Hyphal Fragments Research Articles

Cell wall nanoparticles from hyphae of Alternaria infectoria grown with caspofungin, nikkomycin, or pyroquilon trigger different activation profiles in macrophages.

Alternaria infectoria causes opportunistic human infections and is a source of allergens leading to respiratory allergies. In this work, we prepared cell wall nanoparticles (CWNPs) as a novel approach to study macrophage immunomodulation by fungal hyphal cell walls. A. infectoria was grown in the presence of caspofungin, an inhibitor of β(1,3)-glucan synthesis; nikkomycin Z, an inhibitor of chitin synthases; and pyroquilon, an inhibitor of dihydroxynaphthalene (DHN)-melanin synthesis. Distinct CWNPs were obtained from these cultures, referred to as casCWNPs, nkCWNPs, and pyrCWNPs, respectively. CWNPs are round-shaped particles with a diameter of 70-200 nm diameter particles that when added to macrophages are taken up by membrane ruffling. CWNPs with no DHN-melanin and more glucan (pyrCWNPs) caused early macrophage activation and lowest viability, with the cells exhibiting ultrastructural modifications such as higher vacuolization and formation of autophagy-like structures. CasCWNPs promoted the highest tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) increase, also resulting in the release of partially degraded chitin, an aspect never observed in macrophage-like cells and fungi. After 6 h of interaction with CWNPs, only half were viable, except with control CWNPs. Overall, this work indicates that compounds that modify the fungal cell wall led to CWNPs with new properties that may have implications for the effects of drugs during antifungal therapy. CWNPs provide a new tool to study the interaction of hyphal fungal cell wall components with phagocytic cells and enable to show how the modification of cell wall components in A. infectoria can modulate the response by macrophages.IMPORTANCEAlternaria species are ubiquitous environmental fungi to which the human host can continuously be exposed, through the inhalation of fungal spores but also of fragments of hyphae, from desegregated mycelia. These fungi are involved in hypersensitization and severe respiratory allergies, such as asthma, and can cause opportunistic infections in immunodepressed human host leading to severe disease. The first fungal structures to interact with the host cells are the cell wall components, and their modulation leads to differential immune responses. Here, we show that fungal cells grown with cell wall inhibitors led to cell wall nanoparticles with new properties in their interaction with macrophages. With this strategy, we overcame the limitation of in vitro assays interacting with filamentous fungi and showed that the absence of DNH-melanin leads to higher virulence, while caspofungin leads to cells walls that trigger higher hydrolysis of chitin and higher production of cytokines.

Cyberlindnera fabianii, an Uncommon Yeast Responsible for Gluten Bread Spoilage.

A single strain of yeast was isolated from industrial gluten bread (GB) purchased from a local supermarket. This strain is responsible for spoilage consisting of white powdery and filamentous colonies due to the fragmentation of hyphae into short lengths (dust-type spots), similar to the spoilage produced by chalk yeasts such as Hyphopichia burtonii, Wickerhamomyces anomalus and Saccharomycopsis fibuligera. The isolated strains were identified initially by traditional methods as Wickerhamomyces anomalus, but with genomic analysis, they were definitively identified as Cyberlindnera fabianii, a rare ascomycetous opportunistic yeast species with low virulence attributes, uncommonly implicated in bread spoilage. However, these results demonstrate that this strain is phenotypically similar to Wi. anomalus. Cy. fabianii grew in GB because of its physicochemical characteristics which included pH 5.34, Aw 0.97 and a moisture of about 50.36. This spoilage was also confirmed by the presence of various compounds typical of yeasts, derived from sugar fermentation and amino acid degradation. These compounds included alcohols (ethanol, 1-propanol, isobutyl alcohol, isoamyl alcohol and n-amyl alcohol), organic acids (acetic and pentanoic acids) and esters (Ethylacetate, n-propil acetate, Ethylbutirrate, Isoamylacetate and Ethylpentanoate), identified in higher concentrations in the spoiled samples than in the unspoiled samples. The concentration of acetic acid was lower only in the spoiled samples, but this effect may be due to the consumption of this compound to produce acetate esters, which predominate in the spoiled samples.

