Hydrogen Peroxide and Strigolactones Signaling Are Involved in Alleviation of Salt Stress Induced by Arbuscular Mycorrhizal Fungus in <Emphasis Type="Italic">Sesbania cannabina</Emphasis> Seedlings

The arbuscular mycorrhizal symbiosis can alleviate salt stress in plants by altering strigolactone levels in the host plant. The aim of this study was to investigate the mechanism by which strigolactones enhance salt stress tolerance in arbuscular mycorrhizal Sesbania cannabina seedlings. Strigolactone levels, as determined by means of germination bioassay, gradually increased with treatment time of NaCl applied. Inhibition of NADPH oxidase activity and chemical scavenging of H₂O₂ significantly reduced strigolactone-induced salt tolerance and decreased strigolactone levels. The H₂O₂-induced strigolactone accumulation was accompanied by increased tolerance to salt stress. These results strongly indicated that elevated H₂O₂ concentration resulting from enhanced NADPH oxidase activity regulated strigolactone-induced salt stress tolerance in arbuscular mycorrhizal S. cannabina seedlings.     

田菁根际促生菌的筛选及其促生耐盐效果

【目的】为探究盐生植物田菁及其根际功能微生物改良盐碱地的效果,本研究从黄河三角洲盐碱区田菁根际土壤中分离促生菌,并明确其耐盐促生效果。【方法】采用选择培养方法从田菁根际土壤中分离固氮菌、解磷菌以及解钾菌,并进行16S rRNA分子生物学鉴定。之后对菌株的耐盐及促生特性进行测定,筛选性状优良菌株进行玉米促生作用研究。【结果】共分离得到105株根际促生菌,其中N102兼具多种促生特性且耐盐性达15%。田菁种子发芽试验表明,N102可显著提高田菁发芽率(47%,P<0.05)、芽长(48.5%,P<0.05)和根长(60%,P<0.05);玉米盆栽试验结果表明,N102对盐胁迫下玉米的株高、根长、叶绿素含量、地上部干重以及根干重具有显著的促进作用。经系统发育分析,N102与Enterobacter soli ATCC BAA-2102 (NR117547)序列相似度为99.30%,鉴定属于Enterobacter属。【结论】菌株N102具有多种植物促生耐盐特性,具有开发成有效促进盐碱地作物生长的微生物肥的良好前景。     

Halorubrum rubrum sp. nov., an extremely halophilic archaeon from a Chinese salt lake

Two halophilic archaeal strains, YC87^T and YCA11, were isolated from Yuncheng salt lake in Shanxi, China. Cells of the two strains were observed to be pleomorphic rod-shaped, stained Gram-negative and produced red-pigmented colonies. Strain YC87^T was able to grow at 20–50 °C (optimum 37 °C), at 1.4–4.8 M NaCl (optimum 2.1 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.3 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.0) while strain YCA11 was able to grow at 20–50 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0.01–0.7 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–9.0 (optimum pH 7.5). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 8 % (w/v) for strain YC87^T and 12 % (w/v) for strain YCA11. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether; another major glycolipid and trace amounts of several unidentified lipids were also detected. The 16S rRNA gene sequences of the two strains were 99.8 % identical, showing 93.2–98.2 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB′ gene similarity between strains YC87^T and YCA11 was 99.3 % and showed 87.5–95.2 % similarity to the closest relative members of the genus Halorubrum. The DNA G+C content of strains YC87^T and YCA11 were determined to be 64.9 and 64.5 mol%, respectively. The DNA–DNA hybridization value between strain YC20^T and strain YC77 was 87 % and the two strains showed low DNA–DNA relatedness with Halorubrum cibi JCM 15757^T and Halorubrum aquaticum CGMCC 1.6377^T, the most related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC87^T and YCA11 represent a novel species of the genus Halorubrum, for which the name Halorubrum rubrum sp. nov. is proposed. The type strain is YC87^T (=CGMCC 1.12124^T = JCM 18365^T).     

