Paedogenetic conidiation inNeurospora crassa

Under conditions of growth limitation the germ tubes ofNeurospora crassa macroconidia may convert to functional conidiophores with developing chains of viable, new conidia. The induction of this paedogenetic conidiation in liquid culture does not require exposure to high temperature, as reported for conidia of some other ascomycetous fungi, but it is induced at normal growth temperatures within 12–15 h by germination of the conidia in a dilute, nutrient-poor incubation medium. The inoculum density, temperature of incubation, presence or absence of white light, and genetic strain ofN. crassa also influence the kinetics of conidium formation.     

The role of environmental factors in the conidiation of the predacious rotiferovorous fungus Zoophagus insidians (Zoopagomycota)

Compared to other groups of fungi, the knowledge of freshwater predacious fungi that feed on trapped rotifers and tardigrades is very limited. They are known to spread and survive under adverse conditions by releasing asexual spores (conidia), but the environmental factors that induce their conidiation are unclear. In this study, we investigated the conidiation of the rotiferovorous fungus Zoophagus insidians isolated from activated sludge and maintained under laboratory conditions in spring water (medium). We found that its conidiation can undergo significant changes in response to various environmental factors, such as medium exchange, presence or absence of prey, lighting conditions, and their combination. Our results revealed a surprisingly high flexibility of this obligate predacious fungus, which being constantly exposed to unpredictable availability of prey in an unstable environment is still able to survive and disperse.     

Characterization of an Aspergillus nidulans mutant with abnormal distribution of nuclei in hyphae, metulae, phialides and conidia

The V10 deteriorated variant of Aspergillus nidulans has hyphae, metulae, phialides and conidia with abnormal nuclear distributions. The alterations observed were: increase in the number of nuclei in hyphae, metulae and phialides, presence of anucleate, uninucleate and multinucleate conidia, abnormal vegetative growth and defective conidiation. When 0.5 M NaCl was added to the medium, an increase in the number of conidia was observed but their morphology and number of nuclei were not modified. The gene responsible for these alterations was named anuA1. The anuA1 gene is located on linkage group VII and is possibly involved in nuclear migration to hyphae, metulae, phialides and conidia.     

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.     

Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata

Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose-agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus.     

Conidium formation and germination inNeurospora crassa: Glutamic acid metabolism

In order to assess the role of the large (200 μmol/g) pool of glutamic acid normally found in wild-typeNeurospora crassa conidia, the levels of glutamic acid and other amino acids were manipulated by employing a strain blocked in glutamate synthesis. The double mutant strainen(am)-2;am, which has a lesion in NADP-glutamate dehydrogenase (am) and is auxotrophic for glutamate, was employed in order to obtain conidia with a decreased level of glutamic acid. When cultures of this strain were grown in the presence of 50 or 20 mM glutamate as sole nitrogen source, conidia were obtained with a glutamate pool of 97 and 47 μmole/g, respectively, which represents approximately 57 and 28% of the control values obtained from a prototrophic strain. These observations are interpreted to indicate that conidiation can occur even without the accumulation of the entire pool of glutamate in the conidia. However, when conidia of this double mutant strain were incubated in a minimal medium lacking glutamate, germ tube formation was greatly delayed. Germ tube formation was normal in the presence of glutamate or alanine. During germination of conidia in minimal medium, the levels of glutamate, glutamine, aspartic acid, and γ-aminobutyric acid (GABA) were measured at different times. The results obtained support the view that glutamic acid was catabolized through the GABA pathway during the very early stages of germination.     

Development of Media and Automated Liquid Fermentation Methods to Produce Desiccation-Tolerant Propagules ofTrichoderma harzianum

A suitable medium was developed from modified Richard's medium plus V8 juice (RM8) to produce high levels of desiccation-tolerant conidia ofTrichoderma harzianumstrain 1295-22. The addition of 9% (v/v) glycerol to RM8 improved both biomass production and desiccation tolerance of the conidia ofT. harzianum.This medium was then used in a laboratory scale fermenter (1.5 liter) to determine optimal operating conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the fermenter was 32°C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate was 1000 revolutions per minute. The initial water potential of the medium (with 9% glycerol) was −3.7 MPa, the pH was 6, and neither was controlled during fermentation. Changes in medium pH and dissolved oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to 6.0–6.2 at phialide formation. Intensive conidiation occurred at pH 6.3–6.5 and reached its maximal level at 6.9–7.1. Changes in pH values could be used as indicators to monitor the morphological development and conidiation ofT. harzianumduring fermentation. The use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the time required to complete the shift from vegetative growth to phialide formation. Intensive conidiation occurred immediately after the addition of culture inoculum and reached maximum levels within 68 h of fermentation. Dry weight of biomass increased with the duration of fermentation and was greatest at 96 h. However, no improvements in conidia/gram and CFU/gram were achieved after 72 h of fermentation. The desiccation tolerance of conidia harvested at 72 or 96 h was significantly (P = 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further scale-up of the fermentation process.     

