Fungal Morphology Research Articles

An ex vivo Human Skin Model to Study Superficial Fungal Infections.

Human skin fungal infections (SFIs) affect 25% of the world’s population. Most of these infections are superficial. The main limitation of current animal models of human superficial SFIs is that clinical presentation is different between the different species and animal models do not accurately reflect the human skin environment. An ex vivo human skin model was therefore developed and standardised to accurately model SFIs. In this manuscript, we report our protocol for setting up ex vivo human skin infections and report results from a primary superficial skin infection with Trichophyton rubrum, an anthropophilic fungus. The protocol includes a detailed description of the methodology to prepare the skin explants, establish infection, avoid contamination, and obtain high quality samples for further downstream analyses. Scanning electronic microscopy (SEM), histology and fluorescent microscopy were applied to evaluate skin cell viability and fungal morphology. Furthermore, we describe a broad range of assays, such as RNA extraction and qRT-PCR for human gene expression, and protein extraction from tissue and supernatants for proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS). Non-infected skin was viable after 14 days of incubation, expressed genes and contained proteins associated with proliferative, immune and differentiation functions. The macroscopic damage caused by T. rubrum had a similar appearance to the one expected in clinical settings. Finally, using this model, the host response to T. rubrum infection can be evaluated at different levels.

Live Monitoring and Analysis of Fungal Growth, Viability, and Mycelial Morphology Using the IncuCyte NeuroTrack Processing Module.

Efficient live-imaging methods are pivotal to understand fungal morphogenesis, especially as it relates to interactions with host immune cells and mechanisms of antifungal drugs. Due to the notable similarities in growth patterns of neuronal cells and mycelial networks, we sought to repurpose the NeuroTrack (NT) processing module of the IncuCyte time-lapse microscopy system as a tool to quantify mycelial growth and branching of pathogenic fungi. We showed the robustness of NT analysis to study Candida albicans and five different molds and confirmed established characteristics of mycelial growth kinetics. We also documented high intra- and interassay reproducibility of the NT module for a spectrum of spore inocula and culture periods. Using GFP-expressing Aspergillus fumigatus and Rhizopus arrhizus, the feasibility of fluorescence-based NT analysis was validated. In addition, we performed proof-of-concept experiments of NT analysis for several translational applications such as studying the morphogenesis of a filamentation-defective C. albicans mutant, the effects of different classes of antifungals (polyenes, azoles, and echinocandins), and coculture with host immune cells. High accuracy was found, even at high immune cell-to-fungus ratios or in the presence of fungal debris. For antifungal efficacy studies, addition of a cytotoxicity dye further refined IncuCyte-based analysis, facilitating real-time determination of fungistatic and fungicidal activity in a single assay. Complementing conventional MIC-based assays, NT analysis is an appealing method to study fungal morphogenesis and viability in the context of antifungal compound screening and evaluation of novel immune therapeutics.IMPORTANCE Pathogenic fungi remain a major cause of infectious complications in immunocompromised patients. Microscopic techniques are crucial for our understanding of fungal biology, host-pathogen interaction, and the pleiotropic effects of antifungal drugs on fungal cell growth and morphogenesis. Taking advantage of the morphological similarities of neuronal cell networks and mycelial growth patterns, we employed the IncuCyte time-lapse microscopy system and its NeuroTrack image analysis software package to study growth and branching of a variety of pathogenic yeasts and molds. Using optimized image processing definitions, we validated IncuCyte NeuroTrack analysis as a reliable and efficient tool for translational applications such as antifungal efficacy evaluation and coculture with host immune effector cells. Hence, the IncuCyte system and its NeuroTrack module provide an appealing platform for efficient in vitro studies of antifungal compounds and immunotherapeutic strategies in medical mycology.

