Conidia Formation Research Articles

Efficiency of a new Waitea circinata extract against rice pathogens

ABSTRACT Waitea circinata (Warcup & Talbot) is an orchid antagonist mycorrhizal fungus with biocontrol potential against rice pathogens. This study aimed to optimize the extraction method, obtain a new extract and evaluate its efficiency against rice pathogens in vitro and in vivo, as well as to compare it with other extraction methods and W. circinata. The extracts were obtained and screened for in vitro growth inhibition against the pathogens Cochliobolus miyabeanus, Monographella albescens and Sarocladium oryzae, using the following extracts: mycelial, crude, lyophilized and mycelial mass. An additional in vitro assay was performed with the principal rice pathogen (Magnaporthe oryzae), in order to evaluate the conidial germination and appressorium formation. Based on this evaluation, the lyophilized and mycelial mass extracts were tested in vivo against rice blast (M. oryzae) and compared to the W. circinata mycelial suspension, in different application forms (simultaneous and previous). The mycelial mass extract inhibited all the pathogens, and the crude and lyophilized extracts inhibited C. miyabeanus and M. albescens, respectively. The mycelial mass extract inhibited the M. oryzae conidial germination and appressorium formation by 80 %, and the simultaneous and previous applications suppressed the rice blast by 94 %. These results indicate that the new extract can be used to control rice pathogens.

Whole Transcriptome Analysis Provides Insights Into the Molecular Mechanisms of Chlamydospore-Like Cell Formation in Phanerochaete chrysosporium

Phanerochaete chrysosporium is a white rot fungus naturally isolated from hardwoods and widely used in environmental pollution control because it produces extracellular peroxidases. It forms chlamydospores during nitrogen starvation, which naturally occurs in the habitat of P. chrysosporium. Chlamydospores protect fungi against many stresses; the molecular basis underlying chlamydospore formation in basidiomycetes is poorly explored. Chlamydospores in P. chrysosporium have a different cell wall compared with hyphae, as confirmed by cell wall digestion and microscopy. Furthermore, this study investigated the transcriptome of P. chrysosporium in different life stages, including conidium, hypha, and chlamydospore formation, through RNA sequencing. A total of 2215 differentially expressed genes were identified during these processes. The expression patterns of genes involved in several molecular events critical for chlamydospore formation, including starch and sucrose metabolism, phosphatase and kinase, and transcription factors, were determined. This study serves as a basis for further investigating the function of chlamydospore formation in the biotechnologically relevant fungus P. chrysosporium.

Functional Characterization of Calcineurin-Responsive Transcription Factors Fg01341 and Fg01350 in Fusarium graminearum.

Ca2 +/calmodulin-dependent phosphatase calcineurin is one of the important regulators of intracellular calcium homeostasis and has been investigated extensively in Saccharomyces cerevisiae. However, only a few reports have explored the function of the Crz1 homolog in filamentous fungi, especially in Fusarium graminearum. In this study, we identified Fg01341 as a potential ortholog of yeast Crz1. Fg01341 could interact with calcineurin and initiate nuclear transport in a calcineurin-dependent manner. The ΔFg01341 mutant exhibited normal hyphal growth on basic medium and conidia formation, but sexual reproduction was partially blocked. Pathogenicity assays showed that the virulence of the ΔFg01341 mutant in flowering wheat heads and corn silks dramatically decreased and was thus consistent with the reduction in deoxynivalenol production. Unexpectedly, the sensitivity to osmotic stress of the deletion mutant and that of the wild-type strain did not present any differences. The deletion mutant showed higher sensitivity to tebuconazole than the wild-type strain. Results also showed that the transcription factor Fg01350 might be the calcineurin target and was independent of Crz1. Furthermore, ΔFg01350 showed defects in hyphal growth, sexual production, virulence, and deoxynivalenol production. Collectively, the results indicate that these two proteins functionally redundant and that the calcineurin–Crz1-independent pathway is particularly important in F. graminearum.

Taro koji of Amorphophallus konjac enabling hydrolysis of konjac polysaccharides to various biotechnological interest.

