Impact of natural epizootics of the fungal pathogen Neozygites floridana (Zygomycetes: Entomophthorales) on population dynamics of Tetranychus evansi (Acari: Tetranychidae) in tomato and nightshade

The tomato red spider mite, Tetranychus evansi (Acari: Tetranychidae) was recently introduced in Africa and Europe, where there is an increasing interest in using natural enemies to control this pest on solanaceous crops. Two promising candidates for the control of T. evansi were identified in South America, the fungal pathogen, Neozygites floridana and the predatory mite Phytoseiulus longipes. In this study, population dynamics of T. evansi and its natural enemies together with the influence of environmental conditions on these organisms were evaluated during four crop cycles in the field and in a protected environment on nightshade and tomato plants with and without application of chemical pesticides. N. floridana was the only natural enemy found associated with T. evansi in the four crop cycles under protected environment but only in the last crop cycle in the field. In the treatments where the fungus appeared, reduction of mite populations was drastic. N. floridana appeared in tomato plants even when the population density of T. evansi was relatively low (less than 10 mites/3.14 cm(2) of leaf area) and even at this low population density, the fungus maintained infection rates greater than 50%. The application of pesticides directly affected the fungus by delaying epizootic initiation and contributing to lower infection rates than unsprayed treatments. Rainfalls did not have an apparent impact on mite populations. These results indicate that the pathogenic fungus, N. floridana can play a significant role in the population dynamics of T. evansi, especially under protected environment, and has the potential to control this pest in classical biological control programs. (C) 2009 Elsevier Inc. All rights reserved.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

The effect of host plants on Tetranychus evansi, Tetranychus urticae (Acari: Tetranychidae) and on their fungal pathogen Neozygites floridana (Entomophthorales: Neozygitaceae)

In a series of tritrophic-level interaction experiments, the effect of selected host plants of the spider mites, Tetranychus evansi and Tetranychus urticae, on Neozygites floridana was studied by evaluating the attachment of capilliconidia, presence of hyphal bodies in the infected mites, mortality from fungal infection, mummification and sporulation from fungus-killed mite cadavers. Host plants tested for T. evansi were tomato, cherry tomato, eggplant, nightshade, and pepper while host plants tested for T. urticae were strawberry, jack bean, cotton and Gerbera. Oviposition rate of the mites on each plant was determined to infer host plant suitability while host-switching determined antibiosis effect on fungal activity. T. evansi had a high oviposition on eggplant, tomato and nightshade but not on cherry tomato and pepper. T. urticae on jack bean resulted in a higher oviposition than on strawberry, cotton and Gerbera. Attachment of capilliconidia to the T. evansi body, presence of hyphal bodies in infected T. evansi and mortality from fungal infection were significantly higher on pepper, nightshade and tomato. The highest level of T. evansi mummification was observed on tomato. T. evansi cadavers from tomato and eggplant produced more primary conidia than those from cherry tomato, nightshade and pepper. Switching N. floridana infected T. evansi from one of five Solanaceous host plants to tomato had no prominent effect on N. floridana performance. For T. urticae, strawberry and jack bean provided the best N. floridana performance when considering all measured parameters. Strawberry also had the highest primary conidia production. This study shows that performance of N. floridana can vary with host plants and may be an important factor for the development of N. floridana epizootics. (C) 2011 Elsevier Inc. All rights reserved.

Screening pentachlorophenol degradation ability by environmental fungal strains belonging to the phyla Ascomycota and Zygomycota

Pentachlorophenol (PCP) bioremediation by the fungal strains amongst the cork- colonising community has not yet been analysed. In this paper, the co- and direct metabolism of PCP by each of the 17 fungal species selected from this community were studied. Using hierarchical data analysis, the isolates were ranked by their PCP bioremediation potential. Fifteen isolates were able to degrade PCP under co-metabolic conditions, and surprisingly Chrysonilia sitophila, Trichoderma longibrachiatum, Mucor plumbeus, Penicillium janczewskii and P. glandicola were able to directly metabolise PCP, leading to its complete depletion from media. PCP degradation intermediates are preliminarily discussed. Data emphasise the signiWcance of these fungi to have an interesting potential to be used in PCP bioremediation processes.

