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Volume 8
Issue 12
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J. Fungi, Volume 8, Issue 12 (December 2022) – 82 articles

Cover Story (view full-size image): The ubiquitin–proteasome system (UPS) mediated rapid protein turnover influences various cellular functions in eukaryotic cells, including fungi. As a subunit of the SCF (Skp1-Cullin-F-box protein) E3 ligase, F-box protein is a key component of UPS that determines the substrate specificity. In this study, we identified and characterized all 20 F-box proteins in Cryptococcus neoformans, the leading cause of fungal meningoencephalitis. We highlighted the important cellular functions of three F-box proteins in detail, including their roles in fungal mating, stress response, antifungal resistance, and cell size regulation, as well as fungal virulence. Our study leads to a better understanding of the function of fungal SCF E3 ligase-mediated UPS in fungal development and pathogenesis. View this paper
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24 pages, 11429 KiB  
Article
Lomasomes and Other Fungal Plasma Membrane Macroinvaginations Have a Tubular and Lamellar Genesis
by Igor S. Mazheika, Nadezhda V. Psurtseva and Olga V. Kamzolkina
J. Fungi 2022, 8(12), 1316; https://doi.org/10.3390/jof8121316 - 19 Dec 2022
Viewed by 1807
Abstract
The plasma membrane of filamentous fungi forms large-sized invaginations, which are either tubes or parietal vesicles. Vesicular macroinvaginations at the ultrastructural level correspond to classical lomasomes. There is an assumption that vesicular macroinvaginations/lomasomes may be involved in macrovesicular endocytosis. The original aim of [...] Read more.
The plasma membrane of filamentous fungi forms large-sized invaginations, which are either tubes or parietal vesicles. Vesicular macroinvaginations at the ultrastructural level correspond to classical lomasomes. There is an assumption that vesicular macroinvaginations/lomasomes may be involved in macrovesicular endocytosis. The original aim of this study was to test for the presence of macroendocytosis in xylotrophic basidiomycetes using time-lapse and Z-stacks fluorescent microscopic technologies. However, the results were unexpected since most of the membrane structures labeled by the endocytic tracer (FM4-64 analog) are various types of plasma membrane macroinvaginations and not any endomembranes. All of these macroinvaginations have a tubular or lamellar genesis. Moreover, under specific conditions of a microscopic preparation, the diameter of the tubes forming the macroinvaginations increases with the time of the sample observation. In addition, the morphology and successive formation of the macroinvaginations mimic the endocytic pathway; these invaginations can easily be mistaken for endocytic vesicles, endosomes, and vacuole-lysosomes. The paper analyzes the various macroinvagination types, suggests their biological functions, and discusses some features of fungal endocytosis. This study is a next step toward understanding complex fungal physiology and is a presentation of a new intracellular tubular system in wood-decaying fungi. Full article
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<p>Three types of hyphae in xylotrophic basidiomycetes (in the example of <span class="html-italic">S. hirsutum</span>) differ in uptake of the AM4-64 fluorophore. (<b>A</b>,<b>B</b>)—hyphae of the first type, with an absent or very weak AM4-64 signal in the cytoplasm and endomembranes. PM and its macroinvaginations are labeled. The bright-field photograph (<b>A</b>) clearly shows large vacuoles; on the fluorescent image of the same area of the mycelium (<b>B</b>), there are no vacuoles, but vesicular and tubular invaginations of the PM are visualized. (<b>C</b>–<b>E</b>)—hyphae of the second type with a diffuse signal AM4-64 in the cytoplasm. (<b>C</b>)—relatively weak fluorescence of the cytoplasm; vacuoles are seen either as dark areas (arrowhead) or have a weakly labeled tonoplast (arrows). (<b>D</b>,<b>E</b>)—hypha with a more powerful diffuse signal in the cytoplasm (<b>D</b>) and the same sample region in the bright field (<b>E</b>). (<b>F</b>,<b>G</b>)—the third type—motley hyphae. (<b>F</b>)—arrow points to the motley hypha. (<b>G</b>)—with the help of labeled areas of tonoplasts and the edges of plasmolytic protoplasts, the fungus wrote “ROXY XY”. Scale bars are 5 µm.</p> Full article ">Figure 2
<p>Glomeruli, pendants, and plaques in hyphae of <span class="html-italic">O. olearius</span> (<b>A</b>) and <span class="html-italic">S. hirsutum</span> (<b>B</b>–<b>G</b>). (<b>A</b>)—arrows point to individual glomeruli, and the arrowhead points to plaque. (<b>B</b>)—a large glomerulus is represented (arrow). (<b>C</b>–<b>E</b>)—separate frames from <a href="#app1-jof-08-01316" class="html-app">Video S7</a>, moving a large pendant. (<b>F</b>,<b>G</b>)—fluorescent and bright-field images of one fragment of the hypha; plaques are marked with arrowheads. Scale bars are 5 µm.</p> Full article ">Figure 3
<p>Diagrams showing the quantitative representation of different types of PM macroinvaginations in <span class="html-italic">S. hirsutum</span>. The diagram of each treatment variant or type of invagination consists of three subdiagrams—they correspond to the observation time intervals (5–15 min, 15–30 min, and 30–45 min). Each point on the diagram corresponds to the number of macroinvaginations observed in one biological experiment, calculated per 1000 septa. (<b>A</b>)—all types of macroinvaginations in the basic variant of treatment (without preincubation, CzM, pH 7). The Y-axis scale is non-linear. (<b>B</b>)—diagrams showing the number of glomeruli and pendants formed with different variants of sample treatment. Legend: small square—mean; box—50% standard deviation, the whisker–min/max.</p> Full article ">Figure 4
<p>Diagrams showing the quantitative representation of plaques, glomeruli, and pendants in <span class="html-italic">S. hirsutum</span> and <span class="html-italic">O. olearius</span>. (<b>A</b>,<b>B</b>)—the diagrams reflect the influence of different variants of <span class="html-italic">O. olearius</span> mycelium preincubation on the formation of glomeruli and pendants (<b>A</b>) and plaques (<b>B</b>). (<b>C</b>)—formation of plaques in <span class="html-italic">S. hirsutum</span> with different types of sample treatment. Legend: small square—mean; box—50% standard deviation, the whisker–min/max.</p> Full article ">Figure 5
<p>Small vesicles and thin tubes in the hyphae of <span class="html-italic">O. olearius</span>. Designations: white arrows—small vesicles, light blue arrows—standing thin tubes, blue arrows—longitudinal tubes pressed to the PM, green arrows—longitudinal straight tubes, yellow arrows—longitudinal curving tubes, red arrow—thick tube (&gt;1 μm in diameter), arrowhead—tube of intermediate thickness (700–800 nm). (<b>A</b>,<b>B</b>)—the single focal planes. (<b>C</b>,<b>D</b>)—Z-stacks summation. Scale bars are 5 µm.</p> Full article ">Figure 6
<p>Diagrams showing the quantitative representation of small vesicles and thin tubes in <span class="html-italic">S. hirsutum</span> and <span class="html-italic">O. olearius</span>. (<b>A</b>)—influence of different variants of microscopic sample preparing on the formation of small vesicles and thin tubes in <span class="html-italic">S. hirsutum</span>. (<b>B</b>)—influence of the preincubation time on the formation of small vesicles and thin tubes in <span class="html-italic">O. olearius</span>. Legend: small square—mean; box—50% standard deviation, the whisker–min/max.</p> Full article ">Figure 7
<p>Large vesicles and thick tubes: fluorescent images and diagrams. Designations: white arrows—large vesicles, arrowheads—thick and intermediate tubes. (<b>A</b>,<b>B</b>)—hyphae fragments of <span class="html-italic">S. hirsutum</span> in single focal plane. (<b>C</b>)—Z-stacks summation of <span class="html-italic">O. olearius</span>. Scale bars are 5 µm. (<b>D</b>)—influence of different variants of microscopic sample preparing on the formation of large vesicles and thick tubes in <span class="html-italic">S. hirsutum.</span> Legend: small square—mean; box—50% standard deviation, the whisker–min/max.</p> Full article ">Figure 8
<p>Vacuolar system and colocalization of PM macroinvaginations and vacuoles in <span class="html-italic">S. hirsutum</span>. (<b>A</b>)—AM4-64 labeling, arrowhead—large vesicle (or thick tube), arrow—VLC (vacuole-lysosome candidate). VLC differs from PM macroinvaginations by its often large size and weaker tonoplast signal. (<b>B</b>)—CFDA labeling, large oval vacuoles connected by tubular vacuoles. (<b>C</b>,<b>D</b>)—bright field and CFDA fluorescence images of small round vacuoles (arrowhead) and tubular vacuoles (arrow). (<b>E</b>,<b>F</b>)—labeling with AM4-64 (<b>E</b>); and co-labeling with AM4-64 and CFDA (<b>F</b>). Arrows indicate possible VLCs. It can be seen that only one of the two VLCs carries both labels. (<b>G</b>,<b>H</b>)—the only one of 119 large vesicles analyzed that carries both AM4-64 and CFDA labels (arrow). (<b>I</b>–<b>N</b>)—demonstration of the most common variant, when the vacuolar label and AM4-64 labeled large vesicles and thick tubes do not match. (<b>J</b>,<b>K</b>)—the arrow indicates a large central vacuole, and the arrowhead indicates a large vesicle. (<b>L</b>–<b>N</b>)—the arrow is directed to a large vesicle, and the arrowhead—to a thick tube. Scale bars are 5 µm.</p> Full article ">Figure 9
<p>Transmission electron microscopic images of various types of lomasomes in xylotrophic basidiomycetes corresponding to various AM4-64 labeled PM macroinvaginations. (<b>A</b>)—classic lamellar (bottom) and vesicular (tubular, top) lomasomes corresponding to glomeruli (<span class="html-italic">P. luminescens</span>). (<b>B</b>)—lomasome (glomerulus) consisting of intertwined thin tubules (<span class="html-italic">L. edodes</span>). (<b>C</b>)—a complex lomasome consisting of ordered tubules and lamellae corresponds to a large glomerulus (<span class="html-italic">P. stipticus</span>). (<b>D</b>)—a complex vesicular (tubular) lomasome also corresponds to a large glomerulus (<span class="html-italic">P. luminescens</span>). (<b>E</b>)—a lamellar lomasome on a short stem corresponds to the pendant (<span class="html-italic">L. edodes</span>). (<b>F</b>)—a lamellar-tubular lomasome (<span class="html-italic">S. hirsutum</span>); it can be seen how the lamellae damaged during cutting turned their flat side into the section plane (arrows). (<b>G</b>)—a section through a dolipore septum (<span class="html-italic">L. edodes</span>); the arrow indicates the lamellar lomasome beginning to form; the arrowhead indicates the small vesicle. (<b>H</b>)—the thin longitudinal tube pressed against the PM at the ultrastructural level (<span class="html-italic">L. edodes</span>). (<b>I</b>)—a vesicular (tubular) lomasome corresponding to the small plaque (<span class="html-italic">P. luminescens</span>). Scale bars are 200 nm.</p> Full article ">Figure 10
<p>Three assumed mechanisms for the formation of glomeruli and pendants (same as lomasomes) in basidial xylotrophs. (<b>A</b>–<b>C</b>)—invagination of the PM in the form of a long filamentous tube, then the tube folds into a glomerulus (or a pendant if there is a free tubule-leg between the PM and the body of glomerulus). (<b>D</b>–<b>F</b>)—a thin invagination tube swells into a vesicle and then secondary tubular invaginations are formed from the vesicle membrane, filling the lumen of the maternal vesicle. (<b>F</b>)—the front of the maternal membrane is not drawn. (<b>G</b>,<b>H</b>)—a lamella, usually located across the cell, invaginates and then begins to roll up. The roller can be of different coil densities. Later, the lamella may also begin to swell and form secondary invaginations (not shown in the Figure).</p> Full article ">
23 pages, 5892 KiB  
Article
The Sugar Metabolic Model of Aspergillus niger Can Only Be Reliably Transferred to Fungi of Its Phylum
by Jiajia Li, Tania Chroumpi, Sandra Garrigues, Roland S. Kun, Jiali Meng, Sonia Salazar-Cerezo, Maria Victoria Aguilar-Pontes, Yu Zhang, Sravanthi Tejomurthula, Anna Lipzen, Vivian Ng, Chaevien S. Clendinen, Nikola Tolić, Igor V. Grigoriev, Adrian Tsang, Miia R. Mäkelä, Berend Snel, Mao Peng and Ronald P. de Vries
J. Fungi 2022, 8(12), 1315; https://doi.org/10.3390/jof8121315 - 17 Dec 2022
Cited by 13 | Viewed by 4923
Abstract
Fungi play a critical role in the global carbon cycle by degrading plant polysaccharides to small sugars and metabolizing them as carbon and energy sources. We mapped the well-established sugar metabolic network of Aspergillus niger to five taxonomically distant species (Aspergillus nidulans [...] Read more.
Fungi play a critical role in the global carbon cycle by degrading plant polysaccharides to small sugars and metabolizing them as carbon and energy sources. We mapped the well-established sugar metabolic network of Aspergillus niger to five taxonomically distant species (Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium and Dichomitus squalens) using an orthology-based approach. The diversity of sugar metabolism correlates well with the taxonomic distance of the fungi. The pathways are highly conserved between the three studied Eurotiomycetes (A. niger, A. nidulans, P. subrubescens). A higher level of diversity was observed between the T. reesei and A. niger, and even more so for the two Basidiomycetes. These results were confirmed by integrative analysis of transcriptome, proteome and metabolome, as well as growth profiles of the fungi growing on the corresponding sugars. In conclusion, the establishment of sugar pathway models in different fungi revealed the diversity of fungal sugar conversion and provided a valuable resource for the community, which would facilitate rational metabolic engineering of these fungi as microbial cell factories. Full article
(This article belongs to the Special Issue (Functional) Genomics and Bioinformatics in Fungal Plant Biomass Conversion)
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<p>Conservation of sugar metabolic pathways among <span class="html-italic">A. niger</span>, <span class="html-italic">A. nidulans</span>, <span class="html-italic">T. reesei</span>, <span class="html-italic">P. subrubescens</span>, <span class="html-italic">P. chrysosporium</span> and <span class="html-italic">D. squalens</span>. The size of the dots indicates the number of genes involved in each pathway, and the color indicates the completeness of the pathway. The completeness score of a pathway is defined as the percentage of predicted reactions in a studied fungus compared to the total reactions reported in <span class="html-italic">A. niger</span> for each specific sugar metabolic pathway.</p> Full article ">Figure 2
<p>Sugar metabolic networks of <span class="html-italic">A. niger</span>, <span class="html-italic">A. nidulans</span>, <span class="html-italic">T. reesei</span>, <span class="html-italic">P. subrubescens</span>, <span class="html-italic">P. chrysosporium</span> and <span class="html-italic">D. squalens</span>. The names of the sugar metabolic pathways are shown in blue circles. The solid or dashed lines of the circles indicate whether the corresponding pathway is complete or not, respectively. For enzymes involved in each reaction of each pathway, the corresponding enzyme EC number is shown, and for the reaction with the absence of genes in one or more of our studied species, the EC number is highlighted in red. A color bar was also used to highlight the presence and absence of genes in specific reactions in the studied species. The question marks indicate that the corresponding reaction remains to be discovered.</p> Full article ">Figure 3
<p>Expression profiles of sugar metabolism-related genes in <span class="html-italic">A. nidulans</span> and <span class="html-italic">P. chrysosporium</span> during their growth on diverse monosaccharides. The blue color from light to dark indicates a gene expression level from low to high. Selected genes with specific sugar-induced expression patterns are marked with a red box on the heatmap, and their gene IDs and the associated pathways are displayed. On the right bar, different colors indicate different pathways.</p> Full article ">Figure 4
<p>Protein abundance profiles of the sugar metabolism-related proteins involved in each sugar metabolic pathway in <span class="html-italic">A. nidulans</span> (<b>A</b>) and <span class="html-italic">P. chrysosporium</span> (<b>B</b>). On the boxplot, different colors indicate different monosaccharide growth conditions, and each small circle indicates an individual protein related to each specific sugar metabolic pathway. The metabolic enzymes that were detected in less than one-third of the tested growth conditions were not included in the boxplots. The y-axis represents the abundance of proteins (log2 scaled), and the x-axis depicts different sugar metabolic pathways.</p> Full article ">Figure 5
<p>Correlation between the abundance of metabolites and sugar metabolism-related genes in <span class="html-italic">A. nidulans</span> (<b>A</b>) and <span class="html-italic">P. chrysosporium</span> (<b>B</b>). The y-axis represents Pearson correlation coefficient (PCC ≥ 0.5), and x-axis depicts different metabolites and the related sugar metabolic pathways (highlighted in blue). Each dot represents a gene, and its color indicates the corresponding sugar pathway in which it is involved. The positive and negative correlations were shown in filled and open circles, respectively. Only the names of genes with high correlation (PCC ≥ 0.8) to the analyzed metabolites are displayed.</p> Full article ">Figure 6
<p>Fungal growth profiling on different monosaccharides. All strains were grown on minimal medium (MM) with nine different carbon sources. Growth was performed at 30 °C for the two <span class="html-italic">Aspergilli</span> and 25 °C for other species. If growth on a specific carbon source is the same as with no carbon source, it is considered no growth.</p> Full article ">
16 pages, 5435 KiB  
Article
The SsAtg1 Activating Autophagy Is Required for Sclerotia Formation and Pathogenicity in Sclerotinia sclerotiorum
by Wenli Jiao, Huilin Yu, Xueting Chen, Kunqin Xiao, Dongmei Jia, Fengting Wang, Yanhua Zhang and Hongyu Pan
J. Fungi 2022, 8(12), 1314; https://doi.org/10.3390/jof8121314 - 17 Dec 2022
Cited by 6 | Viewed by 1815
Abstract
Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that produces sclerotia. Sclerotia are essential components of the survival and disease cycle of this devastating pathogen. In this study, we analyzed comparative transcriptomics of hyphae and sclerotia. A total of 1959 differentially expressed genes, 919 [...] Read more.
Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that produces sclerotia. Sclerotia are essential components of the survival and disease cycle of this devastating pathogen. In this study, we analyzed comparative transcriptomics of hyphae and sclerotia. A total of 1959 differentially expressed genes, 919 down-regulated and 1040 up-regulated, were identified. Transcriptomes data provide the possibility to precisely comprehend the sclerotia development. We further analyzed the differentially expressed genes (DEGs) in sclerotia to explore the molecular mechanism of sclerotia development, which include ribosome biogenesis and translation, melanin biosynthesis, autophagy and reactivate oxygen metabolism. Among these, the autophagy-related gene SsAtg1 was up-regulated in sclerotia. Atg1 homologs play critical roles in autophagy, a ubiquitous and evolutionarily highly conserved cellular mechanism for turnover of intracellular materials in eukaryotes. Therefore, we investigated the function of SsAtg1 to explore the function of the autophagy pathway in S. sclerotiorum. Deficiency of SsAtg1 inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. Notably, ΔSsAtg1 was unable to form sclerotia and displayed defects in vegetative growth under conditions of nutrient restriction. Furthermore, the development and penetration of the compound appressoria in ΔSsAtg1 was abnormal. Pathogenicity analysis showed that SsAtg1 was required for full virulence of S. sclerotiorum. Taken together, these results indicate that SsAtg1 is a core autophagy-related gene that has vital functions in nutrient utilization, sclerotia development and pathogenicity in S. sclerotiorum. Full article
(This article belongs to the Section Fungal Pathogenesis and Disease Control)
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<p>DEGs between hypha and sclerotia in <span class="html-italic">S. sclerotiorum</span>. (<b>a</b>) GO annotation enrichment in sclerotia relative to hyphae. (<b>b</b>) Relative expression of genes in sclerotia at different stages verified by RT-qPCR. Samples of wild-type hyphae (S0, 2d), initiation of sclerotia (S1, 3d), developing sclerotia (S2, 5d) and mature sclerotia (S3, 9d) were collected. Genes <span class="html-italic">Sscle_09g070740</span>, <span class="html-italic">Sscle_03g031470</span>, <span class="html-italic">Sscle_06g049830</span> and <span class="html-italic">Sscle_12g087380</span> were selected to assess transcript accumulation dynamics.</p> Full article ">Figure 2
<p><span class="html-italic">SsAtg1</span> is a key gene activating autophagy in <span class="html-italic">S. sclerotiorum</span>. (<b>a</b>) Dendrogram of SsAtg1. Mega 7.0 was used to construct the phylogenetic tree. (<b>b</b>) Sequence information of SsAtg1. The conservation of the STKc_ATG1_ULK_like (cd14009) and Ser/Thr_kinase_C (IPR022708) domains was analyzed by InterPro (<a href="http://www.ebi.ac.uk/interpro" target="_blank">http://www.ebi.ac.uk/interpro</a>, accessed on 18 February 2022) and GPS 2.0 was used for visualization. (<b>c</b>) Alignments of the ATP binding site and active site. Clustal W was used for alignment sequences and conserved amino acids residues are labeled as *. (<b>d</b>) Autophagy analysis of ∆<span class="html-italic">SsAtg1</span>. MDC is a fluorescent pigment used to detect autophagosome formation. Scale bar = 10 μm.</p> Full article ">Figure 3
<p><span class="html-italic">SsAtg1</span> is required for sclerotium formation. (<b>a</b>,<b>b</b>) Colony morphology and colony diameter of ∆<span class="html-italic">SsAtg1</span> and control strains. Strains were inoculated on PDA, CM, MM, MM-N, MM-C and MM-P and colony diameters were measured by the cross method at 48 h. Error bars represent the standard deviations and the letters indicated significant differences (<span class="html-italic">p</span> &lt; 0.05). (<b>c</b>,<b>d</b>) <span class="html-italic">SsAtg1</span> mutation affected sclerotia development. Colony morphology and sclerotia number were recorded after four weeks. Error bars represent mean standard deviation, and statistical significance of data was analyzed using SPSS software (<span class="html-italic">p</span> &lt; 0.05). (<b>e</b>) The expression of <span class="html-italic">SsAtg1</span> was significantly increased in nutrient deficient medium. (<b>f</b>–<b>h</b>) Deficiency of <span class="html-italic">SsAtg1</span> affects the expression of key genes regulating sclerotia development.</p> Full article ">Figure 4
<p>Role of <span class="html-italic">SsAtg1</span> in response to nutritional stress. (<b>a</b>) Colony morphology of wild type and ∆<span class="html-italic">SsAtg1</span> and the genetically complemented strain on different growth media. Strains were inoculated on MM replaced with indicated C sources and N sources. Scale bar = 0.5 cm. (<b>b</b>) Sclerotia number of ∆<span class="html-italic">SsAtg1</span> on different growth media. Colony morphology and sclerotia number were recorded after 4 weeks. The letters indicated significant differences (<span class="html-italic">p</span> &lt; 0.05), error bars represent standard deviation from the mean.</p> Full article ">Figure 5
<p>∆<span class="html-italic">SsAtg1</span> is sensitive to cell wall synthesis inhibitors. (<b>a</b>,<b>b</b>) Colony diameter and morphology of wild type, ∆<span class="html-italic">SsAtg1</span> and the genetically complemented strain. Strains were inoculated on PDA supplemented with CFW, CR and SDS and colony diameters were measured by the cross method at 48 h. (<b>c</b>) Quantification of sclerotia development in response to cell wall synthesis inhibitors. Colony morphology and sclerotia number were recorded after 4 weeks. The letters indicated significant differences (<span class="html-italic">p</span> &lt; 0.05), error bars represent standard deviation from the mean.</p> Full article ">Figure 6
<p><span class="html-italic">SsAtg1</span> affects the virulence of <span class="html-italic">S. sclerotiorum</span>. (<b>a</b>) Pathogenicity of ∆<span class="html-italic">SsAtg1</span> on soybean leaves. (<b>b</b>) Quantification of the infection area. Image J was used to measure the infection area. (<b>c</b>) Pathogenicity of ∆<span class="html-italic">SsAtg1</span> on tobacco. Error bars represent the standard deviations and the asterisk indicated significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 7
<p><span class="html-italic">SsAtg1</span> affected compound appressoria formation and penetration. (<b>a</b>,<b>b</b>) Compound appressoria of strains on hydrophobic interface. Image J was used for analysis of compound appressoria as: adjusted the image to 8 bit format, then selected the part of the compound appressoria by adjust-threshold, then using the area of the agar disk as the control to calculate the area of the compound appressoria. Error bars represent the standard deviations and the asterisk indicated significant differences (<span class="html-italic">p</span> &lt; 0.05). (<b>c</b>) Compound appressoria formation on onion epidermal cells. The red arrow shows the invasion hypha which could not be stained by lactophenol cotton blue solution. Samples were staining by lactophenol cotton blue solution after 30 s, then washed with ddH<sub>2</sub>O and observed under a microscope. Scale bar = 50 μm.</p> Full article ">Figure 8
<p>∆<span class="html-italic">SsAtg1</span> hypersensitivity to ROS. (<b>a</b>) Mycelia growth and morphology of the ∆<span class="html-italic">SsAtg1</span> under different concentrations of H<sub>2</sub>O<sub>2</sub>. (<b>b</b>) Inhibition rate analysis. Inhibition rate = (colony diameter of strain without H<sub>2</sub>O<sub>2</sub> − colony diameter of strain with H<sub>2</sub>O<sub>2</sub>)/ colony diameter of strain without H<sub>2</sub>O<sub>2</sub> × 100%. Error bars represent the standard deviations and the letters indicated significant differences (<span class="html-italic">p</span> &lt; 0.05). (<b>c</b>) Statistics of sclerotia number. Significant difference in data by SPSS software (<span class="html-italic">p</span> &lt; 0.05), error bars represent standard deviation. (<b>d</b>) Expression of reactive oxygen metabolism genes.</p> Full article ">
14 pages, 4035 KiB  
Article
Phenylalanine Promotes Biofilm Formation of Meyerozyma caribbica to Improve Biocontrol Efficacy against Jujube Black Spot Rot
by Qian Deng, Xingmeng Lei, Hongyan Zhang, Lili Deng, Lanhua Yi and Kaifang Zeng
J. Fungi 2022, 8(12), 1313; https://doi.org/10.3390/jof8121313 - 17 Dec 2022
Cited by 17 | Viewed by 1690
Abstract
During storage and transportation after harvest, the jujube fruit is susceptible to black spot rot, which is caused by Alternaria alternata. The present study aimed to evaluate the effectiveness of the yeast Meyerozyma caribbica in controlling A. alternata in postharvest jujube fruits, [...] Read more.
During storage and transportation after harvest, the jujube fruit is susceptible to black spot rot, which is caused by Alternaria alternata. The present study aimed to evaluate the effectiveness of the yeast Meyerozyma caribbica in controlling A. alternata in postharvest jujube fruits, and to explore the biofilm formation mechanism. The results showed that M. caribbica treatment significantly reduced the A. alternata decay in jujube fruits. M. caribbica could rapidly colonize jujube fruit wounds, adhering tightly to hyphae of A. alternata, and accompanied by the production of extracellular secretions. In in vitro experiments, we identified that M. caribbica adhered to polystyrene plates, indicating a strong biofilm-forming ability. Furthermore, we demonstrated that M. caribbica can secrete phenylethanol, a quorum sensing molecule which can affect biofilm development. Phenylalanine (a precursor substance for phenylethanol synthesis) enhanced the secretion of phenylethanol and promoted the formation of M. caribbica biofilms. Meanwhile, phenylalanine enhanced the biological control performance of M. caribbica against jujube black spot rot. Our study provided new insights that enhance the biological control performance of antagonistic yeast. Full article
(This article belongs to the Special Issue Isolation and Control of Fruit and Vegetable Rot Fungi)
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<p>The biocontrol effect of <span class="html-italic">M. caribbica</span> inhibits <span class="html-italic">A. alternata</span> growth in jujube fruit. The (A) DI and (B) LD of jujube fruit were measured after inoculation at 25 °C. Standard errors of the means are indicated using vertical bars, and significant differences are denoted with different letters (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 2
<p>Colonization status of <span class="html-italic">M. caribbica</span> on jujube wounds. Vertical bars denote the standard error of the mean.</p> Full article ">Figure 3
<p>SEM of jujube wound tissues 48 h after inoculation. Wounds were incubated with (A) <span class="html-italic">A. alternata</span>, (B) <span class="html-italic">M. caribbica,</span> and <span class="html-italic">A. alternata</span>; (A1,B1): magnification of 2500×; (A2,B2): magnification of 5000×.</p> Full article ">Figure 4
<p>Biofilm formation ability on <span class="html-italic">M. caribbica</span> stained with crystal violet. The error bar reflects the standard deviation of the mean.</p> Full article ">Figure 5
<p>Effect of various concentrations of CM on (A) cell growth and (B) biofilm formation. Error bars indicate the standard deviation, and different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>Effect of various concentrations of phenylethanol on (A) cell growth and (B) biofilm formation. Error bars indicate the standard deviation, and different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 7
<p>Effect of Phe on phenylethanol production and biofilm formation of <span class="html-italic">M. caribbica</span>. (A) Influence of different Phe concentrations on the phenylethanol content produced by <span class="html-italic">M. caribbica</span>. The vertical bar denotes the standard deviation of the mean. (B) Influence of different Phe concentrations on biofilm formation of <span class="html-italic">M. caribbica.</span> (C) SEM of jujube wound tissues 48 h after inoculation. Wounds were incubated with (C1) SDW, (C2) <span class="html-italic">M. caribbica</span>, (C3) <span class="html-italic">M. caribbica</span> obtained from NYDB medium with 1 mmol/L Phe, and (C4) <span class="html-italic">M. caribbica</span> obtained from NYDB medium with 8 mmol/L Phe. (C1–C4): magnification of 5000×. White arrows indicate the extracellular matrix secreted by <span class="html-italic">M.caribbica</span>. Error bars indicate the standard deviation, and different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 8
<p>The effect of different concentrations of Phe on the biocontrol of jujube black spot rot by <span class="html-italic">M. caribbica</span>. The DI (A) and LD (B) were recorded for jujube black spot rot in fruit stored at 25 °C for 10 d. Error bars indicate the standard deviation, and different letters indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
11 pages, 1614 KiB  
Article
Inhibition of Dopamine Activity and Response of Rhipicephalus microplus Challenged with Metarhizium anisopliae
by Victória Silvestre Bório, Thaís Almeida Corrêa, Jéssica Fiorotti, Emily Mesquita, Laura Nóbrega Meirelles, Mariana Guedes Camargo, Vânia Rita Elias Pinheiro Bittencourt and Patrícia Silva Golo
J. Fungi 2022, 8(12), 1312; https://doi.org/10.3390/jof8121312 - 17 Dec 2022
Viewed by 1646
Abstract
Dopamine modulates ticks and insect hemocytes and links these arthropods’ nervous and immune systems. For the first time, the present study analyzed the effect of a dopamine receptor antagonist on the survival, biological parameters, phagocytic index, and dopamine detection in the hemocytes of [...] Read more.
Dopamine modulates ticks and insect hemocytes and links these arthropods’ nervous and immune systems. For the first time, the present study analyzed the effect of a dopamine receptor antagonist on the survival, biological parameters, phagocytic index, and dopamine detection in the hemocytes of ticks challenged by Metarhizium anisopliae. The survival and egg production index of Rhipicephalus microplus were negatively impacted when ticks were inoculated with the antagonist and fungus. Five days after the treatment, the survival of ticks treated only with fungus was 2.2 times higher than ticks treated with the antagonist (highest concentration) and fungus. A reduction in the phagocytic index of hemocytes of 68.4% was observed in the group inoculated with the highest concentration of the antagonist and fungus compared to ticks treated only with fungus. No changes were detected in the R. microplus levels of intrahemocytic dopamine or hemocytic quantification. Our results support the hypothesis that dopamine is crucial for tick immune defense, changing the phagocytic capacity of hemocytes and the susceptibility of ticks to entomopathogenic fungi. Full article
(This article belongs to the Special Issue New Perspectives on Entomopathogenic and Nematode-Trapping Fungi)
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<p>(<b>A</b>–<b>F</b>) <span class="html-italic">Rhipicephalus microplus</span> hemocytes on slides exposed to <span class="html-italic">Metarhizium anisopliae</span> (MA) or Zymosan (Z) for two hours with or without previous incubation of SCH 23390 dopamine receptor antagonist at 1 nM or 1µM for one hour. (<b>A</b>) Z; (<b>B</b>) SCH 1 nM + Z; (<b>C</b>) SCH 1 µM + MA; (<b>D</b>) MA; (<b>E</b>) SCH 1 nM + MA; (<b>F</b>) SCH 1 µM + MA. Black arrows indicate <span class="html-italic">Metarhizium</span> conidia or Zymosan that were not phagocytosed. Red arrows indicate conidia or Zymosan phagocytosed. The scale bar represents 10 μm. (<b>G</b>) Phagocytic index (%) of <span class="html-italic">R. microplus</span> hemocytes after incubation with <span class="html-italic">M. anisopliae</span> conidia or Saccharomyces cerevisiae (Zymosan A) with or without SCH 23390. Data were analyzed by one-way ANOVA and the Tukey’s test (<span class="html-italic">p</span> &lt; 0.05). Different letters differ statistically. Z: cells exposed to Zymosan A alone; SCH 1 nM + Z: cells exposed to the antagonist at 1 nM followed by Zymosan A; SCH 1 µM + Z: cells exposed to the antagonist at 1 µM followed by Zymosan A; MA: cells exposed to <span class="html-italic">M. anisopliae</span> alone; SCH 1 nM + MA: cells exposed to the antagonist at 1 nM followed by <span class="html-italic">M. anisopliae</span>; SCH 1 µM + MA: cells exposed to the antagonist at 1 µM followed by <span class="html-italic">M. anisopliae</span>.</p> Full article ">Figure 2
<p>(<b>A</b>) Effect of dopamine receptor antagonist SCH23390 on the survival of <span class="html-italic">Rhipicephalus microplus</span> females associated or not with <span class="html-italic">Metarhizium anisopliae</span>. Mean survival (%) and standard deviation of females inoculated with <span class="html-italic">M. anisopliae</span> conidia according to Log-rank (<span class="html-italic">p</span> &lt; 0.0001). A representative experiment of three independent replications, where (*) represents statistical difference between survival averages from MA and SCH 1 nM + MA (<span class="html-italic">p</span> = 0.0253) or MA and SCH 1 µM + MA (<span class="html-italic">p</span> = 0.0291), and (****) represents statistical difference between survival averages from MA and CTR (<span class="html-italic">p</span> &lt; 0.0001) or MA and SCH 1 nM or SCH 1 µM (<span class="html-italic">p</span> &lt; 0.0001). (<b>B</b>) Egg production index (EPI) of <span class="html-italic">R. microplus</span> females inoculated with antagonist SCH 23390 at 1 nM or 1 µM, <span class="html-italic">M. anisopliae</span> LCM S04 at 1.0 × 10<sup>7</sup> conidia/mL and associations. Different letters differ statistically. CTR: untreated ticks; PBS: ticks inoculated with phosphate buffer solution; SCH 1 nM: ticks inoculated with antagonist at 1 nM; SCH 1 µM: ticks inoculated with antagonist at 1 µM; MA: ticks inoculated with <span class="html-italic">M. anisopliae</span>; SCH 1 nM + MA: ticks inoculated with the lowest concentration of antagonist and fungus; SCH 1 µM + MA: ticks inoculated with the highest concentration of antagonist and fungus.</p> Full article ">Figure 3
<p>Average and standard error of <span class="html-italic">Rhipicephalus microplus</span> hemocytes circulating in the hemolymph 24 h after inoculation of the antagonist SCH 23390 and <span class="html-italic">Metarhizium anisopliae</span> LCM S04. Bars with the same letter do not differ statistically according to one-way ANOVA followed by Tukey’s test (<span class="html-italic">p</span> &lt; 0.05). CTR: untreated ticks; PBS: ticks inoculated with phosphate buffer solution; SCH 1 nM: ticks inoculated with the antagonist at 1 nM; SCH 1 µM: ticks inoculated with the antagonist at 1 µM; MA: ticks inoculated with <span class="html-italic">M. anisopliae</span>; SCH 1 nM + MA: ticks inoculated with the lowest concentration of antagonist and fungus; SCH 1 µM + MA: ticks inoculated with the highest concentration of the antagonist and fungus.</p> Full article ">Figure 4
<p>Detection of dopamine in the hemocytes of <span class="html-italic">Rhipicephalus microplus</span> tick females 24 h after inoculation of dopamine receptor antagonist. (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>) Immunofluorescence images and (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>) light microscopy images. (<b>A</b>,<b>B</b>) untreated ticks (CTR); (<b>C</b>,<b>D</b>) ticks inoculated with antagonist (SCH 1 µM); (<b>E</b>,<b>F</b>) ticks inoculated with <span class="html-italic">Metarhizium anisopliae</span> (MA); (<b>G</b>,<b>H</b>) ticks inoculated with antagonist followed by <span class="html-italic">M. anisopliae</span> (SCH 1 µM + MA). The scale bar represents 10 μm. (<b>I</b>) Average fluorescence intensity (marked area) percentage and standard error of dopamine in the hemocytes of <span class="html-italic">R. microplus</span> tick females. Bars with the same letter did not differ statistically according to one-way ANOVA and the Tukey’s test (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
16 pages, 2721 KiB  
Article
Prevalence of Carbendazin Resistance in Field Populations of the Rice False Smut Pathogen Ustilaginoidea virens from Jiangsu, China, Molecular Mechanisms, and Fitness Stability
by Jiehui Song, Zhiying Wang, Yan Wang, Sijie Zhang, Tengyu Lei, You Liang, Qigen Dai, Zhongyang Huo, Ke Xu and Shuning Chen
J. Fungi 2022, 8(12), 1311; https://doi.org/10.3390/jof8121311 - 16 Dec 2022
Cited by 8 | Viewed by 1732
Abstract
Rice false smut (RFS), caused by Ustilaginoidea virens, is an important fungal disease of rice. In China, Methyl Benzimidazole Carbamates (MBCs), including carbendazim, are common fungicides used to control RFS and other rice diseases. In this study, resistance of U. virens to [...] Read more.
Rice false smut (RFS), caused by Ustilaginoidea virens, is an important fungal disease of rice. In China, Methyl Benzimidazole Carbamates (MBCs), including carbendazim, are common fungicides used to control RFS and other rice diseases. In this study, resistance of U. virens to carbendazim was monitored for three consecutive years during 2018 to 2020. A total of 321 U. virens isolates collected from Jiangsu Province of China were tested for their sensitivity to carbendazim on PSA. The concentration at which mycelial growth is inhibited by 50% (EC50) of the carbendazim-sensitive isolates was 0.11 to 1.38 µg/mL, with a mean EC50 value of 0.66 μg/mL. High level of resistance to carbendazim was detected in 14 out of 321 isolates. The resistance was stable but associated with a fitness penalty. There was a statistically significant and moderate negative correlation (r= −0.74, p < 0.001) in sensitivity between carbendazim and diethofencarb. Analysis of the U. virens genome revealed two potential MBC targets, Uvβ1Tub and Uvβ2Tub, that putatively encode β-tubulin gene. The two β-tubulin genes in U. virens share 78% amino acid sequence identity, but their function in MBC sensitivity has been unclear. Both genes were identified and sequenced from U. virens sensitive and resistant isolates. It is known that mutations in the β2-tubulin gene have been shown to confer resistance to carbendazim in other fungi. However, no mutation was found in the Uvβ2Tub gene in either resistant or sensitive isolates. Variations including point mutations, non-sense mutations, codon mutations, and frameshift mutations were found in the Uvβ1Tub gene from the 14 carbendazim-resistant isolates, which have not been reported in other fungi before. Thus, these results indicated that variations of Uvβ1Tub result in the resistance to carbendazim in field isolates of Ustilaginoidea virens. Full article
(This article belongs to the Special Issue Smut Fungi 2.0)
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<p>Geographic origins of <span class="html-italic">Ustilaginoidea virens</span> isolates collected in Jiangsu Province of East China.</p> Full article ">Figure 2
<p>Frequency distribution of EC<sub>50</sub> values of <span class="html-italic">Ustilaginoidea virens</span> isolates to carbendazim. (<b>A</b>) Frequency distribution of EC<sub>50</sub> values of the total of 321 isolates. (<b>B</b>) Frequency distribution of EC<sub>50</sub> values of isolates collected from different years.</p> Full article ">Figure 3
<p>Biological characterization of carbendazim-sensitive and carbendazim-resistant <span class="html-italic">Ustilaginoidea virens</span> isolates. (<b>A</b>) Mycelial growth diameter, (<b>B</b>) mycelial dry weight, (<b>C</b>) conidiation, and (<b>D</b>) conidial germination rate. Five isolates (HWD, JS60-2, JY7b, JY11a, and JY30b) were tested as sensitive isolates, while 14 isolates (GL11, GL12b, GL23, HA17, HA26, JR11, JR12, XH5a, XH7b, XH43b, YD8, YZ11, ZJ7, and ZJ24) were tested as resistant isolates for mycelial growth diameter, mycelial dry weight, and conidiation. Five resistant isolates (GL11, HA17, XH5a, XH7b, and XH43b) were tested for conidial germination rate on PSA and WA media. Asterisks indicate that the difference is statistically significant. Error bars represent standard deviations.</p> Full article ">Figure 4
<p>Sensitivity of resistant isolates GL11 and XH43b to carbendazim after 5 transfers (T5) on fungicide-free PSA. Photos were taken after 28 days of incubation at 27 °C in the dark.</p> Full article ">Figure 5
<p>Correlation of log10-transformed concentration at which mycelial growth is inhibited 50% (EC<sub>50</sub>) values of <span class="html-italic">Ustilaginoidea virens</span> isolates for carbendazim and (<b>A</b>) diethofencarb, (<b>B</b>) azoxystrobin, (<b>C</b>) pyraclostrobin, and (<b>D</b>) tebuconazole.</p> Full article ">Figure 6
<p>Schematic gene structure and alignments of <span class="html-italic">Ustilaginoidea virens Uvβ1Tub</span> and <span class="html-italic">Uvβ2Tub</span>. (<b>A</b>) <span class="html-italic">Uvβ1Tub</span> from twenty-three carbendazim-sensitive isolates is 1825 bp in size interrupted by four introns (with exons from 1–12, 242–265, 334–456, 572–1362, and 1429–1825). Locations of <span class="html-italic">Uvβ1Tub</span> variations in carbendazim-resistant isolates are marked with arrows. <span class="html-italic">Uvβ2Tub</span> from carbendazim-sensitive isolates is 2103 bp in size interrupted by six introns (with exons from 1–12, 157–180, 260–327, 493–547, 647–959, 1080–1378, and 1516–2103). Number indicates nucleotide position of the gene. (<b>B</b>) Alignments of β-tubulin amino acid sequences from <span class="html-italic">Ustilaginoidea virens and Aspergillus nidulans.</span> The sequences of deduced amino acids of β-tubulin from <span class="html-italic">A. nidulans</span> from the NCBI GenBank database AAA3328.1. The shaded letters indicate the conserved residues. Letters above the sequences: substitution amino acids in <span class="html-italic">β2Tub</span> from other resistant field isolates [<a href="#B21-jof-08-01311" class="html-bibr">21</a>]. Letters under sequences: substitution amino acids in <span class="html-italic">β1Tub</span> from resistant laboratory mutants [<a href="#B22-jof-08-01311" class="html-bibr">22</a>,<a href="#B23-jof-08-01311" class="html-bibr">23</a>]. The codon positions where mutations occurred in <span class="html-italic">Uvβ1Tub</span> are marked with arrows.</p> Full article ">Figure 7
<p>Phylogenetic tree generated by the maximum likelihood method with Mega 7.0 software [<a href="#B24-jof-08-01311" class="html-bibr">24</a>] on the basis of deduced amino acid sequences of β-tubulin proteins. The deduced amino acid sequences of Uvβ1Tub and Uvβ2Tub for <span class="html-italic">Ustilaginoidea virens</span> isolate Uv8b and those from other fungal species, with 434 positions in the final dataset. Numbers labeled at each node indicate bootstrap values (%) from 1000 replicates. Stars indicate β1Tub and β2Tub of <span class="html-italic">Ustilaginoidea virens</span> in this study.</p> Full article ">
20 pages, 8380 KiB  
Article
Natural Product Citronellal can Significantly Disturb Chitin Synthesis and Cell Wall Integrity in Magnaporthe oryzae
by Ai-Ai Zhou, Rong-Yu Li, Fei-Xu Mo, Yi Ding, Ruo-Tong Li, Xue Guo, Ke Hu and Ming Li
J. Fungi 2022, 8(12), 1310; https://doi.org/10.3390/jof8121310 - 16 Dec 2022
Cited by 14 | Viewed by 2013
Abstract
Background: Natural products are often favored in the study of crop pests and diseases. Previous studies have shown that citronellal has a strong inhibition effect on Magnaporthe oryzae. The objective of this study was to clarify its mechanism of action against M. [...] Read more.