Due to the indigestibility, utilization of konjac taro, Amorphophallus konjac has been limited only to the Japanese traditional konjac food. Koji preparation with konjac taro was examined to utilize konjac taro as a source of utilizable carbohydrates. Aspergillus luchuensis AKU 3302 was selected as a favorable strain for koji preparation, while Aspergillus oryzae used extensively in sake brewing industry was not so effective. Asp. luchuensis grew well over steamed konjac taro by extending hyphae with least conidia formation. Koji preparation was completed after 3-day incubation at 30°C. D-Mannose and D-glucose were the major monosaccharides found in a hydrolyzate giving the total sugar yield of 50g from 100g of dried konjac taro. An apparent extent of konjac taro hydrolysis at 55°C for 24h seemed to be completed. Since konjac taro is hydrolyzed into monosaccharides, utilization of konjac taro carbohydrates may become possible to various products of biotechnological interest.

Penicillium oxalicum putative methyltransferase Mtr23B has similarities and differences with LaeA in regulating conidium development and glycoside hydrolase gene expression

Putative methyltranferase LaeA and LaeA-like proteins, which are conserved in many filamentous fungi, regulate the sporogenesis and biosynthesis of secondary metabolites. In this study, we reported the biological function of a LaeA-like methyltransferase, Penicillium oxalicum Mtr23B, which contains a methyltransf_23 domain and an S-adenosylmethionine binding domain, in controlling spore pigment formation and in the expression of secondary metabolic gene cluster and glycoside hydrolase genes. Additionally, we compared Mtr23B and LaeA, and determined their similarities and differences in terms of their roles in regulating the above biological processes. mtr23B had the highest transcriptional level among the 12 members of the methyltransf_23 family in P. oxalicum. The colony color of Δmtr23B (deletion of mtr23B) was lighter than that of ΔlaeA, although Δmtr23B produced ~ 19.2-fold more conidia than ΔlaeA. The transcriptional levels of abrA, abrB/yA, albA/wA, arpA, arpB, and aygA, which are involved in the dihydroxynaphtalene–melanin pathway, decreased in Δmtr23B. However, Mtr23B had a little effect on brush-like structures and conidium formation, and had a different function from LaeA. Mtr23B extensively regulated glycoside hydrolase gene expression. The absence of Mtr23B remarkably repressed prominent cellulase- and amylase-encoding genes in the whole culture period, while the effect of LaeA mainly occurred in the later phases of prolonged batch cultures. Similar to LaeA, Mtr23B was involved in the expression of 10 physically linked regions containing secondary metabolic gene clusters; the highest regulatory activities of Mtr23B and LaeA were observed in BrlA-dependent cascades. Although LaeA interacted with VeA, Mtr23B did not interact with VeA directly. We assumed that Mtr23B regulates cellulase and amylase gene transcription by interacting with the CCAAT-binding transcription factor HAP5 and chromatin remodeling complex.

Oligomycins inhibit Magnaporthe oryzae Triticum and suppress wheat blast disease

Oligomycins are macrolide antibiotics, produced by Streptomyces spp. that show antagonistic effects against several microorganisms such as bacteria, fungi, nematodes and the oomycete Plasmopara viticola. Conidiogenesis, germination of conidia and formation of appressoria are determining factors pertaining to pathogenicity and successful diseases cycles of filamentous fungal phytopathogens. The goal of this research was to evaluate the in vitro suppressive effects of two oligomycins, oligomycin B and F along with a commercial fungicide Nativo® 75WG on hyphal growth, conidiogenesis, conidial germination, and appressorial formation of the wheat blast fungus, Magnaporthe oryzae Triticum (MoT) pathotype. We also determined the efficacy of these two oligomycins and the fungicide product in vivo in suppressing wheat blast with a detached leaf assay. Both oligomycins suppressed the growth of MoT mycelium in a dose dependent manner. Between the two natural products, oligomycin F provided higher inhibition of MoT hyphal growth compared to oligomycin B with a minimum inhibitory concentration of 0.005 and 0.05 μg/disk, respectively. The application of the compounds completely halted conidial formation of the MoT mycelium in agar medium. Further bioassays showed that these compounds significantly inhibited MoT conidia germination and induced lysis. The compounds also caused abnormal germ tube formation and suppressed appressorial formation of germinated spores. Interestingly, the application of these macrolides significantly inhibited wheat blast on detached leaves of wheat. This is the first report on the inhibition of mycelial growth, conidiogenesis, germination of conidia, deleterious morphological changes in germinated conidia, and suppression of blast disease of wheat by oligomycins from Streptomyces spp. Further study is needed to unravel the precise mode of action of these natural compounds and consider them as biopesticides for controlling wheat blast.

