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Volatiles from Fusarium verticillioides (Sacc.) nirenb. and their attractiveness to nitidulid beetles.

It is known that sap beetles (Coleoptera: Nitidulidae) can vector the fungus Fusarium verticillioides (Sacc.) Nirenb. (= F. moniliforme Sheldon), which causes an important ear-rot disease in corn and also produces fumonisin mycotoxins. The volatiles produced by this fungus were studied to establish whether they could attract sap beetles. Such an association would suggest more than just an incidental role in transmission of the fungus by the beetles. F. verticillioides consistently produces a blend of five alcohols (ethanol, 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, and 2-methyl-1-butanol), acetaldehyde, and ethyl acetate. Ethanol is the most abundant alcohol. The fungus also produces four phenolic compounds (the most abundant of which is ethylguaiacol), a series of presently unidentified sesquiterpene hydrocarbons, and an unidentified compound that is probably a 10-carbon ketone. Solid-phase microextraction was the key technique used in volatile analysis. The volatile profiles change over time and differ somewhat among fungal strains: The alcohols, aldehyde, and ester always appeared first and were present for each strain. Production of the phenolics lagged by several days, and in some strains these compounds were barely detectable. Volatile production eventually diminished in all strains. All strains were attractive to the sap beetle, Carpophilus humeralis (F.), in wind-tunnel bioassays. Attraction was correlated primarily to the presence of the alcohols, acetaldehyde, and ethyl acetate, rather than to the phenolics. To verify that the identified culture volatiles were responsible for beetle attraction, cultures were quantitatively simulated with synthetic chemicals, and the cultures and corresponding synthetic mixtures were then compared by bioassay. The comparisons were favorable. Volatile emission patterns from cultures were fairly robust with respect to inoculum level or incubation temperature, but some manipulation was possible. For example, after freeze-drying and rehydrating (a rapid simulation of winter/spring conditions), F. verticillioides produced ethyl acetate and other esters at unusually high levels. The fungus produced attractive volatiles following ear inoculation of milk-stage field corn as well as on sterile, mature kernels in the laboratory.

Alcohols↗

Intracellular parasitism by Histoplasma capsulatum: fungal virulence and calcium dependence.

Histoplasma capsulatum is an effective intracellular parasite of macrophages and causes the most prevalent fungal respiratory disease in the United States. A "dimorphic" fungus, H. capsulatum exists as a saprophytic mold in soil and converts to the parasitic yeast form after inhalation. Only the yeasts secrete a calcium-binding protein (CBP) and can grow in calcium-limiting conditions. To probe the relation between calcium limitation and intracellular parasitism, we designed a strategy to disrupt CBP1 in H. capsulatum using a telomeric linear plasmid and a two-step genetic selection. The resultingcbp1 yeasts no longer grew when deprived of calcium, and they were also unable to destroy macrophages in vitro or proliferate in a mouse model of pulmonary infection.

Alleles↗

The role of HLA antigens in the development of paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis is a systemic granulomatous disease that involves primarily the lungs and may disseminate to other organs and systems. It is caused by Paracoccidioides brasiliensis, a fungus that exhibits reversible thermal dimorphism and whose natural habitat is presently unknown. There are two main clinical forms: the acute (subacute) juvenile form and the chronic adult form. The former runs a more rapid course and is more severe than the latter. This mycosis is found throughout Latin America. Brazil accounts for 80% of reported cases. Presumably P. brasiliensis thrives in humid and hot places, especially near forests or farms. The infection is endemic in certain areas, especially in Brazil, Colombia and Venezuela, where nearly 100% of the population show cutaneous paracoccidioidina positive skin tests, indicating previous contact with the fungus, although a small percentage show clinical manifestations of the disease. METHODS: We compared the expression of HLA class I antigens in a healthy group (control) and in a group of patients with paracoccidioidomycosis (chronic adult form) using the Terasaki lymphocytotoxicity test modified by Amos for HLA antigen analysis. AIMS: To discover indications of whether or not individual susceptibility to P. brasiliensis might depend on some specific immunological defect. RESULTS: There is no evidence of association between a specific HLA antigen and paracoccidioidomycosis in the subjects studied. Further investigations are recommended.

