Quantitative PCR analysis of selected Aspergillus, Penicillium and Paecilomyces species

A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of selected Aspergillus, Penicillium and Paecilomyces species. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

Utilisation of soil organic P by agroforestry and crop species in the field,western Kenya

A field experiment in western Kenya assessed whether the agroforestry species Tithonia diversifolia (Hemsley) A. Gray, Tephrosia vogelii Hook f., Crotalaria grahamiana Wight & Arn. and Sesbania sesban (L) Merill. had access to forms of soil P unavailable to maize, and the consequences of this for sustainable management of biomass transfer. The species were grown in rows at high planting density to ensure the soil under rows was thoroughly permeated by roots. Soil samples taken from beneath rows were compared to controls, which included a bulk soil monolith enclosed by iron sheets within the tithonia plot, continuous maize, and bare fallow plots. Three separate plant biomass samples and soil samples were taken at 6-month intervals, over a period of 18 months. The agroforestry species produced mainly leaf biomass in the first 6 months but stem growth dominated thereafter. Consequently, litterfall was greatest early in the experiment (0–6 months) and declined with continued growth. Soil pH increased by up to 1 unit (from pH 4.85) and available P increased by up to 38% (1 g P g^–1) in agroforestry plots where biomass was conserved on the field. In contrast, in plots where biomass was removed, P availability decreased by up to 15%. Coincident with the declines in litterfall, pH decreased by up to 0.26 pH units, plant available P decreased by between 0.27 and 0.72 g g^–1 and P_o concentration decreased by between 8 and 35 g g^–1 in the agroforestry plots. Declines in P_o were related to phosphatase activity (R²=0.65, P<0.05), which was greater under agroforestry species (0.40–0.50 nmol MUB s^–1 g^–1) than maize (0.28 nmol MUB s^–1 g^–1) or the bare fallow (0.25 nmol MUB s^–1 g^–1). Management of tithonia for biomass transfer, decreased available soil P by 0.70 g g^–1 and P_o by 22.82 g g^–1. In this study, tithonia acquired P_o that was unavailable to maize. However, it is apparent that continuous cutting and removal of biomass would lead to rapid depletion of P stored in organic forms.     

Induction of plant responses by a sequestering insect: Relationship of glucosinolate concentration and myrosinase activity

Induction of plant allelochemicals is of particular ecological importance for interactions with herbivores that can make use of induced metabolites by incorporating them for their own defence. Induction patterns in white mustard, Sinapis alba, were investigated following herbivory of the turnip sawfly, Athalia rosae, which sequesters plant glucosinolates. Larvae of different age were allowed to feed for 24 h on young leaves of premature, non-flowering plants. Changes in primary and secondary metabolites were recorded in the damaged leaves (local) and in the adjacent leaves and stems (systemic) for several days. Organ- and time-specific patterns were evident. Local responses included increases in glucosinolate concentrations, soluble and insoluble myrosinase activity and glucose levels, while systemic responses in leaves were restricted to increases in myrosinase activities and glucose. All effects were strongest immediately after feeding and declined mostly within a day. Stems had overall lower constitutive levels of glucosinolates and myrosinase activities than leaves. Feeding by one large larva had a greater impact on the plant's physiology than feeding by three small ones, even though both treatments resulted in quantitatively similar leaf destruction. Local increase in glucosinolate concentration could be beneficial for larvae, while conspecifics feeding on induced adjacent leaves might be negatively affected due to higher myrosinase activity levels. The results are discussed in the context of the ‘optimal defence theory’ and the ‘lethal plant defence paradox’.     