Host-induced silencing of the Colletotrichum gloeosporioides conidial morphology 1 gene (CgCOM1) confers resistance against Anthracnose disease in chilli and tomato.

Host mediated silencing of COM1 gene of Colletotrichum gloeosporioides disables appressorial differentiation and effectively prevents the development of Anthracnose disease in chilli and tomato. Anthracnose disease is caused by the ascomycetes fungal species Colletotrichum, which is responsible for heavy yield losses in chilli and tomato worldwide. Conventionally, harmful pesticides are used to contain anthracnose disease with limited success. In this study, we assessed the potential of Host-Induced Gene Silencing (HIGS) approach to target theColletotrichumgloeosporioides COM1 (CgCOM1) developmental gene involved in the fungal conidial and appressorium formation, to restrict fungal infection in chilli and tomato fruits. For this study, we have developed stable transgenic lines of chilli and tomato expressing CgCOM1-RNAi construct employing Agrobacterium-mediated transformation. Transgenic plants were characterized by molecular and gene expression analyses. Production of specific CgCOM1 siRNA in transgenic chilli and tomato RNAi lines was confirmed by stem-loop RT-PCR. Fungal challenge assays on leaves and fruits showed that the transgenic lines were resistant to anthracnose disease-causing C. gloeosporioides in comparison to wild type and empty-vector control plants. RT-qPCR analyses in transgenic lines revealed extremely low abundance of CgCOM1 transcripts in the C. gloeosporioides infected tissues, indicating near complete silencing of CgCOM1 gene expression in the pathogen. Microscopic examination of the Cg-challenged leaves of chilli-CgCOM1i lines revealed highly suppressed conidial germination, germ tube development, appressoria formation and mycelial growth of C. gloeosporioides, resulting in reduced infection of plant tissues. These results demonstrated highly efficient use of HIGS in silencing the expression of essential fungal developmental genes to inhibit the growth of pathogenic fungi, thus providing a highly precise approach to arrest the spread of disease.

Peptidorhamnomannan from Lomentospora prolificans modulates the inflammatory response in macrophages infected with Candida albicans

BackgroundPeptidorhamnomannan is a glycoconjugate that consists of a peptide chain substituted by O- and N-linked glycans, present on the cell surface of Lomentospora prolificans, a saprophytic fungus which is widely distributed in regions with temperate climates. O-linked oligosaccharides from peptidorhamnomannan isolated from Lomentospora prolificans conidia are recognized by macrophages mediating macrophage - conidia interaction. In this work, peptidorhamnomannan was isolated from L. prolificans mycelium cell wall and its role in macrophage - Candida albicans interaction was evaluated.ResultsPurified peptidorhamnomannan inhibits the reactivity of rabbit immune sera to mycelial and conidia forms of L. prolificans, indicating that this glycoconjugate is exposed on the fungal surface and can mediate interaction with host immune cells. We demonstrated that peptidorhamnomannan leads to TNF-α production in J774 macrophages for 1, 2 and 3 h of incubation, suggesting that this glycoconjugate may have a beneficial role in the response to fungal infections. In order to confirm this possibility, the effect of peptidorhamnomannan on the macrophage - C. albicans interaction was evaluated. Macrophages treated with peptidorhamnomannan led to a lower fungal survival, suggesting that peptidorhamnomannan induces an increased fungicidal activity in macrophages. Furthermore, TNF-α levels were measured in supernatants after macrophage - C. albicans interaction for 1, 2 and 3 h. Peptidorhamnomannan treatment led to a higher TNF-α production at the beginning of the interaction. However, the release of TNF-α was not maintained after 1 h of incubation. Besides, peptidorhamnomannan did not show any inhibitory or fungicidal effect in C. albicans when used at 100 μg/ml but it was able to kill C. albicans at a concentration of 400 μg/ml.ConclusionWe suggest that peptidorhamnomannan acts as a molecular pattern on the invading pathogen, promotes TNF-α production and, thus, increases macrophage fungicidal activity against Candida albicans.