Bioactive molecules isolated from Cymbopogon flexuosus and Monarda citriodora essential oils: Chemical profiling, antimycotic potential, and molecular docking studies against green and blue molds of Kinnow

Among the most alarming postharvest fungal pathogens affecting the shelf life of citrus fruits are green (Penicillium digitatum) and blue (Penicillium italicum) molds. This in vitro study was aimed to evaluate the essential oils (EOs) of Cymbopogon flexuosus (CF) and Monarda citriodora (MC) and their principal components against these fungal pathogens. The composition of selected EOs was analyzed using Gas Chromatography- Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID). Citral (83.81%) and thymol (62.51%) were identified as major components and were isolated by column chromatography from CF and MC EO respectively followed by characterization using spectral techniques such as Fourier Transform Infrared Spectroscopy (FTIR), Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR) and Carbon Nuclear Magnetic Resonance Spectroscopy (13C-NMR). Among EOs and their major components, thymol displayed promising potential against both Penicillium species with MIC values of 50 and 80 mg L-1 respectively. Molecular docking studies also confirmed the stable binding interaction of both components, particularly thymol with the active sites of target proteins i.e. 14-α-demethylase and chitin synthase of P. digitatum and P. italicum with the docking scores of -5.07, -5.41 kcal/mol and -5.78, -5.23 kcal/mol respectively. Optical microscopy studies revealed hyphal thinning and fragmentation at frequent sites with contraction of cytoplasmic content indicating incipient plasmolysis. The tested principal components, citral and thymol were predicted to exhibit similar mechanism of action to that of synthetic recommended molecules as revealed by molecular docking. Therefore, these findings provide empirical support to explore thymol as a promising eco-friendly antifungal agent for postharvest application in Kinnow.

The Wheat Endophyte Epicoccum layuense J4-3 Inhibits Fusarium graminearum and Enhances Plant Growth.

Fungal endophytes are well-known for their ability to promote plant growth and hinder fungal diseases, including Fusarium head blight (FHB) caused by Fusarium graminearum. This study aimed to characterize the biocontrol efficacy of strain J4-3 isolated from the stem of symptomless wheat collected from Heilongjiang Province, China. It was identified as Epicoccum layuense using morphological characteristics and phylogenetic analysis of the rDNA internal transcribed spacer (ITS) and beta-tubulin (TUB). In a dual culture assay, strain J4-3 significantly inhibited the mycelial growth of F. graminearum strain PH-1 and other fungal pathogens. In addition, wheat coleoptile tests showed that lesion symptoms caused by F. graminearum were significantly reduced in wheat seedlings treated with hyphal fragment suspensions of strain J4-3 compared to the controls. Under field conditions, applying spore suspensions and culture filtrates of strain J4-3 with conidial suspensions of F. graminearum on wheat spikes resulted in the significant biocontrol efficacy of FHB. In addition, wheat seedlings previously treated with spore suspensions of strain J4-3 before sowing successfully resulted in FHB reduction after the application of conidial suspensions of F. graminearum at anthesis. More importantly, wheat seedlings treated with hyphal fragments and spore suspensions of strain J4-3 showed significant increases in wheat growth compared to the controls under greenhouse and field conditions. Overall, these findings suggest that E. layuense J4-3 could be a promising biocontrol agent (BCA) against F. graminearum, causing FHB and a growth-promoting fungus in wheat.

Development of Interspecific Hybrids of Pleurotus sajor-caju and P. flabellatus for Better Yield

Existence of variability in morphological traits and growth rate of mycelium of homokaryotic single basidiospores can be exploited for the development of interspecific hybrids of mushroom. To explore this, interspecific hybrids of P. flabellatus, P. sajor-caju having earliness in production, better nutritional qualities and higher yield as compared to their parent strains tried at Uttar Banga Krishi Viswavidyalaya. Eighteen and six monosporus culture of P. flabellatus, P. sajor-caju, respectively were isolated by spore print or serial dilution or hyphal tip fragmentation techniques. Twenty interspecific hybrids were developed from different cross combinations of parents P. flabellatus and P. sajor-caju. The hybrids were grown on malt extract agar media. Highest growth rate shown by the hybrids in media were selected to study qualitative and quantitative response during cultivation. In cultivation spawn running and pin head formation was faster in PF × PSC5 and PF × PSC7. Comparing all the parameters, growth of PF × PSC6 and PF × PSC8 were found to be more efficient. Nitrogen and protein content was good enough in PF × PSC4, PF × PSC7, PF × PSC5 and PF × PSC10. Regarding all the quantitative parameters of the hybrids, biological efficiency and yield of 1st and 2nd harvest of PF × PSC6 and PF × PSC8, PF × PSC5, PF × PSC10 and PF × PSC3 were better than the rest hybrid combinations. In interspecific hybrids of P. flabellatus and P. sajor-caju, the highest yield and biological efficiency was found in PF × PSC6 combination and may be selected for better mushroom production.

