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 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.

Do the Main Components of the Sclerotia of Umbrella Polypore Mushroom, Polyporus umbellatus (Agaricomycetes), Correlate with Armillaria Associates?

Polyporus umbellatus is a traditional Chinese medicinal mushroom. The growth of P. umbellatus sclerotia requires the rhizomorphs of Armillaria spp. to supply nutrition. Whether the main components (MC) of sclerotia of P. umbellatus are related to the phylogeny of Armillaria associates or other environmental factors is largely unknown. In this study, we collected 17 sclerotia and soil samples from northeast to southwest China. In total, 17 Armillaria associates were isolated, and sclerotial MC contents and soil characteristics (total N, P, K, and organic matter) were determined. The analysis revealed that the MC content of P. umbellatus did not resemble a Brownian motion process in phylogeny of Armillaria associates, but were significantly influenced by the total N content of the soil. These results provide clear evidence that sclerotia of P. umbellatus associating with phylogenetic related Armillaria associates possess differing MC content. The mechanisms of nutrient exchange in P. umbellatus-Armillaria associations now require further elucidation.

Draft genomic sequence of Armillaria gallica 012m: insights into its symbiotic relationship with Gastrodia elata.

Armillaria species (Basidiomycota, Physalacriaceae) are well known as plant pathogens related to serious root rot disease on various trees in forests and plantations. Interestingly, some Armillaria species are essential symbionts of the rare Chinese medicinal herb Gastrodia elata, a rootless and leafless orchid used for over 2000 years. In this work, an 87.3-M draft genome of Armillaria gallica 012m strain, which was symbiotic with G. elata, was assembled. The genome includes approximately 23.6% repetitive sequences and encodes 26,261 predicted genes. In comparison with other four genomes of Armillaria, the following gene families related to pathogenicity/saprophytic phase, including cytochrome P450 monooxygenases, carbohydrate-active enzyme AA3, and hydrophobins, were significantly contracted in A. gallica 012m. These characteristics may be beneficial for G. elata to get less injuries. The genome-guided analysis of differential expression between rhizomorph (RH) and vegetative mycelium (VM) showed that a total of 2549 genes were differentially expressed, including 632 downregulated genes and 1917 upregulated genes. In the RH, most differentially expressed genes (DEGs) related to pathogenicity were significantly upregulated. To further elucidate gene function, Gene Ontology enrichment analysis showed that the upregulated DEGs significantly grouped into monooxygenase activity, hydrolase activity, glucosidase activity, extracellular region, fungal cell wall, response to xenobiotic stimulus, response to toxic substance, etc. These phenomena indicate that RH had better infection ability than VM. The infection ability of RH may be beneficial for G. elata to obtain nutrition, because the rhizomorph constantly infected the nutritional stems of G. elata and formed the hyphae that can be digested by G. elata. These results clarified the characteristics of A. gallica 012m and the reason why the strain 012m can establish a symbiotic relationship with G. elata in some extent from the perspective of genomics.

Phylogenetic Analyses of Armillaria Reveal at Least 15 Phylogenetic Lineages in China, Seven of Which Are Associated with Cultivated Gastrodia elata.

Fungal species of Armillaria, which can act as plant pathogens and/or symbionts of the Chinese traditional medicinal herb Gastrodia elata ("Tianma"), are ecologically and economically important and have consequently attracted the attention of mycologists. However, their taxonomy has been highly dependent on morphological characterization and mating tests. In this study, we phylogenetically analyzed Chinese Armillaria samples using the sequences of the internal transcribed spacer region, translation elongation factor-1 alpha gene and beta-tubulin gene. Our data revealed at least 15 phylogenetic lineages of Armillaria from China, of which seven were newly discovered and two were recorded from China for the first time. Fourteen Chinese biological species of Armillaria, which were previously defined based on mating tests, could be assigned to the 15 phylogenetic lineages identified herein. Seven of the 15 phylogenetic lineages were found to be disjunctively distributed in different continents of the Northern Hemisphere, while eight were revealed to be endemic to certain continents. In addition, we found that seven phylogenetic lineages of Armillaria were used for the cultivation of Tianma, only two of which had been recorded to be associated with Tianma previously. We also illustrated that G. elata f. glauca ("Brown Tianma") and G. elata f. elata ("Red Tianma"), two cultivars of Tianma grown in different regions of China, form symbiotic relationships with different phylogenetic lineages of Armillaria. These findings should aid the development of Tianma cultivation in China.