Soybean and sunflower oils increase the infectivity of Colletotrichum gloeosporioides f. sp. aeschynomene to Northern Jointvetch

Soybean and sunflower oils increased the level of infection of northern jointvetch, Aeschynomene virginica, plants by Colletotrichum gloeosporioides f. sp. aeschynomene. Inoculation of seedlings with spore suspensions containing 10% (v:v) soybean oil or 10% sunflower oil resulted in more disease than when inoculated with suspensions of spores in water alone. The lengths of the dew periods required to establish equivalent levels of disease by spore suspensions containing 10% soybean or 10% sunflower oil were approximately 4–8 h less compared to aqueous suspensions. Incubation of spores in 10% soybean oil followed by removal and resuspension in water did not affect the infectivity of spores when compared to spores incubated in aqueous suspensions. Spore germination and appressoria formation were unaffected by either of the oils tested in in vitro assays; however, in in vivo assays, 10% soybean oil and 10% sunflower oil increased spore germination in comparison to spores that were suspended in water.     

Best linear unbiased prediction of host-range of the facultative parasite Colletotrichum gloeosporioides f. sp. salsolae, a potential biological control agent of Russian thistle

Russian thistle or tumbleweed (Salsola tragus L.) is an introduced invasive weed in N. America. It is widely distributed in the US and is a target of biological control efforts.The fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. in Penz. f. sp. salsolae (CGS) is a facultative parasite under evaluation for classical biological control of this weed. Host-range tests were conducted with CGS in quarantine to determine whether the fungus is safe to release in N. America. Ninetytwo accessions were analyzed from 19 families: Aizoaceae, Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Campanulaceae, Chenopodiaceae, Cucurbitaceae, Cupressaceae, Fabaceae, Malvaceae, Nyctaginaceae, Phytolaccaceae, Poaceae, Polygonaceae, Sarcobataceae, and Solanaceae and 10 tribes within the Chenopodiaceae: Atripliceae, Beteae, Camphorosmeae, Chenopodieae, Corispermeae, Halopepideae, Polycnemeae, Salicornieae, Salsoleae, and Suaedeae. These included 62 genera and 120 species. To facilitate interpretation of results, disease reaction data were combined with a relationship matrix derived from internal transcribed spacer DNA sequences and analyzed with mixed model equations to produce Best Linear Unbiased Predictors (BLUPs) for each species. Twenty-nine species (30 accessions) from seven closely-related Chenopodiaceae tribes had significant levels of disease severity as indicated by BLUPs, compared to six species determined to be susceptible with least squares means estimates. The 29 susceptible species were: 1 from Atripliceae, 4 from Camphorosmeae, 1 from Halopepideae, 2 from Polycnemeae, 6 from Salicornieae, 8 from Salsolae, and 7 from Suaedeae. Most species in the genus Salsola, which are all introduced and weedy, were very susceptible and damaged by CGS. Statistical comparisons and contrasts of BLUPs indicated that these Salsola species were significantly more susceptible than non-target species, including 15 species from relatives in the closely-related genera Bassia (=Kochia), Nitrophila, Salicornia, Sarcocornia, and Suaeda. Of the 29 susceptible species, 10 native or commercially important species in N. America were identified as needing additional tests to determine the extent of any damage caused by infection.     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Chaetomium globosum for reducing primary inoculum of Diaporthe phaseolorum f. sp. meridionalis in soil-surface soybean stubble in field conditions

The potential of an antibiotic-producing isolate of Chaetomium globosum (CgA-1) to suppress Diaporthe phaseolorum f. sp. meridionalis (Dpm) in soybean stubble was studied in field microplots of no-tillage, minimum-tillage, and shallow plowing. Mature soybean stems colonized in vitro with Dpm were spread on the soil surface and C. globosum ascospore suspension, without nutrient supply, was sprayed over the entire plot prior to any tillage operation. Perithecial formation and survival of Dpm in soybean stems, concomitantly with colonization by C. globosum, were monitored for a 180-day period (mid-autumn through winter and mid-spring), which is the normal interval between soybean harvest and sowing. The proportion of soybean stem segments occupied by Dpm and number of perithecia formed decreased linearly with time and showed a strong negative correlation with increase in the occupation by C. globosum. At the end of the study, which coincided with the soybean sowing season, the soybean stubble was free from viable Dpm and was colonized by C. globosum. The effectiveness of C. globosum in eliminating the pathogen from surface-borne residue or harrowed-in residue was similar but much slower than in the shallow-plowed microplots. C. globosum successfully competed with major interfering fungi such as, Trichoderma, Nigrospora, and Fusarium in colonizing the soybean stems above and under the soil surface. The data provide strong evidence for use of the antibiotic-producing isolate of C. globosum to control soybean stem canker disease.     

