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PubMedDate not supplied

Antibody engineering--a valuable asset in preventing closed environment epidemics.

Investigations of Mir, Space Shuttle, Skylab and Apollo missions report extensive colonisation of the spacecraft by bacteria and fungi, which can lead to degradative effects on spacecraft equipment and devastating effects on space-grown crops. More than 80% of terrestrial greenhouse epidemics are due to the fungal genera Phytophthora, Pythium and Fusarium, which have been found in life support system test-beds. The advent of recombinant antibody technologies, including ribosome display and phage display, has made it possible to develop antibodies against virtually any toxin or organism and allows for maturation of antibodies by in vitro molecular evolution. These antibodies may play an important role in an integrated pest management regime for life support systems. Efficacy of existing fungal countermeasures could be increased by chemical linkage to antibodies, which target the site of action of the biocide or trap the pathogen in a biofilter. Novel recombinant antibody-biocide fusions can be expressed in situ by plants or symbiotic microbes to create direct disease resistance.

Antibodies, Fungal↗

PubMed2005

A chitinase with antifungal activity from the mung bean.

A chitinase with antifungal activity was isolated from mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, (NH4)2SO4 precipitation, ion-exchange chromatography on CM-Sepharose, high-performance liquid chromatography (HPLC) on Poros HS-20, and gel filtration on Sephadex G-75. The protein exhibited a molecular mass of 30.8 kDa in SDS-polyacrylamide gel electrophoresis. Its pI was 6.3 as determined by isoelectric focusing. The specific activity of the chitinase was estimated to be 3.81 U/mg. The enzyme expressed its optimum activity at pH 5.4 and was stable from 40 to 50 degrees C. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Mycosphaerella arachidicola, Pythium aphanidermatum, and Sclerotium rolfsii.

Antifungal Agents↗

PubMed2004

Universally occurring phenylpropanoid and species-specific indolic metabolites in infected and uninfected Arabidopsis thaliana roots and leaves.

A total of eleven alkali-released, aromatic compounds were identified by HPLC, MS and NMR analyses in cell wall extracts from Arabidopsis thaliana roots. Nine of them together constituted the three complete series of 4-hydroxy-, 4-hydroxy-3-methoxy, and 4-hydroxy-3,5-dimethoxy-substituted benzaldehydes, benzoic acids and cinnamic acids. The other two were indolic metabolites: indole-3-carboxylic acid and indole-3-carbaldehyde. Qualitatively similar, but quantitatively distinct profiles were obtained using cell-wall extracts from A. thaliana leaves. Several of these compounds, particularly indole-3-carboxylic acid, 4-hydroxybenzoic acid and all four aldehydes, increased considerably in concentration upon infection of roots with Pythium sylvaticum, as did at least some of them upon infection of leaves with Pseudomonas syringae pv tomato. Comparison of these results with analogous data on a variety of different plant species suggests a remarkable structural uniformity among the majority of constitutive as well as infection-induced, aromatic cell wall-bound compounds throughout the entire plant kingdom-in sharp contrast to the highly species-specific, chemically highly divers bouquets of soluble aromatic metabolites.

Arabidopsis↗

PubMed2003

Disseminated pythiosis in three horses.

Three cases of equine subcutaneous pythiosis with dissemination to the internal organs were investigated. The subcutaneous lesions were observed on the mammary gland, nostrils and limbs of the infected horses. Histopathological analysis of the infected tissues revealed a strong eosinophilic reaction, with macrophages, mast cells and giant cells. Sparsely septated hyphal filaments of 4-6 microm diameter were identified in the center of the eosinophilic areas. Specific fluorescent antibody against Pythium insidiosum confirmed the hyphae in the infected tissues in three examined horses. In one of the three cases, the DNA sequences amplified from the infected subcutaneous tissues and internal organs, revealed that P. insidiosum's 18S SSU rDNA amplicons shared 100% identity with those sequences deposit in GenBank. This is the first report confirming by immunochemical and genetic techniques that P. insidiosum can disseminated from superficial to deep structures.

Animals↗

PubMed2005

Epizootic cutaneous pythiosis in beef calves.