Identification of a major QTL allele from wild soybean (Glycine soja Sieb. & Zucc.) for increasing alkaline salt tolerance in soybean

Salt-affected soils are generally classified into two main categories, sodic (alkaline) and saline. Our previous studies showed that the wild soybean accession JWS156-1 (Glycine soja) from the Kinki area of Japan was tolerant to NaCl salt, and the quantitative trait locus (QTL) for NaCl salt tolerance was located on soybean linkage group N (chromosome 3). Further investigation revealed that the wild soybean accession JWS156-1 also had a higher tolerance to alkaline salt stress. In the present study, an F₆ recombinant inbred line mapping population (n = 112) and an F₂ population (n = 149) derived from crosses between a cultivated soybean cultivar Jackson and JWS156-1 were used to identify QTL for alkaline salt tolerance in soybean. Evaluation of soybean alkaline salt tolerance was carried out based on salt tolerance rating (STR) and leaf chlorophyll content (SPAD value) after treatment with 180 mM NaHCO₃ for about 3 weeks under greenhouse conditions. In both populations, a significant QTL for alkaline salt tolerance was detected on the molecular linkage group D2 (chromosome 17), which accounted for 50.2 and 13.0% of the total variation for STR in the F₆ and the F₂ populations, respectively. The wild soybean contributed to the tolerance allele in the progenies. Our results suggest that QTL for alkaline salt tolerance is different from the QTL for NaCl salt tolerance found previously in this wild soybean genotype. The DNA markers closely associated with the QTLs might be useful for marker-assisted selection to pyramid tolerance genes in soybean for both alkaline and saline stresses.     

Determining potential probiotic properties of human originated Lactobacillus plantarum strains

In this study, twenty Lactobacillus plantarum strains which were isolated from the fecal samples of humans were investigated in vitro for their characteristics as potential new probiotic strains. The L. plantarum strains were examined for resistance to gastric acidity in simulated gastric juice at pH 2.0, 2.5, 3.0, and 3.5. The growth of test cultures with different pH was monitored after 0, 10, 30, 60, 90, and 120 min of incubation using a spectrophotometer at 550 nm. At the same time, samples were serially diluted in sterile PBS, and counts of viable bacteria were determined by plate counts using MRS agar for each pH and time parameter. The strains were also examined for resistance to 0.4% phenol, production of H₂O₂, adhesion to Caco-2 cell line and antimicrobial activity. It was determined that the artificial gastric juice, even at pH 2.0, did not significantly change the viability of the cultures. Except L. plantarum AA1-2, all strains were detected at 8 ～ 9 log₁₀ CFU/g. It was found that all L. plantarum strains showed good resistance to 0.4% phenol, and only one strain (AC18-82) produced H₂O₂. Good adhesion of L. plantarum strains to Caco-2 cells was observed in this experiment. These selected strains also showed antimicrobial activity.     

Synergistic Interactions Between Salt-tolerant Rhizobia and Arbuscular Mycorrhizal Fungi on Salinity Tolerance of <Emphasis Type="Italic">Sesbania cannabina</Emphasis> Plants

Legumes can host rhizobia and mycorrhizal fungi, and this triple symbiosis might be exploited to improve saline soil fertility. Therefore, a greater understanding of the interaction of rhizobia and arbuscular mycorrhizal fungus during legume growth in saline soil is required. We investigated the efficiency of salt tolerance conferred by rhizobia in mycorrhizal Sesbania cannabina. Greenhouse experiments were conducted in which S. cannabina plants inoculated with Glomus mosseae BGC NM03D (GM), and two rhizobia strains Agrobacterium pusense YIC4105 (4105) and Neorhizobium huautlense YIC4083 (4083), were exposed to 100 and 200 mM NaCl. Under 200 mM NaCl stress, plants inoculated with 4105, rather than 4083, showed significant increases in shoot and root dry mass compared with non-inoculated plants. Simultaneously, a significant increase over GM-inoculated plants in mycorrhizal colonization and dependency was recorded for 4105 + GM-inoculated plants compared with 4083 + GM-inoculated plants. In addition, under NaCl stress, significant increases in the number and mass of nodules, nitrogenase activity, and leghemoglobin content of nodules occurred in 4105 + GM-inoculated plants compared with 4083 + GM-inoculated plants. Furthermore, the activities of antioxidant enzymes in rhizobia-inoculated plants were significantly higher in the GM + 4105 group than the 4083 + GM group. The malondialdehyde content of plants from the 4105 + GM group was significantly lower than in the 4083 + GM group. Thus, the results revealed a synergistic relationship among the 4105 and GM in alleviating salt stress in S. cannabina. Salt-tolerant rhizobia might improve the salinity tolerance of S. cannabina by enhancing the antioxidant system.     

