Variation in SSRs at different genomic regions and implications for the evolution and identification of Armillaria gallica.

Armillaria spp. are devastating forest pathogens. Due to its low pathogenicity and abundant genetic variation, Armillaria gallica exhibited a unique and beneficial symbiosis with Gastrodia elata, which was used as a traditional Chinese medicine. However, the variation and population structure of A. gallica populations have rarely been investigated. Hence, we analyzed the evolution and variation in simple sequence repeats (SSRs) in three Armillaria genomes: A. gallica, A. cepistipes, and A. ostoyae to assess the genetic diversity and population structure of 14 A. gallica strains. Genome analysis revealed that SSRs were more abundant in the intergenic region than the intron and exon region, as was the SSR density. Compared with other two genomes, SSR density was the lowest in exon region and largest in the intron region of A. gallica, with significant variation in genic region. There were 17 polymorphic markers in A. gallica genome was identified, with 26.7% in genic region, which is higher than that of 18.8% in the intergenic region. Moreover, a total of 50 alleles and 42 polymorphic loci were detected among these A. gallica strains. The averaged polymorphism information content (PIC) was 0.4487, ranged from 0.2577 to 0.6786. Both principal coordinate analysis (PCoA) and population structure analyses based on the genotype data of SSRs divided the strains into two clusters. The cluster I included all the strains from high-altitude G. elata producing areas and some low-altitude areas, while the strains in Cluster II originated from low-altitude G. elata producing areas. These results indicated that substantial genome-specific variation in SSRs within the genic region of A. gallica and provide new insights for further studies on the evolution and breeding of A. gallica.

Characteristics and functions of volatile organic compounds in the tripartite symbiotic system of Gastrodia elata-Armillaria gallica-Rahnella aceris HPDA25.

Tripartite interactions among plants, fungi, and bacteria are critical for maintaining plant growth and fitness, and volatile organic compounds (VOCs) play a significant role in these interactions. However, the functions of VOCs within the niche of mycoheterotrophic plants, which represent unique types of interactions, remain poorly understood. Gastrodia elata, a mycoheterotrophic orchid species, forms a symbiotic relationship with specific Armillaria species, serving as a model system to investigate this intriguing issue. Rahnella aceris HPDA25 is a plant growth-promoting bacteria isolated from G. elata, which has been found to facilitate the establishment of G. elata-Armillaria symbiosis. In this study, using the tripartite symbiotic system of G. elata-Armillaria gallica-R. aceris HPDA25, we investigate the role of VOCs in the interaction among mycoheterotrophic plants, fungi, and bacteria. Our results showed that 33 VOCs of HPDA25-inducible symbiotic G. elata elevated compared to non-symbiotic G. elata, indicating that VOCs indeed play a role in the symbiotic process. Among these, 21 VOCs were accessible, and six active VOCs showed complete growth inhibition activities against A. gallica, while R. aceris HPDA25 had no significant effect. In addition, three key genes of G. elata have been identified that may contribute to the increased concentration of six active VOCs. These results revealed for the first time the VOCs profile of G. elata and demonstrated its regulatory role in the tripartite symbiotic system involving G. elata, Armillaria, and bacteria.

Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea.

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.

In vitro co-culture system for investigating Armillaria root rot in Prunus spp. using a fiber-supported liquid approach.