Characterization and functional analysis of ERAD-related AAA+ ATPase Cdc48 in Aspergillus oryzae

Aspergillus oryzae can secrete large amounts of enzymes. However, the production of abundant secretory proteins triggers the unfolded protein response (UPR) in the endoplasmic reticulum (ER), and it is not clear how ER-associated protein degradation (ERAD) contributes to bulk protein production in A. oryzae. Here we identified AoCdc48, the sole A. oryzae ortholog of Saccharomyces cerevisiae AAA+ ATPase Cdc48, a component of the ERAD machinery. We found that AoCdc48 localizes in both nuclei and cytoplasm. Generation of an Aocdc48 conditional mutant showed that Aocdc48 repression leads to reduced cell growth and aberrant hyphal morphology. When Aocdc48-repressed cells were cultured on starch-containing plates, the α-amylase-encoding gene amyB was about 1.3-fold higher expressed. Indeed, a halo produced by secreted amylase was seen on potato starch-containing plates even when there was almost no growth under Aocdc48 repression. Fluorescence microscopy revealed that although AmyB seemed to be secreted, various organelle distributions were aberrant in Aocdc48-repressed cells. We found that D1 AAA domain is crucial for cell viability. Finally, we show that Aocdc48-overexpression also causes defects of cell growth, colonial morphology and conidial formation. Collectively, our results suggest that AoCdc48 is essential for growth and organelle distribution but dispensable for amylase secretion.

Antifungal and antimycotoxigenic effect of the essential oil of Eremanthus erythropappus on three different Aspergillus species

AbstractEssential oils can be considered antifungal agents in controlling food contamination, due to their biological potential. Eremanthus erythropappus essential oil (EEO) was extracted by hydrodistillation, and its chemical composition was evaluated by gas chromatography using mass spectrometer (GC‐MS) and flame ionization detectors (GC‐FID). EEO antifungal and antimycotoxygenic effect was evaluated on different species of Aspegillus genus. The main component of the EEO was α‐bisabolol (98.853%). The essential oil exhibited fungicidal activity at concentrations of 0.98, 15.63, and 15.63 μL/mL against Aspergillus carbonarius, Aspergillus flavus, and Aspergillus ochraceus, respectively. The application of the EEO significantly inhibited ochratoxin A production that ranged from 62.06% to 84.27% and from 68.29% to 98.44% for the fungal species A carbonarius and A ochraceus, respectively, and also inhibited the production qualitatively of aflatoxins B1 and B2. Ergosterol biosynthesis of fungi A carbonarius (76.56%‐92.07%), A flavus (81.28%‐97.75%), and A ochraceus (47.61%‐85.56%) significantly decreased in the presence of EEO. Electromicrographs showed morphological alterations and some damages caused by EEO in the fungal membranes of three fungi species. There was a change in the hyphae uniformity present in the fungi and also loss of conidia integrity in A carbonarius, and no conidia formation in A flavus and A ochraceus, after treatment with the essential oil. Therefore, the results suggested that EEO was capable of inhibit fungal growth, ergosterol biosynthesis, and the production of mycotoxins that can contaminate food.

