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.     

Interaction of the Microbivorous Nematode Teratorhabditis dentifera and the Nematode-Pathogenic Fungus Hirsutella rhossiliensis

The fungus Hirsutella rhossiliensis is an obligate pathogen with a broad host range among nematodes. Microbivorous nematodes are abundant around plant roots and may serve as hosts for the fungus. Our objective was to determine the influence of the bacterial-feeding nematode Teratorhabditis dentifera on the abundance of H. rhossiliensis. Experiments were conducted in a growth chamber with pots containing pasteurized soil, the fungus, and potato plants. The abundance of infectious conidia was compared in pots with and without T. dentifera after 50 or 70 days. The nematode reached high densities (10-40/cm³ soil) but had no effect on the abundance of conidia. Many individuals were dauer juveniles, a stage that acquired conidia but did not become infected. To test whether this life stage could deplete the pool of conidia in soil, different proportions of dauer juveniles with (resistant) and without (susceptible) a sheath were added to H. rhossiliensis-infested soil. The number of conidia in the soil decreased with an increasing proportion of resistant nematodes. Different stages of T. dentifera appear to have opposing effects on H. rhossiliensis; while adults and regular juveniles acquire conidia, become infected, and produce new infectious conidia, dauer juveniles can deplete the supply of conidia.     

Blastobotrys,Sporothrix and Trichosporiella: generic delimitation,new species,and a Stephanoascus teleomorph

The morphological and physiological characters of the species of Blastobotrys, and of a number of similar species of Sporothrix, Trichosporiella and Candida were studied. Blastobotrys is defined as having mother cells (primary conidia) which form distinct, secondary conidia. Sporothix has non-catenulate conidia, or the conidia are catenulate without marked differentiation of conidia of first and second order. Trichosporiella is delimited from Candida by a stronger coherence between hyphal cells, arthroconidia being absent. Four new Blastobotrys species are described, and two unnamed species; two new combinations are proposed in Trichosporiella. Related species in other genera are briefly discussed. A new Stephanoascus teleomorph is described in two strains originally identified as Sp. fungorum. Two diagnostic keys to the described taxa, using either morphological or physiological characters, are given.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

Orbilia blumenaviensis and its Arthrobotrys anamorph

A member of the nematophagous anamorph genus Arthrobotrys was isolated from its teleomorph Orbilia blumenaviensis comb. nov. (= Orbilia fici), a species closely related to O. auricolor but deviating in having lanceolate paraphyses. In the presence of nematodes, the anamorph forms three-dimensional adhesive networks. A trimorphism in its conidia was observed which vary in shape and number of septa. In the first isolate, two types of heteropolar conidia were obtained. These differ markedly from the type strain of A. vermicola, which forms mainly homopolar (fusoid) conidia. In a further ascospore isolate of O. blumenaviensis, however, mainly fusoid conidia referable to A. vermicola occurred. Combining morphological and phylogenetic analysis, we conclude that these isolates with differently shaped conidia and/or differences in the ITS rDNA (ca. 2–4%) belong to a single anamorph species, A. vermicola. Both teleomorph and anamorph are illustrated and described.     

Rice recycling: a simple strategy to improve conidia production in solid-state cultures

Here we studied at a laboratory scale a potential strategy to revalorize the residual rice remaining at the end of a conventional conidia production process in solid-state culture. The conidia production of Trichoderma asperellum Th-T4 (3) and Metarhizium robertsii Xoch-8.1 started with the use of fresh rice (unrecycled rice) as the substrate (cycle one), and continued with the use of recycled rice in successive cycles of conidia production. The rice remaining at the end of the first cycle was reused without any further sterilization or reinoculation. As a result, it was observed that the conidia production and productivity significantly increased in both fungi. Conidia production in T. asperellum Th-T4 (3) increased from 1 × 10⁹ (first cycle) to 2·9 × 10⁹ conidia per gram of initial dry substrate (con⋅gds⁻¹) (second cycle using recycled rice), while in M. robertsii Xoch-8.1, this parameter increased form 5·7 × 10⁸ to 1·4 × 10⁹ con⋅gds⁻¹. Both fungi grew faster and conidiated earlier when recycled rice was used as the substrate, therefore, conidia productivity was also significantly improved. Furthermore, the use of recycled rice did not affect conidia viability. This is the first report about a recycling methodology completely free of extra-processing steps, and useful to increase conidia production and productivity.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Conidia production by Isaria fumosorosea on solid substrates and its pathogenicity towards Bemisia tabaci

