Conidial Development Research Articles

Study of biodegradation of chloramphenicol by endophytic fungi isolated from Bertholletia excelsa (Brazil nuts)

Chloramphenicol (CAP) is a micropollutant that resists to conventional residual water treatment. Therefore, the aim of this study was to assess CAP biodegradability by five endophytic fungi strains isolated from Bertholletia excelsa collected in the Brazilian Amazonia. The fungi strains were screened in solid and liquid medium, and then experimental design was performed to optimize culture conditions. In addition, an environmental toxicology assessment was carried out using the algae Chlorella vulgaris. Results from fungi cultures in solid medium demonstrated that CAP affected the strains growth and interfered in the development of conidia and spores. The biodegradation in liquid culture medium showed that all strains increased the degradation of this antibiotic. The most efficient strain Trichoderma sp. (BIORG 7) was subjected to an experimental design (Box-behnken) consisted of 15 experiments, having as variables: pH, time, and CAP concentration, and reaching 30% biodegradation determined by HPLC-UV analyses (24 h, pH 7.0 and 150 mg L−1). The main metabolite 4-nitrobenzaldehyde was identified by GC-MS analyses and presented a higher ecotoxicity to green algae than CAP.

Evaluation of a novel endophytic Pseudomonas lactis strain for control of boxwood blight1

Abstract Boxwood blight, caused by Calonectria pseudonaviculata (Cps) (Synonyms: Cylindrocladium buxicola, Cylindrocladium pseudonaviculatum), is a devastating disease which affects boxwood (Buxus) in private and public gardens worldwide. SW, an endophyte identified as a strain of Pseudomonas lactis, was isolated along with seven other bacterial strains from symptom-reversed boxwood leaves infected by Cps. SW had the largest population among the 8 isolates. SW inhibited Cps culture growth similar to three other antagonistic isolates but was the strongest suppressor of Cps conidial germination and germling development. When the cell-free supernatant (CFS) from SW liquid cultures was used to treat boxwood plants 48 h prior to inoculation with Cps, it resulted in a 69.4% disease reduction; suggesting involvement of its metabolites in disease suppression. Boxwood blight control efficacy was further evaluated with resuspended SW cell at 108-9 cfu.ml−1 at different treatment lead times. When SW was applied 2 or 10 days before inoculation with Cps, boxwood blight disease decreased by 72% and 67%, respectively. Further extending the lead time to 20 and 30 days, the efficacy was reduced to 26-27%. However, with its features of moderate control efficacy, high population in plant tissue and safety towards humans, SW shows great potential as a biocontrol agent for boxwood blight. Index words: Biocontrol agent, endophyte, plant pretreatment, Calonectria pseudonaviculata suppression. Species used in this study: Bacterium strain SW (Pseudomonas lactis). Plant species: Buxus sempervirens ‘Justin Brouwers'.

The Early Asexual Development Regulator fluG Codes for a Putative Bifunctional Enzyme.

FluG is a long recognized early regulator of asexual development in Aspergillus nidulans. fluG null mutants show profuse aerial growth and no conidial production. Initial studies reported sequence homology of FluG with a prokaryotic type I glutamine synthetase, but catalytic activity has not been demonstrated. In this study, we conducted an in-depth analysis of the FluG sequence, which revealed a single polypeptide containing a putative N-terminal amidohydrolase region linked to a putative C-terminal γ-glutamyl ligase region. Each region corresponded, separately and completely, to respective single function bacterial enzymes. Separate expression of these regions confirmed that the C-terminal region was essential for asexual development. The N-terminal region alone did not support conidial development, but contributed to increased conidial production under high nutrient availability. Point mutations directed at respective key catalytic residues in each region demonstrated that they were essential for biological function. Moreover, the substitution of the N- and C-terminal regions with homologs from Lactobacillus paracasei and Pseudomonas aeruginosa, respectively, maintained functionality, albeit with altered characteristics. Taken together, the results lead us to conclude that FluG is a bifunctional enzyme that participates in an as yet unidentified metabolic or signaling pathway involving a γ-glutamylated intermediate that contributes to developmental fate.

The Zn(II)2Cys6-Type Transcription Factor ADA-6 Regulates Conidiation, Sexual Development, and Oxidative Stress Response in Neurospora crassa.