Enzootic bovine granulomatosis (EBG) is a disease that affects beef calves in the flooded Savanna regions of Venezuela. Although Pythium insidiosum was originally the suspected etiologic agent, nothing was done to demonstrate its presence in the infected animals. The objective of this study was to investigate the etiologic agent causing cutaneous lesions in a group of 63 calves diagnosed with EBG. The collected samples were analyzed by histopathology, immunohistochemistry, serology, and mycological techniques. The clinical manifestations of BEG included ulcerated and swollen granulomatous lesions on the limbs of young calves. Histopathological studies revealed an eosinophilic inflammatory reaction with neutrophils, giant cells and branching hyphae with the Splendore-Hoeppli phenomenon. Immunofluorescence analysis identified the branching hyphae as elements of the fungal-like organism P. insidiosum. Wet mounts in 10% KOH revealed hyaline slender branching hyaline coenocytic hyphae, but cultures from those samples were negative. Anti-P. insidiosum immunoglobulins were also demonstrated in the collected sera of the affected bovines using latex agglutination, immunodiffusion, ELISA, and Western Blot. The histopathological, immunohistochemical, and serological tests showed for the first time that P. insidiosum could be involved in epizootic bovine pythiosis.

Animals↗

PubMed2003

Pythiosis, lagenidiosis, and zygomycosis in small animals.

Pythiosis, lagenidiosis, and zygomycosis affect animals living in temperate, tropical, and subtropical climates, and these diseases are often fatal. Although Pythium insidiosum, Conidiobolus species, and Basidiobolus species have been recognized as pathogens for several years, members of the genus Lagenidium have been identified as a cause of oomycosis in dogs only recently. Pythiosis, lagenidiosis, and zygomycosis share similar clinical and histologic characteristics, making them difficult to distinguish from one another; however, distinguishing between these pathogens is important because of differences in epidemiology, choice and duration of therapy, and prognosis.

Animals↗

PubMed2003

In vitro activities of Maesa lanceolata extracts against fungal plant pathogens.

In vitro tests were carried out using extracts of Maesa lanceolata var. goulungensis weir against a broad range of fungal plant pathogens such as Phytophthora cryptogea, Trichoderma virens, Aspergillus niger, Phoma sp., Fusarium oxysporium, Pythium ultimum, Cochliobolus heterostrophus, Rhizoctonia solani, Sclerotium rolfsii and Pyrenophora teres. M. lanceolata extracts were very active against all the pathogens tested except P. ultimum and R. solani.

Antifungal Agents↗

PubMed1995

Cutaneous pythiosis in the horse.

Pythiosis of horses in an invasive, ulcerative, proliferative, pyogranulomatous disease of the skin and subcutis caused by Pythium insidiosum, a fungus-like oomycete in the order Peronosporales of the kingdom Protista. Pythiosis is a form of "phycomycosis," which is a complex of pyogranulomatous diseases that also includes conidiobolomysosis, basidiobolobysosis, and disorders caused by members of the order Mucorales.

Animals↗

PubMed1996

Crystal structure of a fungal elicitor secreted by Phytophthora cryptogea, a member of a novel class of plant necrotic proteins.

BACKGROUND: Elicitins form a novel class of plant necrotic proteins which are secreted by Phytophthora and Pythium fungi, parasites of many economically important crops. These proteins induce leaf necrosis in infected plants and elicit an incompatible hypersensitive-like reaction, leading to the development of a systemic acquired resistance against a range of fungal and bacterial plant pathogens. No crystal structures of this class of protein are available. The crystal structure determination of beta-cryptogein (CRY), secreted by Phytophthora cryptogea, was undertaken to identify structural features important for the necrotic activity of elicitins. RESULTS: The structure of CRY was determined using the multiwavelength anomalous diffraction technique and refined to 2.2 A resolution. The overall structure has a novel fold consisting of six alpha helices and a beak-like motif, whose sequence is highly conserved within the family, composed of an antiparallel two-stranded beta sheet and an omega loop. This motif is assumed to be a major recognition site for a putative receptor and/or ligand. Two other distinct binding sites seem to be correlated to the level of necrotic activity of elicitins. CONCLUSIONS: The determination of the crystal structure of a member of the elicitin family may make it possible to separate the activity that causes leaf necrosis from that inducing systemic acquired resistance to pathogens, making it feasible to engineer a non-toxic elicitin that only elicits plant defences. Such studies should aid the development of non-toxic agricultural pest control.

Algal Proteins↗

PubMed2003

Synthetic method and biological activities of cis-fused alpha-methylene gamma-lactones.