Systematic characterization of the ubiquitin-specific proteases in Magnaporthe oryzae

Ubiquitin-specific protease (UBP) family is the largest group of deubiquitinases, which plays important roles in eukaryotic organisms. Comprehensive analysis of UBP genes has not been conducted in the plant pathogenic fungi. In this study, 11 putative UBP genes were identified and characterized in the rice blast fungus Magnaporthe oryzae. Expression profile analysis showed that UBP3, UBP6, UBP12 and UBP14 were highly expressed in different tissues of M. oryzae. In all ubp mutants, especially Δubp3, Δubp12 and previously reported Δubp14, the ubiquitination levels were evidently elevated, which is consistent with their molecular roles in de-ubiquitination. The Δubp1, Δubp3, Δubp4, Δubp8 and Δubp14 mutants were reduced in colony growth. Most of the ubp mutants were severely reduced in conidia production capacity, indicating important roles of the UBPs in conidia formation. Except for Δubp2 and Δubp16, all of the other mutants were decreased in virulence to host plants and defective in invasive growth. These ubp mutants also induced massive ROS accumulation in host cells. We also found that the UBPs may function as both positive and negative regulators in stress response and nutrient utilization of M. oryzae. Collectively, UBPs are important for development, stress response, nutrient utilization and infection of M. oryzae.

The Cell Wall Integrity Pathway Contributes to the Early Stages of Aspergillus fumigatus Asexual Development.

Aspergillus fumigatus is a major cause of human disease. The survival of this fungus is dependent on the cell wall organization and function of its components. The cell wall integrity pathway (CWIP) is the primary signaling cascade that controls de novo synthesis of the cell wall in fungi. Abundant conidiation is a hallmark in A. fumigatus, and uptake of conidia by a susceptible host is usually the initial event in infection. The formation of conidia is mediated by the development of fungus-specific specialized structures, conidiophores, which are accompanied by cell wall remodeling. The molecular regulation of these changes in cell wall composition required for the rise of conidiophore from the solid surface and to disperse the conidia into the air is currently unknown. Here, we investigated the role of CWIP in conidiation. We show that CWIP pkcAG579R, ΔmpkA, and ΔrlmA mutants displayed reduced conidiation during synchronized asexual differentiation. The transcription factor RlmA directly regulated the expression of regulators of conidiation, including flbB, flbC, brlA, abaA, and rasB, as well as genes involved in cell wall synthesis and remodeling, and this affected the chitin content in aerial hyphae. Phosphorylation of RlmA and MpkA was increased during asexual differentiation. We also observed that MpkA physically associated with the proteins FlbB, FlbC, BrlA, and RasB during this process, suggesting another level of cross talk between the CWIP and asexual development pathways. In summary, our results support the conclusion that one function of the CWIP is the regulation of asexual development in filamentous fungi.IMPORTANCE A remarkable feature of the human pathogen Aspergillus fumigatus is its ability to produce impressive amounts of infectious propagules known as conidia. These particles reach immunocompromised patients and may initiate a life-threatening mycosis. The conidiation process in Aspergillus is governed by a sequence of proteins that coordinate the development of conidiophores. This process requires the remodeling of the cell wall so that the conidiophores can rise and withstand the chains of conidia. The events regulating cell wall remodeling during conidiation are currently unknown. Here, we show that the cell wall integrity pathway (CWIP) components RlmA and MpkA directly contribute to the activation of the conidiation cascade by enabling transcription or phosphorylation of critical proteins involved in asexual development. This study points to an essential role for the CWIP during conidiation and provides further insights into the complex regulation of asexual development in filamentous fungi.

Cmfhp Gene Mediates Fruiting Body Development and Carotenoid Production in Cordyceps militaris.

Cordyceps militaris fruiting bodies contain a variety of bioactive components that are beneficial to the human body. However, the low yield of fruiting bodies and the low carotenoid content in C. militaris have seriously hindered the development of the C. militaris industry. To elucidate the developmental mechanism of the fruiting bodies of C. militaris and the biosynthesis mechanism of carotenoids, the function of the flavohemoprotein-like Cmfhp gene of C. militaris was identified for the first time. The Cmfhp gene was knocked out by the split-marker method, and the targeted gene deletion mutant ΔCmfhp was obtained. An increased nitric oxide (NO) content, no fruiting body production, decreased carotenoid content, and reduced conidial production were found in the mutant ΔCmfhp. These characteristics were restored when the Cmfhp gene expression cassette was complemented into the ΔCmfhp strain by the Agrobacterium tumefaciens-mediated transformation method. Nonetheless, the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. These results indicated that the Cmfhp gene regulated the biosynthesis of NO and carotenoids, the development of fruiting bodies, and the formation of conidia. These findings potentially pave the way to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.

