Fungal Morphology Research Articles

Expression of F-actin and β-tubulin genes in free mycelia and robust biofilms of the filamentous fungus Aspergillus niger

The morphology and growth of the filamentous fungi are influenced by different factors as the culture conditions and the type of fermentative process. The production and secretion of metabolites by these organisms present a direct relationship with their morphology. The organization of the microtubules and actin in the cytoskeleton is determinant for both the fungal growth and morphology. In this context, this study aimed to analyze the expression of the β-tubulin, F-actin, and glucan synthase in the A. niger mycelia obtained from submerged fermentation and biofilm fermentation through qPCR, as well as the analysis of the nucleus distribution in the hypha. Herein, we showed that β-tubulin and the F-actin gene were more expressed in the biofilm condition, while the glucan synthase was in the submerged condition. No significant difference was observed in the nucleus distribution between the mycelia obtained from both the fermentative processes. In conclusion, the different morphologies observed for the mycelia from submerged fermentation and biofilm fermentation might be influenced by the differential modulation of genes that codify cytoskeleton proteins, which seems to be potentially regulated by mechanosensing during fungal contact with solid supports.

Inactivation of fungal spores from clinical environment by silver bio-nanoparticles; optimization, artificial neural network model and mechanism

The present study aimed to assess the efficiency of silver bio-nanoparticles (Ag-NPs) in inactivating of the Aspergillus fumigatus, A. parasiticus and A. flavus var. columnaris and A. aculeatus spores. The AgNPs were synthesized in secondary metabolic products of Penicillium pedernalens 604 EAN. The inactivation process was optimized by response surface methodology (RSM) as a function of Ag NPs volume (1–10 μL/mL); time (10–120 min); pH (5–8); initial fungal concentrations (log10) (3–6). The artificial neural network (ANN) model was used to understand the behavior of spores for the factors affecting inactivation process. The best conditions to achieved SAL 10−6 of the fungal spores were recorded with 3.46 μl/mL of AgNPs, after 120 min at pH 5 and with 6 log of initial fungal spore concentrations, at which 5.99 vs. 6.09 (SAL 10−6) log reduction was recorded in actual and predicted results respectively with coefficient of 87.00%. The ANN revealed that the timehas major contribution in the inactivation process compare to Ag NPs volume. The fungal spores were totally inactivated (SAL 10−6, 6 log reduction with 99.9999%) after 110 min of the inactivation process, 10 min more was required to insure the irreversible inactivation of the fungal spores. The absence of protease and cellulase enzymes production confirm the total inactivation of the fungal spores. FESEM analysis revealed that the AgNPs which penetrated the fungal spores leading to damage and deform the fungal spore morphology. The AFM analysis confirmed the total spore surface damage. The bands in the range of the Raman spectroscopy from 1300 to 1600 cm−1 in the inactivated spores indicate the presence of CH3, CH2 and the deformation of lipids released outside the spore cytoplasm. These finding indicate that the AgNPs has high potential as a green alternative inactivation process for the airborne fungal spores.

In Vitro Evaluation of the Inhibitory Activity of Different Selenium Chemical Forms on the Growth of a Fusarium proliferatum Strain Isolated from Rice Seedlings.

In this study, the in vitro effects of different Se concentrations (5, 10, 15, 20, and 100 mg kg−1) from different Se forms (sodium selenite, sodium selenate, selenomethionine, and selenocystine) on the development of a Fusarium proliferatum strain isolated from rice were investigated. A concentration-dependent effect was detected. Se reduced fungal growth starting from 10 mg kg−1 and increasing the concentration (15, 20, and 100 mg kg−1) enhanced the inhibitory effect. Se bioactivity was also chemical form dependent. Selenocystine was found to be the most effective at the lowest concentration (5 mg kg−1). Complete growth inhibition was observed at 20 mg kg−1 of Se from selenite, selenomethionine, and selenocystine. Se speciation analysis revealed that fungus was able to change the Se speciation when the lowest Se concentration was applied. Scanning Electron Microscopy showed an alteration of the fungal morphology induced by Se. Considering that the inorganic forms have a higher solubility in water and are cheaper than organic forms, 20 mg kg−1 of Se from selenite can be suggested as the best combination suitable to inhibit F. proliferatum strain. The addition of low concentrations of Se from selenite to conventional fungicides may be a promising alternative approach for the control of Fusarium species.

