlings inoculated with the bacterium were spiked with 5–500mg/L of Ni, Pb and Cu. Shoot and root length, and plant dry biomass were measured after 5days of germination.Bacterial growth was reduced with the increase of Ni and Cu concentrations. However, there was no significant growth effect with Pb in the considered range. The IAA production also followed the same pattern with Pb and Ni, but in the presence of Cu, IAA content was slightly increased and reached an equilibrium. The growth retardations observed in lettuce in the presence of Pb, Ni and Cu were in the decreasing order of Ni>Cu>Pb. Bacterial inoculation reduced the heavy metal stress and increased the shoot and root lengths of lettuce seedlings. FTIR spectrum of the bacterial biomass showed that amine and nitro groups are responsible in metal sorption process. The Pb-treated bacterial biomass showed a considerable difference in the FTIR spectrum compared to other treatments. According to our results, B. japonicum is sensitive to heavy metal stress with a retardation of growth and IAA production, though it is able to enhance the growth of lettuce seedlings under heavy metal stress.

Effects of plasmid curing on Rhizobium spp.

Present study was carried out to determine the difference between the wild type rhizobial strains and plasmid cured strains for some important characteristics such as antibiotic resistance. The rhizobial strains were isolated from locally produced legumes of different varieties such as Pisum sativum, Sesbania aculeata, Vigna mungo, Phaseolus vulgaris, Lens culinaris and Arachis hypogea.They were purified and subjected to various morphological, cultural and biochemical analysis, and also to antibiogram. All the strains showed resistance against amoxicillin and bacitracin, and exhibited higher (50% or more) resistance against ampicillin, erythromycin, nalidixic acid and strptomycin. The isolates were then subjected to plasmid profile analysis. To determine appropriate dose for plasmid curing, Sesbania nodulating rhizobial strain was treated with different concentrations of acridine orange (plasmid curing agent). Rhizobial growth was found in yeast mannitol broth after curing with 30 ?g/ml of acridine orrange. The rhizobial strain was found to be sensitive against amoxicillin, streptomycin and ampicillin after plasmid curing and no exopolysaccharides were found. The present study reveals that the plasmid in rhizobial species might confer the antibiotic resistance and possibly involve in maintaining symbiotic relationships. DOI: http://dx.doi.org/10.3329/sjm.v2i1.15212 Stamford Journal of Microbiology, Vol.2(1) 2012: 34-37

Heterologous expression of Anabaena sp. PCC7120 cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium (Ensifer) meliloti 1021

Sinorhizobium meliloti infects leguminous plants resulting in a nitrogen-fixing symbiosis. Free living cells accumulate poly(3-hydroxybutyrate) (PHB) as carbon and energy source under imbalanced growth conditions. The cphA1 (7120) gene encoding a cyanophycin (CGP) synthetase of Anabaena sp. PCC7120 in plasmids pVLT31::cphA1 (7120) and pBBR1MCS-3::cphA1 (7120) was expressed in the wild-type S. meliloti 1021 and in a phbC-negative mutant generated in this study. Expression of cphA1 (7120) and accumulation of CGP in cells were studied in various media. Yeast mannitol broth (YMB) and pBBR1MCS-3::cphA1 (7120) yielded the highest CGP contents in both S. meliloti 1021 strains. Supplying the YMB medium with isopropyl-β-D-thiogalactopyranoside, aspartic acid, and arginine enhanced CGP contents about 2.5- and 2.8-fold in S. meliloti 1021 (pBBR1MCS-3::cphA1 (7120)) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 (7120)), respectively. Varying the nitrogen-to-carbon ratio in the medium enhanced the CGP content further to 43.8% (w/w) of cell dry weight (CDW) in recombinant cells of S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 (7120)). Cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 (7120)) accumulated CGP up to 39.6% in addition to 12.1% PHB (w/w, of CDW). CGP from the S. meliloti strains consisted of equimolar amounts of aspartic acid and arginine and contained no other amino acids even if the medium was supplemented with glutamic acid, citrulline, ornithine, or lysine. CGP isolated from cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 (7120)) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 (7120)) exhibited average molecular weights between 20 and 25kDa, whereas CGP isolated from Escherichia coli S17-1 (pBBR1MCS-3::cphA1 (7120)) exhibited average molecular weight between 22 and 30kDa. Co-expression of cyanophycinase from Anabaena sp. PCC7120 encoded by cphB1 (7120) in cphA1 (7120)-positive E. coli S17-1, S. meliloti 1021, and its phbC-negative mutant gave cyanophycinase activities in crude extracts, and no CGP granules occurred. A higher PHB content in S. meliloti 1021 (pBBR1MCS-3::cphB1 (7120)::cphA1 (7120)) in comparison to the control indicated that the cells used CGP degradation product (β-aspartate-arginine dipeptide) to fuel PHB biosynthesis.

