Cu/Zn superoxide dismutase (VdSOD1) mediates reactive oxygen species detoxification and modulates virulence in Verticillium dahliae.

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn-SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione-treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more O2- and less H2 O2 than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host-generated extracellular ROS, and contributes significantly to virulence in V. dahliae.

Diversity and function of fungi associated with the fungivorous millipede, Brachycybe lecontii.

Fungivorous millipedes (subterclass Colobognatha) likely represent some of the earliest known mycophagous terrestrial arthropods, yet their fungal partners remain elusive. Here we describe relationships between fungi and the fungivorous millipede, Brachycybe lecontii. Their fungal community is surprisingly diverse, including 176 genera, 39 orders, four phyla, and several undescribed species. Of particular interest are twelve genera conserved across wood substrates and millipede clades that comprise the core fungal community of B. lecontii. Wood decay fungi, long speculated to serve as the primary food source for Brachycybe species, were absent from this core assemblage and proved lethal to millipedes in pathogenicity assays while entomopathogenic Hypocreales were more common in the core but had little effect on millipede health. This study represents the first survey of fungal communities associated with any colobognath millipede, and these results offer a glimpse into the complexity of millipede fungal communities.

Psychoactive plant- and mushroom-associated alkaloids from two behavior modifying cicada pathogens.

Entomopathogenic fungi routinely kill their hosts before releasing infectious spores, but a few species keep insects alive while sporulating, which enhances dispersal. Transcriptomics- and metabolomics-based studies of entomopathogens with post-mortem dissemination from their parasitized hosts have unraveled infection processes and host responses. However, the mechanisms underlying active spore transmission by Entomophthoralean fungi in living insects remain elusive. Here we report the discovery, through metabolomics, of the plant-associated amphetamine, cathinone, in four Massospora cicadina-infected periodical cicada populations, and the mushroom-associated tryptamine, psilocybin, in annual cicadas infected with Massospora platypediae or Massospora levispora, which likely represent a single fungal species. The absence of some fungal enzymes necessary for cathinone and psilocybin biosynthesis along with the inability to detect intermediate metabolites or gene orthologs are consistent with possibly novel biosynthesis pathways in Massospora. The neurogenic activities of these compounds suggest the extended phenotype of Massospora that modifies cicada behavior to maximize dissemination is chemically-induced.

Proteome and metabolome analyses reveal differential responses in tomato -Verticillium dahliae-interactions.

Verticillium dahliae colonizes vascular tissue and causes vascular discoloration in susceptible hosts. Two well-defined races exist in V. dahliae populations from tomato and lettuce. In this study, proteins and metabolites obtained from stems of race 1-incompatible (Beefsteak) and -compatible (Early Pak) tomato cultivars were characterized. A total of 814 and 584 proteins in Beefsteak; and 456 and 637 proteins in Early Pak were identified in stem extracts of plants inoculated with races 1 and 2, respectively. A significant number of defense-related proteins were expressed in each tomato-V. dahliae interaction, as anticipated. However, phenylalanine ammonia-lyase (PAL), an important defense-associated enzyme of the phenylpropanoid pathway, in addition to remorin 1, NAD-dependent epimerase/dehydratase, and polyphenol oxidase were uniquely expressed in the incompatible interaction. Compared with the uninoculated control, significant overexpression of gene ontology terms associated with lignin biosynthesis, phenylpropanoid pathway and carbohydrate methylation were identified exclusively in the incompatible interaction. Phenolic compounds known to be involved in plant defense mechanisms were at higher levels in the incompatible relative to the compatible interactions. Based on our findings, PAL and enzymes involved defense-related secondary metabolism and the strengthening of cell walls is likely critical to confer resistance to race 1 of V. dahliae in tomato. SIGNIFICANCE: Verticillium dahliae, a soilborne fungal pathogen and a widely distributed fungal pathogen, colonizes vascular tissue and causes vascular discoloration in roots and stems, leaf wilting, and death of susceptible plant hosts. It causes billions of dollars in annual crop losses all over the world. The study focused on the proteomic and metabalomic of V. dahliae interactions (incompatible with Beefsteak and compatible with Early Pak tomato cultivars). Based on our findings, PAL and enzymes involved defense-related secondary metabolism and the strengthening of cell walls is likely critical to confer resistance to race 1 of V. dahliae in tomato.