Selection and Identification of a Listeria monocytogenes Target Strain for Pulsed Electric Field Process Optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23°C for 144 μs, inactivation ranged from 0.7 to 3.7 log₁₀ CFU/ml. Inactivation by 72-μs PEF treatments at 37°C ranged from 0.3 to 2.5 log₁₀ CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.     

Hydrogen emission from nodulated soybeans [Glycine max (L.) Merr.] and consequences for the productivity of a subsequent maize (Zea mays L.) crop

Hydrogen (H₂) is a by-product of the symbiotic nitrogen fixation (N₂ fixation) between legumes and root-nodule bacteria (rhizobia). Some rhizobial strains have an uptake hydrogenase enzyme (commonly referred to as Hup⁺) that recycles H₂ within the nodules. Other rhizobia, described as Hup^?, do not have the enzyme and the H₂ produced diffuses from the nodules into the soil where it is consumed by microorganisms. The effect of this phenomenon on the soil biota and on the soil itself, and consequent stimulation of plant growth, has been demonstrated previously. Soybeans [Glycine max (L.) Merr.] cv. Leichhardt, inoculated with either a Hup⁺ strain (CB1809) or one of two Hup^? strains (USDA442 or USDA16) of Bradyrhizobium japonicum and uninoculated soybeans, plus a non-legume control [capsicum (Capsicum annuum L.)] were grown in the field at Ayr, North Queensland, Australia. The objectives were to examine (1) relationships between N₂ fixation and H₂ emission, and (2) the influence H₂-induced changes in soil might have during the legume phase and/or on the performance of a following crop. Strains CB1809 and USDA442 were highly effective in N₂ fixation (“good” fixers); USDA16 was partly effective (“poor” fixer). The soil had a large but non-uniformly distributed naturalised population of B. japonicum and most uninoculated control plants formed nodules that fixed some N₂. These naturalised strains were classified as “poor fixers” of N₂ and were Hup⁺. H₂ emissions from nodules were assessed for all treatments when the soybean crop was 62 days old. Other parameters of symbiotic N₂ fixation and plant productivity were measured when the crop was 62 and 96 days old and at crop maturity. Immediately after final harvest, the land was sown to a crop of maize (Zea mays L.) in order to determine the consequences of H₂ emission from the soybean crop on maize growth. It was estimated that soybeans inoculated with USDA442, the highly effective Hup^– strain of B. japonicum, fixed 117 kg shoot N/ha (or about 195 kg total N/ha if the fixed N associated with roots and nodules was taken into account), and contributed about 215,000 l H₂ gas per hectare to the ecosystem over the life of the crop. The volume of H₂ evolved from soybeans nodulated by the Hup⁺ strain CB1809 was only 6% of that emitted by the USDA442 treatment, but there was no indication that soybean inoculated with USDA442 benefited from the additional H₂ input. The shoot biomass, grain yield, and amounts of N fixed (105 kg shoot N/ha, 175 kg total N/ha) by the CB1809 treatment were little less than for USDA442 plants. Three days after the soybean crop was harvested, the plots were over-sown with maize along the same row lines in which the soybeans had grown. This procedure exposed the maize roots to whatever influence soybean H₂ emission might have had on the soil and/or the soil microflora immediately surrounding soybean nodules. The evidence for a positive effect of soybean H₂ emission on maize production was equivocal. While the consistent differences between those pre-treatments that emitted H₂ and those that did not indicated a trend, only one difference (out of the 12 parameters of maize productivity that were measured) was statistically significant at P?<?0.05. The findings need substantiation by further investigation.     

Elevated CO2 induces differences in nodulation of soybean depending on bradyrhizobial strain and method of inoculation

