Fungicide-Loaded Liposomes for the Treatment of Fungal Diseases in Agriculture: An Assessment of Botrytis cinerea.

In this work, liposomes loaded with the fungicide, Fludioxonil (FLUD), for the containment of fungal diseases in agriculture were developed. Three types of vesicles with different compositions were compared: (I) plain vesicles, composed of soy phosphatidylcholine and cholesterol; (II) PEG-coated vesicles, with an additional polyethylene glycol coating; and (III) cationic vesicles, containing didodecyldimethylammonium bromide. Nanometric-sized vesicles were obtained both by the micelle-to-vesicle transition method and by the extrusion technique, and encapsulation efficiency, drug loading content, and Zeta potential were determined for all the samples. The extruded and PEGylated liposomes were the most stable over time and together with the cationic ones showed a significant prolonged FLUD release capacity. The liposomes' biological activity was evaluated on conidial germination, germ tube elongation and colony radial growth of the ascomycete Botrytis cinerea, a phytopathogenic fungus affecting worldwide many important agricultural crops in the field as well as in the postharvest phase. The extruded and PEGylated liposomes showed greater effectiveness in inhibiting germ tube elongation and colony radial growth of the fungal pathogen, even at 0.01 µg·mL-1, the lowest concentration assessed.

Antifungal effect and some properties of cell-free supernatants of two Bacillus subtilis isolates against Fusarium verticillioides.

Fusarium verticillioides causes significant decrease in corn yield and quality, and produces fumonisins, which represent a serious risk to human and animal health. Bacillus species can be an effective and environmentally friendly alternative for F. verticillioides biological control. In this study, some properties of cell-free supernatants (CFSs) of two Bacillus spp. identified as Bacillus subtilis (NT1, NT2) as well as the antifungal effect against F. verticillioides 97L were evaluated. B. subtilis NT1 and NT2 were isolated from commercially available fermented whole soybeans (Natto). Antifungal activity was observed in both CFSs of B. subtilis isolates (50-59 mm) obtained by co-culture suggesting that antifungal compound production depends on interaction between bacteria and fungi. Cell-free supernatants from the two B. subtilis isolates inhibited mycelial growth (77%-94%) and conidial germination (22%-74%) of F. verticillioides 97L. In addition, CFSs caused significant morphological changes such as distorted and collapsed hyphae with wrinkled surfaces and the presence of a large amount of extracellular material compared to the control without CFSs. Both B. subtilis isolates (NT1 and NT2) produced extracellular proteases, biosurfactants and polar low molecular weight compounds that probably act synergistically and may contribute to the antifungal activity. Antifungal compounds showed heat and pH stability and resistance to proteolytic enzymes. Furthermore, antifungal compounds showed high polarity, high affinity to water and a molecular weight less than 10 kDa. These results indicated that the two B. subtilis (NT1 and NT2) have potential as biocontrol agents for F. verticillioides.

Electron-transferring flavoprotein and its dehydrogenase contributed to growth development and virulence in Beauveria bassiana.

Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.

Iron/Copper/Phosphate nanocomposite as antimicrobial, antisnail, and wheat growth-promoting agent.

