Aerial Hyphal Growth Research Articles

Putative APSES family transcription factor mbp1 plays an essential role in regulating cell wall synthesis in the agaricomycete Pleurotus ostreatus

The clade A APSES family transcription factors (Mbp1, Swi4, and Swi6) contribute to cell wall synthesis regulation in fungi. Herein, evolutionary relationships among these proteins were clarified by phylogenetic analysis using various ascomycetes and basidiomycetes, and then the detailed function of Mbp1 in cell wall synthesis regulation was analyzed in Pleurotus ostreatus. Our phylogenetic analysis revealed that Mbp1 and Swi6 are widely conserved among various fungi, whereas Swi4 is a protein specific for Saccharomycotina. In P. ostreatus, two putative clade A APSES family transcription factors, protein ID 83192 and 134090, were found and identified as Mbp1 and Swi6, respectively. The mbp1 gene was then disrupted through homologous recombination using P. ostreatus 20b strain (Δku80) as a host to obtain mbp1 disruption strains (Δmbp1). Disruption of mbp1 significantly decreased the growth rate and shortened aerial hyphae, suggesting that Mbp1 is involved in mycelial growth, especially aerial hyphal growth. Furthermore, thinner cell walls, decreased relative percentage of β-glucan, and downregulation of all β-glucan synthase genes were observed in Δmbp1 strains. Therefore, Mbp1 plays an essential role in β-glucan synthesis regulation in P. ostreatus. Disruption of mbp1 also impacted the expression profiles of chitin synthase genes, septum formation, and sensitivity to a chitin synthesis inhibitor, suggesting that Mbp1 also regulates chitin synthesis. In conclusion, Mbp1 is responsible for normal mycelial growth and regulates β-glucan and chitin synthesis in P. ostreatus. To the best of our knowledge, this is the first report on the detailed function of Mbp1 in cell wall synthesis regulation in fungi.

Red and far-red light improve the antagonistic ability of Trichoderma guizhouense against phytopathogenic fungi by promoting phytochrome-dependent aerial hyphal growth.

Light as a source of information regulates morphological and physiological processes of fungi, including development, primary and secondary metabolism, or the circadian rhythm. Light signaling in fungi depends on photoreceptors and downstream components that amplify the signal to govern the expression of an array of genes. Here, we investigated the effects of red and far-red light in the mycoparasite Trichoderma guizhouense on its mycoparasitic potential. We show that the invasion strategy of T. guizhouense depends on the attacked species and that red and far-red light increased aerial hyphal growth and led to faster overgrowth or invasion of the colonies. Molecular experiments and transcriptome analyses revealed that red and far-red light are sensed by phytochrome FPH1 and further transmitted by the downstream MAPK HOG pathway and the bZIP transcription factor ATF1. Overexpression of the red- and far-red light-induced fluffy gene fluG in the dark resulted in abundant aerial hyphae formation and thereby improvement of its antagonistic ability against phytopathogenic fungi. Hence, light-induced fluG expression is important for the mycoparasitic interaction. The increased aggressiveness of fluG-overexpressing strains was phenocopied by four random mutants obtained after UV mutagenesis. Therefore, aerial hyphae formation appears to be a trait for the antagonistic potential of T. guizhouense.

Functional Analysis of Genes Specifically Expressed during Aerial Hyphae Collapse as a Potential Signal for Perithecium Formation Induction in Fusarium graminearum.

Fusarium graminearum, the causal agent of Fusarium head blight (FHB) in cereal crops, employs the production of sexual fruiting bodies (perithecia) on plant debris as a strategy for overwintering and dissemination. In an artificial condition (e.g., carrot agar medium), the F. graminearum Z3643 strain was capable of producing perithecia predominantly in the central region of the fungal culture where aerial hyphae naturally collapsed. To unravel the intricate relationship between natural aerial hyphae collapse and sexual development in this fungus, we focused on 699 genes differentially expressed during aerial hyphae collapse, with 26 selected for further analysis. Targeted gene deletion and quantitative real-time PCR analyses elucidated the functions of specific genes during natural aerial hyphae collapse and perithecium formation. Furthermore, comparative gene expression analyses between natural collapse and artificial removal conditions reveal distinct temporal profiles, with the latter inducing a more rapid and pronounced response, particularly in MAT gene expression. Notably, FGSG_09210 and FGSG_09896 play crucial roles in sexual development and aerial hyphae growth, respectively. Taken together, it is plausible that if aerial hyphae collapse occurs on plant debris, it may serve as a physical cue for inducing perithecium formation in crop fields, representing a survival strategy for F. graminearum during winter. Insights into the molecular mechanisms underlying aerial hyphae collapse provides offer potential strategies for disease control against FHB caused by F. graminearum.

