Alleviating salt stress in tomato seedlings using Arthrobacter and Bacillus megaterium isolated from the rhizosphere of wild plants grown on saline–alkaline lands

Salt-induced soil degradation is common in farmlands and limits the growth and development of numerous crop plants in the world. In this study, we isolated salt-tolerant bacteria from the rhizosphere of Tamarix chinensis, Suaeda salsa and Zoysia sinica, which are common wild plants grown on a saline–alkaline land, to test these bacteria's efficiency in alleviating salt stress in tomato plants. We screened out seven strains (TF1–7) that are efficient in reducing salt stress in tomato seedlings. The sequence data of 16S rRNA genes showed that these strains belong to Arthrobacter and Bacillus megaterium. All strains could hydrolyze casein and solubilize phosphate, and showed at least one plant growth promotion (PGP)-related gene, indicating their potential in promoting plant growth. The Arthrobacter strains TF1 and TF7 and the Bacillus megaterium strain TF2 and TF3 could produce indole acetic acid under salt stress, further demonstrating their PGP potential. Tomato seed germination, seedling length, vigor index, and plant fresh and dry weight were enhanced by inoculation of Arthrobacter and B. megaterium strains under salt stress. Our results demonstrated that salt-tolerant bacteria isolated from the rhizosphere of wild plants grown on saline–alkaline lands could be used for alleviating salt stress in crop plants.     

Effects of chaotropic salts on global proteome stability in halophilic archaea: Implications for life signatures on Mars

Halophilic archaea thriving in hypersaline environments, such as salt lakes, offer models for putative life in extraterrestrial brines such as those found on Mars. However, little is known about the effect of the chaotropic salts that could be found in such brines, such as MgCl₂, CaCl₂ and (per)chlorate salts, on complex biological samples like cell lysates which could be expected to be more representative of biomarkers left behind putative extraterrestrial life forms. We used intrinsic fluorescence to study the salt dependence of proteomes extracted from five halophilic strains: Haloarcula marismortui, Halobacterium salinarum, Haloferax mediterranei, Halorubrum sodomense and Haloferax volcanii. These strains were isolated from Earth environments with different salt compositions. Among the five strains that were analysed, H. mediterranei stood out as a results of its high dependency on NaCl for its proteome stabilization. Interestingly, the results showed contrasting denaturation responses of the proteomes to chaotropic salts. In particular, the proteomes of strains that are most dependent or tolerant on MgCl₂ for growth exhibited higher tolerance towards chaotropic salts that are abundant in terrestrial and Martian brines. These experiments bridge together global protein properties and environmental adaptation and help guide the search for protein-like biomarkers in extraterrestrial briny environments.     

Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria

Cell walls were prepared from freeze-dried samples of 7 strains of Methanobacterium by mechanical disintegration of the cells followed by incubation with trypsin. Electron microscopy revealed the presence of sacculi exhibiting the shape of the original cells, on which no surface structure could be detected. Ultrathin sections of the isolated sacculi showed a homogenously electron dense layer of about 10–15 nm in width. The ash content varied between 8 and 18% of dry weight. The sacculi of all the strains contained Lys: Ala: Glu: GlcNAc or GalNAc in a molar ratio of about 1:1.2:2:1. In one strain (M. ruminantium M 1) alanine is replaced by threonine, however. Neutral sugars and-in some strains-additional amounts of the amino sugars were present in variable amounts, and could be removed by formamide extraction or HF treatment without destroying the sacculi. No muramic acid or d-amino acids typical of peptidoglycan were found. Therefore, the sacculi of the methanobacteria consist of a different polymer containing a set of three l-amino acids and one N-acetylated amino sugar. From cells of Methanospirillum hungatii no sacculi, but tube-like sheaths could be isolated, which tend to fracture perpendicularly to the long axis of the sheath along the fibrills seen on the surface. The sheaths consist of protein containing 18 amino acids and small amounts of neutral sugars. They are resistent to the proteinases tested and are not disintegrated by boiling in 2% sodium dodecylsulfate for 30 min.The three Gram-negative strains Black Sea isolate JR-1, Cariaco isolate JR-1 and Methanobacterium mobile do not contain a rigid sacculus, but merely a SDS-sensitive surface layer composed of regularly arranged protein subunits. This evidence indicates that, within the methanogens, different cell wall polymers characteristic of particular groups of organisms may have evolved during evolution, and supports the hypothesis that the evolution of the methanogens was separated from that of the peptidoglycan-containing procaryotic organisms at a very early stage.Non Standard Abbreviations SDS sodium dodecylsulfate - EDTA ethylenediaminetetra acetic acid - DNP dinitrophenyl Dedicated to Prof. Dr. Adolf Butenandt on the occasion of his 75th birthday     

