Hallmarks of Comparative Transcriptome between Rhizomorphs and Hyphae of Armillaria sp. 541 Participating in Fungal Symbiosis with Emphasis on LysM Domains.

Armillaria sp. 541, a genus of root-infecting fungi, forms a symbiosis with traditional Chinese medicine Gastrodia elata (Orchid) and Polyporus umbellatus via extensive networks of durable rhizomorphs. It is not clear the hallmarks of comparative transcriptome between the rhizomorphs and hyphae of Armillaria sp. 541. In the present study, transcriptomic analysis of Armillaria sp. 541 identified 475 differentially expressed genes (DEGs) between Armillaria rhizomorphs (AR) and hyphae (AH). Of them, 285 genes were upregulated and 190 were downregulated. Bioinformatics analyses and tests demonstrated DEGs involved in oxidoreductase activity and peptidoglycan binding were significantly enriched in this process when rhizomorph formed from hyphae. We accordingly obtained 14 gene-encoding proteins containing the LysM domain, and further consensus pattern and phylogenetic analysis indicated that their amino acid sequences were conserved and their biological functions may be peptidoglycan binding for recognition between the fungus and host. Among these genes, one, named Armillaria LysM domain recognition gene (aLDRG), was expressed significantly when rhizomorphs were differentiated from hyphae. It was located in the cortical cells of the rhizomorph by in situ hybridization. Furthermore, biolayer interferometry (BLI) assay demonstrated that aLDRG can bind specifically to chitin oligosaccharide of the fungal cell wall, including N,N',N″-Triacetylchitotriose (CO3) and N,N',N″,N'″,N″″-Pentaacetylchitopentaose (CO5). Therefore, we deduced that Armillaria sp. 541 expressed higher levels of LysM protein aLDRG for better binding of oligosaccharide after rhizomorphs were generated. This study provides functional genes for further studies on the interaction between Armillaria sp. 541 and its host.

New disease and first report of marasmioid fungus, Marasmius palmivorus (Sharples), causing white root rot in Arachis hypogaea L.

Groundnut (Arachis hypogaea L.) plants showing distinct symptoms of necrosis of leaves and severe rotting of roots were observed in an agricultural field at Arakkonam, Tamil Nadu, India. The rhizomorphs of the phytopathogenic fungus were obtained from the rotted roots of the diseased plants and were cultured in the laboratory, and based on the morphological characteristics and nucleotide sequencing analysis of ITS and nLSU region, the phytopathogen was identified as Marasmius palmivorus. The isolated fungus produced distinct fruiting bodies (basidiocarps) when grown under the laboratory conditions. The fungus grew as cottony white colonies on the potato dextrose agar medium and were found to contain septate and clamp connections when examined under light microscope. The pathogenicity of the isolated fungus was assessed by inoculating it on healthy groundnut plant under glasshouse conditions; this resulted in the establishment of typical disease symptoms, which confirmed the pathogenicity of the fungus. The fungal pathogen re-isolated from the artificially inoculated plants was used for molecular characterization and confirmed that the organism was M. palmivorus. To the best of our knowledge, this is the first report on the occurrence of M. palmivorus, causing white root rot disease in A. hypogaea L.

Ultrastructure and development of acanthocytes, specialized cells in Stropharia rugosoannulata, revealed by scanning electron microscopy (SEM) and cryo-SEM.

Acanthocytes are special cells with a distinct spiky shape produced exclusively by the fungi of Stropharia and can be used to defend against nematodes. In the present study, the ultrastructure and development of acanthocytes were revealed by scanning electron microscopy (SEM) and cryo-SEM in S. rugosoannulata, a popular cultivated mushroom both in China and Europe. The acanthocytes were abundant on the surface of rhizomorph, casing soils, and vegetative mycelia of homokaryotic and heterokaryotic strains in S. rugosoannulata. The development of the acanthocyte was investigated with cryo-SEM, which has distinct advantage for observation of the ultrastructure of live, hydrated structures. Three distinct stages, including formation of lateral branch that was covered with patches, spiky structure formation, and maturation of acanthocytes, were identified and described. The irregular patches deposited on the surface of lateral branches and the holes in the spiky branches of the acanthocytes were reported for the first time. The environmental nitrogen level showed impact on acanthocyte production, but it seemed not to be the indispensable factor. Acid medium could delay the initiation of the acanthocyte formation but did not affect the overall morphology and structure, indicating that the central deposit of acanthocytes should be acid soluble. Acanthocytes of S. rugosoannulata have similar hydrophobicity to mycelia. The observation of ultrastructure and development process of acanthocytes provides insights into the ecological function and evolution of this special structure.

Identification and Characterization of Fungi Causing Thread Blight Diseases on Cacao in Ghana.