BACKGROUND: One of the current challenges is to secure wheat crop production to meet the increasing global food demand and to face the increase in its purchasing power. Therefore, the current study aimed to exploit a new synthesized nanocomposite to enhance wheat growth under both normal and drought regime. The effectiveness of this nanocomposite in improving the microbiological quality of irrigation water and inhibiting the snail's growth was also assessed. RESULTS: Upon the employed one-step synthesis process, a spherical Fe/Cu/P nanocomposite was obtained with a mean particle size of 4.35 ± 1.524 nm. Cu2+, Fe2+, and P4+ were detected in the dried nanocomposite at 14.533 ± 0.176, 5.200 ± 0.208, and 34.167 ± 0.203 mg/ml concentration, respectively. This nanocomposite was found to exert antibacterial activity against Escherichia coli and Salmonella typhi. It caused good inhibition percent against Fusarium oxysporum (43.5 ± 1.47%) and reduced both its germination rate and germination efficiency. The lethal concentration 50 (LC50) of this nanocomposite against Lanistes carinatus snails was 76 ppm. The treated snails showed disturbance in their feeding habit and reached the prevention state. Significant histological changes were observed in snail digestive tract and male and female gonads. Drought stress on wheat's growth was mitigated in response to 100 and 300 ppm treatments. An increase in all assessed growth parameters was reported, mainly in the case of 100 ppm treatment under both standard and drought regimes. Compared to control plants, this stimulative effect was accompanied by a 2.12-fold rise in mitotic index and a 3.2-fold increase in total chromosomal abnormalities. CONCLUSION: The finding of the current study could be employed to mitigate the effect of drought stress on wheat growth and to enhance the microbiological quality of irrigation water. This is due to the increased efficacy of the newly synthesized Fe/Cu/P nanocomposite against bacteria, fungi, and snails. This methodology exhibits potential for promoting sustainable wheat growth and water resource conservation.

Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

Coordination of two regulators SscA and VosA in Aspergillus nidulans conidia.

Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans.

SscA is required for fungal development, aflatoxin production, and pathogenicity in Aspergillus flavus.

Fungal spores are specialized dormant cells that act as primary reproductive biological particles and exhibit strong viability under extremely harsh conditions. They contaminate a variety of crops and foods, causing severe health hazards to humans and animals. Previous studies demonstrated that a spore-specific transcription factor SscA plays pivotal roles in the conidiogenesis of the model organism Aspergillus nidulans. In this study, we investigated the biological and genetic functions of SscA in the aflatoxin-producing fungus A. flavus. Deletion of sscA showed reduced conidia formation, lost long-term viability, and exhibited more sensitivity to thermal, oxidative, and radiative stresses. The sscA-deficient strain showed increased aflatoxin B1 production in conidia as well as mycelia. Importantly, the absence of sscA affected fungal pathogenicity on crops. Further transcriptomic and phenotypic studies suggested that SscA coordinates conidial wall structures. Overall, SscA is important for conidial formation, maturation and dormancy, mycotoxin production, and pathogenicity in A. flavus.

A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds.

BACKGROUND: Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. RESULTS: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. CONCLUSIONS: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Temporal Dynamics and Dispersal Patterns of the Primary Inoculum of Coniella diplodiella, the Causal Agent of Grape White Rot.

Grape white rot can cause considerable yield losses in viticulture areas worldwide and is principally caused by Coniella diplodiella. The fungus overwinters in berry mummies on the soil surface or on the trellis and produces pycnidia and conidia that serve as primary inoculum. However, little is known about the temporal dynamics and dispersal pattern of C. diplodiella conidia. In this study, we investigated the production and dispersal of C. diplodiella conidia from a primary inoculum source, namely, affected mummified berries that overwintered in two vineyards in northern Italy in 2021 and 2022. Conidia of C. diplodiella were repeatedly produced in berry mummies from the budburst of vines to harvesting, with approximately 50 and 75% of the total conidia in a season being produced before fruit set and véraison, respectively. The production dynamics of C. diplodiella conidia over time were described by a Weibull equation in which the thermal time is the independent variable, with a concordance correlation coefficient of ≥0.964. A rainfall cutoff of ≥0.2 mm provided an overall accuracy of ≥0.86 in predicting conidial dispersal through rain splashes from berry mummies on the soil surface, with the number of dispersed conidia increasing with the amount of rainfall. The dispersal of conidia from mummies on the trellis by washing with rain required at least 6.1 mm of rain. The proposed mathematical equations and rain cutoffs can be used to predict periods with a high dispersal risk of C. diplodiella.

Conidial mass production of entomopathogenic fungi and tolerance of their mass-produced conidia to UV-B radiation and heat.

