Advances in submerged liquid fermentation and formulation of entomopathogenic fungi.

Entomopathogenic fungi (EPF) can be defined as beneficial multifunctional eukaryotic microorganisms that display pivotal ecological services in pest management, with some species possessing the special ability to establish mutualistic relationships with plants. Mass production of these fungi is critical to support affordable widespread commercialization and worldwide field application. Among the mass production methods explored mainly by industry, submerged liquid fermentation is a robust and versatile technology that allows the formation of different types of propagules designated for various applications in pest control. Many hypocrealean EPF are easily culturable on artificial substrates by producing single-celled structures (hyphal bodies, blastospores, and submerged conidia) or multicellular structures (mycelium and microsclerotia). Less frequently, some EPF may form environmentally resistant chlamydospores, but these structures have almost always been overlooked. A continued research pipeline encompassing screening fungal strains, media optimization, and proper formulation techniques aligned with the understanding of molecular cues involved in the formation and storage stability of these propagules is imperative to unlock the full potential and to fine-tune the development of robust and effective biocontrol agents against arthropod pests and vectors of diseases. Finally, we envision a bright future for the submerged liquid fermentation technology to supplement or replace the traditional solid substrate fermentation method for the mass production of many important EPF. KEY POINTS: • Submerged liquid fermentation (SLF) allows precise control of nutritional and environmental factors • SLF provides a scalable, robust, and cost-effective platform for mycopesticide production • Enhancing formulation, shelf life, and field efficacy of submerged propagules remain crucial • Understanding the molecular mechanisms behind submerged propagule formation is key to advancing SLF technology.

Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Do Growers Using Solo Fungicides Affect the Durability of Disease Control of Growers Using Mixtures and Alternations? The Case of Spot-Form Net Blotch in Western Australia.

Growers often use alternations or mixtures of fungicides to slow down the development of resistance to fungicides. However, within a landscape, some growers will implement such resistance management methods, whereas others do not, and may even apply solo components of the resistance management program. We investigated whether growers using solo components of resistant management programs affect the durability of disease control in fields of those who implement fungicide resistance management. We developed a spatially implicit semidiscrete epidemiological model for the development of fungicide resistance. The model simulates the development of epidemics of spot-form net blotch disease, caused by the pathogen Pyrenophora teres f. maculata. The landscape comprises three types of fields, grouped according to their treatment program, with spore dispersal between fields early in the cropping season. In one field type, a fungicide resistance management method is implemented, whereas in the two others, it is not, with one of these field types using a component of the fungicide resistance management program. The output of the model suggests that the use of component fungicides does affect the durability of disease control for growers using resistance management programs. The magnitude of the effect depends on the characteristics of the pathosystem, the degree of inoculum mixing between fields, and the resistance management program being used. Additionally, although increasing the amount of the solo component in the landscape generally decreases the lifespan within which the resistance management program provides effective control, situations exist where the lifespan may be minimized at intermediate levels of the solo component fungicide. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First Report of Powdery mildew caused by Golovinomyces ambrosiae on Bidens pilosa in Guangdong Province, China.