Inhibition of cell cycle-dependent hyphal and biofilm formation by a novel cytochalasin 19,20‑epoxycytochalasin Q in Candida albicans

Biofilm-mediated drug resistance is a key virulence factor of pathogenic microbes that cause a serious global health threat especially in immunocompromised individuals. Here, we investigated the antihyphal and antibiofilm activity of 19,20‑epoxycytochalasin Q (ECQ), a cytochalasin actin inhibitor isolated from medicinal mushroom Xylaria sp. BCC1067 against Candida albicans. Remarkably, 256 µg/ml of ECQ inhibited over 95% of C. albicans hyphal formation after 24 h-treatment. Combined ECQ and lipid-based biosurfactant effectively enhanced the antihyphal activity, lowering required ECQ concentrations. Hyphal fragmentation and reduction of biofilm biomass, shown by SEM and AFM visualization of ECQ-treated biofilms, were well corelated to the reduced metabolic activities of young and 24 h-preformed C. albicans biofilms. Induced intracellular accumulation of reactive oxygen species (ROS) also occurred in accompany with the leakage of shrunken cell membrane and defective cell wall at increasing ECQ concentrations. Transcriptomic analyses via RNA-sequencing revealed a massive change (> 1300 genes) in various biological pathways, following ECQ-treatment. Coordinated expression of genes, associated with cellular response to drugs, filamentous growth, cell adhesion, biofilm formation, cytoskeleton organization, cell division cycle, lipid and cell wall metabolisms was confirmed via qRT-PCR. Protein–protein association tool identified coupled expression between key regulators of cell division cyclin-dependent kinases (Cdc19/28) and a gamma-tubulin (Tub4). They coordinated ECQ-dependent hyphal specific gene targets of Ume6 and Tec1 during different phases of cell division. Thus, we first highlight the antihyphal and antibiofilm property of the novel antifungal agent ECQ against one of the most important life-threatening fungal pathogens by providing its key mechanistic detail in biofilm-related fungal infection.

Aspergillus Conidia and Allergens in Outdoor Environment: A Health Hazard?

Aspergillus is a genus of saprophytic fungus widely distributed in the environment and associated with soil, decaying vegetation, or seeds. However, some species, such as A. fumigatus, are considered opportunistic pathogens in humans. Their conidia (asexual spores) and mycelia are associated with clinical diseases known as invasive aspergillosis (IA), mainly related to the respiratory tract, such as allergic asthma, allergic bronchopulmonary aspergillosis (ABPA), or hypersensitivity. However, they can also disseminate to other organs, particularly the central nervous system. Due to the dispersal mechanism of the conidia through the air, airborne fungal particle measurement should be used to prevent and control this mold. This study aims to measure the outdoor airborne concentration of Aspergillus conidia and the Asp f 1 allergen concentration in Bellaterra (Barcelona, Spain) during 2021 and 2022, and to compare their dynamics to improve the understanding of the biology of this genus and contribute to a better diagnosis, prevention, and therapeutic measures in the face of possible health problems. The results show that both particles were airborne nearly all year round, but their concentrations showed no correlation. Due to Asp f 1 not being present in the conidia itself but being detectable during their germination and in hyphal fragments, we report the relevance of the aero-immunological analysis as a methodology to detect the potential pathogenic hazard of this fungus.

Biological decay by microorganisms in stems from the Upper Triassic Ischigualasto Formation (San Juan Province, Argentina): A striking microbial diversity in Carnian-Norian terrestrial ecosystems