Adult↗

Paracoccidioides brasilienses isolates obtained from patients with acute and chronic disease exhibit morphological differences after animal passage.

The basis for virulence in Paracoccidioides brasiliensis is not completely understood. There is a consensus that the sequential in vitro subcultivation of P. brasiliensis leads to loss of its pathogenicity, which can be reverted by reisolation from animal passage. Attention to morphological and biochemical properties that are regained or demonstrated after animal passage may provide new insights into factors related to the pathogenicity and virulence of P. brasiliensis. We evaluated morphological characters: the percentage of budding cells, number of buds by cell and the diameter of 100 mother cells of yeast-like cells of 30 P. brasiliensis isolates, before and after animal passage. The isolates were obtained from patients with different clinical forms of paracoccidioidomycosis (PCM): acute form (group A, n=15) and chronic form (group C, n=15). The measurement of the yeast cell sizes was carried out with the aid of an Olympus CBB microscope coupled with a micrometer disc. We measured the major transverse and longitudinal axes of 100 viable cells of each preparation. The percentage of budding cells as also the number of buds by cell was not influenced by animal passage, regardless of the source of the strain (acute or chronic groups). The size values of P. brasiliensis isolates from groups A and C, measured before the animal passage exhibited the same behavior. After animal passage, there was a statistically significant difference between the cell sizes of P. brasiliensis isolates recovered from testicles inoculated with strains from groups A and C. The maximum diameter of mother cells from group A isolates exhibited a size of 42.1 microm in contrast with 32.9 microm exhibited by mother cells from group C (p<0.05). The diameter of 1500 mother cells from group A isolates exhibited a medium size of 16.0 microm (SD +/- 4.0), a value significantly higher than the 14.1 microm (SD = +/- 3.3) exhibited by 1500 mother cells from group C isolates (p<0.05). Our results reinforce the polymorphism exhibited by P. brasiliensis in biological material and the need for further investigations to elucidate the role of morphological parameters of the fungus in the natural history of the disease.

Acute Disease↗

Fourier transform-infrared spectroscopy as a new method for evaluating host resistance in the Dutch elm disease complex.

Resistance of elms (Ulmus spp.) to the pathogenic fungus Ophiostoma novo-ulmi Brasier depends on chemical and anatomical factors that confine the spread of the pathogen in the vascular system of the host. This study focused on detecting chemical differences in 4-year-old Ulmus minor Mill. seedlings before and after inoculation with a virulent O. novo-ulmi isolate. According to symptom development over 60 days, the trees were divided into resistant (0-33% wilting) and susceptible (67-100% wilting) groups. Histochemical tests and Fourier transform-infrared (FT-IR) spectroscopy analysis were performed on transverse sections of 2-year-old twigs, 2 days before and 40 days after inoculation. Although histochemical tests did not clearly discriminate susceptible from resistant elms, chemical differences between resistant, susceptible and control trees were detected by FT-IR. The average spectrum for resistant tree samples had higher absorbance peaks than the spectra from the susceptible and control samples, indicating increased formation of lignin and suberin. The roles of lignin and suberin in the resistance of the elms against O. novo-ulmi and the usefulness and sensitivity of the FT-IR technique for analyzing metabolic changes caused by pathogens in plants are discussed.

Ascomycota↗

Translationally controlled tumor protein from Madurella mycetomatis, a marker for tumorous mycetoma progression.

About 40 years ago Abs against the fungus Madurella mycetomatis were first demonstrated to be present in eumycetoma patients, a disease characterized by tumorous swellings. To date nothing is known about the individual immunoreactive Ags present in this fungus. In the present study, we identify its first immunogenic Ag, a protein homologous to the translationally controlled tumor protein (TCTP), a well-conserved histamine release factor in a range of eukaryotes. The gene for this Ag was demonstrated to be present in two variants in M. mycetomatis, with 13% aa difference between the two proteins encoded. In vitro, TCTP was secreted into the culture medium. In vivo, it was found to be expressed on hyphae present in developing stages of the eumycetoma-characteristic black grain. Significant IgG and IgM immune responses, against the whole protein and selected M. mycetomatis-specific peptides, were determined. The Ab levels correlated with lesion size and disease duration. Overall, the patients with the largest lesions had the highest Ab level, which lowered with decreasing size of the lesion. After 6-15 years of disease duration the Ab levels were the highest. TCTP is the first well-characterized immunogenic Ag, simultaneously the first monomolecular vaccine candidate, identified for the fungus M. mycetomatis.