Rhizosphere bacteria antagonistic to soybean cyst (Heterodera glycines) and root-knot (Meloidogyne incognita) nematodes: Identification by fatty acid analysis and frequency of biological control activity

Rhizosphere bacteria were isolated from roots of young and mature plants with known antagonism to phytopathogenic nematodes, including velvet bean (Mucuna deeringiana), castor bean (Ricinus communis), sword bean (Cannavalia ensiformis), and Abruzzi rye (Secale cereale). Isolates from antagonistic plants were compared to soybean isolates for the frequency of antagonism to the root-knot (Meloidogyne incognita) and soybean cyst (Heterodera schachtii) nematodes in a disease assay with soybean. Bacterial isolates were identified using fatty acid analysis, and isolates which exhibited a significant reduction in incidence of soybean damage from both nematodes were characterized physiologically. The bacterial taxa associated with antagonistic plants were markedly different from soybean bacteria. Isolates from soybean were predominantly Bacillus spp., while those from antagonistic plants included more coryneform and Gram-negative genera. Pseudomonas cepacia and Pseudomonas gladioli were predominant among Gram-negative bacteria on antagonistic plants but were not isolated from soybean. Four to six times the number of bacteria from antagonistic plants, compared to soybean, significantly reduced disease incidence of both nematodes. No single pattern of physiological reactions was common among all these bacteria, suggesting that multiple mechanisms accounted for the observed biological control. The results suggest that rhizospheres of antagonistic plants may be useful sources of potential biological control agents for phytopathogenic nematodes.     

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and were active in [⁵⁵Fe]ferric alcaligin transport assays. Evidence suggests that several C ₂-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.     

Germination-Arrest Factor (GAF): 3. Determination that the herbicidal activity of GAF is associated with a ninhydrin-reactive compound and counteracted by selected amino acids

A novel, naturally-occurring herbicide (Germination-Arrest Factor, GAF), produced by Pseudomonas fluorescens WH6 and several related isolates of rhizosphere bacteria, irreversibly arrests germination of the seeds of a wide range of graminaceous species, including a number of important grassy weed species. GAF activity has been shown previously to be associated with a hydrophilic, low molecular weight compound that contains an acid group. In the present study, thin-layer chromatography (TLC) of extracts of WH6 culture filtrate demonstrated that GAF activity migrates on TLC plates with a particular ninhydrin-reactive compound. This compound was found to be present in GAF-producing P. fluorescens isolates and absent in P. fluorescens strains that lack the ability to produce GAF. Treatments, including mutagenesis, which resulted in the loss of GAF activity in culture filtrates from P. fluorescens WH6 were shown to result in the disappearance of this ninhydrin-reactive compound from extracts of WH6 culture filtrates or in alteration of its appearance on TLC chromatograms. The ninhydrin-reactivity of GAF indicates that it probably contains an amino group, as well as the acid group previously demonstrated, and suggests that GAF may be a small peptide or amino acid analog. Biological investigations motivated by this conclusion demonstrated that the effects of GAF in inhibiting the germination of seeds of annual bluegrass (Poa annua L.) could be counteracted by treatment with alanine or glutamine and, to lesser extent, by several other amino acids, suggesting that this compound may act by interfering with some aspect of amino acid metabolism or function.     

Quantitative analysis of calcium gradients and activity in growing pollen tubes ofLilium longiflorum

Summary Pollen tubes ofLilium longiflorum were loaded with quin-2 to determine the cytoplasmic free calcium. Quin-2-fluorescence was detected at 500 nm with alternating excitation at 340 nm and 360 nm. The calcium²⁺-concentration was obtained using the intensity ratio R=I₃₄₀/I₃₆₀. The analysis exhibits a [Ca²⁺] of nearly 10^–7mol·l^–1 in the tip region and about 2·10^–8 mol·l^–1at the tube base, near the pollen grain. The data give evidence for the existence of a continuous gradient of free calcium within growing pollen tubes of various length.     