We studied degradation patterns and microorganisms preserved in permineralized stems of Rhexoxylon piatnitzky, Protojuniperoxylon ischigualastense, and Agathoxylon argentinum from the Valle de la Luna Member of the Ischigualasto Formation (Upper Triassic, San Juan, Argentina). All three species show loss of middle lamella, thinning, and whitening of tracheid cell walls, and detachment of the S3 layer, consistent with selective delignification by white rot. This type of rot is the product of lignin and cellulosic degradation by Basidiomycetes and Ascomycetes. Rhexoxylon piatnitzkyi also shows small cavities, more or less circular in outline, on the wall of tracheids. This is known as soft rot and can be produced by some Ascomycetes, the anamorphic phases of diverse fungi, and tunneling bacteria. Additionally, abundant and diverse microorganisms were found. Rhexoxylon piatnitzkyi shows fragments of septate hyphae, evidence of bacterial activity, and structures of unknown affinity. Protojuniperoxylon ischigualastense presents structures common to anamorphic phases of diverse fungi, such as chlamydospores and conidiophores. Lastly, Agathoxylon argentinum bears coiled and septate hyphae, and a fan-shaped mycelium, whose significance is discussed. The microbiological study was complemented by the assessment of sedimentary and taphonomic data of the fossil-bearing beds. A gradual increment in humidity for the Valle de la Luna Member is interpreted, which concludes with a humidity peak in its upper section. This contribution represents the first detailed study of xylophagous and saprophyte micro-communities for the Ischigualasto Formation. The abundance and diversity of microorganisms described illustrate the complexity of wood-inhabiting micro-communities from the Carnian-Norian interval of Argentina.

Using qPCR and microscopy to assess the impact of harvesting and weather conditions on the relationship between Alternaria alternata and Alternaria spp. spores in rural and urban atmospheres

Alternaria is a plant pathogen and human allergen. Alternaria alternata is one of the most abundant fungal spores in the air. The purpose of this study was to examine whether Alternaria spp. spore concentrations can be used to predict the abundance and spatio-temporal pattern of A. alternata spores in the air. This was investigated by testing the hypothesis that A. alternata dominates airborne Alternaria spp. spores and varies spatio-temporally. Secondarily, we aimed at investigating the relationship between airborne Alternaria spp. spores and the DNA profile of A. alternata spores between two proximate (~ 7 km apart) sites. These were examined by sampling Alternaria spp. spores using Burkard 7-day and cyclone samplers for the period 2016–2018 at Worcester and Lakeside campuses of the University of Worcester, UK. Daily Alternaria spp. spores from the Burkard traps were identified using optical microscopy whilst A. alternata from the cyclone samples was detected and quantified using quantitative polymerase chain reaction (qPCR). The results showed that either A. alternata or other Alternaria species spores dominate the airborne Alternaria spore concentrations, generally depending on weather conditions. Furthermore, although Alternaria spp. spore concentrations were similar for the two proximate sites, A. alternata spore concentrations significantly varied for those sites and it is highly likely that the airborne samples contained large amounts of small fragments of A. alternata. Overall, the study shows that there is a higher abundance of airborne Alternaria allergen than reported by aerobiological networks and the majority is likely to be from spore and hyphal fragments.

Stem, leaf and cotyledon resistance responses to a prevalent Sclerotinia sclerotiorum pathotype in Australia highlight new opportunities to improve white mould resistance in common bean

Context White mould (Sclerotinia sclerotiorum) inflicts major yield losses on common bean (Phaseolus vulgaris); yet, commercial cultivars known for their high yields and market-adapted grains lack physiological resistance to this disease. Aims This study aimed to test diverse common bean genotypes for resistance in stem, leaf and cotyledon tissues. Methods Thirty-four common bean genotypes with a wide range of agronomic traits and grain types, including genotypes noted previously for susceptible and resistant responses to white mould, were inoculated with the prevalent S. sclerotiorum isolate MBRS-1. Then they were assessed for resistance in stem, leaf and cotyledon tissues under controlled environment conditions, by inoculating plants with a 105 mL−1 hyphal fragment concentration. Key results There was significant (P < 0.001) variation in resistance responses in stem, leaf and cotyledon tissues across the genotypes. Contender, ICA Bunsi, XAN 280 and Taisho-Kintoki showed the highest resistance in stems, whereas Norvell 2558, Pico de Oro, Sanilac, Othelo and Negro Argel exhibited notable resistance in leaves. Metis, Canario 107, Pico de Oro, Pogonion and Jubilejnaja 287 displayed the most resistance in cotyledons. Conclusions This is the first reported attempt to determine the response of common bean germplasm to a prevalent pathotype of S. sclerotiorum in Australia. Bean genotypes exhibiting high-level resistance to white mould identified in this study can be used as parental lines for crosses in common bean breeding programs and/or directly as improved cultivars. Implications The study highlighted both the value of screening under controlled environmental conditions to reliably locate new stem, leaf and/or cotyledon resistances and the possibility of using rapid cotyledon screening to indicate stem resistances because the expression of resistances in cotyledons generally correlated strongly with those in stems.