Conidiation and sexual development are critical for reproduction, dispersal and better-adapted survival in many filamentous fungi. The Neurospora crassa gene ada-6 encodes a Zn(II)2Cys6-type transcription factor, whose deletion resulted in reduced conidial production and female sterility. In this study, we confirmed the positive contribution of ada-6 to conidiation and sexual development by detailed phenotypic characterization of its deletion mutant and the complemented mutant. To understand the regulatory mechanisms of ADA-6 in conidiation and sexual development, transcriptomic profiles generated by RNA-seq from the Δada-6 mutant and wild type during conidiation and sexual development were compared. During conidial development, differential expressed genes (DEGs) between the Δada-6 mutant and wild type are mainly involved in oxidation-reduction process and single-organism metabolic process. Several conidiation related genes are positively regulated by ADA-6, including genes that positively regulate conidiation (fluffy and acon-3), and genes preferentially expressed during conidial development (eas, con-6, con-8, con-10, con-13, pcp-1, and NCU9357), as the expression of these genes were lower in the Δada-6 mutant compared to wild type during conidial development. Phenotypic observation of deletion mutants for other genes with unknown function down-regulated by ada-6 deletion revealed that deletion mutants for four genes (NCU00929, NCU05260, NCU00116, and NCU04813) produced less conidia than wild type. Deletion of ada-6 resulted in female sterility, which might be due to that ADA-6 affects oxidation-reduction process and transmembrane transport process, and positively regulates the transcription of pre-2, poi-2, and NCU05832, three key genes participating in sexual development. In both conidiation and the sexual development process, ADA-6 regulates the transcription of cat-3 and other genes participating in reactive oxygen species production according to RNA-seq data, indicating a role of ADA-6 in oxidative stress response. This was further confirmed by the results that deletion of ada-6 led to hypersensitivity to oxidants H2O2 and menadione. Together, these results proved that ADA-6, as a global regulator, plays a crucial role in conidiation, sexual development, and oxidative stress response of N. crassa.

Transcription factor CgAzf1 regulates melanin production, conidial development and infection in Colletotrichum gloeosporioides.

Rubber anthracnose caused by Colletotrichum gloeosporioides leads to huge economic loss in the natural rubber industry every year. Conidia of C. gloeosporioides are a major infection source but little is known about molecular mechanisms underlying conidial development and infection. In this study, the C. gloeosporioide C2H2 zinc-finger protein transcription factor gene CgAzf1 is shown to be involved in melanin production, conidial development and infection. Deletion of CgAzf1 resulted in decreased melanin production and hydrophilicity of aerial mycelium was increased. The mutants also showed reduced conidiation, low germination rate, and the formation of appressorium lagged too. Virulence assays showed that the CgAzf1 deletion strain could not infect intact rubber tree leaves and had an attenuated virulence on the wounded leaves. Quantitative RT-PCR showed that CgAzf1 regulates expression of genes involved in the MAPK, cAMP-PKA and melanin biosynthesis pathways.

Dual role for fungal-specific outer kinetochore proteins during cell cycle and development in Magnaporthe oryzae.

The outer kinetochore DASH complex (also known as the Dam1 complex) ensures proper spindle structure and chromosome segregation. While DASH complex protein requirement diverges among different yeasts, its role in filamentous fungi has not yet been investigated. We studied the dynamics and role of middle (Mis12) and outer (Dam1 and Ask1) kinetochore proteins in the filamentous fungal pathogen, Magnaporthe oryzae, which undergoes multiple cell cycle-linked developmental transitions. While Mis12 was constitutively present in the nucleus, Dam1 and Ask1 were recruited only during mitosis. Although Dam1 was not required for viability, loss of its function (dam1Δ) delayed mitotic progression, resulting in impaired conidial and hyphal development. Both Dam1 and Ask1 also localised to the hyphal tips, in the form of punctae oscillating back and forth from the growing ends, suggesting that Magnaporthe DASH complex proteins may play a non-canonical role in polarised growth during interphase, in addition to their function in nuclear segregation during mitosis. Impaired appressorial (infection structure) development and host penetration in the dam1Δ mutant suggest that fungus-specific Dam1 complex proteins could be an attractive target for a novel anti-fungal strategy.This article has an associated First Person interview with the first author of the paper.

