Mobile genetic elements explain size variation in the mitochondrial genomes of four closely-related Armillaria species.

BACKGROUND: Species in the genus Armillaria (fungi, basidiomycota) are well-known as saprophytes and pathogens on plants. Many of them cause white-rot root disease in diverse woody plants worldwide. Mitochondrial genomes (mitogenomes) are widely used in evolutionary and population studies, but despite the importance and wide distribution of Armillaria, the complete mitogenomes have not previously been reported for this genus. Meanwhile, the well-supported phylogeny of Armillaria species provides an excellent framework in which to study variation in mitogenomes and how they have evolved over time. RESULTS: Here we completely sequenced, assembled, and annotated the circular mitogenomes of four species: A. borealis, A. gallica, A. sinapina, and A. solidipes (116,443, 98,896, 103,563, and 122,167 bp, respectively). The variation in mitogenome size can be explained by variable numbers of mobile genetic elements, introns, and plasmid-related sequences. Most Armillaria introns contained open reading frames (ORFs) that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Insertions of mobile elements were also evident as fragments of plasmid-related sequences in Armillaria mitogenomes. We also found several truncated gene duplications in all four mitogenomes. CONCLUSIONS: Our study showed that fungal mitogenomes have a high degree of variation in size, gene content, and genomic organization even among closely related species of Armillara. We suggest that mobile genetic elements invading introns and intergenic sequences in the Armillaria mitogenomes have played a significant role in shaping their genome structure. The mitogenome changes we describe here are consistent with widely accepted phylogenetic relationships among the four species.

[Molecular identification of Armillaria gallica by PCR-RFLP analysis].

Armillaria gallica is a symbiotic fungus in the cultivation process of Gastrodia elata and Polyporus.The rhizomorph of A. gallica invades the stalk of the G. elata or the Sclerotium of the Polyporus,and is digested and utilized by the latter,becoming their important source of nutrition. Different nature of A. gallica affects the growth of G. elata and Polyporus. The authors collected A. gallica from 13 commercially available regions and screened two A. gallica,A and B,at the genetic and metabolic levels,in order to distinguish between the two A. gallica market. We have established convenient and effective DNA molecular identification method.By comparing the sequence differences between the A. gallica type A and type B invertase genes,PCR-RFLP primers were designed based on differential fragment. Primer ZTM.F/ZTM.R can amplified A. gallica type A and B,producing a band of about 304 bp in length. The restriction endonuclease EcoR V could recognize the difference sequence of A and B types of A. gallica. The type B was digested to form two fragments,thereby specifically identifying the A. gallica as type B. The established methods of PCR-RFLP is an accurate identification method for A. gallica. Therefore,in the cultivation process of G. elata and Polyporus,suitable strains can be selected according to different needs of variety,growth stage and ecological environment,and the yield and quality can be improved according to local conditions.

[Identification of four Armillaria strains and their effects on quality and yield of Gastrodia elata f. glauca].

In order to improve the quality and yield of Gastrodia elata f. glauca,determine the suitable Armillaria strains for the accompanying experiment in Xiaocaoba,Yiliang,four Armillaria strains were selected. They were used for G. elata cultivation,and the gene sequence,r DNA-ITS,ß-tubulin and EF1-α of four Armillaria strains,were compared and analyzed. The yield was mesured in November which was based on previous laboratory research. The tubers were washed and steamed,then dried and powdered. The content of gastrodin and p-hydroxybenzyl alcohol was determined by UPLC,the polysaccharide was determined by phenol-concentrated sulfuric acid method. The results showed that the strains M1,M2,M3 and M4 were Armillaria gallica group but there were differences in the yield and active ingredient content when they were cultivated with the same G. elata. The yield of G. elata( Jian Ma) was the lowest when cultivated with Armillaria strain M3,but it was not the same when used M1,0. 981 kg·m-2,the highest yield in the four stains.The content of gastrodin was 0. 581%,the total content of gastrodin and p-hydroxybenzyl alcohol was 0. 595%,when accompanied with M1 strains. It was higher than other strains. The content of G. elata polysaccharide was 2. 132%,which was similar to the content of M3 strain,higher than that of M2 and M4 strain. Selecting phylogenesis of Armillaria strians,the content of active ingredient,and the yield as indicators,it was concluded concerned that the M1 strain was the best of four strains. The results will provide a theoretical basis and guidance for higher yield and quality in cultivation of G. elata in Yiliang.

