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PubMed1997

Formation of Di-Isodityrosine and Loss of Isodityrosine in the Cell Walls of Tomato Cell-Suspension Cultures Treated with Fungal Elicitors or H2O2.

About 84% of the hydroxyproline residues in a cell culture of tomato (Lycopersicon esculentum x Lycopersicon peruvianum) were present in phenol-inextractable (i.e. covalently wall-bound) material. Treatment of the cells with any of three fungal elicitors (wall fragments from Phytophthora megasperma and Pythium aphanidermatum and xylanase from Aureobasidium pullulans) or with 1 mM H2O2 had little effect on the quantity of phenolinextractable hydroxyproline per milligram of freeze-dried cells. However, each treatment induced a decrease in the content of phenol-inextractable isodityrosine (Idt) residues. Each treatment, except with the P. megasperma fragments, also induced an increase in phenol-inextractable di- (Di-Idt). The increase in Di-Idt partly accounted for the loss of Idt. We conclude that the elicitors and H2O2 acted to reinforce the existing cross-linking of cell wall (glyco)proteins by evoking oxidative coupling reactions to convert Idt to Di-Idt plus unidentified products. The promotion of cross-linking by elicitor treatment is proposed to be a defensive response that restricts the penetration of pathogens.

Journal Article↗

PubMed1999

Cell-specific production and antimicrobial activity of naphthoquinones in roots of lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on "noninducing" medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and beta-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed "hairy-root" cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.

Journal Article↗

PubMed1999

Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the andean crop Mirabilis expansa.

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

Amino Acid Sequence↗

PubMed1989

Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings.

l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.

Journal Article↗

PubMed2002

Structure of beta-cinnamomin, a protein toxic to some plant species.

Phytophthora and Pythium species are among the most aggressive plant pathogens, as they invade many economically important crops and forest trees. They secrete large amounts of 10 kDa proteins called elicitins that can act as elicitors of plant defence mechanisms. These proteins may also induce a hypersensitive response (HR) including plant cell necrosis, with different levels of toxicity depending on their pI. Recent studies showed that elicitins function as sterol carrier proteins. The crystallographic structure of the highly necrotic recombinant beta-cinnamomin (beta-CIN) from Phytophthora cinnamomi has been determined at 1.8 A resolution using the molecular-replacement method. beta-CIN has the same overall structure as beta-cryptogein (beta-CRY), an elicitin secreted by Phytophthora cryptogea, although it shows a different surface electrostatic potential distribution. The protein was expressed in Pichia pastoris and crystallized in the triclinic space group with two monomers in the asymmetric unit. The interface formed by these two monomers resembles that from beta-CRY dimer, although with fewer interactions.

Algal Proteins↗

PubMed1998

Aeroallergens and viable microbes in sandstorm dust. Potential triggers of allergic and nonallergic respiratory ailments.

Aeroallergens and antigens in sandstorm dust, extracts of which were skin prick test (SPT) positive in allergic patients, were detected by rocket immunoelectrophoresis and ELISA. Fungi and bacteria isolated by agar settle plates and soil dilution and soil washing methods were enumerated and identified. Cat dander, Acacia, Alternaria, Aspergillus, Chenopodium, Cladosporium, Bermuda grass, Pithecellobium, Prosopis, Rumex, cultivated rye, and Washingtonia palm allergens were detected by both methods. Viable microbes including 1892 +/- 325 colony-forming units (cfu) of bacteria, and 869 +/- 75 cfu of fungi were isolated per gram of dust by the soil dilution method. Randomly selected microbial colonies on streaking and subculture were found to consist of between two and seven mixed colonies. Fungi including Alternaria, Aspergillus, Botrytis, Cladosporium, Mortierella, Mucor, Mycelia sterilia, Penicillium, Pythium, Ulocladium, Verticillium, and some yeasts were isolated. Actinomyces, Bacillus, Pseudomonas, and mostly coagulase-negative Staphylococcus species were identified, but the bulk of unidentified bacterial isolates were mainly mixed colonies of rods, cocci, coccobacilli, and some filamentous types. Six-hour agar settle-plate counts during sandstorms were 100 and 40% higher for bacteria and fungi, respectively, than without sandstorms. The most abundant aeroallergens were those of Acacia, Alternaria, Aspergillus, Bermuda grass, Cladosporium, cultivated rye, Prosopis, and cat dander. Pithecellobium dulce, Rumex crispus, and Washingtonia palm allergens were detectable for the first time in Riyadh. IgE reactivities of the dust in man were demonstrated by ELISA using sera from atopic, exposed, and normal subjects. These results indicate that sandstorm dust is a prolific source of potential triggers of allergic and nonallergic respiratory ailments, and the methods mentioned here should be routinely used for quick sampling of the environment.

