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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

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. 653 __ |a Biological control; Mass production; Blastospores; Microsclerotia; Submerged conidia; Bioreactor.

The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus , the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection. Aspergillus fumigatus is a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date of A. fumigatus resting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)–pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designated c onidial c ell wall p rotein A (CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of Δ ccpA resting conidia appeared normal. However, trypsin shaving of Δ ccpA conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen Δ ccpA conidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cells in vitro . In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with Δ ccpA conidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed to A. fumigatus conidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition. IMPORTANCE The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus , the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.

This study reports that bacteria protect plants against filamentous fungal infections via intercellular contacts between the bacterial cells and the conidia of the fungi.

• Ferroptosis, an iron-dependent cell death process, was found to occur in Magnaporthe oryzae, and plays a key role in infection-related development therein. Ferroptosis in the riceblast fungus was confirmed based on five basic criteria. • We confirmed the dependence of ferroptosis on ferric ions, and optimized ratio-fluorescence imaging of C11-BODIPY581/591 as a precise sensor for lipid peroxides that mediate ferroptosis in M. oryzae. We uncovered an important regulatory function for reduced glutathione and NADPH oxidases in modulating the superoxide moieties required for ferroptotic cell death. • We found ferroptosis to be necessary for the developmental cell death of conidia during appressorium maturation in rice blast. Such ferroptotic cell death initiated first in the terminal cell and progressed sequentially to the entire conidium. Iron chelation or chemical inhibition of ferroptosis caused conidial cells to remain viable, and led to strong defects in host invasion by M. oryzae. Ferroptosis induction exclusively in the host severely constrained the invasive growth of M. oryzae. • We found inter-reliant and independent roles for ferroptosis and autophagy in controlling such precise cell death in M. oryzae during pathogenic differentiation. Our study provides significant molecular insights into the role of developmental cell death and iron homeostasis in fungal pathogenesis.

Among the citrus production industry, Mexico represents the fifth largest producer worldwide as of 1 Mar 2022 data (Producción de cítricos en México, Biblioteca de Publicaciones Oficiales del Gobierno de la República, Gobierno, www.gob.mx [last accessed 3 Jun 2022]). Among insect pests affecting citrus orchards, the citrus blackfly Aleurocanthus woglumi Ashby (Hemiptera: Aleyrodidae) causes great economic losses to this industry. Biological control with parasitoids is used principally to manage this pest. In this study, we evaluated a simple and inexpensive technique for citrus blackfly biocontrol in citrus orchards. Conidia from the entomopathogenic fungus Aschersonia sp. were collected from citrus leaves with Aschersonia sp. infecting citrus blackfly nymphs in 1 locality in Quintana Roo State, Mexico. Conidia were suspended with vigorous shaking in a 0.1% Tween solution to achieve a concentration of about 1.0 × 108 conidia per mL and sprayed on citrus blackfly uninfected nymphs in 1 selected (high citrus blackfly population) Quintana Roo locality when relative humidity was about 95%. After about 55 d, fungus had killed the treated nymphs, and had infected and killed other citrus blackflies causing an epizootic in the application area. This technique was scaled up by citrus producers to 30 ha with similar results. During 2019, relative humidity was lower than 60%, and citrus blackfly-uninfected or Aschersonia sp.-infected nymphs were not detected. During 2020, the relative humidity was higher than 80% and we did not detect Aschersonia sp. Infected- or uninfected-citrus blackfly nymphs in the previously treated area. In conclusion, collection and application of Aschersonia sp. conidia on uninfected citrus blackfly nymphs may result in an epizootic, if relative humidity is 80% or higher, and Aschersonia sp. remains and disseminates along with this insect pest. Entre la industria de producción de cítricos, México representa el quinto productor a nivel mundial según datos del 01 de marzo del 2022 (Producción de cítricos en México, Biblioteca de Publicaciones Oficiales del Gobierno de la República, Gobierno, www.gob.mx [último accedido 3 Jun 2022]). Entre las plagas de insectos, la mosca prieta de los cítricos, Aleurocanthus woglumi Ashby (Hemiptera: Aleyrodidae) puede provocar grandes pérdidas económicas a esta industria. El control biológico con parasitoides es el método más utilizado para controlar esta plaga. En este estudio, evaluamos una técnica sencilla y económica en huertos de cítricos para el control biológico de la mosca prieta de los cítricos. Se recolectaron conidios del hongo entomopatógeno Aschersonia sp. de hojas de cítricos con Aschersonia sp. infectando ninfas de mosca prieta de los cítricos, en una localidad del estado de Quintana Roo. Los conidios se suspendieron con agitación vigorosa en una solución de Tween al 0.1% para lograr sobre 1.0 × 108 conidios por mL y se aplicaron mediante aspersión sobre ninfas no infectadas con mosca prieta de los cítricos, en una localidad seleccionada de Quintana Roo (alta población de mosca prieta de los cítricos), cuando la humedad relativa era sobre el 95%. Después de 55 días, el hongo ya habían matado a las ninfas tratadas e infectado y matado a otras mosca prieta de los cítricos, causando una epizootia en el área de aplicación. Se escaló esta técnica por los productores de cítricos en 30 hectáreas, lo que resultó en un biocontrol similar. Durante 2019, la humedad relativa fue inferior al 60% y la mosca prieta de los cítricos y no se detectaron ninfas sin infectar o infectadas por Aschersonia sp. Para 2020, la humedad relativa fue superior al 80% y se detectaron tanto la mosca prieta de los cítricos como ninfas infectadas por Aschersonia sp. en la zona previamente tratada. En conclusión, la recolección y aplicación de conidios de Aschersonia sp. sobre ninfas de mosca prieta de los cítricos no infectadas, podrían provocar una epizootia si la humedad relativa es del 80% o mayor y Aschersonia sp. podría permanecer y diseminarse entre esta plaga de insectos.