Relationship between peroxidase activity and the amount of fully N-methylated compounds in bean plants infected by Pseudomonas savastanoi pv. phaseolicola

Changes in the level of endogenous formaldehyde (HCHO), some N-methylated compounds (choline and trigonelline) and peroxidase activity were examined in the leaves of bean genotypes (Phaseolus vulgaris L.) with different disease-sensitivity during ontogenesis in the stressfree condition and after natural infection by Pseudomonas savastanoi pv. phaseolicola (until the appearance of lesions). HCHO, as its dimedone adduct, and fully N-methylated compounds were determined by overpressured layer chromatography (OPLC) in different developmental stages and in the infected leaves/leaf discs. Peroxidase activity was measured by a spectrophotometric method. HCHO level decreased with ageing of the primary leaf and accordingly in the leaves at different developmental stages, then increased again in both cases due to the demethylation and methylation processes. Concentration of choline and trigonelline as potential HCHO generators decreased considerably while peroxidase activity increased with ageing of the plants. Comparing the symptomless and the Pseudomonas infected leaf discs (with watersoaked lesions) we found a decrease in the level of HCHO, choline and trigonelline and there was detectable increase in the peroxidase activity in the infected leaf tissues. Our findings are in accordance with previously published results that peroxidases play an important role in oxidative demethylation processes. Our hypothesis is that the high level of HCHO in the old leaves can originate from methylated components as the result of peroxidase activity and this high level may lead to the old leaf being resistant to pathogen. This conclusion is supported by the fact that the leaves of susceptible bean genotypes became resistant to Pseudomonas while growing older.     

Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity

The intracellular concentrations of total glutathione, GSSG and protein · S-SG, the total excreted glutathione concentration, and the susceptibility towards GSH-reacting compounds were assayed in strains of Escherichia coli deficient in biosynthesis and/or reduction of glutathione. A deficiency in glutathione reductase displaced the glutathione status towards the oxidized forms. This displacement was more clearly appreciated in strains additionally deficient in glutathione biosynthesis. A deficiency in catalase activity also produced an increase in the oxidation of glutathione. The most severe changes were observed in the concentrations of protein-glutathione mixed disulfides and in the amount of glutathione excreted to the medium. Increased sensitivities towards compounds known to interact with cellular GSH were observed in glutathione reductase deficient strains, although these effects were enhanced in strains additionally deficient in GSH biosynthesis     

Striga seed-germination activity of root exudates and compounds present in stems of Striga host and nonhost (trap crop) plants is reduced due to root colonization by arbuscular mycorrhizal fungi

Root colonization by arbuscular mycorrhizal (AM) fungi reduces stimulation of seed germination of the plant parasite Striga (Orobanchaceae). This reduction can affect not only host plants for Striga, resulting in a lower parasite incidence, but also false hosts or trap crops, which induce suicidal Striga seed germination, thereby diminishing their effectiveness. In order to better understand these AM-induced effects, we tested the influence of root colonization by different AM fungi on the seed-germination activity of root exudates of the Striga hermonthica nonhost plants cowpea and cotton on S. hermonthica. We also tested the effect of AM fungi on the seed-germination activity of the Striga gesnerioides host plant cowpea on S. gesnerioides. Moreover, we studied whether mycorrhization affects the transport of seed-germination activity to above-ground plant parts. Mycorrhization not only resulted in a lower seed germination of S. gesnerioides in the presence of root exudates of the S. gesnerioides host cowpea but also seed germination of S. hermonthica was also lower in the presence of root exudates of the S. hermonthica nonhosts cowpea and cotton. Downregulation of the Striga seed-germination activity occurs not only in root exudates upon root colonization by different AM fungi but also in the compounds produced by stems. The lowered seed-germination activity does not appear to depend on the presence of seed germination inhibitors in the root exudates of mycorrhizal plants. The implication for Striga control in the field is discussed.     

Inhibition of cinnamyl-alcohol-dehydrogenase activity and lignin synthesis in poplar (Populus x euramericana Dode) tissues by two organic compounds

