Isolation and Identification of Nematode-Infecting Microsporidia.

Nematodes are naturally infected by the fungal-related pathogen microsporidia. These ubiquitous eukaryotic parasites are poorly understood, despite infecting most types of animals. Identifying novel species of microsporidia and studying them in an animal model can expedite our understanding of their infection biology and evolution. Nematodes present an excellent avenue for pursuing such work, as they are abundant in the environment and many species are easily culturable in the laboratory. The protocols presented here describe how to isolate bacterivorous nematodes from rotting substrates, screen them for microsporidia infection, and molecularly identify the nematode and microsporidia species. Additionally, we detail how to remove environmental contaminants and generate a spore preparation of microsporidia from infected samples. We also discuss potential pitfalls and provide suggestions on how to mitigate them. These protocols allow for the identification of novel microsporidia species, which can serve as an excellent starting point for genomic analysis, determination of host specificity, and infection characterization. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Gathering samples Support Protocol 1: Generating 10× and 40× Escherichia coli OP50 and seeding NGM plates Basic Protocol 2: Microsporidia screening, testing for Caenorhabditis elegans susceptibility, and sample freezing Basic Protocol 3: DNA extraction, PCR amplification, and sequencing to identify nematode and microsporidia species Basic Protocol 4: Removal of contaminating microbes and preparation of microsporidia spores Support Protocol 2: Bleach-synchronizing nematodes.

Nitric oxide-mediated regulation of Aspergillus flavus asexual development by targeting TCA cycle and mitochondrial function.

Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.

Biological characteristics of Cordyceps militaris single mating-type strains.

Cordyceps militaris has been extensively cultivated as a model cordyceps species for commercial purposes. Nevertheless, the problems related to strain degeneration and breeding technologies remain unresolved. This study assessed the physiology and fertility traits of six C. militaris strains with distinct origins and characteristics, focusing on single mating-type strains. The results demonstrated that the three identified strains (CMDB01, CMSY01, and CMJB02) were single mating-type possessing only one mating-type gene (MAT1-1). In contrast, the other three strains (CMXF07, CMXF09, and CMMS05) were the dual mating type. The MAT1-1 strains sourced from CMDB01, CMSY01, and CMJB02 consistently produced sporocarps but failed to generate ascospores. However, when paired with MAT1-2 strains, the MAT1-1 strains with slender fruiting bodies and normal morphology were fertile. The hyphal growth rate of single mating-type strains (CMDB01, CMSY01, and CMJB02) typically surpassed that of dual mating-type strains (CMXF07, CMXF09, and CMMS05). The growth rates of MAT1-2 and MAT1-1 strains were proportional to their ratios, such that a single mating-type strain with a higher ratio exhibited an increased growth rate. As C. militaris matured, the adenosine content decreased. In summary, the C. militaris strains that consistently produce sporocarps and have a single mating type are highly promising for production and breeding.

The Gene vepN Regulated by Global Regulatory Factor veA That Affects Aflatoxin Production, Morphological Development and Pathogenicity in Aspergillus flavus.

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus.

Cd pollution in agroforestry ecosystems: An abiotic factor affecting the passive and active lethal efficiency of Beauveria bassiana to Lymantria dispar larvae.

This study evaluated the effects of cadmium (Cd) exposure on the passive and active lethal efficiency of Beauveria bassiana (Bb) to Lymantria dispar larvae and analyzed the corresponding mechanism from mycelial vegetative growth, fungal and host nutrient competition, and fungal spore performance. The results showed that the passive lethal efficiency of Bb to Cd-exposed L. dispar larvae was significantly higher than that of larvae not exposed to Cd. After Bb infection, the fungal biomass in living larvae and the mycelium encapsulation index of dead larvae were significantly decreased under Cd exposure. Cd exposure damaged the mycelial structure, as well as inhibited the mycelial growth and sporulation quantity. A total of 15 and 39 differentially accumulated mycotoxin metabolites were identified in Bb mycelia treated with low Cd and high Cd, respectively, and the contents of these differentially accumulated mycotoxins in the low Cd and high Cd treatment groups were overall lower than those in the control group. Nutrient content and energy metabolism-related gene expression were significantly decreased in Cd-exposed larvae, both before and after Bb infection. Trehalose supplementation alleviated the nutritional deficiency of larvae under the combined treatment of Cd and Bb and decreased the larval susceptibility to Bb. Compared with untreated Bb, the lethal efficiency of low Cd-exposed Bb to larvae increased significantly, while high Cd-exposed Bb was significantly less lethal to larvae. Cd exposure promoted at low concentrations but inhibited the hydrophobicity and adhesion of spores at higher concentrations. Spore germination rate and stress resistance of Bb decreased significantly under the treatment of both Cd concentrations. Taken together, heavy metals can be regarded as an abiotic environmental factor that directly affects the lethal efficiency of Bb to insect pests.