One of the main negative effects of climate change on biological pest control is alteration of relationships between insect pests and their natural enemies (both entomophagous and entomopathogenic). Indeed, environmental conditions can have multiple effects on pest control success when using entomopathogenic fungi (EPF), where conidial depletion, inactivation, and loss in virulence and infectivity can all occur. Appropriate mass production and formulation strategies for EPF can partially solve these problems. However, the only strategy to guarantee high virulence and infectivity is selection of environmentally competent fungal strains that are able to persist in the host environment for the required infection period. This review examines the criteria for selection of environmentally competent EPF. While UV radiation, followed by humidity and temperature, is probably the most important propagule depletion and inactivation factors in epigeal habitats, temperature is most critical for reducing the infectivity and virulence of EPF in epigeal and hypogeal habitats. In addition, geographical origin and other biotic and abiotic factors have an important impact which may guarantee the environmental competence of selected entomopathogenic fungal strains and, therefore, farmer willingness to replace chemicals with mycoinsecticides. To achieve this, it is urgent to promote the development of microbial control solutions adapted to relatively uniform climatic zones through more simplified, targeted, and less costly EPF approval and authorization.

Silver nanoparticles (AgNPs) exhibit unusual biocidal properties thanks to which they find a wide range of applications in diverse fields of science and industry. Numerous research studies have been devoted to the bactericidal properties of AgNPs while less attention has been focused on their fungicidal activity. Our studies were therefore oriented toward determining the impact of AgNPs characterized by different physicochemical properties on Fusarium avenaceum and Fusarium equiseti . The main hypothesis assumed that the fungicidal properties of AgNPs characterized by comparable morphology can be shaped by stabilizing agent molecules adsorbed on nanoparticle surfaces. Two types of AgNPs were prepared by the reduction of silver ions with sodium borohydride (SB) in the presence of trisodium citrate (TC) or cysteamine hydrochloride (CH). Both types of AgNPs exhibited a quasi-spherical shape. Citrate-stabilized AgNPs (TCSB-AgNPs) of an average size of 15 ± 4 nm were negatively charged. Smaller (12 ± 4 nm), cysteamine-capped AgNPs (CHSB-AgNPs) were characterized by a positive surface charge and higher silver ion release profile. The phytopathogens were exposed to the AgNPs in three doses equal to 2.5, 5 and 10 mg L −1 over 24 and 240 h. Additionally, the impact of silver ions delivered in the form of silver nitrate and the stabilizing agents of AgNPs on the fungi was also investigated. The response of phytopathogens to these treatments was evaluated by determining mycelial growth, sporulation and changes in the cell morphology. The results of our studies showed that CHSB-AgNPs, especially at a concentration of 10 mg L −1 , strongly limited the vegetative mycelium growth of both species for short and long treatment times. The cell imaging revealed that CHSB-AgNPs damaged the conidia membranes and penetrated into the cells, while TCSB-AgNPs were deposited on their surface. The