Changes in the Biophysical Properties of the Cell Membrane Are Involved in the Response of Neurospora crassa to Staurosporine.

Neurospora crassa is a non-pathogenic filamentous fungus widely used as a multicellular eukaryotic model. Recently, the biophysical properties of the plasma membrane of N. crassa conidia were thoroughly characterized. They evolve during conidial germination at a speed that depends on culture conditions, suggesting an important association between membrane remodeling and the intense membrane biogenesis that takes place during the germinative process. Staurosporine (STS) is a drug used to induce programmed cell death in various organisms. In N. crassa, STS up-regulates the expression of the ABC transporter ABC-3, which localizes at the plasma membrane and pumps STS out. To understand the role of plasma membrane biophysical properties in the fungal drug response, N. crassa was subjected to STS treatment during early and late conidial development stages. Following 1 h treatment with STS, there is an increase in the abundance of the more ordered, sphingolipid-enriched, domains in the plasma membrane of conidia. This leads to higher fluidity in other membrane regions. The global order of the membrane remains thus practically unchanged. Significant changes in sphingolipid-enriched domains were also observed after 15 min challenge with STS, but they were essentially opposite to those verified for the 1 h treatment, suggesting different types of drug responses. STS effects on membrane properties that are more dependent on ergosterol levels also depend on the developmental stage. There were no alterations on 2 h-grown cells, clearly contrasting to what happens at longer growth times. In this case, the differences were more marked for longer STS treatment, and rationalized considering that the drug prevents the increase in the ergosterol/glycerophospholipid ratio that normally takes place at the late conidial stage/transition to the mycelial stage. This could be perceived as a drug-induced development arrest after 5 h growth, involving ergosterol, and pointing to a role of lipid rafts possibly related with an up-regulated expression of the ABC-3 transporter. Overall, our results suggest the involvement of membrane ordered domains in the response mechanisms to STS in N. crassa.

ANXC7 Is a Mitochondrion-Localized Annexin Involved in Controlling Conidium Development and Oxidative Resistance in the Thermophilic Fungus Thermomyces lanuginosus.

Annexins (ANXs) are widely expressed and structurally related proteins which play multiple biological roles in animals, plants, and fungi. Although ANXs have been localized to the cytosol and the cell membrane and the molecular basis of the four annexin repeats is well established, the in vivo roles of these proteins are still far from clear, particularly with regard to the filamentous fungi. Thermomyces lanuginosus, a thermophilic fungus, is widely used in the fermentation industry; however, the role of ANX in this organism is unknown. In this study, a single ANX homologue (ANXC7) was identified and characterized in T. lanuginosus. The expression pattern indicated that ANXC7 is closely associated to conidium development, and it accumulated in the mitochondria of the forming conidia. The deletion of ANXC7 (ΔANXC7) resulted in no obvious phenotype related to colony growth on solid CM medium. However, when ΔANXC7 was grown in CM liquid culture, the mycelium masses appeared to be larger and looser compared to the wild-type. Additionally, the dry weight of the mutant mycelia was significantly increased. Under conditions that compromise cell-wall integrity, ΔANXC7 was less vulnerable than the wild-type with regard to such damage. Moreover, based on a surface hydrophobicity test, the ΔANXC7 strain was clearly less hydrophobic. The growth of ΔANXC7 was inhibited when grown under selected stress conditions, particularly with regard to salt stress; however, the oxidative resistance to exogenous H2O2 in ΔANXC7 was increased, and endogenous H2O2 levels within the ΔANXC7 were lower than in the wild-type, thereby suggesting that the ANXC7 specifically controls oxidative resistance. Based on microscopic observation, 4-day-conidia were more prevalent than 5-day conidia on the conidiophore stalk of ΔANXC7, even though the ΔANXC7 demonstrated an increased production of conidia during these days, indicating precocious conidial maturation and shedding from the conidiophore stalk in this strain. Taken together, our data indicate that ANXC7 localizes to the mitochondria and is involved in controlling conidium development and oxidative resistance in T. lanuginosus.

Evolution of asexual and sexual reproduction in the aspergilli.

