Network Pharmacology Analysis of Liquid-Cultured Armillaria ostoyae Mycelial Metabolites and Their Molecular Mechanism of Action against Gastric Cancer.

Armillaria sp. are traditional edible medicinal mushrooms with various health functions; however, the relationship between their composition and efficacy has not yet been determined. Here, the ethanol extract of liquid-cultured Armillaria ostoyae mycelia (AOME), a pure wild Armillaria sp. strain, was analyzed using UHPLC-QTOF/MS, network pharmacology, and molecular docking techniques. The obtained extract affects various metabolic pathways, such as JAK/STAT and PI3K/AKT. The extract also contains important compounds such as 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide, isoliquiritigenin, and 7-hydroxycoumarin. Moreover, the extract targets key proteins, including EGFR, SCR, and IL6, to suppress the progression of gastric cancer, thereby synergistically inhibiting cancer development. The molecular docking analyses indicated that the main compounds stably bind to the target proteins. The final cell culture experimental data showed that the ethanol extract inhibited MGC-803 gastric cancer cells. In summary, our research revealed the beneficial components of AOME for treating gastric cancer and its associated molecular pathways. However, further research is needed to confirm its effectiveness and safety in gastric cancer patients.

White rot fungal impact on the evolution of simple phenols during decay of silver fir wood by UHPLC-HQOMS.

INTRODUCTION: Silver fir (Abies alba Mill.) is one of the most valuable conifer wood species in Europe. Among the main opportunistic pathogens that cause root and butt rot on silver fir are Armillaria ostoyae and Heterobasidion abietinum. Due to the different enzymatic pools of these wood-decay fungi, different strategies in metabolizing the phenols were available. OBJECTIVE: This work explores the changes in phenolic compounds during silver fir wood degradation. METHODOLOGY: Phenols were analyzed before and after fungus inoculation in silver fir macerated wood after 2, 4 and 6 months. All samples were analyzed using high-performance liquid chromatography coupled to a hybrid quadrupole-orbitrap mass spectrometer. RESULTS: Thirteen compounds, including simple phenols, alkylphenyl alcohols, hydroxybenzoketones, hydroxycinnamaldehydes, hydroxybenzaldehydes, hydroxyphenylacetic acids, hydroxycinnamic acids, hydroxybenzoic acids and hydroxycoumarins, were detected. Pyrocatechol, coniferyl alcohol, acetovanillone, vanillin, benzoic acid, 4-hydroxybenzoic acid and vanillic acid contents decreased during the degradation process. Methyl vanillate, ferulic acid and p-coumaric were initially produced and then degraded. Scopoletin was accumulated. Pyrocatechol, acetovanillone and methyl vanillate were found for the first time in both degrading and non-degrading wood of silver fir. CONCLUSIONS: Despite differences in the enzymatic pool, both fungi caused a significant decrease in the amounts of phenolic compounds with the accumulation of the only scopoletin. Principal component analysis revealed an initial differentiation between the degradation activity of the two fungal species during degradation, but similar phenolic contents at the end of wood degradation.

A Novel Tyrosinase from Armillaria ostoyae with Comparable Monophenolase and Diphenolase Activities Suffers Substrate Inhibition.