Two organic compounds, N-(O-hydroxyphenul)-and N-(O-aminophenyl)sulfinamoyltertiobutyl acetate (OHPAS and NH₂PAS, respectively) have been designed for inhibiting cinnamylalcohol dehydrogenase (EC 1.1.1.2), a zinc metalloenzyme involved specifically in lignification. This paper describes their effects in vitro on the activity of the enzyme isolated from poplar and in vivo on the lignification of poplar tissues. The enzyme inhibition was time- and dose-dependent and pseudoirreversible indicating that these compounds could act as suicide inhibitors. Neither OHPAS nor NH₂PAS exhibited affinity towards other plant zinc metalloenzymes or phenolic enzymes tested. Only NH₂PAS exerted an effect on cinnamoyl: CoA reductase, another specific enzyme of lignification. In addition, these inhibitors, at the concentration of 80 M, reduced the fluxes of lignin synthesis in poplar stems by 45%. These results show that OHPAS and NH₂PAS could become useful tools for reducing lignification in plants.Abbreviations CDH cinnamyl alcohol dehydrogenase - NH₂PAS N (O-aminophenyl) sulfinamoyltertiobutyl acetate - OHPAS N(O-hydroxyphenyl) sulfinamoyltertiobutyl acetate     

Metabolism of phenolic compounds in <Emphasis Type="Italic">Vitis riparia</Emphasis> seeds during stratification and during germination under optimal and low temperature stress conditions

We studied the alterations in phenolic compounds in grape seeds during their stratification and germination under optimal conditions (+25 °C) and at low temperature (+10 °C). Biological materials in the study were seeds of Vitis riparia. Phenolic compounds were extracted from defatted seeds using 80 % methanol or 80 % acetone. The content of total phenolics was determined with the Folin-Ciocalteau reagent, while the content of tannins was determined by vanillin assay and the protein (BSA) precipitation method. The RP-HPLC method was used to determine phenolic compounds (phenolic acids, catechins) in the extracts. High amounts of tannins, catechins, gallic acid and lesser amounts of p-coumaric acid were found in the seeds. The content of total phenolics in acetone extracts was higher than that obtained using methanol. The amounts of phenolic acids and tannins found in V. riparia seeds after stratification were much lower. It may confirm a possible role of these compounds in dormancy of V. riparia seeds. After 72 h of low temperature treatment, inhibition of grape root growth and biochemical changes in seeds were detected. The chilling stimulated increased accumulation of some phenolic compounds (free gallic acid and catechins) in the seeds. These substances can protect plants against some abiotic stressors.     

Stereochemistry of a diastereoisomeric amphiphile and the species of the lipase influence enzyme activity in the transesterification catalyzed by a lipase-co-lyophilizate with the amphiphile in organic media

Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl-propionylamino)-pentanedioic acid didodecyl ester (d- and l-BIG2C₁₂CA); d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (d- and l-2C₁₂GE). Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated. Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19–48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the l-BIG2C₁₂CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68–78 fold.     

Free and glucosylated phenolics, phenol β-glucosyltransferase activity and membrane permeability in cucumber roots affected by derivatives of cinnamic and benzoic acids

Seven-day-old seedlings of cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.01, 0.1 and 0.5 mM solutions of derivatives of cinnamic acid (ferulic and p-coumaric acids) and benzoic acid (p-hydroxybenzoic and vanillic acids) as stress factors. In cucumber roots phenolics (free and glucosylated), phenol β-glucosyltransferase (E.C. 2.4.1.35) activity as well as membrane permeability were examined. The most intensive glucosylation took place in the first hour of stress duration in roots treated with 0.01 mM ferulic and p-coumaric acids and with 0.01 and 0.1 mM p-hydroxybenzoic and vanillic acids. At these concentrations a high phenol β-glucosyltransferase activity was found. The deterioration of capacity for phenolic glucosylation as well as the decrease of the phenol β-glucosyltransferase was observed at the higher concentrations. It was associated with increased membrane permeability.     