Aspergillus nidulans has long-been used as a model organism to gain insights into the genetic basis of asexual and sexual developmental processes both in other members of the genus Aspergillus, and filamentous fungi in general. Paradigms have been established concerning the regulatory mechanisms of conidial development. However, recent studies have shown considerable genome divergence in the fungal kingdom, questioning the general applicability of findings from Aspergillus, and certain longstanding evolutionary theories have been questioned. The phylogenetic distribution of key regulatory elements of asexual reproduction in A. nidulans was investigated in a broad taxonomic range of fungi. This revealed that some proteins were well conserved in the Pezizomycotina (e.g. AbaA, FlbA, FluG, NsdD, MedA, and some velvet proteins), suggesting similar developmental roles. However, other elements (e.g. BrlA) had a more restricted distribution solely in the Eurotiomycetes, and it appears that the genetic control of sporulation seems to be more complex in the aspergilli than in some other taxonomic groups of the Pezizomycotina. The evolution of the velvet protein family is discussed based on the history of expansion and contraction events in the early divergent fungi. Heterologous expression of the A. nidulans abaA gene in Monascus ruber failed to induce development of complete conidiophores as seen in the aspergilli, but did result in increased conidial production. The absence of many components of the asexual developmental pathway from members of the Saccharomycotina supports the hypothesis that differences in the complexity of their spore formation is due in part to the increased diversity of the sporulation machinery evident in the Pezizomycotina. Investigations were also made into the evolution of sex and sexuality in the aspergilli. MAT loci were identified from the heterothallic Aspergillus (Emericella) heterothallicus and Aspergillus (Neosartorya) fennelliae and the homothallic Aspergillus pseudoglaucus (=Eurotium repens). A consistent architecture of the MAT locus was seen in these and other heterothallic aspergilli whereas much variation was seen in the arrangement of MAT loci in homothallic aspergilli. This suggested that it is most likely that the common ancestor of the aspergilli exhibited a heterothallic breeding system. Finally, the supposed prevalence of asexuality in the aspergilli was examined. Investigations were made using A. clavatus as a representative ‘asexual’ species. It was possible to induce a sexual cycle in A. clavatus given the correct MAT1-1 and MAT1-2 partners and environmental conditions, with recombination confirmed utilising molecular markers. This indicated that sexual reproduction might be possible in many supposedly asexual aspergilli and beyond, providing general insights into the nature of asexuality in fungi.

Adaption to stress via Pbs2 during Metarhizium rileyi conidia and microsclerotia development.

The high osmolarity glycerol (HOG) pathway plays important role in Metarhizium rileyi microsclerotia (MS) development. To investigate how M. rileyi transduce growth stress and regulate MS development via mitogen-activated protein kinase kinase (MAPKK) Pbs2, phenotypic characterization of the yeast Pbs2 homolog were performed. Expression of pbs2 peaked when MS formation occurred day 3 in liquid amended medium. Compared with wild-type and complemented strains, deletion mutant of pbs2 (Δpbs2) delayed dimorphic switch and vegetative growth, displayed sensitivities to various stress, and significantly reduced conidial (98%) and MS (40%) yields. Furthermore, transcription analysis showed that other genes of HOG signaling pathway were down-regulated in Δpbs2 mutants. Insect bioassays revealed that Δpbs2 mutants had decreased virulence levels in topical (24%) and injection (53%) bioassays. This study confirmed that Pbs2 play important roles in colony morphology, conidiation, stresses response and MS development in M. rileyi.

Hamatispora, a new genus of aquatic fungi in Microthyriales isolated from fallen leaves in Vietnam

Abstract A new aquatic fungus was isolated from submerged, decaying leaves collected at Phu Quoc Island in Vietnam. The fungus produced hyaline, unique-shaped conidia consisting of a hook-shaped main axis and three arms at the helicoid part of the main axis. Based on its conidial development and morphological characteristics, Hamatispora phuquocensis was newly introduced. Phylogenetic analyses based on LSU nrDNA sequences showed that it clusters with Microthyrium spp. and belongs to Microthyriales. Furthermore, we generated ITS barcode for this hyphomycetous fungus.