DNA base compositions of halophilic and nonhalophilicBacillus firmus strains of marine origin

The mineral salt requirements of 27 strains ofBacillus firmus were determined. Twenty-six of these strains were of marine origin and one terrestrial strain was used as a reference. Three strains demonstrated strictly halophilic behavior, i.e., they showed no growth in media prepared without sodium chloride. Seven strains were nonhalophilic. The growth of 17 strains was stimulated by the addition of sodium chloride, but the cells were able to grow without it. These results were compared with the DNA base compositions of the strains. In contrast to literature data, relationships between the DNA base ratios and the halophilic or nonhalophilic behavior of the cells could not be detected. But strains with guanine plus cytosine values above 41 mol% did grow well at 44°C, and those strains showing poor growth at this temperature had lower guanine plus cytosine percentages.     

Identification and characterization of CMTM4, a novel gene with inhibitory effects on HeLa cell growth through Inducing G2/M phase accumulation

Human CMTM is a novel gene family consisting of CKLF and CMTM1-8. CMTM4 is the most conserved gene and has three RNA splicing forms designated as CMTM4-v1, -v2 and -v3, but in many types of tissue and cell lines, only CMTM4-v1 and -v2 could be detected. CMTM4-v2 is the full length cDNA product, which has been highly conserved during evolution. CMTM4-v1 and -v2 are broadly expressed in normal types of tissue. They are distributed on the cell membrane and across the cytoplasm in a speckled pattern. Overexpression of CMTM4-v1 and -v2 can inhibit HeLa cell growth via G2/M phase accumulation without inducing apoptosis. Therefore, CMTM4 might be an important gene involved in cell growth and cell cycle regulation.     

Effect of salinity on growth of four strains of Rhizobium and their infectivity and effectiveness on two species of Acacia

Two Rhizobium strains (WU1001 and WU1008) were isolated from nodules of Acacia redolens growing in saline areas of south-west Australia, and two strains selected from the University of Western Australia's culture collection (WU429 isolated from A. saligna and WU433 from A. cyclops). The growth of each in buffered, yeast extract mannitol broth culture was largely unaffected by salt up to 300 mM NaCl. A slight increase in lag time occurred at concentrations of 120 mM NaCl and above, but cell number at the static phase was not affected. Each of the four Rhizobium strains tested accumulated Na⁺ but showed decreasing levels of sugar with increasing salt in the external medium. Amino acid levels also increased, in some cases by more than tenfold. However, the relative proportion of each remained fairly constant in the bacteria, irrespective of salt treatment. Only trace quantities of proline were detected and there was no increase in this amino acid with salt. Acidic amino acids (glutamate and aspartate) remained as a constant proportion.Rhizobium strains WU429, WU1001 and WU1008 produced effective nodules on both A. cyclops and A. redolens grown in sand with up to 80 mM NaCl (added in nutrient solutions free of nitrogen). Strain WU433 was highly infective on both Acacia species tested at low salt concentrations (2–40 mM NaCl), but infection was sensitive to salt levels at 120 mM NaCl and above. Nodules formed with strain WU433 were, however, ineffective on both A. redolens and on A. cyclops and showed nil or negligible rates of acetylene reduction at all salt concentrations. Strains WU429, WU1001 and WU1008 in combination with a highly salt-tolerant provenance of A. redolens formed symbioses which did not vary significantly in nodule number and mass, specific nodule activity or total N content irrespective of salt level up to 160 mM NaCl. On a more salt sensitive provenance of A. redolens and on A. cyclops the infectivity and effectivity of the Rhizobium strains tested usually decreased as the external salt concentration increased. These data are interpreted to indicate that tolerance of the legume host was the most important factor determining the success of compatible Rhizobium strains in forming effective symbioses under conditions of high soil salinity.     