We investigated conidial mass production of eight isolates of six entomopathogenic fungi (EPF), Aphanocladium album (ARSEF 1329), Beauveria bassiana (ARSEF 252 and 3462), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium anisopliae sensu lato (ARSEF 2341), Metarhizium pingshaense (ARSEF 1545), and Simplicillium lanosoniveum (ARSEF 6430 and 6651) on white or brown rice at four moisture conditions (75-100%). The tolerance of mass-produced conidia of the eight fungal isolates to UV-B radiation and heat (45 °C) were also evaluated. For each moisture content compared, a 20-g sample of rice in a polypropylene bag was inoculated with each fungal isolate in three replicates and incubated at 28 ± 1 °C for 14 days. Conidia were then harvested by washing the substrate, and conidial concentrations determined by haemocytometer counts. Conidial suspensions were inoculated on PDAY with 0.002% benomyl in Petri plates and exposed to 978 mW m-2 of Quaite-weighted UV-B for 2 h. Additionally, conidial suspensions were exposed to 45 °C for 3 h, and aliquots inoculated on PDAY with benomyl. The plates were incubated at 28 ± 1 °C, and germination was assessed at 400 × magnification after 48 h. Conidial production was generally higher on white rice than on brown rice for all fungal species, except for L. aphanocladii ARSEF 6433, regardless of moisture combinations. The 100% moisture condition provided higher conidial production for B. bassiana (ARSEF 252 and ARSEF 3462) and M. anisopliae (ARSEF 2341) isolates, while the addition of 10% peanut oil enhanced conidial yield for S. lanosoniveum isolate ARSEF 6430. B. bassiana ARSEF 3462 on white rice with 100% water yielded the highest conidial production (approximately 1.3 × 1010 conidia g-1 of substrate). Conidia produced on white rice with the different moisture conditions did not differ in tolerance to UV-B radiation or heat. However, high tolerance to UV-B radiation and heat was observed for B. bassiana, M. anisopliae, and A. album isolates. Heat-treated conidia of S. lanosoniveum and L. aphanocladii did not germinate.

A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis.

Aspergillus fumigatus, an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores (conidia) for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, and the cryptic pathogen Aspergillus lentulus. After identifying 62 proteins uniquely expressed on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding conidial proteins. Deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection.

Investigation on the Influence of Production and Incubation Temperature on the Growth, Virulence, Germination, and Conidial Size of Metarhizium brunneum for Granule Development.

Important for the infection of an insect with an entomopathogenic fungus and its use as a plant protection agent are its growth, conidiation, germination, and virulence, which all depend on the environmental temperature. We investigated not only the effect of environmental temperature but also that of production temperature of the fungus. For this purpose, Metarhizium brunneum JKI-BI-1450 was produced and incubated at different temperatures, and the factors mentioned as well as conidial size were determined. The temperature at which the fungus was produced affects its subsequent growth and conidiation on granule formulation, the speed of germination, and the conidial width, but not its final germination or virulence. The growth and conidiation was at its highest when the fungus was produced at 25 °C, whereas when the germination was faster, the warmer the fungus was produced. The incubation temperature optimum of JKI-BI-1450 in relation to growth, speed of germination, and survival time was 25-30 °C and for conidiation 20-25 °C. Conidial length decreased with increasing incubation temperature. Although the fungus could not be adapted to unfavorable conditions by the production temperature, it was found that the quality of a biological control agent based on entomopathogenic fungi can be positively influenced by its production temperature.

Regulators of the Asexual Life Cycle of Aspergillus nidulans.

The genus Aspergillus, one of the most abundant airborne fungi, is classified into hundreds of species that affect humans, animals, and plants. Among these, Aspergillus nidulans, as a key model organism, has been extensively studied to understand the mechanisms governing growth and development, physiology, and gene regulation in fungi. A. nidulans primarily reproduces by forming millions of asexual spores known as conidia. The asexual life cycle of A. nidulans can be simply divided into growth and asexual development (conidiation). After a certain period of vegetative growth, some vegetative cells (hyphae) develop into specialized asexual structures called conidiophores. Each A. nidulans conidiophore is composed of a foot cell, stalk, vesicle, metulae, phialides, and 12,000 conidia. This vegetative-to-developmental transition requires the activity of various regulators including FLB proteins, BrlA, and AbaA. Asymmetric repetitive mitotic cell division of phialides results in the formation of immature conidia. Subsequent conidial maturation requires multiple regulators such as WetA, VosA, and VelB. Matured conidia maintain cellular integrity and long-term viability against various stresses and desiccation. Under appropriate conditions, the resting conidia germinate and form new colonies, and this process is governed by a myriad of regulators, such as CreA and SocA. To date, a plethora of regulators for each asexual developmental stage have been identified and investigated. This review summarizes our current understanding of the regulators of conidial formation, maturation, dormancy, and germination in A. nidulans.

