Phylogenetic and Morphological Evidence for Three New Species of Diaporthales (Ascomycota) from Fujian Province, China.

Members of the fungal order Diaporthales are sac fungi that include plant pathogens (the notorious chestnut blight fungus), as well as saprobes and endophytes, and are capable of colonizing a wide variety of substrates in different ecosystems, habitats, and hosts worldwide. However, many Diaporthales species remain unidentified, and various inconsistencies within its taxonomic category remain to be resolved. Here, we aimed to identify and classify new species of Diaporthales by using combined morphological and molecular characterization and coupling this information to expand our current phylogenetic understanding of this order. Fungal samples were obtained from dead branches and diseasedleaves of Camellia (Theaceae) and Castanopsis (Fagaceae) in Fujian Province, China. Based on morphological characteristics and molecular phylogenetic analyses derived from the combined nucleotide sequences of loci of the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA gene (LSU), the translation elongation factor 1-α gene (tef1), the partial beta-tubulin gene (tub2), and partial RNA polymerase II second-largest subunit gene (rpb2), three new species of Diaporthales were identified and characterized. They are as follows: Chrysofolia camelliae sp. nov., Dendrostoma castanopsidis sp. nov., and Pseudoplagiostoma wuyishanense sp. nov. They are described and illustrated. This study extends our understanding of species diversity within the Diaporthales.

Characterization of Terpenoids from the Ambrosia Beetle Symbiont and Laurel Wilt Pathogen Harringtonia lauricola.

Little is known concerning terpenoids produced by members of the fungal order Ophiostomales, with the member Harringtonia lauricola having the unique lifestyle of being a beetle symbiont but potentially devastating tree pathogen. Nine known terpenoids, including six labdane diterpenoids (1-6) and three hopane triterpenes (7-9), were isolated from H. lauricola ethyl acetate (EtOAc) extracts for the first time. All compounds were tested for various in vitro bioactivities. Six compounds, 2, 4, 5, 6, 7, and 9, are described functionally. Compounds 2, 4, 5, and 9 expressed potent antiproliferative activity against the MCF-7, HepG2 and A549 cancer cell lines, with half-maximal inhibitory concentrations (IC50s) ~12.54-26.06 µM. Antimicrobial activity bioassays revealed that compounds 4, 5, and 9 exhibited substantial effects against Gram-negative bacteria (Escherichia coli and Ralstonia solanacearum) with minimum inhibitory concentration (MIC) values between 3.13 and 12.50 µg/mL. Little activity was seen towards Gram-positive bacteria for any of the compounds, whereas compounds 2, 4, 7, and 9 expressed antifungal activities (Fusarium oxysporum) with MIC values ranging from 6.25 to 25.00 µg/mL. Compounds 4, 5, and 9 also displayed free radical scavenging abilities towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide (O2-), with IC50 values of compounds 2, 4, and 6 ~3.45-14.04 µg/mL and 22.87-53.31 µg/mL towards DPPH and O2-, respectively. These data provide an insight into the biopharmaceutical potential of terpenoids from this group of fungal insect symbionts and plant pathogens.

Characterization of an α-Amylase from the Honeybee Chalk Brood Pathogen Ascosphaera apis.

The insect pathogenic fungus, Ascosphaera apis, is the causative agent of honeybee chalk brood disease. Amylases are secreted by many plant pathogenic fungi to access host nutrients through the metabolism of starch, and the identification of new amylases can have important biotechnological applications. Production of amylase by A. apis in submerged culture was optimized using the response surface method (RSM). Media composition was modeled using Box-Behnken design (BBD) at three levels of three variables, and the model was experimentally validated to predict amylase activity (R2 = 0.9528). Amylase activity was highest (45.28 ± 1.16 U/mL, mean ± SE) in media composed of 46 g/L maltose and1.51 g/L CaCl2 at a pH of 6.6, where total activity was ~11-fold greater as compared to standard basal media. The enzyme was purified to homogeneity with a 2.5% yield and 14-fold purification. The purified enzyme had a molecular weight of 75 kDa and was thermostable and active in a broad pH range (> 80% activity at a pH range of 7-10), with optimal activity at 55 °C and pH = 7.5. Kinetic analyses revealed a Km of 6.22 mmol/L and a Vmax of 4.21 µmol/mL·min using soluble starch as the substrate. Activity was significantly stimulated by Fe2+ and completely inhibited by Cu2+, Mn2+, and Ba2+ (10 mM). Ethanol and chloroform (10% v/v) also caused significant levels of inhibition. The purified amylase essentially exhibited activity only on hydrolyzed soluble starch, producing mainly glucose and maltose, indicating that it is an endo-amylase (α-amylase). Amylase activity peaked at 99.38 U/mL fermented in a 3.7 L-bioreactor (2.15-fold greater than what was observed in flask cultures). These data provide a strategy for optimizing the production of enzymes from fungi and provide insight into the α-amylase of A. apis.