In vitro co-culture techniques that allow the growth of plants and pathogens under controlled environmental conditions are being used to re-create host plant infection. These approaches reduce infection times, promote reproducibility, and enable a rapid evaluation of plant-pathogen interactions. As a result, these systems have become essential in breeding programs aimed at developing plant resistance to diseases. In this study, we developed and validated an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting Prunus spp. This disease, caused by fungi Armillaria spp. and Desarmillaria caespitosa, poses a severe threat to the stone and nut fruit industry due to the susceptibility of most commercial rootstocks to infection and the lack of effective management options for its control. The system consists of a fiber-supported liquid approach in sterile plastic vessels that allows a fast and reproducible fungal infection under controlled environmental conditions. The floor of the vessels was covered with a polyester-fiber matte and a germination paper that served as an interface between the mycelia and the plant roots. The vessels were subjected to inoculation with Armillaria mellea and D. caespitosa, and three Prunus genotypes ('Guardian®', 'MP-29', and Prunus cerasifera '14-4') were co-cultured with both fungi. Disease progression and plant and fungal biomass were monitored during co-culture. The presented in vitro co-culture approach facilitates the concurrent growth of Armillaria/Desarmillaria spp. and Prunus spp., excluding most of the limitations associated with greenhouses and field experiments. This system provides consistent and reproducible conditions for investigating a prominent plant disease affecting Prunus spp.

Composition and diversity of soil microbial communities change by introducing Phallus impudicus into a Gastrodia elata Bl.-based soil.

BACKGROUND: The Gastrodia elata Bl. is an orchid, and its growth demands the presence of Armillaria species. The strong competitiveness of Armillaria species has always been a concern of major threat to other soil organisms, thus disrupting the equilibrium of soil biodiversity. Introducing other species to where G. elata was cultivated, could possibly alleviate the problems associated with the disequilibrium of soil microenvironment; however, their impacts on the soil microbial communities and the underlying mechanisms remain unclear. To reveal the changes of microbial groups associated with soil chemical properties responding to different cultivation species, the chemical property measurements coupled with the next-generation pyrosequencing analyses were applied with soil samples collected from fallow land, cultivation of G. elata and Phallus impudicus, respectively. RESULTS: The cultivation of G. elata induced significant increases (p < 0.05) in soil pH and NO3-N content compared with fallow land, whereas subsequent cultivation of P. impudicus reversed these G. elata-induced increases and was also found to significantly increase (p < 0.05) the content of soil NH4+-N and AP. The alpha diversities of soil microbial communities were significantly increased (p < 0.01) by cultivation of G. elata and P. impudicus as indicated with Chao1 estimator and Shannon index. The structure and composition of soil microbial communities differed responding to different cultivation species. In particular, the relative abundances of Bacillus, norank_o_Gaiellales, Mortierella and unclassified_k_Fungi were significantly increased (p < 0.05), while the abundances of potentially beneficial genera such as Acidibacter, Acidothermus, Cryptococcus, and Penicillium etc., were significantly decreased (p < 0.05) by cultivation of G. elata. It's interesting to find that cultivation of P. impudicus increased the abundances of these genera that G. elata decreased before, which contributed to the difference of composition and structure. The results of CCA and heatmap indicated that the changes of soil microbial communities had strong correlations with soil nutrients. Specifically, among 28 genera presented, 50% and 42.9% demonstrated significant correlations with soil pH and NO3-N in response to cultivation of G. elata and P. impudicus. CONCLUSIONS: Our findings suggested that the cultivation of P. impudicus might have potential benefits as result of affecting soil microorganisms coupled with changes in soil nutrient profile.

Characterization of Mycoviruses in Armillaria ostoyae and A. cepistipes in the Czech Republic.

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.

Decoding the RNA virome of the tree parasite Armillaria provides new insights into the viral community of soil-borne fungi.

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.

Full-length genome characterization of a novel mitovirus isolated from the root rot fungus Armillaria mellea.

Members of the genus Armillaria belong to the group of pathogenic and facultative saprotrophic fungi that are generally known as one of the causative agents of white root rot in infected plants including deciduous and evergreen trees and shrubs. Although several single-stranded RNA mycoviruses were previously described in different Armillaria species, there is no report on mitoviruses (one of the simplest RNA viruses of fungal hosts) known to infect Armillaria taxa. In this study, a new mitovirus denominated "Armillaria mellea mitovirus 1" (AmMV1) was identified in the sporophore samples of Armillaria mellea, commonly known as honey mushroom. AmMV1 has a genome length of 4440 nucleotides and a G + C content of 48%. It encompasses a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp). Comparison through BLASTp analysis revealed that the RdRp domain of AmMV1 shares a sequence identity ranging from 33.43% to 43.27% with RdRp domains of Duamitovirus genus members, having the highest similarity (43.27%) to Rhizoctonia solani mitovirus 94. According to phylogenetic analysis, AmMV1 is classified as a member of the genus Duamitovirus belonging to the Mitoviridae family. This marks the initial instance of a mitovirus identified in Armillaria spp..