Theobroma cacao (chocolate tree) is currently under serious threat from thread blight disease (TBD), which has been attributed to the causal agent Marasmiellus scandens in other regions of the world. TBD in Ghana has similar symptomology but variable signs. This study sought to determine whether TBD in Ghana was caused by a single agent and whether Marasmiellus scandens was a significant agent of TBD. Forty-eight isolates were collected from eight geographical locations in Ghana for morphological and molecular characterization. Disease signs occurred as vegetative rhizomorphs or hyphal aggregates, which were classified into five morphotypes: A, abundant thin, black, "horse hair"-type rhizomorphs; B, scattered brown rhizomorphs; C, whitish to brownish-white; D, faint cream or dull white; and E, aggregates of shiny or silky white hyphae. Sequencing and analyses of three loci-the internal transcribed spacer region of the nuclear ribosomal repeat, nuclear large subunit, and mitochondrial small subunit-detected four species, all members of the Marasmiaceae, causing TBD-like disease. These were identified as Marasmius crinis-equi (morphotype A), Marasmius tenuissimus (morphotypes B and C), Marasmiellus palmivorus (morphotype E), and Marasmiellus scandens (morphotype D). Marasmius tenuissimus, the most frequently isolated TBD fungus in this study, is primarily an Asian fungus and not previously associated with diseases of cacao. Marasmiellus palmivorus, the second most frequently isolated fungus, is a pan-tropical pathogen with a broad host range; this is the first report of the fungus causing TBD on cacao. Marasmius crinis-equi also has a broad pan-tropical distribution and host range and causes thread blight on several tropical tree crops. Surprisingly, Marasmiellus scandens, the most frequently cited agent of TBD in cacao, made up only 8% of the isolates.

Application of SmartRoot in Phenotype Profile Analysis of Armillaria gallica / 中国实验方剂学杂志

Objective:To establish a research platform for obtaining accurate phenotypic spectrum data is a technical difficulty that needs to be resolved in the research of traditional Chinese medicine resources. For example，the traditional phenotypic characterization method of Armillaria rhizomorph is mostly in a form of descriptive text，which is subjective and empirical. There is an urgent need for an objective and accurate method to characterize the phenotype of honey fungus rhizomorph. Method:Based on the image processing software Image J and the root identification plug-in SmartRoot combined with the Synbiosis ProtoCol 3 image analyzer，the growth picture of Armillaria spp. was analyzed，and the length，growth rate，branching situation，and angle of nascent rhizomorph of Armillaria gallica were measured to establish a measurement system for the phenotypic analysis of Armillaria rhizomorph. Result:Based on the method developed in this paper，the growth length，growth rate，number of branches，angle of nascent rhizomorph，and other phenotypic changes can be analyzed in a real-time manner without affecting the growth of Armillaria gallica. Armillaria spp. grew fastest at 9-12 days after generation，and the angle between the nascent rhizomorph and the parent rhizomorph was nearly vertical. This method had a certain correlation with the dry weight of traditional Armillaria biomass phenotypic parameters，with a high value in practical application. Conclusion:This study has established an objective，accurate，fast and real-time phenotypic analysis and measurement system for Armillaria rhizomorph，which expands the scope of application of SmartRoot and can be used for phenotypic analysis of traditional Chinese medicine resources under controlled experimental conditions.

The Most Extensive Devonian Fossil Forest with Small Lycopsid Trees Bearing the Earliest Stigmarian Roots.

Since the Late Paleozoic, forests have become distributed worldwide and significantly changed the Earth's climate and landscapes, but the record of forests is rare in the Devonian (419-359 Ma in age) when they first appeared. From the Upper Devonian (Famennian with the age of 372-359 Ma) of Xinhang, Anhui, China, we report a very large in situ forest, which includes locally dense stands of lycopsid plants. The Xinhang forest is monospecific with a small tree lycopsid Guangdedendron gen. nov., probably dioecious with monocarpic reproduction. The plant shows the earliest stigmarian rooting system typical of giant tree lycopsids dominating Carboniferous forests. It colonizes coastal clastic wetlands that were influenced by floods. This significantly increases the paleogeographical coverage of in situ Devonian forests, and contributes to our understanding of atmospheric CO2 decline and coastal consolidation.

Genomewide mutation dynamic within a long-lived individual of Armillaria gallica.

Mutation is the ultimate source of all genetic variation in populations and yet they remain unobservable and buried in the past. Long-lived individuals of Armillaria gallica, a common opportunistic fungal pathogen of tree roots in temperate forests of the northern hemisphere, provide a spatial context for examining the mutational dynamic. Each individual of A. gallica arises in a single mating between two haploid gametes and the resulting diploid then grows vegetatively to occupy a discrete spatial territory often including many adjacent tree root systems. In effect, this leaves a spatial record of growth over time within which mutations can be localized. To identify mutations, the entire genomes of three spatially separated samples of one individual of A. gallica approximately 200 × 60 m were sequenced and compared. In this comparison, mutations and regions of loss of heterozygosity (LOH) were identified then assayed in another 20 isolates from the same individual by conventional PCR and Sanger sequencing. The genotype network of all mutations and LOH were without internal conflict. Further, the spatial pattern of genotypes was nonrandom and appeared to reflect the vegetative expansion leading to the present-day individual. The results reflect the spectrum of spontaneous mutation in nature and provide insight into cellular generation times.