Innate and Adaptive Immune Responses Induced by Aspergillus fumigatus Conidia and Hyphae.

Previous research indicated that hyphae of Aspergillus fumigatus (A. fumigatus) rather than conidia could successfully build a pulmonary aspergillosis model in immunocompetent mice. In this study, we compared the immune responses induced by hyphae and conidia to explore the possible mechanism of this striking phenomenon. Herein, a novel method was designed and adopted to quantify hyphal fragments. Murine macrophages RAW264.7 and human peripheral blood mononuclear cells were stimulated by A. fumigatus hyphae and conidia in vitro, respectively, and then immunological reactions were measured. Male C57BL/6 mice were challenged with conidia and hyphae through intratracheal inoculation. Dynamic conditions of mice were recorded, and RNA-seq measured corresponding immune responses. The results of the study confirmed that hyphae could induce more intensive inflammation than conidia in vitro and in vivo. However, macrophages revealed a higher production of ROS and M1 polarisation in response to conidia stimuli. Additionally, conidia could promote Th1 cell differentiation, while hyphae could increase the CD4/CD8 ratio. RNA-seq validated the fact that those multiple immunologically relevant pathways were more strongly activated by hyphae than conidia, which also promoted Th2 cell differentiation and suppressed Th1 signalling. Both hyphae and conidia could activate Th17 signalling. In general, conidia and hyphae induced distinctly different host immune responses, and the immune responses induced by conidia played a better protective effect. Therefore, the unique function of hyphae in the spread and infection of Aspergillus should be emphasised, and more research is required to clarify the underlying mechanisms for better understanding and management of aspergillosis.

Comparison of Hyphal Fragments and Spores to Evaluate the Pathogenicity of the Eucalyptus Leaf and Shoot Pathogen Calonectria pseudoreteaudii.

The genus Calonectria includes many aggressive plant-pathogenic species with a worldwide distribution. Calonectria leaf blight is one of the most prominent diseases of Eucalyptus trees in Southeast Asian and South American plantations. Inoculation trials to evaluate pathogenicity of Calonectria spp. typically use conidial suspensions but this is not possible for species that do not sporulate sufficiently in culture. Calonectria pseudoreteaudii is one of the species that is most aggressive to Eucalyptus in China but most isolates fail to produce conidia in culture, requiring an alternative procedure for artificial inoculation. This study compared inoculations utilizing conidial and hyphal fragment suspensions. Two Eucalyptus genotypes were used, and these were inoculated with different concentrations of hyphal fragments or conidia of three C. pseudoreteaudii isolates. Three days after inoculation, the treated Eucalyptus plants displayed similar disease symptoms, regardless of whether they had been inoculated with conidia or hyphal fragments. This was consistent for all C. pseudoreteaudii isolates and also the different Eucalyptus genotypes. The results demonstrate that hyphal fragment suspensions can be used to provide a reliable indication of C. pseudoreteaudii isolate pathogenicity when conidia are not available for inoculation studies.