Isolation and Characterization of Endomycorrhizal Fungi Associated with Growth Promotion of Blueberry Plants.

Despite their notable root mutualism with blueberries (Vaccinium spp.), studies related to Ericoid mycorrhizal (ERM) are relatively limited. In this study, we report the isolation of 14 endomycorrhizal fungi and their identification by fungal colony morphology characterization combined with PCR-amplified fungal internal transcribed spacer (ITS) sequence analyses. Six of the isolated strains were confirmed as beneficial mycorrhizal fungi for blueberry plants following inoculation. We observed the formation of typical ERM hyphae coil structures—which promote and nutritionally support growth—in blueberry seedlings and significant nitrogen and phosphorous content increases in diverse tissues. QRT-PCRs confirmed changes in VcPHT1s expression patterns. After the formation of ERM, PHT1-1 transcription in roots was upregulated by 1.4- to threefold, whilst expression of PHT1-3 and PHT1-4 in roots were downregulated 72% and 60%, respectively. Amino acid sequence analysis of all four VcPHT1s genes from the blueberry variety “Sharpblue” revealed an overall structural similarity of 67% and predicted transmembrane domains. Cloning and overexpression of PHT1-1 and PHT1-3 genes in transgenic Arabidopsis thaliana plants significantly enriched total phosphorus and chlorophyll content, confirming that PHT1-1 and PHT1-3 gene functions are associated with the transport and absorption of phosphorus.

Arbuscular Mycorrhizal Colonization Status in Selected Alien Invasive Plants of Asteraceae

Invasive alien plants tend to grow much faster than non-invasive plants. The colonization by AM fungi is confirmed by the presence of mycorrhizal structures in the roots of invasive plants. The pattern of hyphae and arbuscules denotes Arum and Paris type of AM fungal morphology. The most common and abundant invasive plants from Asteraceae family were selected for the study. Ageratum conyzoides, Mikania micrantha, Ageratum haustonianum and Bidens Pilosa were selected and eventually showed that they have a high percentage of mycorrhizal colonization. Arum type of morphology is found in three of the plant species and Paris type of morphology is found in one of the species. The extent of AM fungal colonization is fairly high ranging from 60% to 90% among the studied plants. The percentage of arbuscular colonization is highest in Bidens pilosa and the percentage of vesicle and hyphal colonization is highest in Ageratum conyzoides.

Disruption of protein rhamnosylation affects the Sporothrix schenckii-host interaction

Sporotrichosis is a fungal disease caused by the members of the Sporothrix pathogenic clade, and one of the etiological agents is Sporothrix schenckii. The cell wall of this organism has been previously analyzed and thus far is known to contain an inner layer composed of chitin and β -glucans, and an outer layer of glycoproteins, which are decorated with mannose and rhamnose-containing oligosaccharides. The L-rhamnose biosynthesis pathway is common in bacteria but rare in members of the Fungi kingdom. Therefore, in this study, we aimed to disrupt this metabolic route to assess the contribution of rhamnose during the S.schenckii-host interaction. We identified and silenced in S. schenckii a functional ortholog of the bacterial rmlD gene, which encodes for an essential reductase for the synthesis of nucleotide-activated L-rhamnose. RmlD silencing did not affect fungal growth or morphology but decreased cell wall rhamnose content. Compensatory, the β-1,3-glucan levels increased and were more exposed at the cell surface. Moreover, when incubated with human peripheral blood mononuclear cells, the RmlD silenced mutants differentially stimulated cytokine production when compared with the wild-type strain, reducing TNFα and IL-6 levels and increasing IL-1 β and IL-10 production. Upon incubation with human monocyte-derived macrophages, the silenced strains were more efficiently phagocytosed than the wild-type strain. In both cases, our data suggest that rhamnose-based oligosaccharides are ligands that interact with TLR4. Finally, our findings showed that cell wall rhamnose is required for the S. schenckii virulence in the G. mellonella model of infection.