Arabidopsis defense mutant ndr1-1 displays accelerated development and early flowering mediated by the hormone gibberellic acid.

NONRACE-SPECIFIC DISEASE RESISTANCE (NDR1) is a widely characterized gene that plays a key role in defense against multiple bacterial, fungal, oomycete and nematode plant pathogens. NDR1 is required for activation of resistance by multiple NB and LRR-containing (NLR) protein immune sensors and contributes to basal defense. The role of NDR1 in positively regulating salicylic acid (SA)-mediated plant defense responses is well documented. However, ndr1-1 plants flower earlier and show accelerated development in comparison to wild type (WT) Arabidopsis plants, indicating that NDR1 is a negative regulator of flowering and growth. Exogenous application of gibberellic acid (GA) further accelerates the early flowering phenotype in ndr1-1 plants, while the GA biosynthesis inhibitor paclobutrazol attenuated the early flowering phenotype of ndr1-1, but not to WT levels, suggesting partial resistance to paclobutrazol and enhanced GA response in ndr1-1 plants. Mass spectroscopy analyses confirmed that ndr1-1 plants have 30-40% higher levels of GA3 and GA4, while expression of various GA metabolic genes and major flowering regulatory genes is also altered in the ndr1-1 mutant. Taken together this study provides evidence of crosstalk between the ndr1-1-mediated defense and GA-regulated developmental programs in plants.

Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Genome-Wide Identification and Functional Analyses of the CRK Gene Family in Cotton Reveals GbCRK18 Confers Verticillium Wilt Resistance in Gossypium barbadense.

Cysteine-rich receptor-like kinases (CRKs) are a large subfamily of plant receptor-like kinases that play a critical role in disease resistance in plants. However, knowledge about the CRK gene family in cotton and its function against Verticillium wilt (VW), a destructive disease caused by Verticillium dahliae that significantly reduces cotton yields is lacking. In this study, we identified a total of 30 typical CRKs in a Gossypium barbadense genome (GbCRKs). Eleven of these (>30%) are located on the A06 and D06 chromosomes, and 18 consisted of 9 paralogous pairs encoded in the A and D subgenomes. Phylogenetic analysis showed that the GbCRKs could be classified into four broad groups, the expansion of which has probably been driven by tandem duplication. Gene expression profiling of the GbCRKs in resistant and susceptible cotton cultivars revealed that a phylogenetic cluster of nine of the GbCRK genes were up-regulated in response to V. dahliae infection. Virus-induced gene silencing of each of these nine GbCRKs independently revealed that the silencing of GbCRK18 was sufficient to compromise VW resistance in G. barbadense. GbCRK18 expression could be induced by V. dahliae infection or jasmonic acid, and displayed plasma membrane localization. Therefore, our expression analyses indicated that the CRK gene family is differentially regulated in response to Verticillium infection, while gene silencing experiments revealed that GbCRK18 in particular confers VW resistance in G. barbadense.

Heterologous Expression of the Cotton NBS-LRR Gene GbaNA1 Enhances Verticillium Wilt Resistance in Arabidopsis.

Verticillium wilt caused by Verticillium dahliae results in severe losses in cotton, and is economically the most destructive disease of this crop. Improving genetic resistance is the cleanest and least expensive option to manage Verticillium wilt. Previously, we identified the island cotton NBS-LRR-encoding gene GbaNA1 that confers resistance to the highly virulent V. dahliae isolate Vd991. In this study, we expressed cotton GbaNA1 in the heterologous system of Arabidopsis thaliana and investigated the defense response mediated by GbaNA1 following inoculations with V. dahliae. Heterologous expression of GbaNA1 conferred Verticillium wilt resistance in A. thaliana. Moreover, overexpression of GbaNA1 enabled recovery of the resistance phenotype of A. thaliana mutants that had lost the function of GbaNA1 ortholog gene. Investigations of the defense response in A. thaliana showed that the reactive oxygen species (ROS) production and the expression of genes associated with the ethylene signaling pathway were enhanced significantly following overexpression of GbaNA1. Intriguingly, overexpression of the GbaNA1 ortholog from Gossypium hirsutum (GhNA1) in A. thaliana did not induce the defense response of ROS production due to the premature termination of GhNA1, which lacks the encoded NB-ARC and LRR motifs. GbaNA1 therefore confers Verticillium wilt resistance in A. thaliana by the activation of ROS production and ethylene signaling. These results demonstrate the functional conservation of the NBS-LRR-encoding GbaNA1 in a heterologous system, and the mechanism of this resistance, both of which may prove valuable in incorporating GbaNA1-mediated resistance into other plant species.