Synthesis, Characterization, and Antifungal Activity of Benzimidazole-Grafted Chitosan against Aspergillus flavus.

Aspergillus flavus contamination in agriculture and food industries poses threats to human health, leading to a requirement of a safe and effective method to control fungal contamination. Chitosan-based nitrogen-containing derivatives have attracted much attention due to their safety and enhanced antimicrobial applications. Herein, a new benzimidazole-grafted chitosan (BAC) was synthesized by linking the chitosan (CS) with a simple benzimidazole compound, 2-benzimidazolepropionic acid (BA). The characterization of BAC was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (1H and 13C NMR). Then, the efficiency of BAC against A. flavus ACCC 32656 was investigated in terms of spore germination, mycelial growth, and aflatoxin production. BAC showed a much better antifungal effect than CS and BA. The minimum inhibitory concentration (MIC) value was 1.25 mg/mL for BAC, while the highest solubility of CS (16.0 mg/mL) or BA (4.0 mg/mL) could not completely inhibit the growth of A. flavus. Furthermore, results showed that BAC inhibited spore germination and elongation by affecting ergosterol biosynthesis and the cell membrane integrity, leading to the permeabilization of the plasma membrane and leakage of intracellular content. The production of aflatoxin was also inhibited when treated with BAC. These findings indicate that benzimidazole-derived natural CS has the potential to be used as an ideal antifungal agent for food preservation.

Antifungal and Antivirulence Activity of Vanillin and Tannic Acid Against Aspergillus fumigatus and Fusarium solani.

Aspergillus fumigatus and Fusarium solani infections have become severe health threat; both pathogens are considered a priority due to the increasing emergence of antifungal-resistant strains and high mortality rates. Therefore, the discovery of new therapeutic strategies has become crucial. In this study, we evaluated the antifungal and antivirulence effects of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. The minimum inhibitory concentrations of the compounds were determined by the microdilution method in RPMI broth in 96-well microplates according to CLSI. Conidial germination, protease production, biofilm formation, and in vivo therapeutic efficacy assays were performed. The results demonstrated that vanillin and tannic acid had antifungal activity against Aspergillus fumigatus, while tannic acid only exhibited antifungal activity against Fusarium solani. We found that vanillin and tannic acid inhibited conidial germination and secreted protease production and biofilm formation of the fungal pathogens using sub-inhibitory concentrations. Besides, vanillin and tannic acid altered the fungal membrane permeability, and both compounds showed therapeutic effect against aspergillosis and fusariosis in an infection model in Galleria mellonella larvae. Our results highlight the antivirulence effect of vanillin and tannic acid against priority pathogenic fungi as a possible therapeutic alternative for human fungal infections.

Ganoderma spore lipid ameliorates docetaxel, cisplatin, and 5-fluorouracil chemotherapy-induced damage to bone marrow mesenchymal stem cells and hematopoiesis.