Scedosporium and Lomentospora Infections Are Infrequent, Difficult to Diagnose by Histology, and Highly Virulent.

To further characterize the histomorphology and clinicopathologic features of colonization and invasive disease by Scedosporium and Lomentospora. We conducted a 20-year retrospective study. Patients with at least 1 histopathology specimen and concurrent culture were included. Clinical features, histopathology, microbiology, and outcomes were analyzed. Eighteen patients were identified, and all were immunocompromised. Eight patients had colonization, while 10 had invasive disease (pneumonia [n = 3], skin and soft-tissue infections [n = 3], disseminated disease [n = 4]). Scedosporium apiospermum was identified in 15 patients, Lomentospora prolificans in 2 patients, and Scedosporium ellipsoideum in 1 patient. Fungal elements were identified histologically in 11 patients. Granulomatous, suppurative, and necrotizing inflammation with irregular branching hyphae and characteristic microconidia were observed in 9 cases; conidiogenous cells were identified in 4 cases. Seven patients died of invasive disease despite therapy, and 3 recovered after treatment. No deaths were observed in patients with colonization. Scedosporium and Lomentospora are rare, virulent opportunistic fungal pathogens. Fungal morphology may overlap with other hyaline molds, but identification of obovoid conidia should allow a diagnosis of non-Aspergillus hyalohyphomycosis and consideration of Scedosporium and Lomentospora. Histopathologic correlation with culture and polymerase chain reaction is critical for diagnosis and treatment.

Bioprocess Optimisation for High Cell Density Endoinulinase Production from Recombinant Aspergillus niger.

Endoinulinase gene was expressed in recombinant Aspergillus niger for selective and high-level expression using an exponential fed-batch fermentation. The effects of the growth rate (μ), glucose feed concentration, nitrogen concentration and fungal morphology on enzyme production were evaluated. A recombinant endoinulinase with a molecular weight of 66 kDa was secreted. Endoinulinase production was growth associated at μ> 0.04 h-1, which is characteristic of the constitutive gpd promoter used for the enzyme production. The highest volumetric activity (670 U/ml) was achieved at a growth rate of 93% of μmax (0.07 h-1), while enzyme activity (506 U/ml) and biomass substrate yield (0.043 gbiomassDW/gglucose) significantly decreased at low μ (0.04 h-1). Increasing the feed concentration resulted in high biomass concentrations and viscosity, which necessitated high agitation to enhance the mixing efficiency and oxygen. However, the high agitation and low DO levels (ca. 8% of saturation) led to pellet disruption and growth in dispersed morphology. Enzyme production profiles, product (Yp/s) and biomass (Yx/s) yield coefficients were not affected by feed concentration and morphological change. The gradual increase in the concentration of nitrogen sources showed that, a nitrogen limited culture was not suitable for endoinulinase production in recombinant A. niger. Moreover, the increase in enzyme volumetric activity was still directly related to an increase in biomass concentration. An increase in nitrogen concentration, from 3.8 to 12 g/L, resulted in volumetric activity increase from 393 to 670 U/ml, but the Yp/s (10053 U/gglucose) and Yx/s (0.049 gbiomasDWs/gglucose) did not significantly change. The data demonstrated the potential of recombinant A. niger and high cell density fermentation for the development of large-scale endoinulinase production system.