Rhinocerebral zygomycosis in a diabetic patient

Rhinocerebral zygomycosis is the most frequent form of fungal infection caused by members of the Zygomycetes class. A fatal case of rhinocerebral zygomycosis caused by Rhizopus (oryzae) arrhizus with histopathological and mycological diagnosis is reported in a diabetic patient.

Disseminated Rhizopus microsporus infection cured by salvage allogeneic hematopoietic stem cell transplantation, antifungal combination therapy, and surgical resection.

O. Lebeau, C. Van Delden, J. Garbino, J. Robert, F. Lamoth, J. Passweg, Y. Chalandon. Disseminated Rhizopus microsporus infection cured by salvage allogeneic hematopoietic stem cell transplantation, antifungal combination therapy, and surgical resection. Transpl Infect Dis 2010. All rights reserved Abstract: Invasive Zygomycetes infection complicating prolonged neutropenia is associated with high mortality in the absence of immune recovery. We report a patient who developed disseminated zygomycosis due to Rhizopus microsporus during induction chemotherapy for acute myeloid leukemia. Rescue allogeneic hematopoietic stem cell transplantation (allo-HSCT) was performed as her only chance of cure of this infection and to treat refractory leukemia. Posaconazole combined with liposomal amphotericin B contained the zygomycosis during prolonged neutropenia due to allo-HSCT followed by intense immunosuppression for grade IV acute graft-versus-host disease. Surgical removal of all infected sites after immune recovery, with prolonged posaconazole treatment, ultimately cured the infection. New combination antifungal therapies might sufficiently control disseminated zygomycosis to allow allo-HSCT to be performed, assuring life-saving immune recovery. Surgery appears to be necessary for definite cure of these infections.

Invasive mould infections: a multi-disciplinary update.

Systemic fungal infections remain a significant cause of mortality in neutropenic and immunocompromised patients, despite advances in their diagnosis and treatment. The incidence of such infections is rising due to the use of intensive chemotherapy regimens in patients with solid tumours or haematological cancers, the increasing numbers of allogeneic haematopoietic stem cell and solid organ transplants, and the use of potent immunosuppressive therapy in patients with autoimmune disorders. In addition, the epidemiology of systemic fungal infections is changing, with atypical species such as Aspergillus terreus and zygomycetes becoming more common. Treatment has traditionally focused on empirical therapy, but targeted pre-emptive therapy in high-risk patients and prophylactic antifungal treatment are increasingly being adopted. New treatments, including lipid formulations of amphotericin B, second-generation broad-spectrum azoles, and echinocandins, offer effective antifungal activity with improved tolerability compared with older agents; the potential impact of these treatments is reflected in their inclusion in current treatment and prophylaxis guidelines. New treatment strategies, such as aerosolized lipid formulations of amphotericin B, may also reduce the burden of mortality associated with systemic fungal infections. The challenge is to identify ways of coupling potentially effective treatments with early and reliable identification of patients at highest risk of infection.

Failure of voriconazole to cure disseminated zygomycosis in an immunocompromised child.

Voriconazole is increasingly used as a first-line agent for empirical antifungal therapy of prolonged febrile neutropenia in paediatric cancer patients. We describe the case of a 9-year-old patient with stage IV Burkitt lymphoma, who developed pulmonary and splenic zygomycosis while receiving voriconazole for persistent febrile neutropenia. The causative agent, Absidia corymbifera, was identified by broad-range fungal PCR in a lung biopsy sample. The patient was successfully treated with a combination of partial resection of the left upper lobe and antifungal therapy with high-dose liposomal amphotericin B followed by oral itraconazole as demonstrated by resolving pulmonary infiltrates on serial high resolution CT scans. CONCLUSION: This case emphasises that the lack of in vitro activity of voriconazole against zygomycetes is clinically relevant. Failure of voriconazole in suspected fungal infection should be investigated for the possibility of zygomycosis. Broad-range polymerase chain reaction may be able to identify the causative organism when cultures remain sterile.