Resolved phylogeny and biogeography of the root pathogen Armillaria and its gasteroid relative, Guyanagaster.

BACKGROUND: Armillaria is a globally distributed mushroom-forming genus composed primarily of plant pathogens. Species in this genus are prolific producers of rhizomorphs, or vegetative structures, which, when found, are often associated with infection. Because of their importance as plant pathogens, understanding the evolutionary origins of this genus and how it gained a worldwide distribution is of interest. The first gasteroid fungus with close affinities to Armillaria-Guyanagaster necrorhizus-was described from the Neotropical rainforests of Guyana. In this study, we conducted phylogenetic analyses to fully resolve the relationship of G. necrorhizus with Armillaria. Data sets containing Guyanagaster from two collecting localities, along with a global sampling of 21 Armillaria species-including newly collected specimens from Guyana and Africa-at six loci (28S, EF1α, RPB2, TUB, actin-1 and gpd) were used. Three loci-28S, EF1α and RPB2-were analyzed in a partitioned nucleotide data set to infer divergence dates and ancestral range estimations for well-supported, monophyletic lineages. RESULTS: The six-locus phylogenetic analysis resolves Guyanagaster as the earliest diverging lineage in the armillarioid clade. The next lineage to diverge is that composed of species in Armillaria subgenus Desarmillaria. This subgenus is elevated to genus level to accommodate the exannulate mushroom-forming armillarioid species. The final lineage to diverge is that composed of annulate mushroom-forming armillarioid species, in what is now Armillaria sensu stricto. The molecular clock analysis and ancestral range estimation suggest the most recent common ancestor to the armillarioid lineage arose 51 million years ago in Eurasia. A new species, Guyanagaster lucianii sp. nov. from Guyana, is described. CONCLUSIONS: The armillarioid lineage evolved in Eurasia during the height of tropical rainforest expansion about 51 million years ago, a time marked by a warm and wet global climate. Species of Guyanagaster and Desarmillaria represent extant taxa of these early diverging lineages. Desarmillaria represents an armillarioid lineage that was likely much more widespread in the past. Guyanagaster likely evolved from a gilled mushroom ancestor and could represent a highly specialized endemic in the Guiana Shield. Armillaria species represent those that evolved after the shift in climate from warm and tropical to cool and arid during the late Eocene. No species in either Desarmillaria or Guyanagaster are known to produce melanized rhizomorphs in nature, whereas almost all Armillaria species are known to produce them. The production of rhizomorphs is an adaptation to harsh environments, and could be a driver of diversification in Armillaria by conferring a competitive advantage to the species that produce them.

Insights into the phylogeny of Northern Hemisphere Armillaria: Neighbor-net and Bayesian analyses of translation elongation factor 1-α gene sequences.

Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence-based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

North American matsutake: names clarified and a new species described.

Tricholoma matsutake, known widely as "matsutake," has great commercial and cultural significance in Japan. Because Japanese production is insufficient to meet the high domestic demand, morphologically similar mushrooms, thought by many to belong to T. magnivelare, are imported from western North America. However, molecular data produced since the early 2000s have indicated that more than one species of matsutake occur in North America and this raises the question of correct naming for the different species. To address this question, we assessed the phylogenetic diversity within North American matsutake based on nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS] barcode) sequences, including newly obtained sequences from the type collections for Agaricus ponderosus and Armillaria arenicola, and morphological characters. Our results agree with earlier indications that three matsutake species occur in North America and allow us to clarify the correct application of names-T. magnivelare from the eastern USA and Canada, T. murrillianum from the western USA and Canada, and T. mesoamericanum from Mexico, newly described here. The existence of the three North American species is further supported by the results of evolutionary divergence analysis, geographical distributions, and morphological characters.

Characterization of a novel endo-ß-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis.