The zinc finger transcription factors Bbctf1α and Bbctf1β regulate the expression of genes involved in lipid degradation and contribute to stress tolerance and virulence in a fungal insect pathogen.

To initiate insect infection, entomopathogenic fungi produce diverse cuticle-degrading enzymes. Of those, lipolytic enzymes participate in epicuticular lipid hydrolysis and thus facilitate fungal penetration through the outermost cuticular barrier of the insect host. The Far/CTF1-type zinc finger transcription factors play an important role in the regulation of lipolytic activity and fungal pathogenicity in plant pathogens but remain functionally unknown in fungal insect pathogens. Two Far/CTF1-type transcription factor Bbctf1α and Bbctf1β, which are essential for differential expression of genes involved in the fungal lipid degradation, were identified and functionally characterized in a fungal entomopathogen Beauveria bassiana. Disruption of each gene led to drastic losses of extracellular lipolytic activities under lipidic substrate-inducing conditions, followed by remarkable phenotypic defects associated with the fungal biocontrol potential. These defects mainly included severe impairments of mycelial growth and conidium formation, and drastic losses of tolerance to the stresses of oxidation and cell wall perturbation during colony growth under either normal or induction conditions. Bioassays showed that the virulence of each disruption mutant on the greater wax moth was remarkably attenuated in topical immersion. However, there was no significant difference in intrahemolymph injection when the cuticle penetration process was bypassed. Bbctf1α and Bbctf1β are multifunctional transcription factors that play vital roles in the regulation of fungal lipid utilization and contribute to the vegetative growth, sporulation capacity, environmental fitness and pest control potential in B. bassiana. © 2020 Society of Chemical Industry.

Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae.

Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis.

Ubiquitin E3 Ligase AaBre1 Responsible for H2B Monoubiquitination Is Involved in Hyphal Growth, Conidiation and Pathogenicity in Alternaria alternata.

Ubiquitination is one of several post-transcriptional modifications of histone 2B (H2B) which affect the chromatin structure and, hence, influence gene transcription. This study focuses on Alternaria alternata, a fungal pathogen responsible for leaf spot in many plant species. The experiments show that the product of AaBRE1, a gene which encodes H2B monoubiquitination E3 ligase, regulates hyphal growth, conidial formation and pathogenicity. Knockout of AaBRE1 by the homologous recombination strategy leads to the loss of H2B monoubiquitination (H2Bub1), as well as a remarkable decrease in the enrichment of trimethylated lysine 4 on histone 3 (H3K4me3). RNA sequencing assays elucidated that the transcription of genes encoding certain C2H2 zinc-finger family transcription factors, cell wall-degrading enzymes and chitin-binding proteins was suppressed in the AaBRE1 knockout cells. GO enrichment analysis showed that these proteins encoded by the set of genes differentially transcribed between the deletion mutant and wild type were enriched in the functional categories “macramolecular complex”, “cellular metabolic process”, etc. A major conclusion was that the AaBRE1 product, through its effect on histone 2B monoubiquitination and histone 3 lysine 4 trimethylation, makes an important contribution to the fungus’s hyphal growth, conidial formation and pathogenicity.

Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.

The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (ΔSte12) retained wild-type growth, ACT toxin production, and sensitivity to oxidative and osmotic stress. However, pathogenicity tests assayed on detached Dancy leaves revealed a marked reduction in virulence of ΔSte12. Transcriptome and quantitative RT-PCR analyses revealed that many genes associated with Carbohydrate-Active Enzymes (CAZymes) were downregulated in ΔSte12. Two cutinase-coding genes (AaCut3 and AaCut7) regulated by Ste12 were individually and simultaneously inactivated. The AaCut3 or AaCut7 deficiency strain unchanged in cutinase activities and incited wild-type lesions on Dancy leaves. However, the strain carrying an AaCut3 AaCut7 double mutation produced and secreted significantly fewer cutinases and incited smaller necrotic lesions than wild type. Not only is the host-selective toxin (HST) produced by A. alternata required for fungal penetration and lesion formation, but so too are CWDEs required for full virulence. Overall, this study expands our understanding of how A. alternata overcomes citrus physical barriers to carry out successful penetration and colonization.