Tyrosinase is a bifunctional enzyme mediating the o-hydroxylation and two-electron oxidation of monophenols to o-quinones. The monophenolase activity of tyrosinase is much desired for the industrial synthesis of catechols. However, the generally low ratio of monophenolase/diphenolase activity of tyrosinase limited its utilization in the industry. In this study, a novel tyrosinase from Armillaria ostoyae strain C18/9 (AoTyr) was characterized, and the results showed that the enzyme has an optimal temperature of 25°C and an optimal pH of 6. The enzyme has comparable monophenolase and diphenolase activities and exhibits substrate inhibition in both of the activities. In silico analysis and mutagenesis experiments showed that residues 262 and 266 play important roles in modulating the substrate inhibition and enzymatic activities of AoTyr, and the replacement of D262 with asparagine significantly increased the monophenolase/diphenolase catalytic efficiencies (kcat/Km ratios) (1.63-fold) of the enzyme. The results from this study indicated that this novel tyrosinase could be a potential candidate for the industrial biosynthesis of catechols. IMPORTANCE Tyrosinase is able to oxidize various phenolic compounds, and its ability to convert monophenols into diphenols has caught great attention in the research field and industrial applications. However, the utilization of tyrosinase for the industrial synthesis of catechols has been limited due to the fact that the monophenolase activity of most of the known tyrosinases is much lower than the diphenolase activity. In the present study, a novel tyrosinase with comparable monophenolase and diphenolase activities was characterized. The enzyme exhibits substrate inhibition in both monophenolase and diphenolase activities. In silico analysis followed by mutagenesis experiments confirmed the important roles of residues 262 and 266 in the substrate inhibition and activity modulation of the enzyme, and the D262N variant showed an enhanced monophenolase/diphenolase catalytic efficiency ratio compared to the wild-type enzyme.

Susceptibility of Garden Trees and Shrubs to Armillaria Root Rot.

Armillaria root rot (ARR) is a serious disease of woody plants caused by several species of Armillaria. Armillaria isolates from diagnostic samples received in 2017 were identified by genus- and species-specific PCR and compared with isolates from an earlier survey (2004 to 2007). The results were comparable and, therefore, were combined for further analysis. Three species were identified: Armillaria mellea (83%), A. gallica (15%), and A. ostoyae (2%). Their wide host range makes choice of resistant plants in management of the disease difficult. We used the Royal Horticultural Society diagnostic dataset of ARR records from U.K. gardens to compare the susceptibility of different host genera to the disease. The dataset was compared with an earlier experiment at the University of California. An index-based approach was used to separate genera into three categories: 77 low-index (<0.99), 37 medium-index (0.99 to 1.76), and 56 high-index (>1.76) genera were recorded. All three species were associated with both angiosperms and gymnosperms; moreover, A. ostoyae did not show the host preference for gymnosperms that has been reported elsewhere. A. gallica was particularly common on herbaceous perennials and showed a trend to occur on resistant hosts that may be under other stress, supporting its description as an opportunistic pathogen. Four monocotyledons grown as trees or shrubs in U.K. gardens had a very low ARR index according to indices associated with A. mellea and A. ostoyae. Genera in the order Myrtales were almost always low index, while those in the Saxifragales and Fagales were mostly high index. These results provide confidence in the use of host resistance as part of the integrated management of ARR.

High-density genetic mapping identifies the genetic basis of a natural colony morphology mutant in the root rot pathogen Armillaria ostoyae.

Filamentous fungi exhibit a broad spectrum of heritable growth patterns and morphological variations reflecting the adaptation of the different species to distinct ecological niches. But also within species, isolates show considerable variation in growth rates and other morphological characteristics. The genetic basis of this intraspecific variation in mycelial growth and morphology is currently poorly understood. By chance, a growth mutant in the root rot pathogen Armillaria ostoyae was discovered. The mutant phenotype was characterized by extremely compact and slow growth, as well as shorter aerial hyphae and hyphal compartments in comparison to the wildtype phenotype. Genetic analysis revealed that the abnormal phenotype is caused by a recessive mutation, which segregates asa single locus in sexual crosses. In order to identify the genetic basis of the mutant phenotype, we performed a quantitative trait locus (QTL) analysis. A mapping population of 198 haploid progeny was genotyped at 11,700 genome-wide single nucleotide polymorphisms (SNPs) making use of double digest restriction site associated DNA sequencing (ddRADseq). In accordance with the genetic analysis, a single significant QTL was identified for the abnormal growth phenotype. The QTL confidence interval spans a narrow, gene dense region of 87kb in the A. ostoyae genome which contains 37 genes. Overall, our study reports the first high-density genetic map for an Armillaria species and shows its successful application in forward genetics by resolving the genetic basis of a mutant phenotype with a severe defect in hyphal growth.