Biocontrol of Xyleborus affinis (Curculionidae: Scolitinae) Females and Progeny by Beauveria bassiana (Hypocreales: Cordycipitaceae) in a Sawdust Artificial Diet Model.

The ambrosia beetle Xyleborus affinis, recently reported affecting avocado trees in Mexico, represents one of the most widespread insects worldwide. Previous reports have shown that Xyleborus genera members are susceptible to Beauveria bassiana and other entomopathogenic fungus strains. However, their effect on borer beetles' progeny has not been fully investigated. The aim of the present study was to determine the insecticidal activity of B. bassiana on X. affinis adult females and their progeny in an artificial sawdust diet bioassay model. The B. bassiana strains CHE-CNRCB 44, 171, 431, and 485 were individually tested on females at concentrations ranging from 2 × 106 to 1 × 109 conidia mL-1. After 10 d of incubation, diet was evaluated to count laid eggs, larvae, and adults. Insect conidia loss after exposure was determined by attached conidia to each insect after 12 h of exposure. The results showed that females' mortality ranged between 3.4% and 50.3% in a concentration-response manner. Furthermore, we did not observe statistical differences among strains at the highest concentration. CHE-CNRCB 44 showed the highest mortality at the lowest concentration and reduced larvae and laid eggs at the highest concentration (p < 0.01). Strains CHE-CNRCB 44, 431, and 485 significantly decreased larvae, as compared with the untreated control. After 12 h, up to 70% of conidia was removed by the effect of the artificial diet. In conclusion, B. bassiana has the potential to control X. affinis adult females and progeny.

Isolation of Bacillus siamensis B-612, a Strain That Is Resistant to Rice Blast Disease and an Investigation of the Mechanisms Responsible for Suppressing Rice Blast Fungus.

Rice yield can be significantly impacted by rice blast disease. In this investigation, an endophytic strain of Bacillus siamensis that exhibited a potent inhibitory effect on the growth of rice blast was isolated from healthy cauliflower leaves. 16S rDNA gene sequence analysis showed that it belongs to the genus Bacillus siamensis. Using the rice OsActin gene as an internal control, we analyzed the expression levels of genes related to the defense response of rice. Analysis showed that the expression levels of genes related to the defense response in rice were significantly upregulated 48 h after treatment. In addition, peroxidase (POD) activity gradually increased after treatment with B-612 fermentation solution and peaked 48 h after inoculation. These findings clearly demonstrated that the 1-butanol crude extract of B-612 retarded and inhibited conidial germination as well as the development of appressorium. The results of field experiments showed that treatment with B-612 fermentation solution and B-612 bacterial solution significantly reduced the severity of the disease before the seedling stage of Lijiangxintuan (LTH) was infected with rice blast. Future studies will focus on exploring whether Bacillus siamensis B-612 produces new lipopeptides and will apply proteomic and transcriptomic approaches to investigate the signaling pathways involved in its antimicrobial effects.

Comparative proteome analysis identifies species-specific signature proteins in Aspergillus pathogens.

Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. KEY POINTS: • CSPs and exoproteins could differentiate A. flavus and A. fumigatus. • A. fumigatus conidial surface harbored more antigenic proteins than A. flavus. • Identified species-specific signature proteins of A. flavus and A. fumigatus.

Tandem Multimerization Can Enhance the Structural Homogeneity and Antifungal Activity of the Silkworm Protease Inhibitor BmSPI39.