Adult↗

[Pulmonary allescheriosis (author's transl)].

In a patient properly treated for a previous cavitary tuberculosis, we had the surprise, after hemoptyses, to find a sleigh-bell shaped picture suggesting an intra-cavitary aspergilloma with a very special mycosis with Allescheria Boydii. Proof was provided by the presence of the fungus in direct examination and in culture in bronchial secretions. Surgical exeresis did not cure the patient and a secondary recurrence appeared on which antifungal drugs were ineffective. Antigens were made from the strain. They are used for immunoprecipitations tests. Only the patient's serum is positive; 82 serum test (18 concerned patients with visceral mycosis of farmer's lung disease) remained negative. Twenty two observations or pulmonary allescheriosis were recorded in the world. These figures are certainly below the truth. Three practical consequences can be drawn: --for an intra-cavitary mycetoma, with negative anti-aspergillus precipitins, such a disease must be kept in mind and the fungus looked for in bronchial secretions and specific immuno-precipitation test done; --any mycotic growth must be systematically cultured to determine the type of fungus; --for therapy, if diagnosis was made before the intervention, an antifungal treatment either local or general must be associated to the surgical treatment (contrary to aspergilloma treatment).

Aged↗

[Kininases from yeasts (author's transl)].

Practically no studies are available on the presence of kinin-decomposing enzymes in yeasts. Different preparations (intact washed cell suspensions, cell-homogenised blastospores, nutrient media, supernatant of raw fungus suspensions) of Candida strains sampled from foci of disease and from the environment were studied qualitatively and quantitatively fro the presence of kinin-inactivating enzymes. The parameter measured is the time-dependent inactivation of bradykinin by the test strain preparations as determined in the isolated guinea-pig ileum by the water-bath test. Preceding surveys of the basic presence of kinin-decomposing enzyme activity in 10 Candida strains from foci of disease revealed that only undiluted, intact washed suspensions of spores were capable of bradykinin inactivation. Intact washed blastospores from 4 other strains sampled from foci of disease (Candida tropicalis B5, Candida tropicalis B12, Candida albicans C10, Candida albicans A23) diluted 1:10 by volume did not exhibit bradykinin decomposition at the concentration studied. In contrast to this, identical preparations of three strains from the environment (Candida tropicalis E2, Rhodotorula rubra H14, Saccharomyces lactis R15) were exhibiting kinin-inactivation of partially high intensity which was still enhanced by cell homogenisation. The Candida brumptii Q6 strain, however, did not induce kinin breakdown. In the case of Candida tropicalis E2, the enzymes could be demonstrated also in the nutrient diluted 1 : 2. Supernatants obtained by centrifugation of raw fungus suspensions were ineffective in respect of strains from foci of disease as well as such from the environment. Bradykinin was protected against inactivation by treatment of all kinin-decomposing preparations with 1,10 phenanthroline, acid and heat. Thus, the kinin-decomposing enzymes involved were kininases. In a general view, species-specific differences in the presence of kininases among Candida strains were recognizable.

Bradykinin↗

DNA Fingerprinting with a Dispersed Repeated Sequence Resolves Pathotype Diversity in the Rice Blast Fungus.

The poor definition of pathotype variation in the rice blast fungus has historically handicapped strategies for reducing blast disease damage to the world's rice crop. We have employed a probe for a dispersed repeated DNA sequence called MGR [Hamer et al. (1989). Proc. Natl. Acad. Sci. USA 86, 9981-9985] to construct genotype-specific, EcoRl restriction fragment length profiles (MGR-DNA fingerprints) from United States field isolates of this fungus. By using a blind-test design, we demonstrated that MGR-DNA fingerprints distinguished the major pathotypes in the United States, accurately identified the pathotypes of isolates collected over a 30-year period, and defined the organization of clonal lineages within and among pathotype groups. These results resolved a lingering controversy regarding rice blast pathotype stability and illustrated new opportunities for tracking the population dynamics and evolution of this important crop pathogen.