SNARE-Encoding Genes VdSec22 and VdSso1 Mediate Protein Secretion Required for Full Virulence in Verticillium dahliae.

Proteins that mediate cellular and subcellular membrane fusion are key factors in vesicular trafficking in all eukaryotic cells, including the secretion and transport of plant pathogen virulence factors. In this study, we identified vesicle-fusion components that included 22 soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), four Sec1/Munc18 (SM) family proteins, and 10 Rab GTPases encoded in the genome of the vascular wilt pathogen Verticillium dahliae Vd991. Targeted deletion of two SNARE-encoding genes in V. dahliae, VdSec22 and VdSso1, significantly reduced virulence of both mutants on cotton, relative to the wild-type Vd991 strain. Comparative analyses of the secreted protein content (exoproteome) revealed that many enzymes involved in carbohydrate hydrolysis were regulated by VdSec22 or VdSso1. Consistent with a role of these enzymes in plant cell-wall degradation, pectin, cellulose, and xylan utilization were reduced in the VdSec22 or VdSso1 mutant strains along with a loss of exoproteome cytotoxic activity on cotton leaves. Comparisons with a pathogenicity-related exoproteome revealed that several known virulence factors were not regulated by VdSec22 or VdSso1, but some of the proteins regulated by VdSec22 or VdSso1 displayed different characteristics, including the lack of a typical signal peptide, suggesting that V. dahliae employs more than one secretory route to transport proteins to extracellular sites during infection.

The island cotton NBS-LRR gene GbaNA1 confers resistance to the non-race 1 Verticillium dahliae isolate Vd991.

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non-commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS-LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus-induced gene silencing of each of the eight NBS-LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non-functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full-length (∼1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non-race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.

A Verticillium dahliae Extracellular Cutinase Modulates Plant Immune Responses.

Cutinases have been implicated as important enzymes during the process of fungal infection of aerial plant organs. The function of cutinases in the disease cycle of fungal pathogens that invade plants through the roots has been less studied. Here, functional analysis of 13 cutinase (carbohydrate esterase family 5 domain-containing) genes (VdCUTs) in the highly virulent vascular wilt pathogen Verticillium dahliae Vd991 was performed. Significant sequence divergence in cutinase family members was observed in the genome of V. dahliae Vd991. Functional analyses demonstrated that only VdCUT11, as purified protein, induced cell death and triggered defense responses in Nicotiana benthamiana, cotton, and tomato plants. Virus-induced gene silencing showed that VdCUT11 induces plant defense responses in Nicotiana benthamania in a BAK1 and SOBIR-dependent manner. Furthermore, coinfiltration assays revealed that the carbohydrate-binding module family 1 protein (VdCBM1) suppressed VdCUT11-induced cell death and other defense responses in N. benthamiana. Targeted deletion of VdCUT11 in V. dahliae significantly compromised virulence on cotton plants. The cutinase VdCUT11 is an important secreted enzyme and virulence factor that elicits plant defense responses in the absence of VdCBM1.

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.

Verticillium dahliae transcription factor VdFTF1 regulates the expression of multiple secreted virulence factors and is required for full virulence in cotton.