P506 First case of cutaneous mycosis caused by Nannizziopsis sp. in Chelonia

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMThe present report characterized a case of severe superficial and deep mycoses in a yellow-bellied slider (Trachemys scripta scripta) caused by Nannizziopsis sp. detected by histopathology and fungal culture. A female, 4-year-old, yellow-bellied slider (Trachemys scripta scripta) was presented for ulcerative and proliferative, chronic, skin lesions. White to pink, slightly round, with fuzzy borders, 0.5-2.5 cm in diameter, multifocal, proliferative, and ulcerative lesions were evident mainly on the left forelimb, right forelimb, pericloacal, chin, and oral cavity. The left forelimb was severely affected by a nodular subcutaneous mass.Several courses of disinfectant therapy, antibiotic, and antifungal treatments were given with no clinical improvements.Cytological smears of the ulcerative skin lesion revealed macrophages, heterophils, and fragments of septate hyphae. To investigate the cause of the lesions, the left forelimb was excised, fixed in 10% neutral buffered formalin, and submitted for histopathological examination. Biopsies and swabs from left forelimb, right forelimb, chin, and oral lesions were submitted for microbiological analysis.Samples were cultured on Sabouraud Dextrose Agar (SDA) and incubated at 30°C for 10 days. Histopathological examination showed severe pyogranulomatous dermatitis and myositis. Numerous septate, irregularly branched hyphae were evident within the pyogranulomas. Grocott methenamine silver stain (GMS) and Periodic acid-Schiff (PAS) confirmed the presence of filamentous fungi in the examined sections.Pure cultures of the same fungal strain were obtained from all the samples. Mature colonies on SDA showed a powdery aspect and a white felted surface. Microscopically, the hyphae were straight and branched, and conidia were clavate, sessile, or on short stalks.Based on both macroscopic and microscopic features the fungal strain was identified as Nannizziopsis sp.Susceptibility profile of the fungal isolate was tested for griseofulvin, miconazole, fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole. Amplification and sequencing of ITS, 18S, and SSU gene regions have been performed to identify the fungal isolate. The sequences were aligned with the most similar sequences of established species deposited in GenBank in order to define levels of relatedness. Sequences showed that the isolate clustered with Nannizziopsiaceae. The sequence similarities of the isolate were 99% to N. arthrosporioides in ITS (535/543 bps), 99% to N. vriesii in 18S, and SSU (1648/1649 and 970/973 bps, respectively).Based on the histopathological, microbiological, and molecular analysis, Nannizziopsis sp. was the primary cause of severe deep mycoses in the present turtle. Mycoses caused by Chrysosporium-related fungi are an emerging infectious disease affecting reptiles. The fungi of this group were collectively identified as Chrysosporium anamorph of Nannizziopsis vriesii (CAVN) complex by their morphological characteristics. Recent molecular taxonomy studies have now re-classified Chrysosporium-related fungi into three genera: Nannizziopsis, Paranannizziopsis, and Ophidiomyces.Until now, nine species have been isolated and identified from reptiles. Dermatomycosis associated with Nannizziopsis spp. is frequently reported in lizards, terrestrial, and aquatic snakes, and crocodiles, with classical cutaneous ulcerative lesions. Cutaneous mycosis is common in turtles, however, to our knowledge this is the first case of Nannizziopsis cutaneous mycosis in Chelonia.

A Library of Aspergillus niger Chassis Strains for Morphology Engineering Connects Strain Fitness and Filamentous Growth With Submerged Macromorphology.

Submerged fermentation using filamentous fungal cell factories is used to produce a diverse portfolio of useful molecules, including food, medicines, enzymes, and platform chemicals. Depending on strain background and abiotic culture conditions, different macromorphologies are formed during fermentation, ranging from dispersed hyphal fragments to approximately spherical pellets several millimetres in diameter. These macromorphologies are known to have a critical impact on product titres and rheological performance of the bioreactor. Pilot productivity screens in different macromorphological contexts is technically challenging, time consuming, and thus a significant limitation to achieving maximum product titres. To address this bottleneck, we developed a library of conditional expression mutants in the organic, protein, and secondary metabolite cell factory Aspergillus niger. Thirteen morphology-associated genes transcribed during fermentation were placed via CRISPR-Cas9 under control of a synthetic Tet-on gene switch. Quantitative analysis of submerged growth reveals that these strains have distinct and titratable macromorphologies for use as chassis during strain engineering programs. We also used this library as a tool to quantify how pellet formation is connected with strain fitness and filamentous growth. Using multiple linear regression modelling, we predict that pellet formation is dependent largely on strain fitness, whereas pellet Euclidian parameters depend on fitness and hyphal branching. Finally, we have shown that conditional expression of the putative kinase encoding gene pkh2 can decouple fitness, dry weight, pellet macromorphology, and culture heterogeneity. We hypothesize that further analysis of this gene product and the cell wall integrity pathway in which it is embedded will enable more precise engineering of A. niger macromorphology in future.

