Conidial Production Research Articles

FsCGBP, a Cutinase G-Box Binding Protein, Regulates the Growth, Development, and Virulence of Fusarium sacchari, the Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari is a causal agent of sugarcane Pokkah boeng, an important fungal disease that causes a considerable reduction in yield and sugar content in susceptible varieties of sugarcane worldwide. Despite its importance, the fungal factors that regulate the virulence of this pathogen remain largely unknown. In our previous study, mapping of an insertional mutant defect in virulence resulted in the identification of a cutinase G-box binding protein gene, designated FsCGBP, that encodes a C2H2-type transcription factor (TF). FsCGBP was shown to localize in the nuclei, and the transcript level of FsCGBP was significantly upregulated during the infection process or in response to abiotic stresses. Deletion or silencing of FsCGBP resulted in a reduction in mycelial growth, conidial production, and virulence and a delay in conidial germination in the F. sacchari. Cutinase genes FsCUT2, FsCUT3, and FsCUT4 and the mitogen-activated protein kinase (MAPK) genes FsHOG1, FsMGV1, and FsGPMK1, which were significantly downregulated in ΔFsCGBP. Except for FsHOG1, all of these genes were found to be transcriptionally activated by FsCGBP using the yeast one-hybrid system in vitro. The deletion of individual cutinase genes did not result in any of the phenotypes exhibited in the ΔFsCGBP mutant, except for cutinase activity. However, disruption of the MAPK pathway upon deletion of FsMGV1 or FsGPMK1 resulted in phenotypes similar to those of the ΔFsCGBP mutant. The above results suggest that FsCGBP functions by regulating the MAPK pathway and cutinase genes, providing new insights into the mechanism of virulence regulation in F. sacchari.

Synergistic antifungal mechanism of cinnamaldehyde and nonanal against Aspergillus flavus and its application in food preservation

Aspergillus flavus colonization on agricultural products during preharvest and postharvest results in tremendous economic losses. Inspired by the synergistic antifungal effects of essential oils, the aims of this study were to explore the mechanism of combined cinnamaldehyde and nonanal (SCAN) against A. flavus and to evaluate the antifungal activity of SCAN loading into diatomite (DM). Shriveled mycelia were observed by scanning electron microscopy, especially in the SCAN treatment group. Calcofluor white staining, transmission electron microscopy, dichloro-dihydro-fluorescein diacetate staining and the inhibition of key enzymes in tricarboxylic acid cycle indicated that the antifungal mechanism of SCAN against A. flavus was related to the cell wall damage, reactive oxygen species accumulation and energy metabolism interruption. RNA sequencing revealed that some genes involved in antioxidation were upregulated, whereas genes responsible for cell wall biosynthesis, oxidative stress, cell cycle and spore development were significantly downregulated, supporting the occurrence of cellular apoptosis. In addition, compared with the control group, conidia production in 1.5 mg/mL DM/cinnamaldehyde, DM/nonanal and DM/SCAN groups were decreased by 27.16%, 48.22% and 76.66%, respectively, and the aflatoxin B1 (AFB1) contents decreased by 2.00%, 73.02% and 84.15%, respectively. These finding suggest that DM/SCAN complex has potential uses in food preservation.

Copper ion sorption ability of Alternaria alternata (Fr.) Keissl. and Fusarium oxysporum Schltdl. micromycetes