Fungal transcription factors (TFs) implicated in the regulation of virulence gene expression have been identified in a number of plant pathogens. In Verticillium dahliae, despite its agricultural importance, few regulators of transcription have been characterized. In this study, a T-DNA insertion mutant with significantly reduced virulence towards cotton was identified. The T-DNA was traced to VdFTF1, a gene encoding a TF containing a Fungal_trans domain. Transient expression in onion epidermal cells indicated that VdFTF1 is localized to the nucleus. The VdFTF1-deletion strains displayed normal vegetative growth, mycelial pigmentation and conidial morphology, but exhibited significantly reduced virulence on cotton, suggesting that VdFTF1 is required exclusively for pathogenesis. Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes, 233 of which were associated with catalytic processes. These genes encoded 69 potentially secreted proteins, 43 of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton. Targeted gene deletion of one VdFTF1-regulated gene resulted in significantly impaired vascular colonization, as measured by quantitative polymerase chain reaction, as well as aggressiveness and symptom severity in cotton. In conclusion, VdFTF1, which encodes a TF containing a Fungal_trans domain, regulates the gene expression of plant cell wall degradation enzymes in V. dahliae, which are required for full virulence on cotton.

Short-Term Host Selection Pressure Has Little Effect on the Evolution of a Monoclonal Population of Verticillium dahliae Race 1.

Understanding pathogen evolution over time is vital for plant breeding and deployment of host resistance. In the context of a soilborne pathogen, the potential of host-directed evolution of a Verticillium dahliae race 1 isolate and genotypic variation of V. dahliae associated with two major hosts (lettuce and tomato) were determined. In total, 427 isolates were recovered over 6 years from a resistance screening nursery infested with a single V. dahliae race 1 isolate. In a separate study, an additional 206 isolates representing 163 and 43 isolates from commercial lettuce and tomato fields, respectively, were collected. Analyses of isolates recovered from the screening nursery over 6 years revealed no changes in the race and mating type composition but did uncover seven simple sequence repeat (SSR) variant genotypes. No significant genotypic variation in V. dahliae was observed between or within fields of either lettuce or tomato but pathogen populations were significantly differentiated between these two hosts. Replicated virulence assays of variant SSR genotypes on lettuce differential cultivars suggested no significant difference in virulence from the wild-type race 1 isolate introduced into the field. This suggests that deployed race 1 host resistance will be robust against the widespread race 1 populations in lettuce-growing regions at least for 6 years unless novel pathogen genotypes or races are introduced into the system.

Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1.

Glycoside hydrolase 12 (GH12) proteins act as virulence factors and pathogen-associated molecular patterns (PAMPs) in oomycetes. However, the pathogenic mechanisms of fungal GH12 proteins have not been characterized. In this study, we demonstrated that two of the six GH12 proteins produced by the fungus Verticillium dahliae Vd991, VdEG1 and VdEG3 acted as PAMPs to trigger cell death and PAMP-triggered immunity (PTI) independent of their enzymatic activity in Nicotiana benthamiana. A 63-amino-acid peptide of VdEG3 was sufficient for cell death-inducing activity, but this was not the case for the corresponding peptide of VdEG1. Further study indicated that VdEG1 and VdEG3 trigger PTI in different ways: BAK1 is required for VdEG1- and VdEG3-triggered immunity, while SOBIR1 is specifically required for VdEG1-triggered immunity in N. benthamiana. Unlike oomycetes, which employ RXLR effectors to suppress host immunity, a carbohydrate-binding module family 1 (CBM1) protein domain suppressed GH12 protein-induced cell death. Furthermore, during infection of N. benthamiana and cotton, VdEG1 and VdEG3 acted as PAMPs and virulence factors, respectively indicative of host-dependent molecular functions. These results suggest that VdEG1 and VdEG3 associate differently with BAK1 and SOBIR1 receptor-like kinases to trigger immunity in N. benthamiana, and together with CBM1-containing proteins manipulate plant immunity.

PCR Multiplexes Discriminate Fusarium Symbionts of Invasive Euwallacea Ambrosia Beetles that Inflict Damage on Numerous Tree Species Throughout the United States.