Induction of hairy roots by Agrobacterium rhizogenes in relation to L-theanine production in Camellia sinensis

SummaryTheanine is a functional compound synthesised in roots of Camellia spp., especially tea (C. sinensis). Hairy roots were induced by two strains of Agrobacterium rhizogenes (15834 and LBA9402). The amino acid, catechin and caffeine compositions of hairy roots were determined by HPLC and compared with roots grown from tea seed. The results showed that the most suitable conditions to induce hairy roots were to use stem segments cut from 70 d-old plantlets, co-cultivated on YMB (yeast, mannitol broth) medium for 2 d, then induced on MS (Murashige and Skoog) medium for 4 weeks. Induced roots grown on LG0 medium had 23.12 mg theanine g–1 fresh weight, which was 23% higher than in roots grown from tea seed. Low concentrations of catechin, caffeine and epicatechin were detected in both root samples. Polymerase chain reaction (PCR) confirmed that the genes rol A, rol B and rol C of A. rhizogenes had been transferred into the DNA of the hairy root cells. The integrated genes for plant hormone synthesis from A. rhizogenes could increase hormone levels in the induced hairy roots, which in turn stimulated root growth and theanine synthesis. Hairy roots with higher concentrations of theanine, and lower levels of catechins and caffeine, are considered to be a good source for extracting L-theanine which is used as an active ingredient in food and medicine.

Influence of chlorsulfuron on rhizobial growth, nodule formation, and nitrogen fixation with chickpea

Sulfonylurea residues have been found to inhibit the growth of some legume crops and pastures in seasons following application. Negative effects of these herbicides on symbiotic nitrogen fixation by legume crops and pastures have been demonstrated. Reductions in nitrogen fixation may result from a direct effect of the herbicide on rhizobial growth and/or an indirect effect on plant growth. In this study the influence of chlorsulfuron on the growth of chickpea rhizobia [Mesorhizobium ciceri (CC1192)], the growth of chickpea plants, and the extent of nodulation and nitrogen fixation by the chickpea/rhizobia symbiosis were examined. In vitro studies (in yeast mannitol broth and a defined medium) showed that chlorsulfuron applied at double the recommended field application rate did not influence the growth of chickpea rhizobia. An experiment using 14C-labelled chlorsulfuron was conducted to determine if rhizobial cells exposed to chlorsulfuron could deliver the herbicide to the point of root infection and nodule formation. Approximately 1% of the herbicide present in the rhizobial growth medium remained with the cell/inoculum material after rinsing with 1/4 strength Ringer’s solution. This was considered unlikely to affect chickpea growth, nodulation, or nitrogen fixation. A pot experiment was used to define the influence of chlorsulfuron on the growth, nodulation, and nitrogen fixation of chickpeas. The presence of chlorsulfuron in the soil reduced the nodulation and nitrogen fixation of the chickpea plants. Pre-exposing rhizobia to chlorsulfuron before inoculating them into pots with germinating chickpea seeds, reduced the number of nodules formed by 51%. Exposure of chickpeas and chickpea rhizobia to chlorsulfuron can adversely affect the formation and activity of symbiotic nitrogen-fixing nodules, even when only the rhizobial inoculant is exposed briefly to the herbicide.

Growth of alfalfa in sludge-amended soils and inoculated with rhizobia produced in sludge.