Our analysis of metabolic and ecological features of fungi indicates a high bioremediation potential of fungal mycelium. The relevance of the problems solved in this work is conditioned by insufficiently studied mechanisms and conditions of micromycetal activity on heavy metal detoxication. The effect of copper on the development of two micromycete species, namely, melanised Alternaria alternata (Fr.) Keissl., 1912 and hyaline Fusarium oxysporum Schltdl., 1824 cultivated on Czapek agar (with 2 or 3% sucrose) was compared; the ability of mycelium to sorb copper cations when grown in a liquid culture with 0, 0.05, 0.1, 0.25, and 0.5 mg Cu2+ / L was evaluated. F. oxysporum had noticeable advantages in terms of growth rate, tolerance, conidia production on medium with Cu2+. The effective concentration of copper (EC50), indicating the resistance of the fungus, increased for F. oxysporum by 1.5 times with increasing sucrose content in the medium. The melanised culture of A. alternata was superior to F. oxysporum in terms of sorption capacity. The percentage of Cu2+ extraction by fungal mycelium from the medium reached 40% in the case of F. oxysporum and twice as much in the case of A. alternata . The vast majority of copper was sorbed by the cell walls of hyphae and washed away by water. A small amount (a maximum of 0.16 µg Cu2+ /g dry mycelium of A. alternate ) penetrated inside the mycelial cells. F. oxysporum, which had no intracellular protective melanins, accumulated Cu2+ (2 to 14 times) less. The results show that the mechanisms of resistance to Cu2+ and sorption are different in the studied species: in Fusarium they are determined mainly by the barrier functions of hyphae cell walls, while in Alternaria melanin plays a significant role in protection against Cu toxic action.

Functional Roles of Two β-Tubulin Isotypes in Regulation of Sensitivity of Colletotrichum fructicola to Carbendazim.

Colletotrichum fructicola is the major pathogen of anthracnose in tea-oil trees in China. Control of anthracnose in tea-oil trees mainly depends on the application of chemical fungicides such as carbendazim. However, the current sensitivity of C. fructicola isolates in tea-oil trees to carbendazim has not been reported. Here, we tested the sensitivity of 121 C. fructicola isolates collected from Guangdong, Guangxi, Guizhou, Hainan, Hunan, Jiangsu, and Jiangxi provinces in China to carbendazim. One hundred and ten isolates were sensitive to carbendazim, and 11 isolates were highly resistant to carbendazim. The growth rates, morphology, and pathogenicity of three resistant isolates were identical to those of three sensitive isolates, which indicates that these resistant isolates could form a resistant population under carbendazim application. These results suggest that carbendazim should not be the sole fungicide in control of anthracnose in tea-oil trees; other fungicides with different mechanisms of action or mixtures of fungicides could be considered. In addition, bioinformatics analysis identified two β-tubulin isotypes in C. fructicola: Cfβ1tub and Cfβ2tub. E198A mutation was discovered in the Cfβ2tub of three carbendazim-resistant isolates. We also investigated the functional roles of two β-tubulin isotypes. CfΔβ1tub exhibited slightly increased sensitivity to carbendazim and normal phenotypes. Surprisingly, CfΔβ2tub was highly resistant to carbendazim and showed a seriously decreased growth rate, conidial production, pathogenicity, and abnormal hyphae morphology. Promoter replacement mutant CfΔβ2-2×β1 showed partly restored phenotypes, but it was still highly resistant to carbendazim, which suggests that Cfβ1tub and Cfβ2tub are functionally interchangeable to a certain degree.

Roles of two new transcription factors in the synergistic effect of combined cinnamaldehyde and nonanal against Aspergillus flavus

Aspergillus flavus, an opportunistic pathogen, is renowned for colonizing agricultural crops, leading to aflatoxin contamination. In this study, the inhibitory potency of combined cinnamaldehyde and nonanal (SCAN) against A. flavus on stored grains was evaluated, and the mechanism of action at the molecular level was explored. SCAN exhibited synergistic action against A. flavus on wheat, resulting in a 99.17% decrease in spore production. This was clearly a greater decrease than if the individual compounds were used separately. RNA-sequencing and gene knock-out analyses were performed to clarify the synergistic mechanism. Transcriptome data revealed that two new transcription factors (TFs), TF-1 (AFLA_049760) and TF-2 (AFLA_048110), were down-regulated when exposed to 0.03 μL/mL and 0.45 μL/mL SCAN. The TF-1 and TF-2 deletion mutants presented slower growth rates compared with wild-type A. flavus. In addition, conidial production was decreased by down-regulating the conidiation genes brlA and abaA. Stress assays and confocal laser scanning microscopic examinations revealed that more reactive oxygen species accumulated in ΔTF-1 mutants compared with wild-type A. flavus. These two TFs positively regulated the production of aflatoxin B1 biosynthesis and down-regulated the expression of genes, including aflM, aflQ, aflR and aflS, in the aflatoxin gene cluster. More importantly, our results indicated that the mutants were impaired in their ability to infect crop seeds. Thus, the two new TFs, which play key roles in asexual development, aflatoxin B1 biosynthesis and fungal virulence in A. flavus, may be the critical regulators in the fungus after exposure to SCAN.

