Microcycle conidiation — A review

Microcycle conidiation is defined as the germination of spores by the direct formation of conidia without the intervention of mycelial growth, as occurs in most normal life cycles. It is a method of asexual spore formation in which the normal life cycle of the fungus is bypassed. Spores formed through sexual reproduction and species with unicellular thalli are not included in microcycle conidiation. The term secondary conidium or secondary spore is usually, but not always, synonymous with microcycle conidiation. In the laboratory various factors, but especially temperature, can induce the microcycle condition in such fungi asAspergillus niger, Penicillium andNeurospora crassa, providing a useful tool for research. Microcycle conidiation has also been reported in a broad range of species in nature, and comprises a normal part of the life cycle in several groups, including the Entomophthorales, Taphrinales, Clavicipitales, Uredinales, Ustilaginales, Tremellales and Exobasidiales. The presence of a microcycle in such fungi undoubtedly provides a survival mechanisn for spores that encounter unfavorable conditions.     

Carotenogenesis gene cluster and phytoene desaturase catalyzing both three- and four-step desaturations from Rhodobacter azotoformans

In Saccharomyces cerevisiae, Nce102 encodes a 173 amino acid transmembrane protein, which acts as a key player in eisosome assembly and plasma membrane organization. Here, we describe the characterization of Nce102 homologue in the human pathogen, Aspergillus fumigatus. Our results demonstrated that AfuNce102 is continuously expressed during fungal growth. In addition, microscopic examination of an AfuNce102-GFP-expressing transformant confirmed the localization of the fusion protein to the endoplasmic reticulum with higher density fluorescence at the tip of the mycelium. During conidiogenesis, the protein was localized to the conidiophores and the conidia. Abnormal conidiation of AfuNce102 deletion mutant suggests a potential role for AfuNce102 in sporulation process.     

Identification of genes up-regulated during conidiation of Fusarium oxysporum through expressed sequence tag analysis

Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. F. oxysporum produces microconidia and macroconidia in carboxymethyl cellulose-added liquid medium (CMCLM) and exhibits vegetative growth without conidiation in complete liquid medium (CLM). The cDNA libraries were constructed using mRNAs from CLM and CMCLM cultures. A total of 1288 and 1353 clones from CLM (vegetative growth) and CMCLM (conidiation) libraries, respectively, were sequenced, and 641 and 626 unique genes were identified. Of these unique genes, only 130 ( approximately 20%) were common in the two libraries, indicating different patterns of gene expression during vegetative growth and conidiation. The expression levels of 496 CMCLM-specific genes were compared during vegetative growth and conidiation by cDNA dot-blot differential hybridization and real-time quantitative PCR analyses, and 42 genes were identified to display >5-fold increases in mRNA abundance during conidiation. These genes provide ideal candidates for further studies directed at understanding fungal conidiogenesis and its molecular regulation.     

Separate effects of moisture content and water activity on the hyphal extension of Penicillium rubens on porous media

To prevent indoor fungal growth, understanding the moisture relations of fungi is a key element. Indoor moisture is quantified by the relative humidity (RH). RH controls the water activity of the indoor materials that fungi grow on, a well‐studied parameter known to limit fungal growth. RH, however, also controls the amount of water present in these materials, the moisture content. The significance of the moisture content of these materials to indoor fungal growth is currently overlooked. In the work reported here, growth experiments with the indoor fungus Penicillium rubens on gypsum substrates were performed to test whether the moisture content influences growth on porous materials. Second, we report the development of a video microscopy method that for the first time quantified hyphal growth on a porous material. It is found that a higher moisture content leads to earlier colonization and higher hyphal extension rates. This is a fundamental step in unravelling the effect of RH on indoor fungal growth. The real‐time monitoring of colonization of gypsum provides a new view of growth on indoor surfaces.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

veA is required for toxin and sclerotial production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

Genetic and morphological characterization of a Fusarium verticillioides conidiation mutant

Enteroblastic phialidic conidiation by the corn pathogen Fusarium verticillioides (teleomorph Gibberella moniliformis) produces abundant, mostly single-celled microconidia in distinctive long chains. Because conidia might be critical for establishing in planta associations, we characterized a spontaneous F. verticillioides conidiation mutant in which phialides were incapable of enteroblastic conidiogenesis. Instead of producing a conidium, the phialide apex developed a determinate, slightly undulating, germ tube-like outgrowth, in which nuclei rarely were seen. Electron microscopy showed that the apical outgrowth possessed a thick, rough, highly fibrillar outer wall layer that was continuous with the thinner and smoother outer wall layer of the phialide. Both the inner wall layer and plasma membrane also were continuous between the apical outgrowth and phialide. The apical neck region of mutant phialides lacked both a thickened inner wall layer and a wall-building zone, which were critical for conidium initial formation. No indication of septum formation or separation of the apical outgrowth from mutant phialides was observed. These aberrations suggested the apical outgrowth was not a functional conidium of altered morphology. The mutation did not prevent perithecium development and ascosporogenesis. Genetic analyses indicated that a single locus, designated FPH1 (frustrated phialide), was responsible for the mutation. The conidiogenesis mutants were recovered only during certain sexual crosses involving wild-type conidiating parents, and then only in some perithecia, suggesting that mutation of FPH1 might be meiotically induced, perhaps due to mispairing between homologous chromosomes and deletion of the gene from a chromosome.     