Dual RNA-Seq Profiling Unveils Mycoparasitic Activities of Trichoderma atroviride against Haploid Armillaria ostoyae in Antagonistic Interaction Assays.

Armillaria ostoyae, a species among the destructive forest pathogens from the genus Armillaria, causes root rot disease on woody plants worldwide. Efficient control measures to limit the growth and impact of this severe underground pathogen are under investigation. In a previous study, a new soilborne fungal isolate, Trichoderma atroviride SZMC 24276 (TA), exhibited high antagonistic efficacy, which suggested that it could be utilized as a biocontrol agent. The dual culture assay results indicated that the haploid A. ostoyae-derivative SZMC 23085 (AO) (C18/9) is highly susceptible to the mycelial invasion of TA. In the present study, we analyzed the transcriptome of AO and that of TA in in vitro dual culture assays to test the molecular arsenal of Trichoderma antagonism and the defense mechanisms of Armillaria. We conducted time-course analysis and functional annotation and analyzed enriched pathways and differentially expressed genes including biocontrol-related candidate genes from TA and defense-related candidate genes from AO. The results indicated that TA deployed several biocontrol mechanisms when confronted with AO. In response, AO initiated multiple defense mechanisms to protect against the fungal attack. To our knowledge, the present study offers the first transcriptome analysis of a biocontrol fungus attacking AO. Overall, this study provides insights that aid the further exploration of plant pathogen-biocontrol agent interaction mechanisms. IMPORTANCE Armillaria species can survive for decades in the soil on dead woody debris, develop rapidly under favorable conditions, and harmfully infect newly planted forests. Our previous study found Trichoderma atroviride to be highly effective in controlling Armillaria growth; therefore, our current work explored the molecular mechanisms that might play a key role in Trichoderma-Armillaria interactions. Direct confrontation assays combined with time course-based dual transcriptome analysis provided a reliable system for uncovering the interactive molecular dynamics between the fungal plant pathogen and its mycoparasitic partner. Furthermore, using a haploid Armillaria isolate allowed us to survey the deadly prey-invading activities of the mycoparasite and the ultimate defensive strategies of its prey. Our current study provides detailed insights into the essential genes and mechanisms involved in Armillaria defense against Trichoderma and the genes potentially involved in the efficiency of Trichoderma to control Armillaria. In addition, using a sensitive haploid Armillaria strain (C18/9), with its complete genome data already available, also offers the opportunity to test possible variable molecular responses of Armillaria ostoyae toward diverse Trichoderma isolates with various biocontrol abilities. Initial molecular tests of the dual interactions may soon help to develop a targeted biocontrol intervention with mycoparasites against plant pathogens.

The melanized layer of Armillaria ostoyae rhizomorphs: Its protective role and functions.

Armillaria ostoyae (Romagn.) Herink is a highly pathogenic fungus that uses exploratory, cordlike structures called rhizomorphs to seek out new sources of nutrition, posing a parasitic threat to natural stands of trees, orchards, and vineyards. Rhizomorphs are notoriously difficult to destroy, and this resilience is due in large part to a melanized layer that protects the rhizomorph. While this structure has been previously observed, its structural and chemical defenses are yet to be discerned. Research was conducted on both lab-cultured and wild-harvested rhizomorph samples. While both environments produce rhizomorphs, only the wild-harvested rhizomorphs produced the melanized layer, allowing for direct investigation of its structure and properties. Imaging, chemical analysis, mechanical testing, and finite element modeling were used to understand the defense mechanisms provided by the melanized layer. Imaging showed a porous outer layer in both types of rhizomorphs, though the pores were smaller in the harvested melanized layer. This melanized layer contained calcium, which provides chemical defense against both human and natural control methods, but was absent from cultured samples. Nanoindentation resulted in a larger variance of hardness values for cultured rhizomorphs than for wild-harvested. Finite element analysis proved that the smaller pore structure of the melanized porous layer had the best balance between maximum deformation and resulting permanent deformation. These results allow for a better understanding of the defenses of this pathogenic fungus, which may lead to better control methods.

