Strain ofA Research Articles

Relationship of ammonia excretion, dinitrogen fixation and ammonia assimilatory enzymes to encystation in a continuous culture ofAzospirillum brasilense

Continuous culture studies have been carried out on a strain ofA. brasilense to study ammonia excretion, dinitrogen fixation and ammonia assimilatory enzymes (glutamate-ammonia ligase and glutamate synthase (NADPH)) in encysted conditions. High glutamate synthase (NADPH) and low ammonia excretion was observed at the time of induction of cyst formation. Low and oscillating nitrogenase activity was observed throughout the experiment.

Effects of 45 insecticides, acaricides and molluscicides on the rove beetleAleochara bilineata (Col.: Staphylinidae) in the laboratory

Effects of 45 insecticides, acaricides and molluscicides on adult females of the rove beetleAleochara bilineata were investigated in the laboratory. The pesticides were tested in concentrations equivalent to the highest recommended dosages for practical use. Mortality, egg production and hatch of the eggs were measured. The reproducibility of the test and the suitability of the strain ofA. bilineata used are discussed. The toxicity of the cholinesterase inhibitors tested was varying, but in general high. Only pirimicarb and acephate showed low toxicity. The pyrethroids tested were highly toxic, even though fluvalinate allowed a few eggs to be laid. The test did not give conclusive results for insect growth regulators. The ecological relevance of laboratory results is discussed, and it is concluded that only for pesticides with no or low toxicity can the results be used for prediction of effects in the field.

Adhesion of Actinomyces Isolates to Experimental Pellicles

The ability of oral bacteria to adhere to surfaces is associated with their pathogenicity. Actinomyces can adhere to pellicle and cells through extracellular fimbriae. Research on adhesion of actinomyces has been conducted with use of hydroxyapatite (HA) coated with mammalian-derived salivary constituents, whereas the bacterial-derived components of the acquired pellicle have been largely ignored. The influence of the cell-free bacterial enzyme, glucosyltransferase (GTF), on adhesion of human and rodent isolates of Actinomyces viscosus was examined. Cell-free GTF was adsorbed onto parotid saliva-coated hydroxyapatite (sHA). Next, A. viscosus was exposed to the pellicle following the synthesis of glucan formed in situ by GTF. Glucans formed on the pellicle served as binding sites for adhesion of a rodent strain of A. viscosus. Conversely, the presence of in situ glucans on sHA reduced the adhesion of human isolates of A. viscosus compared with their adhesion to sHA. Adhesion of the rodent strains may be facilitated through a dextran-binding protein, since the rodent strains aggregated in the presence of dextrans and mutan. The human isolates were not aggregated by dextran or mutan. Pellicle harboring A. viscosus rodent strains interfered with the subsequent adhesion of Streptococcus mutans to the bacterial-coated pellicle. In contrast, the adhesion of S. mutans to pellicle was not decreased when the pellicle was pre-exposed to a human isolate of A. viscosus. The experimental data suggest that human and the rodent isolates of A. viscosus have distinct glucan adhesion properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved mating efficiency inAcetabularia acetabulum: recovery of 48?100% of the expected zygotes

We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.

Attachment, Chemotaxis, and Multiplication of Agrobacterium tumefaciens Biovar 1 and Biovar 3 on Grapevine and Pea

Tumorigenic (CG49) and nontumorigenic (CG484) strains of Agrobacterium tumefaciens bv. 3 attached to grape roots at a higher level than did a nonpectinolytic mutant of CG49 (CG50) or a tumorigenic strain of A. tumefaciens bv. 1 (CG628). Strains attached equally well to wounded and unwounded grape roots. Strains responded differently to pea plants in that biovar 3 strains consistently attached to unwounded roots at a lower level than they did to wounded roots, whereas CG628 attached equally well regardless of wounding. The lowest levels of attachment to pea roots were consistently observed for CG50. Population curves were calculated for the strains inoculated into wound sites on grape and pea roots. A. tumefaciens bv. 3 wild-type strains developed greater populations at wound sites on grape roots after 100 h (resulting in root decay) than did CG50 or CG628. Population curves for strains at wound sites on pea roots were different from those on grape roots. There were no significant differences in populations after 100 h, and no strains caused root decay. No differences in the chemotaxis of wild-type and mutant A. tumefaciens bv. 3 strains towards grape roots, crown pieces, or root extracts were observed, but the biovar 1 strain, CG628, always migrated the greatest distance towards all substrates. Polygalacturonase production may affect attachment to grape roots and multiplication of A. tumefaciens bv. 3 at wound sites and thus be associated with the specificity of the bacterium for grape.