FgGmtB Plays an Important Role in Growth, Reproduction, Virulence and Deoxynivalenol Biosynthesis of Fusarium graminearum.

GDP-mannose transporters (GMTs) have been implicated in the virulence of some important pathogenic fungi, and guanosine diphosphate (GDP) mannose transporters transport GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation, where mannose attaches to the modified protein. GMTs could be potential targets for new antifungal drugs, as disruption of any step in GDP-mannose biosynthesis can affect fungal viability, growth, or virulence. To date, the GDP-mannose transporter has been extensively studied in yeast, but its biological function in fungi, particularly F. graminearum, is still unclear. In this experimental study, the role of the GDP-mannose transporter in F. graminearum was investigated by analysing the VRG4 gene. FgGmtA and FgGmtB were blastp-derived from their Scvrg4 protein sequences and proved to be their functional homologues. The mutant and complementary strains of FgGmtA, FgGmtB and FgGmtA&B genes were generated and used to evaluate the effect of the two GMTs genes on mycelial growth, asexual reproduction, sexual reproduction, cell wall sensitivity, glyphosate synthesis and drug susceptibility. Only in the FgGmtB and FgGmtA&B mutants was the rate of mycelial growth slowed, conidium production increased, sexual reproduction impaired, cell wall sensitivity increased, glycemic content decreased, and drug sensitivity reduced. The results of the pathogenicity assessment of GMTs showed that only FgGmtB affects the patogenicity of F. graminearum. At the same time, the effect of GMTs on the ability of rhinoceros to synthesise DON toxins was investigated and the results showed that the ability of ΔFgGmtB and ΔFgGmtA&B mutants to produce the DON toxin was significantly reduced, and the expression of toxin-related genes was also reduced.

The histone deacetylase UvHOS2 regulates vegetative growth, conidiation, ustilaginoidin synthesis, and pathogenicity in Ustilaginoidea virens

Ustilaginoidea virens causes rice false smut, one of the most devastating rice diseases. The pathogen produces various types of mycotoxins, such as ustilaginoidins and ustiloxins, which are harmful to both human and animal health. Histone deacetylases in fungi play an important role in regulating chromatin structure and gene expression. However, there is limited knowledge about how histone deacetylases control pathogenicity and mycotoxin biosynthesis in U. virens. Here, we characterize a putative class I histone deacetylase UvHOS2 in U. virens. The UvHos2-deletion mutants exhibit retarded vegetative growth, reduced conidial production and germination, and attenuated virulence. UvHOS2 positively regulates tolerance to various environmental stresses, including cell wall, cell membrane integrity, osmotic and oxidative stresses. UvHOS2 reduces the acetylation levels of histones at multiple Lys sites, including H3K9, H3K14, H3K27, and H3K56. ChIP-PCR assays revealed that UvHOS2-mediated H3K9 deacetylation regulates the expression of ustilaginoidin biosynthesis genes. Consistently, transcriptome analysis indicates that UvHOS2 regulates the expression of the genes involved in secondary metabolism, mycelial growth, conidiogenesis, and pathogenicity, thereby controlling U. virens virulence and mycotoxin (ustilaginoidins and sorbicillinoids) biosynthesis. This study provides a theoretical reference for revealing the epigenetic regulation of pathogenicity and mycotoxin biosynthesis in plant pathogenic fungi.

Production of Escovopsis conidia and the potential use of this parasitic fungus as a biological control agent of leaf-cutting ant fungus gardens.