A new species and new records species of Pluteus from Xinjiang Uygur Autonomous Region, China.

Xinjiang Uyghur Autonomous Region in China embraces a unique geographical and ecological environment, and the macrofungi represent a rich resource. However, few studies on the genus Pluteus have been reported from Xinjiang. In 2021, the macrofungal resources in Xinjiang were surveyed, and 10 specimens belonging to the genus Pluteus were collected. Based on the morphological study and molecular analysis, three species were recognized, P. aletaiensis, P. brunneidiscus, and P. hongoi. Pluteus aletaiensis is proposed as a new species. It is characterized by its bright yellow lamellae and stipe, brittle texture, subfusiform to vesicular pleurocystidia, with short pedicels to broadly lageniform to obtuse at apices, a hymeniderm pileipellis, containing dark brown intracellular pigment, and it grows on the ground. Pluteus brunneidiscus, a new record to China, is characterized by uneven, smooth, grayish brown to brown pileus, with an entire margin, and pointed or flatter apices intermediate cystidia, without apical hooks. Pluteus hongoi, a new record to Xinjiang Uyghur Autonomous Region, China, is characterized by the apical hook's structure (commonly bifid) of pleurocystidia. The nuclear internal transcribed spacer (nrITS) and translation elongation factor 1-alpha (TEF1-a) region were used for the molecular analysis. Phylogenetic trees were constructed using both the maximum likelihood analysis (ML) and Bayesian inference (BI). Detailed descriptions of the three species are presented herein. Finally, a key to the list of eight species of the genus Pluteus knew from Xinjiang is provided.

Purification and properties of a laccase from the mushroom Agaricus sinodeliciosus.

A homogeneous monomeric laccase (ASL) from Agaricus sinodeliciosus, with a molecular mass of 65 kDa, was isolated using ion-exchange chromatography (CM-cellulose and Q-Sepharose) and gel-filtration chromatography (Superdex 75). This laccase exhibited maximum activity at 50 °C and pH 5.0. Hg2+ and Cd2+ significantly inhibited its activity. The laccase displayed a Km value of 0.9 mM toward 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS). In addition to ABTS, ASL exhibited higher affinity toward o-toluidine and benzidine than other substrates. ASL is able to decolorize malachite green and Eriochrome black T.

Deacetylation of chitin oligomers increases virulence in soil-borne fungal pathogens.

Soil-borne fungal pathogens that cause crop disease are major threats to agriculture worldwide. Here, we identified a secretory polysaccharide deacetylase (PDA1) from the soil-borne fungus Verticillium dahliae, the most notorious plant pathogen of the Verticillium genus, that facilitates virulence through direct deacetylation of chitin oligomers whose N-acetyl group contributes to host lysine motif (LysM)-containing receptor perception for ligand-triggered immunity. Polysaccharide deacetylases are widely present in fungi, bacteria, insects and marine invertebrates and have been reported to possess diverse functions in developmental processes rather than virulence. A phylogenetics analysis of more than 5,000 fungal proteins with conserved polysaccharide deacetylase domains showed that the V. dahliae PDA1-containing subtree includes a large number of proteins from the Verticillium genus as well as the Fusarium genus, another group of characterized soil-borne fungal pathogens, suggesting that soil-borne fungal pathogens have adopted chitin deacetylation as a major virulence strategy. We showed that a Fusarium PDA1 is required for virulence in cotton plants. This study reveals a substantial virulence function role of polysaccharide deacetylases in pathogenic fungi and demonstrates a subtle mechanism whereby deacetylation of chitin oligomers converts them to ligand-inactive chitosan, representing a common strategy of preventing chitin-triggered host immunity by soil-borne fungal pathogens.

Pleurotus eryngii Genomes Reveal Evolution and Adaptation to the Gobi Desert Environment.