Characterization of an Unusual New Agrobacterium tumefaciens Strain from Chrysanthemum morifolium Ram

We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean (Glycine max), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6.

Large-scale production ofPlasmodium vivaxsporozoites

Mass-scale production of Plasmodium vivax sporozoites in Anopheles stephensi was achieved using the chimpanzee (Pan troglodytes) as a source of infective blood. Membrane feeding was as successful as feeding mosquitoes directly on the animal so long as the time between drawing the blood and feeding was restricted to 45 min. Longer delays such as 2-3 h resulted in loss of infectivity in terms of oocyst production. The selected strain of A. stephensi was highly susceptible to P. vivax (Chesson strain). A strain of A. stephensi relatively refractory to P. falciparum showed no cross-refractoriness to P. vivax. Mixed infections of P. falciparum and P. vivax did not interfere with each other in their development in A. stephensi. A second normal blood meal to mosquitoes infected with P. vivax increased the yield of salivary gland sporozoites.

Elicitor activity ofAgrobacterium radiobacter in plants

Agrobacterium radiobacter was tested for the ability to induce the accumulation of phytoalexins and hypersensitive necrotic reaction in pea, bean and potato. A live bacterial suspension with a cell concentration of 1/pL and a solution of a crude polysaccharide produced by the bacteria caused the hypersensitive reaction in potato and bean and the production of phytoalexins in all three species of plants. The results obtained are discussed in connection with the previously found protective effect of the studied strain ofA. radiobacter against soil phytopathogenic fungi. A contribution of defense reactions to the determination of host specificity of the pathogenic strains of theAgrobacterium genus has been proposed.

Dinitrogen fixation (C2H2 reduction) by bacterial strains at various temperatures

Acetylene-reducing activities (ARA) of strains ofEnterobacter agglomerans, Azospirillum brasilense, Azotobacter chroococcum, and Bacillus, isolated from temperate or tropical soils, were compared at different temperatures to study temperature adaptability. All Enterobacter strains and Bacillus strain C-11-25 reduced C2H2 at temperatures as low as 5°C. ARA by Enterobacter strains declined sharply above 30°C but ARA by Bacillus strain C-11-25 continued to increase with an increase in temperature.A. brasilense strain sp 245, isolated from wheat roots in Brazil, reduced more C2H2 at lower temperatures than strain Cd, isolated from a Californian soil. Similarly, the temperate strain ofA. chroococcum was a better N2 fixer than the tropicalA. chroococcum strain at lower temperatures. Tropical strains ofA. brasilense andA. chroococcum reduced more C2H2 than temperate strains at higher temperatures. Therefore, it appears that temperate and tropical N2-fixing organisms adapt themselves to their particular environment and should have more potential to benefit crops grown at the particular temperatures favorable to them.

The isolation and identification of some toxic constituents ofAspergillus wentii wehmer

Extraction of a maize culture of a toxinogenic strain ofA. wentii led to the isolation and characterization of three anthraquinones, three bianthrones, a xanthone and a benzophenone. The structures were derived from spectroscopic data and were supported by chemical degradation. Of these, emodin, 1,6-di-0-methylemodin, 5-0-methylsulochrine and 1,3-di-0-methylemodin bianthrone were mildly toxic to ducklings.

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj7 roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.

