Mycoviruses mediate mycotoxin regulation in Aspergillus ochraceus

To date, no demonstration of a direct correlation between the presence of mycoviruses and the quantitative or qualitative modulation of mycotoxins has been shown. In our study, we transfected a virus-free ochratoxin A (OTA)-producing isolate of Aspergillus ochraceus with purified mycoviruses from a different A. ochraceus isolate and from Penicillium aurantiogriseum. Among the mycoviruses tested, only Aspergillus ochraceus virus (AoV), a partitivirus widespread in A. ochraceus, caused a specific interaction that led to an overproduction of OTA, which is regulated by the European Commission and is the second most important contaminant of food and feed commodities. Gene expression analysis failed to reveal a specific viral upregulation of the mRNA of genes considered to play a role in the OTA biosynthetic pathway. Furthermore, AoOTApks1, a polyketide synthase gene considered essential for OTA production, is surprisingly absent in the genome of our OTA-producing isolate. The possible biological and evolutionary implications of the mycoviral regulation of mycotoxin production are discussed.     

Double-Stranded RNA Mycovirus Infection of Aspergillus fumigatus Is Not Dependent on the Genetic Make-Up of the Host

Aspergillus fumigatus is a fungus that causes opportunistic infections in immunocompromised patients, with high morbidity and mortality. In its turn, A. fumigatus can become infected with mycoviruses. Most mycoviruses have a dsRNA genome and can cause fungal hypovirulence. For that reason, mycoviruses could theoretically be used as therapeutic tools to combat fungal infections. We determined if a certain genetic make-up of A. fumigatus was associated with the presence of mycoviruses in 86 clinical A. fumigatus isolates. Mycovirus screening was performed by isolating dsRNA from mycelial cultures using a Trizol/Chloroform method. The genetic relatedness of dsRNA infected A. fumigatus was determined by cell surface protein (CSP) typing and determination of the mating type. Sixteen (18.6%) of the 86 clinical A. fumigatus isolates contained dsRNA. The A. fumigatus collection could be divided into 11 different CSP types. DsRNA infected A. fumigatus isolates had similar CSP types as non-infected isolates. In both cases, the CSP types t01, t02, t03 and t04 were the most prevalent and the distribution comparable to the CSP types observed in other Dutch collections. Mating types MAT1-1 and MAT1-2 were evenly distributed among all A. fumigatus strains, regardless of CSP type. No difference was observed in mycovirus infections between MAT1-1 and MAT1-2 isolates. DsRNA mycovirus infections in A. fumigatus are not related to either CSP or mating type and therefore represent an interesting future therapeutic tool to combat fungal infections.     

Presence of Transposons and Mycoviruses in Botrytis cinerea Isolates Collected from a German Grapevine Growing Region

Transposons and infection of fungal strains with mycoviruses can have significant effects on distinctive phenotypic traits of phytopathogenic fungi such as mycelial growth and sporulation, pathogenicity or fungicide resistance. Two transposable elements (TE), Boty and Flipper, are known to be associated with the ubiquitous fungus Botrytis cinerea. In addition, the presence of two types of ssRNAsRNA viruses, BVX and BVF, has been reported in B. cinerea. In this study, we assessed the genetic diversity of B. cinerea isolates, all sampled within a small‐sized German viticultural area (‘Rheingau’) by examining and classifying them according to the presence of TEs and mycoviruses. A subset of the isolates was further analysed with microsatellite markers to determine the origin of particular isolates with or without one or both mycoviruses. Virtually all isolates (98%) sampled in two different years (2008 and 2010) were screened positive for the presence of a transposon. Presence of one or both B. cinerea mycoviruses was confirmed for 37% of the analysed isolates sampled in 2010, representing the first record of B. cinerea mycoviruses in German isolates. Assignment on individual B. cinerea isolates to different genetic groups was independent of the presence or absence of a mycovirus or a transposable element, respectively. Furthermore, we found no correlation between the presence of either a mycovirus or a transposable element and different viticultural management practices, soil properties or levels of nitrogen fertilization applied to the respective vineyards. However, mycelial growth of B. cinerea strains containing mycovirus BVF was significantly reduced at lower temperatures.     

