Conidial Wall Research Articles

The novel spore-specific regulator SscA controls Aspergillus conidiogenesis.

Filamentous fungi produce myriads of asexual spores, which are the main reproductive particles that act as infectious or allergenic agents. Although the serial of asexual sporogenesis is coordinated by various genetic regulators, there remain uncharacterized transcription factors in Aspergillus. To understand the underlying mechanism of spore formation, integrity, and viability, we have performed comparative transcriptomic analyses on three Aspergillus species and found a spore-specific transcription factor, SscA. SscA has a major role in conidial formation, maturation and dormancy, and germination in Aspergillus nidulans. Functional studies indicate that SscA coordinates conidial wall integrity, amino acid production, and secondary metabolism in A. nidulans conidia. Furthermore, the roles of SscA are conserved in other Aspergillus species. Our findings that the SscA has broad functions in Aspergillus conidia will help to understand the conidiogenesis of Aspergillus species.

ChsA, a Class Ⅱ Chitin Synthase, Contributes to Asexual Conidiation, Mycelial Morphology, Cell Wall Integrity, and the Production of Enzymes and Organic Acids in Aspergillus niger.

Chitin synthases (CHSs) are vital enzymes for the synthesis of chitin and play important and differential roles in fungal development, cell wall integrity, environmental adaptation, virulence, and metabolism in fungi. However, except for ChsC, a class III CHS, little is known about the functions of CHSs in Aspergillus niger, an important fungus that is widely applied in the fermentation industry and food processing, as well as a spoilage fungus of food and a human pathogen. This study showed the important functions of ChsA, a class II CHS, in A. niger using multi-phenotypic and transcriptional analyses under various conditions. The deletion of chsA led to severe defects in conidiation on different media and resulted in the formation of smaller and less compact pellets with less septa in hyphal cells during submerged fermentation. Compared with the WT, the ΔchsA mutants exhibited less chitin content, reduced growth under the stresses of cell wall-disturbing and oxidative agents, more released protoplasts, a thicker conidial wall, decreased production of amylases, pectinases, cellulases, and malic acid, and increased citric acid production. However, ΔchsA mutants displayed insignificant changes in their sensitivity to osmotic agents and infection ability on apple. These findings concurred with the alteration in the transcript levels and enzymatic activities of some phenotype-related genes. Conclusively, ChsA is important for cell wall integrity and mycelial morphology, and acts as a positive regulator of conidiation, cellular responses to oxidative stresses, and the production of malic acid and some enzymes, but negatively regulates the citric acid production in A. niger.

BbWor1, a Regulator of Morphological Transition, Is Involved in Conidium-Hypha Switching, Blastospore Propagation, and Virulence in Beauveria bassiana.

ABSTRACTMorphological transition is an important adaptive mechanism in the host invasion process. Wor1 is a conserved fungal regulatory protein that controls the phenotypic switching and pathogenicity of Candida albicans. By modulating growth conditions, we simulated three models of Beauveria bassiana morphological transitions, including CTH (conidia to hyphae), HTC (hyphae to conidia), and BTB (blastospore to blastospore). Disruption of BbWor1 (an ortholog of Wor1) resulted in a distinct reduction in the time required for conidial germination (CTH), a significant increase in hyphal growth, and a decrease in the yield of conidia (HTC), indicating that BbWor1 positively controls conidium production and negatively regulates hyphal growth in conidium-hypha switching. Moreover, ΔBbWor1 prominently decreased blastospore yield, shortened the G0/G1 phase, and prolonged the G2/M phase under the BTB model. Importantly, BbWor1 contributed to conidium-hypha switching and blastospore propagation via different genetic pathways, and yeast one-hybrid testing demonstrated the necessity of BbWor1 to control the transcription of an allergen-like protein gene (BBA_02580) and a conidial wall protein gene (BBA_09998). Moreover, the dramatically weakened virulence of ΔBbWor1 was examined by immersion and injection methods. Our findings indicate that BbWor1 is a vital participant in morphological transition and pathogenicity in entomopathogenic fungi.IMPORTANCE As a well-known entomopathogenic fungus, Beauveria bassiana has a complex life cycle and involves transformations among single-cell conidia, blastospores, and filamentous hyphae. This study provides new insight into the regulation of the fungal cell morphological transitions by simulating three models. Our research identified BbWor1 as a core transcription factor of morphological differentiation that positively regulates the production of conidia and blastospores but negatively regulates hyphal growth. More importantly, BbWor1 affects fungal pathogenicity and the global transcription profiles within three models of growth stage transformation. The present study lays a foundation for the exploration of the transition mechanism of entomopathogenic fungi and provides material for the morphological study of fungi.