PdStuA Is a Key Transcription Factor Controlling Sporulation, Hydrophobicity, and Stress Tolerance in Penicillium digitatum.

Penicillium digitatum has become one of the main pathogens in citrus due to its high spore production and easy spread. In this study, the function of the APSES transcription factor StuA in P. digitatum was characterized, and the results indicated that it was involved in conidium and conidiophore development. No conidiophores were observed in the mycelium of the ∆PdStuA mutant that had grown for two days, while an abnormal conidiophore was found after another two days of incubation, and only small thin phialides as well as a very small number of spores were formed at the top of the hyphae. Moreover, it was observed that the ∆PdStuA mutant showed various defects, such as reduced hydrophobicity and decreased tolerance to cell wall inhibitors and H2O2. Compared to the original P. digitatum, the colony diameter of the ∆PdStuA mutant was not significantly affected, but the growth of aerial hyphae was obviously induced. In in vivo experiments, the spore production of the ∆PdStuA mutant grown on citrus fruit was remarkably decreased; however, there was no significant difference in the lesion diameter between the mutant and original strain. It could be inferred that less spore production might result in reduced spread in citrus, thereby reducing the green mold infection in citrus fruit during storage. This study provided a gene, PdStuA, which played key role in the sporulation of P. digitatum, and the results might provide a reference for the molecular mechanisms of sporulation in P. digitatum.

GPI-Anchored Protein Homolog IcFBR1 Functions Directly in Morphological Development of Isaria cicadae.

Isaria cicadae is a famous edible and medicinal fungus in China and Asia. The molecular basis of morphogenesis and synnemal formation needs to be understood in more detail because this is the main source of biomass production in I. cicadae. In the present study, a fruiting body formation-related gene with a glycosylphosphatidylinositol (GPI) anchoring protein (GPI-Ap) gene homolog IcFBR1 was identified by screening random insertion mutants. Targeted deletion of IcFBR1 resulted in abnormal formation of synnemata, impairing aerial hyphae growth and sporulation. The IcFBR1 mutants were defective in the utilization of carbon sources with reduced polysaccharide contents and the regulation of amylase and protease activities. Transcriptome analysis of ΔIcfbr1 showed that IcFBR1 deletion influenced 49 gene ontology terms, including 23 biological processes, 9 molecular functions, and 14 cellular components. IcFBR1 is therefore necessary for regulating synnemal development, secondary metabolism, and nutrient utilization in this important edible and medicinal fungus. This is the first report illustrating that the function of IcFBR1 is associated with the synnemata in I. cicadae.

De Novo Purine Nucleotide Biosynthesis Pathway Is Required for Development and Pathogenicity in Magnaporthe oryzae.

Purine nucleotides are indispensable compounds for many organisms and participate in basic vital activities such as heredity, development, and growth. Blocking of purine nucleotide biosynthesis may inhibit proliferation and development and is commonly used in cancer therapy. However, the function of the purine nucleotide biosynthesis pathway in the pathogenic fungus Magnaporthe oryzae is not clear. In this study, we focused on the de novo purine biosynthesis (DNPB) pathway and characterized MoAde8, a phosphoribosylglycinamide formyltransferase, catalyzing the third step of the DNPB pathway in M. oryzae. MoAde8 was knocked out, and the mutant (∆Moade8) exhibited purine auxotroph, defects in aerial hyphal growth, conidiation, and pathogenicity, and was more sensitive to hyperosmotic stress and oxidative stress. Moreover, ∆Moade8 caused decreased activity of MoTor kinase due to blocked purine nucleotide synthesis. The autophagy level was also impaired in ∆Moade8. Additionally, MoAde5, 7, 6, and 12, which are involved in de novo purine nucleotide biosynthesis, were also analyzed, and the mutants showed defects similar to the defects of ∆Moade8. In summary, de novo purine nucleotide biosynthesis is essential for conidiation, development, and pathogenicity in M. oryzae.