Background: Natural products are often favored in the study of crop pests and diseases. Previous studies have shown that citronellal has a strong inhibition effect on Magnaporthe oryzae. The objective of this study was to clarify its mechanism of action against M. oryzae. Results: Firstly, the biological activity of citronellal against M. oryzae was determined by direct and indirect methods, and the results show that citronellal had a strong inhibition effect on M. oryzae with EC50 values of 134.00 mg/L and 70.48 μL/L air, respectively. Additionally, a preliminary study on its mechanism of action was studied. After citronellal treatment, electron microscopy revealed that the mycelium became thin and broken; scanning electron microscopy revealed that the mycelium was wrinkled and distorted; and transmission electron microscopy revealed that the mycelium cell wall was invaginated, the mass wall of mycelium was separated, and the organelles were blurred. The mycelium was further stained with CFW, and the nodes were blurred, while the mycelium was almost non-fluorescent after PI staining, and there was no significant difference in the relative conductivity of mycelium. In addition, chitinase was significantly enhanced, and the expression of chitin synthesis-related genes was 17.47-fold upregulated. Finally, we found that the efficacy of citronellal against the rice blast was as high as 82.14% according to indoor efficacy tests. Conclusion: These results indicate that citronellal can affect the synthesis of chitin in M. oryzae and damage its cell wall, thereby inhibiting the growth of mycelium and effectively protecting rice from rice blasts. Full article
(This article belongs to the Section Fungal Cell Biology, Metabolism and Physiology)
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<p>Chemical structure of citronellal.</p> Full article ">Figure 2
<p>Inhibitory effect of citronellal on <span class="html-italic">M. oryzae.</span> Error bars denote standard error of mean for three independent experiments, and below is the same. (<b>A</b>) Mycelial growth rate method, (<b>B</b>) Indirect activity method, (<b>C</b>) The colony formation of <span class="html-italic">M. oryzae</span> by mycelial growth velocity method.</p> Full article ">Figure 3
<p>The loss rate of mycelium. Different lowercase letters denote statistically significant differences at α = 0.05; below is the same.</p> Full article ">Figure 4
<p>Effects of citronellal on morphology of <span class="html-italic">M. oryzae:</span> 36 h: (<b>A</b>) (CK), (<b>B</b>) (85 mg/L), (<b>C</b>) (134 mg/L), (<b>D</b>) (218 mg/L); 48 h: (<b>E</b>) (CK), (<b>F</b>) (85 mg/L), (<b>G</b>) (134 mg/L), (<b>H</b>) (218 mg/L); 72 h: (<b>I</b>) (CK), (<b>J</b>) (85 mg/L), (<b>K</b>) (134 mg/L), (<b>L</b>) (218 mg/L); 96 h: (<b>M</b>) (CK), (<b>N</b>) (85 mg/L), (<b>O</b>) (134 mg/L), (<b>P</b>) (218 mg/L); 120 h: (<b>Q</b>) (CK), (<b>R</b>) (85 mg/L), (<b>S</b>) (134 mg/L), (<b>T</b>) (218 mg/L). The scale bar is 30 μm.</p> Full article ">Figure 5
<p>Morphology of mycelium after 72 h treatment with different concentrations of citronellal. (<b>A</b>,<b>B</b>): CK; (<b>C</b>,<b>D</b>): 85 mg/L; (<b>E</b>,<b>F</b>): 134 mg/L; (<b>G</b>,<b>H</b>): 218 mg/L.</p> Full article ">Figure 6
<p>Ultrastructure of mycelial cells after 72 h treatment with different concentrations of citronellal: (<b>A</b>,<b>B</b>): CK; (<b>C</b>,<b>D</b>): 85 mg/L; (<b>E</b>,<b>F</b>): 134 mg/L; (<b>G</b>,<b>H</b>): 218 mg/L. The arrows in the figure indicate cell wall (cw) and vacuole (v).</p> Full article ">Figure 7
<p>Effect of citronellal on the cell membrane of <span class="html-italic">M. oryzae</span>. (<b>A</b>,<b>B</b>): PI fluorescence and nonfluorescent effects of mycelia after treatment with citronellal for 72 h: (<b>a</b>,<b>b</b>): CK; (<b>c</b>,<b>d</b>): 85 mg/L; (<b>e</b>,<b>f</b>): 134 mg/L; (<b>g</b>,<b>h</b>): 218 mg/L. (<b>C</b>): Effect of citronellal on mycelial cell membrane permeability of <span class="html-italic">M. oryzae</span>.</p> Full article ">Figure 8
<p>CFW fluorescence effect and nonfluorescent of mycelia after treatment with citronellal for 72 h. (<b>A</b>,<b>B</b>): CK; (<b>C</b>,<b>D</b>): 85 mg/L; (<b>E</b>,<b>F</b>): 134 mg/L; (<b>G</b>,<b>H</b>): 218 mg/L.</p> Full article ">Figure 9
<p>Enzyme activity of mycelium in <span class="html-italic">M. oryzae</span> treating with citronellal. Standard curve of chitinase (<b>A</b>) and β-1,3-glucanase (<b>B</b>); Change trend of chitinase (<b>C</b>) and β-1,3-glucanase (<b>D</b>) activity.</p> Full article ">Figure 10
<p>Relative expression level of gene of chitinase (<b>A</b>) and β-1,3-glucanase (<b>B</b>).</p> Full article ">Figure 11
<p>Indoor control effect of citronellal on rice blast. <b>(A)</b> Rice leaves inoculated with <span class="html-italic">M. oryzae</span>. <b>(B)</b> Control effect. Error bars indicate means ± SD (<span class="html-italic">n</span> = 3).</p> Full article ">
12 pages, 2117 KiB  
Article
A Novel Gammapartitivirus That Causes Changes in Fungal Development and Multi-Stress Tolerance to Important Medicinal Fungus Cordyceps chanhua
by Qiuyan Zhu, Najie Shi, Ping Wang, Yuxiang Zhang, Fan Peng, Guogen Yang and Bo Huang
J. Fungi 2022, 8(12), 1309; https://doi.org/10.3390/jof8121309 - 16 Dec 2022
Cited by 5 | Viewed by 1608
Abstract
Cicada flower, scientifically named Cordyceps chanhua, is an important and well-known Chinese cordycipitoid medicinal mushroom. Although most mycoviruses seem to induce latent infections, some mycoviruses cause host effects. However, the effects of mycovirus on the fungal development and stress tolerance of C. [...] Read more.
Cicada flower, scientifically named Cordyceps chanhua, is an important and well-known Chinese cordycipitoid medicinal mushroom. Although most mycoviruses seem to induce latent infections, some mycoviruses cause host effects. However, the effects of mycovirus on the fungal development and stress tolerance of C. chanhua remain unknown. In this study, we report a novel mycovirus designated Cordyceps chanhua partitivirus 1 (CchPV1) from C. chanhua isolate RCEF5997. The CchPV1 genome comprises dsRNA 1 and dsRNA 2, 1784 and 1563 bp in length, respectively. Phylogenetic analysis using the aa sequences of RdRp revealed that CchPV1 grouped with members of the genus Gammapartitivirus in the family Partitiviridae. We further co-cultivated on PDA donor strain RCEF5997 and recipient C. chanhua strain RCEF5833 (Vf) for 7 days, and we successfully obtained an isogenic line of strain RCEF5833 with CchPV1 (Vi) through single-spore isolation, along with ISSR marker and dsRNA extraction. The biological comparison revealed that CchPV1 infection slows the growth rate of the host, but increases the conidiation and formation of fruiting bodies of the host. Furthermore, the assessment of fungal tolerance demonstrated that CchPV1 weakens the multi-stress tolerance of the host. Thus, CchPV1 infection cause changes in fungal development and multi-stress tolerance of the host C. chanhua. The findings of this study elucidate the effects of gammapartitivirus on host entomogenous fungi and provide a novel strategy for producing high-quality fruiting bodies of C. chanhua. Full article
(This article belongs to the Special Issue Mycoviruses: Emerging Investigations on Virus-Fungal Host Interaction)
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<p>Fungal development and virus infection of <span class="html-italic">Cordyceps chanhua</span> strains. (<b>A</b>) Colony morphology of <span class="html-italic">C. chanhua</span> strains RCEF5997 and RCEF5833 on PDA medium. (<b>B</b>) Diameters of strains grown on SDAY for 10 days. (<b>C</b>) Sporulation capacity of strains RCEF5997 and RCEF5833 growing on PDA medium after 10 days. (<b>D</b>) Electrophoresis of purified dsRNAs extracted from <span class="html-italic">C. chanhua</span> strains RCEF5997 and RCEF5833 in a 1.5% agarose gel. M, DNA marker; lane 1, dsRNA segments of RCEF5997; lane 2, RCEF5833 did not possess any dsRNA. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 2
<p>Characterization of CchPV1. (<b>A</b>) Electrophoresis of purified dsRNAs extracted from <span class="html-italic">Cordyceps chanhua</span> RCEF5997 in a 1.5% agarose gel. M, DNA marker; lane 1, dsRNA segments of RCEF5997. (<b>B</b>) Schematic representation of CchPV1. (<b>C</b>) Sequence alignment information of 5’-UTR and 3’-UTR of CchPV1 (ClustalX was used for sequence alignment). An asterisk indicates a position with a single, fully conserved residue. Colons and periods indicate conservation between groups with strongly similar properties (scoring &gt; 0.5) and weakly similar properties (scoring ≤ 0.5), respectively, in the Gonnet PAM 250 matrix.</p> Full article ">Figure 3
<p>Phylogenetic analysis of RdRp of CchPV1 and the other members of the family <span class="html-italic">Partitiviridae</span>, including members of the genera <span class="html-italic">Alphapartitivirus</span>, <span class="html-italic">Betapartitivirus</span>, <span class="html-italic">Gammapartitivirus</span>, <span class="html-italic">Deltapartitivirus</span>, and <span class="html-italic">Cryspovirus</span>, as well as the proposed genera “<span class="html-italic">Epsilonpartitivirus</span>” and “<span class="html-italic">Zetapartitivirus</span>”, constructed using the maximum likelihood method (ML), with the LG + G + I + F amino-acid substitution model. The scale represents 0.5 amino-acid substitutions at each site, and the numbers on the nodes indicate bootstrap support of more than 50% (1000 repeats).</p> Full article ">Figure 4
<p>Transmission of CchPV1 virus. (<b>A</b>) Strains RCEF5833 and RCEF5997 were genetically distinguished by ISSR markers. (<b>B</b>) Confrontation culture of RCEF5997 and RCEF5833, in which the large colony was RCEF5833 and the small colony was RCEF5997. (<b>C</b>) ISSR analysis of single-spore isolates. (<b>D</b>) dsRNA verification of single-spore isolates, M, DNA marker; lane 1, dsRNA segments of RCEF5997; lane 2, dsRNA segments of RCEF5833 (Vi).</p> Full article ">Figure 5
<p>Fungal development of Vf and Vi strains. (<b>A</b>) Colony morphology of strains Vf and Vi growing on different media. (<b>B</b>) The growth of asexual fruiting on Chinese tussah silkworm pupae. (<b>C</b>) Left: diameters of strains grown for 10 days on PDA, SDAY, and 1/4 SDAY, respectively; Right: sporulation of the strains after 10 days of growth. (<b>D</b>) The production of the fruiting bodies of 1 g of pupae produced by Vi and Vf strains. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p>Resistance of strains to stress. (<b>A</b>) Colony morphology of Vf and Vi in PDA containing different chemicals. (<b>B</b>) The relative inhibition rate of the strains cultured on the medium containing NaCl, H<sub>2</sub>O<sub>2</sub>, and Congo Red after 10 days. (<b>C</b>) Relative germination rate of strains after 24 h UV-B irradiation. (<b>D</b>) The relative germination rate of strains at 40 °C heat shock after 24 h. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
16 pages, 2375 KiB  
Article
Arbuscular Mycorrhizal Fungus Alters Alfalfa (Medicago sativa) Defense Enzyme Activities and Volatile Organic Compound Contents in Response to Pea Aphid (Acyrthosiphon pisum) Infestation
by Yajie Wang, Yingde Li, Zhen Tian and Tingyu Duan
J. Fungi 2022, 8(12), 1308; https://doi.org/10.3390/jof8121308 - 16 Dec 2022
Cited by 3 | Viewed by 1859
Abstract
Pea aphid (Acyrthosiphon pisum) infestation leads to withering, reduced yield, and lower quality of the host plant. Arbuscular mycorrhizal (AM) fungi have been found to enhance their host plants’ nutrient uptake, growth, and resistance to biotic stresses, including pathogen infection and [...] Read more.
Pea aphid (Acyrthosiphon pisum) infestation leads to withering, reduced yield, and lower quality of the host plant. Arbuscular mycorrhizal (AM) fungi have been found to enhance their host plants’ nutrient uptake, growth, and resistance to biotic stresses, including pathogen infection and insect pest infestation. Therefore, we evaluated the effects of AM fungus Rhizophagus intraradices on alfalfa defense responses to pea aphid infestation. Aphid infestation did not affect the colonization of AM fungus. The inoculation of AM fungus, on average, enhanced alfalfa catalase and the contents of salicylic acid and trypsin inhibitor by 101, 9.05, and 7.89% compared with non-mycorrhizal alfalfa, respectively. In addition, polyphenol oxidase activities significantly increased by six-fold after aphid infestation in mycorrhizal alfalfa. Moreover, the fungus significantly (p < 0.05) improved alfalfa shoot N content, net photosynthetic and transpiration rates, and shoot dry weight in aphid infected treatment. The aphid infestation changed the total volatile organic compounds (VOCs) in alfalfa, while AM fungus enhanced the contents of methyl salicylate (MeSA). The co-expression network analysis of differentially expressed genes (DEGs) and differentially expressed VOCs analysis showed that three DEGs, namely MS.gene23894, MS.gene003889, and MS.gene012415, positively correlated with MeSA both in aphid and AM fungus groups. In conclusion, AM fungus increased alfalfa’s growth, defense enzyme activities, hormones, and VOCs content and up-regulated VOC-related genes to enhance the alfalfa’s resistance following aphid infestation. Full article
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<p>(<b>a</b>) Shoot fresh weight, (<b>b</b>) Shoot dry weight, (<b>c</b>) Shoot total N, and (<b>d</b>) Shoot total P of <span class="html-italic">M. sativa</span> inoculated with <span class="html-italic">R. intraradices</span> (AM) and infested by <span class="html-italic">A. pisum</span> (A+) or without inoculation with <span class="html-italic">R. intraradices</span> (NM) and non-infested with <span class="html-italic">A. pisum</span> (A−). All values are shown as means ± SEM of four biological replicates. Different letters above bars (<b>d</b>) or pairs of bars (<b>a</b>–<b>c</b>) represent significant difference in the comparison at <span class="html-italic">p</span> &lt; 0.05, asterisks indicate significant differences between A− and A−, as determined by a Tukey’s HSD test. SEM, standard error of the mean.</p> Full article ">Figure 2
<p>(<b>a</b>) Net photosynthetic rate and (<b>b</b>) transpiration rate of <span class="html-italic">M. sativa</span> inoculated with <span class="html-italic">R. intraradices</span> (AM) and infested by <span class="html-italic">A. pisum</span> (A+) or without inoculation with <span class="html-italic">R. intraradices</span> (NM) and un-infested by <span class="html-italic">A. pisum</span> (A−) before aphid infesting and after aphid infesting. Boxes show first quartile, median, and third quartile. Whiskers extend to the most extreme points within 1.5 × box lengths, and the points are values that fall outside the whiskers. Different letters above bars or pairs of bars represent significant difference between different treatments before aphid infesting or after aphid infesting in the comparison at <span class="html-italic">p</span> &lt; 0.05, as determined by a Tukey’s HSD test.</p> Full article ">Figure 3
<p>(<b>a</b>) PPO activity, (<b>b</b>) POD activity, (<b>c</b>) CAT activity, (<b>d</b>) JA concentration, (<b>e</b>) SA concentration, and (<b>f</b>) Trypsin inhibitor concentration of <span class="html-italic">M. sativa</span> inoculated with <span class="html-italic">R. intraradices</span> (AM) and infested by <span class="html-italic">A. pisum</span> (A+) or without inoculation with <span class="html-italic">R. intraradices</span> (NM) and non-infested with <span class="html-italic">A. pisum</span> (A−). All values are shown as means ± SEM of four biological replicates. Different letters above bars (<b>a</b>–<b>d</b>) or pairs of bars (<b>e</b>,<b>f</b>) represent significant difference in the comparison at <span class="html-italic">p</span> &lt; 0.05, asterisks indicate significant differences between A− and A−, as determined by a Tukey’s HSD test. SEM, standard error of the mean. PPO, polyphenol oxidase; POD, peroxidase; CAT, catalase; JA, jasmonic acid; SA, salicylic acid.</p> Full article ">Figure 4
<p>(<b>a</b>) Total VOC content from <span class="html-italic">M. sativa</span> inoculated with <span class="html-italic">R. intraradices</span> (AM) and infested by <span class="html-italic">A. pisum</span> (A+) or without inoculation with <span class="html-italic">R. intraradices</span> (NM) and non-infested with <span class="html-italic">A. pisum</span> (A−). All values are shown as means ± SEM of four biological replicates. Different letters above pairs of bars represent significant difference in the comparison at <span class="html-italic">p</span> &lt; 0.05 (<b>b</b>) Individual VOCs’ contents in NMA−, NMA+, AMA−, AMA+ treatments. Means ± SEM for the 20 most abundant compounds. The same compounds are present in each treatment, and the relative contents of compounds are similar. A, 2-Hexenal, (E)−; B, 2-Ethyl-1-hexanol; C, 3-Buten-2-one, 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-; D, 2,4-Heptadienal, (E,E)-; E, 1-Hexanol; F, Hexanal; G, 3-Buten-2-one, 4-(2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-; H, 3,5-Octadien-2-one; I, 1-Octen-3-ol; J, Phenylethyl Alcohol; K, 2-Penten-1-ol, (Z)−; L, Benzyl alcohol; M, 1-Cyclohexene-1-carboxaldehyde, 2,6,6-trimethyl-; N, Cyclohexanol; O, 4-Heptanone, 2,6-dimethyl-; P, Pentadecane; Q, Benzaldehyde; R, 2,6-Nonadienal, (E,Z)−; S, Cyclohexanol, 2,6-dimethyl-; T, Octanoic acid, ethyl ester.</p> Full article ">Figure 5
<p>Co-expression network and expression profiles for each module of differentially expressed genes and differentially expressed VOCs in (<b>a</b>) NMA− vs. AMA−, (<b>b</b>) NMA− vs. NMA+, (<b>c</b>) AMA- vs. AMA+, (<b>d</b>) NMA+ vs. AMA+. Nodes colored in ‘red’ and ‘blue’ represent DEMs and differentially expressed VOCs. Edges colored in ‘red’ and ‘blue’ represent positive and negative correlations. VOCs, volatile organic components.</p> Full article ">
16 pages, 891 KiB  
Review
The Use of Host Biomarkers for the Management of Invasive Fungal Disease
by James S. Griffiths, Selinda J. Orr, Charles Oliver Morton, Juergen Loeffler and P. Lewis White
J. Fungi 2022, 8(12), 1307; https://doi.org/10.3390/jof8121307 - 16 Dec 2022
Cited by 3 | Viewed by 2712
Abstract
Invasive fungal disease (IFD) causes severe morbidity and mortality, and the number of IFD cases is increasing. Exposure to opportunistic fungal pathogens is inevitable, but not all patients with underlying diseases increasing susceptibility to IFD, develop it. IFD diagnosis currently uses fungal biomarkers [...] Read more.