Extracellular enzymes of some black aspergilli in Singapore

Ten strains ofAspergillus niger and one strain ofA. awamori showed proteolytic activity and amylolytic and cellulolytic activities were observed in all the strains except inA. niger isolated from rice grains.Aspergillus awamori was shown to be the best producer of amylolytic enzymes but poorest for cellulases. All the strains were shown to be good producers of proteolytic enzymes while the strain from rubber wood seemed to be the best cellulase-producing strain. Both single conidium and hyphal tip inoculation techniques gave similar results. Differences in the levels of extracellular enzymes produced by similar strains were also observed using the two different techniques.

A cosmid for selecting genes by complementation in Aspergillus nidulans : Selection of the developmentally regulated yA locus

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in Aspergillus nidulans. pKBY2 contains the bacteriophage lambda cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC(+) gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, BglII, Mbo I, or Sau3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35- to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA(-)) pabaA(-)trpC(-)Aspergillus strain (FGSC237) to trpC(+) at frequencies of approximately 10 transformants per mug of DNA. Three of approximately 1000 trpC(+)pabaA(-) colonies obtained were putative yA(+) transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced E. coli to ampicillin resistance following treatment in vitro with a lambda packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded trpC(+) transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.

Research on the aflatoxin problem in groundnut at ICRISAT

Aflatoxin contamination of groundnut is a serious problem in most groundnut producing countries and as such is given high research priority by the Groundnut Improvement Program of ICRISAT. Since 1979 we have concentrated on selecting cultivars resistant to seed invasion and colonization by toxigenicAspergillus flavus, and/or to aflatoxin production following invasion by the fungus. Resistance to invasion and colonization byA. flavus of rehydrated, mature seed has been found, and confirmed, in some cultivars. We have also screened several groundnut cultivars for seed resistance in the field, both under natural conditions and with the inoculum of the fungus added to the soil in the pod zone. Some cultivars with resistance to seed colonization also showed resistance to seed invasion byA. flavus. None of the cultivars tested has shown complete resistance to aflatoxin production but significant cultivar differences occurred in the amounts of aflatoxin produced in seeds inoculated with a toxigenic strain ofA. flavus.

Nitrogen fixation byAzospirillum spp. and effect ofAzospirillum lipoferum on the yield and N-uptake of wheat crop

Seven isolates belonged toA. brasilense and 3 belonged toA. lipoferum. Isolates having more denitrifying capacity fixed less nitrogen in nitrogen free semi-solid malate medium. One strain ofA. lipoferum having high nitrogen fixing capacity with negative test for denitrification was tested as inoculant to supplement the nitrogen need of a wheat crop in field condition with different doses of N with and without the inoculant. While control without nitrogen yielded 1260 kg/ha the yield in inoculated treatment was 2070 kg/ha resulting in significant increase. In a treatment receiving 40 kg N/ha the grain yield was 2370 kg/ha as against yield of 3110 kg/ha in a similar treatment receiving fertiliser plus inoculant. Thus increase in yield was about 30%. Further the treatment receiving 80 kg N/ha yielded 2970 kg/ha as against yield of 4150 kg/ha in a treatment receiving inoculant alongwith the above dose of the fertiliser. Thus increase in yield due to application of inoculant was about 36%. Similarly, the uptake of N in different treatments was augmented due to inoculation of seeds with the culture.

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae.

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-DL-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, toDL-5-fluorotryptophan andDL-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-DL-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,DL-p-fluorophenylalanine,DL-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-DL-aspartic acid, α-methyl-DL-methionine,N-o-nitrophenylsulfenyl-L-tryptophan, norleucine, andS-2-aminoethyl-L-cysteine did not show any significant effect. Treatment with 7-azatryptophan,DL-β-hydroxynorvaline,L-methionine-DL-sulfoximine,L-methionine sulfone, and β-2-thienyl-DL-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.