Wastewater treatment and Mycoremediation by P. ostreatus mycelium

Wastewater from industries contains a considerable amount of chemicals and is characterized by high biological oxygen demand (BOD), heavy metals, intense color and are thus toxic to the environment. White-rot fungi such as Oyster mushrooms are comprised of high biomass and various degradative enzymes that add to another dimension of use, such as mycoremediation. The mushroom was maintained in vitro in mycelial form. Remazol Brilliant Blue R (RBBR), Methyl Red (M.R.), and Malachite Green (M.G.) dyes, which are recalcitrant dyes that are often used for the preparation of many polymeric dyes, and textile dyeing, were used to test the decolorization potential of natural isolates of oyster mushroom. Mycelium was grown in the presence of RBBR, MR, and M.G. at a concentration of 100μM. It was found to decolorize more than 70%, 95%, and 80% of RBBR, MR, and M.G. dye, respectively, within 6 days. Mushroom mycelium was also tested for its lead tolerance and heavy metal biosorption potential. Lead tolerance was studied up to 500mg/L, which shows species′ latent growth with fungal morphology changes. Biosorption ability was observed by the use of Inductively Coupled Plasma spectrometry, which shows lowered concentrations of heavy metals (Pb, Cr, Ni) after 5 days of mycelial growth in the presence of these metals. The isolated strain was also found to have significant decolorizing and degrading potential for synthetic dyes and phenolic compounds in industrial effluents. This study focuses on edible oyster mushrooms′ ability to develop environmentally less aggressive processes in the treatment of industrial effluents.

Tenebrio molitor as an Alternative Model to Analyze the Sporothrix Species Virulence.

BackgroundSporotrichosis is an increasing threat for humans, affecting mainly skin and subcutaneous tissues but that can cause disseminated infection in immunocompromised patients. Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main etiological agents of this mycosis, and each species show different virulence levels. The gold standard to assess fungal virulence is the mouse model that is expensive and time-consuming. Thus, invertebrate models have been reported as an alternative for the evaluation of fungal virulence. Here, we assessed whether Tenebrio molitor larvae could be a new alternative to study Sporothrix spp. virulence.MethodsT. molitor larvae were inoculated with different doses of S. schenckii, S. brasiliensis, and S. globosa, and animal mortality, cytotoxicity, and immunological parameters were analyzed, including the ability to stimulate immunological priming.ResultsMortality curves demonstrated that yeast-like cells were the best fungal morphology to kill larvae and showed a similar ranking in virulence than that reported in other animal models, ie, being S. brasiliensis and S. globosa the species with the highest and lowest virulence, respectively. The usefulness of this model was validated with the analysis of several S. schenckii strains with different virulence degrees, and changes in cytotoxicity, humoral and cellular immunological parameters. Low-virulence strains stimulated low levels of cytotoxicity, phenoloxidase activity, and hemocyte countings, and these immunological cells poorly uptake fungi. Moreover, using recombinant Gp70 from S. schenckii immunological priming was stimulated in larvae and this protected against a lethal dose of fungal cells from any of the three species under study.ConclusionThe study demonstrated that T. molitor larvae are an appropriate alternative invertebrate model to analyze the virulence of S. schenckii, S. brasiliensis, and S. globosa. Additionally, hemocyte levels, phenoloxidase activity, cytotoxicity, uptake by hemocytes, and immunological priming are biological parameters that can be used to study the Sporothrix–T. molitor interaction.

Pseudomonas Strains Induce Transcriptional and Morphological Changes and Reduce Root Colonization of Verticillium spp.

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection.

Genetic diversity and virulence spectrum of Rhizoctonia solani, the incitant of banded leaf and sheath blight of maize