Innate and adaptive immune interactions in Coccidioides infection

Abstract Protective immunity against the fungal pathogen Coccidioides requires specific T helper cellular responses. Mouse infection and vaccine studies have defined CD4 T helper (Th)1 and Th17 cells in the resolution of infection and in effective protection, while patients with chronic Coccidioides infection demonstrate reduced cellular responses. Several gaps in knowledge preclude our ability to develop new diagnostics and therapeutics for treating this infection. Little is known about the microenvironment in human lung tissue during infection, and the types of immune cells that promote effective control of the fungus within this specialized location. How variations on local immune populations alter disease severity and ability of the fungus to disseminate have not been explored. We recently identified several peripheral blood biomarkers during acute infection that distinguish pediatric patients that resolve infection from those that develop chronic disease. Patients with chronic disease had reduced Th17 and elevated Treg frequencies compared to patients that resolved disease. The inability to clear Coccidioides infection may be a result of elevated Treg frequency and functional capacity, or of skewed effector differentiation. We use mouse Coccidioides infection and in vitro culture assays to uncover the host immune players that promote effective immunity, and to identify fungal morphology changes and fungal burden differences at the innate and adaptive immune interface.

Influence of the construction of porous spargers on lovastatin production by Aspergillus terreus ATCC 20,542 in a laboratory bubble column.

In bubble column bioreactors, the hydrodynamic behavior like mixing time, bubble size and morphology of filamentous fungi are influenced by the construction of spargers. Sparger pore size is an important factor influencing formation of bubbles. In this study for the first time, a 5-L bubble column bioreactor with different porous spargers was used to investigate the effect of mean air bubble diameter (at 0.36, 0.18 and 0.09cm) on fungal growth, broth viscosity, fungal pellet morphology and lovastatin production by the filamentous fungus Aspergillus terreus. All cultivations were carried out at air flow rate equal to 0.5 Lair L-1min-1. The viscosity of the broth was influenced by both biomass concentration and size of the fungal pellets. The highest values of viscosity were observed at bubbles of 0.09cm diameter after 192h of cultivation. The largest fluffy pellets and the highest yield of lovastatin (443mg/L) were obtained at air bubbles diameter of 0.18cm. Lovastatin yield on biomass growth in this condition was, respectively, 1.7-fold and 3.5-fold higher than in the cultivations performed with air bubbles of 0.36 and 0.09cm diameters. These laboratory scale experiment indicates that air bubble diameter has the impact on lovastatin production and A. terreus culture conditions.

First detection of Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) on Cavendish banana in India

Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), is regarded as one of the most devastating diseases of bananas globally. Foc consists of three races, of which Foc races 1 and 2 occur in India. Foc race 4 has not been found in the country despite the importance and extensive cultivation of Cavendish cultivars. In 2015, a banana grower from Barari village in the Bihar State of India reported the wilting of Cavendish banana cvs. Robusta and Grand Naine. The incidence of the disease ranged from 2 to 26.6%. Symptoms included yellowing and browning of older leaves, which progressed from the oldest to the youngest leaves. Samples were collected from pseudostems and the responsible fungus isolated. The cultures were then identified using fungal morphology, vegetative compatibility group (VCG) analysis, volatile production and PCR with Foc tropical race (TR) 4-specific primers. Pathogenicity tests were conducted using Grand Naine plantlets produced by micropropagation. The cultures were identified as volatile-producing F. oxysporum strains, and pathogenicity tests confirmed their status as the banana pathogen Foc. VCG analysis, and the use of race-specific PCR markers, identified them as members of VCG 01213/16 and Foc TR4, respectively. This indicated that the fungus responsible for Fusarium wilt in the Bihar state of India is Foc TR4. Most Indian farmers use suckers as planting material, and are not aware of the impact of the disease. If the disease spreads to other states in India it can lead to an unemployment problem for farm labourers and the change in production to non-remunerative crops that may reduce profits.