Effect of different isolates of Beauveria bassiana on field populations of Lygus hesperus

Lygus hesperus (Knight) (Hemiptera: Miridae) is a particularly damaging pest of many crops in the Western United States. Current control tactics are chemically based and there is some concern over resistance building up in populations. Based on previous laboratory studies conducted in California and Mississippi, USA, two new isolates of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) were selected for field-testing against L. hesperus in California. Alfalfa plots were treated with one of three isolates of B. bassiana (a commercial isolate, an isolate from CA (WTPB2) or an isolate from MS (TPB3)) or the chemical pesticide Warrior T. More than 75% of the adults collected from plots 3 days after application with B. bassiana were infected but no differences in percentage infection occurred among fungal treatments. In addition, approximately 30% of the insects collected from control plots or plots treated with Warrior T were also infected. PCR analysis using SSR markers revealed that the isolate causing most of the infections in fungus treated plots was the isolate applied. A mix of infections was found in control plots and plots treated with Warrior T. Despite high levels of infection, no significant reductions of adult populations occurred until 10–14 days after application when plots treated with Warrior T or B. bassiana had about half the numbers of adult L. hesperus as the control plots.     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.     

Studies on (K + H)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A (K⁺ + H⁺)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, n = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Utilization of chemical inducers of resistance and Cryptococcus flavescens OH 182.9 to reduce Fusarium head blight under greenhouse conditions

Four chemicals [salicylic acid (SA), sodium salt of salicylic acid (NaSA), isonicotinic acid (INA), and DL-β-amino-n-butyric acid (BABA)] and the yeast antagonist Cryptococcus flavescens (=C. nodaensis nomen nudum) OH 182.9 were evaluated separately or together for the ability to reduce Fusarium head blight (FHB) of wheat in the greenhouse. When sprayed onto wheat heads at 3 days prior to pathogen challenge with Gibberella zeae, NaSA and INA at 10 mM significantly reduced FHB severity compared to the non-treated disease control. Applied at concentrations of 1 and 5 mM at 3 days before pathogen challenge, NaSA or INA in combination with OH 182.9 did not significantly reduce FHB severity compared to either treatment alone, though the lowest disease severity values frequently were associated with the combination treatments. When sprayed onto wheat heads just beginning to emerge from boot at 10 days prior to pathogen inoculation, NaSA, INA, and BABA at 1 mM significantly reduced FHB severity indicating that induced systemic resistance was at least partially responsible for the reduction of FHB disease. Induced FHB resistance was achieved by treating wheat with INA at concentrations as low as 0.1 mM. In only one instance was 100-kernel weight affected by any chemical or combination of chemicals with OH 182.9 treatment. Data from our studies in the greenhouse suggest that chemical inducers can induce resistance in wheat against FHB, and that further efforts are warranted to explore the potential of improved control of FHB disease by incorporating chemical inducers with the FHB biocontrol agent OH 182.9.     

Impact of Crown Rust (Puccinia coronata f. sp. avenae) on Competitive Interactions between Wild Oats (Avena fatua) and Stipa (Nassella pulchra)

Crown rust of oats (Puccinia coronata f. sp. avenae) was investigated as a biocontrol agent for wild oats (Avena fatua) on San Clemente Island, California. Successful restoration of the native habitats of this island will involve the reduction of wild oats and revegetation with native grasses such as stipa (Nassella pulchra). Determination of the outcome of interference between wild oats and stipa is important in the prediction of the success of the biocontrol agent. An addition series design was used to investigate these interactions, with densities of each species ranging from 0 to 2000 seeds per m². Eight replicates were established, four of which were randomly chosen and infected with the pathogen. After 75 days, plant height, dry weight, and seed production were measured. The data were fit to a hyperbola surface model by use of a nonlinear regression procedure. Results indicate that wild oat is the superior competitor at the seedling stage; however, early rust infection greatly reduces fitness of wild oats, causing an increased fitness for stipa. Simulations with a plant community model constructed from the results of the greenhouse experiment and information in the literature indicated that an equilibrium may be established between wild oat and stipa if high initial seeding rates of stipa are used in revegetation.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Cell volume change of Escherichia coli under stringent control apparent increase of K in rel cells