Bidens pilosa L., an annual herbaceous plant with a wide distribution, possesses novel medicinal properties. In January 2021, a powdery mildew disease outbreak was documented on B. pilosa plants located in the roadside areas in Shenzhen, Guangdong Province, China, with 60 to 80% disease incidence. Initial symptoms manifested as small, irregular white powdery patches, primarily on the adaxial surfaces of leaves. Subsequently, the colonies expanded, forming coalescent colonies that spread across the leaves, petioles, and stems, eventually leading to the distortion and senescence of leaves. Hyphae are hyaline, flexuous to straight, septate, with thin walls and a width ranging from 2 to 8 µm. Hyphal appressoria are nipple-shaped. Conidophores are erect or slightly flexuous, ranging from 80 to 150 µm in length and 12 to 18 µm in width (n = 30). Typically, these conidophores bear chains of 2 to 5 immature conidia, displaying a sinuate outline. Foot-cells, located at the base of conidophores, are cylindrical and erect, approximately 33 to 100 µm in length and 6 to 10 µm in width (n = 30). Conidia are hyaline, ellipsoid-ovoid to barrel-shaped, and lack fibrosin bodies. Primary conidia are ellipsoid-ovoid in shape, characterized by a rounded apex and a subtruncate base, 25 to 40 µm × 15 to 22 µm in width. Secondary conidia are barrel-shaped with truncate or subtruncate ends, 27 to 35 µm × 15 to 20 µm in width. Germ tubes exhibit a longitubus pattern and are prominently produced at the perihilar or apical region of the conidia. No chasmothecia were observed in the collected samples. In order to conduct a molecular-level identification, mycelium and conidia were collected from B. pilosa leaves. Genomic DNA was subsequently extracted from these samples. The internal transcribed spacer (ITS), intergenic spacer (IGS) and beta-tubulin (tub2) sequences were performed using primer pairs ITS1/ITS4, IGS-12a/NS1R, and tub2, respectively (Carbone and Kohn 1999; Scholin et al. 1994; White et al.,1990). A 568-bp ITS, a 366-bp IGS, and a 354-bp tub2 sequences (GenBank accession nos. OR647592, OR978282 and OR978283) were obtained. The ITS sequence exhibited over 99.6% similarity to Golovinomyces ambrosiae (MT929773) and G. cichoracearum (MH590731). The IGS sequence displayed 100% similarity to G. ambrosiae (MK383490) and G. ambrosiae (OK349420). The tub2 sequence displayed 100% similarity to G. ambrosiae (MW981257) and G. ambrosiae (MW981256). Phylogenetic analysis of IGS, ITS and tub2 also grouped obtained sequences within the G. ambrosiae complex. Based on the analysis of morphological characteristics and sequence identity, the pathogen was identified as G. ambrosiae. In order to satisfy Koch's postulates, an infected leaf was carefully pressed onto leaves of six healthy young B. pilosa plants, each grown in a separate pot. Additionally, a control group consisted of six non-inoculated plants. All plants were placed in a greenhouse: 25°C, 14/10-h light/dark photoperiod, and relative humidity 50%. After 10 days, the inoculated leaves exhibited powdery mildew colonies similar to those observed in the original infected plants. At 16 days, the inoculated leaves exhibited discoloration and premature leaf drop. The pathogenicity test was conducted twice. Microscopic observation and sequencing confirmed that isolated fungus was identical to the original pathogen. G. ambrosiae has previously been documented on B. pilosa in Fuzhou, Fujian Province, China (Mukhtar et al., 2022). However, to the best of our knowledge, this study represents the first report of powdery mildew caused by G. ambrosiae on B. pilosa in Shenzhen, Guangdong Province, China.

Adhesive solid-state fermentation producing Aspergillus niger conidia on stainless-steel dixon ring supports.

An adhesive solid-state fermentation (adSSF) mode was developed to produce Aspergillus niger conidia, which used a stainless-steel Dixon ring as the support and water-retaining adhesive to load nutritional media on its surface. To obtain high conidia yields, the components of the water-retaining adhesive were screened, optimized by single-factor optimization and response surface methodology, and the optimal dosages of the main components were: wheat bran powder 0.023 g·cm-3bed, cassava starch 0.0022 g·cm-3bed, and xanthan gum 0.0083 g·cm-3bed. The experimentally tested conidia yield was 4.2-fold that without water-retaining adhesive but was 3.7% lower than the maximum yield predicted by the model. The observed double-side growth of A. niger on the Dixon ring supports improved space utilization of the fermentation bed, and the void fraction can increase with the shrinkage of the gel layer. In 1.6 L tray reactors with three-point online temperature monitoring, the inner-bed temperature of adSSF was at most 4 °C lower than the adsorbed carrier solid-state fermentation (ACSSF) mode, and the conidia yield was 1.68 × 108 conidia.cm-3bed, 61.5% higher than that of the ACSSF bed at the same time, but when the fermentation time was extended to 168 h, the conidia yield of the adSSF bed and ACSSF bed were close to each other. The results revealed that the high voidage of the adSSF bed was the main reason for low bed temperature, which can benefit the inner-bed natural convection and water evaporation.

Improvement of the production and quality of Cordyceps javanica conidia for the control of Diaphorina citri adults.