Ceratobasidium orchid mycorrhizal fungi reveal intraspecific variation and interaction with different nutrient media in symbiotic germination of Prasophyllum (Orchidaceae)

Understanding how nutrient requirements of orchid mycorrhizal fungi (OMF) affect symbiotic germination is essential for the ex situ conservation of threatened orchids and their mycorrhizal symbioses. Yet the influence of isolate-level variation in OMF nutrient preferences on orchid germination is unknown. We tested germination of Prasophyllum frenchii (Orchidaceae) on 15 different media of varying carbon and macronutrient compositions with three Ceratobasidium isolates of the same operational taxonomic unit (OTU) as determined with internal transcribed spacer locus sequencing. There was a significant interaction between media and fungal isolate on percentage germination, with each isolate recording its highest percentage germination on different nutrient media (Isolate 9.3: 5.2 ± 1.4% on MOM–S; Isolate 8.2: 5.4 ± 1.1% on MOM + S; Isolate 4.3: 2.2 ± 0.5% on 1.25 g/L wheat bran agar). Across all isolates, germination (percentage germination > 0) occurred more frequently on wheat bran agar media (39.7% of plates) than on oatmeal agar media (6.0% of plates). There was also an effect of media type on aerial hyphal growth behaviour of the OMF isolate. All isolates supported growth through to adult flowering plants. We demonstrated that symbiotic germination of Prasophyllum is affected by media composition. Further, percentage germination and aerial hyphal growth behaviour differed significantly among OMF isolates of the same OTU. This illustrates that a diversity of functionally significant fungal strains occurs within a single OTU, a previously unknown aspect of OMF research with important ecological and conservation implications.

A Perilipin Affects Lipid Droplet Homeostasis and Aerial Hyphal Growth, but Has Only Small Effects on Virulence in the Insect Pathogenic Fungus Beauveria bassiana.

Lipid assimilation, storage, and turnover impact growth, development, and virulence in many microbial pathogens including fungi. Perilipins are proteins associated with lipid droplets (LDs) that mediate their assembly and turnover. Here, we characterized the Beauveria bassiana (BbPlin1) perilipin. BbPlin1 expression was higher in minimal media than in rich media, and, using a BbPlin1::eGFP fusion protein, the protein was shown to be co–localized to LDs, with the high expression seen during infection and proliferation within the insect (Galleria mellonella) host that dramatically decreased to almost no expression during fungal outgrowth on cadavers including in conidia, but that BbPlin1 production resumed in the conidia once placed in nutrient–containing media allowing for germination and growth. Characterization of a targeted gene deletion strain (ΔBbPlin1) revealed a dramatic (>30%) reduction in cellular LD content, promotion of aerial hyphal growth, and a small decrease in virulence, with little to no effects on vegetative growth and stress responses. However, in the ΔBbPlin1 strain, expression of the complementary LD–associated caleosin gene, BbCal1, was enhanced under nutrient–poor conditions, although no changes in BbPlin1 expression were seen in a ΔBbCal1 strain and the expression of BbPlin1 in the ΔBbCal1 strain did not change LD patterns in cells. Transcriptome and RT–PCR analyses indicated increased expression of lipid metabolism–related genes, including triacylglyercol lipase 3, enoyl–CoA isomerase, and diacylglycerol–O–acetyl transferase in the BbPlin1 deletion mutant. Lipid profile analyses confirmed that the loss of BbPlin1 significantly reduced the cellular levels of contents of triacylglycerol, diacylglycerol, and phosphatidylethanolamine as compared to the wild–type strain. These results demonstrate the involvement of the B. bassiana perilipin in mediating lipid homeostasis, fungal aerial hyphal growth, and virulence, revealing critical cycling from high expression during nutrient utilization within host cadavers to low expression during growth on the surface of the cadaver during the infection process.