Two distinct non-ribosomal peptide synthetase-independent siderophore synthetase gene clusters identified in Armillaria and other species in the Physalacriaceae.

Siderophores are important for ferric iron solubilization, sequestration, transportation, and storage, especially under iron-limiting conditions such as aerobic conditions at high pH. Siderophores are mainly produced by non-ribosomal peptide synthetase-dependent siderophore pathway, non-ribosomal peptide synthetase-independent siderophore synthetase pathway, or the hybrid non-ribosomal peptide synthetases/non-ribosomal peptide synthetases-independent siderophore pathway. Outcompeting or inhibition of plant pathogens, alteration of host defense mechanisms, and alteration of plant-fungal interactions have been associated with fungal siderophores. To understand these mechanisms in fungi, studies have been conducted on siderophore biosynthesis by ascomycetes with limited focus on the basidiomycetes. Armillaria includes several species that are pathogens of woody plants and trees important to agriculture, horticulture, and forestry. The aim of this study was to investigate the presence of non-ribosomal peptide synthetases-independent siderophore synthetase gene cluster(s) in genomes of Armillaria species using a comparative genomics approach. Iron-dependent growth and siderophore biosynthesis in strains of selected Armillaria spp. were also evaluated in vitro. Two distinct non-ribosomal peptide synthetases-independent siderophore synthetase gene clusters were identified in all the genomes. All non-ribosomal peptide synthetases-independent siderophore synthetase genes identified putatively encode Type A' non-ribosomal peptide synthetases-independent siderophore synthetases, most of which have IucA_IucC and FhuF-like transporter domains at their N- and C-terminals, respectively. The effect of iron on culture growth varied among the strains studied. Bioassays using the CAS assay on selected Armillaria spp. revealed in vitro siderophore biosynthesis by all strains irrespective of added FeCl3 concentration. This study highlights some of the tools that Armillaria species allocate to iron homeostasis. The information generated from this study may in future aid in developing molecular based methods to control these phytopathogens.

A practical approach to genome assembly and annotation of Basidiomycota using the example of Armillaria.

Technological advancements in genome sequencing, assembly and annotation platforms and algorithms that resulted in several genomic studies have created an opportunity to further our understanding of the biology of phytopathogens, including Armillaria species. Most Armillaria species are facultative necrotrophs that cause root- and stem-rot, usually on woody plants, significantly impacting agriculture and forestry worldwide. Genome sequencing, assembly and annotation in terms of samples used and methods applied in Armillaria genome projects are evaluated in this review. Infographic guidelines and a database of resources to facilitate future Armillaria genome projects were developed. Knowledge gained from genomic studies of Armillaria species is summarized and prospects for further research are provided. This guide can be applied to other diploid and dikaryotic fungal genomics.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

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.

Characterization of the transcriptional responses of Armillaria gallica 012m to GA3.

Gastrodia elata needs to establish a symbiotic relationship with Armillaria strains to obtain nutrients and energy. However, the signaling cross talk between G. elata and Armillaria strains is still unclear. During our experiment, we found that the vegetative mycelium of Armillaria gallica 012m grew significantly better in the media containing gibberellic acid (GA3) than the blank control group (BK). To explore the response mechanism, we performed an RNA-sequencing experiment to profile the transcriptome changes of A. gallica 012m cultured in the medium with exogenous GA3. The transcriptome-guided differential expression genes (DEGs) analysis of GA3 and BK showed that a total of 1309 genes were differentially expressed, including 361 upregulated genes and 948 downregulated genes. Some of those DEGs correlated with the biological process, including positive regulation of chromosome segregation, mitotic metaphase/anaphase transition, attachment of mitotic spindle microtubules to kinetochore, mitotic cytokinesis, and nuclear division. These analyses explained that GA3 actively promoted the growth of A. gallica to some extent. Further analysis of protein domain features showed that the deduced polypeptide contained 41 candidate genes of GA receptor, and 27 of them were expressed in our samples. We speculate that GA receptors exist in A. gallica 012m. Comparative studies of proteins showed that the postulated GA receptor domains of A. gallica 012m have a higher homologous correlation with fungi than others based on cluster analysis.