High CO₂ has been shown to increase plant growth and to affect symbiotic activity in many legumes species, including soybean (Glycine max [L.] Merr.). In order to assess the interaction between elevated CO₂ and rhizobial symbionts on soybean growth and nodulation, we combined the effects of CO₂ with those of different bradyrhizobial strains and methods of inoculation. Soybean seeds were sown in agricultural soil in pots and inoculated with three strains of Bradyrhizobium japonicum (5Sc2 and 12NS14 indigenous to Quebec soils, and 532c, a reference strain), the inoculum being either applied directly to the seed or incorporated into the soil. Plants were grown in growth chambers (22/17ºC) for 6 weeks, under either near ambient (400 μmol mol^?1) or elevated (800 μmol mol^?1) concentrations of CO_2. Elevated CO₂ increased mass (63%) and number (50%) of soybean nodules, particularly medium and large, allowed a deeper nodule development, and increased shoot dry weight (+30%), shoot C uptake (+33%) and shoot N uptake (+78%), compared to ambient CO₂. The two indigenous strains induced more medium and large nodules under elevated CO₂ than the reference strain and showed the greatest increases in shoot dry weight. Soil inoculation induced higher number of small nodules than seed inoculation, specifically for the two indigenous strains, but did not affect plant growth parameters. We conclude that soybean yield enhancements due to elevated CO₂ are associated with the production of large and medium-size nodules and a deep nodulation, that the two indigenous strains better respond to elevated CO₂ than the reference strain, and that the method of inoculation has little influence on this response.     

ATP-dependent proton-pumping activities of zucchini fruit microsomes. A study of tonoplast and plasma membrane activities

The mesocarp tissue of zucchini (Cucurbita pepo L. cv. Black Beauty, zucchini) fruit exhibits ATP-dependent H⁺-pumping activities associated with tonoplast (nitrate-sensitive) and plasma membrane (vanadate-sensitive) vesicles. The two activities are easily separated on step gradients with isopycnic densities lower than usually reported (< 20% (w/w) sucrose for tonoplast; 25–35% (w/w) sucrose for plasma membrane). The tonoplast is relatively impermeable to H⁺ (the half-time for equilibration of a pH gradient is 23–36 min) compared to plasma membrane (half-time of 4–6 min). Anion permeability was measured by adding ATP in the absence of an accompanying K⁺ salt, then measuring the increase in the pH gradient caused by the addition of a K⁺ salt. The increase in the pH gradient is presumably due to alleviation of the Δψ component (positive inside) and consequent increase in the Δ pH component (acid inside) of the electrochemical gradient by movement of the anion into the vesicle interior. Cl⁻ and NO₃⁻ are permeable, SO₄²⁻ is not. The anion permeabilities of the tonoplast and plasma membrane were similar. This is inconsistent with the marked difference in the H⁺ permeabilities, but might be explained by the presence of anion channel(s) associated with tonoplast-derived vesicles.     

Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

Identification of quantitative trait loci controlling linolenic acid concentration in PI483463 (Glycine soja)

Key message

The QTLs controlling alpha-linolenic acid concentration from wild soybean were mapped on nine soybean chromosomes with various phenotypic variations. New QTLs for alpha-linolenic acid were detected in wild soybean.

Abstract

Alpha-linolenic acid (ALA) is a polyunsaturated fatty acid desired in human and animal diets. Some wild soybean (Glycine soja) genotypes are high in ALA. The objective of this study was to identify quantitative trait loci (QTLs) controlling ALA concentration in a wild soybean accession, PI483463. In total, 188 recombinant inbred lines of F_5:6, F_5:7, and F_5:8 generations derived from a cross of wild soybean PI483463 (~15 % ALA) and cultivar Hutcheson (~9 % ALA) were planted in four environments. Harvested seeds were used to measure fatty acid concentration. Single nucleotide polymorphism markers of the universal soybean linkage panel (USLP 1.0) and simple sequence repeat markers were used for molecular genotyping. Nine putative QTLs were identified that controlled ALA concentration by model-based composite interval mapping and mapped to different soybean chromosomes. The QTLs detected in four environments explained 2.4–7.9 % of the total phenotypic variation (PV). Five QTLs, qALA5_3, qALA6_1, qALA14_1, qALA15_1, and qALA17_1, located on chromosomes 5, 6, 14, 15, and 17 were identified by model-based composite interval mapping and composite interval mapping in two individual environments. Among them, qALA6_1 showed the highest contribution to the PV with 10.0–10.2 % in two environments. The total detected QTLs for additive and epistatic effects explained 52.4 % of the PV for ALA concentration. These findings will provide useful information for understanding genetic structure and marker-assisted breeding programs to increase ALA concentration in seeds derived from wild soybean PI483463.     