El 1er Mini Foray de la British Mycological Society a Catalunya (1985)

Es presenten els resultats del ler «Mini Foray» de la British Mycological Society a Catalunya, celebrat a Viladrau (Girona) a la tardor de 1985, amb visites a diverses localitats del Vallès Oriental, Baix Llobregat, Ripollès, La Selva i Osona. Es van recol lectar un total de 407 espècies de fongs, repartides en els diferents grups: Acrasiomycetes (1); Ceratiomyxomycetes (2); Myxomycetes (44); Zygomycetes (1); Ascomycetes (36); Basidiomycetes (319); Deuteromycetes (4).

Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges

Conidiobolomycosis is a granulomatous disease caused by the fungus Conidiobolus spp. in humans and animals. Traditional technique for diagnosis of the disease is isolation of the agent associated with the presence of typical clinical signs and pathological conditions. The aim of this study was to describe the development of a specific polymerase chain reaction (PCR) test for Conidiobolus lamprauges to detect the fungus in clinical samples. Samples from suspected animals were collected and submitted to isolation, histopathological analysis and amplification by PCR. DNA from tissues was subjected to PCR with fungi universal primers 18S rDNA gene, and specific primers were designed based on the same gene in C. lamprauges that generated products of about 540 bp and 222 bp respectively. The culture was positive in 26.6% of clinical samples. The PCR technique for C. lamprauges showed amplification of DNA from fresh tissues (80%) and paraffin sections (44.4%). In conclusion, the PCR technique described here demonstrated a high sensitivity and specificity for detection of fungal DNA in tissue samples, providing a tool for the rapid diagnosis of C. lamprauges.

Meningoencephalitis caused by a zygomycete fungus (Basidiobolus) associated with septic shock in an immunocompetent patient: 1-year follow-up after treatment

Zygomycosis is an infection caused by opportunistic fungi of the Zygomycetes class, specifically those from the Mucorales and Entomophthorales orders. It is an uncommon disease, mainly restricted to immunocompromised patients. We report a case of a 73-year-old male patient with a history of fever (39°C) lasting for 1 day, accompanied by shivering, trembling, and intense asthenia. The patient was admitted to the intensive care unit with complex partial seizures, and submitted to orotracheal intubation and mechanical ventilation under sedation with midazolam. The electroencephalogram showed evidence of non-convulsive status epilepticus. There is no fast specific laboratory test that permits confirmation of invasive fungal disease. Unless the physician suspects this condition, the disease may progress rapidly while the patient is treated with broad-spectrum antibiotics. Differential diagnosis between fungal and bacterial infection is often difficult. The clinical presentation is sometimes atypical, and etiological investigation is not always successful. In the present case, the histopathological examination of the biopsy obtained from the right temporal lobe indicated the presence of irregular, round, thick-walled fungi forming papillae and elongated structures of irregular diameter, with no septa, indicative of zygomycete (Basidiobolus). Treatment with liposomal amphotericin B and fluconazole was initiated after diagnosis of meningoencephalitis by zygomycete, with a successful outcome.

Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Immuno-gluorescence study of five zygomycetous fungi with two chitin-binding probes

A polyclonal antiserum was prepared against a purified microsomal chitinase isolated from the fungus Choanephora cucurbitarum. Indirect immunofluorescence was used to localize chitinase at various developmental stages of five zygomycetous fungi and during abiotrophic mycoparasite interaction with a susceptible and resistant host. This was compared to localization of oligomers of N-acetylglucosamine with the lectin wheat germ agglutinin (WGA). Dotimmunoblot and Western blot techniques revealed that the anti-serum reacted strongly with the antigen from which it was derived. Cross reactivity of the antiserum was found with WGA and another chitin binding lectin, Phyto/acca americana agglutinin (PAA). Immuno-fluorescence results showed the direct involvement of chitinase in spore swelling, germination, sporangium development and response during mechanical injury. There appeared to be no involvement of chitinase during apical hyphal growth or new branch initiation in any of the fungi tested despite mild proteolysis and permeabilization of the cell surface prior to labelling. Binding with WGA revealed similar patterns of fluorescence to that of chitinase localization but differed by showing fluorescence and therefore chitin localization at the apex and new branch initiation when tested at different developmental stages. There was no difference between chitinase localization and binding with WGA in a susceptible host and resistant host challenged with the mycoparasite, Piptocephalis virginiana. Differences in binding ability of antichitinase and lectin WGA suggests that the latter is not a suitable indicator for indirect localization of the lytic enzyme, chitinase.