The bZIP transcription factor FpAda1 is essential for fungal growth and conidiation in Fusarium pseudograminearum

Fusarium pseudograminearum is an important pathogen of Fusarium crown rot and Fusarium head blight, which is able to infect wheat and barley worldwide, causing great economic losses. Transcription factors (TFs) of the basic leucine zipper (bZIP) protein family control important processes in all eukaryotes. In this study, we identified a gene, designated FpAda1, encoding a bZIP TF in F. pseudograminearum. The homolog of FpAda1 is also known to affect hyphal growth in Neurospora crassa. Deletion of FpAda1 in F. pseudograminearum resulted in defects in hyphal growth, mycelial branching and conidia formation. Pathogenicity assays showed that virulence of the Δfpada1 mutant was dramatically decreased on wheat coleoptiles and barley leaves. However, wheat coleoptile inoculation assay showed that Δfpada1 could penetrate and proliferate in wheat cells. Moreover, the FpAda1 was required for abnormal nuclear morphology in conidia and transcription of FpCdc2 and FpCdc42. Taken together, these results indicate that FpAda1 is an important transcription factor involved in growth and development in F. pseudograminearum.

Current Status of Soybean Anthracnose Associated with Colletotrichum truncatum in Brazil and Argentina.

Brazil and Argentina have a combined soybean area of 53.6 million hectares, which accounts for over half of the total global production. The soybean crop in South America extends from latitude 8–10° S to 32–36° S. Such a vast, almost contiguous area imposes a serious sanitary risk to the crop. Currently, the prevalence of anthracnose is increasing, with recurring reports of severe epidemics and expressive yield losses. Soybean anthracnose is mainly associated with Colletotrichum truncatum, although other Colletotrichum species have also been reported as causal agents of this disease. Knowledge about the morphological, cultural, and molecular variability of C. truncatum in South America is crucial for disease management. Here, we present data on the molecular, morphological, biological, cultural, and pathogenicity of C. truncatum isolates collected in Brazil and Argentina. Light microscopy and randomly-amplified polymorphic DNA (RAPD) analysis were used for estimating the variability of isolates. Colletotrichum truncatum displayed three types of conidiogenesis, viz. conidial formation from conidiogenous cells on hyphal extremities, in conidiomas in acervuli, and directly from fertile setae (a mechanism yet-unreported for C. truncatum). RAPD profiling was effective in revealing the genetic diversity among C. truncatum isolates. The intra-group similarity was greater among the Argentinian isolates when compared to the Brazilian group. Furthermore, the results indicated a strong correlation between geographical origin and molecular grouping, with the exclusive or semi-exclusive assembling of Brazilian and Argentinian isolates in distinct clades. Finally, a preliminary account of the reaction of soybean accessions to C. truncatum is also included.

The velvet repressed vidA gene plays a key role in governing development in Aspergillus nidulans.

Fungal development is regulated by a variety of transcription factors in Aspergillus nidulans. Previous studies demonstrated that the NF-κB type velvet transcription factors regulate certain target genes that govern fungal differentiation and cellular metabolism. In this study, we characterize one of the VosA/VelB-inhibited developmental genes called vidA, which is predicted to encode a 581-amino acid protein with a C2H2 zinc finger domain at the C-terminus. Levels of vidA mRNA are high during the early and middle phases of asexual development and decrease during the late phase of asexual development and asexual spore (conidium) formation. Deletion of either vosA or velB results in increased vidA mRNA accumulation in conidia, suggesting that vidA transcript accumulation in conidia is repressed by VosA and VelB. Phenotypic analysis demonstrated that deletion of vidA causes decreased colony growth, reduced production of asexual spores, and abnormal formation of sexual fruiting bodies. In addition, the vidA deletion mutant conidia contain more trehalose and β-glucan than wild type. Overall, these results suggest that VidA is a putative transcription factor that plays a key role in governing proper fungal growth, asexual and sexual development, and conidia formation in A. nidulans.