​Diversity of Actinomycetes in Tomato Plants

Background: Tomato plant holds great biodiversity of actinomycetes. This diversity can be explored for new potential actinomycetes strains. Methods: Two different methods were followed for actinomycetes isolation (Serial dilution and direct streaking method). The direct streaking method was found to be the best method where it gave a higher number of actinomycetes compared to the plant extract method. All the actinomycetes were isolated by ISP2 medium supplemented with nystatin and cycloheximide (at 50 µg/ml). All the endophytic actinomycetes strains were isolated and purified based on the difference in appearances such as colony morphology, growth pattern, aerial hyphae growth, filamentous appearance and pigment production. Result: The direct streak method, yielded 192 isolates and only 48 isolates by plant extract method. Thus, a total of 240 strains were isolated. Plants from Madurai yielded a maximum of 130 isolates (54%) and a minimum from Tuticorin 20 isolates (8%). The highest viable count of 80.01±14.24 (CFU g-1) x 103 were recorded in the agro- fields of Dindugal followed by Madurai, Theni, Tirunelveli and Tuticorin. Out of 240 strains, 49.5% exhibited different aerial hyphae and 36.25% showed different filamentous appearances of growth and 5.8% produced pigments. The endophytic actinomycetes species isolated from tomato plants in different locations possess significant morphologically varied actinobacterial diversity. We can explore further on how these diverse actinomycetes are involved in plant growth promotion and protection against pathogens.

Evaluating the Mode of Antifungal Action of Heat-Stable Antifungal Factor (HSAF) in Neurospora crassa

Heat-stable antifungal factor (HSAF) isolated from Lysobacter enzymogenes has shown a broad-spectrum of antifungal activities. However, little is known about its mode of action. In this study, we used the model filamentous fungus Neurospora crassa to investigate the antifungal mechanism of HSAF. We first used HSAF to treat the N. crassa strain at different time points. Spore germination, growth phenotype and differential gene expression analysis were conducted by utilizing global transcriptional profiling combined with genetic and physiological analyses. Our data showed that HSAF could significantly inhibit the germination and aerial hyphae growth of N. crassa. RNA-seq analysis showed that a group of genes, associated with cell wall formation and remodeling, were highly activated. Screening of N. crassa gene deletion mutants combined with scanning electron microscopic observation revealed that three fungal cell wall integrity-related genes played an important role in the interaction between N. crassa and L. enzymogens. In addition, Weighted Gene Co-Expression Network Analysis (WGCNA), accompanied by confocal microscopy observation revealed that HSAF could trigger autophagy-mediated degradation and eventually result in cell death in N. crassa. The findings of this work provided new insights into the interactions between the predatory Lysobacter and its fungal prey.

The CfMK1 Gene Regulates Reproduction, Appressorium Formation, and Pathogenesis in a Pear Anthracnose-Causing Fungus.

Colletotrichum fructicola, the causal agent of pear anthracnose, causes significant annual economic losses. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that play a crucial role in mediating cellular responses to environmental and host signals in plant pathogenic fungi. In this study, we identified an ortholog of the FUS3/KSS1-related MAPK gene, CfMK1, and characterized its function in C. fructicola. The Cfmk1 deletion mutants exhibited poorly developed aerial hyphae, autolysis, no conidial mass or perithecia on solid plates. However, the conidiation of the Cfmk1 mutant in PDB liquid medium was normal compared with that of the wild type (WT). Conidia of the Cfmk1 mutant exhibited a reduced germination rate on glass slides or plant surfaces. The Cfmk1 deletion mutants were unable to form appressoria and lost the capacity to penetrate plant epidermal cells. The ability of the Cfmk1 mutants to infect pear leaves and fruit was severely reduced. Moreover, RNA sequencing (RNA-seq) analysis of the WT and Cfmk1 mutant was performed, and the results revealed 1886 upregulated and 1554 downregulated differentially expressed genes (DEGs) in the mutant. The DEGs were significantly enriched in cell wall and pathogenesis terms, which was consistent with the defects of the Cfmk1 mutant in cell wall integrity and plant infection. Overall, our data demonstrate that CfMK1 plays critical roles in the regulation of aerial hyphal growth, asexual and sexual reproduction, autolysis, appressorium formation, and pathogenicity.

MoCpa1-mediated arginine biosynthesis is crucial for fungal growth, conidiation, and plant infection of Magnaporthe oryzae.