AbstractBanded leaf and sheath blight (BLSB) of maize caused by Rhizoctonia solani is one of the most important yield‐limiting factors faced by growers in India. The pathogen is known to be highly diverse, and several species are known to cause BLSB. In the current study, forty‐four isolates of R. solani associated with maize were collected from different agro‐ecological zones of Punjab, a north Indian state. These were characterized genetically and phenotypically and for their virulence behaviour. Using internal transcribed spacer (ITS)‐based species‐specific markers, we confirmed the association of two species viz., solani (100%) and oryzae‐sativae (18.18%) with BLSB with later being detected in mixed infection. All the isolates belonged to anastomosis group AG1‐IA with a typical amplicon size of 265 bp. Genetic variation in the associated Rhizoctonia sp. was analysed using 27 inter simple sequence repeats (ISSR) markers which revealed high degree of polymorphism (PIC value ranging from 0.819 to 0.921) and clustered the Rhizoctonia population into two major groups. Based on fungal morphology, the hierarchical cluster analysis placed 44 Rhizoctonia isolates into six major groups (M1–M6). The virulence of each isolate studied on four hybrids and seven inbred maize lines revealed that maize hybrid JH3459 and inbred LMDR‐2 were less susceptible to Punjab population of Rhizoctonia species. Partial correlation was also obtained between genetic diversity, pathological variation and geographical origins of isolates. This is the first attempt to study the diversity of Rhizoctonia sp. associated with banded leaf and sheath blight of maize in Punjab state.

The Indigenous Volatile Inhibitor 2-Methyl-2-butene Impacts Biofilm Formation and Interspecies Interaction of the Pathogenic Mucorale Rhizopus arrhizus.

2-Methyl-2-butene has recently been reported to be a quorum-based volatile self-inhibitor of spore germination and growth in pathogenic Mucorale Rhizopus arrhizus. The present study aimed to elucidate if this compound can influence R. arrhizus biofilm formation and interspecies interaction. The compound was found to significantly decrease R. arrhizus biofilm formation (p < 0.001), with nearly 25% and 50% lesser biomass in the biofilms cultured with exposure to 4 and 32µg/ml of 2-methyl-2-butene, respectively. The growth of pre-formed biofilms was also impacted, albeit to a lesser extent. Additionally, 2-methyl-2-butene was found to self-limit R. arrhizus growth during interspecies interaction with Staphylococcus aureus and was detected at a substantially greater concentration in the headspace of co-cultures (2338.75µg/ml) compared with monocultures (69.52µg/ml). Some of the C5 derivatives of this compound (3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-1-butyne) were also observed to partially mimic its action, such as inhibition of spore germination, but did not impact R. arrhizus biofilm formation. Finally, the treated R. arrhizus displayed changes in fungal morphology suggestive of cytoskeletal alterations, such as filopodia formation, blebs, increased longitudinal folds and/or corrugations, and finger-like and sheet-like surface protrusions, depending upon the concentration of the compound(s) and the planktonic or biofilm growth mode.

Biological control activity of Trichoderma asperelloides PSU-P1 against gummy stem blight in muskmelon (Cucumis melo)

This study aimed to test the antifungal activity of Trichoderma species against gummy stem blight caused by Stagonosporopsis cucurbitacearum in muskmelon plants. Eight isolates of Trichoderma were screened for their inhibitory effect on S. cucurbitacearum, on potato dextrose agar (PDA) by dual culture assay and pre-colonized methods. The results show that Trichoderma sp. PSU-P1 was superior in inhibiting the fungal growth of S. cucurbitacearum. Molecular identification of internal transcribed spacers (ITS), RNA polymerase II (rpb2), and translation elongation factor 1-α (tef1-α) gene sequences revealed the isolate PSU-P1 as Trichoderma asperelloides. To clarify the defense response, enzyme assays of the defense-related enzymes peroxidase (POD) and polyphenol oxidase (PPO) and the cell wall degrading enzymes chitinase and β-1,3-glucanase were conducted for muskmelon seedlings inoculated with T. asperelloides PSU-P1. The results show that the activities of POD, PPO, chitinase and β-1,3-glucanase were higher than in the control. Crude metabolites extracted from T. asperelloides PSU-P1 inoculated muskmelon plants caused abnormal fungal morphology. The inoculation test (in vivo) revealed that muskmelon seedlings treated with T. asperelloides PSU-P1 showed resistance to gummy stem blight with a disease score of 0.2 and disease severity index (DSI) of 10%, lower than those of the control, which had a disease score of 2.8 and 75% DSI. The results obtained in this study reveal that T. asperelloides PSU-P1 exhibited antifungal activity by mycoparasitism and enhanced defense response in muskmelon through the production of defense-related enzymes and cell wall degrading enzymes. High activities of the enzymes are associated with systemic resistance in muskmelon against gummy stem blight.