BACKGROUND: A triplet chemotherapy regimen of docetaxel, cisplatin, and 5-fluorouracil (TPF) is used to treat head and neck squamous cell carcinoma; however, it is toxic to bone marrow mesenchymal stem cells (BMSCs). We previously demonstrated that Ganoderma spore lipid (GSL) protect BMSCs against cyclophosphamide toxicity. In this study, we investigated the protective effects of GSL against TPF-induced BMSCs and hematopoietic damage. METHODS: BMSCs and C57BL/6 mice were divided into control, TPF, co-treatment (simultaneously treated with GSL and TPF for 2 days), and pre-treatment (treated with GSL for 7 days before 2 days of TPF treatment) groups. In vitro, morphology, phenotype, proliferation, senescence, apoptosis, reactive oxygen species (ROS), and differentiation of BMSCs were evaluated. In vivo, peripheral platelets (PLTs) and white blood cells (WBCs) from mouse venous blood were quantified. Bone marrow cells were isolated for hematopoietic colony-forming examination. RESULTS: In vitro, GSL significantly alleviated TPF-induced damage to BMSCs compared with the TPF group, recovering their morphology, phenotype, proliferation, and differentiation capacity (p < 0.05). Annexin V/PI and senescence-associated ß-galactosidase staining showed that GSL inhibited apoptosis and delayed senescence in TPF-treated BMSCs (p < 0.05). GSL downregulated the expression of caspase-3 and reduced ROS formation (p < 0.05). In vivo, GSL restored the number of peripheral PLTs and WBCs and protected the colony-forming capacity of bone marrow cells (p < 0.05). CONCLUSIONS: GSL efficiently protected BMSCs from damage caused by TPF and recovered hematopoiesis.

[Evaluation of Toll-Like Receptor Gene Expressions in Encephalitozoon intestinalis Infection]. / Encephalitozoon intestinalis Enfeksiyonunda Toll-Like Reseptör Gen Ekspresyonlarinin Degerlendirilmesi.

Microsporidia are obligate intracellular pathogens that can infect many vertebrate and invertebrate hosts. While the Microsporidia phylum was defined as protozoa until the 1990s, it has been associated with fungi in line with the data obtained as a result of phylogenetic and molecular analyzes in recent years. Although approximately 200 genera and 1400 Microsporidia species related to these genera have been reported to date, only 14 species are known to cause infection in humans. Encephalitozoon intestinalis is one of the most frequently detected species in humans and causes serious clinical conditions in immunosuppressed individuals. Little information is available about the immunology of this infection. This study was aimed to investigate the changes in Toll-Like receptor (TLR) gene expressions in Madin-Darby canine kidney (MDCK) cells treated with E.intestinalis spores. Three groups were formed in the study. In the first group, only the medium prepared for E.intestinalis was added to the MDCK cells. In the second group, 108 live spores waiting at +4 °C were added. In the third group, 108 heat-inactivated spores were added. All three groups were incubated at 37ºC with 5% CO2 . RNA isolation and cDNA synthesis were performed from samples taken from these groups at the 1st, 3rd, 6th, 12th and 24th hours. Expression of TLR1-10 genes from the obtained cDNAs was evaluated by real-time polymerase chain reaction (Rt-PCR). GAPDH and ACTB genes were used as housekeeping genes in the study. Target genes were normalized by taking the average of these two genes and statistical analysis was performed by applying the 2-ΔΔCt formula. Genes detected above the threshold value (threshold 1) were considered to have increased expression. Genes detected below the threshold value were considered to have decreased expression. The growth of the live and inactive spores were followed simultaneously with the experimental groups. Approximately two weeks after the start of the culture, it was observed that E.intestinalis grew in the culture with live spore, but did not grow in the culture with inactivated spores. No statistically significant change was observed in gene expressions in the inactivated spore group. In the live spore group, a significant increase was seen in the expression of only two genes. These genes were TLR3 and TLR4. It was observed that there was a significant increase in TLR3 gene expression at the first hour (1.6-fold of control group) but the expression level started to decrease at the third hour (1.4-fold of control group) and returned to the control level at the sixth hour. It was observed that TLR4 gene expression continued parallel to the control until the 24th hour and increased significantly (2.1-fold of control group) at the 24th hour. In conclusion, this study is the f irst report in which the changes in ten different TLR gene expressions were evaluated at different times in MDCK cells stimulated with E.intestinalis and the change in TLR3 gene expression.

