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Cloning, characterization, and transcription of three laccase genes from Gaeumannomyces graminis var. tritici, the take-all fungus.

Gaeumannomyces graminis var. tritici, a filamentous ascomycete, is an important root pathogen of cereals that causes take-all disease and results in severe crop losses worldwide. Previously we identified a polyphenol oxidase (laccase) secreted by the fungus when induced with copper. Here we report cloning and partial characterization of three laccase genes (LAC1, LAC2, and LAC3) from G. graminis var. tritici. Predicted polypeptides encoded by these genes had 38 to 42% amino acid sequence identity and had conserved copper-binding sites characteristic of laccases. The sequence of the LAC2 predicted polypeptide matched the N-terminal sequence of the secreted laccase that we purified in earlier studies. We also characterized expression patterns of these genes by reverse transcription-PCR. LAC1 was transcribed constitutively, and transcription of LAC2 was Cu inducible. All three genes were transcribed in planta; however, transcription of LAC3 was observed only in planta or in the presence of host (wheat) plant homogenate.

Amino Acid Sequence↗

The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type pseudomonas spp.bacteria.

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.

Endoribonucleases↗

Identification and Ds-tagged isolation of a new gene at the Cf-4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5.

Leaf mould disease in tomato is caused by the biotrophic fungus Cladosporium fulvum. An Ac/Ds targeted transposon tagging strategy was used to isolate the gene conferring resistance to race 5 of C. fulvum, a strain expressing the avirulence gene Avr4. An infection assay of 2-week-old seedlings yielded five susceptible mutants, of which two had a Ds element integrated in the same gene at different positions. This gene, member of a gene family, showed high sequence homology to the C. fulvum resistance genes Cf-9 and Cf-2. The gene is predicted to encode an extracellular transmembrane protein containing a divided domain of 25 leucine-rich repeats. Three mutants exhibited a genomic deletion covering most of the Lycopersicon hirsutum introgressed segment, including the Cf-4 locus. Southern blot analysis revealed that this deletion includes the tagged gene and five homologous sequences. To test whether the tagged gene confers resistance to C. fulvum via Avr4 recognition, the Avr4 gene was expressed in planta. Surprisingly, expression of the Avr4 gene still triggered a specific necrotic response in the transposon-tagged plants, indicating that the tagged resistance gene is not, or is not the only gene, involved in Avr4 recognition. Mutants harbouring the genomic deletion did not show this Avr4-specific response. The deleted segment apparently contains, in addition to the tagged gene, one or more other genes, which play a role in the Avr4 responses. The tagged gene is present at the Cf-4 locus, but it does not necessarily recognize Avr4 and is therefore designated Cf-4A.

Amino Acid Sequence↗

Lung weight parallels disease severity in experimental coccidioidomycosis.

Evidence provided by histopathological study of lesions is a valuable adjunct for evaluating chemotherapeutic efficacy in experimental animal models, In addition, this should be correlated with a measure of disease severity in the same animal. The latter could be obtained by homogenization of infected organs and quantitative enumeration of viable cells of the etiological agent, but this would preclude histopathological studies in the same animal. Progression of disease in pulmonary infection is associated with replacement of air space by fluid, cells, and cellular debris. Therefore, an increase in lung weight should reflect severity of disease. Results with the murine model of coccidioidomycosis demonstrate that increasing lung weight parallels the increasing census of fungus cells in the lungs of both treated and nontreated infected mice. This was supported with evidence obtained from microscopic studies of lesions indicating that specific chemotherapy limited spread of the infection and inhibited multiplication of the fungus in the lung. Therefore, lung weight can be used as a measure of disease severity in the murine model of coccidioidomycosis.

Amphotericin B↗

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↗

Evaluation of the fungus Beauveria bassiana as a potential biological control agent against phlebotomine sand flies in Colombian coffee plantations.

In Colombia, the entomopathogenic fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes) is widely used to control the coffee berry borer Hypothenemus hampei (Coleoptera: Scolytidae) in coffee plantations. Recent studies suggested that this fungus is also pathogenic to several important vectors of disease, including Phlebotomus papatasi and Lutzomyia longipalpis (Diptera: Psychodidae). The present study evaluated the use of B. bassiana as a potential biological control agent against phlebotomine sand flies in Colombian coffee plantations. Histopathologic examination indicates that B. bassiana is unable to infect sand flies under natural conditions, although dead sand flies were shown to be readily infected. In addition, laboratory bioassays where flies were exposed to the fungus applied onto coffee plants (though not filter paper) showed lower mean survival times than the control.

Animals↗

The genetics and biology of Phytophthora infestans: modern approaches to a historical challenge.