A reliable method was developed for the synthesis of cis-fused alpha-methylene gamma-lactones via alpha-methyl gamma-lactones. Bromination of alpha-methyl gamma-lactones with LDA/CBr(4) or TMSOTf/PTAB and successive dehydrobromination with DBU or TBAF of the resulting alpha-bromo-alpha-methyl gamma-lactones gave the desired alpha-methylene gamma-lactones in high yield. This method was successfully applied to the synthesis of biologically active compounds. alpha-Methylene gamma-lactone derivatives 1c, 2c, 4c, and 17 showed cell growth inhibitory activity to P388 lymphocytic leukemia. They also showed significant activities to crop diseases. Thus, alpha-methylene gamma-lactone 1c showed preventive activity in controlling scab of apple caused by Venturia inaequalis. alpha-Methylene gamma-lactones 2c, 4c, 17, and 18 also showed significant preventive activities in controlling damping off of cucumber caused by Pythium aphanidermatum.

Animals↗

PubMed1998

Penicilliosis marneffei and pythiosis: emerging tropical diseases.

Penicilliosis marneffei and pythiosis insidiosi are emerging infections in subtropical, tropical, and temperate areas of the world. Penicilliosis marneffei is endemic in several Southeast Asian countries and may be carried to other areas of the world by residents of these countries or visitors. Pythiosis occurs in humans and animals who frequent the aquatic habitats that harbor Pythium insidiosum. Although early diagnosis is important because of the high morbidity or mortality associated with these two diseases, the diagnosis of these infections can be difficult because their clinical and histologic features are not pathognomonic. Prompt diagnosis is a prerequisite to their appropriate treatment. Laboratory testing, involving cultural, histologic and immunologic methods, is necessary to establish an unequivocal diagnosis. The clinical presentation, epidemiology, diagnosis and treatment of these diseases are discussed.

AIDS-Related Opportunistic Infections↗

PubMed1998

Cutaneous Pythiosis insidiosi in calves from the Pantanal region of Brazil.

Two cases of cutaneous Pythiosis insidiosi were diagnosed in cattle from the Pantanal region, Brazil. The lesions were observed in the limbs of two 8-month-old beef calves. Close examination showed local swelling and focal ulceration of the skin. Microscopically, there was multifocal granulomatous dermatitis with intralesional Pythium insidivosum hyphae. The diagnosis was based on the morphological aspects, immunohistochemical findings and culture of the etiologic agent.

Animals↗

PubMed2001

Isolation of the first putative peroxidase cDNA from a conifer and the local and systemic accumulation of related proteins upon pathogen infection.

Peroxidases are associated with the active defence reactions in higher plants in response to foreign organisms. They are involved in the oxidation of phenolic compounds in cell walls, polymerization of lignin and suberin, and in several other oxidation processes but the exact function of individual peroxidases is not known. We have isolated a cDNA encoding the putative defence-related and basic plant peroxidase SPI2 (spruce pathogen-induced 2), with an estimated molecular mass of 34 kDa, from roots of Norway spruce (Picea abies) seedlings. This is the first description of the isolation of a complete cDNA encoding a putative peroxidase from a gymnosperm. The transcript was present in the roots of healthy seedlings, and during infection with the pathogen Pythium dimorphum there was a rapid initial increase followed by a dramatic reduction of the transcript. The 34 kDa mature SPI2 protein was detected in both the developing root and shoot of healthy seedlings and increased amounts of SPI2 and increased accumulation of highly basic peroxidase isoforms was observed in roots after infection. In addition, two SPI2-related proteins with apparent molecular masses of 38 and 39 kDa, were also detected. Both these proteins accumulated in roots only after infection, and the 39 kDa protein was in addition detected in shoots of root-infected seedlings. Thus, both SPI2 and the SPI2-related proteins accumulate as a local response, in roots, and as a systemic response to infection the 39 kDa protein accumulates in the shoot.

Amino Acid Sequence↗

PubMed2004

Antifungal activities of four fatty acids against plant pathogenic fungi.