Journal Article↗

Recurring challenges from a necrotrophic fungal plant pathogen: a case study with Leptosphaeria maculans (causal agent of blackleg disease in brassicas) in Western Australia.

BACKGROUND: Blackleg disease of Brassica napus, caused by the necrotrophic fungus Leptosphaeria maculans, causes severe yield losses in Australia, Europe and Canada. In Western Australia, it nearly destroyed the oilseed rape industry in 1972 when host genotypes and conducive environmental conditions favoured severe epidemics. The introduction of cultivars with polygenic resistance and the adoption of sound cultural practices two decades later helped to manage the disease. These were abandoned by many farmers in recent years in favour of the effective but ephemeral resistance conferred by the single dominant gene-based resistance derived from B. rapa ssp. sylvestris. Recently, several cultivars carrying this gene have collapsed widely within a period of 3 years after their commercial release. An environment conducive to the disease and the association of the pathogen with susceptible hosts in Western Australia for over 80 years together have led to the proliferation of L. maculans races, amounting to half of all races delineated to date from Europe, including the United Kingdom, Canada and Australia. SCOPE: This review demonstrates the problems that emerge when traditional cultural practices employed, along with cultivars containing polygenic resistance to a serious necrotrophic pathogen, are discarded in preference to the exclusive deployment of effective but ephemeral single dominant gene-based resistance to the disease across Southern Australia. CONCLUSIONS: Single dominant gene-based resistance currently available, on its own, will not confer durable resistance to blackleg disease in oilseed rape. Return to earlier management practices, including reliance upon polygenic resistance and induced resistance, may be the best currently available options to maintain production in regions across Southern Australia predisposed to severe epidemics.

Ascomycota↗

Meningoencephalitis caused by Histoplasma capsulatum: occurrence in a renal transplant recipient and a review of the literature.

A case of meningoencephalitis caused by histoplasmosis in a renal transplant patient is described. The diagnosis was made postmortem. The clinicopathological features of 39 additional cases of central nervous system (CNS) invasion by histoplasmosis were reviewed. In the great majority of instances (92.1%), CNS involvement occurred in the disseminated form of the disease. Diagnosis was proved by culturing the fungus from bone marrow, blood, lymph nodes, or liver. Neurological symptoms and signs and cerebrospinal fluid (CSF) changes did not occur until extensive brain damage had resulted. Difficulty in culturing the organism in the (CSF) caused a further delay in making an early diagnosis of CNS involvement. The use of meningeal and brain biopsy specimens in conjunction with the electroencephalogram (EEG) may help in making an earlier diagnosis of CNS involvement.

Brain↗

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.

Electrophoresis, Gel, Two-Dimensional↗

Purified recombinant A. fumigatus allergens induce different responses in mice.

Aspergillus fumigatus an opportunistic fungus is associated with a number of diseases in humans. Allergy resulting from exposure to the A. fumigatus allergens has been recognized frequently. The damage caused by the disease is very striking in patients with atopy and those with cystic fibrosis. Avoidance to exposure is not feasible because A. fumigatus spores are ubiquitously distributed in the environment. Hence, immunotherapeutic regimens in severe forms of A. fumigatus allergy may have a high potential. However, before such forms of therapy can be envisaged, it is essential to understand the immunopathogenesis. In the present study, we investigated the role of purified A. fumigatus allergens in the development of allergic asthma in mice. We have used four major recombinant A. fumigatus allergens in the murine model. Mice exposed to Asp f 1, f 3, and f 4 showed inflammatory changes in the lungs and airway hyperreactivity. The immune responses, including elevated serum IgE, enhanced eosinophils, recruitment in the peripheral blood and lungs, and expression of regulatory cytokines, are characteristic of a Th2 response. Asp f 6 demonstrated only a reduced response in these animals. The results suggest that the pathology induced by crude A. fumigatus extract results from the cumulative effects of the allergens and the individual responses varied considerably with different purified antigens.