Arginine is an important amino acid involved in processes such as cell signal transduction, protein synthesis, and sexual reproduction. To understand the biological roles of arginine biosynthesis in pathogenic fungi, we used Cpa1, the carbamoyl phosphate synthase arginine-specific small chain subunit in Saccharomyces cerevisiae as a query to identify its ortholog in the Magnaporthe oryzae genome and named it MoCpa1. MoCpa1 is a 471-amino acid protein containing a CPSase_sm_chain domain and a GATase domain. MoCpa1 transcripts were highly expressed at the conidiation, early-infection, and late-infection stages of the fungus. Targeted deletion of the MoCPA1 gene resulted in a ΔMocpa1 mutant exhibiting arginine auxotrophy on minimum culture medium (MM), confirming its role in de novo arginine biosynthesis. The ΔMocpa1 mutant presented significantly decreased sporulation with some of its conidia being defective in morphology. Furthermore, the ΔMocpa1 mutant was nonpathogenic on rice and barley leaves, which was a result of defects in appressorium-mediated penetration and restricted invasive hyphal growth within host cells. Addition of exogenous arginine partially rescued conidiation and pathogenicity defects on the barley and rice leaves, while introduction of the MoCPA1 gene into the ΔMocpa1 mutant fully complemented the lost phenotype. Further confocal microscopy examination revealed that MoCpa1 is localized in the mitochondria. In summary, our results demonstrate that MoCpa1-mediated arginine biosynthesis is crucial for fungal development, conidiation, appressorium formation, and infection-related morphogenesis in M. oryzae, thus serving as an attractive target for mitigating obstinate fungal plant pathogens. KEY POINTS: • MoCpa1 is important for aerial hyphal growth and arginine biosynthesis. • MoCpa1 is pivotal for conidial morphogenesis and appressorium formation. • MoCpa1 is crucial for full virulence in M. oryzae.

Two regulators of G-protein signaling (RGS) proteins FlbA1 and FlbA2 differentially regulate fumonisin B1 biosynthesis in Fusarium verticillioides.

Fumonisins are a group of mycotoxins produced by maize pathogen Fusarium verticillioides that pose health concerns to humans and animals. Yet we still lack a clear understanding of the mechanism of fumonisins regulation during pathogenesis. The heterotrimeric G protein complex, which consists of canonical subunits and various regulators of G-protein signaling (RGS) proteins, plays an important role in transducing signals under environmental stress. Earlier studies demonstrated that Gα and Gβ subunits are positive regulators of fumonisin B1 (FB1) biosynthesis and that two RGS genes, FvFlbA1 and FvFlbA2, were highly upregulated in Gβ deletion mutant ∆Fvgbb1. Notably, FvFlbA2 has a negative role in FB1 regulation. While many fungi contain a single copy of FlbA, F. verticillioides harbors two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While ∆FvflbA1 deletion mutant exhibited no significant defects, ∆FvflbA2 and ∆FvflbA2/A1 mutants showed thinner aerial hyphal growth while promoting FB1 production. FvFlbA2 is required for proper expression of key conidia regulation genes, including putative FvBRLA, FvWETA, and FvABAA, while suppressing FUM21, FUM1, and FUM8 expression. Split luciferase assays determined that FvFlbA paralogs interact with key heterotrimeric G protein components, which in turn will lead altered G-protein-mediated signaling pathways that regulate FB1 production and asexual development in F. verticillioides.

An Exo-Polygalacturonase Pgc4 Regulates Aerial Hyphal Growth and Virulence in Fusarium oxysporum f. sp. cubense race 4.

Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 kDa based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis. We further performed its sequence analysis and biochemical characterization. The two pgc4 genes encoding Pgc4 from Foc4 and Foc1 were 1434 bp in length and encoded 477 amino acids with differences, due to some nucleotide differences between the two. The Km and Vmax values of Pgc4 purified from Foc4 were determined to be 0.45 mg/mL and 105.26 Units·mg·protein−1 ·min−1, respectively. The recombinant proteins, r-Foc1-Pgc4 and r-Foc4-Pgc4, were expressed and purified from Pichia pastoris and showed optimal Pgc4 activity at 55 °C and pH 4.0; both could induce tissue maceration and necrosis in the “Guangfen-1” and “Baxi” varieties of banana but to a different extent. Phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Therefore, our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4.

A MADS-box transcription factor FoRlm1 regulates aerial hyphal growth, oxidative stress, cell wall biosynthesis and virulence in Fusarium oxysporum f. sp. cubense

The fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt that affects banana plants. However, the detailed molecular mechanisms of Foc virulence determinants have not been elucidated. In this study, we identified the MADS-box transcription factor FoRlm1 that is conserved among mitogen-activated protein kinases. Our data revealed that FoRlm1 is essential for aerial hyphal growth and virulence. Transcriptional analysis revealed that FoRlm1 deletion altered the expression of anti-oxidant enzymes, chitin synthases, fusaric acid (FA), and beauvericin biosynthesis genes. Furthermore, FoRlm1 deletion promoted tolerance to Congo red and increased sensitivity to hydrogen peroxide. Transcriptome analysis of ΔFoRlm1 mutant and wild-type strain indicated that the expression of many genes associated with fungal physiology and virulence was up- or down-regulated. Overall, these results suggested that FoRlm1 plays a critical role in the regulation of hyphal growth, anti-oxidation mechanisms, cell wall biosynthesis, transcription of mycotoxin biosynthetic genes encoding FA and beauvericin, and virulence in Foc.