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.

Comparing resistance of bacterial spores and fungal conidia to pulsed light and UVC radiation at a wavelength of 254 nm.

Pulsed light (PL) inactivates microorganisms by UV-rich, high-irradiance and short time pulses (250 µs) of white light with wavelengths from 200 nm to 1100 nm. PL is applied for disinfection of food packaging material and food-contact equipment. Spores of seven Bacillus ssp. strains and one Geobacillus stearothermophilus strain and conidia of filamentous fungi (One strain of Aspergillus brasiliensis, A. carbonarius and Penicillium rubens) were submitted to PL (fluence from 0.23 J/cm2 to 4.0 J/cm2) and UVC (at λ = 254 nm; fluence from 0.01 J/cm2 to 3.0 J/cm2). One PL flash at 3 J/cm2 allowed at least 3 log-reduction of all tested microorganisms. The emetic B. cereus strain F4810/72 was the most resistant of the tested spore-forming bacteria. The PL fluence to 3 log-reduction (F3 PL) of its spores suspended in water was 2.9 J/cm2 and F3 UVC was 0.21 J/cm2, higher than F3 PL and F3 UVC of spores of B. pumilus SAFR-032 2.0 J/cm2 and 0.15 J/cm2, respectively), yet reported as a highly UV-resistant spore-forming bacterium. PL and UVC sensitivity of bacterial spores was correlated. Aspergillus spp. conidia suspended in water were poorly sensitive to PL. In contrast, PL inactivated Aspergillus spp. conidia spread on a dry surface more efficiently than UVC. The F2 PL of A. brasiliensis DSM1988 was 0.39 J/cm2 and F2 UVC was 0.83 J/cm2. The resistance of spore-forming bacteria to PL could be reasonably predicted from the knowledge of their UVC resistance. In contrast, the sensitivity of fungal conidia to PL must be specifically explored.

[Initial study of trap and identification of environmental pollens and fungi spores in Samborondon, Ecuador]. / Estudio inicial de captación e identificación de pólenes y hongos ambientales en Samborondón, Ecuador.

OBJETIVO: To identify and registry the most important aeroallergens trapped at the aerobiology station in the city of Samborondon, Ecuador. METHODS: Pollen grains and fungal spore counts were performed according to the standardized technique with a Hirst-type collection equipment, Burkard spore trap for seven days, following the recommendations of the National Allergy Bureau (NAB) of the American Academy of Allergy Asthma and Immunology (AAAAI). The equipment was installed on the roof of the Universidad de Especialidades Espiritu Santo (UEES), 25 m above ground level, coordinates: 2°07 ́57 ́ ́S 79°52 ́06 ́ ́W, in the city of Samborondon. The sampling period was performed from November 2022 to April 2023. RESULTS: We identified the following pollen families: Poaceae (258 grains/m3), Apocynaceae (Plumeria rubra pc) (214 grains/m3), Lamiaceae (180 grains/m3), Asteraceae Ambrosía spp.- (60 grains/m3), Chenopodiacea (27 grains/m3), Myrtaceae (17 grains/m3), Pinaceae (11 grains/m3), Betulaceae (7 grains/m3). Also identified fungical spores: Fuzariella spp./Leptosphaeria spp. (1899/m3), Cladosporium spp. (1407/m3), Nigrospora spp. (1183/m3), Dreschlera/Helmintosporum spp. (329/m3), Alternaria spp. (98/m3), Pithomyces spp. (79/m3), Curvularia spp. (48/m3), Stemphylium spp. (46/m3). CONCLUSIONS: We reported the first study of aerobiology (capture and identification of environmental pollens and fungi) in the city of Samborondon. The inhabitants of this area are exposed to different aeroallergens with a predominance of Poaceaes pollen and Fuzzariella spp./Leptosphaeria spp. spores. The identified allergens should be part of the usual allergy studies. The results of this first preliminary study should be compared with information from the forthcoming years, which will help to identify variations in the concentration of seasonal aeroallergens, annual fluctuations, and extend the traps to other parts of the city.