The efficiency of rhizobial inoculants produced in wastewater sludge used as a growth medium and as a carrier was compared with that of inoculants produced in yeast mannitol broth (YMB) medium and by using peat as a carrier. Alfalfa (Medicago sativa L.) plants were inoculated with solid and liquid Sinorhizobium meliloti inoculants and grown in pots containing two soil types (Kamouraska clay soil and Saint-André sandy soil). The effect of various levels of sludge amendment (60 and 120 kg N/ha) and nitrogen fertilizer (60 kg N/ ha) was also studied. The sludge-based inoculants showed the same symbiotic efficiency (nodulation and plant yield) as YMB-based inoculants. The inoculation increased the nodulation indexes from 4-6 to 8-12, and the rhizobial number from 10(3) (uninoculated soils) to 10(6)-10(7) cells/g in inoculated soils. However, the shoot dry weights and the nitrogen contents were not increased significantly by the inoculation. Applying sludge as an amendment enhanced the rhizobial number in soils from 10(3) to 10(4) cells/g and improved significantly the plant growth (shoot dry weights and nitrogen contents). This improvement increased with sludge rate and with the cut (three cuts). Compared with sludge, N fertilizer gave lower plant yields. The nodulation was not affected by sludge and N-fertilizer application. The texture and physico-chemical properties of soil were found to affect the yield and nitrogen content of the plants. In this study, macroelements and heavy metals were at acceptable levels and were not considered to be negative factors.

Production of growth-promoting substances and high colonization ability of rhizobacteria enhance the nitrogen fixation of soybean when coinoculated with Bradyrhizobium japonicum

Understanding the interaction mechanisms between plant growth-promoting rhizobacteria (PGPR), leguminous crops, and rhizobia is necessary to effectively use PGPR in increasing the biological nitrogen fixation of legumes. We determined the coinoculation effects of Bradyrhizobium japonicum A1017 and a gusA-marked strain of Pseudomonas fluorescens 2137, P. fluorescens WCS365, Azomonas agilis 125, and Azospirillum lipoferum 137 on soybean [Glycine max (L.) Merr] cv. Enrei grown under axenic conditions. The gusA-marked rhizobacteria effectively colonized the root tips and surfaces near the roots tips with a colonization rate ranging from 7.50 to 8.62 log colony forming units (cfu) gfw–1. P. fluorescens 2137 had the highest colonization activity on soybean roots whether inoculated alone or coinoculated with B. japonicum A1017. Coinoculation of P. fluorescens 2137 and B. japonicum A1017 increased the colonization of B. japonicum A1017 on soybean roots, nodule number, and acetylene reduction activity (ARA) at 10 and 20 days after inoculation. Moreover, the addition of sterile spent medium of P. fluorescens 2137 increased the growth of B. japonicum A1017 in yeast mannitol broth (YMB), indicating that P. fluorescens 2137 may have released substances that increased the rhizobial population. The results of this study suggest that the enhanced nodulation and ARA of soybean due to the high colonization of P. fluorescens on soybean roots could depend on the production of growth-promoting substances that stimulate the growth of B. japonicum. However, coinoculation with P. fluorescens WCS365 decreased the nodule number and ARA, despite its slight stimulation of the growth of B. japonicum on the roots, indicating that coinoculation effects are strain dependent.

Regulation of nodule formation in soybean-Bradyrhizobium symbiosis is controlled by shoot or/and root signals

Two strains of Bradyrhizobium japonicum wereevaluated with five commercial cultivars of soybean(Clark, Crauford, Davis, Centaur, and Nessen) and onehypernodulating mutant NOD1-3. The hypernodulatingNOD1-3 produced 30–50 times more nodules thancommercial cultivars either inoculated with B.japonicum strain USDA 123 or RCR 3409. The currentexperiments were extended to determine if therestricted nodulation of commercial cultivars could be overcome by grafting them to a hypernodulated shoot (NOD1-3). Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. The shoots of Clark, Davis, Centaur and Nessen significantlyinhibited nodule formation on the root of NOD1-3,while Crauford shoots did not alter nodule formationon the roots of NOD1-3 as compared with self-grafts ofNOD1-3. It appears that the shoot of NOD1-3 has theability to alter autoregulatory control of nodulationof Clark and Davis cultivars, but did not withCrauford, Centaur and Nessen. The results suggestedthat the regulation of nodulation in soybean cultivarsClark and Davis is controlled by the shoot factors,while the Crauford was root controlled.Reciprocal-grafts between NOD1-3 and Centaur or Nessenindicate that both shoot and root factors involved inregulation of nodulation and the regulation ofnodulation did not depend on bradyrhizobial strains. Isoflavonoid analyses from extracts of grafted plantsshowed that NOD1-3 shoots had markedly higher rootisoflavonoid concentrations in roots of both Clark andNOD1-3. The shoot control of hypernodulation may becausally related to differential root isoflavonoidlevels, which are also controlled by the shoot. Thecurrent work was extended to investigate the effect ofapplication of an isoflavonoid (daidzein) on nodulationand nitrogen fixation of soybean cultivars Clark andCentaur as well as in vitro growth of Bradyrhizobium japonicum. Application of theisoflavonoid (daidzein) significantly enhanced thenodulation and nitrogenase activity of Clark but notof Centaur indicating that this character is notrelated to isoflavonoids. Therefore, autoregulationin Clark and Centaur plants may be separate events inlegume-rhizobia symbiosis and regulated by differentkinds of signals. Addition of daidzein to yeastmannitol broth medium promoted the growth of B.japonicum strain USDA 123 and RCR 3409. It seemsthat this compound is able to help the nodulation ofsoybean cv Clark by a Bradyrhizobium strain. Understanding the signaling pathways between rhizobiaand their host plants may allow modifications of thisinteraction to improve symbiotic performance.

Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil.

A method was developed for the fast screening and selection of high-temperature tolerant rhizobial strains from root nodules of Prosopis juliflora growing in alkaline soils. The high-temperature tolerant rhizobia were selected from 2,500 Rhizobium isolates with similar growth patterns on yeast mannitol agar plates after 72 h incubation at 30 and 45 degrees C, followed by a second screening at 47.5 degrees C. Seventeen high-temperature tolerant rhizobial strains having distinguishable protein band patterns were finally selected for further screening by subjecting them to temperature stress up to 60 degrees C in yeast mannitol broth for 6 h. The high-temperature tolerant strains were NBRI12, NBRI329, NBRI330, NBRI332, and NBRI133. Using this procedure, a large number of rhizobia from root nodules of P. juliflora were screened for high-temperature tolerance. The assimilation of several carbon sources, tolerance to high pH and salt stress, and ability to nodulate P. juliflora growing in a glasshouse and nursery of the strains were studied. All five isolates had higher plant dry weight in the range of 29.9 to 88.6% in comparison with uninoculated nursery-grown plants. It was demonstrated that it is possible to screen in nature for superior rhizobia exemplified by the isolation of temperature-tolerant strains, which established effective symbiosis with nursery-grown P. juliflora. These findings indicate a correlation between strain performance under in vitro stress in pure culture and strain behavior under symbiotic conditions. Pure culture evaluation may be a useful tool in search for Rhizobium strains better suited for soil environments where high temperature, pH, and salt stress constitutes a limitation for symbiotic biological nitrogen fixation.

RHIZOBIUM GROWTH AND SURVIVAL AS INFLUENCED BY TEMPERATURE

The number of viable cells produced by Rhizobium on solid media gives an indication of the strains survivability when used as an inoculant. This study was conducted to determine the viability of different strains of Bradyrhizobium. and their ability to nodulate pigeonpea (Calanus calan) and cowpea (Vlgna unguiculata) genotypes at two temperature regimes: 20/10°C and 30/20°C (day/night). Bradyrhizobium strains USDA 3278, 3362 and 3458 were cultured in yeast mannitol broth for 7 days from which a 10% dilution was made. From this dilution, 0.1 ml of a further 10 dilution was allowed to grow on yeast mannitol agar plates at the test temperatures. Strains cultured at 30/20°C had a higher number of viable cells than those cultured at 20/10°C for 4 days. While there was no difference between Rhizobium strains 3278 and 3362, strain 3458 had the highest number of cells per ml of 7-day old broth culture. Pigeonpea and cowpea plants inoculated at 20/10°C with the three Rhizobium strains and those inoculated with 3362 at 30/20°C had no nodules at 30 days after planting. At 30/20°C, plgeonpea and cowpea plants Inoculated with strain 3458 produced a higher number of nodules than those inoculated with strain 3278. These results indicate that temperature Influences the Rhizobium strains and their ability to nodulate plgeonpea and cowpea.