Apc.LaeA and Apc.VeA of the velvet complex govern secondary metabolism and morphological development in the echinocandin-producing fungus Aspergillus pachycristatus.

The impact of the global secondary metabolite regulators LaeA and VeA on echinocandin B production and morphological development was evaluated in the industrial production strain Aspergillus pachycristatus NRRL 11440. Other representative secondary metabolites were examined as well to determine if the velvet complex functions as in A. nidulans and other species of fungi. Genetic methods used for gene manipulations in A. nidulans were applied to A. pachycristatus. Separate deletions of genes Apc.laeA and Apc.veA resulted in similar yet differing phenotypes in strain NRRL 11440. Disruption of Apc.laeA and Apc.veA significantly reduced, but did not eliminate, the production of echinocandin B. Similar to what has been observed in A. nidulans, the production of sterigmatocystin was nearly eliminated in both mutants. Quantitative reverse transcription PCR analyses confirmed that selected genes of both the echinocandin B and sterigmatocystin gene clusters were down-regulated in both mutant types. The two mutants differed with respect to growth of aerial hyphae, pigmentation, development of conidiophores, conidial germination rate, and ascospore maturation. Further functional annotation of key regulatory genes in A. pachycristatus and related Aspergillus species will improve our understanding of regulation of echinocandin production and co-produced metabolites.

First Report of Fusarium oxysporum Causing Root Rot on Polygonatum sibiricum in Chizhou, China

Polygonatum sibiricum, which produces active ingredients including steroidal saponins, polysaccharide, and flavones, is used as a Chinese traditional medicine to alleviate palpitation, dry cough, and diabetes mellitus, and is widely cultivated in China. During the past 2 years, a root rot disease was identified that seriously affected the production of P. sibiricum, resulting in approximately 20 to 50% yield loss each year, in Chizhou city of China. To investigate the causal agents of this disease, root rot samples were collected from a field in this area in March 2019. Most infected plants had reddish brown to dark brown lesions on the roots, which thereafter expanded in the depth of root tuber and make them putrid. The diseased tissues were surface disinfested in 2% sodium hypochlorite for 2 min, rinsed three times using sterilized water, and plated on potato dextrose agar (PDA) at 28°C in darkness for 3 to 4 days. Fungal colonies were obtained from infectious tissues and nine single-spore isolates were purified, with white to violet fluffy aerial hyphae growth on PDA. On these colonies, two types of conidia developed; the slightly falcate macroconidia (3.96 to 5.93 × 10.26 to 31.25 μm) have 3 to 5 septa, while the ovoid or ellipse microconidia (2.05 to 4.56 × 4.55 to 9.96 μm) have 0 to 1 septum. Chlamydospores were terminal or intercalary, in pairs or in chains, and measured 5.63 to 9.46 µm in diameter. Such typical morphological characteristics suggests the identification of the nine single-spore isolates to be Fusarium oxysporum (Leslie and Summerell 2006). To confirm pathogenicity, Koch’s postulate was carried out. Five 1-year-old P. sibiricum seedlings with healthy growth were planted together in a pot with sterilized soil (diameter = 30 cm), and a 20 ml conidial suspension of each isolate was irrigated into the soil near the roots (1 × 10⁵ conidia/ml, 4 ml for each plant). As a control, five 1-year-old P. sibiricum seedlings were treated with distilled water. Inoculated seedlings were grown in a greenhouse at 28°C with an 8-h photoperiod of fluorescent light. Similar lesions were observed on the inoculated roots but not on control ones after 3 weeks. To further confirm the identification of this pathogen, we reisolated the F. oxysporum fungus from the infected roots and characterized them by DNA sequencing. The internal transcribed spacer (ITS) and translation elongation factor (TEF-1α) gene regions were amplified using the primers ITS4/ITS5 (Guo et al. 2012; Wang et al. 2016) and EF-1/EF-2 (O’Donnell et al. 1998), respectively, and sequences were deposited in GenBank (ITS, accession no. MK811209; TEF-1α, accession no. MK830060). The sequences shared 99.61% and 100% similarity with F. oxysporum for ITS (MK681347 and 31 others) and TEF-1α (MH828020 and 99 others), respectively. Based on the above data, all nine isolates were identified as F. oxysporum. To our knowledge, this is the first report of F. oxysporum as causal agent of root rot on P. sibiricum in Chizhou, China. F. oxysporum infections pose a serious threat to P. sibiricum production and thus need to be considered when developing effective control strategies.