Large amounts of conidia must be recovered to use Isaria fumosorosea as a biological control agent. Aerial conidia are mass-produced in solid substrate culture. Conidia obtained from growth on different substrates must be similar in quantity and quality (viability, purity and virulence). In the present work, several solid substrates were evaluated: rice, sorghum, sugarcane, corncob, plantain and wheat (derived from the production of Trichogramma sp.) to find alternatives that are economical and available in different regions. The highest conidia production was obtained on rice, plantain and corncob substrates, which yielded 5.33 × 10⁹ conidia/g, 5.24 × 10⁹ conidia/g and 4.8 × 10⁹ conidia/g substrate, respectively. At concentrations of 1 × 10⁷ conidia/mL, the mortality rates obtained with conidia developed on rice, plantain and corncob substrates were 98%, 97% and 88%, respectively. The lethal concentration 50 (LC₅₀₎ values of conidia obtained from rice and plantain were 7.2 × 10⁴ conidia/mL and 5.1 × 10⁴ conidia/mL, respectively.     

Conidial water affinity is an important characteristic for thermotolerance in entomopathogenic fungi

The interspecific thermotolerance of several species of entomopathogenic fungi was evaluated based on the conidial water affinity. The species were divided between hydrophilic and hydrophobic conidia. The species with hydrophobic conidia were Beauveria bassiana (ARSEF 252), Metarhizium brunneum (ARSEF 1187), Metarhizium robertsii (ARSEF 2575), Isaria fumosorosea (ARSEF 3889) and Metarhizium anisopliae s.l. (ARSEF 5749). The species with hydrophilic conidia were Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), Simplicillium lanosoniveum (ARSEF 6430), Lecanicillium aphanocladii (ARSEF 6433), S. lanosoniveum (ARSEF 6651), Aschersonia placenta (ARSEF 7637) and Aschersonia aleyrodis (ARSEF 10276). The conidial surface tension of each isolate was also studied. Conidial suspensions were exposed to 38, 41 or 45 °C. After exposure, the suspensions were inoculated on media and conidial germination was evaluated. Considerable differences in thermotolerance were found among the 12 entomopathogenic fungal species. Species with hydrophobic conidia were generally more thermotolerant than species with hydrophilic conidia. All isolates with hydrophobic conidia showed higher conidial surface tension than the isolates with hydrophilic conidia.     

Production of conidia of Beauveria bassiana in solid-state culture: current status and future perspectives

Beauveria bassiana is an important entomopathogenic fungus widely commercialized in the world. Recent progress and achievements on conidia production have focused on a yield goal of 10⁹ to 10¹⁰ conidia per gram of dry substrate. Due to cost-competitive perspectives, these yields should be associated with better production rates or productivities. This study presents a review of relevant studies of B. bassiana conidia production on solid-state cultures and the parameters that should be taken into account to maintain constant quality in the product to be commercialized. Conditions for maximizing production and infectivity of B. bassiana conidia are also analysed.     

A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata

Summary A diffusible self-inhibitor of germination of conidia of Glomerella cingulata appears to act as a regulator of protein synthesis. Both uptake of labeled amino acids and their incorporation into protein are reduced by the inhibitor or by crowding. Compared to conidia incubated without self-inhibitor, conidia incubated with self-inhibitor incorporated no labeled amino acids into protein in the first hour and 80% less in 6h. Thoroughly washed conidia were more permeable to amino acids and incorporated 6 times more precursor into proteins than unwashed conidia. At high density in nutrient medium, conidia of G. cingulata preferentially form secondary conidia instead of germ tubes and a mycelium. This inhibition of germination of conidia and regulation of development is mimicked by exposing them to an auto-inhibitor extracted from used culture medium and conidial washings. Germination of conidia of G. cingulata involves two steps, an initial step of 5 h duration which continues unaffected by crowing (1.7×10⁸/ml) and a subsequent 2 h step which conidia do not take unless they are sufficiently diluted. It is this step for which protein synthesis may be required.Non-Standard Abbreviations CHM cyloheximide - NM Neurospora minimal medium - psi pound per square inch - RPH reconstituted algal protein hydrolysate - TCA trichloroacetic acid     