Conidiation ofTrichoderma atroviride isolate during submerged cultivation in a laboratory stirred-tank fermenter

Conditions for conidiation of a natural isolate of Trichoderma atroviride during submerged cultivation in Erlenmeyer flasks and in a laboratory stirred-tank fermenter were optimized. From the simple sugars tested, cellobiose was the best substrate for conidia production while cellulose fines from paper mill waste proved to be a suitable cheap complex carbon source. Optimum temperature for conidiation was 24-26 degrees C, and the required dissolved oxygen level was > 40% saturation. After initial slight decrease during the 1st d after inoculation, the pH of the culture medium constantly increased throughout the sporulation period. Attempts to regulate the pH during fermentation did not improve the spore yields. The most intense formation of conidia took place between 2nd and 3rd d of growth and the overall volumetric productivity of conidia was 4.1-8.2 x 10(9) conidia per L/h.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidiogenesis.

A cultivation system has been developed for Penicillium urticae which yields 'microcycle' conidiation in submerged culture. Spherical growth of spores was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged spores to a nitrogen-poor medium at 35 degrees C results in synchronous germination and limited outgrowth followed by roughly synchronous conidiation. A study of the conidiation stage showed that a phialide and an immature conidium began to form at the tip of all germ tubes 18 h after the temperature shift. By 24 h additional phialides commonly appeared as a branch near the tip of the germ tube and the more mature conidia exhibited increasing refractility. The earliest ultrastructural signs of conidiation were various round invaginations in the plasma membrane and a thickening and rounding of the new spore wall which appeared as an inner extension of the phialide cell wall. Upon segregation of the conidium from the phialide cell by conidial wall formation, 'trench-like' invaginations gradually appeared in the plasma membrane and a disorganized rodlet pattern was formed on the outer surface of the maturing conidial wall. Continued maturation involved the formation of chains of conidia and phialide senescence which was characterized by a general degradation of intracellular structure. A comparison with standard surface and submerged culture conidiation indicated that 'microcycle' conidiation, while less prolific, was essentially identical.     

Cyclic AMP-dependent protein kinase A negatively regulates conidia formation by the tangerine pathotype of Alternaria alternata

The necrotrophic fungal pathogen Alternaria alternata causes brown spot diseases in many citrus cultivars. The FUS3 and SLT2 mitogen-activated protein kinases (MAPK)-mediated signaling pathways have been shown to be required for conidiation. Exogenous application of cAMP to this fungal pathogen decreased conidia formation considerably. This study determined whether a cAMP-activated protein kinase A (PKA) is required for conidiation. Using loss-of-function mutations in PKA catalytic and regulatory subunit-coding genes, we demonstrated that PKA negatively regulates conidiation. Fungal mutants lacking PKA catalytic subunit gene (PKA ^cat ) reduced growth, lacked detectable PKA activity, and produced higher amounts of conidia compared to wild-type. Introduction of a functional copy of PKA ^cat into a null mutant partially restored PKA activity and produced wild-type level of conidia. In contrast, fungi lacking PKA regulatory subunit gene (PKA ^reg ) produced detectable PKA activity, exhibited severe growth reduction, formed swelling hyphal segments, and produced no mature conidia. Introduction of the PKA ^reg gene to a regulatory subunit mutant restored all phenotypes to wild type. PKA ^reg -null mutants induced fewer necrotic lesions on citrus compared to wild-type, whereas PKA ^cat mutant displayed wild-type virulence. Overall, our studies indicate that PKA and FUS3-mediated signaling pathways apparently have very different roles in the regulation of conidia production and A. alternata pathogenesis in citrus.     

Synchronous Conidiation of Piricularia oryzae by the Replacement Culture

For the purpose of studying the mechanism of conidiogenesis in Piricularia oryzae, methods were developed to separate the phase of mycelial growth from that of conidiation and also to evaluate them quantitatively.