Airway Resistance↗

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection.

Endochitinases (E.C.3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.

Base Sequence↗

Introgression molecular analysis of a leaf rust resistance gene from Coffea liberica into C. arabica L.

Leaf rust caused by the fungus Hemileia vastatrix is the most devastating disease of arabica coffee ( Coffea arabica). Therefore, developing leaf rust-resistant varieties has been a breeding objective of the highest priority in many countries. The purpose of the present work was to gain insight into the mechanism of introgression into C. arabica of a leaf rust resistance gene from C. liberica (i.e. S(H)3 resistance factor) and to identify associated molecular markers. An F(2) progeny (i.e. 101 individuals) derived from a cross between Matari, an arabica accession and liberica-introgressed line S.288, was evaluated for resistance against three different races of H. vastatrix. The progeny segregated for the S(H)3 gene in a 3:1 ratio, as expected for a single dominant gene. Amplified fragment length polymorphism analysis of a population subset using 80 different primer combinations revealed that at least half of the total polymorphism observed in the population is associated with introgression of C. liberica chromosome fragments. Furthermore, 15 primer combinations generating candidate marker bands associated with the S(H)3 resistance gene were used to analyse the whole F(2) population. A total of 34 marker bands originating from S.288 and attributable to introgression were scored. None exhibited segregation distortion. Linkage analysis revealed only three distinct introgressed fragments corresponding to a total length of 52.8 cM. Twenty-one markers were strongly associated (LOD score >14) with the S(H)3 gene and were grouped together in a single linkage group of 6.3 cM. The results are discussed in relation to the efficient use of genetic resources in arabica breeding.

Basidiomycota↗

Molecular mapping of two cultivar-specific avirulence genes in the rice blast fungus Magnaporthe grisea.

Rice blast, caused by the fungus Magnaporthe grisea, is a globally important disease of rice that causes annual yield losses. The segregation of genes controlling the virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in the interaction of rice and M. grisea. The segregation of avirulence and virulence was studied in 87 M. grisea F(1) progeny isolates from a cross of two isolates, Guy11 and JS153, using resistance-gene-differential rice cultivars. The segregation ratio indicated that avirulence and virulence in the rice cultivars Aichi-asahi and K59, respectively, are controlled by single major genes. Genetic analyses of backcrosses and full-sib crosses in these populations were also performed. The chi(2 )test of goodness-of-fitness for a 1:1 ratio indicated that one dominant gene controls avirulence in Aichi-asahi and K59 in this population. Based on the resistance reactions of rice differential lines harboring known resistance genes to the parental isolates, two genetically independent avirulence genes, AVR-Pit and AVR-Pia, were identified. Genetic linkage analysis showed that the SSR marker m355-356 is closely linked to AVR-Pit, on the telomere of chromosome 1 at a distance of approximately 2.3 cM. The RAPD marker S487, which was converted to a sequence-characterized amplified region (SCAR) marker, was found to be closely linked to AVR-Pia, on the chromosome 7 telomere at a distance of 3.5 cM. These molecular markers will facilitate the positional cloning of the two AVR genes, and can be applied to molecular-marker-assisted studies of M. grisea populations.

Chromosome Mapping↗

Exserohilum: an emerging human pathogen.

Exserohilum is a dematiaceous fungus that may cause a spectrum of diseases in humans, including skin and corneal infection, invasive disease, and allergic fungal sinusitis. The aim of this work is to describe two new cases of Exserohilum infection and to review the literature. The review yielded 33 cases of Exserohilum infection, of which 23 were reported since 1993. Most occurred in regions with hot climates, such as India, Israel, and the southern USA. Impaired immunity was present in the majority of patients with invasive and skin infections, whereas local trauma and atopy were the predisposing factors in those with corneal infections and allergic fungal sinusitis, respectively. Surgical debridement was the principal mode of therapy for allergic fungal sinusitis. Amphotericin B was the initial single antifungal agent used in all cases of invasive disease; the response rate was low but improved with the addition of triazole agents. Outcome appeared to be better than for other mold infections and depended mainly on the underlying diseases.

Adolescent↗