A novel endo-ß-1,4-glucanase (EG)-producing strain was isolated and identified as Armillaria gemina KJS114 based on its morphology and internal transcribed spacer rDNA gene sequence. A. gemina EG (AgEG) was purified using a single-step purification by gel filtration. The relative molecular mass of AgEG by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 65 kDa and by size exclusion chromatography was 66 kDa, indicating that the enzyme is a monomer in solution. The pH and temperature optima for hydrolysis were 5.0 and 60 °C, respectively. Purified AgEG had the highest catalytic efficiency with carboxymethylcellulose (k(cat)/K(m) = 3,590 mg mL⁻¹ s⁻¹) unlike that reported for any fungal EG, highlighting the significance of the current study. The amino acid sequence of AgEG showed homology with hydrolases from the glycoside hydrolase family 61. The addition of AgEG to a Populus nigra hydrolysate reaction containing a commercial cellulase mixture (Celluclast 1.5L and Novozyme 188) showed a stimulatory effect on reducing sugar production. AgEG is a good candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.

[Isolation and characterization of 27 polymorphic microsatellite markers in Armillaria luteo-virens ( Physalacriaceae)].

[OBJECTIVE] The objectives of this study were to use Roche 454 GS FLX system to develop SSR markers for Armillaria luteo-virens. These datasets will be valuable for detecting genetic diversity and population structure of this species. [METHODS] We collected Armillaria luteo-virens samples from Yushu in Qinghai province, China. Total RNA was isolated by using the TRIzol reagent, after that we constructed cDNA library and performed one quarter plate of the whole run 454 pyrosequencing. We selected 98 primer pairs randomly from the 321 SSRs to evaluate their application and the polymorphism across 66 individuals (Armillaria luteo-virens) representing 3 wild populations. [RESULTS] Roche 454 sequencing yielded 197,121 reads with a total nucleotide size of 88,585,965bp. 27 of 98 SSRs loci were polymorphic. Numbers of alleles (Na) ranged from 2 to 8. Expected heterozygosity (HE) ranged from less than 0. 001 to 0. 810 at locus ALV65, while observed heterozygosity (Ho) from 0 at loci AIV64 and AIV92 to 0. 900 at loci ALV8. We found no evidence of linkage disequiliburium, however 10 of 27 SSR markers showed significant deviation from Hardy-weinberg equilibrium. [CONCLUSION] These remaining 17 pairs of Armillaria luteo-virens SSR markers will be valuable for future research on detecting population structure and conservation of this species.

Genomic and proteomic dissection of the ubiquitous plant pathogen, Armillaria mellea: toward a new infection model system.

Armillaria mellea is a major plant pathogen. Yet, no large-scale "-omics" data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the A. mellea genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (n = 629 unique) and secreted (n = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (n = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the A. mellea proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that A. mellea exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive A. mellea proteins (n = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms.

A new multilocus approach for a reliable DNA-based identification of Armillaria species.

In this paper we highlight and critically discuss limitations to molecular methods for identification of fungi via the example of the basidiomycete genus Armillaria. We analyzed a total of 144 sequences of three DNA regions commonly used for identifying fungi (ribosomal IGS-1 and ITS regions, translation elongation factor-1 alpha gene) from 48 specimens of six Armillaria species occurring in Europe (A. cepistipes, A. ostoyae, A. gallica, A. borealis, A. mellea, A. tabescens). Species were identified by comparing newly obtained sequences with those from the NCBI database, phylogenetic analyses and PCR-RFLP analyses of the three regions considered. When analyzed separately, no single gene region could unambiguously identify all six Armillaria species because of low interspecific and high intrasequence variability. We therefore developed a multilocus approach, which involves the stepwise use of the three regions. Following this scheme, all six species could be clearly discriminated. Our study suggests that, to improve the reliability of DNA-based techniques for species identification, multiple genes or intergenic regions should be analyzed.

Armillaria altimontana, a new species from the western interior of North America.

Armillaria altimontana, previously considered North American biological species (NABS) X, is described as new. To date, it appears that A. altimontana prefers higher-elevation, mesic sites within the dry, conifer forest zone of western interior North America. This species has been found on hardwoods and conifers and is associated most commonly with Abies-dominated forest types in southern British Columbia, Washington, Oregon, Idaho and northern California. Partial elongation factor 1-alpha (tef1) sequences were generated from six isolates of A. altimontana originating from three locations in northern Idaho. Phylogenetic analyses of all 10 North American Armillaria species were carried out with maximum parsimony and maximum likelihood. Results indicate that isolates of A. altimontana formed a monophyletic group and clustered with A. calvescens, A. cepistipes, A. gallica and A. nabsnona, which is in agreement with recent phylogenetic studies of Armillaria.

Taxonomy of Armillaria in the Patagonian forests of Argentina.