The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana

Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A bar-specific short guide RNA was used to target and cause a double-strand break in bar, and a donor DNA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation.

Juglanconis gen. nov. on Juglandaceae, and the new family Juglanconidaceae (Diaporthales).

Molecular phylogenetic analyses of ITS-LSU rDNA sequence data demonstrate that Melanconis species occurring on Juglandaceae are phylogenetically distinct from Melanconis s.str., and therefore the new genus Juglanconis is described. Morphologically, the genus Juglanconis differs from Melanconis by light to dark brown conidia with irregular verrucae on the inner surface of the conidial wall, while in Melanconis s.str. they are smooth. Juglanconis forms a separate clade not affiliated with a described family of Diaporthales, and the family Juglanconidaceae is introduced to accommodate it. Data of macro- and microscopic morphology and phylogenetic multilocus analyses of partial nuSSU-ITS-LSU rDNA, cal, his, ms204, rpb1, rpb2, tef1 and tub2 sequences revealed four distinct species of Juglanconis. Comparison of the markers revealed that tef1 introns are the best performing markers for species delimitation, followed by cal, ms204 and tub2. The ITS, which is the primary barcoding locus for fungi, is amongst the poorest performing markers analysed, due to the comparatively low number of informative characters. Melanconium juglandinum (= Melanconis carthusiana), M. oblongum (= Melanconis juglandis) and M. pterocaryae are formally combined into Juglanconis, and J. appendiculata is described as a new species. Melanconium juglandinum and Melanconis carthusiana are neotypified and M. oblongum and Diaporthe juglandis are lectotypified. A short description and illustrations of the holotype of Melanconium ershadii from Pterocarya fraxinifolia are given, but based on morphology it is not considered to belong to Juglanconis. A key to all treated species of Juglanconis is provided.

A flavoprotein supports cell wall properties in the necrotrophic fungus Alternaria brassicicola

BackgroundFlavin-dependent monooxygenases are involved in key biological processes as they catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. Flavoprotein monooxygenases are frequently encountered in micro-organisms, most of which require further functional and biocatalytic assessment. Here we investigated the function of the AbMak1 gene, which encodes a group A flavin monooxygenase in the plant pathogenic fungus Alternaria brassicicola, by generating a deficient mutant and examining its phenotype.ResultsFunctional analysis indicates that the AbMak1 protein is involved in cell wall biogenesis and influences the melanization process. We documented a significant decrease in melanin content in the Δabmak1 strain compared to the wild-type and complemented strains. We investigated the cell wall morphology and physical properties in the wild-type and transformants using electron and atomic force microscopy. These approaches confirmed the aberrant morphology of the conidial wall structure in the Δabmak1 strain which had an impact on hydrophilic adhesion and conidial surface stiffness. However, there was no significant impairment in growth, conidia formation, pathogenicity or susceptibility to various environmental stresses in the Δabmak1 strain.ConclusionThis study sheds new light on the function of a fungal flavin-dependent monooxygenase, which plays an important role in melanization.