3株罗伊氏乳杆菌生物学特性的分析比较

【目的】对3株罗伊氏乳杆菌的生物学特性进行分析比较,为后期生产应用提供一定的参考。【方法】对实验室保藏的3株罗伊氏乳杆菌的生长曲线、pH曲线、耐受人工胃液能力、耐受猪胆盐能力、黏附能力、抑菌能力和对抗生素的耐药性等特性进行了分析比较。【结果】3株菌生长趋势大致相同;3株菌对人工胃液均具有良好的耐受性,且可以有效地抑制大肠杆菌和金黄色葡萄球菌的生长;菌株L0和L2对高胆盐的环境耐受性较差,菌株L1则对高胆盐环境具有极强的耐受性;菌株L1和L2具有很强的黏附能力;3株菌对20种抗生素表现出不同的耐受性。【结论】菌株L1的生物学特性明显优于其他两株菌株,有利于后期的生产应用。     

The colicin A pore-forming domain fused to mitochondrial intermembrane space sorting signals can be functionally inserted into the Escherichia coli plasma membrane by a mechanism that bypasses the Tol proteins

Colicin A is a pore-forming bacteriocin that depends upon the Tol proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c₁ (pc1) as well as the first 167 amino acids of cytochrome b₂ (pb2) were fused to the pore-forming domain of colicin A (pfColA). Both hybrid proteins (pc1-pfColA and pb2-pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no lethal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities were related to their pore-forming activities. The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins. In addition, we showed that both the cytochrome C₁ presequence fused to the dihydrofolate reductase (pc1-DHFR) and the cytochrome c, presequence moiety of pc1-pfColA were translocated across inverted membrane vesicles. Our results indicated that: (i) pc1-pfColA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore-forming domain had a channel activity; and (ii) this channel activity was inhibited within the membrane by the immunity protein.     

Strain-specific salt tolerance and chemotaxis ofAzospirillum brasilense and their associative N-fixation with finger millet in saline calcareous soil

Three salt-tolerantAzospirillum brasilense strains were isolated from the roots of finger millet grown in saline calcareous soil and characterized. The effect of various salts on growth and N₂ase activity of these strains was tested and strain STR1 was found more tolerant at higher concentrations of Cl^-, SO₄ ² and HCO₃ ^-. Bicarbonate was found to be the most toxic. The content and concentrations of root exudates of finger millet genotypes were different and chemotaxis to sugars, amino acids, organic acids and root exudates was strain specific. Under salt stress, significant interactions between strains and genotypes of finger millet resulted in different responses of N₂ase activity, endo- and exorhizospheric population, dry weight of root, shoot and grain yield.     

The effect of AmtR on growth and amino acids production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, plays important roles in nitrogen metabolism. To investigate the influence of AmtR on amino acids production in C. glutamicum ATCC 13032, the amtR deletion strain C. glutamicum Q1 was constructed and cultured in modified CGXII minimal medium for 60 h. The ammonium consumption rates as well as amino acids production of both strains cultured in modified CGXII minimal medium were determined. The amtR deletion in C. glutamicum caused an obvious growth defect in the exponential growth phase, but both strains had the same biomass in the stationary phases. Maybe the less α-oxoglutarate was used for the tricarboxylic acid cycle to influence the growth of strains. During 12 h, the rate of ammonium consumption and the concentration of Glu, Pro, Arg and Ser were higher but Asp, Gly, He, Leu, Lys were lower in the mutation strain. During 48 h, the Q1 had higher levels of Asp, Lys, Pro, Ala and Val, and lower levels of Glu, Arg, Leu and Ile, compared to the wild. The more Glu was synthesized by the activated GS/GOGAT pathway in Q1, and then the accumulation of relative amino acids (Pro, Arg and Ser) were up-regulated within 12 h growth. After 48 h growth, the amtR deletion obviously influenced accumulation of Ala, Asp and Pro. The amtR deletion could influence the growth and amino acids production, which could be useful to the production of amino acids.     