The structure of carbonyl horseradish peroxidase: spectroscopic and kinetic characterization of the carbon monoxide complexes of His-42 → Leu and Arg-38 → Leu mutants

Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the involvement of two key distal heme cavity residues, histidine 42 and arginine 38, in the formation and structure of the carbon monoxide complex of HRPC (carbonyl HRPC). The rates of CO binding to the wild-type glycosylated and non-glycosylated recombinant (HRPC*) ferrous enzymes were essentially identical and exhibited the same pH dependence with pK _as at 7.4 and 4.0. Data obtained with the His-42?→?Leu [(H42L)HRPC*)] and Arg-38?→?Leu [(R38L)HRPC*] mutants allowed the pK _a at 7.4 in ferrous HRPC to be assigned to His-42. The infra-red and electronic absorption spectra of HRPC-CO, HRPC*-CO, (R38L)HRPC*-CO and (H42L)HRPC*-CO have been investigated over the pH range 3.0–10.0. HRPC*-CO exhibited two ν?(CO) bands at 1934?cm^–1 and 1905?cm^–1 whose relative intensity changed with pH, showing an acidic and a basic pK _a as previously reported for HRPC [IE Holzbaur; AM English, AA Ismail (1996) J Am Chem Soc 118?:?3354–3359]. (H42L)HRPC*-CO and (R38L)HRPC*-CO exhibited single infra-red bands at 1924.2?cm^–1 (pH?7.0) and 1941.5?cm^–1 (pH?5.0) respectively. Acidic and alkaline pK _as were determined from shifts in the infra-red frequencies and by UV-visible spectrophotometry at the Söret maxima. (H42L)HRPC*-CO exhibited a pK _a at ～pH?4.0 but no alkaline pK _a. (R38L)HRPC*-CO exhibited a single pK _a at pH?6.5. Shifts of 2–3?cm^–1 in ν?(CO) with (H42L)HRPC*-CO in D₂O show that a distal residue is H-bonding to the CO in this variant at both pD?7.5 and 3.9. However, with (R38L)HRPC*-CO, only a small shift of the ν?(CO) band was observed at pD?5.5. The results are consistent with the involvement of Arg-38 in H-bonding to the CO ligand in HRPC and with His-42 modulating the distribution of carbonyl HRPC conformers below pH?8.7. These data are discussed in terms of the importance of distal pocket polarity in HRPC. It is concluded that His-42 can have a pK _a between 4.0 and 8.7 depending on its environment and the nature of the distal ligand at position 38. This enables His-42 to carry out multiple functions during the catalytic cycle of HRPC.     

Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the K_m (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the K_m (MgPEP) was found to be significantly lower than the respective K_m (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism.     

<Emphasis Type="Italic">Virgibacillus kimchii</Emphasis> sp. nov., a halophilic bacterium isolated from kimchi

A Gram-stain-positive, halophilic, rod-shaped, non-motile, spore forming bacterium, strain NKC1-2^T, was isolated from kimchi, a Korean fermented food. Comparative analysis based on 16S rRNA gene sequence demonstrated that the isolated strain was a species of the genus Virgibacillus. Strain NKC1-2^T exhibited high level of 16S rRNA gene sequence similarity with the type strains of Virgibacillus xinjiangensis SL6-1^T (96.9%), V. sediminis YIM kkny3^T (96.8%), and V. salarius SA-Vb1^T (96.7%). The isolate grew at pH 6.5–10.0 (optimum, pH 8.5–9.0), 0.0–25.0% (w/v) NaCl (optimum, 10–15% NaCl), and 15–50°C (optimum, 37°C). The major menaquinone in the strain was menaquinone-7, and the main peptidoglycan of the strain was meso-diaminopimelic acid. The predominant fatty acids of the strain were iso-C_14:0, anteisio-C_15:0, iso- C_15:0, and iso-C_16:0 (other components were < 10.0%). The polar lipids consisted of diphosphatidylglycerol and phosphatidylglycerol. The genomic DNA G + C content of NKC1-2^T was 42.5 mol%. On the basis of these findings, strain NKC1-2^T is proposed as a novel species in the genus Virgibacillus, for which the name Virgibacillus kimchii sp. nov. is proposed (=KACC 19404^T =JCM 32284^T). The type strain of Virgibacillus kimchii is NKC1-2T.     