Invasive fungal disease (IFD) causes severe morbidity and mortality, and the number of IFD cases is increasing. Exposure to opportunistic fungal pathogens is inevitable, but not all patients with underlying diseases increasing susceptibility to IFD, develop it. IFD diagnosis currently uses fungal biomarkers and clinical risk/presentation to stratify high-risk patients and classifies them into possible, probable, and proven IFD. However, the fungal species responsible for IFD are highly diverse and present numerous diagnostic challenges, which culminates in the empirical anti-fungal treatment of patients at risk of IFD. Recent studies have focussed on host-derived biomarkers that may mediate IFD risk and can be used to predict, and even identify IFD. The identification of novel host genetic variants, host gene expression changes, and host protein expression (cytokines and chemokines) associated with increased risk of IFD has enhanced our understanding of why only some patients at risk of IFD actually develop disease. Furthermore, these host biomarkers when incorporated into predictive models alongside conventional diagnostic techniques enhance predictive and diagnostic results. Once validated in larger studies, host biomarkers associated with IFD may optimize the clinical management of populations at risk of IFD. This review will summarise the latest developments in the identification of host biomarkers for IFD, their use in predictive modelling and their potential application/usefulness for informing clinical decisions. Full article
(This article belongs to the Special Issue Novel Technologies for Diagnosis of Fungal Infection)
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<p><b>Incorporating host biomarkers into IFD diagnostic strategies.</b> Prospective IFD diagnostic strategy that utilizes the latest advancements in IFD-associated host biomarkers alongside conventional fungal biomarkers and clinical risk to better target anti-fungal prophylaxis [<a href="#B65-jof-08-01307" class="html-bibr">65</a>,<a href="#B85-jof-08-01307" class="html-bibr">85</a>,<a href="#B90-jof-08-01307" class="html-bibr">90</a>,<a href="#B96-jof-08-01307" class="html-bibr">96</a>,<a href="#B97-jof-08-01307" class="html-bibr">97</a>,<a href="#B98-jof-08-01307" class="html-bibr">98</a>]. Blue dotted boxes represent current investigations into IFD susceptibility and incidence. Red dotted boxes represent potential new host biomarker investigations into IFD susceptibility and incidence. Solid boxes represent patient pathway and potential stratification into high, intermediate, and low IFD risk cohorts.</p> Full article ">
13 pages, 954 KiB  
Article
Cryptococcus neoformans Causing Meningoencephalitis in Adults and a Child from Lima, Peru: Genotypic Diversity and Antifungal Susceptibility
by Carolina Firacative, Natalia Zuluaga-Puerto and José Guevara
J. Fungi 2022, 8(12), 1306; https://doi.org/10.3390/jof8121306 - 16 Dec 2022
Cited by 1 | Viewed by 2086
Abstract
Cryptococcosis, caused predominantly by Cryptococcus neoformans, is a potentially fatal, opportunistic infection that commonly affects the central nervous system of immunocompromised patients. Globally, this mycosis is responsible for almost 20% of AIDS-related deaths, and in countries like Peru, its incidence remains high, [...] Read more.
Cryptococcosis, caused predominantly by Cryptococcus neoformans, is a potentially fatal, opportunistic infection that commonly affects the central nervous system of immunocompromised patients. Globally, this mycosis is responsible for almost 20% of AIDS-related deaths, and in countries like Peru, its incidence remains high, mostly due to the annual increase in new cases of HIV infection. This study aimed to establish the genotypic diversity and antifungal susceptibility of C. neoformans isolates causing meningoencephalitis in 25 adults and a 9-year-old girl with HIV and other risk factors from Lima, Peru. To identify the genotype of the isolates, multilocus sequence typing was applied, and to establish the susceptibility of the isolates to six antifungals, a YeastOne® broth microdilution was used. From the isolates, 19 were identified as molecular type VNI, and seven as VNII, grouped in eight and three sequence types, respectively, which shows that the studied population was highly diverse. Most isolates were susceptible to all antifungals tested. However, VNI isolates were less susceptible to fluconazole, itraconazole and voriconazole than VNII isolates (p < 0.05). This study contributes data on the molecular epidemiology and the antifungal susceptibility profile of the most common etiological agent of cryptococcosis, highlighting a pediatric case, something which is rare among cryptococcal infection. Full article
(This article belongs to the Special Issue Fifty Years of Fungi: A Special Issue Honoring the Contributions of Dr. June Kwon-Chung to the Field of Medical Mycology)
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<p>A dendrogram depicting the genetic relationship between clinical <span class="html-italic">Cryptococcus neoformans</span> isolates from this study (triangles) and other clinical (red) and environmental (green) isolates from Argentina (ARG), Brazil (BRA), Colombia (COL) and Peru (PER), as reported in former <span class="html-italic">C. neoformans</span> genotyping studies [<a href="#B10-jof-08-01306" class="html-bibr">10</a>,<a href="#B18-jof-08-01306" class="html-bibr">18</a>,<a href="#B19-jof-08-01306" class="html-bibr">19</a>,<a href="#B20-jof-08-01306" class="html-bibr">20</a>,<a href="#B21-jof-08-01306" class="html-bibr">21</a>,<a href="#B22-jof-08-01306" class="html-bibr">22</a>,<a href="#B23-jof-08-01306" class="html-bibr">23</a>]. The VNII sequence types (ST), ST664, newly identified, and ST43, belonging to an isolate from a 9-year-old girl, are highlighted in blue and yellow, respectively. The dendrogram is based on the analysis of the seven concatenated ISHAM consensus MLST loci using the program MEGA 11 [<a href="#B24-jof-08-01306" class="html-bibr">24</a>]. Bootstrap values above 75 are indicated on the branches.</p> Full article ">
10 pages, 1513 KiB  
Article
Mycorrhizal Effects on Growth and Expressions of Stress-Responsive Genes (aquaporins and SOSs) of Tomato under Salt Stress
by Sheng-Min Liang, Qiu-Shuang Li, Ming-Yang Liu, Abeer Hashem, Al-Bandari Fahad Al-Arjani, Mekhled M. Alenazi, Elsayed Fathi Abd_Allah, Pandiyan Muthuramalingam and Qiang-Sheng Wu
J. Fungi 2022, 8(12), 1305; https://doi.org/10.3390/jof8121305 - 16 Dec 2022
Cited by 6 | Viewed by 2585
Abstract
Environmentally friendly arbuscular mycorrhizal fungi (AMF) in the soil can alleviate host damage from abiotic stresses, but the underlying mechanisms are unclear. The objective of this study was to analyze the effects of an arbuscular mycorrhizal fungus, Paraglomus occultum, on plant growth, [...] Read more.
Environmentally friendly arbuscular mycorrhizal fungi (AMF) in the soil can alleviate host damage from abiotic stresses, but the underlying mechanisms are unclear. The objective of this study was to analyze the effects of an arbuscular mycorrhizal fungus, Paraglomus occultum, on plant growth, nitrogen balance index, and expressions of salt overly sensitive genes (SOSs), plasma membrane intrinsic protein genes (PIPs), and tonoplast intrinsic protein genes (TIPs) in leaves of tomato (Solanum lycopersicum L. var. Huapiqiu) seedlings grown in 0 and 150 mM NaCl stress. NaCl stress severely inhibited plant growth, but P. occultum inoculation significantly improved plant growth. NaCl stress also suppressed the chlorophyll index, accompanied by an increase in the flavonoid index, whereas inoculation with AMF significantly promoted the chlorophyll index as well as reduced the flavonoid index under NaCl conditions, thus leading to an increase in the nitrogen balance index in inoculated plants. NaCl stress regulated the expression of SlPIP1 and SlPIP2 genes in leaves, and five SlPIPs genes were up-regulated after P. occultum colonization under NaCl stress, along with the down-regulation of only SlPIP1;2. Both NaCl stress and P. occultum inoculation induced diverse expression patterns in SlTIPs, coupled with a greater number of up-regulated TIPs in inoculated versus uninoculated plants under NaCl stress. NaCl stress up-regulated SlSOS2 expressions of mycorrhizal and non-mycorrhizal plants, while P. occultum significantly up-regulated SlSOS1 expressions by 1.13- and 0.45-fold under non-NaCl and NaCl conditions, respectively. It was concluded that P. occultum inoculation enhanced the salt tolerance of the tomato, associated with the nutrient status and stress-responsive gene (aquaporins and SOS1) expressions. Full article
(This article belongs to the Special Issue Physiological and Biochemical Mechanisms of Arbuscular Mycorrhiza under Stress)
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<p>Root colonization of tomato plants by <span class="html-italic">Paraglomus occultum</span>.</p> Full article ">Figure 2
<p>Changes in leaf chlorophyll index (Chi) (<b>a</b>), flavonoid index (Flav) (<b>b</b>), and nitrogen balance index (Nbi) (<b>c</b>) of tomato plants colonized by <span class="html-italic">Paraglomus occultum</span> under 0 and 150 mM NaCl stress. Data (means ± SD, <span class="html-italic">n</span> = 6) followed by different letters on the bar indicate significant differences among treatments at the 0.05 level. See <a href="#jof-08-01305-t001" class="html-table">Table 1</a> for abbreviations.</p> Full article ">Figure 3
<p>Changes in leaf <span class="html-italic">SlPIP1</span> and <span class="html-italic">SlPIP2</span> expressions of tomato plants colonized by <span class="html-italic">Paraglomus occultum</span> under 0 and 150 mM NaCl stress. Data (means ± SD, <span class="html-italic">n</span> = 3) followed by different letters on the bar indicate significant differences among treatments at the 0.05 level. See <a href="#jof-08-01305-t001" class="html-table">Table 1</a> for abbreviations.</p> Full article ">Figure 4
<p>Changes in leaf <span class="html-italic">SlTIP1</span> and <span class="html-italic">SlTIP2</span> expressions of tomato plants colonized by <span class="html-italic">Paraglomus occultum</span> under 0 and 150 mM NaCl stress. Data (means ± SD, <span class="html-italic">n</span> = 3) followed by different letters on the bar indicate significant differences among treatments at the 0.05 level. See <a href="#jof-08-01305-t001" class="html-table">Table 1</a> for abbreviations.</p> Full article ">Figure 5
<p>Changes in leaf <span class="html-italic">SlSOS1</span> and <span class="html-italic">SlSOS2</span> expressions of tomato plants colonized by <span class="html-italic">Paraglomus occultum</span> under 0 and 150 mM NaCl stress. Data (means ± SD, <span class="html-italic">n</span> = 3) followed by different letters on the bar indicate significant differences among treatments at the 0.05 level. See <a href="#jof-08-01305-t001" class="html-table">Table 1</a> for abbreviations.</p> Full article ">
15 pages, 7052 KiB  
Article
Cloning and Molecular Characterization of CmOxdc3 Coding for Oxalate Decarboxylase in the Mycoparasite Coniothyrium minitans
by Yuping Xu, Mingde Wu, Jing Zhang, Guoqing Li and Long Yang
J. Fungi 2022, 8(12), 1304; https://doi.org/10.3390/jof8121304 - 16 Dec 2022
Cited by 4 | Viewed by 1706
Abstract
Coniothyrium minitans (Cm) is a mycoparasitic fungus of Sclerotinia sclerotiorum (Ss), the causal agent of Sclerotinia stem rot of oilseed rape. Ss can produce oxalic acid (OA) as a phytotoxin, whereas Cm can degrade OA, thereby nullifying the toxic [...] Read more.
Coniothyrium minitans (Cm) is a mycoparasitic fungus of Sclerotinia sclerotiorum (Ss), the causal agent of Sclerotinia stem rot of oilseed rape. Ss can produce oxalic acid (OA) as a phytotoxin, whereas Cm can degrade OA, thereby nullifying the toxic effect of OA. Two oxalate decarboxylase (OxDC)-coding genes, CmOxdc1 and CmOxdc2, were cloned, and only CmOxdc1 was found to be partially responsible for OA degradation, implying that other OA-degrading genes may exist in Cm. This study cloned a novel OxDC gene (CmOxdc3) in Cm and its OA-degrading function was characterized by disruption and complementation of CmOxdc3. Sequence analysis indicated that, unlike CmOxdc1, CmOxdc3 does not have the signal peptide sequence, implying that CmOxDC3 may have no secretory capability. Quantitative RT-PCR showed that CmOxdc3 was up-regulated in the presence of OA, malonic acid and hydrochloric acid. Deletion of CmOxdc3 resulted in reduced capability to parasitize sclerotia of Ss. The polypeptide (CmOxDC3) encoded by CmOxdc3 was localized in cytoplasm and gathered in vacuoles in response to the extracellular OA. Taken together, our results demonstrated that CmOxdc3 is a novel gene responsible for OA degradation, which may work in a synergistic manner with CmOxdc1. Full article
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Multiple sequence alignment of oxalate decarboxylases (OxDCs) showing the evolutionary status of CmOxDC3. (<b>A</b>) Multiple amino acid sequences of OxDCs. Red letters indicate the region for the secretion signal peptides; Two dashed line boxes represent the cupin domains; Grey areas show the conserved motifs in the Cupin domains; Red areas indicate amino acid residues for Mn<sup>2+</sup>-binding sites; *, the same amino acid residues; chemically-similar amino acid residues; (<b>B</b>) Phylogenetic tree for 39 OxDCs. Red dots for CmOxDC1 to 3. The bootstrap values from 1000 replications are given at the branches of the tree. The domains and signal peptides were predicted by SMART and SignalP 5.0, and were displayed on the right of the tree.</p> Full article ">Figure 2
<p>The transcript pattern of <span class="html-italic">CmOxdc3</span> and <span class="html-italic">CmOxdc1</span> in response to oxalic acid (<b>A</b>) and different acids adjusted to pH 3 (<b>B</b>) as well as under different ambient pH conditions (<b>C</b>). <span class="html-italic">Cmactin</span> was used as a reference. ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 3
<p>Identification of the subcellular localization of CmOxDC3 (green fluorescent color) in hyphal cells of <span class="html-italic">Coniothyrium minitans</span>. Strain ZS-1-E1 constitutively expressing eGFP was used as the control.</p> Full article ">Figure 4
<p>Effect of disruption of <span class="html-italic">CmOxdc3</span> and/or <span class="html-italic">CmOxdc1</span> in <span class="html-italic">C. minitans</span> on its sensitivity to oxalic acid (OA). (<b>A</b>) Colony morphology of the disruption mutants and the wild type (WT) on PDA amended with OA; (<b>B</b>) Histogram showing mycelial growth-inhibition rates of the mutants and WT in response to OA. Means ± S.D. (<span class="html-italic">n</span> = 3) labeled with the same letters for each concentration of OA indicate no significant difference (<span class="html-italic">p</span> &gt; 0.01) according to Duncan’s multiple range test.</p> Full article ">Figure 5
<p>Histogram showing rates of degradation of oxalic acid (OA) by the wild type (WT) and mutants of <span class="html-italic">C. minitans</span>. Means ± S.D (<span class="html-italic">n</span> = 3) labeled with the same letters for each concentration of OA indicate no significant difference (<span class="html-italic">p</span> &gt; 0.01).</p> Full article ">Figure 6
<p>Mycoparasitism of the mutants and the wild type of <span class="html-italic">C. minitans</span> on hyphae and sclerotia of <span class="html-italic">S. sclerotiorum</span> (20 °C, 30 d). (<b>A</b>) Dual cultures between <span class="html-italic">C. minitans</span> and <span class="html-italic">S. sclerotiorum</span>. A schematic diagram showing the number of agar disks that gave rise to either <span class="html-italic">C. minitans</span>, <span class="html-italic">S. sclerotiorum</span> or both. Each circle represents a colony developed from a mycelial agar disk sampled from Zones I, II, III or IV in the dual cultures (see the schematic diagram on the left). Red dash lines on the dual-culture plates roughly differentiate the yellow areas from the blue areas. Yellow means low pH due to oxalic acid (OA) secreted by <span class="html-italic">S. sclerotiorum</span> and blue represents high pH likely due to OA degradation by <span class="html-italic">C. minitans</span>. (<b>B</b>) Petri dishes on the top line with the sclerotia of <span class="html-italic">S. sclerotiorum</span> infected by different strains of <span class="html-italic">C. minitans</span>; The sclerotia in the middle line indicate the typical symptoms of the <span class="html-italic">C. minitans</span>-infected sclerotia; and Sections of the <span class="html-italic">C. minitans</span>-infected sclerotia on the bottom line. (<b>C</b>) Histogram showing sclerotial rot indices caused by different strains of <span class="html-italic">C. minitans</span>. Means ± SD (<span class="html-italic">n</span> = 3) labeled with the same letters indicate no significant difference (<span class="html-italic">p</span> &gt; 0.01).</p> Full article ">
16 pages, 3482 KiB  
Article
Fungal-Modified Lignin-Enhanced Physicochemical Properties of Collagen-Based Composite Films
by Alitenai Tunuhe, Pengyang Liu, Mati Ullah, Su Sun, Hua Xie, Fuying Ma, Hongbo Yu, Yaxian Zhou and Shangxian Xie
J. Fungi 2022, 8(12), 1303; https://doi.org/10.3390/jof8121303 - 16 Dec 2022
Cited by 1 | Viewed by 1848
Abstract
Renewable and biodegradable materials have attracted broad attention as alternatives to existing conventional plastics, which have caused serious environmental problems. Collagen is a potential material for developing versatile film due to its biosafety, renewability, and biodegradability. However, it is still critical to overcome [...] Read more.