The taxonomy of Armillaria in southern South America has received little attention since the work of Singer and others. In this study we examine the morphological traits and cultural features for taxa representing the lineages revealed based on molecular phylogeny, and we link them to previously described taxa based on morphology. Lineages I-IV were identified as Armillaria novae-zelandiae, A. montagnei, A. umbrinobrunnea comb. nov. and A. sparrei respectively. They could be differentiated morphologically based on dimension, features of the epicutis, annulus, stipe, hymenophoral trama and flavor and characteristics in culture. Furthermore there was no evidence of host preference for the species recognized. This is the first study integrating the phylogeny and morphology of Armillaria species from Patagonia, and it provides a foundation for future research on these fungi in South America.

Sequence-based identification of Japanese Armillaria species using the elongation factor-1 alpha gene.

We analyzed the sequences of three DNA regions-the translation elongation factor-1 alpha (EF-1 alpha) gene and the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA-to compare their accuracy in identifying species of Japanese Armillaria. We studied 49 isolates of eight Armillaria species, A. mellea, A. ostoyae, A. nabsnona, A. cepistipes, A. gallica, A. sinapina, A. tabescens and the biological species Nagasawa E (Nag. E). Phylogenetic analyses of the ITS and IGS data helped in identifying A. mellea, A. ostoyae, A. nabsnona, A. tabescens and Nag. E but could not be used to identify A. gallica, A. cepistipes and A. sinapina. Nevertheless our analysis showed that the EF-1 alpha gene was clearly different in the eight examined species. Restriction fragment length polymorphisms (RFLP) of the IGS-1 region could be used to distinguish most species, but the RFLP profiles of some isolates of A. cepistipes and A. sinapina were the same even with four different restriction enzymes. In conclusion, among the techniques examined in this study, analyzing the EF-1 alpha sequence was found to be the most suitable method for identifying different species of Japanese Armillaria.

Armillaria.

Lingering in forests around the world, some of the largest and oldest terrestrial organisms on earth hide in plain sight. In this Quick Guide, Sipos et al. shed light on the biology of the Armillaria fungi.

Armillaria mexicana, a newly described species from Mexico.

Armillaria mexicana (Agaricales, Physalacriaceae) is described as a new species based on morphology, DNA sequence data, and phylogenetic analyses. It clearly differs from previously reported Armillaria species in North, Central, and South America. It is characterized by the absence of fibulae in the basidioma, abundant cheilocystidia, and ellipsoidal, hyaline basidiospores that are apparently smooth under light microscope, but slightly to moderately rugulose under scanning electron microscope. It is differentiated from other Armillaria species by macromorphological characters, including annulus structure, pileus and stipe coloration, and other structures. DNA sequence data (nuc rDNA internal transcribed spacers [ITS1-5.8S-ITS2 = ITS], 28S D-domain, 3' end of 28S intergenic spacer 1, and translation elongation factor 1-α [TEF1]) show that A. mexicana sequences are quite distinct from sequences of analogous Armillaria species in GenBank. In addition, sequences of ITS of the A. mexicana ex-type culture reveal an ITS1 of 1299 bp and an ITS2 of 582 bp, the longest ITS regions reported thus far in fungi. Phylogenetic analysis based on TEF1 sequences place A. mexicana in a well-separated, monophyletic clade basal to the polyphyletic A. mellea complex.

Genetic analysis reveals efficient sexual spore dispersal at a fine spatial scale in Armillaria ostoyae, the causal agent of root-rot disease in conifers.

Armillaria ostoyae (sometimes named Armillaria solidipes) is a fungal species causing root diseases in numerous coniferous forests of the northern hemisphere. The importance of sexual spores for the establishment of new disease centres remains unclear, particularly in the large maritime pine plantations of southwestern France. An analysis of the genetic diversity of a local fungal population distributed over 500 ha in this French forest showed genetic recombination between genotypes to be frequent, consistent with regular sexual reproduction within the population. The estimated spatial genetic structure displayed a significant pattern of isolation by distance, consistent with the dispersal of sexual spores mostly at the spatial scale studied. Using these genetic data, we inferred an effective density of reproductive individuals of 0.1-0.3 individuals/ha, and a second moment of parent-progeny dispersal distance of 130-800 m, compatible with the main models of fungal spore dispersal. These results contrast with those obtained for studies of A. ostoyae over larger spatial scales, suggesting that inferences about mean spore dispersal may be best performed at fine spatial scales (i.e. a few kilometres) for most fungal species.