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.

Whole genome sequencing and analysis of Armillaria gallica Jzi34 symbiotic with Gastrodia elata.

BACKGROUND: Armillaria species are plant pathogens, but a few Armillaria species can establish a symbiotic relationship with Gastrodia elata, a rootless and leafless orchid, that is used as a Chinese herbal medicine. Armillaria is a nutrient source for the growth of G. elata. However, there are few reports on the molecular mechanism of symbiosis between Armillaria species and G. elata. The genome sequencing and analysis of Armillaria symbiotic with G. elata would provide genomic information for further studying the molecular mechanism of symbiosis. RESULTS: The de novo genome assembly was performed with the PacBio Sequel platform and Illumina NovaSeq PE150 for the A. gallica Jzi34 strain, which was symbiotic with G. elata. Its genome assembly contained ~ 79.9 Mbp and consisted of 60 contigs with an N50 of 2,535,910 bp. There were only 4.1% repetitive sequences in the genome assembly. Functional annotation analysis revealed a total of 16,280 protein coding genes. Compared with the other five genomes of Armillaria, the carbohydrate enzyme gene family of the genome was significantly contracted, while it had the largest set of glycosyl transferase (GT) genes. It also had an expansion of auxiliary activity enzymes AA3-2 gene subfamily and cytochrome P450 genes. The synteny analysis result of P450 genes reveals that the evolutionary relationship of P450 proteins between A. gallica Jzi34 and other four Armillaria was complex. CONCLUSIONS: These characteristics may be beneficial for establishing a symbiotic relationship with G. elata. These results explore the characteristics of A. gallica Jzi34 from a genomic perspective and provide an important genomic resource for further detailed study of Armillaria. This will help to further study the symbiotic mechanism between A. gallica and G. elata.

[Screening of an Armillaria gallica strain for Gastrodia elata cultivation].

This study aims to screen a strain from Armillaria for the cultivation of Gastrodia elata. Specifically, Armillaria strains were isolated from different producing areas of G. elata and identified. Based on the growth characteristics of the strains and the experiment on the cultivation of G. elata, an optimal A. gallica strain was screened out. The specific process is as follows. The fungus-gro-wing materials of G. elata were collected from four producing areas and the Armillaria strains were isolated(G,Y,S,H). The strains were then identified based on morphological observation and phylogeny analysis and the commonly used strains were determined. The sucrase genotypes of the strains were identified according to our previous research findings, and the growth characteristics of the strains, such as growth rate, diameter, dry weight, and polysaccharide content of the rhizomorphs, were measured. According to the biological characteristics and sucrase genotypes, two strains were selected for the cultivation of G. elata. The tuber yield and the content of gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata were measured to select the optimal strain. The results showed that the four strains were all A. gallica. The rhizomorphs of strains G and H of the same sucrase genotype had larger/higher length, growth rate, diameter, branch number, dry weight, and polysaccharide content than those of strains S and Y of the same sucrase genotype. The tuber yield and the total content of gastrodin and p-hydroxybenzyl alcohol in tuber of G. elata cultivated with strain H were 6.528 kg·m~(-2) and 0.566%, respectively, which were 4.58 and 1.30 folds those of G. elata cultivated with strain S. Strains H and S were screened out from four strains of A. gallica based on the growth characteristics and sucrase genotype. According to the tuber yield and content of total gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata, strain H was identified as the optimal one. The findings in this study are expected to lay a basis for cultivating G. elata with high yield and quality of tubers.