Pleurotus eryngii (King Oyster) is one of the most highly prized edible mushrooms. Among the diverse varieties within P. eryngii, P. eryngii var. eryngii is the commonest one, with a worldwide distribution, while P. eryngii var. ferulae is only distributed in Europe and China, and is especially adapted to the Gobi Desert in Xinjiang Autonomous Region of China. However, little is known about the genome-wide pattern of evolution and adaptation to the divergent environments of P. eryngii. Here, we present the high-quality genome sequences of P. eryngii var. eryngii strain PEE81 originating from Europe and P. eryngii var. ferulae strain PEF12 originating from the Gobi Desert of China. The assembled genome sizes of PEE81 and PEF12 were 53.6 and 48.0 Mbp, respectively, which are larger than other reported genomes in the genus Pleurotus. We propose that the selective amplification of long terminal repeat (LTR) retrotransposons increases the genome size of the genus Pleurotus, and may play a key role in driving their rapid species diversification. Molecular clock analyses of five Pleurotus species, namely PEE81, PEF12, P. tuoliensis, P. ostreatus and P. cf. floridanus suggest that the divergence estimates of the genus Pleurotus over time scales ranged from ∼4 to ∼38 million years ago (Mya), and PEE81 and PEF12 diverged at ∼13 Mya. The whole genome resequencing of 33 geographically diverse strains of P. eryngii var. eryngii and var. ferulae was then performed and the genome variation among and within these two populations were investigated. Comparative analyses of these two populations detected several candidate genes related to stress responses and DNA repair that are putatively involved in adaptation to the Gobi Desert environment. These findings offer insights into genome evolution of the genus Pleurotus and provide valuable genomic resources for King Oyster mushroom breeding.

Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from Verticillium dahliae.

Verticillium dahliae Kleb. is a hemibiotrophic, phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. Successful host colonization by hemibiotrophic pathogens requires the induction of plant cell death to provide the saprophytic nutrition for the transition from the biotrophic to the necrotrophic stage. In this study, we identified a necrosis-inducing Phytophthora protein (NPP1) domain-containing protein family containing nine genes in a virulent, defoliating isolate of V. dahliae (V592), named the VdNLP genes. Functional analysis demonstrated that only two of these VdNLP genes, VdNLP1 and VdNLP2, encoded proteins that were capable of inducing necrotic lesions and triggering defense responses in Nicotiana benthamiana, Arabidopsis, and cotton plants. Both VdNLP1 and VdNLP2 induced the wilting of cotton seedling cotyledons. However, gene-deletion mutants targeted by VdNLP1, VdNLP2, or both did not affect the pathogenicity of V. dahliae V592 in cotton infection. Similar expression and induction patterns were found for seven of the nine VdNLP transcripts. Through a comparison of the conserved amino acid residues of VdNLP with different necrosis-inducing activities, combined with mutagenesis-based analyses, we identified several novel conserved amino acid residues, in addition to the known conserved heptapeptide GHRHDWE motif and the cysteine residues of the NPP domain-containing protein, that are indispensable for the necrosis-inducing activity of the VdNLP2 protein.

A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity.

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. The life cycle of V. dahliae includes three vegetative phases: parasitic, saprophytic and dormant. The dormant microsclerotia are the primary infectious propagules, which germinate when they are stimulated by root exudates. In this study, we report the first application of Agrobacterium tumefaciens-mediated transformation (ATMT) for construction of insertional mutants from a virulent defoliating isolate of V. dahliae (V592). Changes in morphology, especially a lack of melanized microsclerotia or pigmentation traits, were observed in mutants. Together with the established laboratory unimpaired root dip-inoculation approach, we found insertional mutants to be affected in their pathogenicities in cotton. One of the genes tagged in a pathogenicity mutant encoded a glutamic acid-rich protein (VdGARP1), which shared no significant similarity to any known annotated gene. The vdgarp1 mutant showed vigorous mycelium growth with a significant delay in melanized microsclerotial formation. The expression of VdGARP1 in the wild type V529 was organ-specific and differentially regulated by different stress agencies and conditions, in addition to being stimulated by cotton root extract in liquid culture medium. Under extreme infertile nutrient conditions, VdGARP1 was not necessary for melanized microsclerotial formation. Taken together, our data suggest that VdGARP1 plays an important role in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. Overall, our findings indicate that insertional mutagenesis by ATMT is a valuable tool for the genome-wide analysis of gene function and identification of pathogenicity genes in this important cotton pathogen.