The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei

BackgroundOn-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates.ResultsEnzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation.ConclusionCultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

Rapid Detection of Pine Pathogens Lecanosticta acicola, Dothistroma pini and D. septosporum on Needles by Probe-Based LAMP Assays

Needle blights are serious needle fungal diseases affecting pines both in natural and productive forests. Among needle blight agents, the ascomycetes Lecanosticta acicola, Dothistroma pini and D. septosporum are of particular concern. These pathogens need specific, fast and accurate diagnostics since they are regulated species in many countries and may require differential management measures. Due to the similarities in fungal morphology and the symptoms they elicit, these species are hard to distinguish using morphological characteristics. The symptoms can also be confused with those caused by insects or abiotic agents. DNA-based detection is therefore recommended. However, the specific PCR assays that have been produced to date for the differential diagnosis of these pathogens can be applied only in a well-furnished laboratory and the procedure takes a relatively long execution time. Surveillance and forest protection would benefit from a faster diagnostic method, such as a loop-mediated isothermal amplification (LAMP) assay, which requires less sophisticated equipment and can also be deployed directly on-site using portable devices. LAMP assays for the rapid and early detection of L. acicola, D. pini and D. septosporum were developed in this work. Species-specific LAMP primers and fluorescent assimilating probes were designed for each assay, targeting the beta tubulin (β-tub2) gene for the two Dothistroma species and the elongation factor (EF-1α) region for L. acicola. Each reaction detected its respective pathogen rapidly and with high specificity and sensitivity in DNA extracts from both pure fungal cultures and directly from infected pine needles. These qualities and the compatibility with inexpensive portable instrumentation position these LAMP assays as an effective method for routine phytosanitary control of plant material in real time, and they could profitably assist the management of L. acicola, D. pini and D. septosporum.

Sequence analysis of 18SrDNA gene from sagoplast degrading fungi

The bioplastic can be made from sago flour and known as sagoplast. It was widely known that for making bioplastic, the addition of acetic acid and glycerol are needed. Products that are air-dried are easy to grow fungi within a few weeks. This makes the basis for researchers to undestand more about the character and identity of the sagoplast degrading fungi. Characterization and identification were carried out by observed morphology and analyzing the 18SrDNA gene sequence of fungal isolates that had grown on the sagoplast. Fungal isolates morphology showed yellowish-orange color with white thread-like mycelia and a blackish brown mace with white thread-shaped mycelia. These characters of fungal morphology that similar with Aspergillus. The gene sequences of the fungal isolates were aligned with reference gene sequences of the fungi obtained from the Gen Bank of the National Center for Biotechnology Information (NCBI). Sequence data analysis was performed by using the Clustal X program to determine the kinship and taxonomy of the fungal isolates that able to degrade sagoplast. The result showed that two fungal isolates, DFSP.J1 and DFSP.J4, were found and demonstrated their ability for degrading sagoplast. Isolate DFSP.J1 is related to Aspergillus flavus strain PSU2 LC127086.1, while isolate DFSP.J4 is related to Aspergillus niger IFO4033 D63697.1.