The oomyceteous fungus Phytophthora infestans, which causes the late blight diseases of potato and tomato, has a history that is closely associated with that of mycology and plant pathology. Nevertheless, P. infestans and other oomycetes remain poorly understood relative to fungi in other groups. A resurgence in the worldwide impact of late blight has recently increased interest in the species. Fortunately, over the past decade improved tools for laboratory analysis have been developed which provide an opportunity to advance our understanding of this important pathogen. Since oomycetes do not have a close taxonomic affinity with well-characterized organisms such as ascomycetes and basidiomycetes, it is likely that studies of P. infestans will yield novel biological findings. This review provides an update on the status of research into the fundamental aspects of the biology, genetics, and pathology of P. infestans and describes prospects for future advances.

Genome, Fungal↗

Molecular mapping of genes conferring field resistance to South American Leaf Blight ( Microcyclus ulei) in rubber tree.

The South American Leaf Blight (SALB), caused by the fungus Microcyclus ulei, is the major rubber tree disease in all Central and South America. A population of 192 progeny individuals derived from a cross between a resistant clone and a susceptible cultivated clone was planted in a field trial in French Guiana in order to evaluate the resistance parameters under real infestation conditions. The resistance type (RT), the presence of stromata (ST) and the level of attack (AT) were observed 20-times on a 22-months period, and semi-quantitative evaluation of stromata was registered only once. The search for QTLs was performed using the Kruskal-Wallis test, Interval Mapping and the Composite Interval Mapping method. One major QTL located on linkage group g13 was detected on the RO 38 map, responsible for 36 to 89% of the phenotypic variance of resistance. This resistance QTL corresponds to one that had previously been detected under controlled conditions of infestation and we called it M13-1bn. Surprisingly, the effect of this QTL was larger under natural conditions of infestation than under controlled inoculation. Other minor QTLs (four on the RO38 map and one on the PB 260 map) were also detected. The type of resistance brought by M13-1bn, as well as its durability, are discussed. Applications for rubber tree breeding programs are considered.

Bacteria↗

Analysis of epidermis- and mesophyll-specific transcript accumulation in powdery mildew-inoculated wheat leaves.

Powdery mildew is an important disease of wheat caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. This pathogen invades exclusively epidermal cells after penetrating directly through the cell wall. Because powdery mildew colonizes exclusively epidermal cells, it is of importance not only to identify genes which are activated, but also to monitor tissue specificity of gene activation. Acquired resistance of wheat to powdery mildew can be induced by a previous inoculation with the non-host pathogen B. graminis f. sp. hordei, the causal agent of barley powdery mildew. The establishment of the resistant state is accompanied by the activation of genes. Here we report the tissue-specific cDNA-AFLP analysis and cloning of transcripts accumulating 6 and 24 h after the resistance-inducing inoculation with B. graminis f. sp. hordei. A total of 25,000 fragments estimated to represent about 17,000 transcripts were displayed. Out of these, 141 transcripts, were found to accumulate after Bgh inoculation using microarray hybridization analysis. Forty-four accumulated predominantly in the epidermis whereas 76 transcripts accumulated mostly in mesophyll tissue.

Ascomycota↗

Disseminated Scedosporium apiospermum infection in a cystic fibrosis patient after double-lung transplantation.

Scedosporium apiospermum is a saprophytic ubiquitous filamentous fungus. It can cause a wide spectrum of diseases, from localized to invasive infections. S apiospermum has been described as one of the major fungal agents of chronic colonization of airways in cystic fibrosis (CF) patients. Invasive infections due to S apiospermum are only rarely reported in CF after lung transplantation. A 26-year-old woman with CF and chronic bronchial colonization by S apiospermum developed bilateral chorioretinitis and subcutaneous nodules 4 weeks after double-lung transplantation (LTx). Isolates of S apiospermum from sputum samples before and after LTx and from vitreal fluid were typed by random amplification of polymorphic DNA (RAPD). The patient was treated with voriconazole (VRC). The patient improved with VRC given orally for 6 months. Two days after VRC discontinuation, she developed sub-acute meningitis (isolation of S apiospermum from the cerebrospinal fluid). She was again given VRC, but died 23 days later from uncontrolled fungal infection. Molecular typing of clinical isolates of S apiospermum performed by RAPD demonstrated that all isolates belonged to the same genotype. S apiospermum is a frequent, but late colonizing fungal agent in CF patients. In the case of LTx, these patients can develop invasive infection due to the colonizing strain, as confirmed by molecular typing.

Adult↗

An immunohistochemical study of canine nasal aspergillosis.