Characterization of nicking of the nontoxic-nonhemagglutinin components of Clostridium botulinum types C and D progenitor toxin

Clostridium botulinum C and D strains produce two types of progenitor toxins, M and L. Previously we reported that a 130-kDa nontoxic-nonhemagglutinin (NTNHA) component of the M toxin produced by type D strain CB16 was nicked at a unique site, leading to a 15-kDa N-terminal fragment and a 115-kDa C-terminal fragment. In this study, we identified the amino acid sequences around the nicking sites in the NTNHAs of the M toxins produced by C. botulinum type C and D strains by analysis of their C-terminal and N-terminal sequences and mass spectrometry. The C-terminus of the 15-kDa fragments was identified as Lys127 from these strains, indicating that a bacterial trypsin-like protease is responsible for the nicking. The 115-kDa fragment had mixtures of three different N-terminal amino acid sequences beginning with Leu135, Val139, and Ser141, indicating that 7–13 amino acid residues were deleted from the nicking site. The sequence beginning with Leu135 would also suggest cleavage by a trypsin-like protease, while the other two N-terminal amino acid sequences beginning with Val139 and Ser141 would imply proteolysis by an unknown protease. The nicked NTNHA forms a binary complex of two fragments that could not be separated without sodium dodecyl sulfate.     

Influence of Incubation Atmosphere on Growth and Amino Acid Requirements of Streptococcus mutans

The growth response of Streptococcus mutans representing antigenic type a or d in a chemically defined medium was influenced by the oxygen concentration of the growth atmosphere. Under controlled aerobic (1.5% O(2)) conditions these cultures attained a greater density than when the atmosphere contained 0.006% O(2) or less. The growth of S. mutans strains representing antigenic types b or c in the defined medium was independent of the oxygen concentration of the growth environment. Under the conditions used in this study, none of the strains tested could utilize ammonium ion as a sole source of nitrogen for growth. The requirement for certain amino acids and inhibition by other amino acids varied with antigenic type and relative oxygen concentration of the growth environment. Under conditions where the atmospheric oxygen was reduced to 0.0006% O(2) or less, the amino acid requirements of the cultures became either more numerous or more stringent. S. mutans strains of type c generally required the least number of amino acids, whereas cultures of type d had more numerous requirements. Nearly every culture tested under the anaerobic atmosphere was inhibited by one of the branched-chain amino acids, leucine, valine, or isoleucine. Methionine and lysine were also found to be inhibitory, particularly toward the type c strains.     

Aspects of physiology of Histoplasma capsulatum (A review)

Yeast and mycelial forms of several strains of Histoplasma capsulatum have been analysed with respect to their ability to grow on a defined medium with or without the amino acid supplement. It appeared that whereas mycelial cells of all strains tested were prototrophic, the yeast cells of most strains stringently required L-cysteine for growth. This was due to the absence from these cells of an active form of an enzyme, sulfite reductase, normally needed for cysteine biosynthesis. We have found that the yeast cells of two strains (Downs and G 184 B) can grow without cysteine supplement if L-serine is added to the medium. These cells have an active sulfite reductase but the enzyme disappears when cysteine is added. Thus, the regulation of sulfite reductase is different in mycelium and yeast — the enzyme is constitutive or repressible, respectively.Examination of RNA synthetic components of H. capsulatum revealed that the major proportion of RNA polymerase of the yeast form is sensitive to inhibition by -amanitin. The sensitivity to the toxin disappears completely upon conversion to mycelial phase. The yeast cells possess an unusual enzyme capable of synthesizing oligonucleotides without the aid of a DNA template. The enzyme stimulates DNA synthesis in the reaction catalyzed by DNA polymerase from H. capsulatum or Escherichia coli. The above data are discussed in terms of regulatory mechanisms involved in the process of morphological conversion. It is proposed that efforts be directed toward the identification and isolation of specific gene products so that qualitative and quantitative analysis of the conversion could be carried out.presented, in part, at the 1st International Histoplasmosis Conference, held on April 10–12, 1978 in Atlanta, Georgia, U.S.A.     