A phylogenomics approach to resolving fungal evolution, and phylogenetic method development

Bien que les champignons soient régulièrement utilisés comme modèle d'étude des systèmes eucaryotes, leurs relations phylogénétiques soulèvent encore des questions controversées. Parmi celles-ci, la classification des zygomycètes reste inconsistante. Ils sont potentiellement paraphylétiques, i.e. regroupent de lignées fongiques non directement affiliées. La position phylogénétique du genre Schizosaccharomyces est aussi controversée: appartient-il aux Taphrinomycotina (précédemment connus comme archiascomycetes) comme prédit par l'analyse de gènes nucléaires, ou est-il plutôt relié aux Saccharomycotina (levures bourgeonnantes) tel que le suggère la phylogénie mitochondriale? Une autre question concerne la position phylogénétique des nucléariides, un groupe d'eucaryotes amiboïdes que l'on suppose étroitement relié aux champignons. Des analyses multi-gènes réalisées antérieurement n'ont pu conclure, étant donné le choix d'un nombre réduit de taxons et l'utilisation de six gènes nucléaires seulement. Nous avons abordé ces questions par le biais d'inférences phylogénétiques et tests statistiques appliqués à des assemblages de données phylogénomiques nucléaires et mitochondriales. D'après nos résultats, les zygomycètes sont paraphylétiques (Chapitre 2) bien que le signal phylogénétique issu du jeu de données mitochondriales disponibles est insuffisant pour résoudre l'ordre de cet embranchement avec une confiance statistique significative. Dans le Chapitre 3, nous montrons à l'aide d'un jeu de données nucléaires important (plus de cent protéines) et avec supports statistiques concluants, que le genre Schizosaccharomyces appartient aux Taphrinomycotina. De plus, nous démontrons que le regroupement conflictuel des Schizosaccharomyces avec les Saccharomycotina, venant des données mitochondriales, est le résultat d'un type d'erreur phylogénétique connu: l'attraction des longues branches (ALB), un artéfact menant au regroupement d'espèces dont le taux d'évolution rapide n'est pas représentatif de leur véritable position dans l'arbre phylogénétique. Dans le Chapitre 4, en utilisant encore un important jeu de données nucléaires, nous démontrons avec support statistique significatif que les nucleariides constituent le groupe lié de plus près aux champignons. Nous confirmons aussi la paraphylie des zygomycètes traditionnels tel que suggéré précédemment, avec support statistique significatif, bien que ne pouvant placer tous les membres du groupe avec confiance. Nos résultats remettent en cause des aspects d'une récente reclassification taxonomique des zygomycètes et de leurs voisins, les chytridiomycètes. Contrer ou minimiser les artéfacts phylogénétiques telle l'attraction des longues branches (ALB) constitue une question récurrente majeure. Dans ce sens, nous avons développé une nouvelle méthode (Chapitre 5) qui identifie et élimine dans une séquence les sites présentant une grande variation du taux d'évolution (sites fortement hétérotaches - sites HH); ces sites sont connus comme contribuant significativement au phénomène d'ALB. Notre méthode est basée sur un test de rapport de vraisemblance (likelihood ratio test, LRT). Deux jeux de données publiés précédemment sont utilisés pour démontrer que le retrait graduel des sites HH chez les espèces à évolution accélérée (sensibles à l'ALB) augmente significativement le support pour la topologie « vraie » attendue, et ce, de façon plus efficace comparée à d'autres méthodes publiées de retrait de sites de séquences. Néanmoins, et de façon générale, la manipulation de données préalable à l'analyse est loin d’être idéale. Les développements futurs devront viser l'intégration de l'identification et la pondération des sites HH au processus d'inférence phylogénétique lui-même.