Morphological Characterization of Biocontrol Isolates of <i>Trichoderma</i> to Study the Correlation between Morphological Characters and Biocontrol Efficacy

The morphological characterization was carried out for 5 isolates of Trichoderma harzianum and 7 isolates of Trichoderma viride and tested for their biocontrol efficacy. The isolates belonging to T.harzianum were analogous in colony colour, culture smell, mycelial colour, conidiation, conidial shape, conidial wall and conidial colour. Correspondingly the isolates of T.viride showed certain similarity in colony colour, colony edge, culture smell, conidiophore branching, conidial wall, conidial colour and chlamydospores. Inter specific differences through cluster analysis based on morphological characters grouped the twelve isolates into three major clusters where all the isolates of T.harzianum formed a single cluster while the isolates of T.viride were bifurcated into two groups. The clustering was substantiated by similarity index which showed maximum similarity among T.harzianum isolates with only less than 20% variation among themselves. Similarly the clusters having isolates of T.viride also had less variation within them. The biocontrol efficacy of these twelve isolates of Trichoderma was experimented by dual culture test under laboratory condition and there existed some relation between the biocontrol efficacy of these isolates and morphology.

Morphological Characterization of Biocontrol Isolates of <i>Trichoderma</i> to Study the Correlation between Morphological Characters and Biocontrol Efficacy

The morphological characterization was carried out for 5 isolates of Trichoderma harzianum and 7 isolates of Trichoderma viride and tested for their biocontrol efficacy. The isolates belonging to T.harzianum were analogous in colony colour, culture smell, mycelial colour, conidiation, conidial shape, conidial wall and conidial colour. Correspondingly the isolates of T.viride showed certain similarity in colony colour, colony edge, culture smell, conidiophore branching, conidial wall, conidial colour and chlamydospores. Inter specific differences through cluster analysis based on morphological characters grouped the twelve isolates into three major clusters where all the isolates of T.harzianum formed a single cluster while the isolates of T.viride were bifurcated into two groups. The clustering was substantiated by similarity index which showed maximum similarity among T.harzianum isolates with only less than 20% variation among themselves. Similarly the clusters having isolates of T.viride also had less variation within them. The biocontrol efficacy of these twelve isolates of Trichoderma was experimented by dual culture test under laboratory condition and there existed some relation between the biocontrol efficacy of these isolates and morphology.

Effect of pH on the ultrastructure of the conidial wall ofAspergillus niger aggregate conidia and its relationship to zearalenone adsorption

The purpose of the present study was to evaluate the effect of pH on the ultrastructure of the conidial wall of dead conidia harvested from the non-toxicogenicAspergillus niger aggregate strain RC084 and its relationship to zearalenone (ZEA) adsorption capacity. Moreover, mathematical models were applied to explain the interaction between conidia and ZEA absorbance. A ZEA adsorption test was performed using a concentration of 1×107 dead conidia/ml at pH 2 and 6 at 37 °C for 30 min. Unbound ZEA was quantified by high-pressure liquid chromatography. The ZEA adsorption was strongly dependent on the pH of the medium; the highest values were 2.2×10-6 μg ZEA/conidia at pH 6. Isotherms representing the amount of bound ZEA as a function of ZEA concentration in equilibrium after adsorption have typical S- or L-shapes. The experimental data could be fitted to the Frumkin-Fowler-Guggenheim (FFG) and Hill theoretical models. The ultrastructure of the conidial wall was studied by infrared spectroscopy (IR) and transmission electron microscopy (TEM). The representative IR spectra showed that pH did not produce significant changes in the different chemical groups. However, ultrastructure studies by TEM detected considerable changes in the organisation of the conidial wall. This is the first study showing that the loss of the outermost electron dense layer, responsible for the ornamentations on the conidial surface. Adsorption is favoured at pH 6".

Intra- and interspecific diversity of ultrastructural markers in Scedosporium

Ultrastructural features of conidia, lateral walls of aerial and submerged hyphal cells, and of septal pore apparatus of Scedosporium apiospermum, S. boydii, Pseudallescheria angusta and Scedosporium aurantiacum were studied. Submerged hyphal cells possessed a thick extracellular matrix. Crystalline satellites accessory to the septal pore apparatus were revealed. Fundamental ultrastructural features appeared to be heterogeneous at low taxonomic levels. The closely interrelated members of the S. apiospermum complex showed quantitative ultrastructural variability, but the unambiguously different species S. aurantiacum deviated qualitatively by markers of conidial wall structure, Woronin bodies morphology and presence/absence of crystalline satellites of the septal pore apparatus.