Asian Euwallacea ambrosia beetles vector Fusarium mutualists. The ambrosial fusaria are all members of the ambrosia Fusarium clade (AFC) within the Fusarium solani species complex (FSSC). Several Euwallacea-Fusarium mutualists have been introduced into nonnative regions and have caused varying degrees of damage to orchard, landscape, and forest trees. Knowledge of symbiont fidelity is limited by current identification methods, which typically requires analysis of DNA sequence data from beetles and the symbionts cultured from their oral mycangia. Here, polymerase chain reaction (PCR)-based diagnostic tools were developed to identify the six Fusarium symbionts of exotic Euwallacea spp. currently known within the United States. Whole-genome sequences were generated for representatives of six AFC species plus F. ambrosium and aligned to the annotated genome of F. euwallaceae. Taxon-specific primer-annealing sites were identified that rapidly distinguish the AFC species currently within the United States. PCR specificity, reliability, and sensitivity were validated using a panel of 72 Fusarium isolates, including 47 reference cultures. Culture-independent multiplex assays accurately identified two AFC fusaria using DNA isolated from heads of their respective beetle partners. The PCR assays were used to show that Euwallacea validus is exclusively associated with AF-4 throughout its sampled range within eastern North America. The rapid assay supports federal and state agency efforts to monitor spread of these invasive pests and mitigate further introductions.

Epitypification of Fusisporium (Fusarium) solani and its assignment to a common phylogenetic species in the Fusarium solani species complex.

Fusisporium solani was described as the causal agent of a dry rot of potato in Germany in the mid 19th century. As Fusarium solani, the species became known as a plurivorous plant pathogen, endophyte, decomposer, and opportunistic pathogen of humans and nutritional symbiont of insects. In parallel, it became evident that the morphologically defined species F. solani represents a phylogenetically and biologically complex group of often morphologically cryptic species that has come to be known in part as the F. solani species complex (FSSC), accommodating several formae speciales and mating populations/biological species. The FSSC currently includes more than 60 phylogenetic species. Several of these have been named, but the majority remains unnamed and the identity of F. solani sensu stricto is unclear. To promote further taxonomic developments in the FSSC, lectoand epitypification is proposed for Fusisporium solani Although no type material for F. solani is known to exist, the species was abundantly illustrated in the protologue. Thus, a relevant illustration provided by von Martius is selected as the lectotype. The epitype selected here originates from a rotting potato collected in a field in Slovenia. This strain causes a dry rot of artificially inoculated potatoes. It groups in the heretofore unnamed phylogenetic species 5, which is nested within clade 3 of the FSSC (FSSC 5). Members of this phylogenetic species have a wide geographic distribution and include soil saprotrophs and plant and opportunistic human pathogens. This typification is consistent with the original description of Fusisporium solani and the concept of F. solani as a widely distributed soil inhabitant and pathogen.

New Fungus-Insect Symbiosis: Culturing, Molecular, and Histological Methods Determine Saprophytic Polyporales Mutualists of Ambrosiodmus Ambrosia Beetles.

Ambrosia symbiosis is an obligate, farming-like mutualism between wood-boring beetles and fungi. It evolved at least 11 times and includes many notorious invasive pests. All ambrosia beetles studied to date cultivate ascomycotan fungi: early colonizers of recently killed trees with poor wood digestion. Beetles in the widespread genus Ambrosiodmus, however, colonize decayed wood. We characterized the mycosymbionts of three Ambrosiodmus species using quantitative culturing, high-throughput metabarcoding, and histology. We determined the fungi to be within the Polyporales, closely related to Flavodon flavus. Culture-independent sequencing of Ambrosiodmus minor mycangia revealed a single operational taxonomic unit identical to the sequences from the cultured Flavodon. Histological sectioning confirmed that Ambrosiodmus possessed preoral mycangia containing dimitic hyphae similar to cultured F. cf. flavus. The Ambrosiodmus-Flavodon symbiosis is unique in several aspects: it is the first reported association between an ambrosia beetle and a basidiomycotan fungus; the mycosymbiont grows as hyphae in the mycangia, not as budding pseudo-mycelium; and the mycosymbiont is a white-rot saprophyte rather than an early colonizer: a previously undocumented wood borer niche. Few fungi are capable of turning rotten wood into complete animal nutrition. Several thousand beetle-fungus symbioses remain unstudied and promise unknown and unexpected mycological diversity and enzymatic innovations.