Sequence analysis of 18SrDNA gene from sagoplast degrading fungi

The bioplastic can be made from sago flour and known as sagoplast. It was widely known that for making bioplastic, the addition of acetic acid and glycerol are needed. Products that are air-dried are easy to grow fungi within a few weeks. This makes the basis for researchers to undestand more about the character and identity of the sagoplast degrading fungi. Characterization and identification were carried out by observed morphology and analyzing the 18SrDNA gene sequence of fungal isolates that had grown on the sagoplast. Fungal isolates morphology showed yellowish-orange color with white thread-like mycelia and a blackish brown mace with white thread-shaped mycelia. These characters of fungal morphology that similar with Aspergillus. The gene sequences of the fungal isolates were aligned with reference gene sequences of the fungi obtained from the Gen Bank of the National Center for Biotechnology Information (NCBI). Sequence data analysis was performed by using the Clustal X program to determine the kinship and taxonomy of the fungal isolates that able to degrade sagoplast. The result showed that two fungal isolates, DFSP.J1 and DFSP.J4, were found and demonstrated their ability for degrading sagoplast. Isolate DFSP.J1 is related to Aspergillus flavus strain PSU2 LC127086.1, while isolate DFSP.J4 is related to Aspergillus niger IFO4033 D63697.1.

Phytochemical characterization and inhibition of Candida sp. by the essential oil of Baccharis trimera (Less.) DC.

This study aimed to investigate the chemical composition and antifungal potential of the essential oil of Baccharis trimera (Less.) DC. against Candida strains. The half maximal inhibitory concentration (IC50) was assessed by the microdilution method using the essential oil at a concentration range of 8192 to 8μg/mL. The minimum fungicide concentration (MFC) was determined by subculture in solid medium. The ability of the essential oil to modulate the activity of antifungals was determined in wells treated simultaneously with the oil at a subinhibitory concentration (MFC/16) and fluconazole (FCZ). The fungal morphology was analyzed by microscopy. Gas chromatography coupled with mass spectrometry (GC/MS) was used to identify the chemical composition. The essential oil presented an CI50 of 11.24 and 1.45μg/mL, which was found to potentiate the effect of FCZ against Candida albicans. On the other hand, this combined treatment resulted in antagonism against Candida tropicalis and no evident modulation against Candida krusei was observed. The essential oil significantly inhibited hyphae growth. However, with a MFC ≥ 16,384μg/mL, it is assumed that it has a fungistatic action. The antifungal properties demonstrated in this study might be related to the presence of sesquiterpenes and monoterpenes, and the interaction between them. In conclusion, Baccharis trimera showed promising anti-Candida effects, in addition to potentiating the activity of FCZ against Candida albicans, affecting its morphological transition. Therefore, this species constitutes a source of chemical compounds with the potential to be used in the combat of fungal infections.

Promotion of monacolin K production in Monascus extractive fermentation: the variation in fungal morphology and in the expression levels of biosynthetic gene clusters.

Monacolin K, an important secondary metabolite of Monascus, possesses a cholesterol-lowering effect and is widely used in the manufacture of antihypertensive drugs. In the present study, we constructed an extractive fermentation system by adding non-ionic surfactant and acquired a high monacolin K yield. The mechanism was determined by examining both cell morphology and the transcription levels of the related mokA-I genes in the monacolin K biosynthetic gene cluster. The monacolin K yield was effectively increased to 539.59 mg L-1 during extraction, which was an increase of 386.16% compared to that in the control group fermentation. The non-ionic surfactant showed good biocompatibility with Monascus. Electron scanning microscopy revealed alterations in the morphology of Monascus. The loosened mycelial structure and increased number of cell surface wrinkles were found to be related to the increased cell-membrane permeability and extracellular accumulation of monacolin K. Gene expression levels were measured via a quantitative reverse transciptase-polymerase chain reaction. By contrast, in the control group, mokA, mokB, mokC, mokD and mokF showed higher-level and longer-term expression in the extractive fermentation group, whereas mokE and mokG did not present a similar trend. The expression levels of mokH and mokI, encoding a transcription factor and efflux pump, respectively, were also higher than the control levels. The addition of a non-ionic surfactant to Monascus fermentation effectively increases the yield of monacolin K by transforming the fungus morphology and promoting the expression of monacolin K biosynthesis genes. © 2021 Society of Chemical Industry.