The Aspergillus fumigatus septins play pleiotropic roles in septation, conidiation, and cell wall stress, but are dispensable for virulence

Septins are a conserved family of GTPases that regulate important cellular processes such as cell wall integrity, and septation in fungi. The requirement of septins for virulence has been demonstrated in the human pathogenic yeasts Candida albicans and Cryptococcus neoformans, as well as the plant pathogen Magnaporthe oryzae. Aspergillus spp. contains five genes encoding for septins (aspA–E). While the importance of septins AspA, AspB, AspC, and AspE for growth and conidiation has been elucidated in the filamentous fungal model Aspergillus nidulans, nothing is known on the role of septins in growth and virulence in the human pathogen Aspergillus fumigatus. Here we deleted all five A. fumigatus septins, and generated certain double and triple septin deletion strains. Phenotypic analyses revealed that while all the septins are dispensable in normal growth conditions, AspA, AspB, AspC and AspE are required for regular septation. Furthermore, deletion of only the core septin genes significantly reduced conidiation. Concomitant with the absence of an electron-dense outer conidial wall, the ΔaspB strain was also sensitive to anti-cell wall agents. Infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, but no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Although the deletion of aspB resulted in increased release of TNF-α from the macrophages, no significant inflammation differences in lung histology was noted between the ΔaspB strain and the wild-type strain. Taken together, these results point to the importance of septins in A. fumigatus growth, but not virulence in a murine model.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

OPTIMIZATION OF HYDROLYSIS TO PRODUCE WATER-SOLUBLE CHITOSAN AND ITS ANTIFUNGAL EFFECT ON POSTHARVEST ANTHRACNOSE IN MANGO

Water-soluble chitosan, the product of chitosan hydrolysis with particular biological characteristics, has been used in the fields of food storage and plant protection. Hydrogen peroxide was used to optimally degrade the crude chitosan into water-soluble chitosan (WSC). The mathematical model between independent factors (H2O2 level, time and temperature) and the recovery of WSC was constructed to optimize the degradation conditions using response surface methodology. The optimal conditions for highest recovery of WSC were 5.4% of H2O2, 47.1°C and 3.4 h, with the predicted recovery of 87.2%. The fungal strain Colletotrichum gloeosporioides, isolated from mango naturally infected by typical anthracnose, was used to study the antifungal activity of WSC in vitro. Results indicated that C. gloeosporioides was much sensitive to WSC, the inhibitory effect increased as the WSC increased. The concentration of 0.8% WSC completely inhibited the mycelial growth of C. gloeosporioides on PDA media and the 50% PIRG (percentage inhibition of radial growth) value was achieved with the concentration of 0.1% WSC. The micrograph study showed that WSC can cause distortion, rupture, and thinning of the conidial cell wall of C. gloeosporioides. These results indicate the high potential of WSC as an antifungal agent for future commercial application.

Cell wall modifications during conidial maturation of the human pathogenic fungus Pseudallescheria boydii.

Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses.

English

This study shows the conidiomatal development of Pestalotiopsis disseminata (Thuen.) Stey., using light and transmission electron microscope. Light microscopic study showed the presence of non-ostiolate pycnidial conidiomata in culture, whereas it is known to produce only acervular conidiomata on leaves. Interestingly, the fungus showed the ultrastructure of the conidial wall in the coloured cells as well as the basal and apical hyaline cells with the appendages. P. disseminata was studied for the first time on the development of the conidiomata and conidia by light and transmission electron microscopes.   Key words: Coelomycetes, Conidiomata, conidium ontogeny, appendages.