Anti-aging mechanism of polysaccharide from rhizomorph of Armillaria mellea in Caenorhabditis elegans / 中草药

Objective: To explore the anti-aging mechanism of polysaccharide from rhizomorph of Armillaria mellea (AMP) in Caenorhabditis elegans. Methods: With C. elegans as living model, the life span and the average number of offsprings were determined under normal culture conditions; The survival rate under oxidative stress and the expression of heat shock protein-16.2 (HSP-16.2) and superoxide dismutase-3 (SOD-3) were determined. Results: Under the normal culture conditions, the life span of C. elegans was significantly extended by AMP-1 and AMP-2 without the damage of the reproductive capacity; The expression of HSP-16.2 and SOD-3 was increased in C. elegans under the oxidative stress. Conclusion: The possible mechanism of AMP for anti-aging of C. elegans may be caused by increasing of the capacity of stress resistance.

Polysaccharide from the rhizomorph of Armillaria mella(AMP-1)protects INS-1 cells from alloxan injury / 中国药理学通报

Aim To investigate the protective effects of polysaccharides from the rhizomorph of Armillaria mella(AMP-1) on alloxan injured INS-1 cells.Methods Graded concentrations of AMP-1(2,10,50,100,500,1 000 mg?L-1) were added into the culture medium of alloxan injured INS-1 cells.The survival rate was measured by MTT assay.The amount of glucose-stimulated insulin secretion in different concentrations of AMP-1 was determined by radioimmunoassay(RIA).SOD and NOS activity,NO,MDA and GSH production were assayed colorimetrically.Results AMP-1 could reduce oxidative injuries induced by alloxan in INS-1 cells.The survival rate of cells treated with AMP-1 increased significantly.In the presence of 5.6 mmol?L-1 or 16.7 mmol?L-1 glucose,AMP-1(50,100,500,1 000 mg?L-1)increased glucose-induced insulin secretion in INS-1 cells in a dose-dependent manner.NOS levels and the production of NO and MDA decreased significantly by AMP-1,while SOD levels and the production of GSH increased.Conclusions AMP-1 promoted glucose-induced insulin secretion in INS-1 cells by increasing the abilities of scavenging the free radicals induced by alloxan.

Sclerotial Development of Grifola umbellata

Sclerotial development of Grifola umbellata (Pers. : Fr.) Donk was investigated through microscopic examinations. The sclerotium of G. umbellata was bumpy and rugged, multi-branched, and dark-brown to black in color. The sclerotial development of G. umbellata was categorized into three stages such as sclerotial initial, development and maturation. Sclerotium development was initiated as the white fungal mass. The superficial part of white sclerotium changed into gray, light brown and then black as its development proceeded further. As a distinctive characteristic of this fungus, a large number of crystals were observed in the medulla layer of sclerotium during its maturation. For development of new sclerotium, G. umbellata formed a white sclerotial primordium on the matured sclerotium. Development of sclerotium in G. umbellata was intimately associated with rhizomorphs of Armillariella mellea and the developing sclerotia were often penetrated by rhizomorphs of A. mellea into medulla layer.

Anti-aging effects of polysaccharides from rhizomorph of Armillaria mellea / 中成药

AIM: To study the anti-aging effects of polysaccharides from rhizomorph of Armillaria mellea(ARP) on fruit fly and mouse. METHODS: Regarding fruit files and D-galactose induced aging mice as research objects,observing the fruit flies lifespan;The ability to learn and memorize on model mice were observed by jumping platform test and maze test,the body weight,thymus and spleen index,SOD and GSH-Px activities、MDA and NO levels in serum,liver and brain were measured. RESULTS: ARP could significantly lengthen the lifespan of fruit flies;Obviously improve the ability to learn and memorize,markedly increase the body weight and resist the atrophy of thymus and spleen of model mice,SOD and GSH-Px activities increased obviously,MDA and NO levels decreased dramatically on model mice. CONCLUSION: ARP has anti-aging effects by immune-regulatory and eliminating free radicals.

A New Method of Armillaria mellea Isolation-Gastrodia elata Tissue Isolating Method / 微生物学通报

This paper reported a new method of Armillaria mellea isolation-Gastrodia elata tissue isolating. Compared with normal isolating method-rhizomorph isolating method, it showed that the success rate of new method (78% ) was higher than the rhizomorph isolating method (16% ) , besides this, the new method was easier, and growth characteristic of obtained strain was superior to that obtained from rhizomorph isolating method.