Virulence of Different Isolates and Formulations of Beauveria bassiana for House Flies and the Parasitoid Muscidifurax raptor

Adult house flies (Musca domestica) were susceptible (94-100% mortality) to Beauveria bassiana when conidia of the Hf88 isolate were applied to plywood boards at 10⁷ conidia/cm²; a starch dust formulation was more effective than a liquid suspension. Adult flies were also susceptible (90-99% mortality) to this isolate when they were con fined with treated water (10⁸ conidia/ml) or food (10⁸ conidia/100 mg). House fly larvae were not affected by treatments with up to 10⁸ conidia/cm³ of rearing medium. A 2-year survey of house flies cm from New York dairies indicated that most natural infections of house flies occurred in September and October, although prevalence rates never exceeded 1%. Thirteen single-fly isolates obtained during this survey were compared with the Hf88 isolate for virulence against flies; the 2 most virulent isolates were slightly more virulent for flies than for the fly parasitoid Muscidifurax raptor. Incorporation of conidia into a sucrose bait (10⁸ conidia/100 mg) in cages gave high levels of house fly control (78-100% mortality) 5 days after exposure.     

Bambusicolous fungi in Japan (8): a new species of <Emphasis Type="Italic">Pseudolachnella from</Emphasis> Yakushima Island,southern Japan

A coelomycetous fungus occurring on culms of Pleioblastus sp. in Yakushima Island, southern Japan, is described and illustrated as a new species, Pseudolachnella yakushimensis. The species is characterized by cupulate, superficial black setose conidiomata, and cylindrical 3-septate conidia with two to five appendages at each end. Pseudolachnella yakushimensis is similar to P. indica and P. scolecospora by its 3-septate conidia, but is different from them by its smaller conidia with more than two appendages.     

Three new species and a new record of Diplococcium from plant debris in Spain

Diplococcium dimorphosporum sp. nov., D. racemosum sp. nov., D. singulare sp. nov. and D. pulneyense Subram. & Sekar collected from plant debris in natural areas of Spain are described and illustrated. The first species is characterized principally by the production of branched conidiophores and short chains of conidia. Diplococcium singulare has unbranched conidiophores, and conidia produced usually at the tip of conidiophores and from lateral spherical conidiogenous cells. In addition, both species develop a Selenosporella synanamorph with narrow falcate conidia. Diplococcium racemosum produces branched, verrucose conidiophores, and verrucose conidia in long branched chains. Diplococcium pulneyense is the second record, being described for first time on the natural substratum and re-described in pure culture. A key to currently accepted species of Diplococcium is provided.     

Infectivity of two members of theEntomophthora muscae complex [Zygomycetes: Entomophthorales] forMusca domestica [Dipt.: Muscidae]

Dose-mortality studies were conducted with 2 members of theEntomophthora muscae (Cohn) Fresenius complex from southern California (CA) and Denmark (DA) infecting house flies,Musca domestica L., from southern California. Primary conidia of the DA form were significantly more infective (LC₅₀=34 conidia/mm²) than primary conidia of the CA form (LC₅₀=67 conidia/mm²) for 2–7 days old (‘young’) flies. Infectivity (single experimental trial) for 10–14 day old (‘old’) flies of primary conidia of the CA form (LC₅₀=34 conidia/mm²) was similar to that of the DA form for young flies. Secondary conidia of the CA form were markedly more infective (LC₅₀=0.36 conidia/mm²) than primary conidia of either form. Dose had no significant effect on incubation period for primary conidia of either form, but increased doses resulted in significantly shorter incubation periods for flies exposed to secondary conidia of the CA form.      