Transcriptomic analysis of entomopathogenic fungus Beauveria bassiana infected by a hypervirulent polymycovirus BbPmV-4

Polymycoviridae is a recently established family of mycoviruses. Beauveria bassiana polymycovirus 4 (BbPmV-4) was previously reported. However, the effect of the virus on host fungus B. bassiana was not clarified. Here, a comparison between virus-free and virus-infected isogenic lines of B. bassiana revealed that BbPmV-4 infection of B. bassiana changes morphology and could lead to decreases in conidiation and increases in virulence against Ostrinia furnacalis larvae. The differential expression of genes between virus-free and virus-infected strains was compared by RNA-Seq and was consistent with the phenotype of B. bassiana. The enhanced pathogenicity may be related to the significant up-regulation of genes encoding mitogen activated protein kinase, cytochrome P450, and polyketide synthase. The results enable studies of the mechanism of interaction between BbPmV-4 and B. bassiana.     

Characterization of Mycoviruses and Analyses of Chitinase Secretion in the Biocontrol Fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae is the best-characterized entomopathogen and is used to control insect pests in sugar cane plantations in Brazil on a commercial scale. We have previously reported the infection of some M. anisopliae strains by dsRNA mycoviruses. Here we describe the purification and characterization of the viruses (MaV-A1, MaV-M5, MaV-RJ) in terms of dsRNA content, capsid proteins, electron microscopy, Western blot, and hybridization patterns. One spontaneous mutant lost some of the high molecular weight dsRNA components and showed significant alterations in colony morphology and spore production, suggesting that viral genes interfere with fungal phenotype. A comparison between dsRNA mycovirus-free and infected M. anisopliae isolates showed that virus-free isolates have increased endochitinase secretion. By comparing the following parameters: the buoyant density in CsCl of the presumed virions; the number and estimated molecular weight of the dsRNA components and the molecular mass of the capsid proteins to other mycoviruses previously described, we suggest the inclusion of MaV-A1 and MaV-M5 in the family Totiviridae and MaV-RJ in the family Partitiviridae. Received: 25 September 2001 / Accepted: 1 February 2002     

Virus Community Dynamics in the Conifer Pathogenic Fungus Heterobasidion parviporum Following an Artificial Introduction of a Partitivirus

Viruses infecting the conifer pathogenic fungus Heterobasidion annosum sensu lato are intracellular and spread via anastomosis contacts. In the laboratory, these viruses transmit readily even between somatically incompatible isolates, but their dispersal capacity in natural conditions has not been previously studied. We introduced a mycovirus to a heavily diseased forest site by inoculating Norway spruce stumps with heartrot decay using a mycelial suspension of Heterobasidion parviporum strain RT3.49C hosting the partitivirus strain HetRV4-pa1. The Heterobasidion population at the sample plot was screened for mycoviruses prior to and after the inoculation. Based on sequence analysis, the resident H. parviporum strains harbored six different strains of the virus species Heterobasidion RNA virus 6 (HetRV6) and one strain of HetRV4 prior to the inoculation. After three growth seasons, the inoculated H. parviporum host strain was not detected, but the introduced virus had infected two resident H. parviporum genets. The presence of a preexisting HetRV6 infection did not hinder spread of the introduced partitivirus but resulted in coinfections instead. The resident HetRV6 virus population seemed to be highly stable during the incubation period, while the single indigenous HetRV4 infection was not detected after the inoculation. In laboratory infection experiments, the introduced virus could be transmitted successfully into all of the resident H. parviporum genets. This study shows for the first time transmission of a Heterobasidion virus between somatically incompatible hosts in natural conditions.     

Phlebiopsis gigantea and associated viruses survive passing through the digestive tract of Hylobius abietis

Phlebiopsis gigantea (Fr.) Jül. is one of the most common fungal species in coniferous forests and commonly used as a biological control agent to prevent aerial infections of conifers by Heterobasidion fungi. We used feeding experiments to examine whether Hylobius abietis L. (Coleoptera: Curculionidae) could serve as a vector for P. gigantea and associated viruses by disseminating the fungus in its faecal pellets. Two different P. gigantea strains were used in the experiments: the virus-free Rotsop biocontrol strain and P. gigantea 93073 infected with the virus strain PgLV-1. The Rotstop strain showed 100% viability during insect feeding, while the viability of the virus-infected strain 93073 was only 35%. Virus persistence was 100% during the passage of the host fungus through the alimentary tract. Based on growth experiments using virus-infected and virus-free strains obtained by hyphal tip or oidial isolation, the presence of PgLV-1 did not significantly affect the growth rate of the host fungus.     

Airborne Aspergillus fumigatus conidia: a risk factor for aspergillosis

Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis, a usually fatal infection. The disease has risen in prominence in recent years due to the increasing numbers of severely immunocompromised patients becoming infected. The fungus is ubiquitous in the environment, producing large numbers of conidia that are dispersed in the air. Humans inhale numerous conidia everyday, but infections are not seen in healthy individuals. As inhalation of conidia is the main route of infection, considerable efforts are required to prevent infection in susceptible patients. This review summarises the current knowledge on airborne concentrations of A. fumigatus conidia, their background levels in outdoor air and seasonal distribution patterns. New and established methods of air sampling for airborne A. fumigatus conidia are discussed. Common environmental sources of the fungus are reviewed, including its presence in compost heaps. Finally, the lack of stringent guidelines on the monitoring and control of airborne A. fumigatus concentrations in hospitals is discussed.     