Fungal Spore Diversity over Garbage Depot of Pimpri Chinchwad, Pune, M.S., India

Aerobiology deals with the airborne particles of biological origin and their effect on living organisms. The atmosphere of the big city including Pimpri Chinchwad is dominated by biopollutants such as grain pollen, fungal grains and piles of dust particles. These days garbage disposal is a major problem in various cities. Many nearby villages are experiencing serious health problems due to elevated levels of aerobiopollutants. Air monitoring within garbage depot has been very rare. Therefore, it was felt that if a continuous survey of aerobiocomponents is conducted, it may be helpful in solving or suggesting a suitable solution. The present study deals with the assessment of aeromicrobiota over the garbage depot of Moshi, Pune for a year (Jan 2019 to Dec 2019). The study aimed to monitor the concentration of various biocomponents in the atmosphere over garbage depot. While scanning the prepared slides of the garbage depot for a year, fungal spores, pollen, hyphal fragments, insect parts, etc. were observed. However, during the present investigation, more emphasis has been given to the fungal components of airspora. The outcome of the present study would ultimately help allergy clinicians in the treatment of allergies and to the Municipal Corporation for designing the dumping site for garbage and developing better garbage disposal mechanisms ultimately bringing comfort to the population of the adjoining area.

Temporal variations in fungal spores in Mardin city atmosphere, upper Mesopotamia, SE-Turkey

The present study aimed to determine the fungal spores and their concentrations in the atmosphere of Mardin (southeast Turkey) in 2014 and 2015 using the volumetric method. A total of 43 taxa, hyphal fragments, and single septate ascospores were identified by light microscopy. It was determined that the dominant fungal spores included Cladosporium (51.5%), Ustilaginales species spores (13.9%), Alternaria (6.9%), Pucciniales species spores (1.6%), Agrocybe (1.2%), Pleospora (1.1%) and hyphal fragments (15.5%). The highest atmospheric spore concentrations were observed in May and the lowest levels were determined in February in both years. Dry air spores constituted a significant ratio of the total count (73.9%) in the atmosphere of Mardin. There were significant positive correlations between atmospheric concentrations of Alternaria, Cladosporium, Ustilaginales, and Pucciniales spores and temperatures. There were significant negative correlations between airborne fungal spore concentrations and daily mean humidity. There was a significant negative correlation between Agrocybe spore concentrations and daily mean temperature and a significant positive correlation between the same and humidity and precipitation. No significant correlation was determined between the meteorological variables and Pleospora and Aspergillus/Penicillium spore concentrations.

First Report of Rhizoctonia solani AG2-1 on roots of wheat in Kazakhstan.

In June 2019, approximately 20 tillers of wheat (Triticum aestivum L.) were sampled at the ripening stage (Feekes scale 11) from four different fields in Almaty, Kazakhstan. Brown lesions (3-5 mm in length) were present on the roots of sampled plants, with 20% incidence. To determine the causal agent, diseased roots were surface disinfected in sodium hypochlorite solution (1%) for 3 min, rinsed triple with sterile distilled water, air-dried in a laminar flow hood, and plated onto one-fifth strength potato dextrose agar (PDA) supplemented with 50 ppm chloramphenicol. After three days, the hyphal fragments that developed from the sections were transferred to fresh PDA and incubated at 23°C with 12-h photoperiod for 7 days to obtain pure cultures. Brown pigmented fungal colonies with a constriction at the base of hyphal branches, septa near the branching point, and right-angled branching resembling Rhizoctonia solani were observed. The identification anastomosis group (AG) of a representative isolate for each field was conducted by sequencing the internal transcribed spacer (ITS) region of rDNA with the universal primers ITS4 and ITS5 (White et al. 1990). The resulting sequences of 693 bp length were deposited in GenBank (accession nos. MW898143:MW898146). These sequences were 100% identical to the isolate 8Rs of R. solani AG2-1 (accession no. AF354063). To confirm the pathogenicity of the four isolates, the colonized wheat kernels method described by Demirci (1998) was used to inoculate a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v) into which wheat (cv. Seri) was planted. Control pots were inoculated with sterile wheat kernels using the same procedure. Wheat plants were left to grow for four weeks under controlled environmental conditions with a 23°C temperature regime. During the period that the plants remained in the glasshouse, the typical light regime was 16 h. Brown lesions were observed on the roots of plants in the inoculated pots whereas no symptoms were observed on plants grown in the control pots. R. solani was consistently reisolated from symptomatic plants, thereby confirming Koch's postulates. To our knowledge, this is the first report of R. solani AG2-1 on roots of wheat in Kazakhstan. R. solani AG2-1 isolates have been previously reported to be a weak pathogen to wheat (Roberts and Sivasithamparam 1986; Sturrock et al. 2015; Jaaffar et al. 2016; Özer et al. 2019). We suggest further studies are required to characterize the impact of R. solani AG2-1 in wheat. Considering crop rotation, the selection of non-host crops to this AG group is important to pathogen management, by reducing the amount of inoculum in the soil.