Morphometry and growth of three <Emphasis Type="Italic">Synechococcus</Emphasis>-like picoplanktic cyanobacteria at different culture conditions

Three phycocyanin-rich strains of Synechococcus-like picoplanktic cyanobacteria, isolated from the plankton of Czech oligotrophic to eutrophic freshwater reservoirs, were investigated in crossed gradients of light and temperature and in combination with two different culture media (BG-11 and WC). The strains exhibited similar growth and reproduction patterns and displayed overlapping ranges of cell size (1.5 × 0.8 μm) under standardized laboratory conditions (18 μmol m⁻² s⁻¹; 20°C). However, strains behavior differed in the crossed gradients. All strains preferred BG-11 medium, where also remarkable size changes could be observed. Length, width, cell abundance and growth rate of two strains were positively correlated with temperature and nutrients, whereas the impact of light intensity was insignificant. Maximum cell elongation (involution cells up to 19 μm) occurred in two strains only in BG-11 medium at highest temperature (28°C) and highest irradiance (53 μmol m⁻² s⁻¹). Cell dimensions in WC medium were constant under most conditions given. The third strain was influenced by all three factors, from which light and nutrients played pivotal role. The length of the lag-phase for all strains appeared to be temperature dependent (negative correlation). Despite the fact that the cell volume in all strains increased more than five times under the lowest light and low temperature (6 μmol m⁻² s⁻¹, <15°C) in both media, the length/width ratio remained unchanged. The strains differed in the degree of cell enlargement and cell division symmetry as well as in optimum temperature and light dependence. Based on this experimental work two strains could be identified as Synechococcus sp. and one as Cyanobium sp., which can be used as a support for the following genetical analyses.     

Growth Conditions Influence Expression of Cell Surface Hydrophobicity of Staphylococci and Other Wound Infection Pathogens

The initial adhesion of microbes to tissue and solid surfaces can be mediated by hydrophobic interaction. Expression of microbial cell surface hydrophobicity (CSH) is influenced by growth conditions, and often best expressed after growth under nutrient-poor conditions, or “starvation.” In the present study, the CSH of 133 strains of Enterobacteriaceae, Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, group A streptococcus, Pseudomonas aeruginosa, Clostridium perfringens, Bacteroides fragilis, Peptococcus magnus, and of 8 Candida albicans strains was measured by the salt aggregation test after growth on hematin agar in a 5% CO₂ atmosphere, or under anaerobiosis. Cells of all but 8 strains expressed pronounced or moderate CSH, i.e., they aggregated in 0.01-2 M ammonium sulfate. When the agar surface was covered by human serum (diluted 1:5) to mimic growth conditions in a wound, 94 strains expressed higher CSH, and 44 strains the same CSH as after growth without serum. The CSH of 12 strains of different species was measured after growth on blood, hematin and PDM agar, with or without serum, and in an aerobic or a 5% CO₂ atmosphere. The highest CSH was expressed after growth in 5% CO₂ with serum, and the lowest growth after on blood agar in aerobic atmosphere. Identical results were obtained with native and heat-inactivated (56 C, 20 min) serum. The reduced surface tension obtained in 5% CO₂, as well as yet unidentified serum factors, promotes expression of CSH.     

Development of a chemically defined medium for growth ofNeisseria gonorrhoeae

A chemically defined medium satisfactory for growth of a number of laboratory strains and recent isolates ofNeisseria gonorrhoeae has been devised. It contains inorganic salts, dextrose, guanine, cytosine, B-vitamin supplement, and the following amino acids:l-arginine,l-aspartic acid,l-cystine,l-isoleucine,l-leucine,l-proline,l-threonine, andl-valine.Nine of the eleven strains grew satisfactorily in this medium without being provided supplemental CO₂ during incubation, and a tenth strain grew in the medium supplemented with glutamine. No single B-vitamin or purine or pyrimidine base was essential for growth of any of the strains, but some combinations of them were stimulatory. Riboflavin, however, was inhibitory. The strains showed variations in requirements for amino acids. The amino acids which were either essential or stimulatory for one or more of the strains were included in the medium. Those to which the strains responded differently were used at concentrations intermediate between those optimal for growth of one strain and inhibitory for another. Conventional agar was inhibitory, but a purified agar, having a gel strength twice that of conventional agar, was satisfactory. An aqueous solution of 0.1% cysteine and 0.86% NaCl was satisfactory for preparation of inocula.This investigation was supported by a Public Health Service Predoctoral Fellowship (F-FI-GM-24-755-01A1) from the National Institute of General Medical Sciences of the United States Public Health Service to the senior author.     