Polymorphism and phylogenetic relationships among species in the genus Oryza as determined by analysis of nuclear RFLPs

Summary Ninety-three accessions representing 21 species from the genus Oryza were examined for restriction fragment length polymorphism. The majority (78%) of the accessions, for which five individuals were tested, were found to be monomorphic. Most of the polymorphic accessions segregated for only one or two probes and appeared to be mixed pure lines. For most of the Oryza species tested, the majority of the genetic variation (83%) was found between accessions from different species with only 17% between accessions within species. Tetraploid species were found to have, on average, nearly 50% more alleles (unique fragments) per individual than diploid species reflecting the allopolyploid nature of their genomes.Classification of Oryza species based on RFLPs matches remarkably well previous classifications based on morphology, hybridization and isozymes. In the current study, four species complexes could be identified corresponding to those proposed by Vaughan (1989): the O. ridleyi complex, the O. meyeriana complex, the O. officinalis complex and the O. sativa complex. Within the O. sativa complex, accessions of O. rufipogon from Asia (including O. nivara) and perennial forms of O. rufipogon from Australia clustered together with accessions of cultivated rice O. sativa. Surprisingly, indica and japonica (the two major subspecies of cultivated rice) showed closer affinity with different accessions of wild O. Rufipogon than to each other, supporting a hypothesis of independent domestication events for these two types of rice. Australian annual wild rice O. meridionalis (previously classified as O. rufipogon) was clearly distinct from all other O. rufipogon accessions supporting its recent reclassification as O. meridionalis (Ng et al. 1981). Using genetic relatedness as a criterion, it was possible to identify the closest living diploid relatives of the currently known tetraploid rice species. Results from these analyses suggest that BBCC tetraploids (O. malampuzhaensis, O. punctata and O. minuta) are either of independent origins or have experienced introgression from sympatric C-genome diploid rice species. CCDD tetraploid species from America (O. latifolia, O. alta and O. grandiglumis) may be of ancient origin since they show a closer affinity to each other than to any known diploid species. Their closest living diploid relatives belong to C genome (O. eichingeri) and E genome (O. Australiensis) species. Comparisons among African, Australian and Asian rice species suggest that Oryza species in Africa and Australia are of polyphyletic origin and probably migrated to these regions at different times in the past.Finally, on a practical note, the majority of probes used in this study detected polymorphism between cultivated rice and its wild relatives. Hence, RFLP markers and maps based on such markers are likely to be very useful in monitoring and aiding introgression of genes from wild rice into modern cultivars.     

Differentiation of six sibling species in the Saccharomyces sensu stricto complex by multilocus enzyme electrophoresis and UP-PCR analysis

UP-PCR analysis and multilocus enzyme electrophoresis were used to characterize 37 strains of the sibling species Saccharomyces cerevisiae, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae and S. paradoxus. The results demonstrate that both molecular approaches are useful for discriminating between these phenotypically indistinguishable Saccharomyces species. The data obtained are in excellent agreement with previously reported genetic analyses, sequencing of the 18S rRNA and ITS regions, and DNA-DNA reassociation data. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modification of 3,5-diiodo-4-hydroxybenzoic acid (DIHB) activity and stimulation of ethylene production by small concentrations of oxygen in the root environment

The apical 2 cm of seedling roots of oilseed rape (Brassica napus L., cv. Primor) produced more ethylene than adjacent, older tissue. Treatment with 5 × 10^–3 mol m^–3 3,5-diiodo4-hydroxybenzoic acid (DIHB), a presumed inhibitor of ethylene action, failed to stimulate root extension. Larger concentrations were inhibitory. Ethylene, applied as ethephon decreased root extension but DIHB (5 × 10^–3 mol m^–3) partially overcame this effect. Oxygen concentrations below that present in air also inhibited root extension but this was not ameliorated by DIHB.Roots of barley seedlings (Hordeum vulgare L., cv. Midas) evolved ethylene more slowly than roots of oilseed rape. DIHB (10^–3–10^–2 mol m^–3) stimulated root extension in the absence of ethephon. Ethephon alone retarded root extension but DIHB partially overcame this inhibition. Small concentrations of oxygen also inhibited root extension but DIHB failed to ameliorate the effect even though the slow growth of oxygen-deficient roots (3–5% oxygen) was associated with abnormally fast rates of endogenous ethylene production.Extension growth in different oxygen concentrations was more closely associated with rates of oxygen consumption than with the amount of ethylene produced. Thus respiration rather than ethylene appeared to limit root extension under oxygen deficiency. This may explain why DIHB was unable to offset this form of environmental stress.