With several pairs of rel⁺ and rel⁻ strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K⁺ and the rate of uptake of ⁴²K⁺ were investigated. In the early phase of the experiments, the intracellular concentration of K⁺ was estimated by the conventional method in which the cell volume per A₆₆₀ value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K⁺ concentration of rel⁺ cells was kept almost constant, while that of rel⁻ cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A₆₆₀ changed only slightly in CP78 (rel⁺) cells, while it increased markedly in CP79 (rel⁻) cells after the exposure to amino acid starvation. Reestimation of the K⁺ concentrations based on the estimated respective values of cell volumes per A₆₆₀ revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel⁺ cells was arrested upon amino acid starvation whereas that in the rel⁻ cells was not. The ⁴²K⁺ uptake by the rel⁺ cells was depressed upon amino acid starvation, whereas that by the rel⁻ cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K⁺ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A₆₆₀ of the culture, was pointed out.     

Temperature effects on kinetic properties of plasma membrane ATPase from the yeast Saccharomyces cerevisiae

The reaction of plasma membrane ATPase from yeast with Mg²⁺ and Mg · ATP was studied in a temperature range of 10 – 30°C. The random mechanism of activation by Mg²⁺ and the pseudocompetitive inhibition at higher concentrations was not altered when the temperature was varied, nor were the kinetic constants representing substrate binding. However, at low temperature, the affinity of the enzyme for Mg²⁺ is greatly reduced. The Arrhenius plot of log V vs. 1/T shows straight lines with an inflection point at 24°C, which disappears in the presence of detergent. Calorimetric studies of the plasma membranes show a transition point at the same temperature. From these findings we suppose that Mg²⁺ is bound at a regulatory site of the ATPase, which is influenced by the surrounding phospholipids.     

Hemispheric asymmetry of rapid chloride responses to inositol trisphosphate and calcium in Xenopus oocytes

Shallow injection of inositol 1,4,5-trisphosphate (IP₃) near the animal pole of the Xenopus oocyte resulted in a large depolarizing current that decayed rapidly. A similar injection near the vegetal pole produced a much smaller response characterized by a significantly slower rate of decay. Injection of CaCl₂ near the animal pole of the oocyte resulted in a large depolarizing current characterized by rapid rise and decay times. Injection near the vegetal pole of the cell produced responses that exhibited similar amplitudes but much longer rise and decay times. The protein kinase C (PK-C) activator, β-phorbol 12-myristate 13-acetate (PMA), significantly enhanced the rapid responses to IP₃ injections at either hemisphere but did not affect the amplitudes of the responses to CaCl₂. The PK-C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) had no effect on the responses to CaCl₂. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.     

Nature of the (Ca + Mg)-ATPase activator protein which associates with human erythrocyte membranes

(Ca²⁺ + Mg²⁺)-ATPase activator protein associated with human erythrocyte membranes could be extracted with EDTA under isotonic condition at pH 7.6. No activator was released, however, using isotonic buffer alone. Like calmodulin, the activator in the EDTA extract migrated as a fast moving band on polyacrylamide gel electrophoresis. It was also heat-stable, was capable of stimulating active calcium transport and could stimulate (Ca²⁺ + Mg²⁺)-ATPase to the same extent. When chromatographed on a Sephacryl S-200 column, it was eluted in the same position as calmodulin and a membrane associated (Ca²⁺ + Mg²⁺)-ATPase activator prepared according to Mauldin and Roufogalis (Mauldin, D. and Roufogalis, B.D. (1980) Biochem. J. 187, 507–513). Furthermore, both Mauldin and Roufogalis protein and the activator in the EDTA extract exhibited calcium-dependent binding to a fluphenazine-Sepharose affinity column. On the basis of these data, it is concluded that the activator protein released from erythrocyte membranes by EDTA is calmodulin. A further pool of the ATPase activator could be released by boiling but not by Triton X-100 treatment of the EDTA-extracted membranes. This pool amounted to 8.9% of the EDTA-extractable pool.     