Characterisation of novel-cell-wall LysM-domain proteins LdpA and LdpB from the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus, a filamentous fungus that is ubiquitous in the environment, causes several human pulmonary disorders, including chronic and acute invasive infections and allergic diseases. Lysin motif (LysM) is a small protein domain that binds chitin, a major component of fungal cell wall polysaccharides. Several secreted LysM-domain proteins without catalytic function (LysM effectors) have been identified. They act as virulence factors in plant pathogenic fungi by preventing the immune response induced by chitin; however, LysM proteins in mammalian pathogenic fungi remain largely unexplored. We describe two novel LysM-domain proteins, LdpA and LdpB, in A. fumigatus. Functional analyses of single and double knockouts revealed no significant effects on cell wall chitin content, cell wall integrity, fungal morphology and fungal growth. Fluorescent signals from LdpA-green fluorescent protein (GFP) and LdpB-GFP were observed in cell wall and extracellular matrix. In a mouse model of invasive pulmonary aspergillosis, survival did not differ between ΔldpA/B and wild-type infection; however, further studies are required to reveal their functions in fungal−host interactions.

Arbuscular mycorrhiza and dark septate endophyte fungal associations of Oryza sativa L. under field condition: colonization features and their occurrence

The present study was aimed to study monthly colonization of arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal associations in rice. The presence of mycorrhizal structures in the roots confirms the colonization by AM fungi. The pattern of hyphae and arbuscules denotes Arum type of AM fungal morphology. The presence of dark coloured septate hyphae running frequently on the epidermal layer and in root cortex and the occurrence of microsclerotia marks the colonization by DSE fungi. The co-occurrence of both AM and DSE fungi ensure dual colonization by two distinct fungal groups. There was significant increase in arbuscules, vesicles and hyphal percentages from first to third month in both the samples collected from two sites. In the third month, AM colonization significantly higher in both the sites. DSE colonization percentages do not differ significantly in first to third month. A total of nine AM fungal species were recovered from two sites. This study is an effort to make aware the local farmers about the usefulness of these native AM mycobiota which can be a preferable choice over chemical fertilizers leading to ecofriendly organic farming.

Application of Aluminum Oxide Nanoparticles in Aspergillus terreus Cultivations: Evaluating the Effects on Lovastatin Production and Fungal Morphology.

Aluminum oxide nanoparticles were supplemented to Aspergillus terreus ATCC 20542 precultures and the outcomes of the process were evaluated relative to the results of microparticle-enhanced and standard cultivations. The selected morphological parameters of fungal pellets (projected area, elongation, convexity, and shape factor) were monitored throughout the experiment, together with biomass, lactose, and lovastatin concentration. The qualitative and quantitative chemical analysis was performed with the use of liquid chromatography coupled with high resolution mass spectrometry. The results of the study indicated that the application of nanoparticles was indeed associated with morphological consequences, most notably the decreased pellet size. However, it turned out that the term “nanoparticle-enhanced cultivation” could not be used in the context of lovastatin production, as no marked increase of product titer was observed in nanoparticle-influenced variants relative to standard and microparticle-enhanced cultivation. In addition, the concentration of biomass in the nanoparticle-influenced runs was relatively low. Comparative analysis of total ion chromatograms revealed the presence of a molecule of unknown structure that could be detected solely in broths from standard and microparticle-containing cultures. This study represents the first evaluation of nanoparticles as the tools of morphological engineering aimed at enhanced lovastatin biosynthesis in A. terreus cultures.

Effects of Cotton Seed Powder as the Seed Medium Nitrogen Source on the Morphology and Pneumocandin B0 Yield of Glarea lozoyensis.

Pneumocandin B0 is an important antifungal drug precursor produced by filamentous fungus Glarea lozoyensis. The high broth viscosity of cultures of this organism results in lower oxygen solubility and higher energy consumption for agitation and aeration, which mostly caused by the morphologies of filamentous fungi in submerged culture. In this study, the effects of different seed medium nitrogen sources on morphology and fermentation behavior of G. lozoyensis were investigated, and cotton seed powder resulted in small, compact pellets. Moreover, pneumocandin B0 yield in Erlenmeyer flasks were increased by 22.9%. Furthermore, pneumocandin B0 yield in a 50-L fermenter reached 2,100 mg/L and the dissolved oxygen maintained above 30%. Additionally, activities of phosphofructokinase (PFK), isocitrate dehydrogenase (ICDH), glucose 6-phosphate dehydrogenase (G6PDH), and malic enzyme (ME) were increased by 87.5, 50, 41.6, and 10.7%, respectively. This study demonstrates the feasibility and advantages of using cotton seed powder for controlling the fungal morphology and improving the product yield in pneumocandin fermentations.