When P. oryzae was grown on media with low carbon : nitrogen ratio and high nitrogen concentration, conidiation did not take place, in spite of its vegetative growth. Conidiation occurred in a very short period of time when the above mycelia were replaced on nutritionally poor media. Cellophane membrane was overlaid on solid medium and conidia were spread uniformly. Evenly grown mycelial mat which could be easily transferred onto the post-culture medium was obtained. As the preculture medium, MSA medium with carbon: nitrogen ratio of 6.3 and nitrogen concentration of 1.5 g/liter was used. The evenly grown mycelial mat was cut into small square mats of 1.44 cm² each and the small square mycelial mats were transferred onto the post-culture medium together with the cellophane membrane. The conidiation took place in the post-culture and the vegetative growth in the preculture and the conidiation in the post-culture could be observed separately and quantitatively.

Conidiation did not occur at all in the preculture and the degree of conidiation which took place in the post-culture varied according to the precultural conditions. This means that it is a certain state of physiological condition in the preculture which determines the degree of conidiation in the post-culture. The authers designated this state of physiological condition as the “latent activity of conidiation” (LAC). For the purpose of the quantitative estimation of this activity, we expressed LAC in terms of the degree of conidiation in the post-culture under a defined cultural condition. The LAC was subject to change very easily, declined rapidly and disappeared upon prolonged preculture. Only young mycelia showed to have this activity.

The influences of the precultural condition on the development of the LAC and vegetative growth were generally parallel. However, the LAC was generally more sensitive to the environmental condition than the vegetative growth, especially to the temperature change.

The conidia formed were uniform in size and had high rate of germination. Several strains of P. oryzae tested showed very similar behavior.     

Microscopic and Cytochemical Analysis of Extracellular Matrices and Endogenous Reserves of Conidia of Colletotrichum truncatum Harvested from Carbon- and Nitrogen-limited Cultures

The effects of nutrition during conidiation on extracellular matrices (ECM) and endogenous reserves of conidia of the promising mycoherbicide Colletotrichum truncatum have been examined. Transmission electron microscopy showed no ECM on the conidia, regardless of the C:N ratio (10:1, 30:1 or 80:1) of the medium on which they were produced. Cytochemical analysis using fluorochrome-labelled lectins revealed the presence of specific sugars in ECM around the germlings. Furthermore, ECM-containing amino groups (basic protein) were detected using colloidal gold. However, nutrition during conidiation had no effect on the patterns of lectin labelling or the pattern of colloidal gold staining of germlings. The relative amounts of trehalose, glycerol and mannitol in conidia produced in a liquid medium with a C:N of 10:1 were more than those obtained when the C:N was 30:1 or 80:1. Thus, internal carbohydrates such as trehalose and polyols may play an important role in viability of conidia during long-term storage.     

The effect of pH and amino acids on conidiation and pigment production of Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 were cultivated aerobically on media containing nitrate or ammonium as nitrogen source to which the following modifications were made: (1) pH adjusted to 2.5 before sterilization; (2) addition of yeast extract; (3) addition of amino acids in identical proportions and concentrations to those found in yeast extract; (4) adjustment of pH to 2.5 after addition of amino acids. The addition of amino acids in the form of yeast extract increased mycelium formation and reduced conidiation and pigment production. The addition of an amino acid mixture did not increase mycelium formation to the same extent as yeast extract but increased the number of conidia, while pigment production was reduced, especially when nitrate was the nitrogen source. As the amino acids are taken up after conidial formation has started, it would appear that it is not the amino acids themselves which are directly responsible for the induction of conidiation. The addition of amino acids inhibits nitrate and ammonium uptake suggesting the need for an early intracellular nitrogen limitation to induce conidiation. Lowering the pH inhibits the formation of conidia and increases pigment production; also the effect of amino acid addition is totally annulled. The pH of the medium is all important in regulating the formation of conidia and pigment production. The possible effects of the pH on the uptake of certain medium components is discussed, as well as their possible control of certain metabolic pathways which ultimately determines the availability of intermediates for conidiation and pigment production.     

