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.     

A spindle pole body-associated protein, SNAD, affects septation and conidiation in Aspergillus nidulans

The nudA1 mutation in the cytoplasmic dynein heavy chain gene inhibits nuclear migration, colony growth and asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans. It also alters the location of the first cell division event (septation) and prevents nucleation of tip cells. We showed previously that a suppressor of nudA1, snaD290, partially reversed the nuclear migration defect and partially restored colony growth. We have now demonstrated that the snaD290 mutation also delays septation and restores the septum to its normal position, allowing tip cells to be nucleated. Although snaD290 does not affect nuclear migration or vegetative hyphal growth, it almost completely inhibits conidiation. We propose that the SNAD protein participates in septation, and is essential for asexual spore formation. SnaD encodes a novel 76-kDa coiled-coil protein (SNAD) that is located at the spindle pole body throughout the cell cycle. Therefore, our results suggest that proteins at the spindle pole body are likely to be involved in temporal regulation of septation in A. nidulans. Received: 5 October 1999 / Accepted: 28 December 1999     

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.     

The Role,Interaction and Regulation of the Velvet Regulator VelB in Aspergillus nidulans

The multifunctional regulator VelB physically interacts with other velvet regulators and the resulting complexes govern development and secondary metabolism in the filamentous fungus Aspergillus nidulans. Here, we further characterize VelB’s role in governing asexual development and conidiogenesis in A. nidulans. In asexual spore formation, velB deletion strains show reduced number of conidia, and decreased and delayed mRNA accumulation of the key asexual regulatory genes brlA, abaA, and vosA. Overexpression of velB induces a two-fold increase of asexual spore production compared to wild type. Furthermore, the velB deletion mutant exhibits increased conidial germination rates in the presence of glucose, and rapid germination of conidia in the absence of external carbon sources. In vivo immuno-pull-down analyses reveal that VelB primarily interacts with VosA in both asexual and sexual spores, and VelB and VosA play an inter-dependent role in spore viability, focal trehalose biogenesis and control of conidial germination. Genetic and in vitro studies reveal that AbaA positively regulates velB and vosA mRNA expression during sporogenesis, and directly binds to the promoters of velB and vosA. In summary, VelB acts as a positive regulator of asexual development and regulates spore maturation, focal trehalose biogenesis and germination by interacting with VosA in A. nidulans.     

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.     

Autophagy during conidiation and conidial germination in filamentous fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)-AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.     

Deletion of the Aspergillus flavus Orthologue of A. nidulans fluG Reduces Conidiation and Promotes Production of Sclerotia but Does Not Abolish Aflatoxin Biosynthesis

The fluG gene is a member of a family of genes required for conidiation and sterigmatocystin production in Aspergillus nidulans. We examined the role of the Aspergillus flavus fluG orthologue in asexual development and aflatoxin biosynthesis. Deletion of fluG in A. flavus yielded strains with an approximately 3-fold reduction in conidiation but a 30-fold increase in sclerotial formation when grown on potato dextrose agar in the dark. The concurrent developmental changes suggest that A. flavus FluG exerts opposite effects on a mutual signaling pathway for both processes. The altered conidial development was in part attributable to delayed expression of brlA, a gene controlling conidiophore formation. Unlike the loss of sterigmatocystin production by A. nidulans fluG deletion strains, aflatoxin biosynthesis was not affected by the fluG deletion in A. flavus. In A. nidulans, FluG was recently found to be involved in the formation of dehydroaustinol, a component of a diffusible signal of conidiation. Coculturing experiments did not show a similar diffusible meroterpenoid secondary metabolite produced by A. flavus. These results suggest that the function of fluG and the signaling pathways related to conidiation are different in the two related 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.     

Characterization of the velvet regulators in Aspergillus fumigatus

Fungal development and secondary metabolism is intimately associated via activities of the fungi‐specific velvet family proteins. Here we characterize the four velvet regulators in the opportunistic human pathogen Aspergillus fumigatus. The deletion of AfuvosA, AfuveA and AfuvelB causes hyperactive asexual development (conidiation) and precocious and elevated accumulation of AfubrlA during developmental progression. Moreover, the absence of AfuvosA, AfuveA or AfuvelB results in the abundant formation of conidiophores and highly increased AfubrlA mRNA accumulation in liquid submerged culture, suggesting that they act as repressors of conidiation. The deletion of AfuvosA or AfuvelB causes a reduction in conidial trehalose amount, long‐term spore viability, conidial tolerance to oxidative and UV stresses, and accelerated and elevated conidial germination regardless of the presence or absence of an external carbon source, suggesting an interdependent role of them in many aspects of fungal biology. Genetic studies suggest that AfuAbaA activates AfuvosA and AfuvelB expression during the mid to late phase of conidiation. Finally, the AfuveA null mutation can be fully complemented by Aspergillus nidulans VeA, which can physically interact with AfuVelB and AfuLaeA in vivo. A model depicting the similar yet different roles of the velvet regulators governing conidiation and sporogenesis in A. fumigatus is presented.     

基于转录组的冬虫夏草菌微循环产孢研究

为研究冬虫夏草菌（Ophiocordyceps sinensis）微循环产孢过程中的分子机理,采用高通量测序技术,对微循环产孢前后冬虫夏草菌的转录组进行研究。筛选获得差异表达基因6 902个。Gene Ontology（GO）功能聚类分析表明,差异表达基因主要参与分子功能、胞内运输、核糖核蛋白复合体等生物学通路。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG)功能富集分析结果表明,差异表达基因参与了核糖体、嘧啶代谢、蛋白酶体、嘌呤代谢、甘氨酸代谢等过程。Mmc、Mcb、MaH1、MaPP5、MaAGA、Pyk等候选基因不同程度的参与了冬虫夏草菌微循环产孢过程,其中Mcb家族基因可能起到关键作用。通过qRT PCR验证差异表达基因,定量结果与转录组测序结果一致。本研究为进一步揭示冬虫夏草菌微循环产孢分子机制以及关键基因的调控提供了重要信息。     

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.     

The circadian conidiation rhythm inNeurospora crassa

The filamentous fungusNeurospora crassais one of the best organisms for analysing the molecular basis of the circadian rhythm observed in asexual spore formation, conidiation. Many clock mutants in which the circadian conidiation rhythm has different characteristics compared to those in the wild-type strain have been isolated since the early 1970s. With the cloning of one of these clock genes,frq, the molecular basis of the circadian clock inNeurosporahas become gradually clearer. Physiological and pharmacological studies have also contributed to our understanding of the physiological basis of the circadian clock inNeurospora. These studies strongly indicate that the circadian clock is based on or is closely related to a network of metabolic processes for cellular activities. Based on these studies, it may be possible to isolate new types of clock mutants which should contribute to a better understanding of the molecular basis of the circadian clock inNeurospora.