Morphological Characteristic Regulation of Ligninolytic Enzyme Produced by Trametes polyzona

A newly isolated white rot fungal strain KU-RNW027 was identified as Trametes polyzona, based on an analysis of its morphological characteristics and phylogenetic data. Aeration and fungal morphology were important factors which drove strain KU-RNW027 to secrete two different ligninolytic enzymes as manganese peroxidase (MnP) and laccase. Highest activities of MnP and laccase were obtained in a continuous shaking culture at 8 and 47 times higher, respectively, than under static conditions. Strain KU-RNW027 existed as pellets and free form mycelial clumps in submerged cultivation with the pellet form producing more enzymes. Fungal biomass increased with increasing amounts of pellet inoculum while pellet diameter decreased. Strain KU-RNW027 formed terminal chlamydospore-like structures in cultures inoculated with 0.05 g/L as optimal pellet inoculum which resulted in highest enzyme production. Enzyme production efficiency of T. polyzona KU-RNW027 depended on fungal pellet morphology as size, porosity, and formation of chlamydospore-like structures.

The afterlife effects of fungal morphology: Contrasting decomposition rates between diffuse and rhizomorphic necromass

Microbial necromass is now recognized as an important input into stable soil organic matter pools in terrestrial ecosystems. While melanin and nitrogen content have been identified as factors that influence the decomposition rate of fungal necromass, the effects of mycelial morphology on necromass decomposition remain largely unknown. Using the fungus Armillaria mellea, which produces both diffuse and rhizomorphic biomass in pure culture, we assessed the effects of necromass morphology on decomposition in a 12 week field experiment in Pinus and Quercus dominated forests in Minnesota, USA. Diffuse and rhizomorphic necromass was incubated for 2, 4, 6, and 12 weeks to assess differences in decay rates and changes in residual necromass chemistry. Rhizomorphic necromass decomposed significantly slower than diffuse necromass in both forest types. This difference was correlated with initial necromass chemistry, particularly nitrogen content, but not with hydrophobicity. Over the course of the incubation, there was a greater change in the chemistry of diffuse versus rhizomorphic necromass, with both becoming more enriched in recalcitrant compounds. Given that many fungi with both saprotrophic and mycorrhizal ecologies produce rhizomorphs, these results suggest that mycelial morphology should be explicitly considered as an important functional trait influencing the rate of fungal necromass decomposition.

High-Throughput Screening Identifies Genes Required for Candida albicans Induction of Macrophage Pyroptosis.

ABSTRACTThe innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.