Comparative studies on glucoamylases from three fungal sources

Five commercial preparations of glucoamylases (three fromAspergillus niger, one each fromAspergillus foetidus andAspergillus candidus) were purified by ultrafiltration, Sepharose-gel filtration and DEAE-sephadex chromatography. Two forms of the enzyme, namely glucoamylase I and glucoamylase II were obtained from the fungi except from one strain ofA. Niger. All the enzymes appeared homogeneous by electrophoresis and ultracentrifugation. The specific activities varied between 85 and 142 units. The pH and temperature optima were between 4 and 5, and 60‡C respectively. The molecular weight as determined by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis ranged from 75,000 to 79,000 for glucoamylase I and 60,000 to 72,000 for glucoamylase II. OnlyA. niger glucoamylases contained phenylalanine at the N-terminal end. The amino acid composition of the enzymes was generally similar. However,A. niger andA. foetidus glucoamylases, in contrast toA. candidus enzymes, contained greater percentage of acidic than of basic amino acids. The enzymes contained 15 to 30% carbohydrate and 49 to 57 residues of monosaccharides per mol.A. niger enzymes contained mannose, glucose, galactose, xylose and glucosamine but theA. candidus enzyme lacked xylose and glucose and only xylose was absent inA, foetidus enzymes. Majority of the carbohydrate moieties were O-glycosidically linked through mannose to the hydroxyl groups of seline and threonine of the polypeptide chain.

Intergeneric transfer of streptomycin-resistance marker between two blue-green algae

Intergeneric transfer of streptomycin-resistance marker from a unicellular blue-green algaAnacystis nidulans to a filamentous blue-green algaAnabaena doliolum was demonstrated. Mutants ofA. nidulans resistant to streptomycin, occurring spontaneously or mutagenically-induced could be isolated easily. Naturally occurring streptomycin-resistant mutants ofA. doliolum could not be detected. Attempts at isolating such mutants either in nitrogen-free medium or in nitrate containing medium were unsuccessful. However, a streptomycin-resistant strain (recombinant) ofA. doliolum could be isolated in a mixed culture of streptomycin-sensitiveA. doliolum and streptomycin-resistantA. nidulans.

In Vitro Synergism Between Carbenicillin and Aminoglycosidic Aminocyclitols Against Acinetobacter calcoaceticus var. anitratus

Acinetobacter caleoaceticus var. anitratus is a nonfermentative, gram-negative bacillus that has been demonstrated to cause severe infections, usually in hospitalized patients. Since mild to moderate resistance of A. calcoaceticus to one or more aminoglycosidic aminocyclitols has been noted to occur, a study was undertaken to evaluate the activity of combinations of carbenicillin with either kanamycin, tobramycin, or gentamicin against 28 isolates of A. calcoaceticus obtained from clinical sources. Synergism (defined as at least 100-fold-increased killing at 24 h by the combination as compared with the most efficacious of the individual antibiotics) was demonstrated against 26 of 28 strains of A. calcoaceticus with carbenicillin plus kanamycin and carbenicillin plus tobramycin and against 25 of 28 strains with carbenicillin plus gentamicin. The median increased killing for the 28 strains was 4.2 log(10) with carbenicillin plus kanamycin and with carbenicillin plus tobramycin and 3.1 log(10) with carbenicillin plus gentamicin. The most important determinant of synergistic potential of each combination was the level of resistance of each strain of A. calcoaceticus to the aminoglycoside component of the combination.

Susceptibility of Serratia, Pseudomonas, and Enterobacter to acetic acid.

Agrocin 84, produced by Agrobacterium radiobacter K84, inhibited ribonucleic acid, deoxyribonucleic acid, and protein synthesis and amino acid transport in a susceptible, virulent strain of A. tumefaciens H-38-9. Cell motility was immediately stopped by action of the agrocin, 50% of the cells were killed within 15 min of contact, and the remainder were inhibited. Agrocin 84 is trypsin and pepsin resistant, but chemical analysis indicated a small peptide with a molecular weight of 2,500 containing six different amino acids, including nine molecules of glutamine or glutamic acid and seven molecules of serine.