Aspergillus Spp. as an important occupational risk factor among susceptible individuals

The prevalence of different species of aspergilli was studied for two consecutive years (from April 1989 to March 1991) in a mechanised bakery, a poultry farm and a large municipal hospital in Delhi, India using an Andersen six stage volumetric sampler. A rich diversity of aspergilli (26 species) were recorded in each environment. The major contributors in each environment were Aspergillus flavus, A. niger, A. versicolor, A. sydowi, A. fumigatus, A. japonicus. The concentration of aspergilli was higher inside the work place compared to the outside air, with 25% to 45% contribution to the total fungal load. The concentration of A. flavus was higher in the storage section of the bakery and in the shed of the poultry farm, while A. niger was characteristic of the packing section and the hatchery. The hospital, which was naturally ventilated with a continuous mixing of indoor and outdoor air had a significantly high concentration of A. flavus (p < 0.05) inside the ward. Some of the frequently encountered species of Aspergillus are A. janus, A. ochraceous, A. nidulans, A. variecolor, A. luchuensis and A. terreus. Other species of Aspergillus had a very low occurrence, accounting for less than 1% of the total fungal load.     

Deodorization of pig slurry and characterization of bacterial diversity using 16S rDNA sequence analysis

Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.     

Morphogenetic and developmental functions of the Aspergillus nidulans homologues of the yeast bud site selection proteins Bud4 and Axl2

The yeast bud site selection system represents a paradigm for understanding how fungal cells regulate the formation of a polarity axis. In Saccharomyces cerevisiae, Bud4 and Axl2 are components of the axial bud site marker. To address the possibility that these proteins regulate cellular morphogenesis in filamentous fungi, we have characterized homologues of Bud4 and Axl2 in Aspergillus nidulans. Our results show that Bud4 is involved in septum formation in both hyphae and developing conidiophores. Whereas Axl2 appears to have no obvious role in hyphal growth, it is required for the regulation of phialide morphogenesis during conidiation. In particular, Axl2 localizes to the phialide-spore junction, where it appears to promote the recruitment of septins. Furthermore, the developmental regulators BrlA and AbaA control the expression of Axl2. Additional studies indicate that Axl2 is also involved in the regulation of sexual development, not only in A. nidulans, but also in the phylogenetically unrelated fungus Fusarium graminearum. Our results suggest that Axl2 plays a key role in phialide morphogenesis and/or function during conidiation in the aspergilli.     

Light-independent conidiation in Trichoderma spp.: a novel approach to microcycle conidiation

Microcycle conidiation in Trichoderma hamatum and T. harzianum has been achieved in complete darkness for the first time. The time required for mass conidiation without intervening vegetative growth was decreased to 24 h instead of 6 to 7 days. The conidia produced by microcycle conidiation were viable and had pigmentation and antagonistic behaviour similar to those of the parental stock cultures.N. Khurana, R.K. Saxena, R. Gupta and R.C. Kuhad are with the Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India.     

Heterotrimeric Galpha protein Pga1 of Penicillium chrysogenum controls conidiation mainly by a cAMP-independent mechanism.

Fungal heterotrimeric G proteins regulate different processes related to development, such as colony growth and asexual sporulation, the main mechanism of propagation in filamentous fungi. To gain insight into the mechanisms controlling growth and differentiation in the industrial penicillin producer Penicillioum chrysogenum, we investigated the role of the heterotrimeric Galpha subunit Pga1 in conidiogenesis. A pga1 deleted strain (Deltapga1) and transformants with constitutively activated (pga1G42R) and inactivated (pga1G203R) Pga1 alpha subunits were obtained. They showed phenotypes that clearly implicate Pga1 as an important negative regulator of conidiogenesis. Pga1 positively affected the level of intracellular cAMP, which acts as secondary messenger of Pga1-mediated signalling. Although cAMP has some inhibitory effect on conidiation, the regulation of asexual development by Pga1 is exerted mainly via cAMP-independent pathways. The regulation of conidiation by Pga1 is mediated by repression of the brlA and wetA genes. The Deltapga1 strain and transformants with the constitutively inactive Pga1G203R subunit developed a sporulation microcycle in submerged cultures triggered by the expression of brlA and wetA genes, which are deregulated in the absence of active Pga1. Our results indicate that although basic mechanisms for regulating conidiation are similar in most filamentous fungi, there are differences in the degree of involvement of specific pathways, such as the cAMP-mediated pathway, in the regulation of this process.     

veA Is Required for Toxin and Sclerotial Production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection-related fungal development, and is required for pathogenicity of M. grisea.     

Fungal colonization of synthetic substrates for use in space craft

Summary Materials being used or considered for use in space flight were examined for their susceptibility to fungal colonization. The materials included soft goods (clothing) and insulation and fabrication products such as Velcro^® attachments and elastic cord binders. Materials were exposed for at least 28 days in a highhumidity chamber colonized with over 50 species of fungi, including those species recommended for determining recalcitrance of materials to fungal biodegradation. At least nine of 25 products demonstrated extensive microscopic colonization by fungi, mostly byAcremonium obclavatum. Challenge procedures that rely on observations with the unaided eye, or 40×magnification of growth by a restricted number of fungal species with a cellulosic substrate as a positive control, are insufficient for determining the resistance of synthetic substrates to fungal colonization.