fungistatic (lethal) effect was demonstrated only for silver ions at the highest concentration for the F. equiseti species in the 240 h treatment. The number of spores of both Fusarium species was significantly reduced independently of the type of silver compounds used. Generally, it was found that the positively charged CHSB-AgNPs were more fungicidal than negatively charged TCSB-AgNPs. Thereby, it was established that the stabilizing agents of AgNPs and surface charge play a crucial role in the shaping of their fungicidal properties.

This study assessed the nontarget effect of entomopathogenic fungi on the Western honey bee Apis mellifera L. and the African stingless bee Meliponula ferruginea Cockrell (Hymenoptera: Apidae). Pathogenicity of five Metarhizium anisopliae (ICIPE 7, ICIPE 20, ICIPE 62, ICIPE 69, and ICIPE 78) (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) and one of Beauveria bassiana (ICIPE 284) (Balsamo) Vuillemin (Hypocreales: Cordicipitaceae) isolates were evaluated on bees at 108 conidia/ml. Conidial acquisition was evaluated immediately after exposure. Apis mellifera acquired more conidia (2.8 × 104–1.3 × 105 conidia per bee) compared to M. ferruginea (1.1 × 104–2.3 × 104 conidia per bee). In the bioassay with A. mellifera, ICIPE 7, ICIPE 20, and ICIPE 69 moderately reduced the survival by 16.9, 17.4, 15.3%, with lethal times LT10 = 7.4, 7.6, 8.1 d and LT25 = 8.7, 10.0, 9.9 d, respectively. The three isolates caused A. mellifera mycosis of 11.6–18.5%. None of the isolates had a significant effect on M. ferruginea. The tested isolates are nontoxic to bees according to the International Organization of Biological Control (IOBC) classification. However, the effect of ICIPE 7, ICIPE 20, and ICIPE 69 merits further studies on bee colonies, especially those of A. mellifera, under field conditions. Graphical Abstract

The polyphagous moth, Thaumatotibia leucotreta (Meyrick) is among the major constraints to the horticultural industry in East Africa. In a search of sustainable control methods, the pathogenicity of the dry conidia of 17 Metarhizium anisopliae (Metschn.) and five Beauveria bassiana (Bals.) (Vuill.) isolates were tested against T. leucotreta moths. The conidial uptake by a single moth, horizontal transmission, and effect of fungal inoculant on egg-laying and hatchability for the pest were also assessed. The findings from this study showed that tested fungi were virulent to the moths, with 12 isolates causing mortality that ranged between 58.8 and 94.2% for M. anisopliae and three isolates between 57.6 and 84.6% for B. bassiana. The entomopathogenic fungi isolates, M. anisopliae ICIPE 69 and B. bassiana ICIPE 279 were highly virulent with low lethal times to 50% mortality (LT50) of 3.81 and 5.13 d, respectively. The isolate, ICIPE 69 conidia acquisition by a single moth were approximately 4.58 × 106 and 3.51 × 106 for males and females, respectively. The lowest survival rate (3.33 %) caused by inoculum transmission was recorded in donor males compared to 16.67% in recipient males. Fecundity was reduced by 33.6 and 25.9% for donor and recipient females, respectively. The pest can acquire conidia from the treated substrate and transmission is then possible between adults. ICIPE 69 is virulent and reduces the reproduction potential of the pest after transmission. The findings are discussed in the prospects of microbial control of this pest using an autodissemination technique.