Effects of Conidia of Various Aspergillus Species on Apoptosis of Human Pneumocytes and Bronchial Epithelial Cells

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species. F. Féménia and D. Huet made an equal contribution to this work.     

烟曲霉黑色素的研究进展

烟曲霉(Aspergillus fumigatus)是一种分布于世界各地的腐生真菌,属于人类临床常见的三大机会性致病真菌之一,是侵袭性曲霉菌病的主要病原菌。烟曲霉可以产生DHN-黑色素(dihydroxynaphthalene melanin)和脓黑素(pyomelanin)这2种类型黑色素。本综述介绍烟曲霉黑色素产生的遗传代谢途径、功能以及与宿主免疫系统相互作用的最新认识,有助于更好地理解烟曲霉的病理生理特征,为烟曲霉感染快速诊断技术和新型抗真菌药物的研发提供理论依据。     

Silencing of mitochondrial alternative oxidase gene of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.     

Effects of fungicides on Aspergillus fumigatus

Aspergillus fumigatus was the most frequently isolated thermophilous fungus from green leaf surfaces. The application of fungicides significantly reduced the frequency of its occurrence there. A. fumigatus was relatively tolerant to fungicides. On Captan-, Thiram-, and Verdasan-treated leaves, A. fumigatus constituted 66%–80% of the total number of isolates obtained at 45°C from each treatment while Dicloran did not depress the percentages. At 45°C, A. fumigatus was found to be strongly cellulolytic with a slow rate of radial extension on YpSs agar and rapid rate of mycelial growth in Czapek Dox liquid medium. Increasing concentrations of all four fungicides reduced or prevented growth, sporulation, starch depletion and cellulose clearing of A. fumigatus. The fungus could tolerate higher concentrations of HgCl₂ than of Verdasan. 0.5 g/ml of the four fungicides altered the rates of mycelial growth but not the maximum amount of mycelial dry weight attained.     

HdaA, a class 2 histone deacetylase of Aspergillus fumigatus, affects germination and secondary metabolite production

Histone deacetylases (HDACs) play an important role in regulation of gene expression through histone modifications. Here we show that the Aspergillus fumigatus HDAC HdaA is involved in regulation of secondary metabolite production and is required for normal germination and vegetative growth. Deletion of the hdaA gene increased the production of several secondary metabolites but decreased production of gliotoxin whereas over-expression hdaA increased production of gliotoxin. RT-PCR analysis of 14 nonribosomal peptide synthases indicated HdaA regulation of up to nine of them. A mammalian cell toxicity assay indicated increased activity in the over-expression strain. Neither mutant affected virulence of the fungus as measured by macrophage engulfment of conidia or virulence in a neutropenic mouse model.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

Azole Resistance in Aspergillus fumigatus—Current Epidemiology and Future Perspectives

Azole resistance in Aspergillus fumigatus isolates is increasingly reported in different nosologic contexts with variable prevalence in different countries. Mutations in the target of triazoles are widely described in azole-resistant clinical isolates. The recovery of mutated/resistant isolates is described either in patients undergoing long-term azole treatment or after inhalation of environmentally acquired resistant isolates. Acquisition in patients during azole therapy highlights the capacity of this fungus to adapt to its environment, but it has a low impact in terms of public health, as interhuman transmission of A. fumigatus is uncommon. Environmentally acquired resistant isolates may propagate and affect populations at risk. The use of triazoles as first-line therapy or prophylaxis could lead to selection of resistant isolates in patients, because most isolates harbor azole cross-resistance. Although mold-active triazoles have provided major progress in the prophylaxis and treatment of Aspergillus infection, the increase of azole resistance could question their use in humans.     

Effect of amphotericin B on the metabolism of Aspergillus fumigatus

Action of amphotericin B on the growth and metabolism of Aspergillus fumigatus has been investigated. The fungus proved to be very sensitive to amphotericin B, showing complete inhibition of growth at 0.5 units/ml. Amphotericin B suppressed the exogenous and endogenous respiration and glycolysis of A. fumigatus as well as the assimilation of various glycolysis and TCA cycle intermediates. Addition of cations and cholesterol failed to reverse the action of amphotericin B. The treated mycelium released a variety of cellular constituents and it is inferred that the antibiotic effects the permeability of A. fumigatus cells. In experiments with ³²P labelled mycelium phosphorus compounds leached out in concentrations which were dependent on the antibiotic dose, period of contact, incubation temperature and metabolic state of the fungus.     