Comparative Study of Biological Agents, Trichoderma harzianum (Th- Azad) and Trichoderma viride (01PP) for Controlling Wilt Disease in Pigeon Pea

The paper aims to study the morphological, physiological, molecular characterization and bio-formulation of Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315, an effective fungicide and a biological control agent too that protects the plants and seeds from other pathogenic fungi. The physiologic study is done in an attempt to find the effective management of the disease caused by soil borne pathogens. Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315 isolated from the infected soil samples of Pigeon pea fields and grown at preferable temperatures, pH and different solid and liquid culture media. Most preferable temperature for the growth and sporulation of Trichoderma harzianum (Th Azad) and Trichoderma viride 01PP-8315 has been observed up to 30oC (210.5 mg dry weight of mycelium). A detailed morphology of the strain is done in this study including colony growth rate, colony color, colony edge, mycelial form, conidiation, conidiophore branching, conidial wall, conidial color, etc. The molecular characterization of the strain is carried out using an 18S rRNA gene sequence with the help of a universal Internal Transcribed Spacer marker that gives an amplicon of a total of 1173 base pairs and 546 bp of the 18S rRNA gene was sequenced and used for the identification of isolated fungal strains that is later sequenced and allotted with Gene Bank Accession no. JX119211 and KC800922 respectively. To check the presence of endochitinase gene in two of the potential strains of Trichoderma species viz. T. harzianum (Th Azad) and T. viride 01PP an ech42 primer was used. A talc based bio-formulation has been prepared with this strain where the population of the spores was found to decline after 180 days.

P144 Pathogen recognition by the long pentraxin PTX3: Roles of glycosylation and interplay with complement

Introduction The long pentraxin PTX3 is a soluble Pattern Recognition Molecule (sPRM) with non-redundant roles in resistance to selected pathogens, such as the opportunistic fungus Aspergillus fumigatus . PTX3 facilitates recognition and phagocytosis of fungal conidia mainly by polymorphonuclear neutrophils (PMNs) via Fc γ receptors (Fc γ Rs) and alternative complement pathway (ACP) mechanisms. The human PTX3 protein contains a single N-glycosylation site with complex type sialylated oligosaccharides that has been involved in a number of PTX3 functions [1] . The present study was designed to characterize the role of this post translational modification in the PTX3-depedent recognition and phagocytosis of A. fumigatus . This microorganism has developed several strategies to evade the immune response, such as attracting onto the conidial wall inhibitors of the ACP, like factor H (FH, a ligand of PTX3). Here we present preliminary evidences on the effect of PTX3 on FH engagement by A. fumigatus . Methods Recombinant as well as natural human PTX3 (i.e., from PMNs) was purified by immunoaffinity chromatography and the attached oligosaccharides were analyzed using glycosidases, lectin blotting and mass spectrometry. A range of PTX3 glycoforms were made in vitro by enzymatic hydrolysis of terminal monosaccharides of the recombinant protein. The opsonic and pro-phagocytic activities of these variants were characterized in buffer solutions and whole human peripheral blood, respectively, by flow cytometry. The influence of PTX3 on FH binding to conidia was studied in competition assays using microtiter plates. Results Complex-type sugars were found on PTX3 of both natural and recombinant origins. These showed heterogeneity with regard to the relative content of terminal sialic acid (SA) and bi-, tri- and tetrantennary glycans, depending on the cellular source. Enzymatic removal of SA caused ∼50% reduction of PTX3 binding to A. fumigatus conidia, with no further change due to additional deglycosylation. Conversely, saccharide hydrolysis had no effect on PTX3 binding to PMNs, where Fc γ Rs have been proposed as putative receptors of this pentraxin [1] . However, consistent with binding data from conidia, desialylated PTX3 showed reduced potentiation of conidia phagocytosis by PMNs as compared to the unmodified protein. Also, we observed that PTX3 inhibits the binding of FH to A. fumigatus conidia, even when these were pre-opsonised with the long pentraxin and FH was applied at physiologically relevant concentrations. Conclusion Different pathogens express neuraminidases on their surface during infection and natural immunity cells, such as PMNs, mobilize intracellular neuraminidases to the plasma membrane upon inflammation. Here, we report that the glycosylation status of the sPRM PTX3 changes, with respect to SA, depending on cellular source and perhaps inducing stimuli. We describe that SA modulates the PTX3-dependent recognition and phagocytosis of A. fumigatus , which points to glycosylation as a strategy to tune PTX3 functions in innate immunity. Furthermore, we provided evidences that PTX3 impairs the interaction of FH with A. fumigatus , where this might link to a new mechanism to counterbalance the complement evasion strategy exploited by this pathogen.