A MYST Histone Acetyltransferase Modulates Conidia Development and Secondary Metabolism in Pestalotiopsis microspora, a Taxol Producer

Reverse genetics is a promising strategy for elucidating the regulatory mechanisms involved in secondary metabolism and development in fungi. Previous studies have demonstrated the key role of histone acetyltransferases in transcriptional regulation. Here, we identified a MYST family histone acetyltransferase encoding gene, mst2, in the filamentous fungus Pestalotiopsis microspora NK17 and revealed its role in development and secondary metabolism. The gene mst2 showed temporal expression that corresponded to the conidiation process in the wild-type strain. Deletion of mst2 resulted in serious growth retardation and impaired conidial development, e.g., a delay and reduced capacity of conidiation and aberrant conidia. Overexpression of mst2 triggered earlier conidiation and higher conidial production. Additionally, deletion of mst2 led to abnormal germination of the conidia and caused cell wall defects. Most significantly, by HPLC profiling, we found that loss of mst2 diminished the production of secondary metabolites in the fungus. Our data suggest that mst2 may function as a general mediator in growth, secondary metabolism and morphological development.

The Myosin5-mediated actomyosin motility system is required for Verticillium pathogenesis of cotton.

The vascular wilt fungus Verticillium dahliae is one of the most destructive pathogens of cotton (Gossypium hirsutum) and many other economically important dicot plants. Fungal pathogens require Myosin-mediated actomyosin motility system for colonization of their host plants; however, the mechanisms underlying this process have not been fully characterized for V. dahliae. Here, in a knock-out experiment, we characterized the role of VdMyo5, a member of the Myosin V family, before and during infection of cotton and Arabidopsis thaliana. The VdMyo5 deletion mutant (ΔVdmyo5) fungi showed obvious defects in the development of conidia and the polarized elongation of vegetative hyphae, but no inhibition of host root penetration. Overall, the ΔVdmyo5 fungi exhibited dramatically reduced virulence in cotton and Arabidopsis, with almost no colonization in sections of host vascular tissue. We found labelled Myosin5-GFP to be specifically enriched at the hyphal tip, co-localized with FM4-64 labelled Spitzenkörper, which is the vesicle supply centre in filamentous fungi. Comparative secretome analysis revealed that proteins associated with cell wall modification and degradation of reactive oxygen species were significantly altered in mutant strains. Our results indicate that Myosin5 is required for vegetative growth and full virulence, possibly by regulating vesicle transport. The findings provide important insight into the cellular mechanisms of Verticillium pathogenesis.

Impact of Cationic and Neutral Gemini Surfactants on Conidia and Hyphal Forms of Aspergillus brasiliensis.

This study investigates the biological activity of two cationic gemini surfactants, hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) C6 and pentamethylene-1,5-bis-(N,N-dimethyl-N-dodecylammonium bromide) C5, and their two neutral analogs, hexamethylene-1,6-bis-(N-methyl-N-dodecylamine) (A6) and pentamethylene-1,5-bis-(N-methyl-N-dodecylamine) (A5). Experiments were performed with Aspergillus brasiliensis, which is used in the standard tests for biocides. The minimal inhibitory concentration (MIC) values for conidia and mycelium were determined using the dilution method. The viability of the conidia was evaluated using the plate count method. The dry mass of the mycelium was determined using the thermogravimetric method. Ergosterol was extracted from the mycelium and evaluated by gas chromatography. The effect of gemini surfactants on fungal morphology was observed using scanning electron microscopy. Cationic gemini surfactants were found to be active at lower concentrations compared to their non-ionic analogues, rapidly reducing the total number of conidia that were able to grow. They also decreased both the ergosterol content in the mycelium and its dry weight. These results suggest that cationic gemini surfactants C6 and C5 could have a wide range of practical applications as active compounds. However, it should be remembered that usage at too low concentrations, below the MIC, will only lead to short-term disturbances in the development of conidia and mycelium.

Two members of the velvet family, VmVeA and VmVelB, affect conidiation, virulence and pectinase expression in Valsa mali.