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. on their root colonization and growth promotion of cucumber (Cucumis sativus L.)

Interaction between arbuscular mycorrhizal fungus Glomus mosseae and plant growth promoting fungus Phoma sp. was studied for its effect on their root colonization and plant growth of cucumber. Two isolates of Phoma sp. (GS8-2 and GS8-3) were tested with G. mosseae. The percent root length colonized by G. mosseae was not adversely affected by the presence of Phoma isolates. In contrast, the root colonization of both isolates GS8-2 and GS8-3 in 4-week-old plants was significantly reduced (80.7% and 84.3%, respectively) by added G. mosseae. Inoculating plants with each Phoma isolate significantly increased the shoot dry weight. However, dual inoculation of each Phoma isolate with G. mosseae had no significant effect on growth enhancement.     

Isolation and toxigenicity of Aspergillus fumigatus from moldy silage

Thirty-nine silage samples were collected from various siloson Terceira Island in the Azores. Samples were examined for the presence of total fungi, and isolates of Aspergillus fumigatus were analyzed for their ability to produce fumitremorgens B and C, fumigaclavines B and C, and gliotoxin. Thirty-four silage samples (87%) were contaminated with fungi, and A. fumigatus was isolated from 27 samples (69%). Samples that were taken from the surface of silos had significantly higher populations of both total fungi and A. fumigatus than did samples taken from the middle of silos. Analysis of 27 A. fumigatus isolates (one representing each positive sample) showed that 59.3% produced fumitremorgen B; 33.3% produced fumitremorgen C; 29.6% produced fumigaclavine B; 7.4% produced fumigaclavine C; and 11.1% produced gliotoxin. Fifty-two percent of the isolates produced multiple toxins, and 25.9% did not produce any of these toxins. Gliotoxin and fumigaclavine C were always produced in combination with other toxins. Because of the demonstrated potential of these A. fumigatus isolates to producemycotoxins, it is important to properly construct and manage silos to prevent their contamination with A. fumigatus.This revised version was published online in October 2005 with corrections to the Cover Date.     

Degradation and corrosive activities of fungi in a diesel–mild steel–aqueous system

The fungi Aspergillus fumigatus, Hormoconis resinae and Candida silvicola were isolated from the fuel/water interfacial biomass in diesel storage tanks in Brazil. Their corrosive activities on mild steel ASTM A 283-93-C, used in storage tanks for urban diesel, were evaluated after various times of incubation at 30 °C in a modified Bushnell–Haas mineral medium (without chlorides) with diesel oil as sole source of carbon. Their ability to degrade diesel oil was evaluated after growth for 30 and 60 days. The fungus Aspergillus fumigatus and the consortium of all three organisms showed the highest production of biomass; A. fumigatus gave the greatest value for steel weight loss and produced the greatest reduction in pH of the aqueous phase. Solid phase microextraction (SPME) showed that the main acid present in the aqueous phase after 60 days incubation with A. fumigatus was propionic acid. Polarization curves indicated that microbial activity influenced the anodic process, probably by the production of corrosive metabolites, and that this was particularly important in the case of A. fumigatus. This fungus preferentially degraded aliphatic hydrocarbons of chain lengths C₁₁--C₁₃ in the diesel, producing 47.7, 37.5 and 51% reductions in C₁₁, C₁₂ and C₁₃, respectively. It produced more degradation than the consortium after 60 days incubation. It is likely that the presence of other species in the consortium inhibited the growth of A. fumigatus, thus resulting in a lower rate of diesel fuel degradation.     

The effect of asparagus virus infection on asparagus tissue culture

The impact of asparagus virus I (AV-I), a potyvirus, and asparagus virus II (AV-II), an ilarvirus, on micropropagation of field-grown asparagus was studied. Apical shoot tips excised from singly or doubly-infected plants were slow to develop roots and had a 15 to 75% reduction in survival in culture, respectively, compared to those excised from virus-free plants. The four virus infection groups were ranked: virus-free >AV-II>AV-I>AV-I & II for capacity of explants to both root and survivein vitro. Micropropagated plants infected with AV-II exhibited slight reductions in fresh and dry weights, with greater reductions associated with infection with AV-I and double infection, compared to the virus-free controls. Eighty-one virus-infected apical shoot tips yielded 7 (8.6%) virus-free clones, as determined by rub inoculation on indicator plants.