OBJETIVO: Identificar y registrar los aeroalérgenos más importantes captados en la estación de aerobiología en la ciudad de Samborondón, Ecuador. MÉTODOS: Los conteos de granos de polen y esporas de hongos se realizaron según la técnica estandarizada, con un equipo colector tipo Hirst, Burkard spore trap for seven days, siguiendo las recomendaciones de la National Allergy Bureau (NAB) de la American Academy Allergy Asthma and Immunology (AAAAI). El equipo se instaló en la azotea de la Universidad Espíritu Santo (UEES), en la ciudad de Samborondón, a 25 m de altura desde el nivel del suelo, 2°07´57´´S 79°52´06´´O. El periodo de captación se llevó a cabo entre noviembre de 2022 y abril de 2023. RESULTADOS: Identificamos las siguientes familias polínicas: Poaceae (258 granos/m3), Apocynaceae (Plumeria rubra pc) (214 granos/m3), Lamiaceae (180 granos/m3), Asteraceae Ambrosía spp.- (60 granos/m3), Chenopodiacea (27 granos/m3), Myrtaceae (17 granos/m3), Pinaceae (11 granos/m3), Betulaceae (7 granos/m3). Además esporas fúngicas: Fuzariella spp./Leptosphaeria spp. (1899/m3), Cladosporium spp. (1407/m3), Nigrospora spp. (1183/m3), Dreschlera/Helmintosporum spp. (329/m3), Alternaria spp. (98/m3), Pithomyces spp. (79/m3), Curvularia spp. (48/m3), Stemphylium spp. (46/m3). CONCLUSIONES: Se reporta el primer estudio de aerobiología (captación e identificación de pólenes y hongos ambientales), en la ciudad de Samborondón. Los habitantes de esta zona están expuestos a diferentes aeroalérgenos con predominancia al polen de Poaceaes y esporas de Fuzzariella spp./Leptosphaeria spp. Los alérgenos identificados deberían formar parte de los estudios alergológicos habituales. Los resultados de este primer estudio preliminar deberían ser comparados con información de los siguientes años para ayudar a identificar las variaciones de concentración de aeroalérgenos estacionales, las fluctuaciones anuales, y extender los captadores a otros puntos de la ciudad.

[Identification and sensitization to environmental fungal spores in Lima City, Peru]. / Identificación y sensibilización a hongos ambientales en Lima, Perú.

OBJECTIVE: To identify and registry the most important fungal spores trapped in our aerobiology station, as well as to report the prevalence of skin sensitization to these allergens. METHODS: The pollen counts were made according to standardized technique with a Burkard seven days spore trap, following the American Academy of Allergy, Asthma and Immunology (AAAAI) through National Allergy Bureau (NAB) recommendations. The trap was installed on the roof of Clinica SANNA, El GOLF, San Isidro, which is 20 m high, 12°5'54"S 77°3'6"W in the west-south of the Lima urban area. The sampling period was performed from September 2020 to October 2021. Skin prick tests were carried out according to the recommendations of the Spanish Society of Allergology and Clinical Immunology (SEAIC) in 200 patients (18 to 60 years old) with symptoms of rhinoconjunctivitis and/or asthma, who were evaluated in the Allergology Service of Clinica SANNA el Golf. Allergenic extracts were applied, dust mites (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), cat and dog danders, cockroach (Periplaneta americana), grass 6 mix, weed mix, molds (Cladosporium herbarum, Alternaria alternata, Aspergillus fumigatus, Penicillium notatum, Nigrospora spp.), INMUNOTEK-Spain provided the extracts. We also tested other fungal allergens such as Fusarium spp, Stemphylium spp, Curvularia spp, a mixture of Helmintosporum/Dreschlera spp. from the DIATER-Argentina laboratory. RESULTS: We identified spores of Alternaria alternata, Cladosporium spp., Nigrospora spp., Stemphylium spp., Fusarium spp., Curvularia spp., Dreschlera/Helmintosporum spp. The patients showed sensitization to Cladosporium herbarum (14%), Fusarium spp. (13,5%), Nigrospora spp. (8%), Alternaria Alternata (7%), Stemphylium (6%), Dreschlera/Helmintosporium spp. (5,5%), Curvularia spp. (3%), Aspergillus fumigatus (2,5%). CONCLUSIONS: The inhabitants of the south-western area of the urban city of Lima are exposed to different fungal spores with allergenic potential, with a higher concentration being identified during the summer/autumn months. Cutaneous sensitization is demonstrated in variable percentages to the fungal spores identified in this aerobiological sampling. The results of this study should be expanded and compared with data in the forthcoming years, identify seasonal and annual fluctuations and extend the traps to other locations in Lima.