This study was carried out to investigate the use of different substrates for the production of Escovopsis conidia and verify the virulence of four different isolates cultured on four types of substrates using a novel bioassay. Escovopsis isolates were molecularly identified, based on Internal Transcribed Spacer (ITS) nucleotide sequences. To evaluate conidial production, suspensions (1 × 106 conidia mL-1) of each Escovopsis isolate were inoculated onto four substrates (parboiled rice, white rice, rolled oats, and corn grits). After 14days, conidial yields were assessed. The virulence of each isolate cultured on the four substrates was tested against Leucoagaricus fungus garden fragments, by directly applying 500µL of each conidial suspension (1 × 107 conidia mL-1), and the development of the parasite was monitored daily until it completely colonized the fungus garden. It was observed that rolled oats were the best substrate for conidial production, with a yield of1.7 × 107 to 2.0 × 108 conidia mL-1. Furthermore, isolate AT-01 produced the highest number of conidia when compared with the other isolates. Regardless of the substrate used to produce AT-01 conidia, this isolate completely colonized the fungus garden 6days post inoculation (dpi), followed by AT-02, AC-01, and AC-2. High levels of both conidial production and virulence against the leaf-cutting ant fungus garden were observed here.

Liamocins overproduction via the two-pH stage fermentation and anti-Aspergillus flavus activity of Massoia lactone.

Aureobasidium melanogenum was found to be grown the best at the constant pH 7.0 and to produce the highest amount of liamocins at the constant pH 3.0. Therefore, the wild type strain A. melanogenum 9-1 and the engineered strain V33 constructed in the laboratory were grown at the constant pH 7.0 for 48 h, then, they were continued to be cultivated at the constant pH 3.0. Under such conditions, A. melanogenum 9-1 produced 36.51 ± 0.55 g L-1 of liamocin and its cell mass was 27.43 ± 0.63 and 6.00 ± 0.11 g L-1 of glucose was left in the finished medium within 168 h while the engineered strain V33 secreted 70.86 ± 2.04 g L-1 of liamocin, its cell mass was 31.63 ± 0.74 g L-1 , 0.16 ± 0.01 g L-1 of glucose was maintained in the finished medium. Then, Massoia lactone was released from the produced liamocins. The released Massoia lactone loaded in the nanoemulsions could be used to actively damage cell wall and cell membrane of both spores and mycelia of Aspergillus flavus, leading to its cell necrosis. Massoia lactone loaded in the nanoemulsions also actively inhibited cell growth of A. flavus, its conidia production and aflatoxin biosynthesis on peanuts, indicating that Massoia lactone loaded in the nanoemulsions had highly potential application in controlling cell growth of A. flavus and aflatoxin biosynthesis in foods and feedstuffs.

Antifungal potential of commercial silver nanoparticles against rice blast pathogen Magnaporthe oryzae

The rice blast caused by Magnaporthe oryzae is one of the major and recurrent threats to sustainable rice production in Bangladesh. To mitigate this problem, the present study was undertaken with the aim to investigate the inhibitory effects of commercially available silver nanoparticles (Ag-NPs) on the growth and development of Magnaporthe oryzae, and control of rice blast disease. In vitro experiments demonstrated that Ag-NPs significantly suppressed the radial growth of fungal mycelium, with the highest inhibition (83.54%) at a concentration of 250 ppm, while Trooper 75 WP at 400 ppm completely inhibited the fungal mycelial growth. The production of conidia and colony formation were also reduced significantly by Ag-NPs treatment. Furthermore, Ag-NPs exhibited a substantial inhibitory effect on the germination of M. oryzae conidia, with complete inhibition observed at concentrations between 200 and 250 ppm. Comparative analyses revealed that the fungicidal effect of Ag-NPs was superior to silver nitrate (AgNO3), suggesting the augmented activity of the nano properties. The sensitivity of M. oryzae to Ag-NPs was determined by calculating EC50 and EC95 values, which indicated a mean EC50 value of 131.5 ppm and a mean EC95 value of 276 ppm. Additionally, in vivo experiments showed that preventive and curative treatments with Ag-NPs significantly reduced disease incidence and severity in rice plants infected with M. oryzae, with efficacy comparable to that of the fungicide Trooper 75 WP. At higher concentrations of Ag-NPs, preventive treatment was found to be more effective than curative treatment in disease control. These findings highlighted the potential of Ag-NPs as an effective alternative for the management of rice blast. Ann. Bangladesh Agric. (2023) 27 (1): 17-30