Transcriptome analysis of Armillaria gallica 012 m in response to auxin.

Gastrodia elata is an achlorophyllous and fully mycoheterotrophic orchid which obtains carbon and other nutrients from Armillaria species in its life cycle. Many researchers suggested that plant hormones, as signing molecules, play a central role in the plant-fungi interaction. In the process of Armillaria gallica 012 m cultivation, both exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) distinctly stimulated the growth of mycelia in solid media. The differential expression genes (DEGs) of A. gallica 012 m with IAA versus blank control (BK) and IBA versus BK were investigated. The results showed that more than 80% of DEGs of the IAA group were coincident with the DEGs of the IBA group, and more than half of upregulated DEGs and most of the downregulated DEGs of the IAA group coincided with those DEGs of the IBA group. Above research implied that A. gallica 012 m could perceive IAA and IBA, and possess similar responses and signaling pathways to IAA and IBA. The overlapping differential genes of the IAA group and IBA group were analyzed by GO term, and the results showed that several DEGs identified were related to biological processes including positive regulation of the biological process and biological process. The downregulated NmrA-like and FKBP_C genes might be benefit to the growth of mycelia. Those results can explain that exiguous IAA and IBA improved the growth of A. gallica to some extent. We speculate that IAA and IBA are signaling molecules, and regulate the expression of growth-related genes of A. gallica 012 m by the same signaling pathway.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity.

Genomic Comparisons of Two Armillaria Species with Different Ecological Behaviors and Their Associated Soil Microbial Communities.

Armillaria species show considerable variation in ecological roles and virulence, from mycorrhizae and saprophytes to important root pathogens of trees and horticultural crops. We studied two Armillaria species that can be found in coniferous forests of northwestern USA and southwestern Canada. Armillaria altimontana not only is considered as a weak, opportunistic pathogen of coniferous trees, but it also appears to exhibit in situ biological control against A. solidipes, formerly North American A. ostoyae, which is considered a virulent pathogen of coniferous trees. Here, we describe their genome assemblies and present a functional annotation of the predicted genes and proteins for the two Armillaria species that exhibit contrasting ecological roles. In addition, the soil microbial communities were examined in association with the two Armillaria species within a 45-year-old plantation of western white pine (Pinus monticola) in northern Idaho, USA, where A. altimontana was associated with improved tree growth and survival, while A. solidipes was associated with reduced growth and survival. The results from this study reveal a high similarity between the genomes of the beneficial/non-pathogenic A. altimontana and pathogenic A. solidipes; however, many relatively small differences in gene content were identified that could contribute to differences in ecological lifestyles and interactions with woody hosts and soil microbial communities.

Transcriptome analysis reveals the regulatory mode by which NAA promotes the growth of Armillaria gallica.

A symbiotic relationship is observed between Armillaria and the Chinese herbal medicine Gastrodia elata (G. elata). Armillaria is a nutrient source for the growth of G. elata, and its nutrient metabolism efficiency affects the growth and development of G. elata. Auxin has been reported to stimulate Armillaria species, but the molecular mechanism remains unknown. We found that naphthalene acetic acid (NAA) can also promote the growth of A. gallica. Moreover, we identified a total of 2071 differentially expressed genes (DEGs) by analyzing the transcriptome sequencing data of A. gallica at 5 and 10 hour of NAA treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these unigenes were significantly enriched in the metabolism pathways of arginine, proline, propanoate, phenylalanine and tryptophan. The expression levels of the general amino acid permease (Gap), ammonium transporter (AMT), glutamate dehydrogenase (GDH), glutamine synthetase (GS), Zn(II) 2Cys6 and C2H2 transcription factor genes were upregulated. Our transcriptome analysis showed that the amino acid and nitrogen metabolism pathways in Armillaria were rapidly induced within hours after NAA treatment. These results provide valuable insights into the molecular mechanisms by which NAA promotes the growth of Armillaria species.