Spot Blight on Tobacco (Nicotiana tabacum) Caused by Stagonosporopsis cucurbitacearum in Guizhou Province of China.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China accounts for approximately 40% of the global tobacco production. In August 2017, small (2 to 8 mm in diameter) water-soaked lesions were observed on the leaves of tobacco cultivar Yunyan 85 in Zheng’an (Guizhou, China, 28.56 N, 107.43 E). Subsequently, the affected areas turned tan-colored and formed irregular leaf spots with conspicuous yellow borders between symptomatic and healthy tissues. The spots frequently coalesced, commonly resulting in wilting of entire leaves within 20 to 30 days. The symptoms were observed in 30% of the tobacco plants in a 3-ha field. Symptomatic leaf samples were surface sterilized with 70% ethanol for 2 min, followed by 1% NaOCl for 40 s, and then rinsed three times with sterile distilled water. These leaf pieces were placed on potato dextrose agar medium and incubated at 28°C in the dark for 5 days. Pure cultures were obtained using a modified method (Chilvers et al. 2014), and single spores were obtained after 5 days of incubation on 10% V8 medium. One isolate (H-2-3) was selected for identification. Perithecia were globose to oblate and 80 to 130 μm in diameter. Asci measured 45 to 70 × 14 to 25 µm, were straight or slightly curved, bitunicate, and clavate. Pseudoparaphyses were filiform. Ascospores were 22 to 43 × 8 to 14 µm, fusoid to ellipsoidal, uniseptate, and hyaline. Conidia were globose and 18 to 35 µm in diameter. Based on these characteristics, the fungus was identified as Stagonosporopsis cucurbitacearum (Fr.) Aveskamp, Gruyter & Verkley (syn. Phoma cucurbitacearum [Fr.] Sacc.) in Punithalingam et al. (1972). Species verification was completed with representative isolate H-2-3 by polymerase chain reaction amplification of internal transcribed spacer (ITS) rDNA and β-tubulin (TUB) with universal primers ITS1F/ITS4 and Bt2a/Bt2b (Stewart et al. 2015). The ITS region was sequenced and showed 100% identity to S. cucurbitacearum (GenBank MG195974). The TUB region was 100% homologous to those of S. cucurbitacearum (GenBank MG866169). Pathogenicity of the fungus was confirmed by performing Koch’s postulates as follows. The agar plugs of three isolates (H-1-2, H-2-1, and H-2-3) and healthy tobacco (cv. Yunyan 85) leaves were prepared. Plugs were directly placed onto the adaxial surface of a leaf, and agar plugs without fungus were handled as a control treatment. For each treatment of each isolate, three plugs were conducted. After inoculation, leaves were maintained at 25°C, 100 Lux, >70% relative humidity, and 16-h light/day, and monitored for 7 days for symptoms. Symptoms similar to the original ones occurred on the points of the leaves, whereas control leaves remained symptomless. By morphological characteristics study, S. cucurbitacearum was reisolated from all symptomatic leaves. Based on the fungal morphology and sequence analysis of the ITS and TUB, this fungus was identified as S. cucurbitacearum, which has been reported from luohanguo (Siraitia grosvenorii) (Jiang et al. 2015) and water spinach (Ipomoea aquatica) (Liu et al. 2017) in China. However, to the best of our knowledge, this is the first report of S. cucurbitacearum causing spot blight on tobacco in China, and more attention should be given to other potentially susceptible host plants in the area.

Innate Immune and Fungal Model of Alzheimer's Disease.

Various fungi and bacteria can colonize in the brain and produce physical alterations seen in Alzheimer’s disease (AD). Environmental and genetic factors affect the occurrence of fungal colonization, and how fungi can grow, enter the brain, and interact with the innate immune system. The essence of AD development is the defeat of the innate immune system, whether through vulnerable patient health status or treatment that suppresses inflammation by suppressing the innate immune system. External and mechanical factors that lead to inflammation are a door for pathogenic opportunity. Current research associates the presence of fungi in the etiology of AD and is shown in cerebral tissue at autopsy. From the time of the discovery of AD, much speculation exists for an infective cause. Identifying any AD disease organism is obscured by processes that can take place over years. Amyloid protein deposits are generally considered to be evidence of an intrinsic response to stress or imbalance, but instead amyloid may be evidence of the innate immune response which exists to destroy fungal colonization through structural interference and cytotoxicity. Fungi can remain ensconced for a long time in niches or inside cells, and it is the harboring of fungi that leads to repeated reinfection and slow wider colonization that eventually leads to a grave outcome. Although many fungi and bacteria are associated with AD affected tissues, discussion here focuses on Candida albicans as the archetype of human fungal pathology because of its wide proliferation as a commensal fungus, extensive published research, numerous fungal morphologies, and majority proliferation in AD tissues.