Optimization of mineral salts in medium for enhanced production of pullulan by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> HP-2001 using an orthogonal array method

Based on intuitive analyses and statistical calculations using data from orthogonal array experiments, the optimal concentrations of K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄ in cell growth medium of Aureobasidium pullulans HP-2001 were measured as 7.5, 1.0, 0.1, and 2.4 g/L, respectively, whereas those for the production of pullulan were 2.5, 0.25, 0.8, and 0.3 g/L, respectively. The most important factor for cell growth and production of pullulan by A. pullulans HP-2001 was identified as K₂HPO₄. Optimal concentrations of glucose and yeast extract, along with the initial pH of the cell growth medium of A. pullulans HP-2001 containing optimized salt concentrations, were found to be 100.0, 10.0, and 6.0 g/L, respectively, whereas those for the production of pullulan were 100.0, 2.5, and 6.0 g/L, respectively. Conversion rates of pullulan from 10.0, 25.0, 50.0, 75.0, and 100.0 g/L of glucose in the presence of optimized salt concentrations were 26.0, 25.2, 22.4, 17.9, and 14.1%, respectively, whereas those in the presence of previously reported salt concentrations were 26.6, 25.2, 19.9, 14.3, and 11.7%, respectively. Optimal salt concentrations for the production of pullulan by A. pullulans HP-2001 varied according to the concentrations of the carbon and nitrogen sources, especially at higher concentrations.     

The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO_(x)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO_(x) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent K_m for nitrite of 0.49 millimolar, and an apparent K_m for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO_(x) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH₂) and reduced methyl viologen (MV) can serve as electron donors for NO_(x) evolution activity. The NADPH-, FMNH₂-, and reduced MV-NO_(x) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH₂ and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO_(x). NADPH eluted both NR and NO_(x) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.     

Potential of ochratoxin A production by Aspergillus carbonarius strains isolated from grapes at different ecological factors

Aspergillus carbonarius is known to colonize and produce ochratoxin A (OTA) on grapes and its derived products which is harmful to humans. We screened and tested A. carbonarius strains which isolated from grapes for production of OTA and selected three high OTA producing strains (ACSP1, ACSP2, ACSP3) for this study. These strains were further tested for their ability to produce OTA at different ecological factors [temperature 15, 25, 30, 35°C; water activity (a_w) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0]. Out of the three strains tested, A. carbonarius ACSP3 produced high levels of OTA than ACSP2 and ACSP1 in all the ecological factors. At 30°C A. carbonarius strains produced the highest OTA compared with other temperature regimes. With reference to water activity, a_w 0.98 favoured mycelial growth and accumulation of more OTA with all the three A. carbonarius strains. Further, pH 4.0 was encouraged the greatest production of OTA in all the strains. No growth was observed at a_w 0.88 and pH 10.0 in all the three strains except the strain ACSP3 at high pH. Our work demonstrated that temperature 30°C, a_w 0.98 and pH 4.0 is optimum for growth and production of OTA by A. carbonarius strains. Maximum amounts of OTA were found at earlier growth stages (7–9 days of incubation) in all the strains of A. carbonarius. The present study revealed that different ecological factors had great impact on OTA production by A. carbonarius which is useful for understanding OTA contamination and to develop proper management practices in future research programmes.     

Anaerobic treatment of saline wastewater by Halanaerobium lacusrosei

An upflow anaerobic packed bed reactor was operated continuously with synthetic saline wastewater at different initial COD concentrations (COD₀ = 1900–6300 mg/L), salt concentrations (0–5%, w/v) and hydraulic retention times (θ_H = 11–30 h) to investigate the effect of those operating parameters on COD removal from saline synthetic wastewater. Anaerobic salt tolerant bacteria, Halanaerobium lacusrosei, were used as dominant microbial culture in the process. The percent COD removal reached up to 94% at COD₀ = 1900 mg/L, 19 h hydraulic retention time and 3% salt concentration. No substrate inhibition effect was observed at high feed CODs. Increasing hydraulic retention time from 11 h to 30 h resulted in a substantial improvement in the COD removal from 60% to 84% at around COD₀ = 3400 mg/L and 3% salt concentration. Salt inhibition effect on COD utilization was observed at above 3% salt concentration. Modified Stover–Kincannon model was applied to the experimental data to determine the biokinetic coefficients. Saturation value constant, and maximum utilization rate constant of Stover–Kincannon model for COD were determined as K_B = 5.3 g/L day, U_max = 7.05 g/L day, respectively.