Previous studies have shown that BmSPI39, a serine protease inhibitor of silkworm, can inhibit virulence-related proteases and the conidial germination of insect pathogenic fungi, thereby enhancing the antifungal capacity of Bombyx mori. The recombinant BmSPI39 expressed in Escherichia coli has poor structural homogeneity and is prone to spontaneous multimerization, which greatly limits its development and application. To date, the effect of multimerization on the inhibitory activity and antifungal ability of BmSPI39 remains unknown. It is urgent to explore whether a BmSPI39 tandem multimer with better structural homogeneity, higher activity and a stronger antifungal ability can be obtained by protein engineering. In this study, the expression vectors of BmSPI39 homotype tandem multimers were constructed using the isocaudomer method, and the recombinant proteins of tandem multimers were obtained by prokaryotic expression. The effects of BmSPI39 multimerization on its inhibitory activity and antifungal ability were investigated by protease inhibition and fungal growth inhibition experiments. In-gel activity staining and protease inhibition assays showed that tandem multimerization could not only greatly improve the structural homogeneity of the BmSPI39 protein, but also significantly increase its inhibitory activity against subtilisin and proteinase K. The results of conidial germination assays showed that tandem multimerization could effectively enhance the inhibitory ability of BmSPI39 on the conidial germination of Beauveria bassiana. A fungal growth inhibition assay showed that BmSPI39 tandem multimers had certain inhibitory effects on both Saccharomyces cerevisiae and Candida albicans. The inhibitory ability of BmSPI39 against these the above two fungi could be enhanced by tandem multimerization. In conclusion, this study successfully achieved the soluble expression of tandem multimers of the silkworm protease inhibitor BmSPI39 in E. coli and confirmed that tandem multimerization can improve the structural homogeneity and antifungal ability of BmSPI39. This study will not only help to deepen our understanding of the action mechanism of BmSPI39, but also provide an important theoretical basis and new strategy for cultivating antifungal transgenic silkworms. It will also promote its exogenous production and development and application in the medical field.

Essential Role of WetA, but No Role of VosA, in Asexual Development, Conidial Maturation and Insect Pathogenicity of Metarhizium robertsii.

Conidial maturation, which is crucial for conidial quality, is controlled by the asexual development activator WetA and the downstream, velvety protein VosA in Aspergillus. Their orthologs have proved functional in conidial quality control of Beauveria bassiana, as seen in Aspergillus, but are functionally unexplored, in Metarhizium robertsii, another hypocrealean insect pathogen. Here, WetA and VosA prove essential and nonessential for M. robertsii's life cycle, respectively. Disruption of wetA increased hyphal sensitivity to oxidative stress and Congo red-induced cell wall stress, but had little impact on radial growth. The ΔwetA mutant was severely compromised in conidiation capacity and conidial quality, which was featured by slower germination, decreased UV resistance, reduced hydrophobicity, and deformed hydrophobin rodlet bundles that were assembled onto conidial coat. The mutant's virulence was greatly attenuated via normal infection due to a blockage of infection-required cellular processes. All examined phenotypes were unaffected for the ΔvosA mutant. Intriguingly, mannitol was much less accumulated in the 7- and 15-day-old cultures of ΔwetA and ΔvosA than of control strains, while accumulated trehalose was not detectable at all, revealing little a link of intracellular polyol accumulation to conidial maturation. Transcriptomic analysis revealed differential regulation of 160 genes (up/down ratio: 72:88) in ΔwetA. These genes were mostly involved in cellular component, biological process, and molecular function but rarely associated with asexual development. Conclusively, WetA plays a relatively conserved role in M. robertsii's spore surface structure, and also a differentiated role in some other cellular processes associated with conidial maturation. VosA is functionally redundant in M. robertsii unlike its ortholog in B. bassiana. IMPORTANCE WetA and VosA regulate conidiation and conidial maturation required for the life cycle of Beauveria bassiana, like they do in Aspergillus, but remain functionally unexplored in Metarhizium robertsii, another hypocrealean pathogen considered to have evolved insect pathogenicity ~130 million years later than B. bassiana. This study reveals a similar role of WetA ortholog in asexual development, conidial maturation, and insect pathogenicity, and also its distinctive role in mediating some other conidial maturation-related cellular events, but has functional redundancy of VosA in M. robertsii. The maturation process vital for conidial quality proves dependent on a role of WetA in spore wall assembly but is independent of its role in intracellular polyol accumulation. Transcriptomic analysis reveals a link of WetA to 160 genes involved in cellular component, biological process, and molecular function. Our study unveils that M. robertsii WetA or VosA is functionally differential or different from those learned in B. bassiana and other ascomycetes.