Construction and Analyses of Mutant <Emphasis Type="Italic">ftsH</Emphasis> Alleles of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Involving the ATPase- and Zn-Binding Domains

The ftsH gene, present in all eubacterial species, is anchored in the cytoplasmic membrane and contains an ATP- and a Zn-binding domain that are both part of a metalloprotease activity. The Bacillus subtilis ftsH is not essential, but null mutants exhibit a pleiotropic phenotype including filamentous growth; hypersensitivity towards heat and salt stress and a failure to sporulate. To find out whether one or the other functional domain is involved in these different phenotypes, point mutations were introduced into the coding region for both domains leading to a replacement of conserved amino acid residues. The mutant alleles were fused to a xylose-inducible promoter and integrated ectopically into two different strains, one expressing the wild-type ftsH allele and the other carrying a ftsH knockout. While none of the strains exhibited a growth defect in rich medium at 37°C, those strains expressing only the mutant alleles did not resume growth after heat or salt stress challenge. Furthermore, none of the mutant alleles promoted sporulation. While only those purified mutant FtsH proteins with an intact Walker A box exhibited ATPase activity, all of them failed to degrade -casein.     

The Interactive Effects of Temperature and Osmotic Potential on the Growth of Aquatic Isolates of Fusarium culmorum

The mycelial growth of 10 Fusarium culmorum strains isolated from water of the Andarax riverbed in the provinces of Granada and Almeria in southeastern Spain was tested on potato-dextrose-agar adjusted to different osmotic potentials with either KCl or NaCl (?1.50 to ?144.54 bars) at 10°C intervals ranging from 15° to 35°C. Fungal growth was determined by measuring colony diameter after 4 d of incubation. Mycelial growth was maximal at 25°C. The quantity and capacity of mycelial growth of F. culmorum were similar at 15 and 25°C, with maximal growth occurring at ?13.79 bars water potential and a lack of growth at 35°C. The effect of water potential was independent of salt composition. The general growth pattern of Fusarium culmorum growth declined at potentials below ?13.79 bars. Fungal growth at 25°C was always greater than growth at 15°C, at all of the water potentials tested. Significant differences were observed in the response of mycelia to water potential and temperature as main and interactive effects. The number of isolates that showed growth was increasingly inhibited as the water potential dropped, but some growth was still observable at ?99.56 bars. These findings could indicate that F. culmorum strains isolated from water have a physiological mechanism that permits survival in environments with low water potential. Propagules of Fusarium culmorum are transported long distances by river water, which could explain the severity of diseases caused by F. culmorum on cereal plants irrigated with river water and its interaction under hydric stress or moderate soil salinity. The observed differences in growth magnitude and capacity could indicate that the biological factors governing potential and actual growth are affected by osmotic potential in different ways.     

Combined Use of Ribotyping,PFGE Typing and IS431 Typing in the Discrimination of Nosocomial Strains of Methicillin-Resistant Staphylococcus aureus

We have previously reported the phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clinical strains isolated in Malaya University Hospital in the period 1987 to 1989 using antibiogram, coagulase typing, plasmid profiles, and phage typing. Here, we report the analysis of the same strains with three genotyping methods; ribotyping, pulsed-field gel electrophoresis (PFGE) typing, and IS431 typing (a restriction enzyme fragment length polymorphism analysis using an IS431 probe). Ribotyping could discriminate 46 clinical MRSA strains into 5 ribotypes, PFGE typing into 22 types, and IS431 typing into 15 types. Since the differences of the three genotyping patterns from strain to strain were quite independent from one another, the combined use of the three genotyping methods could discriminate 46 strains into 39 genotypes. Thus, the powerful discriminatory ability of the combination was demonstrated.