Air Pollutants↗

PubMed2006

A new, broad-spectrum azole antifungal: posaconazole--mechanisms of action and resistance, spectrum of activity.

Posaconazole, a new triazole antifungal, exerts principally the same mechanism of action as the other azole derivatives, i.e. it inhibits the ergosterol production by binding and inhibiting the lanosterol-14alpha-demethylase which is present in almost all fungi except Pneumocystis and Pythium. Posaconazole has an exquisitely high affinity to this target. Since posaconazole has a chemical structure different from fluconazole and voriconazole, it can interact with an additional domain of the target so that it may inhibit even mutated strains resistant to fluconazole and voriconazole. In addition posaconazole is a bad substrate for efflux pumps in fungi, so it can stay active when other azoles are already inactive. Furthermore, the spectrum of posaconazole is rather large including also some zygomycetes resistant to other azoles. In conclusion, posaconazole is actually the most potent azole derivative used in medicine. A combination of posaconazole with other groups of antifungals may have a favourable effect. There are several methods to test the in vitro activities of posaconazole including the E-test, though interpretive breakpoints are still lacking.

Antifungal Agents↗

PubMed2005

Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray.

Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown.

Agriculture↗

PubMed1999

Chitinolytic activity of Pseudomonas fluorescens isolates from barley and sugar beet rhizosphere.

Twelve isolates of Pseudomonas fluorescens, isolated from barley and sugar beet rhizosphere as antagonists towards the plant pathogenic microfungi Rhizoctonia solani and Pythium ultimum, showed chitinolytic activity in batch cultures when grown in media without exogenous chitin. Enzyme tests in cultures demonstrated a complete array of chitinolytic enzymes. Endochitinase and chitobiosidase activities seemed to be tightly coupled in the isolates, while there was no relationship to N-acetyl-glucosaminidase activity. Endochitinase activity, showing hydrolysis of chitin polymers, was found to be extracellular in all isolates, although the most active isolates also retained a cell-bound fraction. Isoelectric focusing gel electrophoresis of supernatants containing extracellular enzyme activity showed that all isolates produced one native endochitinase in logarithmic phase. This enzyme was subsequently modified into several isozymes by extracellular processing as the cultures aged in stationary phase. The 12 isolates could be grouped into seven isozymic patterns. Detailed studies of three selected isolates showed that extracellular release of endochitinase activity also took place in stationary phase. The results, however, indicated that in stationary phase regulation of both the overall production of native enzyme and the subsequent formation of isozymes were different among the P. fluorescens isolates.

Journal Article↗

PubMed2006

Functional characterization of front-end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan.

A survey of the three kinetoplastid genome projects revealed the presence of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Delta8, Delta5 and Delta6 desaturases for L. major, and Delta6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Delta6 desaturases could have Delta4 desaturase activity, based mainly on the conserved HX(3)HH motif for the second histidine box, when compared with Delta4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L. major and T. brucei. Our prediction about the presence of Delta4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Delta5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Delta8 desaturase turned out to be a functional Delta6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareDelta6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very-long-chain polyunsaturated fatty acid biosynthesis is functional in L. major using Delta6, Delta5 and Delta4 desaturases. In trypanosomes, only Delta4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.

Amino Acid Sequence↗

PubMed1984

Phycomycosis in a dog.

A case of phycomycosis, with skin lesions, in a dog is reported. Hyphomyces destruens (Pythium gracile) was identified from aspirate collected from a regional lymph node, and characteristic hyphae were seen in stained sections of a biopsy specimen of affected skin. Incidence, pathogenesis and therapy are discussed.

Animals↗

PubMedDate not supplied

Clinicopathologic findings associated with Lagenidium sp. infection in 6 dogs: initial description of an emerging oomycosis.