Phylogenetic constrains on Polyporus umbellatus-Armillaria associations.

It has been well established that some Armillaria species are symbionts of Polyporus umbellatus, However, little is known about the evolutionary history of P. umbellatus-Armillaria associations. In this research, we used an analysis based on the strength of the phylogenetic signal to investigate P. umbellatus-Armillaria associations in 57 sclerotial samples across 11 provinces of China. We isolated Armillaria strains from the invasion cavity inside the sclerotia of P. umbellatus and then phylogenetically analyzed these Armillaria isolates. We also tested the effect of P. umbellatus and Armillaria phylogenies on the P. umbellatus-Armillaria associations. We isolated forty-seven Armillaria strains from 26 P. umbellatus sclerotial samples. All Armillaria isolates were classified into the 5 phylogenetic lineages found in China except for one singleton. Among the 5 phylogenetic lineages, one lineage (lineage 8) was recognized by delimitation of an uncertain phylogenetic lineage in previous study. Results of simple Mantel test implied that phylogenetically related P. umbellatus populations tend to interact with phylogenetically related Armillaria species. Phylogenetic network analyses revealed that the interaction between P. umbellatus and Armillaria is significantly influenced by the phylogenetic relationships between the Armillaria species.

Genetic variation and phylogenetic relationships of the ectomycorrhizal Floccularia luteovirens on the Qinghai-Tibet Plateau.

Floccularia luteovirens, as an ectomycorrhizal fungus, is widely distributed in the Qinghai-Tibet Plateau. As an edible fungus, it is famous for its unique flavor. Former studies mainly focus on the chemical composition and genetic structure of this species. However, the phylogenetic relationship between genotypes remains unknown. In this study, the genetic variation and phylogenetic relationship between the genotypes of F. luteovirens in Qinghai-Tibet Plateau was estimated through the analysis on two protein-coding genes (rpb1 and ef-1α) from 398 individuals collected from 24 wild populations. The sample covered the entire range of this species during all the growth seasons from 2011 to 2015. 13 genotypes were detected and moderate genetic diversity was revealed. Based on the results of network analysis, the maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) analyses, the genotypes H-1, H-4, H-6, H-8, H-10, and H-11 were grouped into one clade. Additionally, a relatively higher genotype diversity (average h value is 0.722) and unique genotypes in the northeast edge of Qinghai- Tibet plateau have been found, combined with the results of mismatch analysis and neutrality tests indicated that Southeast Qinghai-Tibet plateau was a refuge for F. luteovirens during the historical geological or climatic events (uplifting of the Qinghai-Tibet Plateau or Last Glacial Maximum). Furthermore, the present distribution of the species on the Qinghai-Tibet plateau has resulted from the recent population expansion. Our findings provide a foundation for the future study of the evolutionary history and the speciation of this species.

Rapid identification of Armillaria species by PCR-DGGE.

The molecular approach has been used to distinguish between Armillaria species because of the ambiguity when using morphological traits. PCR-DGGE using the fungal primers ITS3GC-ITS4 enables a selective separation of amplicons and results in a simple and rapid approach to discriminate in one-step the most common Armillaria species in Europe.

Comparative bioluminescence dynamics among multiple Armillaria gallica, A. mellea, and A. tabescens genets.

Bioluminescence is well known among white-spored species of Basidiomycota including several species of the white-rot wood decay genus Armillaria. Previous work demonstrated consistent differences among A. gallica, A. mellea, and A. tabescens in luminescence magnitude and in luminescence expression relative to environmental stimuli. In the present studies, temporal fluctuations in mycelial luminescence were quantitatively characterized using genets matched for geographical location. All genets derived from rhizomorphs or basdiomata were constitutively luminescent while six of 13 genets originating from mycelial fans were inconsistently luminescent. Using time series of 1000 consecutive measurements over 800 ms intervals, fluctuation patterns had significantly quantifiable structure and were not simply 'white noise'. Fluctuation patterns were qualitatively similar with alternating periods of rapid fluctuation and relative stability, regardless of luminescence magnitude. Anomalous spikes or shifts in luminescence were recorded for several genets suggesting further work to identify the transient stimuli which elicited these altered luminescence patterns.