RHO-3 plays a significant role in hyphal extension rate, conidiation, and the integrity of the Spitzenkörper in Neurospora crassa

The Rho family of monomeric GTPases act as signaling proteins to establish and maintain cell polarity and other essential cellular processes. Rho3 is a GTPase of the Rho family that is exclusive of fungi that regulate cell polarity in yeast. However, studies have yet to explore its function in filamentous fungi. In this work, we investigated the role of RHO-3 in the model organism Neurospora crassa. Confocal microscopy analysis revealed that RHO-3 localizes in the outer region of the Spitzenkörper (Spk), in the plasma membrane from region II to the beginning of region III, and in the septa of mature hyphae. The phenotypic effect of the rho-3 deletion was analyzed. The results revealed that the rho-3 null strain showed severe defects in growth rate, aerial hyphae length, and conidia production. The organization of the Spk is also affected in the absence of RHO-3. Co-expression analysis of GFP-RHO-3 with glucan synthase 1 (GS-1-mChFP) and chitin synthase 1 (CHS-1-mChFP) revealed that RHO-3 localizes in the external region of the Spk in the macrovesicles zone. In summary, our results suggest that RHO-3 is not essential for the polarized growth of hyphae but plays a significant role in hyphal extension rate, conidiation, sexual reproduction and the integrity of the Spk, possibly regulating the delivery of macrovesicles to the apical dome.

StRAB4 gene is required for filamentous growth, conidial development, and pathogenicity in Setosphaeria turcica.

Setosphaeria turcica, the fungal pathogen responsible for northern corn leaf blight in maize, forms specialized infectious structures called appressoria that are critical for fungal penetration of maize epidermal cells. The Rab family of proteins play a crucial role in the growth, development, and pathogenesis of many eukaryotic species. Rab4, in particular, is a key regulator of endocytosis and vesicle trafficking, essential for filamentous growth and successful infection by other fungal pathogens. In this study, we silenced StRAB4 in S. turcica to gain a better understanding the function of Rab4 in this plant pathogen. Phenotypically, the mutants exhibited a reduced growth rate, a significant decline in conidia production, and an abnormal conidial morphology. These phenotypes indicate that StRab4 plays an instrumental role in regulating mycelial growth and conidial development in S. turcica. Further investigations revealed that StRab4 is a positive regulator of cell wall integrity and melanin secretion. Functional enrichment analysis of differentially expressed genes highlighted primary enrichments in peroxisome pathways, oxidoreductase and catalytic activities, membrane components, and cell wall organization processes. Collectively, our findings emphasize the significant role of StRab4 in S. turcica infection and pathogenicity in maize and provide valuable insights into fungal behavior and disease mechanisms.

Identification and characterization of FpRco1 in regulating vegetative growth and pathogenicity based on T-DNA insertion in Fusarium pseudograminearum

Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot (FCR) in wheat and poses a significant threat to wheat production in terms of grain yield and quality. However, the mechanism by which F. pseudograminearum infects wheat remains unclear. In this study, we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F. pseudograminearum. By screening this mutant library, we identified nine independent mutants that displayed impaired pathogenesis in barley leaves. Among these mutants, one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1, encoding essential component of the Rpd3S histone deacetylase complex in F. pseudograminearum. To further investigate the role of FpRCO1 in F. pseudograminearum, we employed a split-marker approach to knock out FpRCO1 in F. pseudograminearum WZ-8A. FpRCO1 deletion mutants exhibit reduced vegetative growth, conidium production, and virulence in wheat coleoptiles and barley leaves, whereas the complementary strain restores these phenotypes. Moreover, under stress conditions, the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl, sorbitol, and SDS, but possessed reduced sensitivity to H2O2 compared to these characteristics in the wild-type strain. RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression (particularly the downregulation of TRI gene expression), thus resulting in significantly reduced deoxynivalenol (DON) production. In summary, our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development, asexual reproduction, DON production, and pathogenicity of F. pseudograminearum. This study provides valuable insights into the molecular mechanisms underlying F. pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.