In this study, histochemistry and immunohistochemistry were used to characterize the phenotype and distribution of leucocytes in the distal nasal mucosa of 15 dogs with nasal aspergillosis. The most consistent histopathological finding was a severe, predominantly lymphoplasmacytic, inflammatory infiltration of the lamina propria. Fungal hyphae were not observed to invade the mucosa but were found at the mucosal surface and within material collected from the nasal cavity. The main immunohistochemical findings were (1) a predominance of IgG(+) plasma cells over IgA(+) and IgM(+) plasma cells, (2) significant numbers of macrophages and dendritic cells expressing MHC class II molecules, (3) macrophages and neutrophils expressing L1 antigen and (4) a mixture of CD4(+) and CD8(+) T cells. These findings are consistent with a dominant Th1-regulated cell-mediated immune response. The nature of the inflammatory infiltrate and the lack of invasiveness of the mucosa by the fungus, together with the clinical course of the disease and the apparent immunocompetence of the affected dogs, suggest that canine nasal aspergillosis resembles the chronic erosive non-invasive fungal sinusitis described in human patients.

Animals↗

Novel hosts of the Eucalyptus canker pathogen Chrysoporthe cubensis and a new Chrysoporthe species from Colombia.

The pathogen Chrysoporthe cubensis (formerly Cryphonectria cubensis) is best known for the important canker disease that it causes on Eucalyptus species. This fungus is also a pathogen of Syzygium aromaticum (clove), which is native to Indonesia, and like Eucalyptus, is a member of Myrtaceae. Furthermore, C. cubensis has been found on Miconia spp. native to South America and residing in Melastomataceae. Recent surveys have yielded C. cubensis isolates from new hosts, characterized in this study based on DNA sequences for the ITS and beta-tubulin gene regions. These hosts include native Clidemia sericea and Rhynchanthera mexicana (Melastomataceae) in Mexico, and non-native Lagerstroemia indica (Pride of India, Lythraceae) in Cuba. Isolates from these hosts and areas group in the sub-clade of C. cubensis accommodating the South American collections of the fungus. This sub-clade also includes isolates recently collected from Eucalyptus in Cuba, which are used to epitypify C. cubensis. New host records from Southeast Asia include exotic Tibouchina urvilleana from Singapore and Thailand and native Melastoma malabathricum (Melastomataceae) in Sumatra, Indonesia. Consistent with their areas of occurrence isolates from the latter collections group in the Asian sub-clade of C. cubensis. DNA sequence comparisons of isolates from Tibouchina lepidota in Colombia revealed that they represent a new sub-clade within the greater Chrysoporthe clade. Isolates in this clade are described as Chrysoporthe inopina sp. nov., based on distinctive morphological differences.

Ascomycota↗

The mycotoxin fumonisin B1 inhibits integrin-mediated cell-matrix adhesion.

Fumonisin B1 (FB1), a mycotoxin produced by the corn fungus Fusarium moniliforme, causes a variety of animal diseases and is a suspected human carcinogen. The FB1 molecule bears remarkable structural resemblance to the long-chain sphingoid base backbones of sphingolipids. The toxicity and carcinogenicity of FB1 has been ascribed to its ability to inhibit ceramide synthase, a key enzyme in the metabolism of complex sphingolipids. In this study we have investigated whether the exposure of B16-BL6 mouse melanoma cells to FB1 affects cell growth and integrin-mediated cell matrix adhesion. Cell treatment with the highest tested dose (75 microM) of FB1 for 72 h induced an about 20% inhibition of cell growth. FB1 strongly affected B16-BL6 cell adhesion to immobilized fibronectin, by causing a dose-dependent inhibition of cell attachment to this substrate. FB1 also inhibited in a dose-dependent manner the adhesion of B16-BL6 cells to the immobilized anti-fibronectin receptor antibody, whereas it affected only to a low extent cell attachment to concanavalin A. Our results demonstrate that FB1 treatment alters integrin adhesive activity, thus affecting all cellular integrin-dependent functions.

Animals↗

Chromoblastomycosis--a clinical and mycological study of 71 cases from Sri Lanka.

Chromoblastomycosis, a well-documented chronic fungal infection, represents a specific clinical entity with typical warty cutaneous nodules and a worldwide distribution. Although more prevalent in tropical and subtropical regions, only a few reports are available from Sri Lanka or from Asia. Five etiologic agents of chromoblastomycosis have been recognized worldwide. Of these the majority of infections have been caused by Fonsecaea pedrosoi. During the period from 1952 to 1962, only twelve culturally proven cases of this disease had been recorded from Sri Lanka. The fungus responsible was F. pedrosoi. The present report presents a study of the clinical and mycological features of 71 Sri Lankan patients with chromoblastomycosis for the 16-year period from 1978 to 1993. It documents three etiological agents. Culture identification was made in 69 cases. The three fungal species were Fonsecaea pedrosoi (64), Phialophora verrucosa (3) and a fungus compatible morphologically with F. compacta (2). The isolation of a fungus morphologically compatible with F. compacta is of significance since only 12 cases have been documented in the world's literature so far.

Adolescent↗