Ecology and impact of Allorhogas sp. (Hymenoptera: Braconidae) and Apion sp. (Coleoptera: Curculionoidea) on fruits of Miconia calvescens DC (Melastomataceae) in Brazil

Two fruit-feeding insects, a gall wasp, Allorhogas sp. (Hymenoptera: Braconidae), and a beetle, Apion sp. (Coleoptera: Curculionoidea), were evaluated in their native habitat in Brazil as potential biological control agents of Miconia calvescens DC (Melastomataceae). Allorhogas sp. occurred at two out of three field sites with native populations of M. calvescens, and Apion sp. occurred at all three sites. Both species exhibited aggregated distributions among M. calvescens trees sampled at each site. Allorhogas sp. infested 9.0% and 3.8% of fruits at each of two sites. The number of larvae and pupae of Allorhogas sp. and/or an unidentified parasitoid (Hymenopetera: Eulophidae: Tetrastichinae) ranged from one to five per infested fruit. Fruits infested with Allorhogas sp. were 20% larger and had 79% fewer seeds than healthy fruits. Although adults of Apion sp. were found on leaves and inflorescences of M. calvescens at all three sites, larvae and pupae were found in fruits at only one site, where a maximum of 1.4% of fruits were infested. Fruits infested by Apion sp. contained only one larva or pupa, and were 15% smaller and had 62% fewer seeds than healthy fruits. While a variety of apionids have been used for biological control in the past, this is the first time a braconid wasp has been considered for biological control of a weed.     

Cylindrocarpon pauciseptatum sp. nov., with notes on Cylindrocarpon species with wide, predominantly 3-septate macroconidia

A Cylindrocarpon species with up to 10 μm wide, straight and predominantly 3-septate macroconidia, subglobose to ovoidal microconidia and chlamydospores, is described as Cyl. pauciseptatum. It is most similar to Cyl. austrodestructans but no chlamydospores and microconidia are formed in the latter. Similar macroconidia also occur in Cyl. theobromicola, which forms oval to ellipsoidal microconidia at least sparsely and has slightly curved macroconidia, and Cyl. destructans var. crassum, which forms abundant 1-celled microconidia. DNA sequence data of the internal transcribed spacer regions 1 and 2 plus the 5.8S rDNA and the partial beta-tubulin gene were used for phylogenetic inferences. Cylindrocarpon pauciseptatum and Cyl. macrodidymum are monophyletic and are closely related to other species of Cylindrocarpon sensu stricto including members of the Cyl. destructans (teleomorph, Neonectria radicicola) species complex, which accommodates Cyl. liriodendri (teleomorph, Neon. liriodendri), Cyl. destructans var. crassum and Cyl. austrodestructans (teleomorph, Neonectria austroradicicola comb. nov.). Cylindrocarpon theobromicola is distantly related to species of Cylindrocarpon sensu stricto or Neonectria sensu stricto. It clustered among cylindrocarpon-like species with curved macroconidia, of which some belong to the Neon. mammoidea group. Relatively voluminous cells in sporodochial conidiophores of Cyl. theobromicola resembled those described for Campylocarpon, which is closely related to members of the Neon. mammoidea group including Cyl. theobromicola. Cylindrocarpon pauciseptatum has been isolated from roots of Vitis spp. in South-eastern Europe (Slovenia) as well as New Zealand, where it also occurs on roots of Erica melanthera.     

Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation

The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA^Phe was studied. Removal of domain 8 of the β subunit resulted in drastic reduction of the dissociation constant of the FRS·tRNA^Phe complex. Neither the removal of arginine 2 of the β subunit, which makes the only major contact between domains β1–5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1–5 of the β subunit may not be essential for efficient aminoacylation of tRNA^Phe by the FRS and rather be involved in other functions.     

Plasmodium vivax: Prevalence of mutations associated with sulfadoxine–pyrimethamine resistance in Plasmodium vivax clinical isolates from Pakistan

The main aim of the present study was to investigate the frequency of SNPs-haplotypes of dhfr and dhps genes associated to sulfadoxine–pyrimethamine (SP) resistance in Plasmodium vivax clinical isolates circulating in a malaria endemic area, Pakistan. All 164 collected isolates were analyzed for SNPs-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of pvdhfr and 383 and 553 of pvdhps genes using PCR–RFLP methods. All examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 15.2% and 53.6% of isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B (79.3%) was the most prevalent variant. The combination of pvdhfr and pvdhps haplotypes demonstrated nine distinct haplotypes. The three most prevalent haplotypes were I₁₃P₃₃F₅₇S₅₈T₆₁S₁₁₇I₁₇₃/A₃₈₃A₅₅₃ (43.9%), I₁₃P₃₃F₅₇S₅₈T₆₁N₁₁₇I₁₇₃/A₃₈₃A₅₅₃ (33.6%) and I₁₃P₃₃F₅₇R₅₈T₆₁N₁₁₇I₁₇₃/A₃₈₃A₅₅₃ (12.2%). The presence of mutant haplotypes is worrying and indicates the emergence of drug tolerant/resistant P. vivax isolates in Pakistan in near future.