Mitochondrial Outer Membrane Translocase MoTom20 Modulates Mitochondrial Morphology and Is Important for Infectious Growth of the Rice Blast Fungus Magnaporthe oryzae.

Mitochondria are highly dynamic organelles that constantly change their morphology to adapt to the cellular environment through fission and fusion, which is critical for a cell to maintain normal cellular functions. Despite the significance of this process in the development and pathogenicity of the rice blast fungus Magnaporthe oryzae, the underlying mechanism remains largely elusive. Here, we identified and characterized a mitochondrial outer membrane translocase, MoTom20, in M. oryzae. Targeted gene deletion revealed that MoTom20 plays an important role in vegetative growth, conidiogenesis, penetration, and infectious growth of M. oryzae. The growth rate, conidial production, appressorium turgor, and pathogenicity are decreased in the ΔMotom20 mutant compared with the wild-type and complemented strains. Further analysis revealed that MoTom20 localizes in mitochondrion and plays a key role in regulating mitochondrial fission and fusion balance, which is critical for infectious growth. Finally, we found that MoTom20 is involved in fatty-acid utilization, and its yeast homolog ScTom20 is able to rescue the defects of ΔMotom20 in mitochondrial morphology and pathogenicity. Overall, our data demonstrate that MoTom20 is a key regulator for mitochondrial morphology maintenance, which is important for infectious growth of the rice blast fungus M. oryzae. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Co-infection with two novel mycoviruses affects the biocontrol activity of Trichoderma polysporum

This study reports the whole genome sequence of two double-stranded RNA viruses (mycoviruses) that co-infected an isolate of Trichoderma polysporum NFCF205 and the interactions between fungus and virus. Based on genome size, organization, sequence similarity, and phylogenic analysis, one of these is best characterized as a new member of the proposed family “Fusagraviridae”, and accordingly suggest that it be referred to as Trichoderma polysporum fusagravirus 1 (TpFGV1). The other is best classified as a new strain of the genus Alphapartitivirus in the family Partitiviridae, and should be designated Trichoderma polysporum partitivirus 1 from T. polysporum NFCF205 (TpPV1). By single spore isolation, virus-free or singly-infected isogenic strains were obtained and compared to determine the biological functions of the viruses. The colony morphology of the co-infected parental strain showed the characteristic spiraled hairy and fluffy mycelia, while both the singly-infected and virus-free strain rarely showed the conspicuous aerial mycelia. The growth rate of the co-infected strain was slightly, but nevertheless statistically significantly, higher on PDA. Interestingly, conidia production was observed to have significantly increased in the co-infected strain while no difference was seen in the others. Growth inhibition measured by dual-culturing with Rhizoctonia solani revealed a significant difference between the co-infected strain and a TpPV1-infected strain. However, the secreted soluble compounds in the co-infected strain showed the least antimicrobial activity. Moreover, antimicrobial activities via volatile organic compounds (VOCs), which signify strong biocontrol activity by Trichoderma spp., was the lowest in the co-infected strain. The profiles and amount of representative VOCs in the assessed strains, as measured by gas chromatography-mass spectrometry (GC/MS/MS), were affected by virus-infection. Virus infection affected colony morphology, conidia production, and biocontrol activities of T. polysporum. Most interestingly, virus-virus interaction was observed and measured in the co-infected strain, as their symptoms differed from those of the singly-infected strains.

VdPT1 Encoding a Neutral Trehalase of Verticillium dahliae Is Required for Growth and Virulence of the Pathogen.

Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene (VdPT1) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants (ΔVdPT1) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae. Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae. Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.

Pleiotropic functions of SscA on the asexual spore of the human pathogenic fungus Aspergillus fumigatus

ABSTRACT Asexual spores, called conidia, are key reproductive fungal particles that enable survival in harsh environmental conditions or host systems. The conidia can infect humans, animals, and plants to cause various fungal diseases. Transcription factors, including VosA, WetA, and SscA, have key roles in conidia formation and long-term survival in Aspergillus nidulans. Herein, we report the pleiotropic functions of SscA in the conidia of the human pathogen A. fumigatus. The deletion of sscA increased conidia formation despite decreased fungal growth. Absence of sscA impaired long-term survival and reduced spore resistance to various stresses, including heat, UV, and oxidation. Transcriptomic analyses showed that SscA involved the mRNA expression of cell wall organisation-related genes. Importantly, the sscA deletion mutant conidia contained an increased amount of β-glucan and chitin compared to wild type conidia. In addition, conidial gliotoxin production was decreased in the sscA deletion strain. Overall, SscA has pleiotropic roles in conidia formation, maturation and dormancy and mycotoxin production in A. fumigatus.