Inhibitory Effect of Flower-Shaped Zinc Oxide Nanostructures on the Growth and Aflatoxin Production of a Highly Toxigenic Strain of Aspergillus flavus Link.

Flower-shaped zinc oxide (ZnO) nanostructures were prepared via a simple aqueous precipitation strategy at room temperature. The as-grown nanostructures were characterized by UV–vis spectroscopy, UV–vis diffuse reflectance spectroscopy (DRS), spectrofluorometry, Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR), X-ray diffraction (XRD), and field emission scanning electron microscopy (FESEM). The antifungal and anti-aflatoxigenic activities of the ZnO nanostructures were further investigated using a highly toxigenic strain of Aspergillus flavus Link under in vitro and in situ conditions. The results showed that the A. flavus isolate was inhibited to various extents by different concentrations of ZnO nanostructures, but the best inhibitions occurred at 1.25, 2.5, and 5 mM in the culture media. At these concentrations, suppression of aflatoxin biosynthesis (99.7%) was also observed. Moreover, a reasonable reduction in the aflatoxin content (69%) was observed in maize grains treated with the lowest ZnO concentration that exhibited the strongest inhibitory activity in the liquid media. SEM micrographs clearly indicate multiple degenerative alterations in fungal morphology after treatment with ZnO such as damage of the tubular filaments, loss of hyphae shape, as well as hyphae rupture. These results suggest that flower-shaped ZnO nanostructures exhibit strong antifungal and anti-aflatoxigenic activity with potential applications in the agro-food system.

Comparative transcriptome profiling of Blumeria graminis f. sp. tritici during compatible and incompatible interactions with sister wheat lines carrying and lacking Pm40.

Blumeria graminis f. sp. tritici (Bgt) is an obligate biotrophic fungus that causes wheat powdery mildew, which is a devastating disease in wheat. However, little is known about the pathogenesis of this fungus, and differences in the pathogenesis of the same pathogen at various resistance levels in hosts have not been determined. In the present study, leaf tissues of both Pm40-expressing hexaploid wheat line L658 and its Pm40-deficient sister line L958 were harvested at 0 (without inoculation), 6, 12, 24, 48 and 72 hours post-inoculation (hpi) with Bgt race 15 and then subjected to RNA sequencing (RNA-seq). In addition, we also observed changes in fungal growth morphology at the aforementioned time points. There was a high correlation between percentage of reads mapped to the Bgt reference genome and biomass of the fungus within the leaf tissue during the growth process. The percentage of mapped reads of Bgt in compatible interactions was significantly higher (at the p<0.05 level) than that of reads in incompatible interactions from 24 to 72 hpi. Further functional annotations indicated that expression levels of genes encoding H+-transporting ATPase, putative secreted effector proteins (PSEPs) and heat shock proteins (HSPs) were significantly up-regulated in compatible interactions compared with these levels in incompatible interactions, particularly at 72 hpi. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that genes involved in the endocytosis pathway were also enriched in compatible interactions. Overall, genes encoding H+-transporting ATPase, PSEPs and HSPs possibly played crucial roles in successfully establishing the pathogenesis of compatible interactions during late stages of inoculation. The study results also indicated that endocytosis is likely to play a potential role in Bgt in establishing compatible interactions.

Disruption of gul-1 decreased the culture viscosity and improved protein secretion in the filamentous fungus Neurospora crassa

BackgroundThe cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. However, large scale applications are hindered by its filamentous growth. Although previous investigations have shown that mycelial morphology in submerged culture can be controlled by altering physical factors, there is little knowledge available about the potential for morphology control by genetic modification.ResultsIn this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only two mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Transcriptional profiling showed that expression of genes encoding eight GPI-anchored cell wall proteins was lowered in Δgul-1 while expression of genes associated with two non-anchored cell wall proteins was elevated. Meanwhile, the expression levels of two hydrophobin genes were also significantly altered. These results suggested that GUL-1 affected the transcription of cell wall-related genes, thereby influencing cell wall structure and mycelial morphology. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical applications, deletion of gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth.ConclusionsUsing the model filamentous fungus N. crassa, genes that affect mycelial morphology in submerged culture were explored through systematic screening of morphological mutants. Disrupting several candidate genes altered viscosities in submerged culture. This work provides an example for controlling fungal morphology in submerged fermentation by genetic engineering, and will be beneficial for industrial fungal strain improvement.