This study aimed to evaluate the use of palm kernel meal (PKM) in the traditional solid-state fermentation system to improve the production and quality of Cordyceps javanica conidia. The impact of PKM was determined by measuring conidia yield, viability, hydrophobicity, shelf life, and conidia pathogenicity against Diaphorina citri adults. By supplementing rice grains with 5% palm kernel meal increased the conidial yield by up to 40%, without compromising conidia viability and hydrophobicity. In addition, conidia caused higher levels of mortality by mycosis against D. citri adults (90%), relative to conidia harvested from rice (52%). The conidia recovered from rice/palm kernel meal mixtures also retained viability greater than 90% after storage for 10 months at 4 °C, while the conidia produced on rice reached 80%. Thus, conidia produced in the presence of palm kernel meal can be consumed immediately or in the medium term. Some process advantages of the palm kernel meal as co-substrate in the traditional production system of C. javanica are also mentioned. These results are attractive for improving the mycoinsecticide production process, with excellent cost-benefit and minimal changes in infrastructure and process.

The sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) coordinately regulate the response of Neurospora crassa to the springtail Sinella curviseta (Collembola: Entomobryidae) attack.

IMPORTANCE: Understanding the regulatory pathways by which fungi respond to environmental signals through interlinked genes provides insights into the interactions between fungi and insects. The coordinated optimization of the regulatory networks is necessary for fungi to adapt to their habitats. We demonstrated that the synergistic regulation of sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) plays a critical role in regulating the fungal response to Sinella curviseta stress. Furthermore, we found that the enhanced production of trehalose, carotenoids, and 5-MTHF plays crucial role in the resistance to the fungivore. Our results provide insights into the understanding of the adaptation of N. crassa to environmental stimuli.

Gcc1 homologs regulate growth, oxidative stress, conidiation and appressorium formation in Colletotrichum siamense and Colletotrichum graminicola.

The Zn2Cys6 transcription factor is a fungal-specific zinc finger protein, which plays an important role in regulating growth, development and pathogenicity of pathogenic fungi. In this study, we characterized two Zn2Cys6 transcription factors, CsGcc1 and CgrGcc1 in Colletotrichum siamense and C. graminicola, respectively, which are homologous to Gcc1 in Magnaporthe oryzae. Both CsGcc1 and CgrGcc1 contain a typical GAL4 DNA-binding domain. Deletion of CsGCC1 or CgrGCC1 decreased the growth rate and lowered the tolerance to H2O2. In addition, disrupting CsGCC1 reduced conidial yield and lowered the germination rate and appressorium formation rate of C. siamense. Cellophane assays showed that deletion of CsGCC1 also weakened the penetration ability of appressoria. In C. graminicola, CgrGcc1 did not affect the production and germination of oval conidia, but its deletion significantly decreased the yield of the falcate conidium, and led to abnormal appressorium formation. In terms of pathogenicity, CsGcc1 slightly reduced the virulence of C. siamense, while deleting CgrGcc1 did not affect virulence of C. graminicola. In conclusion, the Zn2Cys6 transcription factors CsGcc1 and CgrGcc1 are involved in the regulation of vegetative growth, oxidative stress, conidial/falcate conidial production and appressorium formation in C. siamense and C. graminicola.

α-Tomatine degradation to tomatidine by food-related Aspergillus species belonging to the section Nigri.

α-Tomatine is a steroidal glycoalkaloid in tomato plants and degrades with ripening. The aglycone form, tomatidine, is reported to have beneficial effects. In this study, the ability of food-related microorganisms to produce tomatidine from α-tomatine was evaluated. A total of 11 strains of Aspergillus species belonging to the section Nigri exhibited tomatinase activity, and Aspergillus luchuensis JCM 22302 was selected for optimization due to its high activity in its mycelia, conidia, and non-mycotoxin-producing property. Next, using A. luchuensis JCM22302 conidia, the highest yield was obtained in a 24-h reaction with 50 m m of acetic acid-sodium acetate buffer (pH 5.5) at 37 °C. Similar to the tomato pathogen Fusarium oxysporum f. lyceopersici, the time course analysis suggested that A. luchuensis JCM 22302 removed the entire sugar moiety in a single step. Future research will focus on utilizing conidia for large-scale tomatidine production because of their high tolerance and manageability.