Isolation and Identification of Bacillus subtilis LY-1 and Its Antifungal and Growth-Promoting Effects.

Peanut root rot, caused by Fusarium spp., is a devastating fungal disease. As part of a program to obtain a biocontrol agent to control peanut root rot in the field, a bacterial strain LY-1 capable of inhibiting the growth of the fungus in vitro was isolated from rhizosphere soil samples collected from wild mint by agar disk dilution and dual-culture assay. Strain LY-1 was identified as Bacillus subtilis based on morphological characteristics, 16S rDNA, and gyrA sequence analyses. The bacterial suspension and cell-free culture filtrate of LY-1 could significantly inhibit the growth of Fusarium oxysporum, Fusarium proliferatum and Fusarium solani, but volatile organic compounds from the cultures had only a weak effect on mycelial growth. The percentage inhibition of 20% concentration of the cell-free culture filtrate of LY-1 on conidium production of each of the three Fusarium species was greater than 72.38%, and the percentage inhibition by the culture filtration on the germination of conidia of the three species was at least 62.37%. The production of extracellular enzyme activity by LY-1 was studied in functional assays, showing protease, cellulase, amylase, chitinase, and β-1,3-glucanase activity, while LY-1 contained a gene encoding iturin, an antifungal lipopeptide. In addition, under pot culture in a greenhouse, culture filtrate of LY-1 significantly promoted the growth of peanut, increasing the fresh and dry mass of the plant by 30.77% and 27.27%, respectively, in comparison with the no-filtrate control. The culture filtrate of LY-1 increased the resistance of peanut plants to F. oxysporum, with the biocontrol efficiency reaching 44.71%. In conclusion, B. subtilis LY-1, a plant-growth-promoting rhizobacterium, was able to protect peanuts from Fusarium spp. infection.

Biological Characteristics of Beauveria majiangensis Strain MJ1015 and Optimization of Solid Medium Technology for Sporulation.

The entomopathogenic fungus Beauveria majiangensis strain MJ1015, recently isolated from white grubs on a blueberry farm in Guizhou, China, could be used as a biocontrol agent. As a first step toward determining the effect of different solid culture media, temperature, and pH on colony growth rate and sporulation, we evaluated the optimum solid medium for mycelial growth and conidia production on a commercial scale. Subsequently, we also used single-factor analysis and response surface optimization to optimize the composition of the solid culture medium. On potato dextrose agar (PDA) medium, MJ1015 grew fastest and produced the highest spore yield at 29°C and pH 5. The best solid medium for the growth and sporulation of strain MJ1015 comprised 64.70 g/l of rice, 13.00 g/l of wheat, 0.30 g/l of NaNO3, 0.36 g/l of K2HPO4 · 3H2O, and 1.00 g/l of CaCO3. Rice, NaNO3, and K2HPO4 · 3H2O were the main influencing factors. The predicted value of cultured spores using the optimal medium was 4.56 x 1010 conidia/l. The validation test results showed that the average growth rate of strain MJ1015 on the optimal medium was 85% and 96% faster than that on Sabouraud dextrose agar with yeast extracts medium (SDAY) and PDA, respectively. Sporulation was 43.90 times and 9.65 times of that produced on SDAY and PDA, respectively. Our findings provide a theoretical basis for the commercial production of B. majiangensis to control white grubs.

Cytological studies reveal high variation in ascospore number and shape and conidia produced directly from ascospores in Morchella galilaea.

Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely results in asci with fewer than eight ascospores. This study provides new insights into the poorly understood life cycle of Morchella species and more broadly improves knowledge of conidia formation and reproductive strategies in Pezizomycetes.