Factors affecting the determination of the moisture characteristics of peat by the filter paper method

The filter paper method of Fawcett and Collis-George (1967) for determining soil moisutre characteristics was modified for use with finely milled peat; a carrier used in the production of legume inoculants. The filter paper method proved to be as accurate as the standard method using pressure plate apparatus. Fungal growth in the peat and on the filter papers was overcome by using fungicides or by sterilising the peat. However, these preventative measures all significantly affected the resultant moisture characteristic curves (mcc's) by underestimating the amount of moisture required to adjust peat to any specified moisture potential. Substituting either sterile yeast mannitol (YM) broth or YM broth containing Rhizobium leguminosarum bv. trifolii strain TAI had a similar effect. Reducing the equilibration time of the filter papers from 7 to as little as 2 days gave consistent results and avoided fungal growth. The mcc of solid-based carriers for bacterial inoculants should be determined after they have been subjected to the same treatments as used in the production process.

GERMINATION, SEEDLING DEVELOPMENT AND MODULATION OF COWPEA AND PIGEONPEA AT LOW TEMPERATURE

There is an increasing number of tropical legumes presently grown under temperate conditions with varying amounts of success, This growth chamber study examines the germination, modulation and N2 fixation of two cowpea and one pigeonpea genotypes at two temperature regimes, 15/10° C and 20/10° C, day/night. Prior to planting, surface sterilized seeds were inoculated by soaking in yeast mannitol broth containing approximately 2 × 106 cells ml-1 Bradyrhizobium (USDA 3384). Uninoculated control seeds were soaked in sterile water before planting. Air temperature of 15/10°C, day/night delayed seed germination, nodule initiation, and seedling development. Inoculated cowpea seeds planted at the 20/10° C regime attained 50% germination within 9 days, while inoculated pigeonpea took 13 days under similar regime. Bradyrhizobium persistence was not significantly affected by low temperature. The results indicate that nodule development for both crops were inhibited chiefly by a lack of developing root hairs at low soil temperature,

Influence of host cultivars and Bradyrhizobium strains on the growth and symbiotic performance of soybean under salt stress

This paper examines the importance of salt tolerance of host cultivars, Bradyrhizobium strains, and host-Bradyrhizobium combinations on the symbiotic nitrogen fixation potential of soybean under NaCl and KCl salt stress. Plants were grown in a soil medium, and the experiments were conducted under controlled environment growth room conditions. Bradyrhizobium growth was examined in yeast-mannitol broth andB. japonicum strains tolerant of NaCl and KCl (80 mM) stress were identified. Soybean cultivar Williams, which was sensitive to salt stress, performed poorly both in growth and symbiotic nitrogen fixation, irrespective of whether it was matched with a tolerant or sensitive Bradyrhizobium strain. Tolerant cultivar Manchu sustained nodulation and nitrogen fixation, irrespective of whether it was matched with a tolerant or sensitive Bradyrhizobium strain. Evidence presented here suggests a need, first to select soybean cultivars that are tolerant to salt stress, and then to match them with tolerant and effective Bradyrhizobium strains.

Large-Scale Production of Rhizobium meliloti on Whey

Whey, a by-product of the cheese industry, can sustain the growth of fast-growing rhizobia. To avoid any latency of growth, rhizobial inoculum must be prepared under inducing conditions. In unsupplemented whey, the number of cells of Rhizobium meliloti Balsac reached 5 x 10 CFU/ml in 48 h of incubation. This is comparable to the yield obtained with yeast-mannitol broth, the standard medium for the growth of rhizobia. In raw whey supplemented with yeast extract (1.0 g/liter) and phosphate (0.5 g/liter), the number of cells reached 10 CFU/ml in 48 h of incubation. This is a twofold increase compared with the population normally obtained in industrial production. Whey represents a relatively inexpensive and efficient substrate medium for the large-scale production of fast-growing rhizobia.

Salinity tolerance of aRhizobium meliloti strain isolated from salt affected soils

The salinity tolerance of aRhizobium meliloti strain isolated from salt-affected soils was examined. Growth of the strain on yeast—mannitol broth containing 0–1.2% NaCl exhibited in all cases the same generation time and simultaneous onset of the stationary phase while the total viable number of cells was the same for three continuous generations. The nodulation, plant yield and elemental composition ofMedicago sativa plants grown on agar slopes, inoculated with cultures from the third generation grown on broth containing 0–1.2% NaCl responded identically to all inocula. The salinity tolerance of the strain in fixing nitrogen was furthermore demonstrated withM. sativa plants grown on either nitrogen-free agar slopes containing 0.2–1.2% NaCl or soil-agar slopes with saline soil in which 0.15 and 0.3% additional NaCl was used.