Temperature-dependent sporulation of the fungus Coniella diplodiella, the causal agent of grape white rot.

White rot, caused by the fungus Coniella diplodiella, can severely reduce grapevine yields worldwide. Currently, white rot control mainly relies on fungicides applied on a calendar basis or following hailstorms that favor disease outbreak; however, the control achieved with this strategy is often inconsistent or otherwise unsatisfactory. Realizing more rational control requires an improved understanding of white rot epidemiology. Toward this end, we conducted experiments with grapevine berries of two Vitis vinifera cultivars (either injured or not before artificial inoculation with a conidia suspension of C. diplodiella) to determine the effect of temperature on the length of latency (i.e., the time between infection and onset of mature pycnidia on berries) and the production of pycnidia and conidia. Sporulation occurred between 10°C and 35°C, with the optimum detected at 20°C. The latency period was shorter at 25-35°C than at lower temperatures; the shortest latency period was 120 h at 30°C on injured berries. Affected berries produced abundant conidia at 15-30℃ (the optimum was 20℃) for more than two months following inoculation. Mathematical equations were developed that fit the data, with strong associations with temperature for latency period (R2 = 0.831) and for the production dynamics of secondary conidia (R2 = 0.918). These equations may contribute to the development of a risk algorithm to predict infection periods, which can inform risk-based rather than calendar-based disease control strategies.

Novel Harzia ixtarensis Fungus on Annona cherimola Fruit in Mexico and Its Synergistic Relationship with Colletotrichum fragariae.

Since 2005 in Íxtaro, Michoacán, symptoms of Harzia infection have been observed on immature Annona cherimola fruit with Colletotrichum fragariae-induced anthracnose lesions and mummified fruit. This study aimed to identify the Harzia sp. and evaluate its pathogenicity. Four isolates were obtained from fruit exhibiting symptoms, cultured in four types of agar under various conditions, and characterized based on concatenated internal transcribes spacer (ITS) + large subunit and ITS + small subunit sequences. Additionally, the isolates were compared with two CBS species (two-type strains and two isolates) of Harzia patula and H. tenella under the same conditions as the Harzia isolates, and all known Harzia spp. in culture were included in two phylogenetic analyses. H. ixtarensis sp. nov. was proposed. Compared with H. patula CBS isolate 121524 which was the most closely phylogenetically related species, H. ixtarensis was characterized by slower colony growth (white to salmonish-beige), different percentages of two forms of conidia (elongated and globose; unicellular and hyaline to subhyaline), and smaller conidia. The conidia mainly germinated with two hyaline tubes without an appressorium. In situ inoculations (1 × 106 ml-1 conidia suspension) of fruit showed that fruit with wounds developed larger lesions than those without wounds. Harzia inoculation on anthracnose lesions (induced by prior inoculation with C. fragariae) produced larger anthracnose lesions than C. fragariae alone. When C. fragariae or H. ixtarensis was inoculated alone, the lesion size was 51 and 99% smaller, respectively, indicating synergy between C. fragariae and H. ixtarensis. Thus, H. ixtarensis may have a parasitic-synergistic and necrotrophic lifestyle, and exhibited symptoms on anthracnose lesions.

Effects of Temperature on Spore Germination and Mycelial Growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata Isolates Associated with Sweet Cherry Canker Diseases.

Although fungal canker diseases constitute a limiting factor to orchard productivity and longevity, little is known about the effects of temperature on spore germination and mycelial growth of the fungal causal agents. Accordingly, the germination of spores and colony growth of Calosphaeria pulchella, Cytospora sorbicola, and Eutypa lata were evaluated after incubation on 2% water agar and 4% potato dextrose agar, respectively, at 5, 10, 15, 20, 25, 30, 35, and 40°C. Temperature optima for spore germination and mycelial growth were derived from nonlinear models fitted to germination rates and colony diameter data. The optimal temperatures for spore germination of Cal. pulchella were 28.5°C for ascospores and 29.2°C for conidia. The optimal temperatures for Cyt. sorbicola conidia and E. lata ascospore germination were 25.8 and 23.1°C, respectively. The germination of ascospores and conidia of Cal. pulchella at temperatures below 15°C required an incubation time of at least 72 h. Ascospores of E. lata and conidia of Cyt. sorbicola germinated at 10°C after 36 h. The optimal temperature for colony growth of Cal. pulchella was 24.6°C, whereas it was 21.7°C for both Cyt. sorbicola and E. lata. Our study indicates that temperature requirements for basic biological functions are higher for Cal. pulchella than for Cyt. sorbicola and E. lata. The overall higher temperatures of California relative to other cherry-producing regions in the United States or worldwide could explain the prevalence of Calosphaeria canker in the state. Conversely, Cyt. sorbicola and E. lata appear better adapted to cooler temperatures.