OBJETIVO: Identificar y registrar las esporas de hongos más importantes captadas en nuestra estación de aerobiología, además reportar la prevalencia de sensibilización cutánea a estos alérgenos. MÉTODOS: La identificación y los conteos de esporas de hongos se realizaron según la técnica estandarizada con un equipo colector Burkard Spore Trap For Seven Days, siguiendo las recomendaciones de la National Allergy Bureau (NAB), de la American Academy Allergy Asthma and Immunology (AAAAI). El equipo se instaló a 20 m de altura desde el nivel del suelo, en la azotea de la Clínica SANNA El Golf, distrito de San Isidro, (12°5'54"S 77°3'6"O), en la zona sur-oeste del área urbana de Lima. El periodo de captación se llevó a cabo entre septiembre de 2020 y octubre de 2021. Se realizaron estudios de pruebas cutáneas (skin prick-test), según recomendaciones de la Sociedad Española de Alergología e Inmunología Clínica (SEAIC), en 200 pacientes (entre 18 y 60 años), con sintomatología de rinoconjuntivitis y/o asma. Fueron evaluados en el servicio de Alergología de la Clínica SANNA El Golf. Se aplicaron extractos alergénicos de ácaros del polvo (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), epitelios de gato y perro, Periplaneta americana, mezclas de seis gramíneas, mezclas de malezas, hongos ambientales (Cladosporium herbarum, Alternaria alternata, Aspergillus fumigatus, Penicillium notatum, Nigrospora spp.), extractos del laboratorio INMUNOTEK-España. Además, testeamos otros alérgenos fúngicos de Fusarium spp, Stemphylium spp, Curvularia spp, una mezcla de Helmintosporum/Dreschlera spp. del laboratorio DIATER-Argentina. RESULTADOS: Identificamos esporas de Alternaria alternata, Cladosporium spp., Nigrospora spp., Stemphylium spp., Fusarium spp., Curvularia spp., Dreschlera/Helmintosporum spp. Los pacientes mostraron sensibilización a Cladosporium herbarum (14%), Fusarium spp. (13,5%), Nigrospora spp. (8%), Alternaria Alternata (7%), Stemphylium (6%), Dreschlera/Helmintosporium spp. (5,5%), Curvularia spp. (3%) y Aspergillus fumigatus (2,5%). CONCLUSIONES: Los habitantes de la zona sur-oeste de la ciudad urbana de Lima están expuestos a distintas esporas de hongos con potencial alergénico, identificándose mayor concentración durante los meses de verano y otoño. Se demuestra sensibilización cutánea en porcentajes variables a las esporas fúngicas identificadas en este muestreo aerobiológico. Los resultados de este estudio deberían ampliarse y ser comparados con data en los años siguientes, identificar fluctuaciones estacionales y anuales y extender los captadores a otras locaciones en Lima.

Nucleoside Diphosphate Kinase FgNdpk Is Required for DON Production and Pathogenicity by Regulating the Growth and Toxisome Formation of Fusarium graminearum.