Engineering fungal morphology for enhanced production of hydrolytic enzymes by Aspergillus oryzae SBS50 using microparticles.

Effect of microparticles and silver nanoparticles was studied on the production of hydrolytic enzymes by a potent phytase-producing mould, Aspergillus oryzae SBS50. Addition of microparticles, viz. talc powder and aluminum oxide enhanced phytase production from 2894 to 3903 and 2847 to 4204U/L, cellulase from 2529 to 4931 and 2455 to 3444U/L, xylanase from 9067 to 9642 and 9994 to 14,783U/L, amylase from 5880 to 11,000 and 6130 to 13,145U/L, respectively. Fungal morphology was also engineered by the use of microparticles. Fungal pellet size was significantly reduced (~ 90%) by the addition of microparticles. Fermentation time was reduced from 4 to 3 days after the addition of microparticles, thus increasing the productivity of the enzymes significantly. These results confirmed the importance of microparticles in engineering fungal morphology for enhanced production of hydrolytic enzymes.

Nanohexaconazole: synthesis, characterisation and efficacy of a novel fungicidal nanodispersion.

Here, the authors describe a simple method to formulate the nanodispersion of hexaconazole (hexa); henceforth, referred to as nanohexaconazole (N-hexa) that is water soluble and effective against several species of Aspergillus. Size and shape of the prepared nanocomposite was determined with high-resolution transmission electron microscopy and field-emission scanning electron microscopy. Nanohexaconazole structure was further confirmed by Fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry. The antifungal efficacy of nanohexaconazole (N-hexa) was studied in vitro, compared with micronised hexaconazole (M-hexa) at different doses (5 ppm, 10 ppm and control) against two food pathogenic fungi: Aspergillus niger (MTCC 282, MTCC 2196 and BDS 113) and Aspergillus fumigatus through poisoned food technique. A dose-dependent significant growth inhibition was observed in nanohexaconazole (N-hexa) treated fungal sample compared with that of micronised hexaconazole (M-hexa). Micrographic studies for the morphological analysis of control and nanohexaconazole (N-hexa) treated fungal samples were done, exhibited an alternation in fungal morphology. Results showed that nanohexaconazole (N-hexa) is more efficacious than commercially available micronised hexaconazole (M-hexa). In future nanohexaconazole (N-hexa) could be a possible candidate for modern medical science and also reduce damage to the environment from injudicious use of pesticides.

Adaptive Divergence in a Defense Symbiosis Driven from the Top Down.

Most studies of adaptive radiation in animals focus on resource competition as the primary driver of trait divergence. The roles of other ecological interactions in shaping divergent phenotypes during such radiations have received less attention. We evaluate natural enemies as primary agents of diversifying selection on the phenotypes of an actively diverging lineage of gall midges on tall goldenrod. In this system, the gall of the midge consists of a biotrophic fungal symbiont that develops on host-plant leaves and forms distinctly variable protective carapaces over midge larvae. Through field studies, we show that fungal gall morphology, which is induced by midges (i.e., it is an extended phenotype), is under directional and diversifying selection by parasitoid enemies. Overall, natural enemies disruptively select for either small or large galls, mainly along the axis of gall thickness. These results imply that predators are driving the evolution of phenotypic diversity in symbiotic defense traits in this system and that divergence in defensive morphology may provide ecological opportunities that help to fuel the adaptive radiation of this genus of midges on goldenrods. This enemy-driven phenotypic divergence in a diversifying lineage illustrates the potential importance of consumer-resource and symbiotic species interactions in adaptive radiation.