Species Identification and Fungicide Sensitivity of Fungi Causing Alternaria Leaf Blight and Head Rot in Cole Crops in the Eastern United States.

Alternaria leaf blight and head rot is an important disease of broccoli and other cole crops. With no resistant host varieties, fungicides are utilized to manage this disease. However, anecdotal evidence suggests that, in southeastern U.S. broccoli-producing states, there is a loss of disease control through the use of quinone outside inhibitor (QoI) fungicides. To understand why there is a reduced sensitivity to QoI fungicides in these states, we isolated Alternaria spp. from symptomatic lesions on cole crops from Georgia and Virginia (two states with observations of loss of fungicide sensitivity) as well as New York (a state with no observations of loss of fungicide sensitivity). Using multilocus sequencing and phylogenetic analysis, we identified two species, Alternaria brassicicola and A. japonica. Whereas A. brassicicola was isolated in all states, A. japonica was only isolated in Georgia. Next, we wanted to determine the sensitivity of these isolates to azoxystrobin-an active ingredient in some QoI fungicides-by estimating the effective concentration at which only 50% of spores germinate (EC50). The EC50 of A. brassicicola ranged from 0.01 to 0.17 ppm, whereas that of A. japonica was 8.1 to 28.1 ppm. None of the known target-site mutations that confer resistance to QoI fungicides were identified during screening of either species. A. japonica was first reported on the east coast of the United States in 2020 in South Carolina. The substantially higher EC50 value suggests that its emergence in the southeastern United States may play at least a part in the observed loss of disease control. However, further in planta and field studies are needed to thoroughly test this hypothesis.

Light-Driven Tetra- and Octa-ß-substituted Cationic Zinc(II) Phthalocyanines for Eradicating Fusarium oxysporum Conidia.

Photodynamic inactivation (PDI) is an emerging therapeutic approach that can effectively inactivate diverse microbial forms, including vegetative forms and spores, while preserving host tissues and avoiding the development of resistance to the photosensitization procedure. This study evaluates the antifungal and sporicidal photodynamic activity of two water-soluble amphiphilic tetra- and octa-ß-substituted zinc(II) phthalocyanine (ZnPc) dyes with dimethylaminopyridinium groups at the periphery (ZnPcs 1, 2) and their quaternized derivatives (ZnPcs 1a, 2a). Tetra(1, 1a)- and octa(2, 2a)-ß-substituted zinc(II) phthalocyanines were prepared and assessed as photosensitizers (PSs) for their effects on Fusarium oxysporum conidia. Antimicrobial photoinactivation experiments were performed with each PS at 0.1, 1, 10, and 20 µM under white light irradiation at an irradiance of 135 mW·cm-2, for 60 min (light dose of 486 J·cm-2). High PDI efficiency was observed for PSs 1a, 2, and 2a (10 µM), corresponding to inactivation until the method's detection limit. PS 1 (20 µM) also achieved a considerable reduction of >5 log10 in the concentration of viable conidia. The quaternized PSs (1a, 2a) showed better PDI performance than the non-quaternized ones (1, 2), even at the low concentration of 1 µM, and a light dose of 486 J·cm-2. These cationic phthalocyanines are potent photodynamic drugs for antifungal applications due to their ability to effectively inactivate resistant forms, like conidia, with low concentrations and reasonable energy doses.

In Vitro Photoinactivation of Fusarium oxysporum Conidia with Light-Activated Ammonium Phthalocyanines.