Nucleoside diphosphate kinases (NDPKs) are nucleotide metabolism enzymes that play different physiological functions in different species. However, the roles of NDPK in phytopathogen and mycotoxin production are not well understood. In this study, we showed that Fusarium graminearum FgNdpk is important for vegetative growth, conidiation, sexual development, and pathogenicity. Furthermore, FgNdpk is required for deoxynivalenol (DON) production; deletion of FgNDPK downregulates the expression of DON biosynthesis genes and disrupts the formation of FgTri4-GFP-labeled toxisomes, while overexpression of FgNDPK significantly increases DON production. Interestingly, FgNdpk colocalizes with the DON biosynthesis proteins FgTri1 and FgTri4 in the toxisome, and coimmunoprecipitation (Co-IP) assays show that FgNdpk associates with FgTri1 and FgTri4 in vivo and regulates their localizations and expressions, respectively. Taken together, these data demonstrate that FgNdpk is important for vegetative growth, conidiation, and pathogenicity and acts as a key protein that regulates toxisome formation and DON biosynthesis in F. graminearum.

Enhancing biological control of postharvest green mold in lemons: Synergistic efficacy of native yeasts with diverse mechanisms of action.

Argentina is among the most important lemon fruit producers in the world. Penicillium digitatum is the primary lemon fungal phytopathogen, causing green mold during the postharvest. Several alternatives to the use of synthetic fungicides have been developed, being the use of biocontrol yeasts one of the most promising. Although many of the reports are based on the use of a single yeast species, it has been shown that the combination of agents with different mechanisms of action can increase control efficiency through synergistic effects. The combined use of native yeasts with different mechanisms of action had not been studied as a biological control strategy in lemons. In this work, the mechanisms of action of native yeasts (Clavispora lusitaniae AgL21, Clavispora lusitaniae AgL2 and Clavispora lusitaniae AcL2) with biocontrol activity against P. digitatum were evaluated. Isolate AgL21 was selected for its ability to form biofilm, colonize lemon wounds, and inhibit fungal spore germination. The compatibility of C. lusitaniae AgL21 with two killer yeasts of the species Kazachstania exigua (AcL4 and AcL8) was evaluated. In vivo assays were then carried out with the yeasts applied individually or mixed in equal cell concentrations. AgL21 alone was able to control green mold with 87.5% efficiency, while individual killer yeasts were significantly less efficient (43.3% and 38.3%, respectively). Inhibitory effects were increased when C. lusitaniae AgL21 and K. exigua strains were jointly applied. The most efficient treatment was the combination of AgL21 and AcL4, reaching 100% efficiency in wound protection. The combination of AgL21 with AcL8 was as well promising, with an efficiency of 97.5%. The combined application of native yeasts showed a synergistic effect considering that the multiple mechanisms of action involved could hinder the development of green mold in lemon more efficiently than using single yeasts. Therefore, this work demonstrates that the integration of native yeasts with diverse modes of action can provide new insights to formulate effective microbial consortia. This could lead to the development of tailor-made biofungicides, allowing control of postharvest fungal diseases in lemons while remaining competitive with traditionally used synthetic chemicals.

Microsclerotia from Metarhizium robertsii: Production, ultrastructural analysis, robustness, and insecticidal activity.

Microsclerotia (MS) are considered one of the most promising propagules for use as active ingredients in biopesticides due to their tolerance to abiotic factors and ability to produce infective conidia for the control of pests. Therefore, the objective of this research was to establish the conditions required to induce the formation of microsclerotia in Metarhizium robertsii Mt004 and to study its development process, tolerance to abiotic factors and insecticidal activity of MS-derived conidia. M. robertsii started to form hyphal aggregates after 2 days and looked more compact after 8 days. MS were mature and pigmented after 20 days. The final yield was 2.0 × 103 MS/mL and MS size varied between 356.9 and 1348.4 µm. Ultrastructure analysis revealed that mature MS contained only a few live cells embedded in an extracellular matrix. Mature MS were more tolerance to UV-B radiation, heat and storage trials than conidia from Solid State Fermentation. MS-derived conidia were as virulent as conidia against Diatraea saccharalis larvae. These results showed that MS are promising propagules for the development of more persistent and efficient biopesticides for harsh environmental conditions. Our findings provide a baseline for production and a better understanding of microsclerotia development in M. robertsii strains.

Investigating the effect of acoustic waves on spoilage fungal growth and shelf life of strawberry fruit.