The potential of Beauveria bassiana inoculum formulated into a polymeric matrix for a microbial control of spruce bark beetle

Two local strains of Beauveria bassiana originally isolated from naturally infected spruce bark beetles in Slovakia were tested for their virulence to Ips typographus (IT) and for their compatibility with a polymeric matrix composed of low-molecular polyethylene. Conidia could be homogenously immobilized in the low-molecular polyethylene matrix with no adverse effect on their viability and infectivity. At constant temperature (25°C), viability of immobilized conidial decreased only by 1–2% after 7 or 14 days when compared with non-formulated conidia. In field conditions, viability of conidia formulated in the matrix was even significantly higher than non-formulated conidia 35 days after their application in traps. Conidia incorporated into the polymeric matrix were infective to IT adults in laboratory bioassays. Mean values of LC₅₀ for native conidia (0.72–2.05?×?10⁶ conidia?ml^?1) and conidia immobilized in the polymeric matrix (0.64–1.03?×?10⁵ conidia?mm^?2) demonstrated high virulence. The efficacy of the local strains was significantly higher than that of B. bassiana strains from mycoinsecticides (Boverol^®, Botanigard^® ES and Naturalis-L^®). Results showed potential of this polymeric material for its use in microbial control of IT when mixed with conidia of B. bassiana.     

Aspergillus fumigatus adhesion factors in dormant conidia revealed through comparative phenotypic and transcriptomic analyses

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K‐means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high‐adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin‐like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.     

Impact of culture age on conidial germination,desiccation and UV tolerance of entomopathogenic fungi

The impact of culture age on conidial yields, germination and tolerance to UV exposure of freshly harvested and dry conidia produced by five entomopathogenic fungal (EPF) isolates was studied. Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii and Lecanicillium muscarium were grown on potato dextrose agar (PDA) medium for 7 or 14 days at 25°C. While the age of cultures had a significant impact on the germination rate of conidia produced by isolates L. lecanii CBS 122.175 and B. bassiana LMSA 1.01.093, other EPF isolates germinated at the same rate regardless of the culture age. When exposed to UV radiation, conidia produced by all isolates germinated at a lower rate compared to the non-irradiated conidia, although this decrease in germination (20–80% decrease) was unaffected by the culture age. Air-drying had only a slight impact on conidial germination (0–60% decrease). Under the conditions of this study, the stability of irradiated conidia produced by M. anisopliae LMSA 1.01.197 and B. bassiana CBS 110.25 was significantly increased when conidia were dried prior to UV exposure. This increase in tolerance to stress of dried conidia might be caused, at least partially, by the low metabolic activity associated with dehydration.     

Brown spot ofRheum officinale in indonesia with special reference to the variation in conidia of the causal fungus

Brown spot disease ofRheum officinale, a traditional medicinal plant in South East Asia, was newly recorded from the highland area of West Java Province. After confirmation of its pathogenicity towardRheum officinale and comparison of its morphology with hitherto known species, the casual fungus was identified asAscochyta rhei. It produces widely varied conidia from unicellular and bacillar conidia (Phoma-type), 1-septate oblong conidia (Ascochyta-type) to 2–3-septate cylindric conidia. These conidia were sometimes formed within the same pycnidium. They could germinate easily and produce the similar colonies to each other. Supported by “Joint Study Project for Strengthening Research on Diseases of Industrial Crops in Indonesia, Japan International Cooperation Agency (JICA)” in BALITTRO.     

Bambusicolous fungi in Japan (7): a new coelomycetous genus, <Emphasis Type="Italic">Versicolorisporium</Emphasis>

A new genus, Versicolorisporium, is established for the coelomycetous fungus collected in Japan on dead culms of the bamboos Pleioblastus chino and Sasamorpha borealis. The type species of the genus, V. triseptatum, is characterized by the production of holoblastic, 3-septate, obovoid, versicolored conidia. Versicolorisporium is similar to Toxosporiella, Neohendersonia, Toxosporiopsis, and Scolicosporium in having versicolored conidia, but differs from these genera by the uniloculate pycnidial conidiomata with a periphysate ostiole, lacking paraphyses, and the conidia without black-banded septa. A BLAST search using LSU nrDNA sequence indicates that the new genus is a member of Pleosporales, Dothideomycetes.