Conidiation in Neurospora crassa: vegetative reproduction by a model fungus.

Asexual development, conidiation, in the filamentous fungus Neurospora crassa is a simple developmental process that starts with the growth of aerial hyphae. Then, the formation of constrictions and subsequent maturation gives rise to the mature conidia that are easily dispersed by air currents. Conidiation is regulated by environmental factors such as light, aeration and nutrient limitation, and by the circadian clock. Different regulatory proteins acting at different stages of conidiation have been described. The role of transcription factors such as FL, and components of signal transduction pathways such as the cAMP phosphodiesterase ACON-2 suggest a complex interplay between differential transcription and signal transduction pathways. Comparisons between the molecular basis of conidiation in N. crassa and other filamentous fungi will help to identify common regulatory elements.

FgIlv3a is crucial in branched-chain amino acid biosynthesis, vegetative differentiation, and virulence in Fusarium graminearum.

Dihydroxyacid dehydratase (DHAD), encoded by ILV3, catalyses the third step in the biosynthetic pathway of branched-chain amino acids (BCAAs), which include isoleucine (Ile), leucine (Leu), and valine (Val). Enzymes involved in BCAA biosynthesis exist in bacteria, plants, and fungi but not in mammals and are therefore attractive targets for antimicrobial or herbicide development. In this study, three paralogous ILV3 genes (FgILV3A, FgILV3B, and FgILV3C) were identified in the genome of Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Deletion of FgILV3A alone or combined with FgILV3B or FgILV3C indicated an important role for FgILV3A in BCAA biosynthesis. FgILV3A deletion mutants lost the ability to grow on medium lacking amino acids. Exogenous supplementation of 1 mM Ile and Val rescued the auxotrophy of ΔFgIlv3A, though 5 mM was required to recover the growth defects in ΔFgIlv3AB and ΔFgIlv3AC strains, indicating that FgIlv3b and FgIlv3c exhibit redundant but accessory roles with FgIlv3a in BCAA biosynthesis. The auxotrophy of ΔFgIlv3A resulted in pleiotropic defects in aerial hyphal growth, in conidial formation and germination, and in aurofusarin accumulation. In addition, the mutants showed reduced virulence and deoxynivalenol production. Overall, our study demonstrates that FgIlv3a is crucial for BCAA biosynthesis in F. graminearum and a candidate fungicide target for FHB management.

A Colletotrichum gloeosporioides cerato-platanin protein, CgCP1, contributes to conidiation and plays roles in the interaction with rubber tree.

Colletotrichum gloeosporioides is the causal agent of rubber tree anthracnose and leads to serious losses of natural rubber production. The pathogenesis of C. gloeosporioides on rubber tree remains unknown. Cerato-platanin proteins are small, secreted cysteine-rich proteins that contribute to virulence and function in plant-fungal interactions. A gene encoding cerato-platanin protein, CgCP1, was identified in C. gloeosporioides. In silico analysis indicated that CgCP1 belongs to a new branch of the cerato-platanin protein family. The CgCP1 knockout mutants (ΔCgCP1) and complementary strain (Res-ΔCgCP1) were generated to investigate its biological function. The results showed that the speed of growth of aerial hyphae was not significantly different among the wild-type (WT), ΔCgCP1, and Res-ΔCgCP1 strains, but conidiation of ΔCgCP1 significantly decreased in comparison with the WT. The pathogenicity test proved that the severity of symptoms caused by ΔCgCP1 was reduced significantly compared with those caused by the Res-ΔCgCP1 and WT strains. Additionally, CgCP1 induced necrosis-like cell death on tobacco leaf and accumulation of reactive oxygen species in rubber tree mesophyll protoplasts. Altogether, these data indicate the involvement of C. gloeosporioides CgCP1 in conidiation and the interaction with rubber tree.