Frequent diploidisation of haploid Armillaria ostoyae strains in an outdoor inoculation experiment.

Very little is known about the biology and ecology of haploid Armillaria strains in nature. In this outdoor inoculation experiment, we assessed the virulence of six haploid Armillariaostoyae strains along with their diploid parent towards 2-year-old seedlings and 4-year-old saplings of Norway spruce (Picea abies), and determined their ability to colonise freshly cut stumps. As inoculum source an Armillaria-colonised hazelnut (Corylus avellana) stem segment was inserted into the soil substrate. Re-isolations from mycelial fans at the root collar of infected trees or stumps were made. Surprisingly, not a single haploid re-isolate could be recovered. Microsatellite genotyping of 133 re-isolates suggests that the inoculated haploid strains were diploidised either by mating propagules (basidiospores or haploid mycelia) already present in the soil substrate or naturally disseminated in the course of the experiment from nearby forests. Consequently, no conclusion about the infectious ability of haploid Armillaria mycelia under natural conditions can be drawn. Nonetheless, the diploid half-sib families resulting from the diploidisation showed varying degrees of virulence, with a high correlation between the experiment with 2-year-old seedlings and 4-year-old saplings. Despite extensive genotyping of re-isolates, no evidence for somatic recombination between haploid mating propagules and diploidised mycelia was detected, suggesting that this is an uncommon phenomenon in A. ostoyae.

Molecular properties and antioxidant activities of polysaccharides isolated from alkaline extract of wild Armillaria ostoyae mushrooms.

This study aims to discover novel and bioactive polysaccharides (PS) from wild Armillaria ostoyae, a honey mushroom species. Two PS designated AkPS1V-1 (66.6 kDa) and AkPS1V-2 (15.3 kDa) were isolated and fractionated by anion ion exchange (IEC) and size exclusion chromatography (SEC) from the alkaline extract of A. ostoyae mushrooms. AkPS1V-1 was a glucan composed of solely glucose residues and AkPS1V-2 a heteropolysaccharide composed of glucose and galactose at 6:1 molar ratio. AkPS1V-2 exhibited higher antioxidant activities than AkPS1V-1 based on reducing power, radical scavenging and metal chelating assays. The structure of AkPS1V-2 was further analyzed and elucidated as a branched galactoglucan with a backbone composed of (1→6)-ß-D-glucopyranosyl, (1→3)-ß-D-glucopyranosyl, (1→3)-α-D-galactopyranosyl and (1→3,6)-ß-D-glucopyranosyl residues at 3:1:1:1 ratio, and side chain of (1→3)-ß-D-glucopyranosyl residue. This is the first report on a pure PS structure and its antioxidant activities from this mushroom species.

The physiology of basidiomycete linear organs: III. Uptake and translocation of radiocaesium within differentiated mycelia of Armillaria spp. growing in microcosms and in the field.

Autoradiography and quantitative image analysis were used to measure (137) Cs distribution and translocation through mycelia of A. gallica Marxmuller & Romagn. and A. ostoyae (Romagn.) Herink growing in small microcosms in the laboratory. Rhizomorphs of A. gallica were labelled with (134) Cs in the field, and the translation of radiolabel measured after excavation and destructive harvesting. The flux of radiocaesium through rhizomorphs was estimated to be greater than through undifferentiated hyphae, and greater through rhizomorphs growing in the field than through rhizomorphs grown across homogeneous media in the laboratory'. Differentiation within mycelia resulting in melanization or rhizomorph formation cave rise to increased heterogeneity in the (137) Cs distribution through laboratory microcosms compared with that through microcosms containing undifferentiated mycelia. Radiocaesium leaked from undifferentiated hyphae into the surrounding medium, but melanized regions of the mycelium appeared to be conservative for radiocaesium. These findings provide further evidence for the importance of filamentous fungi in determining the distribution and rate of release of radiocaesium currently present in the environment as a result of weapons testing and the accident at the Chernobyl nuclear reactor.