The effects of acoustic waves on growth inhibition of food spoilage fungi (Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus and Botrytis cinerea) on the medium and strawberry surfaces were investigated. Firstly, single-frequency sound waves (250, 500, 1000, 2000, 4000, 8000, 12,000 and 16,000 Hz) were induced on inoculated medium with fungi spores for 24 h and growth diameter of each mold was evaluated during the incubation period. In the second stage, the sound waves with two frequencies of 250 Hz and 16,000 Hz were induced on inoculated strawberries with fungi spores at 5 °C for different times (2, 4, 6, 8 and 10 days). The results from the first stage indicated that the sound waves inhibited the growth of A. niger (20.02%) at 250 Hz and B. cinerea (4/64%) at 4000 Hz on potato dextrose agar (PDA) surface. Also, comparison of the growth diameter of some species of Aspergillus revealed various responses in presence of 250 Hz frequency. In the second stage, applying a frequency of 250 Hz over a period of 10 days proved to be more effective in inhibiting the growth of A. niger and B. cinerea on strawberries inoculated with fungal spores. Consequently, the shelf lives of the strawberries significantly increased to 26 days and 18 days, respectively, under this treatment. Based on the findings, it is concluded that sounding with acoustic waves can be used as a green and cheap technology along with other technologies to improve food safety.

MoMkk1 and MoAtg1 dichotomously regulating autophagy and pathogenicity through MoAtg9 phosphorylation in Magnaporthe oryzae.

Autophagy is a central biodegradation pathway critical in eliminating intracellular cargo to maintain cellular homeostasis and improve stress resistance. At the same time, the key component of the mitogen-activated protein kinase cascade regulating cell wall integrity signaling MoMkk1 has an essential role in the autophagy of the rice blast fungus Magnaporthe oryzae. Still, the mechanism of how MoMkk1 regulates autophagy is unclear. Interestingly, we found that MoMkk1 regulates the autophagy protein MoAtg9 through phosphorylation. MoAtg9 is a transmembrane protein subjected to phosphorylation by autophagy-related protein kinase MoAtg1. Here, we provide evidence demonstrating that MoMkk1-dependent MoAtg9 phosphorylation is required for phospholipid translocation during isolation membrane stages of autophagosome formation, an autophagic process essential for the development and pathogenicity of the fungus. In contrast, MoAtg1-dependent phosphorylation of MoAtg9 negatively regulates this process, also impacting growth and pathogenicity. Our studies are the first to demonstrate that MoAtg9 is subject to MoMkk1 regulation through protein phosphorylation and that MoMkk1 and MoAtg1 dichotomously regulate autophagy to underlie the growth and pathogenicity of M. oryzae.IMPORTANCEMagnaporthe oryzae utilizes multiple signaling pathways to promote colonization of host plants. MoMkk1, a cell wall integrity signaling kinase, plays an essential role in autophagy governed by a highly conserved autophagy kinase MoAtg1-mediated pathway. How MoMkk1 regulates autophagy in coordination with MoAtg1 remains elusive. Here, we provide evidence that MoMkk1 phosphorylates MoAtg9 to positively regulate phospholipid translocation during the isolation membrane or smaller membrane structures stage of autophagosome formation. This is in contrast to the negative regulation of MoAtg9 by MoAtg1 for the same process. Intriguingly, MoMkk1-mediated MoAtg9 phosphorylation enhances the fungal infection of rice, whereas MoAtg1-dependant MoAtg9 phosphorylation significantly attenuates it. Taken together, we revealed a novel mechanism of autophagy and virulence regulation by demonstrating the dichotomous functions of MoMkk1 and MoAtg1 in the regulation of fungal autophagy and pathogenicity.

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.

The role of the Flb protein family in the life cycle of Aspergillus niger.

Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger. Strains ΔflbA, ΔflbB and ΔflbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain ΔflbA was more sensitive to H2O2, DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD, and ΔflbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H2O2. Enzyme secretion was impacted when the Δflb strains were grown on xylose. Strain ΔflbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the ΔflbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the ΔflbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion.