The genus Armillaria is a broad group of basidiomycete (Agaricales, Physalacriaceae) plant pathogens that can have detrimental effects on woody hosts in forested, urban, and horticultural landscapes. Several species are known as aggressive root pathogens on both conifers and deciduous woody plants. Armillaria species are considered white rot fungi due to their capacity to degrade both lignin and cellulose in woody tissues. Armillaria mexicana was recently described as a new species found in peach (Prunus persica) orchards of Coatepec Harinas, State of Mexico. However, a subsequent study identified A. mexicana on avocado (Persea americana) and pine (Pinus sp.), suggesting that A. mexicana may be able to infect diverse hosts that are planted in disturbed areas following deforestation. We assembled a reference genome for A. mexicana consisting of 38 contigs constructed using PacBio and Illumina sequencing reads. Genome annotation and comparison with A. mellea, a phylogenetic sister taxon, revealed notable differences, including a larger number of interspersed repeats in A. mexicana and the absence of small RNAs, which were detected in A. mellea. While A. mexicana (49 Mb) has a roughly 30% smaller genome than A. mellea (70 Mb), it has a similar number of genes encoding pectinases and nonribosomal peptide synthetase (NRPS) and NPRS-like secondary metabolites, which may influence the pathogenicity of A. mexicana. This reference genome of A. mexicana allows future genomic comparisons that can help characterize the evolutionary history and enhance our understanding of the molecular mechanisms involved in pathogenicity and wood decomposition of Armillaria species.

Basidiomycetous fungi are prolific producers of terpenoids, yet many biosynthetic pathways remain unexplored. Here, we report the discovery of two novel terpene synthases: PoTS6 from the oyster mushroom (Pleurotus ostreatus) and AgTS2 from the bulbous honey fungus (Armillaria gallica). Both produce a unique terpene scaffold, pleostene (1), whose structure was elucidated by using NMR spectroscopy and GC-MS analysis. Despite sharing only 44% sequence identity, PoTS6 and AgTS2 exhibit highly similar three-dimensional conformations, including conserved active-site residues and their spatial arrangement within the substrate-binding pockets. Our finding highlights the importance of structural conservation over sequence similarity in product specificity. Phylogenetic analysis reveals two distinct clades of homologous enzymes, suggesting pleostene biosynthesis may be widespread among Agaricales fungi. Functional analysis demonstrates that the C-terminal region of PoTS6 (residues 331-407) is dispensable for catalytic activity. Genomic context analysis predicts that PoTS6 is not part of a gene cluster, whereas AgTS2 resides within a putative biosynthetic gene cluster containing 3 cytochrome P450 enzymes. These P450s may oxidize pleostene into bioactive derivatives such as clitocybulols and nebucanes, known for their cytotoxic and antimicrobial activities. These findings expand our understanding of fungal terpenoid biosynthesis and offer new opportunities for natural product biosynthesis.

The genus Armillaria (Basidiomycota, Agaricales, Physalacriaceae) comprises pathogenic fungi that cause root-rot disease in plants, as well as species with low pathogenicity, some of which are hosts of the fully mycoheterotrophic orchid plant Gastrodia elata (Orchidaceae). To investigate the mechanisms underlying such special interactions between Armillaria fungi and G. elata, it is crucial to establish genetic transformation platforms for the Armillaria fungi and G. elata. In this study, an Armillaria strain Arm37 was isolated from G. elata, which can form symbiosis with G. elata in axenic culture under laboratory conditions. A vector pYT-EV containing a cassette for hygromycin-resistance selection and a cassette for expressing or silencing target genes was constructed. An Agrobacterium tumefaciens-mediated transformation (ATMT) system for Arm37 was successfully developed and optimized to achieve a transformation efficiency of 32%. The ATMT system was successfully used to express the reporter genes eGFP encoding enhanced green fluorescent protein and GUS encoding β-glucuronidase and to effectively silence the endogenous gene URA3 encoding orotidine-5'-phosphate decarboxylase in Arm37. This ATMT system established for Arm37 provides an efficient genetic tool for exploring the Arm37 genes that are involved in the unique interaction between the Armillaria fungi and fully mycoheterotrophic plant G. elata.

Higher fungi of the genus Armillaria belonging to the phylum Basidiomycota produce bioactive sesquiterpenoid aryl esters called melleolides. A bioactivity-guided discovery process led to the identification of the new melleolide 5'-methoxyarmillane (1) in organic extracts from the mycelium of Armillaria ostoyae. Remarkably, supplementation of rapeseed oil to the culture medium potato dextrose broth increased the production of 1 by a factor of six during the course of the 35 days fermentation. Compound 1 was isolated and its structure elucidated by UHPLC-QTOF-HR-MS/MS and NMR spectroscopy. It showed toxicity against Madin-Darby canine kidney II (MDCK II, IC50 19.2 μg/mL, 44.1 μM) and human lung cancer Calu-3 cells (IC50 15.2 μg/mL, 34.9 μM) as well as moderate bioactivity against Mycobacterium tuberculosis (MIC 8 mg/mL, 18.4 μM) and Mycobacterium smegmatis (MIC 16 mg/mL, 36.8 μM), but not against Staphylococcus aureus, Escherichia coli, Candida albicans, and Septoria tritici. No inhibitory effects of 1 against the influenza viruses H3N2, H1N1pdm, B/Malaysia, and B/Massachusetts were observed.

The genus Armillaria is an essential component of forest ecosystems playing very important ecological role in dead wood decomposition, but it often becomes a serious pathogen causing white root rot in trees. It is also known that Armillaria species significantly differ in the level of pathogenicity. Thus, accurate identification of Armillaria is critical for assessing the risk of tree disease. In this study we analyzed 28 Armillaria isolates from Siberia and the Far East using nucleotide sequences of ITS, IGS-1-1 and TEF-1α gene regions and generated phylogenetic trees based on maximum likelihood method. In total, four Armillaria species were identified: A borealis, A. cepistipes, A. ostoyae and A. gallica. A. borealis was the most frequent among collected isolates (18 out of 28 isolates). A. gallica, A. cepistipes and A. ostoyae were much less frequent with two, five and three isolates out of 28, respectively. Thedistribution of Armillaria species in Siberia and the Far East was described for the first time. It is concluded that further studies are necessary to determine the role of Armillaria in trees pathological dieback, and A. borealis should be a key focus.

Members of the genus Armillaria are widespread forest pathogens against which effective protection has not yet been developed. Due to their longevity and the creation of large-scale cloning of Armillaria individuals, the use of mycoviruses as biocontrol agents (BCAs) against these pathogens could be an effective alternative. This work describes the detection and characterization of viruses in Armillaria spp. collected in the Czech Republic through the application of stranded total RNA sequencing. A total of five single-stranded RNA viruses were detected in Armillaria ostoyae and A. cepistipes, including viruses of the family Tymoviridae and four viruses belonging to the recently described "ambivirus" group with a circular ambisense genome arrangement. Both hammerhead (HHRz) and hairpin (HpRz) ribozymes were detected in all the ambiviricot sequences. Armillaria viruses were compared through phylogenetic analysis and confirmed their specific host by direct RT-PCR. One virus appears to infect both Armillaria species, suggesting the occurrence of interspecies transmission in nature.

Armillaria korhonenii, designated as Chinese biological species Q (CBSQ) here, is described as a new species based on phylogenetic and morphological evidence as well as the results of mating tests. This species is the sixteenth biological species of Armillaria reported from China following the discovery of 15 previously known biological species. Phylogenetically, A. korhonenii is the earliest divergent lineage of the genus Armillaria as inferred from the nucleotide sequences of the Elongation Factor 1-Alpha (EF1a). Morphologically, it is characterized by its ellipsoid to elongate basidiospores, yellowish orange to honey yellow pileus with fibrillose squamules. Mating tests indicated that A. korhonenii is a tetrapolar heterogeneous species and is inter-sterile with all the tested species except partial compatibility with CBSH.

The globally distributed basidiomycete genus Armillaria includes wood decomposers that can act as opportunistic parasites, causing deadly root rot on woody plants. To test whether RNA viruses are involved in this opportunistic behaviour, a large isolate collection of five Armillaria species collected over 40 years in Switzerland from trees, dead wood and soil was analysed. De novo assembly of RNA-Seq data revealed 21 viruses, 14 of which belong to putative new species. Two dsRNA viruses and an unclassified Tymovirales are formally described for the first time for Armillaria. One mitovirus occurred with a high prevalence of 71.1%, while all other viruses were much less prevalent (0.6%-16.9%). About half of all viruses were found only in one fungal species, others occurred in 2-6 fungal species. Co-infections of 2-7 viruses per isolate were not uncommon (34.9%), and most viruses persisted circulating within fungal populations for decades. Some viruses were related to viruses associated with other Armillaria species, supporting the hypothesis that virus transmission can occur between different fungal species. Although no specific correlation between viruses and the fungal trophic strategy was found, this study opens new insights into viral diversity hidden in the soil microbiome.

The majority study of Armillaria globally had been focused on A. mellea. The Indonesian data of genus Armillaria remains poor. During our fungus foray in West Java (Indonesia), some basidiomata of supa jambu (local name) consumed by the local people were collected. In the field, the specimens were morphologically identified as Armillaria. The present study was to confirm the species identity of our specimens based on morphological and molecular evidence. The fresh basidiomata were used for the examination. The combination of morphological and molecular analyses confirmed our specimen as Armillaria novae-zelandiae. Armillaria novae-zelandiae FIPIA-DEP50 was identified by its viscid, thin, striate, honey-yellow pileus, semi-bulbous to bulbous stipe which darken to black with age towards the base. Spore moderately thick walled, ellipsoid, to elongate-ellipsoid, smooth with some internal structure. Armillaria novae-zelandiae FIPIA-DEP50 is generally smaller in stature than the other closely related species (A. sparrei and A. hinnulea). The BLAST result revealed that our specimen exhibits 98% similarity to Armillaria novae-zelandiae as the top hit. The phylogenetic tree nested our specimens with A. novae-zelandiae clade (BS 100%) and A. mellea as its sister clade. The current study provides the first edibility information and herbarium collection of A. novae-zelandiae for Indonesia.

The Armillaria response to Gastrodia elata is partially mediated by strigolactone-induced changes in reactive oxygen species

Disorders of purine metabolism are the main cause of hyperuricemia. Current drugs for the treatment of hyperuricemia usually cause a degree of cardiovascular damage. This study aimed to investigate the therapeutic effects of Armillaria mellea fruiting body (AFB), Armillaria rhizomorph (AR) and Armillaria mellea fermentation product (after rhizomorphs removal) (AFP) on hyperuricemic mice. The hyperuricemia mouse model was established by oral administration of potassium oxonate 0.9 g⋅kg-1 and hypoxanthine 0.5 g⋅kg-1 for two weeks. Starting from the third week, the intragastric administration of the intervention drug group was as follows: Allopurinol 0.013 g⋅kg-1, AFB (3.9 and 7.8 g⋅kg-1), AR (3.9 and 7.8 g⋅kg-1), AFP (1.95 and 3.9 g⋅kg-1) once daily for 14 days. Results showed that AFB, AR, and AFP reduced the contents of serum uric acid, serum creatinine, and blood urea nitrogen in hyperuricemic mice and the mechanism of action might be through up-regulation of the expression levels of organic anion transporter 1/organic anion transporter 3 proteins in kidney tissue. AR and AFP both exhibited better uric acid-lowering effects than AFB, which may be due to the higher purine content of AFB. Armillaria mellea and its fermentation products can treat hyperuricemia by up-regulating OAT1 protein and OAT3 protein, reducing uric acid content in mice.

Protoilludene-type sesquiterpene aryl esters are a unique and very diverse compound class that were exclusively isolated from members of the genus Armillaria (Agaricomycetes, Physalacriaceae) up to this point. Herein, we describe the isolation and structural characterization of 5′-O-methyl-14-hydroxyarmillane (1), a new armillane-type derivative, that was obtained from submerged cultures of Guyanagaster necrorhiza (CBS 138623) together with the known congeners melleolide G (2), melleolide B (3), and 10-dehydroxy-melleolide B (4). ROESY data and coupling constants assigned the relative configurations of 1, while common absolute configurations were confirmed from comparison of its ECD spectrum to the one of 10-hydroxy-5′-methoxy-6′-chloroarmillane (5). Additionally, the configuration of melleolide G (2) was revised based on observed ROESY correlations. It is the first time that protoilludene-type sesquiterpene aryl esters were isolated from another genus, namely, Guyanagaster, that is closely related to Armillaria. 1–4 were evaluated for their biological activities in a serial dilution assay against several yeast, fungi, and bacteria, as well as in a cytotoxicity assay against different cell lines. Compound 4 was moderately active against Bacillus subtilis, Staphylococcus aureus, and Mucor hiemalis. Furthermore, 1, 3, and 4 showed weak cytotoxic effects against the mouse fibroblast cell line L929 and the cervix carcinoma cell line KB3.1.

During the period of the oak forests’ mass desiccation, Armillaria root rot became widespread, exacerbating the state of depression. The infection of stands by white sapwood rot caused by fungi of the genus Armillaria was more often detected in Bug-Polesia and Berezina-Pre-Polesia forest growth areas. The incidence of the disease in the oak forests of Belarus increases with the raising of the stands age and the decrease in their density. More often, the affliction with Armillaria root rot occurs in floodplain oak forests, as well as in the dry lands forest types with the most fertile soils. The oak’s proportion in the stand’s composition does not significantly affect the occurrence of the infection. Under the conditions of massive weakening of the Belarussian oak forests, facultative parasites from the Armillaria genus can occur as dangerous secondary pathogens that accelerate the death of weakened oak trees. Their pathogenicity persists in the north of the republic, where the negative role of other pathological factors in oak forests decreases. After the end of the depression period, the transition of Armillaria from a parasitic to a predominantly saprotrophic strategy was recorded. The affliction of oak forests by Armillaria root rot can act as one of the oak formation’s condition indicators, marking the depression phase in oak stands. Therefore, the spread of foci of Armillaria root rot should be monitored while conducting forest pathology surveys and monitoring the condition of oak forests.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

On Norway spruce (Picea abies L. [H.] Karst.) seedlings, influence of three treatments – (i) inoculation with Ectovit® preparation containing ectomycorrhizal fungi (INOCULATION), (ii) Ectovit® + Conavit® fertilizer (INOCULATION + FERTILIZATION), and (iii) untreated group (CONTROL) – were evaluated in this study. In autumn 2021 (3.5 years after inoculation), 100 seedlings/treatment (300 seedlings in total) were sampled to evaluate seedlings survival percentage and growth characteristics, and 10 live seedlings/treatment (30 seedlings in total) to evaluate dry weight and the number of seedlings with Armillaria rhizomorphs. By relating these values to the previous evaluation (autumn 2019), values for period autumn 2019 – autumn 2021 were obtained for statistical analyses. Increment of the seedling height, increment of the root collar diameter and increment of the seedling aboveground dry weight were significantly higher in INOCULATION compared to INOCULATION + FERTILIZATION, the first and second named variables were also significantly higher in INOCULATION compared to CONTROL. There were no significant differences in seedlings survival percentage nor number of seedlings with Armillaria rhizomorphs (all identified as Armillaria ostoyae [Romagn.] Herink). Contrary to the previous evaluation (1.5 years after inoculation), the protective effect of artificial ectomycorrhizal inoculation against infection of seedling by honey fungus was not confirmed.

ABSTRACT The genus Armillaria has high edible and medical values, with zones of antagonism often occurring when different species are paired in culture on agar media, while the antagonism-induced metabolic alteration remains unclear. Here, the metabolome of mycelial exudates of two Chinese Armillaria biological species, C and G, co-cultured or cultured separately was analysed to discover the candidate biomarkers and the key metabolic pathways involved in Armillaria antagonists. A total of 2,377 metabolites were identified, mainly organic acids and derivatives, lipids and lipid-like molecules, and organoheterocyclic compounds. There were 248 and 142 differentially expressed metabolites between group C-G and C, C-G, and G, respectively, and fourteen common differentially expressed metabolites including malate, uracil, Leu-Gln-Arg, etc. Metabolic pathways like TCA cycle and pyrimidine metabolism were significantly affected by C-G co-culture. Additionally, 156 new metabolites (largely organic acids and derivatives) including 32 potential antifungal compounds, primarily enriched into biosynthesis of secondary metabolites pathways were identified in C-G co-culture mode. We concluded that malate and uracil could be used as the candidate biomarkers, and TCA cycle and pyrimidine metabolism were the key metabolic pathways involved in Armillaria antagonists. The metabolic changes revealed in this study provide insights into the mechanisms underlying fungal antagonists.

Background: Aflatoxin is one of the most problematic fungal-produced toxins as it is responsible for massive global agricultural losses and is deleterious to both human and animal health. Contamination of crops by certain strains of Aspergillus fungi accumulate aflatoxin in both post- and pre-harvest conditions. Methods: In this report, we tested the aflatoxin-degradation efficiency of an endogenously expressed enzyme in harvested maize kernels. In post-harvest conditions, equivalent loads of A. flavus were used to infect harvested maize kernels previously engineered to express an aflatoxin-degrading enzyme from the Honey mushroom fungus. Results: No measurable, or significantly reduced, levels of aflatoxin were detected in all the enzyme-expressing harvested kernels initially and then 3 days post –harvest the transgenic kernels amassed aflatoxin. Conclusions: This is the first report of an enzyme degradation of aflatoxin in a crop in harvested kernels. This demonstrates the potential of this strategy to aid in the mitigation of aflatoxin in post-harvest conditions.

Background: Aflatoxin is one of the most problematic fungal-produced toxins as it is responsible for massive global agricultural losses and is deleterious to both human and animal health. Contamination of crops by certain strains of Aspergillus fungi accumulate aflatoxin in both post- and pre-harvest conditions. Methods: In this report, we tested the aflatoxin-degradation efficiency of an endogenously expressed enzyme in harvested maize kernels. In post-harvest conditions, equivalent loads of A. flavus were used to infect harvested maize kernels previously engineered to express an aflatoxin-degrading enzyme from the Honey mushroom fungus. Results: No measurable, or significantly reduced, levels of aflatoxin were detected in all the enzyme-expressing harvested kernels initially and then 3 days post –harvest the transgenic kernels amassed aflatoxin. Conclusions: This is the first report of an enzyme degradation of aflatoxin in a crop in harvested kernels. This demonstrates the potential of this strategy to aid in the mitigation of aflatoxin in post-harvest conditions.

ABSTRACTArmillaria 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.

Honey mushroom, Armillaria mellea, is an edible and medicinal lignicolous basidiomycete. In this study, we investigated the chemical composition and bioactive properties of its methanolic and acetonic extracts. The chemical characterization of extracts was done with the HPLC-DAD-MS/MS method. The results showed that potassium was the most abundant mineral; chlorogenic acid was the most abundant polyphenol; malic acid was the most abundant organic acid; and among carbohydrates, the most abundant were sorbitol, glucose, fructose, and saccharose. Antioxidative activity was assessed by DPPH (IC50 of the methanolic extract was 608.32 μg/mL and of the acetonic extract 595.71 μg/mL) and reducing power assays (the results ranged between 0.034 and 0.102 μg/mL). Total phenolic content was determined as gallic acid equivalent (methanolic extract: 4.74 mg GAE/g; acetonic extract: 5.68 mg GAE/g). The microdilution assay was used to evaluate the antimicrobial activity of the extracts, and the results ranged from 1.25 to 20 mg/mL. The antidiabetic effect of the extracts was tested by the α-amylase (the results ranged from 34.90% to 41.98%) and α-glucosidase assays (the results were in the range of 0.55-2.79%). The neuroprotective activity was explored by the acetylcholinesterase inhibition assay (the results were in the range of 1.94-7.76%). The microtetrazolium assay was used to investigate the cytotoxic activity of the extracts (the IC50 values ranged from 212.06 to > 400 μg/mL). Although some results suggest that some activities of the extracts are relatively moderate, the honey mushroom can still be considered an excellent source of food and bioactive compounds with medicinal value.