Renewable and biodegradable materials have attracted broad attention as alternatives to existing conventional plastics, which have caused serious environmental problems. Collagen is a potential material for developing versatile film due to its biosafety, renewability, and biodegradability. However, it is still critical to overcome the low mechanical, antibacterial and antioxidant properties of the collagen film for food packaging applications. To address these limitations, we developed a new technology to prepare composite film by using collagen and fungal-modified APL (alkali pretreatment liquor). In this study, five edible and medical fungi, Cunninghamella echinulata FR3, Pleurotus ostreatus BP3, Ganoderma lucidum EN2, Schizophyllum commune DS1 and Xylariaceae sp. XY were used to modify the APL, and that showed that the modified APL significantly improved the mechanical, antibacterial and antioxidant properties of APL/Collagen composite films. Particularly, the APL modified by BP3, EN2 and XY showed preferable performance in enhancing the properties of the composite films. The tensile strength of the film was increased by 1.5-fold in the presence of the APL modified by EN2. To further understand the effect of fungal-biomodified APL on the properties of the composite films, a correlation analysis between the components of APL and the properties of composite films was conducted and indicated that the content of aromatic functional groups and lignin had a positive correlation with the enhanced mechanical and antioxidant properties of the composite films. In summary, composite films prepared from collagen and fungal biomodified APL showed elevated mechanical, antibacterial and antioxidant properties, and the herein-reported novel technology prospectively possesses great potential application in the food packaging industry. Full article
(This article belongs to the Special Issue Fungal Applications in Bioenergy, Bioremediation, Biomedicine, Biocontrol, and Biomaterials)
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<p>Contents of sugar with different molecular weights (<b>A</b>) and contents of soluble solids (<b>B</b>) in APL after fungal biomodification. Polysaccharides of different molecular weights were graded through a 200D dialysis bag. AC (none-modified APL); AF (<span class="html-italic">C. echinulate</span> FR3-modified APL); AB (<span class="html-italic">P. ostreatus</span> BP3-modified APL); AE (<span class="html-italic">G. lucidum</span> EN2-modified APL); AD (<span class="html-italic">S. commune</span> DS1-modified APL); AX (<span class="html-italic">Xylariaceae</span> sp. XY-modified APL). For the comparison between different biological modifications, different lowercase letters represent significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 2
<p>The SEM images (400×) of the collagen film surfaces (<b>A</b>–<b>G</b>) and cross-sections (<b>a</b>–<b>g</b>). <b>A/a</b>: Control (collagen film); <b>B/b</b>: collagen with none-modified APL film; <b>C/c</b>: collagen with <span class="html-italic">C. echinulate</span> FR3-modified APL film; <b>D/d</b>: collagen with <span class="html-italic">G. lucidum</span> EN2-modified APL film; <b>E/e</b>: collagen with <span class="html-italic">P. ostreatus</span> BP3-modified APL film; <b>F/f</b>: collagen with <span class="html-italic">S. commune</span> DS1-modified APL film; <b>G/g</b>: collagen with <span class="html-italic">Xylariaceae</span> sp. XY-modified APL film.</p> Full article ">Figure 3
<p>ATR−FTIR spectrum (<b>A</b>–<b>C</b>) and X-ray diffraction spectrum (<b>D</b>) of APL with collagen films. (<b>A</b>): wavenumber from 500 to 4000 cm<sup>−1</sup> of the different fungi-modified APL; (<b>B</b>): wavenumber from 500 to 2000 cm<sup>−1</sup>of the different fungi-modified APL; (<b>C</b>): wavenumber from 500 to 4000 cm<sup>−1</sup> of the different fungi-modified APL with collagen film. C (collagen film); C−AC (collagen with none−modified APL film); C−AF (collagen with <span class="html-italic">C. echinulate</span> FR3−modified APL film); C−AE (collagen with <span class="html-italic">G. lucidum</span> EN2−modified APL film); C−AB (collagen with <span class="html-italic">P. ostreatus</span> BP3−modified APL film); C−AD (collagen with <span class="html-italic">S. commune</span> DS1−modified APL film); C−AX (collagen with <span class="html-italic">Xylariaceae</span> sp. XY−modified APL film).</p> Full article ">Figure 4
<p>Different films co-cultured with <span class="html-italic">E. coli</span> after 48h and observation of effects on the growth inhibition of <span class="html-italic">E. coli</span>. (<b>A</b>) <span class="html-italic">E. coli</span>; (<b>B</b>) collagen film; (<b>C</b>) collagen with none-modified APL film; (<b>D</b>) collagen with <span class="html-italic">C. echinulate</span> FR3-modified APL film; (<b>E</b>) collagen with <span class="html-italic">G. lucidum</span> EN2-modified APL film; (<b>F</b>) collagen with <span class="html-italic">P. ostreatus</span> BP3-modified APL film; (<b>G</b>) collagen with <span class="html-italic">S. commune</span> DS1-modified APL film; (<b>H</b>) collagen with <span class="html-italic">Xylariaceae</span> sp. XY-modified APL film.</p> Full article ">Figure 5
<p>The ABTS+ scavenging rate (<b>A</b>) and WCA (<b>B</b>) of the collagen-based films. C (collagen film); C-AC (collagen with none-modified APL film); C-AF (collagen with <span class="html-italic">C. echinulate</span> FR3-modified APL film); C-AE (collagen with <span class="html-italic">G. lucidum</span> EN2-modified APL film); C-AB (collagen with <span class="html-italic">P. ostreatus</span> BP3-modified APL film); C-AD (collagen with <span class="html-italic">S. commune</span> DS1-modified APL film); C-AX (collagen with <span class="html-italic">Xylariaceae</span> sp. XY-modified APL film). For the comparison between different biological modifications, different lowercase letters represent significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>The digital photos (<b>A</b>) and the transmittances (<b>B</b>) of the collagen-based films. (<b>a</b>): collagen film; (<b>b</b>): collagen with non-modified APL film; (<b>c</b>): collagen with <span class="html-italic">C. echinulate</span> FR3-modified APL film; (<b>d</b>): collagen with <span class="html-italic">G. lucidum</span> EN2-modified APL film; (<b>e</b>): collagen with <span class="html-italic">P. ostreatus</span> BP3-modified APL film; (<b>f</b>): collagen with <span class="html-italic">S. commune</span> DS1-modified APL film; (<b>g</b>): collagen with <span class="html-italic">Xylariaceae</span> sp. XY-modified APL film.</p> Full article ">Figure 7
<p>Mechanical properties of collagen-based film. TS = tensile strength, EAB = elongation at break. C (collagen film); C-AC (collagen with none-modified APL film); C-AF (collagen with <span class="html-italic">C. echinulate</span> FR3-modified APL film); C-AE (collagen with <span class="html-italic">G. lucidum</span> EN2-modified APL film); C-AB (collagen with <span class="html-italic">P. ostreatus</span> BP3-modified APL film); C-AD (collagen with <span class="html-italic">S. commune</span> DS1-modified APL film); C-AX (collagen with <span class="html-italic">Xylariaceae</span> sp. XY-modified APL film). For the comparison between different biological modifications, different lowercase letters represent significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 8
<p>The heatmap combined with the Pearson correlation coefficient among the different factors. Factors including hemicellulose, lignin, acid-soluble lignin, acid insoluble lignin and cellulose from soluble solids; phenolic hydroxyl, total carbohydrate, total hydroxyl and aliphatic hydroxyl by 31P NMR and aromatic compounds by GC−MS were related to the antioxidant properties (ORAC), hydrophobicity (WCA), and mechanical properties (TS and EAB). Different color blocks represent different correlation coefficient values of the corresponding variables, using the color scale as a reference. ORAC (antioxidant activity obtained by measuring ABTS<sup>+</sup> clearance), WCA (water contact angle), TS (tensile strength), EAB (elongation at break).</p> Full article ">
20 pages, 6819 KiB  
Article
Phylogenetic Analyses of Hydnobolites and New Species from China
by Shan-Ping Wan, Lan-Lan Huang, Meng-Jin Cui, Cheng-Jin Yu, Wei Liu, Rui Wang, Xiao-Fei Shi and Fu-Qiang Yu
J. Fungi 2022, 8(12), 1302; https://doi.org/10.3390/jof8121302 - 15 Dec 2022
Cited by 1 | Viewed by 1531
Abstract
Hydnobolites is an ectomycorrhizal fungal genus with hypogeous ascomata in the family Pezizaceae (Pezizales). Molecular analyses of Hydnobolites using both single (ITS) and concatenated gene datasets (ITS-nLSU) showed a total of 223 sequences, including 92 newly gained sequences from Chinese specimens. [...] Read more.
Hydnobolites is an ectomycorrhizal fungal genus with hypogeous ascomata in the family Pezizaceae (Pezizales). Molecular analyses of Hydnobolites using both single (ITS) and concatenated gene datasets (ITS-nLSU) showed a total of 223 sequences, including 92 newly gained sequences from Chinese specimens. Phylogenetic results based on these two datasets revealed seven distinct phylogenetic clades. Among them, the ITS phylogenetic tree confirmed the presence of at least 42 phylogenetic species in Hydnobolites. Combined the morphological observations with molecular analyses, five new species of Hydnobolites translucidus sp. nov., H. subrufus sp. nov., H. lini sp. nov., H. sichuanensis sp. nov. and H. tenuiperidius sp. nov., and one new record species of H. cerebriformis Tul., were illustrated from Southwest China. Macro- and micro-morphological analyses of ascomata revealed a few, but diagnostic differences between the H. cerebriformis complex, while the similarities of the ITS sequences ranged from 94.4 to 97.2% resulting in well-supported clades. Full article
(This article belongs to the Special Issue Edible and Medicinal Macrofungi)
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<p>The consensus phylogram of the Genus <span class="html-italic">Hydnobolites</span> obtained in RAxML of ITS rDNA. Nodes were annotated if supported by &gt; 70% ML BP or &gt; 0.90 Bayesian PP, but non-significant support values are exceptionally represented inside parentheses. New species are in bold font. Complex is reported as red thick branch.</p> Full article ">Figure 2
<p>The consensus phylogram of the Genus <span class="html-italic">Hydnobolites</span> obtained in MrBayes of ITS-nrLSU. Nodes were annotated if supported by &gt; 70% ML BP or &gt; 0.90 Bayesian PP, but non-significant support values are exceptionally represented inside parentheses. New species are in bold font. Complex is reported as red thick branch.</p> Full article ">Figure 3
<p><span class="html-italic">Hydnobolites translucidus</span> (Holotype, HKAS95861). (<b>a</b>) Fresh ascomata and gleba; (<b>b</b>) dried ascoma; (<b>c</b>) peridium; (<b>d</b>) paraphyses; (<b>e</b>) asci and ascospores under LM; (<b>f</b>) ascospore under SEM. Scale bars: a = 1 cm; b = 1 mm; c–e = 20 μm; f = 5 μm.</p> Full article ">Figure 4
<p><span class="html-italic">Hydnobolites subrufus</span> (Holotype, HKAS95869). (<b>a</b>) Fresh ascomata and gleba; (<b>b</b>) dried ascoma; (<b>c</b>,<b>d</b>) peridium and paraphyses; (<b>e</b>) asci and ascospores under LM; (<b>f</b>) ascospore under SEM. Scale bars: a = 1 cm; b = 1 mm; c–e = 20 μm; f = 5 μm.</p> Full article ">Figure 5
<p><span class="html-italic">Hydnobolites lini</span> (Holotype, YNAU0860). (<b>a</b>) Fresh ascomata and gleba; (<b>b</b>) dried ascoma; (<b>c</b>) peridium; (<b>d</b>) paraphyses; (<b>e</b>) ascus and ascospores under LM; (<b>f</b>) ascospore under SEM. Scale bars: a = 1 cm; b = 1 mm; c–e = 20 μm; f = 5 μm.</p> Full article ">Figure 6
<p><span class="html-italic">Hydnobolites sichuanensis</span> (Holotype, YNAU0705). (<b>a</b>) Fresh ascoma and gleba; (<b>b</b>) dried ascoma; (<b>c</b>) peridium; (<b>d</b>) paraphyses; (<b>e</b>) asci and ascospores under LM; (<b>f</b>) ascospore under SEM. Scale bars: a = 1 cm; b = 1 mm; c–e = 20 μm; f = 5 μm.</p> Full article ">Figure 7
<p><span class="html-italic">Hydnobolites tenuiperidius</span> (Holotype, YNAU0899). (<b>a</b>) Fresh ascoma and gleba; (<b>b</b>) surface of dried ascoma; (<b>c</b>) peridium; (<b>d</b>) paraphyses; (<b>e</b>) ascus and ascospores under LM; (<b>f</b>) ascospore under SEM. Scale bars: a = 1 cm; b = 0.1 mm; c–e = 20 μm; f = 5 μm.</p> Full article ">Figure 8
<p><span class="html-italic">Hydnobolites cerebriformis</span> (YNAU0318). (<b>a</b>) Fresh ascomata; (<b>b</b>) dried ascoma; (<b>c</b>,<b>e</b>) peridium; (<b>d</b>) paraphyses; (<b>f</b>) hyphae of gleba. Scale bars: a = 1 cm; b = 1 mm; c–f = 20 μm.</p> Full article ">
36 pages, 7496 KiB  
Article
Taxonomy and Multigene Phylogeny of Diaporthales in Guizhou Province, China
by Si-Yao Wang, Eric H. C. McKenzie, Alan J. L. Phillips, Yan Li and Yong Wang
J. Fungi 2022, 8(12), 1301; https://doi.org/10.3390/jof8121301 - 15 Dec 2022
Cited by 7 | Viewed by 2289
Abstract
In a study of fungi isolated from plant material in Guizhou Province, China, we identified 23 strains of Diaporthales belonging to nine species. These are identified from multigene phylogenetic analyses of ITS, LSU, rpb2, tef1, and tub2 gene sequence data coupled [...] Read more.
In a study of fungi isolated from plant material in Guizhou Province, China, we identified 23 strains of Diaporthales belonging to nine species. These are identified from multigene phylogenetic analyses of ITS, LSU, rpb2, tef1, and tub2 gene sequence data coupled with morphological studies. The fungi include a new genus (Pseudomastigosporella) in Foliocryphiaceae isolated from Acer palmatum and Hypericum patulum, a new species of Chrysofolia isolated from Coriaria nepalensis, and five new species of Diaporthe isolated from Juglans regia, Eucommia ulmoides, and Hypericum patulum. Gnomoniopsis rosae and Coniella quercicola are newly recorded species for China. Full article
(This article belongs to the Special Issue Polyphasic Identification of Fungi 2.0)
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<p>Phylogram generated from RAxML analysis of a concatenated ITS-LSU-<span class="html-italic">rpb2</span>-<span class="html-italic">tef1</span>-<span class="html-italic">tub2</span> sequence dataset to represent the phylogenetic relationships of taxa in <span class="html-italic">Foliocryphiaceae</span> and <span class="html-italic">Cryphonectriaceae</span>. The tree was rooted with <span class="html-italic">Dwiroopa lythri</span> (CBS 109755, ex-type strain) and <span class="html-italic">Dw. punicae</span> (CBS 143163, ex-type strain). Bootstrap support values for ML and MP equal to or greater than 70% and Bayesian posterior probabilities equal to or higher than 0.95 PP are indicated above the nodes as ML/MP/PP. Support values lower than 70% ML/MP and 0.95 PP are indicated by a hyphen (-). The newly generated sequences are indicated in red.</p> Full article ">Figure 2
<p>Results of the pairwise homoplasy index (PHI) test of closely related species using both LogDet transformation and splits decomposition. Our strains are indicated in red, other involved strains are indicated in blue. (<b>a</b>) <span class="html-italic">Foliocryphiaceae</span>. (<b>b</b>–<b>f</b>) <span class="html-italic">Diaporthe</span>. (<b>g</b>) <span class="html-italic">Gnomoniopsis</span>. (<b>h</b>) <span class="html-italic">Coniella</span>.</p> Full article ">Figure 3
<p>Phylogram generated from RAxML analysis of a concatenated ITS-<span class="html-italic">tef1</span>-<span class="html-italic">tub2</span> sequence dataset to represent the phylogenetic relationships of taxa in <span class="html-italic">Diaporthe</span>. The tree was rooted with <span class="html-italic">Diaporthella corylina</span> (CBS 121124, ex-type strain) and <span class="html-italic">Di. cryptica</span> (CBS 140348, ex-neotype strain). Bootstrap support values for ML and MP equal to or greater than 70% and Bayesian posterior probabilities equal to or higher than 0.95 PP are indicated above the nodes as ML/MP/PP. Support values lower than 70% ML/MP and 0.95 PP are indicated by a hyphen (-). The newly generated sequences are indicated in red.</p> Full article ">Figure 4
<p>Phylogram generated from RAxML analysis of a concatenated ITS-LSU-<span class="html-italic">rpb2</span>-<span class="html-italic">tef1</span>-<span class="html-italic">tub2</span> sequence dataset to represent the phylogenetic relationships of taxa in <span class="html-italic">Gnomoniopsis</span>. The tree was rooted with <span class="html-italic">Sirococcus tsugae</span> (CBS 119626). Bootstrap support values for ML and MP equal to or greater than 70% and the Bayesian posterior probabilities equal to or higher than 0.95 PP are indicated above the nodes as ML/MP/PP. Support values lower than 70% ML/MP and 0.95 PP are indicated by a hyphen (-). The newly generated sequences are indicated in red.</p> Full article ">Figure 5
<p><span class="html-italic">Pseudomastigosporella guizhouensis</span> (GUCC 406.6). Hosts: (<b>a</b>) <span class="html-italic">Hypericum patulum</span>; (<b>b</b>) <span class="html-italic">Acer palmatum</span>. (<b>c</b>) Colony on PDA after 2 wk at 25 °C (left: above, right: reverse). (<b>d</b>) Colony on OA after 2 wk at 25 °C (left: above, right: reverse). (<b>e</b>) Mass of conidia. (<b>f</b>) Conidioma. (<b>g</b>,<b>h</b>) Conidiomata and conidiogenous cells. (<b>i</b>–<b>l</b>) Conidia. Scale bars: (<b>f</b>–<b>i</b>) = 50 µm; (<b>j</b>–<b>l</b>) = 10 µm.</p> Full article ">Figure 6
<p><span class="html-italic">Chrysofolia coriariae</span> (GUCC 416.4). (<b>a</b>,<b>b</b>) Host: <span class="html-italic">Coriaria nepalensis</span>. (<b>c</b>) Colony on PDA after 2 wk at 25 °C (left: above, right: reverse). (<b>d</b>) Colony on OA after 2 wk at 25 °C (left: above, right: reverse). (<b>e</b>,<b>f</b>) Conidial masses. (<b>g</b>,<b>h</b>) Conidiomata. (<b>i</b>,<b>j</b>) Conidiogenous cells. (<b>k</b>,<b>l</b>) Conidia. Scale bars: (<b>g</b>–<b>i</b>) = 50 µm; (<b>j</b>–<b>l</b>) = 10 µm.</p> Full article ">Figure 7
<p><span class="html-italic">Diaporthe juglandigena</span> (GUCC 422.16). (<b>a</b>) Host: <span class="html-italic">Juglans regia</span>. (<b>b</b>) Colony on PDA after 2 wk (left: above, right: reverse). (<b>c</b>) Colony on OA after 2 wk (left: above, right: reverse). (<b>d</b>,<b>e</b>) Mass of conidia. (<b>f</b>,<b>g</b>) Conidiogenous cells. (<b>h</b>,<b>i</b>) Alpha conidia. (<b>j</b>,<b>k</b>) Beta conidia. Scale bars: (<b>f</b>–<b>k</b>) = 10 µm.</p> Full article ">Figure 8
<p><span class="html-italic">Diaporthe eucommiigena</span> (GUCC 420.9). (<b>a</b>,<b>b</b>) Host: <span class="html-italic">Eucommia ulmoides</span>. (<b>c</b>) Colony on PDA after 2 wk at 25 °C (left: above, right: reverse). (<b>d</b>) Colony on OA after 2 wk at 25 °C (left: above, right: reverse). (<b>e</b>–<b>g</b>) Mass of conidia. (<b>h</b>) Conidiogenous cells. (<b>i</b>,<b>j</b>) Alpha conidia. (<b>k</b>,<b>l</b>) Beta conidia. (<b>m</b>,<b>n</b>) Gamma conidia. Scale bars: (<b>h</b>–<b>n</b>) = 10 µm.</p> Full article ">Figure 9
<p><span class="html-italic">Diaporthe dejiangensis</span> (GUCC 421.2). (<b>a</b>) Host: <span class="html-italic">Juglans regia</span>. (<b>b</b>) Colony on PDA after 2 wk at 25 °C (left: above, right: reverse). (<b>c</b>) Colony on OA after 2 wk at 25 °C (left: above, right: reverse). (<b>d</b>–<b>f</b>) Mass of conidia. (<b>g</b>,<b>h</b>) Conidiogenous cells. (<b>i</b>,<b>j</b>) Alpha conidia. Scale bars: (<b>g</b>–<b>j</b>) = 10 µm.</p> Full article ">Figure 10
<p><span class="html-italic">Diaporthe tongrensis</span> (GUCC 421.10). (<b>a</b>) Host: <span class="html-italic">Juglans regia</span>. (<b>b</b>) Colony on PDA after 2 wk at 25 °C (left: above, right: reverse). (<b>c</b>) Colony on OA after 2 wk at 25 °C (left: above, right: reverse). (<b>d</b>,<b>e</b>) Mass of conidia. (<b>f</b>,<b>g</b>) Conidiogenous cells. h Alpha conidia. (<b>i</b>,<b>j</b>) Beta conidia. Scale bars: (<b>f</b>–<b>j</b>) = 10 µm.</p> Full article ">Figure 11
<p><span class="html-italic">Diaporthe hyperici</span> (GUCC 414.4). (<b>a</b>,<b>b</b>) Host: <span class="html-italic">Hypericum patulum</span>. (<b>c</b>) Colony on PDA after 2 wk at 25 °C (left: above, right: reverse). (<b>d</b>) Colony on OA after 2 wk at 25 °C (left: above, right: reverse). (<b>e</b>,<b>f</b>) Mass of conidia. (<b>g</b>–<b>i</b>) Conidiogenous cells. (<b>j</b>) Alpha conidia. (<b>k</b>) Alpha and beta conidia. (<b>l</b>,<b>m</b>) Beta conidia. Scale bars: (<b>g</b>,<b>h</b>) = 50 µm; (<b>i</b>–<b>m</b>) = 10 µm.</p> Full article ">Figure 12
<p><span class="html-italic">Gnomoniopsis rosae</span> (GUCC 408.7). (<b>a</b>) Host: <span class="html-italic">Rose</span> sp. (<b>b</b>) Colonies on PDA after 2 wk at 25 °C (left: above, right: reverse). (<b>c</b>) Colony on OA after 2 wk at 25 °C (left: above, right: reverse). (<b>d</b>) Mass of conidia. (<b>e</b>) Conidiomata. (<b>f</b>,<b>g</b>) Conidiogenous cells. (<b>h</b>–<b>j</b>) Conidia. Scale bars: (<b>e</b>–<b>j</b>) = 10 µm.</p> Full article ">Figure 13
<p><span class="html-italic">Coniella quercicola</span> (GUCC 412.3). Hosts. (<b>a</b>) <span class="html-italic">Acer palmatum</span>; (<b>b</b>) <span class="html-italic">Aralia chinensis</span>; (<b>c</b>) <span class="html-italic">Hypericum patulum</span>. (<b>d</b>) Colony on PDA after 2 wk 25 °C (left: above, right: reverse). (<b>e</b>) Colony on OA after 2 wk 25 °C (left: above, right: reverse). (<b>f</b>) Mass of conidia. (<b>g</b>) Conidioma. (<b>h</b>–<b>j</b>) Conidiogenous cells. (<b>k</b>–<b>n</b>) Conidia. Scale bars: (<b>g</b>) = 50 µm; (<b>h</b>–<b>n</b>) = 10 µm.</p> Full article ">Figure 14
<p>Phylogram generated from RAxML analysis of a concatenated ITS-LSU-<span class="html-italic">tef1</span> sequence dataset to represent the phylogenetic relationships of taxa in <span class="html-italic">Coniella</span>. The tree was rooted with <span class="html-italic">C. fragariae</span> (CBS 172.49, ex-type strain) and <span class="html-italic">C. nigra</span> (CBS 165.60, ex-type strain). Bootstrap support values for ML and MP equal to or greater than 70% and the Bayesian posterior probabilities equal to or higher than 0.95 PP are indicated above the nodes as ML/MP/PP. Support values lower than 70% ML/MP and 0.95 PP are indicated by a hyphen (-). The newly generated sequences are indicated in red.</p> Full article ">
15 pages, 2853 KiB  
Article
Fusarium oxysporum Casein Kinase 1, a Negative Regulator of the Plasma Membrane H+-ATPase Pma1, Is Required for Development and Pathogenicity
by Melani Mariscal, Cristina Miguel-Rojas, Concepción Hera, Tânia R. Fernandes and Antonio Di Pietro
J. Fungi 2022, 8(12), 1300; https://doi.org/10.3390/jof8121300 - 15 Dec 2022
Cited by 3 | Viewed by 2030
Abstract
Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pH [...] Read more.
Like many hemibiotrophic plant pathogens, the root-infecting vascular wilt fungus Fusarium oxysporum induces an increase in the pH of the surrounding host tissue. How alkalinization promotes fungal infection is not fully understood, but recent studies point towards the role of cytosolic pH (pHc) and mitogen-activated protein kinase (MAPK) signaling. In fungi, pHc is mainly controlled by the essential plasma membrane H+-ATPase Pma1. Here we created mutants of F. oxysporum lacking casein kinase 1 (Ck1), a known negative regulator of Pma1. We found that the ck1Δ mutants have constitutively high Pma1 activity and exhibit reduced alkalinization of the surrounding medium as well as decreased hyphal growth and conidiation. Importantly, the ck1Δ mutants exhibit defects in hyphal chemotropism towards plant roots and in pathogenicity on tomato plants. Thus, Ck1 is a key regulator of the development and virulence of F. oxysporum. Full article
(This article belongs to the Special Issue Signal Transductions in Fungi 2.0)
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Figure 1

Figure 1
<p>Loss of <span class="html-italic">ck1</span> strongly impacts colony growth of <span class="html-italic">F. oxysporum</span>. (<b>A</b>) Aliquots of 5 × 10<sup>4</sup> fresh microconidia of the indicated strains were spot-inoculated on minimal medium agar (MMA) or complete medium agar (YPDA) plates. Colonies were imaged 3 days after inoculation. The plates shown are representative of three independent experiments with three plates each. (<b>B</b>) The increase in colony area was calculated after spot-inoculation of the indicated strains on MMA and YPGA and normalized to that of the wt strain (100%). <span class="html-italic">p</span> &lt; 0.05 (*), <span class="html-italic">p</span> &lt; 0.0001 (****) and not significant (ns) versus wt, according to Welch’s <span class="html-italic">t</span>-test. versus wt, according to Welch’s <span class="html-italic">t</span>-test. Data show the mean and standard deviation from at least three independent experiments.</p> Full article ">Figure 2
<p>Loss of <span class="html-italic">ck1</span> severely affects growth and conidiation in a liquid medium. (<b>A</b>) Growth of the indicated strains was monitored in microwell plates containing liquid yeast extract-dextrose (YD) medium by measuring absorbance at 600 nm. Values were normalized to time 0. <span class="html-italic">p</span> &lt; 0.01 (**) and <span class="html-italic">p</span> &lt; 0.0001 (****), ns, not significant versus wt, according to Welch’s <span class="html-italic">t</span>-test. Data presented are the mean ±and standard deviation of three replicate microwells from one representative experiment. The experiment was performed twice with similar results. (<b>B</b>) The percentage of germinated microconidia was determined 15 h after inoculation in PDB medium. ns, not significant versus wt, according to Welch’s <span class="html-italic">t</span>-test. The data presented are the mean and standard deviation of two biological replicates with three technical replicates each. (<b>C</b>) Microconidia production by the indicated strains was determined after 48 h growth in liquid PDB medium and normalized to the wt strain (100%). <span class="html-italic">p</span> &lt; 0.0001 (****) versus wt, according to Welch’s <span class="html-italic">t</span>-test. The data presented are the mean and standard deviation of two independent experiments with three technical replicates each.</p> Full article ">Figure 3
<p>Ck1 contributes to the response to membrane and hyperosmotic stress. Aliquots of 10<sup>5</sup>, 10<sup>4</sup>, and 10<sup>3</sup> fresh microconidia of the indicated strains were spot-inoculated on YPGA plates in the absence or presence of 0.075% (<span class="html-italic">w/v</span>) SDS, 1.2 M NaCl, or 1.2 M KCl. The plates were imaged after two days of incubation at 28 °C. Images are from one representative experiment. Experiments were performed twice, each with three independent plates per growth condition.</p> Full article ">Figure 4
<p>Ck1 negatively regulates Pma1 activity and controls pH homeostasis. (<b>A</b>) Pma1 ATPase activity was assayed in total membrane fractions isolated from germlings of the indicated strains after 15 h incubation (see <a href="#sec2-jof-08-01300" class="html-sec">Section 2</a>). The activity was normalized to that of the wt strain. <span class="html-italic">p</span> &lt; 0.001 (***), ns, not significant versus wt according to Welch’s test. Data shown are the mean and standard deviation from three independent experiments with three technical replicates each. (<b>B</b>) Aliquots of 5 × 10<sup>4</sup> fresh microconidia of the indicated strains were spot-inoculated on minimal medium agar (MMA) plates and incubated for 7 days. At the indicated time points, 4 mm<sup>2</sup> squares were cut from the center of each colony, homogenized in 50 µL of ultrapure water, and the pH was measured with a microelectrode. <span class="html-italic">p</span> &lt; 0.0001(****), <span class="html-italic">p</span> &lt; 0.001 (***), and <span class="html-italic">p</span> &lt; 0.01 (**) versus wt according to two-way ANOVA and Turkey Test. Data presented are the mean and standard deviation from three independent biological experiments, each with three colonies analyzed. (<b>C</b>) Cytosolic pH (pH<sub>c</sub>) was monitored in germlings of the indicated strains expressing the ratiometric pH probe pHluorin Microconidia were germinated 15 h in YD medium buffered at pH 7.4, germlings were washed and resuspended in KSU buffer at pH 6.0, aliquoted into microwells and pre-incubated 60 min at 28 °C, before monitoring pH<sub>c</sub> spectrophluorometrically every 5 min for 2 h. For pH<sub>c</sub> measurements, the ratio between the emission intensities (collected at 510 nm) after excitation at 395 nm and 475 nm was calculated. Data show the mean and standard deviation of three independent replicate wells from one representative experiment. Experiments were performed three times with similar results.</p> Full article ">Figure 5
<p>The <span class="html-italic">ck1</span>Δ mutant displays increased resistance to acetic acid-induced cell death. (<b>A</b>) The pH<sub>c</sub> was monitored in <span class="html-italic">F. oxysporum</span> wt germlings expressing pHluorin. Microconidia were germinated during 15 h in liquid PDB, germlings were washed and transferred to KSU buffered at pH 3.0, aliquoted into microwells and pre-incubated 60 min at 28 °C before adding or not 40 mM acetic acid (AA). Samples were monitored spectrophluorometrically every 5 min starting 20 min before AA addition. pH<sub>c</sub> was calculated as the ratio between the emission intensities at 510 nm after excitation at 395 nm and 475 nm and normalized to the standard curve. Data show the mean and standard deviation from three replicate microwells from one representative experiment. Experiments were performed at least twice with similar results. (<b>B</b>) Microconidia of the indicated strains were germinated during 15 h in PDB, then the pH of the medium was adjusted to 3.0 with diluted HCl and germlings were pre-incubated for 1 h at 28 °C before adding 0.4 mM AA. Germlings collected before (time 0) or at the indicated times after AA addition (min) were diluted and plated on complete medium plates. After two days of incubation at 28 °C, the number of colonies was counted, and survival was calculated. <span class="html-italic">p</span> &lt; 0.0001(****), <span class="html-italic">p</span> &lt; 0.001 (**), ns, not significant versus wt according to two-way ANOVA and Bonferroni Test. Bars show the mean and standard deviation from three independent biological experiments.</p> Full article ">Figure 6
<p>Ck1 is required for the chemotropic response to an acid pH gradient. The directed growth of germ tubes of the indicated <span class="html-italic">F. oxysporum</span> strains was determined after 8 h exposure to opposing gradients of 25 mM HCl and NaOH. <span class="html-italic">p</span> &lt; 0.0001 (****), ns, not significant versus wt according to Welch’s <span class="html-italic">t</span>-test. Data show the mean and standard deviation from three independent experiments with three replicates (n = 500 germ tubes per experiment).</p> Full article ">Figure 7
<p>The <span class="html-italic">ck1</span>Δ mutant shows altered MAPK phosphorylation levels. Microconidia of the indicated <span class="html-italic">F. oxysporum</span> strains were germinated in PDB for 15 h at 28 °C; then germlings were washed and resuspended in KSU buffer, pH 6.0. After 1 h at 28 °C, 0.5 mM of the specific Pma1 inhibitor diethylstilbestrol (DES) was added. Protein extracts collected either before (time 0) or at the indicated times after DES addition, were subjected to immunoblot analysis with anti-phospho-p44/42 MAPK antibody, which specifically detects the phosphorylated version of the Mpk1 and Fmk1 MAPKs. Anti-α-tubulin (α-Tub) antibody was used as a loading control.</p> Full article ">Figure 8
<p>Ck1 is required for invasive hyphal growth and pathogenicity on tomato plants. (<b>A</b>) Aliquots of 5 × 10<sup>4</sup> fresh microconidia of the indicated strains were spot-inoculated on top of cellophane membranes placed on MMA plates that were either unbuffered of buffered to pH 7.0 or 5.0 with 100 mM MES. Plates were imaged after 3 days incubation at 28 °C (before), then the cellophane membrane with the fungal colony was removed, and plates were incubated for an additional day to visualize the presence of mycelium on the plate, indicative of penetration through the cellophane (after). The images shown are representative of three independent experiments, each with three plates per treatment. (<b>B</b>) Kaplan–Meier plots showing the survival of tomato plants (cv. Moneymaker) inoculated by dipping roots into a suspension of 5 × 10<sup>6</sup> fresh microconidia/mL of the indicated fungal strains. Survival of tomato plants was recorded for 40 days. Ten plants were used per treatment. <span class="html-italic">p</span> &lt; 0.0001 (****) versus wt according to the log-rank test. The data shown are from one representative experiment. Experiments were performed three times with similar results.</p> Full article ">
12 pages, 2451 KiB  
Article
Alumina as an Antifungal Agent for Pinus elliottii Wood
by Andrey P. Acosta, Ezequiel Gallio, Nidria Cruz, Arthur B. Aramburu, Nayara Lunkes, André L. Missio, Rafael de A. Delucis and Darci A. Gatto
J. Fungi 2022, 8(12), 1299; https://doi.org/10.3390/jof8121299 - 14 Dec 2022
Cited by 1 | Viewed by 1583
Abstract
This work deals with the durability of a Pinus elliotti wood impregnated with alumina (Al2O3) particles. The samples were impregnated at three different Al2O3 weight fractions (c.a. 0.1%, 0.3% and 0.5%) and were then exposed to [...] Read more.
This work deals with the durability of a Pinus elliotti wood impregnated with alumina (Al2O3) particles. The samples were impregnated at three different Al2O3 weight fractions (c.a. 0.1%, 0.3% and 0.5%) and were then exposed to two wood-rot fungi, namely white-rot fungus (Trametes versicolor) and brown-rot fungus (Gloeophyllum trabeum). Thermal and chemical characteristics were evaluated by Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric (TG) analyses. The wood which incorporated 0.3 wt% of Al2O3 presented a weight loss 91.5% smaller than the untreated wood after being exposed to the white-rot fungus. On the other hand, the highest effectiveness against the brown-rot fungus was reached by the wood treated with 5 wt% of Al2O3, which presented a mass loss 91.6% smaller than that of the untreated pine wood. The Al2O3-treated woods presented higher antifungal resistances than the untreated ones in a way that: the higher the Al2O3 content, the higher the thermal stability. In general, the impregnation of the Al2O3 particles seems to be a promising treatment for wood protection against both studied wood-rot fungi. Additionally, both FT-IR and TG results were valuable tools to ascertain chemical changes ascribed to fungal decay. Full article
(This article belongs to the Special Issue Eco-Physiology of Wood Decay Fungi: Basics and Applications)
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<p>Mass losses of the treated and untreated <span class="html-italic">P. elliottii</span> (P<sub>E</sub>) woods exposed to the <span class="html-italic">G. trabeum</span> (<b>A</b>) and <span class="html-italic">T. versicolor</span> (<b>B</b>) fungi, where different letters above the bars indicate statistically different means.</p> Full article ">Figure 2
<p>FT−IR spectra (<b>A</b>) and lignin/carbohydrate ratios (<b>B</b>) of the untreated <span class="html-italic">P. elliottii</span> (P<sub>E</sub>) woods exposed to the <span class="html-italic">G. trabeum</span> and <span class="html-italic">T. versicolor</span> fungi. Where: Different letters above the bars indicate statistically different means for each lignin/carbohydrate ratio.</p> Full article ">Figure 3
<p>FT−IR spectra (<b>A</b>) and lignin/carbohydrate ratios (<b>B</b>) of the <span class="html-italic">P. elliottii</span> (P<sub>E</sub>) woods treated with 0.1% of Al<sub>2</sub>O<sub>3</sub> exposed to the <span class="html-italic">G. trabeum</span> and <span class="html-italic">T. versicolor</span> fungi. Where: Different letters above the bars indicate statistically different means for each lignin/carbohydrate ratio.</p> Full article ">Figure 4
<p>FT−IR spectra (<b>A</b>) and lignin/carbohydrate ratios (<b>B</b>) of the <span class="html-italic">P. elliottii</span> (P<sub>E</sub>) woods treated with 0.3% of Al<sub>2</sub>O<sub>3</sub> exposed to the <span class="html-italic">G. trabeum</span> and <span class="html-italic">T. versicolor</span> fungi. Where: Different letters above the bars indicate statistically different means for each lignin/carbohydrate ratio.</p> Full article ">Figure 5
<p>FT−IR spectra (<b>A</b>) and lignin/carbohydrate ratios (<b>B</b>) of the <span class="html-italic">P. elliottii</span> (P<sub>E</sub>) woods treated with 0.5% of Al<sub>2</sub>O<sub>3</sub> exposed to the <span class="html-italic">G. trabeum</span> and <span class="html-italic">T. versicolor</span> fungi. Where: Different letters above the bars indicate statistically different means for each lignin/carbohydrate ratio.</p> Full article ">Figure 6
<p>TG curves and main thermal parameters of the treated and untreated <span class="html-italic">P. elliottii</span> (P<sub>E</sub>) woods exposed to the <span class="html-italic">G. trabeum</span> (<b>A</b>,<b>C</b>) and <span class="html-italic">T. versicolor</span> (<b>B</b>,<b>D</b>) fungi.</p> Full article ">
17 pages, 2436 KiB  
Article
Characterization of Defensin-like Protein 1 for Its Anti-Biofilm and Anti-Virulence Properties for the Development of Novel Antifungal Drug against Candida auris
by Majid Rasool Kamli, Jamal S. M. Sabir, Maqsood Ahmad Malik and Aijaz Ahmad
J. Fungi 2022, 8(12), 1298; https://doi.org/10.3390/jof8121298 - 14 Dec 2022
Cited by 3 | Viewed by 2061
Abstract
Candida auris has emerged as a pan-resistant pathogenic yeast among immunocompromised patients worldwide. As this pathogen is involved in biofilm-associated infections with serious medical manifestations due to the collective expression of pathogenic attributes and factors associated with drug resistance, successful treatment becomes a [...] Read more.
Candida auris has emerged as a pan-resistant pathogenic yeast among immunocompromised patients worldwide. As this pathogen is involved in biofilm-associated infections with serious medical manifestations due to the collective expression of pathogenic attributes and factors associated with drug resistance, successful treatment becomes a major concern. In the present study, we investigated the candidicidal activity of a plant defensin peptide named defensin-like protein 1 (D-lp1) against twenty-five clinical strains of C. auris. Furthermore, following the standard protocols, the D-lp1 was analyzed for its anti-biofilm and anti-virulence properties. The impact of these peptides on membrane integrity was also evaluated. For cytotoxicity determination, a hemolytic assay was conducted using horse blood. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values ranged from 0.047–0.78 mg/mL and 0.095–1.56 mg/mL, respectively. D-lp1 at sub-inhibitory concentrations potentially abrogated both biofilm formation and 24-h mature biofilms. Similarly, the peptide severely impacted virulence attributes in the clinical strain of C. auris. For the insight mechanism, D-lp1 displayed a strong impact on the cell membrane integrity of the test pathogen. It is important to note that D-lp1 at sub-inhibitory concentrations displayed minimal hemolytic activity against horse blood cells. Therefore, it is highly useful to correlate the anti-Candida property of D-lp1 along with anti-biofilm and anti-virulent properties against C. auris, with the aim of discovering an alternative strategy for combating serious biofilm-associated infections caused by C. auris. Full article
(This article belongs to the Special Issue Candida Pathogenicity Mechanisms)
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<p>Antibiofilm activity of D-lp1 against <span class="html-italic">C. auris</span> biofilms. (<b>A</b>) Adherance inhibition; (<b>B</b>) Inhibition of biofilm; (<b>C</b>) Inhibition of mature biofilm. The figure shows the anti-biofilm potency of the test agent against different stages of biofilms formed by clinical strains of <span class="html-italic">C. auris</span>. XTT assay calculated the metabolic activity of cells embedded in biofilms, and readings were recorded at 490 nm. NC, negative control. Statistical difference between test concentrations relative to the negative control was evaluated using a two-way ANOVA, with post hoc Dunnet’s test for multiple comparisons **** <span class="html-italic">p</span> value &lt; 0.0001; *** <span class="html-italic">p</span> value = 0.0004; ** <span class="html-italic">p</span> value = 0.0027; * <span class="html-italic">p</span> value = 0.0121.</p> Full article ">Figure 2
<p>SEM images of <span class="html-italic">C. auris</span> MRL6057. The figure demonstrates the disruptive ability of D-lp1 against 24-h mature biofilm formed by <span class="html-italic">C. auris</span> MRL6057. (<b>A</b>) represents untreated control, (<b>B</b>) represents abrogated <span class="html-italic">C. auris</span> biofilm after treatment with D-lp1 at 3.12 mg/mL. The image at higher magnification has been placed in the upper left corner.</p> Full article ">Figure 3
<p>D-lp1 affects <span class="html-italic">C. auris</span> MRL6057 cell viability. The figure shows <span class="html-italic">C. auris</span> viability profile. Negative control: unexposed <span class="html-italic">C. auris</span> cells; positive control: H<sub>2</sub>O<sub>2</sub> exposed cells; <span class="html-italic">C. auris</span> exposed to varied MIC values of the test peptide; 0.39 mg/mL (0.5 × MIC); 0.78 mg/mL (MIC) of 1.56 mg/mL (MFC).</p> Full article ">Figure 4
<p>Uptake of PI by <span class="html-italic">C. auris</span> MRL6057. Exposure of yeast cells by D-lp1 at various concentrations (<b>C</b>–<b>E</b>). (<b>A</b>) Untreated cells were a negative control for intact <span class="html-italic">C. auris</span> plasma membrane. (<b>B</b>) Exposure to H<sub>2</sub>O<sub>2</sub> caused compromised cell membrane resulting in cellular uptake of PI. Exposure of <span class="html-italic">C. auris</span> at (<b>C</b>) 0.39 mg/mL (0.5 × MIC); (<b>D</b>) 0.78 mg/mL (MIC); (<b>E</b>) 1.56 mg/mL (MFC).</p> Full article ">Figure 5
<p>Inhibition of SAP production in <span class="html-italic">C. auris</span> MRL6057 by the D-lp1. The figure represents the average Pz value (diameter of colony/total diameter) in exposed and unexposed (NC) cells. 0.19 mg/mL, 0.25 × MIC; 0.39 mg/mL, 0.5 × MIC; 0.78 mg/mL, MIC. Student’s unpaired two-tailed <span class="html-italic">t</span>-tests assuming unequal variance, ** <span class="html-italic">p</span> value = 0.003.</p> Full article ">Figure 6
<p>Adherence of <span class="html-italic">C. auris</span> MRL6057 to buccal epithelial cells. (<b>A</b>) Negative control shows the attachment of unexposed <span class="html-italic">C. auris</span> to the epithelial cells. The anti-adherence property of D-lp1 at various concentrations, (<b>B</b>) 0.19 mg/mL, 0.25 × MIC; (<b>C</b>) 0.39 mg/mL, 0.5 × MIC; (<b>D</b>) 0.78 mg/mL, MIC. The arrow points out the attached yeast cells to the epithelial cells. (<b>E</b>) represents the number of yeast cells attached to the buccal epithelial cells in the negative control (NC) and at various concentrations of D-lp1.</p> Full article ">Figure 7
<p>D-lp1 modulates extracellular efflux of R6G. The image displays the extracellular quantity of R6G effluxed by <span class="html-italic">C. auris</span> MRL6057. The absorbance of extracellular R6G was recorded at 527 nm. NC, negative control. The values are the average of three independent experiments. 0.39 mg/mL, 0.5 × MIC; 0.78 mg/mL, MIC. Student’s unpaired two-tailed <span class="html-italic">t</span>-tests assuming unequal variance, * <span class="html-italic">p</span> value &lt; 0.05.</p> Full article ">Figure 8
<p>Intracellular accumulation of R6G in <span class="html-italic">C. auris</span>. In the figure, <span class="html-italic">C. auris</span> MRL6057 was observed under bright field and fluorescence microscopy. (<b>A</b>) The figure shows untreated <span class="html-italic">C. auris</span> cells with effluxed R6G dye during incubation with glucose. Whereas treatment with 0.39 mg/mL, 0.5 × MIC (<b>B</b>), and 0.78 mg/mL MIC (<b>C</b>) resulted in the accumulation of R6G inside the cells after incubation with glucose, which was represented by high fluorescence inside the cells.</p> Full article ">Figure 9
<p>Hemolytic activity of D-lp1 against horse blood. Positive control (PC) 1% Triton X-100, Negative control (NC). Hemolytic activity of D-lp1 at varied concentrations; *** <span class="html-italic">p</span> value &lt; 0.0004.</p> Full article ">
15 pages, 6259 KiB  
Article
Two New Species and a New Record of Microdochium from Grasses in Yunnan Province, South-West China
by Ying Gao, Guang-Cong Ren, Dhanushka N. Wanasinghe, Jian-Chu Xu, Antonio Roberto Gomes de Farias and Heng Gui
J. Fungi 2022, 8(12), 1297; https://doi.org/10.3390/jof8121297 - 14 Dec 2022
Cited by 5 | Viewed by 2009
Abstract
Microdochium species are frequently reported as phytopathogens on various plants and also as saprobic and soil-inhabiting organisms. As a pathogen, they mainly affect grasses and cereals, causing severe disease in economically valuable crops, resulting in reduced yield and, thus, economic loss. Numerous asexual [...] Read more.
Microdochium species are frequently reported as phytopathogens on various plants and also as saprobic and soil-inhabiting organisms. As a pathogen, they mainly affect grasses and cereals, causing severe disease in economically valuable crops, resulting in reduced yield and, thus, economic loss. Numerous asexual Microdochium species have been described and reported as hyphomycetous. However, the sexual morph is not often found. The main purpose of this study was to describe and illustrate two new species and a new record of Microdochium based on morphological characterization and multi-locus phylogenetic analyses. Surveys of both asexual and sexual morph specimens were conducted from March to June 2021 in Yunnan Province, China. Here, we introduce Microdochium graminearum and M. shilinense, from dead herbaceous stems of grasses and report M. bolleyi as an endophyte of Setaria parviflora leaves. This study improves the understanding of Microdochium species on monocotyledonous flowering plants in East Asia. A summary of the morphological characteristics of the genus and detailed references are provided for use in future research. Full article
(This article belongs to the Special Issue Polyphasic Identification of Fungi 2.0)
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<p>Phylogenetic tree of <span class="html-italic">Microdochium</span> species based on maximum likelihood analysis of a combined multigene alignment (LSU, ITS, <span class="html-italic">tub2</span>, and <span class="html-italic">rpb2</span>). Bootstrap support values for ML higher than 70% and Bayesian posterior probabilities (PP) higher than 0.95 are indicated at the node. <span class="html-italic">Idriella lunata</span> (CBS 204.56) was used as the outgroup. Ex-type strains are in bold font; the newly generated sequences are denoted in blue.</p> Full article ">Figure 2
<p><span class="html-italic">Microdochium graminearum</span> (HKAS 123200, holotype). (<b>a</b>,<b>b</b>) Appearance of immersed ascomata on the host; (<b>c</b>,<b>d</b>) vertical section of the ascoma; (<b>e</b>) peridium; (<b>f</b>) paraphyses; (<b>g</b>–<b>k</b>) asci; (<b>l</b>) asci stained by Melzer’s reagent, showing a refractive ring around cytoplasmic protrusion (black circle); (<b>m</b>–<b>p</b>) ascospores; (<b>q</b>) surface of colony on PDA; and (<b>r</b>) reverse of colony on PDA. Scale bars (<b>c</b>) 50 μm; (<b>d</b>) 30 μm; (<b>e</b>,<b>f</b>) 20 μm; (<b>g</b>–<b>k</b>) 15 μm; (<b>l</b>) 10 μm; and (<b>m</b>–<b>p</b>) 5 μm.</p> Full article ">Figure 3
<p><span class="html-italic">Microdochium shilinense</span> (HKAS 123198, holotype). (<b>a</b>,<b>b</b>) Appearance of immersed ascomata on the ©t; (<b>c</b>) vertical section of the ascoma; (<b>d</b>) peridium. (<b>e</b>) paraphyses; (<b>f</b>–<b>l</b>) asci; (<b>m</b>–<b>q</b>) ascospores; (<b>r</b>) germinated ascospores; (<b>s</b>) surface of the colony on PDA; and (<b>t</b>) reverse of the colony on PDA. Scale bars, (<b>c</b>–<b>f</b>) 20 μm; (<b>g</b>–<b>j</b>) 10 μm; (<b>k</b>,<b>l</b>) 20 μm; and (<b>m</b>–<b>r</b>) 10 μm.</p> Full article ">Figure 4
<p><span class="html-italic">Microdochium bolleyi</span> (HKAS 123195) on leaves of healthy <span class="html-italic">Setaria parviflora</span>. (<b>a</b>) The surface of the colony on PDA; (<b>b</b>) the reverse of the colony on PDA; (<b>d</b>) hyaline mycelium; (<b>c</b>,<b>e</b>,<b>f</b>) conidiophores and conidiogenous cells; (<b>g</b>,<b>h</b>) conidia; and (<b>i</b>,<b>j</b>) chlamydospores. Scale bars, (<b>c</b>) 5 μm; (<b>d</b>–<b>g</b>) 10 μm; (<b>h</b>,<b>i</b>) 5 μm; and (<b>j</b>) 15 μm.</p> Full article ">
12 pages, 1973 KiB  
Article
Eco-Physiological Adaptations of the Xylotrophic Basidiomycetes Fungi to CO2 and O2 Mode in the Woody Habitat
by Victor A. Mukhin and Daria K. Diyarova
J. Fungi 2022, 8(12), 1296; https://doi.org/10.3390/jof8121296 - 13 Dec 2022
Cited by 2 | Viewed by 1824
Abstract
The aim of this research is to study of eco-physiological adaptations of xylotrophic fungi (Basidiomycota, Agaricomycetes) to hypoxia, anoxia and hypercapnia as the main environmental factors that determine the activity of fungi in woody habitat. The study was carried out on seven species [...] Read more.
The aim of this research is to study of eco-physiological adaptations of xylotrophic fungi (Basidiomycota, Agaricomycetes) to hypoxia, anoxia and hypercapnia as the main environmental factors that determine the activity of fungi in woody habitat. The study was carried out on seven species of polypore fungi widespread in the preforest-steppe pine-birch forests of the Central Urals, including both white (D. tricolor, D. septentrionalis, F. fomentarius, H. rutilans, T. biforme) and brown (F. betulina, F. pinicola) rot. Their CO2 and O2 gas exchange were analyzed in natural samples of woody substrates (Betula pendula, Pinus sylvestris) and basidiocarps by the chamber method using a CO2/O2 gas analyzer. It was shown that the intensity of O2 gas exchange is positively related to the oxygen concentration but is not very sensitive to a decrease in its content in the woody habitat. Xylotrophic fungi are able to completely exhaust the O2 in the habitat, and this process is linear, indicating that they do not have threshold values for oxygen content. Oxygen consumption is accompanied by an adequate linear increase in CO2 concentration up to 18–19%. At a concentration of 5–10%, carbon dioxide does not affect the gas exchange of xylotrophic fungi and can even enhance it, but at 20% it significantly reduces its intensity. Xylotrophic fungi are resistant to high CO2 concentrations and remain viable at 100% CO2 concentration and are capable of growth under these conditions. In an oxygen-free habitat, anaerobic CO2 emissions are recorded; when O2 appears, its consumption is restored to the level preceding anoxia. Xylotrophic fungi are the specialized group of saprotrophic microaerophilic and capnophilic facultative anaerobes adapted to develop at low oxygen and high carbon dioxide concentration, anoxia. Full article
(This article belongs to the Special Issue Eco-Physiology of Wood Decay Fungi: Basics and Applications)
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Figure 1
<p><span class="html-italic">Daedaleopsis tricolor</span> (<b>a</b>), <span class="html-italic">D. septentrionalis</span> (<b>b</b>), <span class="html-italic">Fomitopsis pinicola</span> (<b>c</b>), <span class="html-italic">Hapalopilus rutilans</span> (<b>d</b>), <span class="html-italic">F. betulina</span> (<b>e</b>), <span class="html-italic">Fomes fomentarius</span> (<b>f</b>), <span class="html-italic">Trichaptum pargamenum</span> (<b>g</b>); substrates: <span class="html-italic">Pinus sylvestris</span> (<b>h</b>), <span class="html-italic">Betula pendula</span> (<b>i</b>).</p> Full article ">Figure 2
<p>Dynamics of O<sub>2</sub> consumption and CO<sub>2</sub> accumulation in exposure chambers with basidiocarps <span class="html-italic">Fomes fomentarius</span> (<b>a</b>), <span class="html-italic">Fomitopsis pinicola</span> (<b>b</b>) and substrates <span class="html-italic">Fomes fomentarius</span> (<b>c</b>), <span class="html-italic">Fomitopsis betulina</span> (<b>d</b>). Dark columns—O<sub>2</sub>, light columns—CO<sub>2</sub>, R<sup>2</sup>—coefficient of determination.</p> Full article ">Figure 3
<p>O<sub>2</sub> concentration (1) and intensity of its consumption (2) by <span class="html-italic">Fomes fomentarius</span> (<b>a</b>), <span class="html-italic">Fomitopsis pinicola</span> (<b>b</b>) basidiocarps and <span class="html-italic">Fomes fomentarius</span> (<b>c</b>), <span class="html-italic">Fomitopsis betulina</span> (<b>d</b>) substrates. R<sup>2</sup>—coefficient of determination.</p> Full article ">Figure 4
<p>CO<sub>2</sub> concentration (1) and intensity of its emission (2) by <span class="html-italic">Fomes fomentarius</span> (<b>a</b>), <span class="html-italic">Fomitopsis pinicola</span> (<b>b</b>) basidiocarps and <span class="html-italic">Fomes fomentarius</span> (<b>c</b>), <span class="html-italic">Fomitopsis betulina</span> (<b>d</b>) substrates. R<sup>2</sup>—coefficient of determination.</p> Full article ">Figure 5
<p>Growth of dikaryotic mycelium of <span class="html-italic">Daedaleopsis septentrionalis</span> in 100% CO<sub>2</sub> (<b>a</b>) and in 100% N<sub>2</sub> (<b>b</b>). I—initial size, II—after 3 days exposure in oxygen-free medium, III—after the next 3 days in air; control in air (<b>c</b>), initial size (I), after 3 (II) and 6 (III) days in air.</p> Full article ">
14 pages, 3871 KiB  
Article
β-Xylosidase SRBX1 Activity from Sporisorium reilianum and Its Synergism with Xylanase SRXL1 in Xylose Release from Corn Hemicellulose
by Yuridia Mercado-Flores, Alejandro Téllez-Jurado, Carlos Iván Lopéz-Gil and Miguel Angel Anducho-Reyes
J. Fungi 2022, 8(12), 1295; https://doi.org/10.3390/jof8121295 - 13 Dec 2022
Cited by 3 | Viewed by 1840
Abstract
Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-β-1,4-xylanases and one with α-L-arabinofuranosidase activity. [...] Read more.
Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-β-1,4-xylanases and one with α-L-arabinofuranosidase activity. In this study, the native protein extracellular with β-xylosidase activity, called SRBX1, produced by this basidiomycete was analyzed by performing production kinetics and its subsequent purification by gel filtration. The enzyme was characterized biochemically and sequenced. Finally, its synergism with Xylanase SRXL1 was determined. Its activity was higher in a medium with corn hemicellulose and glucose as carbon sources. The purified protein was a monomer associated with the sr16700 gene, with a molecular weight of 117 kDa and optimal activity at 60 °C in a pH range of 4–7, which had the ability to hydrolyze the ρ-nitrophenyl β-D-xylanopyranoside and ρ-Nitrophenyl α-L-arabinofuranoside substrates. Its activity was strongly inhibited by silver ions and presented Km and Vmax values of 2.5 mM and 0.2 μmol/min/mg, respectively, using ρ-nitrophenyl β-D-xylanopyranoside as a substrate. The enzyme degrades corn hemicellulose and birch xylan in combination and in sequential synergism with the xylanase SRXL1. Full article
(This article belongs to the Special Issue Smut Fungi 2.0)
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<p>β-xylosidase production from <span class="html-italic">S. reilianum</span> in different culture media. (<b>a</b>) Minimum medium with glucose; (<b>b</b>) minimum medium with corn hemicellulose; and (<b>c</b>) minimum medium with corn hemicellulose and glucose. Green line: biomass determined by the increase in the absorbance at 660 nm. Purple line: β-xylosidase activity.</p> Full article ">Figure 2
<p>Purification of β-xylosidase SRBX1 from <span class="html-italic">S. reilianum</span>. (<b>a</b>) Chromatographic step in the gel filtration column. Green line: enzymatic activity. Purple line: absorbance at 260 nm. (<b>b</b>) Silver-stained SDS-PAGE. Line 1: molecular weight marker; line 2: without sample; lines 3 and 4: fractions with activity obtained by gel filtration chromatography.</p> Full article ">Figure 3
<p>Effects of temperature and pH on β-xylosidase SRBX1 from <span class="html-italic">S. reilianum</span>. (<b>a</b>) Optimal temperature and pH. (<b>b</b>) Temperature and pH stability. Green line: temperature. Purple line: pH. Solid line: McIlvaine buffer (pH 2–7); dashed line: Tris–HCl buffer (pH 7–10); and dotted line: glycine–NaOH buffer (pH 9–11.0).</p> Full article ">Figure 4
<p>Effect of metal ions and chemical substances on the activity of β-xylosidase SRBX1 from <span class="html-italic">S. reilianum</span>: green: 0 mM; brown: 2 mM; and yellow: 10 mM. *** <span class="html-italic">p</span> &lt; 0.001. EDTA: ethylenediaminetetraacetic acid; SDS: sodium dodecyl sulfate; and βME: β-mercaptoethanol.</p> Full article ">Figure 5
<p>Structural modeling of the β-xylosidase SRBX1 protein from S. reilianum. (<b>a</b>,<b>b</b>) Three-dimensional structures. (<b>c</b>) Location of the glycosylation sites. The protein has 12 possible glycosylation sites. Except for site number 11, all others are located on the protein’s surface (indicated in blue). (<b>d</b>) The location of the active site is in yellow in the dashed box. (<b>e</b>) Amino acids of the active site. ASP315, which theoretically participates in enzymatic catalysis, is shown in the letters red. The structural modeling was performed using the bioinformatic web-server AlphaFold Colab (<a href="https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb#scrollTo=XUo6foMQxwS2" target="_blank">https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb#scrollTo=XUo6foMQxwS2</a>, accessed on 5 November 2022).</p> Full article ">Figure 6
<p>Xylose release and degree of synergism between xylanase SRXL1 and β-xylosidase SRBX1 activity from <span class="html-italic">S. reilianum</span> in the degradation of corn hemicellulose (<b>a</b>) and birch xylan (<b>b</b>). Sequence 1: xylanase was added first, followed by β-xylosidase. Sequence 2: β-xylosidase was added first, followed by xylanase. Amounts of 0.5 and 2.0 U of xylanase and the β-xylosidase were used, respectively. The same numbers roman indicates that no statistically significant difference was observed; <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 7
<p>Xylose release and degree of synergism between the actions of xylanase SRXL1 and β-xylosidase SRBX1 from <span class="html-italic">S. reilianum</span> using different enzymatic units in the degradation of corn hemicellulose (<b>a</b>) and birch xylan (<b>b</b>). The same numbers roman indicates indicate that no statistically significant difference was observed; <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">
3 pages, 204 KiB  
Editorial
Fungal Taxonomy, Phylogeny, and Ecology: A Themed Issue Dedicated to Academician Wen-Ying Zhuang
by Cheng Gao and Lei Cai
J. Fungi 2022, 8(12), 1294; https://doi.org/10.3390/jof8121294 - 11 Dec 2022
Viewed by 1802
Abstract
We are honored and privileged to edit this Special Issue, “Fungal Taxonomy, Phylogeny, and Ecology: A Themed Issue Dedicated to Academician Wen-Ying Zhuang” [...] Full article
(This article belongs to the Special Issue Fungal Taxonomy, Phylogeny, and Ecology: A Themed Issue Dedicated to Academician Wen-Ying Zhuang)
12 pages, 2837 KiB  
Article
Heterologous Expression of CFL1 Confers Flocculating Ability to Cutaneotrichosporon oleaginosus Lipid-Rich Cells
by Silvia Donzella and Concetta Compagno
J. Fungi 2022, 8(12), 1293; https://doi.org/10.3390/jof8121293 - 11 Dec 2022
Viewed by 1645
Abstract
Lipid extraction from microbial and microalgae biomass requires the separation of oil-rich cells from the production media. This downstream procedure represents a major bottleneck in biodiesel production, increasing the cost of the final product. Flocculation is a rapid and cheap system for removing [...] Read more.
Lipid extraction from microbial and microalgae biomass requires the separation of oil-rich cells from the production media. This downstream procedure represents a major bottleneck in biodiesel production, increasing the cost of the final product. Flocculation is a rapid and cheap system for removing solid particles from a suspension. This natural characteristic is displayed by some microorganisms due to the presence of lectin-like proteins (called flocculins/adhesins) in the cell wall. In this work, we showed, for the first time, that the heterologous expression of the adhesin Cfl1p endows the oleaginous species Cutaneotrichosporon oleaginosus with the capacity of cell flocculation. We used Helm’s test to demonstrate that the acquisition of this trait allows for reducing the time required for the separation of lipid-rich cells from liquid culture by centrifugation without altering the productivity. This improves the lipid production process remarkably by providing a more efficient downstream. Full article
(This article belongs to the Special Issue New Perspectives for Oleaginous Fungi)
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<p>Structure of linearized plasmid for Cfl1 expression.</p> Full article ">Figure 2
<p>Screening of recombinant clones by Helm’s test. The percentage of flocculation is expressed as fold change in comparison to the control strain (wild-type ATCC 20509), which is set to 1.</p> Full article ">Figure 3
<p>(<b>A</b>): Wild-type cells (ATCC 20509). (<b>B</b>): Recombinant 31R cells forming aggregates.</p> Full article ">Figure 4
<p>Lipid content (expressed in % of dry weight) produced by the recombinant strains after 65 h of growth.</p> Full article ">Figure 5
<p>Helm’s test after 24, 48, and 65 h of growth in B medium. The percentage of flocculation is expressed as fold change in comparison to the control strain (wild-type ATCC 20509), which is set to 1.</p> Full article ">Figure 6
<p>Undiluted cultures collected after 65 h of growth and rested for 3 h.</p> Full article ">Figure 7
<p>Time course of dry weight (DW, g/L), lipid accumulation (g/L), and flocculation rate (%) of the wild type, 31R and 65L. The flocculation rate was calculated using the Helm’s test and expressed in % (see Materials and Methods).</p> Full article ">Figure 8
<p>(<b>A</b>) OD<sub>660</sub> of the upper part after centrifugation for different times at 4000 rpm. *: <span class="html-italic">p</span> &lt; 0.05. **: <span class="html-italic">p</span> &lt; 0.01. (<b>B</b>) Pellets of cultures collected after 90 h of process on medium B and centrifuged for 10 min at 4000 rpm.</p> Full article ">
16 pages, 1425 KiB  
Article
Essential Mineral Content (Fe, Mg, P, Mn, K, Ca, and Na) in Five Wild Edible Species of Lactarius Mushrooms from Southern Spain and Northern Morocco: Reference to Daily Intake
by Alejandro R. López, Marta Barea-Sepúlveda, Gerardo F. Barbero, Marta Ferreiro-González, José Gerardo López-Castillo, Miguel Palma and Estrella Espada-Bellido
J. Fungi 2022, 8(12), 1292; https://doi.org/10.3390/jof8121292 - 10 Dec 2022
Cited by 3 | Viewed by 2066
Abstract
Mushroom consumption has increased in recent years due to their beneficial properties to the proper functioning of the body. Within this framework, the high potential of mushrooms as a source of essential elements has been reported. Therefore, the present study aims to determine [...] Read more.
Mushroom consumption has increased in recent years due to their beneficial properties to the proper functioning of the body. Within this framework, the high potential of mushrooms as a source of essential elements has been reported. Therefore, the present study aims to determine the mineral content of seven essential metals, Fe, Mg, Mn, P, K, Ca, and Na, in twenty samples of mushrooms of the genus Lactarius collected from various locations in southern Spain and northern Morocco, by FAAS, UV-Vis spectroscopy, and ICP-OES after acid digestion. Statistics showed that K was the macronutrient found at the highest levels in all mushrooms studied. ANOVA showed that there were statistically significant differences among the species for K, P, and Na. The multivariate study suggested that there were differences between the accumulation of the elements according to the geographic location and species. Furthermore, the intake of 300 g of fresh mushrooms of each sample covers a high percentage of the RDI, but does not meet the recommended daily intake (RDI) for any of the metals studied, except for Fe. Even considering these benefits, the consumption of mushrooms should be moderated due to the presence of toxic metals, which may pose health risks. Full article
(This article belongs to the Special Issue Edible and Medicinal Macrofungi)
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<p>Five wild seasonal species of the genus <span class="html-italic">Lactarius</span> studied: (<b>a</b>) <span class="html-italic">L. deliciosus</span>; (<b>b</b>) <span class="html-italic">L. semisanguifluus</span>; (<b>c</b>) <span class="html-italic">L. sanguifluus</span>; (<b>d</b>) <span class="html-italic">L. rugatus</span>; (<b>e</b>) <span class="html-italic">L. vinosus</span>.</p> Full article ">Figure 2
<p>Graphical display of the resulting HCA dendrogram using Euclidean distance and Ward’s method to identify clustering trend patterns among the studied mushroom species based on the content of the seven macronutrients determined.</p> Full article ">Figure 3
<p>Representation of the toxic metal content of mushrooms of the genus <span class="html-italic">Lactarius</span> and the recommended daily limits.</p> Full article ">
16 pages, 4193 KiB  
Article
Antifungal Mechanism of Metabolites from Newly Isolated Streptomyces sp. Y1-14 against Banana Fusarium Wilt Disease Using Metabolomics
by Miaomiao Cao, Qifeng Cheng, Bingyu Cai, Yufeng Chen, Yongzan Wei, Dengfeng Qi, Yuqi Li, Liu Yan, Xiaojuan Li, Weiqiang Long, Qiao Liu, Jianghui Xie and Wei Wang
J. Fungi 2022, 8(12), 1291; https://doi.org/10.3390/jof8121291 - 9 Dec 2022
Cited by 11 | Viewed by 2702
Abstract
Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is one of the most destructive banana diseases in the world, which limits the development of the banana industry. Compared with traditional physical and chemical practices, biological control [...] Read more.
Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is one of the most destructive banana diseases in the world, which limits the development of the banana industry. Compared with traditional physical and chemical practices, biological control becomes a promising safe and efficient strategy. In this study, strain Y1-14 with strong antagonistic activity against Foc TR4 was isolated from the rhizosphere soil of a banana plantation, where no disease symptom was detected for more than ten years. The strain was identified as Streptomyces according to the morphological, physiological, and biochemical characteristics and the phylogenetic tree of 16S rRNA. Streptomyces sp. Y1-14 also showed a broad-spectrum antifungal activity against the selected 12 plant pathogenic fungi. Its extracts inhibited the growth and spore germination of Foc TR4 by destroying the integrity of the cell membrane and the ultrastructure of mycelia. Twenty-three compounds were identified by gas chromatography–mass spectrometry (GC-MS). The antifungal mechanism was investigated further by metabolomic analysis. Strain Y1-14 extracts significantly affect the carbohydrate metabolism pathway of Foc TR4 by disrupting energy metabolism. Full article
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<p>Isolation and identification of strain Y1-14 with strong antifungal activity against Foc TR4. (<b>A</b>) Strain Y1-14 inhibiting mycelial growth of Foc TR4. (<b>B</b>) Strain Y1-14 extracts inhibiting mycelial growth of Foc TR4. (<b>C</b>) Morphological characteristics of aerial mycelia and spores of strain Y1-14 using SEM. (<b>D</b>) Phylogenetic tree of strain Y1-14 based on 16S rRNA gene sequence analysis. The bootstrap values (%) at the branches were calculated from 1000 replications.</p> Full article ">Figure 2
<p>(<b>A</b>) A broad-spectrum antifungal activity of <span class="html-italic">Streptomyces</span> sp. Y1-14 against the selected twelve phytopathogenic fungi. (<b>B</b>) The mycelial inhibition (%) of Foc TR4 after treatment with <span class="html-italic">Streptomyces</span> sp. Y1-14. (<b>C</b>) Effects of <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts on mycelial growth of Foc TR4. Different lowercase letters indicated a significant difference according to the Duncan’s multiple range test (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 3
<p>Inhibition of <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts on spore germination of Foc TR4. (<b>A</b>) Spore germination characteristics of Foc TR4 after treatment with 1 ×, 2 ×, 4 ×, or 8 × EC<sub>50</sub> extracts. A 10% concentration of DMSO was used as a control. Bar = 20 µm. (<b>B</b>) The spore germination rate (%) of Foc TR4 after treatment with different dose extracts. (<b>C</b>) The germ tube length (µm) of Foc TR4 after treatment with different dose extracts. (<b>D</b>) Mycelial cell ultrastructure of Foc TR4 observed under TEM. (<b>a</b>) Ultrastructure characteristics of Foc TR4. (<b>b</b>–<b>d</b>) Ultrastructure characteristics of Foc TR4 treated with 4 × EC<sub>50</sub> of <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts. Bar = 2 µm. (<b>E</b>) Mycelial morphology of Foc TR4 observed under SEM. (<b>a</b>,<b>b</b>) Mycelial characteristics of Foc TR4 in the absence of stain Y1-14 extracts. (<b>c</b>,<b>d</b>) Mycelial morphology of Foc TR4 treated with 4 × EC<sub>50</sub> of <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts. Bar = 10 µm (<b>F</b>) Mycelial morphology of Foc TR4 observed under fluorescence microscope. (<b>a</b>) Mycelial characteristics of Foc TR4 in the absence of <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts. (<b>b</b>) Mycelial morphology of Foc TR4 treated with 8 × EC<sub>50</sub> of <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts. The GFP gene disap-peared site was marked with the red frame. Bar = 20 µm.</p> Full article ">Figure 4
<p>Antifungal activity of VOCs produced by <span class="html-italic">Streptomyces</span> sp. Y1-14 against Foc TR4. (<b>A</b>) Double plate assay was used to evaluate the antagonistic activity of <span class="html-italic">Streptomyces</span> sp. Y1-14. The blue circle represented the disc of <span class="html-italic">Streptomyces</span> sp. Y1-14 and the yellow circle represented the disc of Foc TR4. (<b>B</b>) VOCs produced by <span class="html-italic">Streptomyces</span> sp. Y1-14 inhibiting mycelial growth of Foc TR4.</p> Full article ">Figure 5
<p>Effect of <span class="html-italic">Streptomyces</span> sp. Y1-14 on secondary metabolism of Foc TR4. (<b>A</b>) Heatmap based on the metabolome data of Foc TR4 treated with extracts of <span class="html-italic">Streptomyces</span> sp. Y1-14 at 0 h, 6 h, and 12 h. Three biological replications were carried out for analyzing the metabolite profiling. (<b>B</b>,<b>C</b>) Volcanic map of differential metabolites. Each dot represents a metabolite, the abscissa represents the group contrast ratio of each material change (with 2 logs base), the vertical ordinate represents the <span class="html-italic">p</span>-<span class="html-italic">t</span> test value (in the logarithm of 10 at the bottom), and the size of scatter represents the VIP value of the OPLS-DA model, the larger the scatter, the greater the VIP value; the differential expressed metabolites obtained by screening were more reliable. Blue dots in the figure represent downregulated DEMs, red dots represent upregulated DEMs, and grey dots represent no significantly different metabolites. In addition, the top 5 qualitative metabolites were selected and labeled in the figure after sorting by <span class="html-italic">p</span> value. (<b>D</b>) Venn diagram of the metabolite numbers. Ck-12 h vs. T-12 h represents DEMs of Foc TR4 treated with <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts compared with the control at 12 h. Ck-6 h vs. T-6 h represents DEMs of Foc TR4 treated with <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts compared with the control at 6 h. (<b>E</b>) Determination of enzyme activities of Foc TR4 after treatment with <span class="html-italic">Streptomyces</span> sp. Y1-14 extracts.</p> Full article ">
11 pages, 619 KiB  
Review
A Review of Antifungal Susceptibility Testing for Dermatophyte Fungi and It’s Correlation with Previous Exposure and Clinical Responses
by Sidra Saleem Khan, Roderick James Hay and Ditte Marie Lindhardt Saunte
J. Fungi 2022, 8(12), 1290; https://doi.org/10.3390/jof8121290 - 9 Dec 2022
Cited by 9 | Viewed by 2798
Abstract
Background: An increase in the number of recurrent and recalcitrant dermatophytoses calls for a tool to guide the clinician to correlate in vitro minimum inhibitory concentration (MIC) data, antifungal treatment with clinical outcomes. This systematic review aims to explore a possible correlation between [...] Read more.
Background: An increase in the number of recurrent and recalcitrant dermatophytoses calls for a tool to guide the clinician to correlate in vitro minimum inhibitory concentration (MIC) data, antifungal treatment with clinical outcomes. This systematic review aims to explore a possible correlation between one aspect of this, previous antifungal exposure, and clinical outcomes. Methods: A systematic literature search for articles on previous antifungal treatment, treatment outcome, susceptibility methods used, organism (genus/species), and MIC values was conducted. Results: A total of 720 records were identified of which 19 articles met the inclusion criteria. Forty percent of the cases had contact with or travel to India, 28% originated from or had traveled to other countries where treatment unresponsive tinea infections had been reported. Tinea corporis was the most common clinical presentation and the species involved were Trichophyton (T.) indotineae and T. rubrum, followed by T. mentagrophyte/interdigitale complex and T. tonsurans. Nearly all patients had previously been exposed to one or more antifungals. The studies were too heterogeneous to perform a statistical analysis to test if previous antifungal exposure was related to resistance. Conclusions: Only a few studies were identified, which had both sufficient and robust data on in vitro susceptibility testing and clinical treatment failure. Further research on the value of susceptibility testing to improve clinical practice in the management of dermatophyte infections is needed. Full article
(This article belongs to the Special Issue Fungal Infections: From Diagnostics to Treatments)
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<p>PRISMA Flow Diagram, Flowchart of literature search.</p> Full article ">
8 pages, 4218 KiB  
Article
Eumycetoma Caused by Madurella pseudomycetomatis in a Captive Tiger (Panthera tigris)
by Margherita Orlandi, Giuseppe Giglia, Patrizia Danesi, Piero Laricchiuta and Francesca Abramo
J. Fungi 2022, 8(12), 1289; https://doi.org/10.3390/jof8121289 - 9 Dec 2022
Viewed by 2016
Abstract
A captive-kept adult male tiger presented with a large cutaneous and subcutaneous mass on the thigh with a fistula. During sedation, multiple nodules were detected and samples for a histopathological exam were collected. Histologically, granulomatous panniculitis and dermatitis were seen around dense aggregates [...] Read more.
A captive-kept adult male tiger presented with a large cutaneous and subcutaneous mass on the thigh with a fistula. During sedation, multiple nodules were detected and samples for a histopathological exam were collected. Histologically, granulomatous panniculitis and dermatitis were seen around dense aggregates of pigmented fungal hyphae, and a diagnosis of phaeohyphomycosis was made; considering the clinical features, it was classified as a eumycotic mycetoma. This is a rarely reported subcutaneous fungal infection in humans and animals, caused by dematiaceous fungi. Clinically, it is characterized by tumefaction, fistulous sinus tracts, and the formation of macroscopically visible grains. In the literature, only a few infections in wild felids have been reported. In this case, Fontana–Masson staining better showed pigmentation and panfungal PCR and sequencing identified Madurella pseudomyectomatis (OP623507) as the causative agent. Systemic therapy with oral administration of itraconazole was planned, but the patient died during the first period of treatment. The animal was not submitted for post-mortem examination. Visceral dissemination of the agent cannot be excluded. To the authors’ knowledge, this is the first report of eumycotic mycetoma by Madurella pseudomycetomatis in a captive tiger. Full article
(This article belongs to the Special Issue Fungal Diseases in Animals)
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<p>Skin histopathology. (<b>a</b>) In the superficial dermis, two individual nodules composed of pyogranulomatous reactions centered on dark-pink fungal aggregates (HE stain). (<b>b</b>) Serial unstained section demonstrating the light-brownish pigmentation of the fungal aggregates. (<b>c</b>) Serial section stained with Fontana–Masson stain showing highly melanized fungal aggregates.</p> Full article ">Figure 2
<p>Histopathology and histochemistry of granules. HE stain (<b>a</b>) shows the presence of hyphal aggregates within the granules; unstained (<b>b</b>) and Fontana–Masson-stained (<b>c</b>) sections show the presence of a faint brownish pigment (<b>b</b>) or a very dark melanized background within the granules; Grocott staining (<b>d</b>) allows better definition of hyphal characteristics; short hyphae (arrows) and cystic dilations (asterisks) are indicated in the inset.</p> Full article ">Figure 3
<p>Phylogenetic tree based on portions of the 28S LSU sequences of various members of the order Sordariales. In boldface, PAR21-5180-2 was the sequence produced in our study. The <span class="html-italic">Pleospora herbarum</span> sequence was used as an outgroup. The maximum likelihood method was used to construct the tree. Bootstrap values shown at the main nodes represent the probabilities based on 1000 replicates. (T) = type strains.</p> Full article ">
19 pages, 1902 KiB  
Review
Lysinibacilli: A Biological Factories Intended for Bio-Insecticidal, Bio-Control, and Bioremediation Activities
by Qazi Mohammad Sajid Jamal and Varish Ahmad
J. Fungi 2022, 8(12), 1288; https://doi.org/10.3390/jof8121288 - 8 Dec 2022
Cited by 12 | Viewed by 3476
Abstract
Microbes are ubiquitous in the biosphere, and their therapeutic and ecological potential is not much more explored and still needs to be explored more. The bacilli are a heterogeneous group of Gram-negative and Gram-positive bacteria. Lysinibacillus are dominantly found as motile, spore-forming, Gram-positive [...] Read more.
Microbes are ubiquitous in the biosphere, and their therapeutic and ecological potential is not much more explored and still needs to be explored more. The bacilli are a heterogeneous group of Gram-negative and Gram-positive bacteria. Lysinibacillus are dominantly found as motile, spore-forming, Gram-positive bacilli belonging to phylum Firmicutes and the family Bacillaceae. Lysinibacillus species initially came into light due to their insecticidal and larvicidal properties. Bacillus thuringiensis, a well-known insecticidal Lysinibacillus, can control many insect vectors, including a malarial vector and another, a Plasmodium vector that transmits infectious microbes in humans. Now its potential in the environment as a piece of green machinery for remediation of heavy metal is used. Moreover, some species of Lysinibacillus have antimicrobial potential due to the bacteriocin, peptide antibiotics, and other therapeutic molecules. Thus, this review will explore the biological disease control abilities, food preservative, therapeutic, plant growth-promoting, bioremediation, and entomopathogenic potentials of the genus Lysinibacillus. Full article
(This article belongs to the Special Issue Plant Protection: New Green Antifungal Agents)
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<p>Potential applications of <span class="html-italic">Lysinibacillus</span> species in human welfare.</p> Full article ">Figure 2
<p>Phylogenic evaluation of <span class="html-italic">Lysinibacillus</span> species.</p> Full article ">Figure 3
<p>Effect of VOCs emitted by strain CT32 on the growth of 8 phytopathogenic fungi in vitro. In the sealed plates test, fungi in the control groups were cultured on potato dextrose agar (PDA) medium. The mycelial growth of fungi in the treatment groups was suppressed upon exposure to volatiles emitted by strain CT32. (<b>a</b>) <span class="html-italic">V. dahliae</span>; (<b>b</b>) <span class="html-italic">F. oxysporum</span> f. sp. <span class="html-italic">fragariae</span>; (<b>c</b>) <span class="html-italic">F. oxysporum</span>, f. sp. <span class="html-italic">niveum</span>; (<b>d</b>) <span class="html-italic">F. oxysporum</span>, f. sp. <span class="html-italic">cucumerinum</span>; (<b>e</b>) <span class="html-italic">B. cinerea</span>; (<b>f</b>) <span class="html-italic">T. cucumeris</span>; (<b>g</b>) <span class="html-italic">G. cingulata</span>; (<b>h</b>) <span class="html-italic">B. dothidea</span>. Adapted from Li et al., 2020 [<a href="#B69-jof-08-01288" class="html-bibr">69</a>].</p> Full article ">Figure 4
<p>The silver nanoparticles synthesized by <span class="html-italic">Lysinibacillus</span> sp. and their potential possible applications.</p> Full article ">
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Brief Report
The Calcium Chloride Responsive Type 2C Protein Phosphatases Play Synergistic Roles in Regulating MAPK Pathways in Magnaporthe oryzae
by Wilfred M. Anjago, Jules Biregeya, Mingyue Shi, Yixiao Chen, Yupeng Wang, Zonghua Wang, Yonghe Hong and Meilian Chen
J. Fungi 2022, 8(12), 1287; https://doi.org/10.3390/jof8121287 - 8 Dec 2022
Cited by 1 | Viewed by 1639
Abstract
Reversible protein phosphorylation is essential in cellular signal transduction. The rice blast fungus Magnaporthe oryzae contains six putative type 2C protein phosphatases, namely MoPtc1, MoPtc2, MoPtc5, MoPtc6, MoPtc7, and MoPtc8. The major functions of MoPtc1 and MoPtc2 have been reported recently. In this [...] Read more.
Reversible protein phosphorylation is essential in cellular signal transduction. The rice blast fungus Magnaporthe oryzae contains six putative type 2C protein phosphatases, namely MoPtc1, MoPtc2, MoPtc5, MoPtc6, MoPtc7, and MoPtc8. The major functions of MoPtc1 and MoPtc2 have been reported recently. In this communication, we found that MoPtc1 and MoPtc2 were induced by calcium chloride. We also found that the deletion of both MoPtc1 and MoPtc2 resulted in the overstimulation of both the high-osmolarity glycerol (Hog1) and pathogenicity MAP kinase 1 (Pmk1) pathways in M. oryzae. MoPtc1 was recruited directly to Osm1 (the osmotic stress-sensitive mutant) by the adaptor protein MoNbp2 to inactivate the Osm1 during hypoosmotic stress, distinct from the budding yeast. Moreover, we showed that MoPtc1 and MoPtc2 were localized in different cellular compartments in the fungal development. Taken together, we added some new findings of type 2C protein phosphatases MoPtc1 and MoPtc2 functions to the current knowledge on the regulation of MAPK signaling pathways in M. oryzae. Full article
(This article belongs to the Special Issue Signal Transductions in Fungi 2.0)
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Figure 1

Figure 1
<p>Transcript expression patterns of MoPTC1 and MoPTC2 in different stress-causing agents. In this assay, β-tubulin was used as the reference gene. The error bars represent the mean standard error from three independent replicates. The double asterisks indicate the adjusted <span class="html-italic">p</span>-value of 0.0013 and the quadruple asterisks show the adjusted <span class="html-italic">p</span>-value of &lt;0.0001.</p> Full article ">Figure 2
<p>MoPtc1 and MoPtc2 regulate MAPK signaling cascades in <span class="html-italic">M. oryzae</span>. (<b>A</b>) The Western blot assay shows the Pmk1 phosphorylation level in Guy11, <span class="html-italic">∆Moptc1</span>, and <span class="html-italic">∆Moptc2</span> and <span class="html-italic">∆Moptc1∆Moptc2</span> strains. (<b>B</b>) The extent of Pmk1 phosphorylation was estimated by calculating the amount of phosphorylated Pmk1 (P-Pmk1) compared to the total amount of Pmk1. (<b>C</b>) Western blot images quantify the Osm1-MAPK phosphorylation in Guy11 and <span class="html-italic">∆Mopt</span>c1, <span class="html-italic">∆Moptc2</span>, <span class="html-italic">∆Moptc1∆Moptc2</span>, <span class="html-italic">∆Moptc1_Com</span> (Complementation), <span class="html-italic">∆Moptc2_Com</span> (Complementation), and <span class="html-italic">∆Moosm1</span>. (<b>D</b>) The band intensity ratio between phosphorylated Osm1 (P-Osm1) and β-actin. The single asterisk (*) represents statistical significance with an adjusted <span class="html-italic">p</span>-value of 0.0338.</p> Full article ">Figure 3
<p>MoPtc1 interacts with MoPmk1 and MoOsm1 in <span class="html-italic">M. oryzae</span>. The yeast two-hybrid assay shows the interactions between (<b>A</b>) MoPtc1 and MoNbp2, between (<b>B</b>) MoNbp2 and MoPmk1, and between (<b>C</b>) MoNbp2 and MoOsm1 in rice blast fungus. The pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T vectors were used as positive and negative controls, respectively. The co-transformation of prey and bait constructs into the yeast strain resulted in the transcription activation of reporter genes and growth on the selective medium -Leu/-Trp/-His/-Ade. The yeast colonies turned blue after the secretion of β-galactosidase (LacZ) in the selective medium and the hydrolysis of X-α-gal. The co-immunoprecipitation assay confirms the interactions between (<b>D</b>) MoPtc1 and MoNbp2, between (<b>E</b>) MoNbp2 and MoPmk1, and between (<b>F</b>) MoNbp2 and MoOsm1 in <span class="html-italic">M. oryzae</span>.</p> Full article ">Figure 4
<p>The subcellular localization of MoPtc1 and MoPtc2 protein phosphatases in <span class="html-italic">M. oryzae</span>. (<b>A</b>) The colocalization of MoPtc1 and MoPtc2 in hyphal (HY) stages of development (<b>B</b>) The localization pattern of MoPtc1 in conidia (CO). (<b>C</b>) The localization of MoPtc2 in conidia (CO). (<b>D</b>,<b>E</b>) The localization of MoPtc1 and MoPtc2 in appressorium (AP) development. (<b>F</b>) The colocalization of MoPtc1 and MoPtc2 in invasive hypha (IH). H1_RFP is the Histone 1 fused with red fluorescence protein. The live cell images were visualized by Nikon Air Laser confocal microscopy (scale bar = 20 µm).</p> Full article ">Figure 5
<p>A model showing regulation of MAPK signaling pathways by MoPtc1 and MoPtc2 in <span class="html-italic">M. oryzae</span>. The Pmk1 pathway is essential for appressorium formation and invasive growth in this fungus. We identified MoPtc1 and MoPtc2 as negative regulators of the MoPmk1-MAPK in <span class="html-italic">M. oryzae</span>. The Osm1 pathway was activated during high osmolarity and oxidative stress and played crucial role in osmotic stress response. This pathway was negatively regulated by MoPtc1 and positively regulated by MoPtc2. The MoPtc1 interacted with MoOsm1-MAPK through the adaptor protein MoNbp2 both in vitro and in vivo.</p> Full article ">
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