Antimicrobial photodynamic therapy (aPDT) has been explored as an innovative therapeutic approach because it can be used to inactivate a variety of microbial forms (vegetative forms and spores) without causing significant damage to host tissues, and without the development of resistance to the photosensitization process. This study assesses the photodynamic antifungal/sporicidal activity of tetra- and octasubstituted phthalocyanine (Pc) dyes with ammonium groups. Tetra- and octasubstituted zinc(II) phthalocyanines (1 and 2) were prepared and tested as photosensitizers (PSs) on Fusarium oxysporum conidia. Photoinactivation (PDI) tests were conducted with photosensitizer (PS) concentrations of 20, 40, and 60 µM under white-light exposure at an irradiance of 135 mW·cm-2, applied during 30 and 60 min (light doses of 243 and 486 J·cm-2). High PDI efficiency corresponding to the inactivation process until the detection limit was observed for both PSs. The tetrasubstituted PS was the most effective, requiring the lowest concentration and the shortest irradiation time for the complete inactivation of conidia (40 µM, 30 min, 243 J·cm-2). Complete inactivation was also achieved with PS 2, but a longer irradiation time and a higher concentration (60 µM, 60 min, 486 J·cm-2) were necessary. Because of the low concentrations and moderate energy doses required to inactivate resistant biological forms such as fungal conidia, these phthalocyanines can be considered potent antifungal photodynamic drugs.

Aspergillus terreus Species Complex.

Infections due to Aspergillus species are an acute threat to human health; members of the Aspergillus section Fumigati are the most frequently occurring agents, but depending on the local epidemiology, representatives of section Terrei or section Flavi are the second or third most important. Aspergillus terreus species complex is of great interest, as it is usually amphotericin B resistant and displays notable differences in immune interactions in comparison to Aspergillus fumigatus. The latest epidemiological surveys show an increased incidence of A. terreus as well as an expanding clinical spectrum (chronic infections) and new groups of at-risk patients being affected. Hallmarks of these non-Aspergillus fumigatus invasive mold infections are high potential for tissue invasion, dissemination, and possible morbidity due to mycotoxin production. We seek to review the microbiology, epidemiology, and pathogenesis of A. terreus species complex, address clinical characteristics, and highlight the underlying mechanisms of amphotericin B resistance. Selected topics will contrast key elements of A. terreus with A. fumigatus. We provide a comprehensive resource for clinicians dealing with fungal infections and researchers working on A. terreus pathogenesis, aiming to bridge the emerging translational knowledge and future therapeutic challenges on this opportunistic pathogen.

Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905.

Marine-derived Aspergillus terreus produces a variety of structurally novel secondary metabolites, most of which show unique biological activities. However, the lack of efficient genetic tools limits the discovery of new compounds, the elucidation of involved biosynthesis mechanism, as well as the strain engineering efforts. Therefore, in this study, we first established both an effective PEG-mediated chemical transformation system of protoplasts and an electroporation system of conidia in a marine-derived fungus A. terreus RA2905. To overcome the insensitivity of RA2905 to fungicides, the uracil auxotrophy strain (pyrG gene deletion mutant, ΔpyrG) was constructed using PEG-mediated transformation system, and using ΔpyrG as the genetic background, the methyltransferase gene laeA-overexpression transformants were further constructed through both PEG- and electroporation-mediated transformations, which showed enhanced terrein production. Besides, in this study, an efficient CRISPR/Cas9 genome-editing system was established for the first time in A. terreus, and a higher gene deletion efficiency of 71% for APSES transcription factor gene stuA could be achieved when using short homologous arms compared with conventional long homologous ones. In addition, using a non-integrative Cas9 plasmid, another efficient and marker-free genome-editing system was established, which allowing repeatable and unlimited genetic manipulation in A. terreus. Using the marker-free genome-editing system, we successfully developed the ΔpyrGΔku70 double-deletion mutant in RA2905, which could further improve gene deletion efficiency. In conclusion, efficient genetic manipulation systems along with a variety of functional mutants were developed in this study, which would significantly expedite both theoretical and applied researches in not only A. terreus but also other marine-derived filamentous fungi.

The Cell Wall of the Human Fungal Pathogen Aspergillus fumigatus: Biosynthesis, Organization, Immune Response, and Virulence.