Factors affecting sporulation by Phomopsis phaeolorum

Light and nutrition are the important factors in the production of pycnidia and conidia by cowpea isolates of the Phomopsis state of Diaporthe phaseolorum. The highest number of pycnidia and conidia were produced on plant tissue exposed to cool-white fluorescent light. In semisynthetic media more pycnidia were formed at high glucose concentrations, but they matured more slowly than those formed at lower glucose concentrations. Both the level of conidiation and the percentage of pycnidia that formed conidia were higher at lower glucose concentrations. The best artificial medium for inducing a high number of pycnidia containing abundant conidia was one that contained 0.4% glucose and 0.4% NaNO₃. A number of carbon sources could replace glucose in this medium.     

Conidiation of Hirsutella thompsonii var. synnematosa in submerged culture

Fourteen monosporal isolates of Hirsutella thompsonii grew vegetatively in various liquid media producing typical phialidic-like conidiophores. Hirsutella thompsonii var. synnematosa from Ivory Coast (HtIC) was the only pathotype which produced true conidia in submerged culture. HtIC began producing conidia after 3 days incubation reaching a peak ranging from 6.8 × 10⁵ to 9.7 × 10⁷ conidia/ml between 6 and 11 days. A concentration of 10 g/liter of corn steep liquor and 0.2% Tween 80 were essential for maximum conidiation. Submerged conidia had a smooth but somewhat rugose conidial walls, whereas aerially formed conidia were distinctly verrucose. Germination of submerged conidia ranged from 5.2 to 12.9% and were virulent causing 32.5% infection to adult citrus rust mites when sprayed on citrus foliage at 1.2 × 10⁹ conidia/ml.     

Developmental regulators in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

The filamentous fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing severe and usually fatal invasive aspergillosis in immunocompromised patients. This fungus produces a large number of small hydrophobic asexual spores called conidia as the primary means of reproduction, cell survival, propagation, and infectivity. The initiation, progression, and completion of asexual development (conidiation) is controlled by various regulators that govern expression of thousands of genes associated with formation of the asexual developmental structure conidiophore, and biogenesis of conidia. In this review, we summarize key regulators that directly or indirectly govern conidiation in this important pathogenic fungus. Better understanding these developmental regulators may provide insights into the improvement in controlling both beneficial and detrimental aspects of various Aspergillus species.     

Conidiogenous cell development inHelminthosporium carbonum

We devised a procedure to propagate selectively the vegetative and asexual-reproductive states ofHelminthosporium carbonum so that we could characterize morphological and subcellular events associated with the onset of conidiation. Solidified agar media were uniformly inoculated with an overlay of conidia suspended in molten agar. After the overlay solidified, it was covered with a sheet of Miracloth. When incubated in the dark, cultures produced abundant aerial hyphae that grew through the Miracloth layer and conidiation was suppressed for 48 to 50 h. Hyphae were easily harvested from the surface of the Miracloth with a spatula. When cultures were placed in the light after 38 h of growth in the dark, differentiation was detected in 90% of the hyphal tips within 8 to 10 h. The initial response of the hyphal tips, comprising early stages in conidiophore development, was rapid and highly synchronized. The behavior of nuclei during conidiogenous cell development and the initiation of conidia was similar to that reported for other fungi that form blastic conidia. One-dimensional gel electrophoresis ofin vitro translation products confirmed differences in poly(A) RNA populations from dark-grown and light-induced cultures.     

Carbon Concentration and Carbon-to-Nitrogen Ratio Influence Submerged-Culture Conidiation by the Potential Bioherbicide Colletotrichum truncatum NRRL 13737

We assessed the influence of various carbon concentrations and carbon-to-nitrogen (C:N) ratios on Colletotrichum truncatum NRRL 13737 conidium formation in submerged cultures grown in a basal salts medium containing various amounts of glucose and Casamino Acids. Under the nutritional conditions tested, the highest conidium concentrations were produced in media with carbon concentrations of 4.0 to 15.3 g/liter. High carbon concentrations (20.4 to 40.8 g/liter) inhibited sporulation and enhanced the formation of microsclerotiumlike hyphal masses. At all the carbon concentrations tested, a culture grown in a medium with a C:N ratio of 15:1 produced more conidia than cultures grown in media with C:N ratios of 40:1 or 5:1. While glucose exhaustion was often coincident with conidium formation, cultures containing residual glucose sporulated and those with high carbon concentrations (>25 g/liter) exhausted glucose without sporulation. Nitrogen source studies showed that the levels of C. truncatum NRRL 13737 conidiation were similar for all protein hydrolysates tested. Reduced conidiation occurred when amino acid and inorganic nitrogen sources were used. Of the nine carbon sources evaluated, acetate as the sole carbon source resulted in the lowest level of sporulation.