An oomycotic pathogen in the genus Lagenidium was isolated from tissues obtained from 6 dogs with progressive cutaneous disease. Initial clinical findings in 5 dogs included multifocal cutaneous lesions, subcutaneous lesions, or both associated with regional lymphadenopathy: the 6th dog initially was presented for evaluation of mandibular lymphadenopathy. Cutaneous lesions were ulcerated, exudative regions (often with necrosis and draining tracts) or multiple firm dermal or subcutaneous nodules. Two dogs subsequently developed hemoabdomen from great vessel rupture and died acutely. Four dogs were euthanized because of progression of subcutaneous lesions or lymphadenopathy. On postmortem examination, regional granulomatous lymphadenitis was found in all 6 dogs, great vessel invasion in 3 dogs, pulmonary lesions in 2 dogs. ureteral obstruction in 1 dog, mediastinal lymphadenitis in 1 dog, and hilar lymphadenitis with invasion of the distal esophagus and trachea in 1 dog. Histologically, lesions were similar to those associated with pythiosis and zygomycosis and were characterized by severe eosinophilic granulomatous inflammation (often with numerous large multinucleated giant cells) centered around broad (7-25 micro), infrequently septate hyphae. Immunoblot analysis of the serologic response of 4 dogs to a soluble mycelial extract of Lagenidium giganteum indicated that each dog's serum recognized at least 10 different antigens of L. giganteum. Culture of infected tissues yielded rapid growth of colorless to white submerged colonies. Microscopically, mature hyphae in culture were broad (25-40 micro), segmented, and occasionally branching and produced motile laterally biflagellate zoospores in water culture. This report is the 1st description of infection caused by an oomycete other than Pythium insidiosum in any mammalian species.

Animals↗

PubMed1968

Cytomembrane differentiation in the endoplasmic reticulum-Golgi apparatus-vesicle complex.

Diversity of cytomembrane types is confirmed in hyphae of the fungus Pythium ultimum by electron microscopy. A transition in membrane morphology across stacks of dictyosome cisternae (from endoplasmic reticulumlike at one pole to plasma membrane-like at the opposite pole) suggests that dictyosomes of the Golgi apparatus are sites of membrane interconversion.

Endoplasmic Reticulum↗

PubMed1989

Rhizobacteria of cotton and their repression of seedling disease pathogens.

During the 1983 field season, the rhizobacteria (including organisms from rhizosphere soil and the root rhizoplane) of cotton plants at one location in Mississippi were inventoried at different plant growth stages. Isolates (1,000) were identified to the genus level and characterized for repression of Pythium ultimum and Rhizoctonia solani. Cotton seedlings were initially colonized by bacteria of many different genera, and populations quickly reached 10 CFU/g of root tissue. As the season progressed, the bacterial populations declined as root mass increased and the roots became more woodlike in consistency. Fluorescent pseudomonads were the most numerous gram-negative rhizobacterial isolates of those that were randomly collected and identified, and they provided the largest number of isolates with fungal repressive activity. Several other gram-negative bacterial genera were recovered throughout the growing season, and some gram-positive bacteria were also isolated routinely, but at lower numbers. There was no correlation between the proportion of rhizobacterial isolates that possessed fungal repressive activity and the plant growth stage from which the isolates were obtained. Approximately twice as many bacterial isolates demonstrated fungal repression in the agar assay compared with the inplanta assay, and isolates were found more frequently with fungal repressive activity against P. ultimum than against R. solai.

Journal Article↗

PubMed1991

Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain.

Pseudomonas aureofaciens Q2-87 produces the antibiotic 2,4-diacetophloroglucinol (Phl), which inhibits Gaeumannomyces graminis var. tritici and other fungi in vitro. Strain Q2-87 also provides biological control of take-all, a root disease of wheat caused by this fungus. To assess the role of Phl in the antifungal activity of strain Q2-87, a genetic analysis of antibiotic production was conducted. Two mutants of Q2-87 with altered antifungal activity were isolated by site-directed mutagenesis with Tn5. One mutant, Q2-87::Tn5-1, did not inhibit G. graminis var. tritici in vitro and did not produce Phl. Two cosmids were isolated from a genomic library of the wild-type strain by probing with the mutant genomic fragment. Antifungal activity and Phl production were coordinately restored in Q2-87::Tn5-1 by complementation with either cosmid. Mobilization of one of these cosmids into two heterologous Pseudomonas strains conferred the ability to synthesize Phl and increased their activity against G. graminis var. tritici, Pythium ultimum, and Rhizoctonia solani in vitro. Subcloning and deletion analysis of these cosmids identified a 4.8-kb region which was necessary for Phl synthesis and antifungal activity.

Antifungal Agents↗

PubMed1992

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.

Anti-Bacterial Agents↗

PubMed1993

Isolation and characterization of actinomycete antagonists of a fungal root pathogen.

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum.

Journal Article↗

PubMed1993

Enhanced growth and activity of a biocontrol bacterium genetically engineered to utilize salicylate.

Plasmid NAH7 was transferred from Pseudomonas putida PpG7 to P. putida R20 [R20(NAH7)], an antagonist of Pythium ultimum. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 mug/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 x 10 CFU/g to approximately 3 x 10 CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 x 10 CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 mug of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10 CFU/g of soil by 48 h of incubation, with the fastest increase at 100 mug/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 mug/g; at 1,000 mug/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 mug of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (P = 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.

Journal Article↗