More than 90% of the cell wall of the filamentous fungus Aspergillus fumigatus comprises polysaccharides. Biosynthesis of the cell wall polysaccharides is under the control of three types of enzymes: transmembrane synthases, which are anchored to the plasma membrane and use nucleotide sugars as substrates, and cell wall-associated transglycosidases and glycosyl hydrolases, which are responsible for remodeling the de novo synthesized polysaccharides and establishing the three-dimensional structure of the cell wall. For years, the cell wall was considered an inert exoskeleton of the fungal cell. The cell wall is now recognized as a living organelle, since the composition and cellular localization of the different constitutive cell wall components (especially of the outer layers) vary when the fungus senses changes in the external environment. The cell wall plays a major role during infection. The recognition of the fungal cell wall by the host is essential in the initiation of the immune response. The interactions between the different pattern-recognition receptors (PRRs) and cell wall pathogen-associated molecular patterns (PAMPs) orientate the host response toward either fungal death or growth, which would then lead to disease development. Understanding the molecular determinants of the interplay between the cell wall and host immunity is fundamental to combatting Aspergillus diseases.

Mrada3 is required for sexual reproduction and secondary metabolite production in industrial fungi Monascus strain.

AIMS: Monascus spp. are valuable industrial fungi for producing beneficial compounds. Because sporulation is often coupled with the production of secondary metabolites, the current study was performed to investigate how Mrada3 regulated asexual and sexual development and the production of edible pigments and mycotoxin. METHODS AND RESULTS: The functional characteristics of Mrada3 were identified by gene deletion and overexpression in Monascus ruber M7 (the wild-type, WT). The results revealed that the ΔMrada3 strain aborted sexual development, but it produced many more conidia than WT. RNA-seq data showed that the deletion of Mrada3 altered the expression levels of partial genes involved in sexual and asexual development. In addition, the deletion of Mrada3 also resulted in slower growth, lower pigment production and increased citrinin yield during the late period. For the Mrada3-overexpressed strain, the number of ascospores and pigment content were significantly higher than those of WT, but citrinin was slightly lower than that of WT. CONCLUSIONS: The Mrada3 gene plays a vital role in the sporulation development and secondary metabolism of Monascus species. SIGNIFICANCE AND IMPACT OF THE STUDY: Mrada3 is first identified as an essential regulator for sexual development in Monascus species, enriching the regulatory knowledge of sexual development in filamentous fungi.

Antifungal effects of Conyza bonariensis (L.) Cronquist essential oil against pathogenic Colletotrichum musae and its incorporation in gum Arabic coating to reduce anthracnose development in banana during storage.

AIM: This study evaluated the inhibitory effects on mycelial growth and damage on membrane integrity and enzymatic activity caused by Conyza bonariensis essential oil (CBEO) on distinct pathogenic Colletotrichum musae isolates, as well as the preventive and curative effects of coatings with gum Arabic (GA) and CBEO to reduce anthracnose development in banana during room temperature storage. The effects of GA-CBEO coatings on some physicochemical parameters of banana were investigated during room temperature storage. METHOD AND RESULTS: CBEO (0.4-1 µl ml-1 ) inhibited the mycelial growth of C. musae isolates in laboratory media. The exposure of C. musae conidia to CBEO (0.6 µl ml-1 ) for 3 and 5 days resulted in high percentages of conidia with damaged cytoplasmic membrane and without enzymatic activity. Coatings with GA (0.1 mg ml-1 ) and CBEO (0.4-1 µl ml-1 ) reduced the anthracnose development in banana artificially contaminated with C. musae during storage. In most cases, the disease severity indexes found for GA-CBEO-coated banana were lower than or similar to those for banana treated with commercial fungicide. GA-CBEO-coated banana had reduced alterations in physicochemical parameters during storage, indicating more prolonged storability. CONCLUSION: The application of GA-CBEO coatings is effective to delay the anthracnose development in banana during storage, which should help to reduce the amount of fungicides used to control postharvest diseases in this fruit. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing the efficacy of coatings formulated with GA and CBEO to delay the development of anthracnose in banana, as well as to decrease alterations in physicochemical parameters indicative of postharvest quality of this fruit during storage. In a practical point of view, GA-CBEO coatings could be innovative strategies to delay the anthracnose development and postharvest losses in banana.