The effect of the fatty acids linolenic acid, linoleic acid, erucic acid and oleic acid on the growth of the plant pathogenic fungi Rhizoctonia solani, Pythium ultimum, Pyrenophora avenae and Crinipellis perniciosa were examined in in vitro studies. Linolenic and linoleic acids exhibited activity against all of the fungi. However, whereas linolenic acid reduced mycelial growth of R. solani and C. perniciosa at 100 microM, the concentration had to be increased to 1000 microM before any effect on mycelial growth of P. ultimum and P. avenae was observed. Linoleic acid only reduced mycelial growth of R. solani, P. ultimum and P. avenae at 1000 microM, but led to a significant reduction in growth of C. perniciosa at 100 microM. In contrast, oleic acid had no significant effect on growth of R. solani or P. avenae, but gave significant reductions in mycelial growth of P. ultimum at 100 microM and reduced growth of C. perniciosa significantly at 1000 microM. All of the fatty acids reduced biomass production by all of the fungi significantly in liquid culture when added to the media at 100 microM. Erucic acid had no effect on fungal growth at any concentration examined. The antifungal activities exhibited by linolenic, linoleic and oleic acids may be useful in the search for alternative approaches to controlling important plant pathogens, such as those examined in this study.

Ascomycota↗

PubMed2001

Survival, root colonisation and biocontrol capacities of Pseudomonas fluorescens F113 LacZY in dry alginate microbeads.

Cells of Pseudomonas fluorescens F113 LacZY were encapsulated in alginate and their survival and ability to colonise sugar beet were evaluated. To assess survival, the formulation, composed of dry alginate microbeads of 300- to 700-microm diameter, was stored 1 year at 28+/-2 and 4+/-2 degrees C and then tested against pathogenic fungi Pythium ultimum and Rhizoctonia solani in in vitro inhibition experiments. The same material was also used as inoculant for protection of sugar beet against Py. ultimum in microcosm experiments. The results obtained indicated that, although drying alginate beads resulted in a significant reduction of bacterial viability, the use of microbeads enabled a satisfactory level of root colonisation and protection, at least under microcosm conditions. The capability of the encapsulated cells to produce the antifungal metabolite 2,4-diacetylphloroglucinol (Phl) was not significantly affected by 12 months storage.

Alginates↗

PubMed1999

Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54.

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi-preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H-, 13C-NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Antifungal Agents↗

PubMed2004

Orbital pythiosis: a non-fungal disease mimicking orbital mycotic infections, with a retrospective review of the literature.

In the past decade there have been four well-documented cases of orbital pythiosis caused by Pythium insidiosum. All were recorded in apparently healthy children. Although pythiosis seems to be a rare infection in humans, we recently conducted a review of the medical literature to investigate misdiagnosed cases of orbital pythiosis in the past 100 years in children. To track putative cases of orbital pythiosis, we first identified orbital cases initially diagnosed as fungal infections. We were particularly interested in cases (a) involving apparently young healthy hosts, (b) the presence of hyaline, aseptate hyphal elements in the infected tissues, (c) the morphological features of the hyphal elements, (d) the presence of an eosinophilic granulomatous reaction with the Splendore-Hoeppli phenomenon around the mycelial elements, (e) resistance to antifungal therapy, (f) outcome after therapy, if any, and (g) cultural strategies. This study showed that indeed, there had been five other recorded cases of orbital infections, all in young children in the USA, with characteristics consistent with infections caused by P. insidiosum. The reports had described those cases of orbital-cranial-arterial diseases as patients with aspergillosis (one case), penicilliosis infection (one case), and zygomycosis (three cases). We reviewed those anomalous cases and discuss details about their clinical, pathologic, therapeutic, and etiologic evidence used to reclassify them as putative cases of orbital pythiosis.

Aspergillosis↗

PubMed1999

Genetic basis in plants for interactions with disease-suppressive bacteria.

Plant health depends, in part, on associations with disease-suppressive microflora, but little is known about the role of plant genes in establishing such associations. Identifying such genes will contribute to understanding the basis for plant health in natural communities and to new strategies to reduce dependence on pesticides in agriculture. To assess the role of the plant host in disease suppression, we used a genetic mapping population of tomato to evaluate the efficacy of the biocontrol agent Bacillus cereus against the seed pathogen Pythium torulosum. We detected significant phenotypic variation among recombinant inbred lines that comprise the mapping population for resistance to P. torulosum, disease suppression by B. cereus, and growth of B. cereus on the seed. Genetic analysis revealed that three quantitative trait loci (QTL) associated with disease suppression by B. cereus explained 38% of the phenotypic variation among the recombinant inbred lines. In two cases, QTL for disease suppression by B. cereus map to the same locations as QTL for other traits, suggesting that the host effect on biocontrol is mediated by different mechanisms. The discovery of a genetic basis in the host for interactions with a biocontrol agent suggests new opportunities to exploit natural genetic variation in host species to enhance our understanding of beneficial plant-microbe interactions and develop ecologically sound strategies for disease control in agriculture.

Bacillus cereus↗