GAR-transferase contributes to purine synthesis and mitochondrion function to maintain fungal development and full virulence of Penicillium digitatum.

Penicillium digitatum is one of the most critical phytopathogens during the citrus postharvest period. However, the molecular mechanism of pathogenesis remains to be further explored. Purine is a multiple functional substance in organisms. To verify the role of the de novo purine biosynthesis (DNPB) pathway in P. digitatum, we investigated the third gene Pdgart, glycinamide ribonucleotide (GAR)-transferase, of this pathway in this study. The deletion mutant ΔPdgart was generated in the principle of homologous recombination via Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotypic assay indicated that the ΔPdgart mutant displayed severe defects in hyphae growth, conidiation and germination, which can be rescued by the addition of exogenous ATP and AMP. Compared with wild-type strain N1, the ATP level of strain ΔPdgart was detected to be sharply declined during conidial germination, and this was resulted from the damage to purine synthesis and aerobic respiration. The pathogenicity assay suggested that mutant ΔPdgart infected citrus fruit but attenuated disease, which was owing to its reduced production of organic acids and activities of cell wall degradation enzymes. Additionally, the ΔPdgart mutant showed altered sensitivity to stress agents and fungicides. Taken together, the present study provides insights into the essential functions of Pdgart, and paves the way for further study and novel fungicide development.

The Entomopathogenic Fungus Beauveria bassiana Employs Autophagy as a Persistence and Recovery Mechanism during Conidial Dormancy.

Many filamentous fungi develop a conidiation process as an essential mechanism for their dispersal and survival in natural ecosystems. However, the mechanisms underlying conidial persistence in environments are still not fully understood. Here, we report that autophagy is crucial for conidial lifespans (i.e., viability) and vitality (e.g., stress responses and virulence) in the filamentous mycopathogen Beauveria bassiana. Specifically, Atg11-mediated selective autophagy played an important, but not dominant, role in the total autophagic flux. Furthermore, the aspartyl aminopeptidase Ape4 was found to be involved in conidial vitality during dormancy. Notably, the vacuolar translocation of Ape4 was dependent on its physical interaction with autophagy-related protein 8 (Atg8) and associated with the autophagic role of Atg8, as determined through a truncation assay of a critical carboxyl-tripeptide. These observations revealed that autophagy acted as a subcellular mechanism for conidial recovery during dormancy in environments. In addition, a novel Atg8-dependent targeting route for vacuolar hydrolase was identified, which is essential for conidial exit from a long-term dormancy. These new insights improved our understanding of the roles of autophagy in the physiological ecology of filamentous fungi as well as the molecular mechanisms involved in selective autophagy. IMPORTANCE Conidial environmental persistence is essential for fungal dispersal in ecosystems while also serving as a determinant for the biocontrol efficacy of entomopathogenic fungi during integrated pest management. This study identified autophagy as a mechanism to safeguard conidial lifespans and vitality postmaturation. In this mechanism, the aspartyl aminopeptidase Ape4 translocates into vacuoles via its physical interaction with autophagy-related protein 8 (Atg8) and is involved in conidial vitality during survival. The study revealed that autophagy acted as a subcellular mechanism for maintaining conidial persistence during dormancy, while also documenting an Atg8-dependent targeting route for vacuolar hydrolase during conidial recovery from dormancy. Thus, these observations provided new insight into the roles of autophagy in the physiological ecology of filamentous fungi and documented novel molecular mechanisms involved in selective autophagy.