Potential Application in Animal Feed of Phytase Produced from Agro-Industrial Residues by Aspergillus japonicus

of the plant wall cell, as cellulose, hemicellulose, lignin and pectins are degraded by enzymatic systems produced by a wide variety of saprophytic and phythopathogenic microorganisms, including bacteria and fungi. The aim of this work was to produce avicelase, carboxymethylcellulase, xylanase and pectinase from the exo-1 mutantNeurospora crassa, FGSC 2256 and thewild typeNeurospora crassa, FGSC 424. The exo-1 mutant has a pleiotropic mutation which confers derepression of alpha-amylase, glucoamylase, betafructofuranosidase, and trehalase, as previously documented. This mutation appears to also affect the composition of the cell wall, since segregants resulting from the backcross of exo-1 to the wild-type strain from which it derived are altered in the ratio of galactosamine to glucosamine in the cell wall. Conidial cell walls of exo-1 mutant exhibit a marked decrease in the amount of galactosamine. In this work, increased levels (approximately sixfold) of pectinasewere found in conidia of exo-1 as comparedwith those of thewild type andalso thepolysaccharide-degrading activitieswere preferentially induced and secreted by the exo-1mutant compared with wild type cells, in the presence of inducers. Then, in culture media supplemented with 1% avicel from the exo-1 mutant were observed higher levels of avicelase (10.6-fold), carboxymethylcellulase (8.92-fold) and xylanase (4.74-fold) than in the wild type. Galactose was better inducer of pectinase than pectin and the exo1 mutant produced three-fold more polygalacturonase than the wild type. Constitutive levels of polygalacturonase and cellulolytic enzymes were detected in medium devoid of carbon source. This results show the importance of composition of the fungal cell wall in the regulation and secretion of industrial enzymes with application in the biomass treatment and biofuel biotechnology. Support: FAPESP and CNPq

Hydrophobicity-Related Protein Contents and Surface Areas of Aerial Conidia are Useful Traits for Formulation Design of Fungal Biocontrol Agents

To clarify the potential use of hydrophobicity-related traits of aerial conidia in formulation design of fungal biocontrol agents, hydrophobicity rates (H (r)) and surface areas (S (a)) of aerial conidia were assessed with 48 strains of Beauveria bassiana, Isaria fumosorosea and Metarhizium spp. Inter- or intra-specific variation was large in H (r) (59.7-92.2%) and S (a) (7.9-25.3 microm(2) conidium(-1)) measurements, which were significantly correlated (r (2) = 0.55). Six isolates of the three fungi with distinguished H (r) and S (a) were further studied. Conidial wall proteins of these isolates were sequentially extracted with sodium dodecyl sulfate (SDS), formic acid (FA) and trifluoroacetic acid (TFA). Their H (r) values were significantly correlated to the contents (P (c)) of TFA-soluble, but FA-insoluble, proteins (2.7-44.8 microg per 10(7) conidia; r (2) = 0.79) and reduced drastically by the FA/TFA treatments, which eliminated the hydrophobin-based rodlet layers of conidial surfaces. However, the SDS treatments had no effect on either H (r) or rodlet layers. The dispersancy of a tested emulsifier to oil formulations of the six isolates in water was adversely correlated to their H (r) (r (2) = 0.94). The results indicate that both P (c) and S (a) are inherent hydrophobicity-related traits and can be utilized to select fungal biocontrol candidates for improved formulation and application.

A new non-hydrophobic cell wall protein (CWP10) of Metarhizium anisopliae enhances conidial hydrophobicity when expressed in Beauveria bassiana

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.