In vitro and in silico approach of fungal growth inhibition by Trichoderma asperellum HbGT6-07 derived volatile organic compounds

The species of Trichoderma are one of the most frequently used natural biocontrol agent. This study, we identified isolate HbGT6-07 of Trichoderma asperellum and evaluated the antimicrobial effects both in vitro and in silico approaches. Tested 10% concentrated culture filtrate of HbGT6-07 inhibited 98% of colony radial growth in B. cinerea (B05.10) as well as 91% of S. sclerotiorum (A367). HbGT6-07 was detected to produce volatile organic compounds (VOCs) with antifungal activity. In in-vitro dish-within-dish method (DwD), The HbGT6-07 VOCs effectively reduced colonial diameter, growth rate and sclerotia production by two virulent fungal pathogens. Moreover, the hyphal fragments of HbGT6-07 demonstrated successful mycelia growth suppression (97%) against infection oilseed rape leaves by hyphae of the two virulent fungal pathogens through competition. The mixed culture assay, exhibited that the isolate T. asperellum HbGT6-07 was significantly reduced the production and weight of sclerotia. The GC-MS analysis identified 32 VOCs derived from HbGT6-07. In addition, VOCs derived from HbGT6-07 were assessed against targeted protein of three fungal species; Aspergillus oryzae, Saccharomyces cerevisiae, Candida albicans via molecular docking. Butylated hydroxytolune and Beta-Cedrene had energy (−5.3 and −5.7 Kcal/mol) for targeted protein of Aspergillus oryzae and (−6.8 and −8.0 Kcal/mol) for Saccharomyces cerevisiae, whereas alpha-bergamotene and Beta-Cedrene exihbit energy (−7.5 and −7.4 Kcal/mol), respectively. The molecular dynamics study confirms the structural stability and rigidity of the docked complex through multiple descriptors from simulation trajectories. The above findings indicated that HbGT6-07 could attain competitive progress via production of VOCs and comprehensive mycelial growth.

Activity of Fengycin and Iturin A Isolated From Bacillus subtilis Z-14 on Gaeumannomyces graminis Var. tritici and Soil Microbial Diversity.

Bacillus subtilis Z-14 can inhibit phytopathogenic fungi, and is used as a biocontrol agent for wheat take-all disease. The present study used the soil-borne fungus Gaeumannomyces graminis var. tritici (Ggt), which causes wheat take-all disease, and the soil microbial community as indicators, and investigated the antifungal effects of fengycin and iturin A purified from strain Z-14 using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, respectively. The results showed that fengycin destroyed the internal structure of Ggt cells by digesting the cytoplasm and organelles, forming vacuoles, and inducing hyphal shrinkage and distortion. Iturin A induced cell wall disappearance, membrane degeneration, intracellular material shrinkage, and hyphal fragmentation. A biocontrol test demonstrated a 100% control effect on wheat take-all when wheat seedlings were treated with fengycin at 100 μg/ml or iturin A at 500 μg/ml. Iturin A and fengycin both reduced the relative abundance of Aspergillus and Gibberella. At the genus level, iturin A reduced the relative abundance of Mortierella and Myrothecium, while fengycin reduced that of Fusarium. Only fengycin treatment for 7 days had a significant effect on soil bacterial diversity.

Depiction of chitinase extracted from Serratia marcescens SU05 on Agriculturally important Fungi

Traditional method of controlling fungi and use of chemical pesticides, which have already been proved harmful to the environment. Hence is a potential alternative in biological control using chitinase enzyme. Thus, the necessity to achieve large scale, cost effective production of active preparations of this biopesticides has been increased. Serratia marcescens is a Gram-negative bacilli isolated from soil and identified using basic biochemical test and 16s rRNA sequencing and accession number KX002030 was obtained, that produces chitinase. This enzyme will be optimized for maximum production at various factors and media components. The extracted and partially purified enzyme (ammonium sulphate and dialysis) will be used in the analysis of for the effect of cell wall destruction of fungi pathogens. Biocontrol potential of Chitinase, dual plate assay, hyphal fragmentation test were analyzed against Alternaria alternata.