Velvet protein family members are important fungal-specific regulators which are involved in conidial development, secondary metabolism and virulence. To gain a broader insight into the physiological functions of the velvet protein family of Valsa mali, which causes a highly destructive canker disease on apple, we conducted a functional analysis of two velvet protein family members (VmVeA and VmVelB) via a gene replacement strategy. Deletion mutants of VmVeA and VmVelB showed increased melanin production, conidiation and sensitivity to abiotic stresses, but exhibited reduced virulence on detached apple leaves and twigs. Further studies demonstrated that the regulation of conidiation by VmVeA and VmVelB was positively correlated with the melanin synthesis transcription factor VmCmr1. More importantly, transcript levels of pectinase genes were shown to be decreased in deletion mutants compared with those of the wild-type during infection. However, the expression of other cell wall-degrading enzyme genes, including cellulase, hemi-cellulase and ligninase genes, was not affected in the deletion mutants. Furthermore, the determination of pectinase activity and immunogold labelling of pectin demonstrated that the capacity for pectin degradation was attenuated as a result of deletions of VmVeA and VmVelB. Finally, the interaction of VmVeA with VmVelB was identified through co-immunoprecipitation assays. VmVeA and VmVelB play critical roles in conidiation and virulence, probably via the regulation of the melanin synthesis transcription factor VmCmr1 and their effect on pectinase gene expression in V.mali, respectively.

Zinc Transporter Protein (tzn-1) May Also Play a Role in Conidiation Pathway of Neurospora crassa: An Insight Using Proteogenomic Approach.

Zinc transporter (tzn-1) of Neurospora crassa plays a crucial role in conidiation pathway, as its removal results in aconidiation which was reported in our earlier studies. The main objective of this study was to analyze the role of tzn-1 in conidiation process, by comparing knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) with wild oak ridge (OR) 74 'A' strain by 'Proteo-genomic' approach. To identify the commonly expressed protein spots in knockout (KO) mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) by comparing with wild oak ridge (OR) 74 'A' strain. Two sets (Δtzn-1 to wild and Δacon-3 to wild) were analyzed by combining 2- Dimensional gel electrophoresis (2DE) with Matrix Associated Laser Desortion/Ionization mass spectrometry -Peptide Mass Fingerprint (MALDI-PMF). Then, the peptide sequences which were obtained by MASCOT (database software) were identified by FGSC BLASTp search analysis. Finally, to evaluate the expression of the KO mutants zinc transporter KO (Δtzn-1) and aconidiating gene KO (Δacon-3) in comparison to wild (OR) 74 'A' type was analyzed by Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) studies. 2DE and MALDI-PMF has shown the nine commonly overexpressed protein spots from the two sets (Δtzn-1 to wild and Δacon-3 to wild). Peptide sequences were obtained by MASCOT (database software) analysis and peptide sequences were identified by FGSC BLASTp search. Eight sequences have shown the similarities with the genes involved during the early stages of conidial and sexual development. Our qRT-PCR analysis has shown that tzn-1 gene was upregulated in contrast to acon-3 gene in absence of iron concentration and down regulated with increase in iron concentrations in wild samples. With increase in zinc supplements, the tzn-1 gene is normally regulated and shown contrasting feature in absence of zinc and acon-3 gene is normally regulated both in presence and absence of zinc. At regular time intervals, declined growth rate was observed after 18hours of induction. Thus, we conclude that tzn-1 and acon-3 genes were actively participating in early stages of conidial process and metal ions play some crucial role in the development of the organism.

Comparative colonisation by virulent versus avirulent Pyricularia oryzae on wild Oryza australiensis

Pyricularia oryzae (rice blast) conidial development at pre-penetration stage determines success or otherwise of infection inside the rice host plants. Studies on conidial germination and growth on the leaf surface in commercial rice (Oryza sativa) report differently, dependent upon host type and level of blast resistance. Although wild rice (O. australiensis) is known to be an alternative host of blast, the interaction between P. oryzae conidia and wild O. australiensis on its leaf surface has not been previously studied. We found significant (P < 0.001) differences in conidial development between two blast isolates with different virulence in terms of conidial germination, germ tube growth and appressoria formation on both wild and cultivated rice. Conidial germination at 6 h post-inoculation (hpi) for the virulent isolate was significantly (P < 0.001) delayed. Germ tubes of the avirulent isolate conidia grew significantly (P < 0.001) faster and with significantly (P < 0.001) longer germ tubes than from virulent conidia. Appressoria development for the virulent isolate was significantly (P < 0.001) faster at its later growth stages of 12 and 18 hpi when approximately 100% of germ tubes formed appressoria. In contrast, formation rate of appressoria for the avirulent isolate was significantly (P < 0.001) slower and only reached 76% of germ tubes forming appressoria. Appressoria formation on O. australiensis was significantly (P < 0.001) greater than the formation on O. sativa for both virulent and avirulent P. oryzae at 12 hpi, a clear indication that host type influences the extent of appressoria formation.

Differences in early hyphal development of Podosphaera xanthii on Cucumis sativus leaves acclimatized to high or low relative humidity

This research investigated early hyphal development of cucurbit powdery mildew (Podosphaera xanthii) on leaves of cucumber (Cucumis sativus L.) acclimatized to different relative humidities to clarify the factors that limit it. Cucumber seedlings were acclimatized to a low (10%) or high (90%) relative humidity (RH) at 30 °C from germination. When the cotyledons had expanded and the first true leaf had begun to emerge, the plants were inoculated, and initial conidial invasion, hyphal development, and secondary haustorial formation were evaluated. The initial invasion of P. xanthii into the epidermis was slower on the low-RH leaves than on the high-RH leaves, suggesting that structural properties, such as greater leaf mass per area, physically limited the development of P. xanthii. The hyphal development and secondary haustoria of conidia that had successfully infected the epidermis were also slower on the low-RH leaves. These post-invasion behaviors indicate that conidial development was also suppressed by nonstructural properties of low-RH leaves such as nutrients, which correlate with nonstructural defense. The reduced leaf N content of the low-RH leaves may in part have inhibited the post-invasion behaviors of P. xanthii.

Oxaloacetate hydrolase gene links the cytoplasmic route of oxalate formation to differentiation and virulence of entomopathogenic fungus Beauveria bassiana

Oxalic acid is used as a functional molecule by most filamentous fungi, and produced via cytoplasmic pathway and mitochondrial pathway. The cytoplasmic pathway of oxalate production from oxaloacetate is a one-step reaction catalyzed by oxaloacetate hydrolase. The entomopathogenic fungus Beauveria bassiana contains a unique oxaloacetate hydrolase gene (BbOAH). The role of cytoplasmic pathway of oxalate production in B. bassiana development and virulence was studied via construction of a targeted gene disruption mutant of BbOAH. Disruption of BbOAH resulted in a slight decrease (~30%) in oxalate production, but has no significant influence on fungal growth. The mutant strain displayed a significant delay at early stage of conidial development, and a significant defect in dimorphic transition. Additionally, bioassay using the greater waxmoth as host indicated a slight (~20%) decrease in mortality caused by the gene disruption strain. The phenotypic defects of the ΔBbOAH strain could be restored by ectopic integration of BbOAH. Our findings indicate that BbOAH gene connects the cytoplasmic route of oxalate production to fungal development and virulence, although the cytoplasmic route is not indispensable for oxalate production in B. bassiana.

The Fungal bZIP Transcription Factor AtfB Controls Virulence-Associated Processes in Aspergillus parasiticus.

Fungal basic leucine zipper (bZIP) transcription factors mediate responses to oxidative stress. The ability to regulate stress response pathways in Aspergillus spp. was postulated to be an important virulence-associated cellular process, because it helps establish infection in humans, plants, and animals. Previous studies have demonstrated that the fungal transcription factor AtfB encodes a protein that is associated with resistance to oxidative stress in asexual conidiospores, and AtfB binds to the promoters of several stress response genes. Here, we conducted a gene silencing of AtfB in Aspergillus parasiticus, a well-characterized fungal pathogen of plants, animals, and humans that produces the secondary metabolite and carcinogen aflatoxin, in order to determine the mechanisms by which AtfB contributes to virulence. We show that AtfB silencing results in a decrease in aflatoxin enzyme levels, the down-regulation of aflatoxin accumulation, and impaired conidiospore development in AtfB-silenced strains. This observation is supported by a decrease of AtfB protein levels, and the down-regulation of many genes in the aflatoxin cluster, as well as genes involved in secondary metabolism and conidiospore development. Global expression analysis (RNA Seq) demonstrated that AtfB functionally links oxidative stress response pathways to a broader and novel subset of target genes involved in cellular defense, as well as in actin and cytoskeleton arrangement/transport. Thus, AtfB regulates the genes involved in development, stress response, and secondary metabolism in A. parasiticus. We propose that the bZIP regulatory circuit controlled by AtfB provides a large number of excellent cellular targets to reduce fungal virulence. More importantly, understanding key players that are crucial to initiate the cellular response to oxidative stress will enable better control over its detrimental impacts on humans.