Transcriptome analysis of Ganoderma lingzhi (Agaricomycetes) response to Trichoderma hengshanicum infection.

Green mold caused by Trichoderma spp. has become one of the most serious diseases which threatening the production of Ganoderma lingzhi. To understand the possible resistance mechanism of the G. lingzhi response to T. hengshanicum infection, we examined the G. lingzhi transcript accumulation at 0, 12, and 24 h after T. hengshanicum inoculation. The gene expression analysis was conducted on the interaction between G. lingzhi and T. hengshanicum using RNA-seq and digital gene expression (DGE) profiling methods. Transcriptome sequencing indicated that there were 162 differentially expressed genes (DEGs) at three infection time points, containing 15 up-regulated DEGs and 147 down-regulated DEGs. Resistance-related genes thaumatin-like proteins (TLPs) (PR-5s), phenylalanine ammonia-lyase, and Beta-1,3-glucan binding protein were significantly up-regulated. At the three time points of infection, the heat shock proteins (HSPs) genes of G. lingzhi were down-regulated. The down-regulation of HSPs genes led to the inhibition of HSP function, which may compromise the HSP-mediated defense signaling transduction pathway, leading to G. lingzhi susceptibility. Pathway enrichment analyses showed that the main enriched pathways by G. lingzhi after infection were sphingolipid metabolism, ether lipid metabolism, and valine, leucine and isoleucine degradation pathway. Overall, the results described here improve fundamental knowledge of molecular responses to G. lingzhi defense and contribute to the design of strategies against Trichoderma spp.

Morphological characteristics and phylogenetic analyses revealed four new species of Agaricales from China.

Four new species of Agaricales from China viz. Hohenbuehelia tomentosa, Rhodophana qinghaiensis, Rhodophana aershanensis, and Spodocybe tomentosum are described based on their unique morphological features and molecular evidence. Hohenbuehelia tomentosa is mainly characterized by its dark brown pileus with finely dense pure white tomentum, dirty white, decurrent lamellae, eccentric stipe, smooth spores, and fusiform metuloid cystidia. The characteristics of Rhodophana qinghaiensis are glabrous, smooth, reddish-brown pileus, gray-orange lamellae, and initially light orange becoming reddish brown stipe. The unique morphological characteristics of Rhodophana aershanensis are reddish brown pileus with age, brown-orange toward the margin, light orange lamellae and stipe dark brown at first, and reddish-brown with age. Spodocybe tomentosum is characterized by subclitocyboid and small basidiomes, finely dense pure white tomentum on the pileus surface, and broadly ellipsoid to ellipsoid and smaller basidiospores. Phylogenetic analysis showed that Hohenbuehelia tomentosa, Rhodophana qinghaiensis, Rhodophana aershanensis, and Spodocybe tomentosum formed an independent lineage. Full descriptions, illustrations, and phylogenetic trees of the four new species are provided in this study.

Analysis on the Structure and Function of the Bacterial Community in the Replanting Soil of Basswood of Ganoderma lingzhi Medicinal Mushroom (Agaricomycetes).

This study aimed to investigate the impact of the replanting of basswood Ganoderma lingzhi on the soil bacterial community and reveal the obstacle phenomenon of replanting basswood G. lingzhi. In this study, the soil bacterial community of wild 20 cm (N0a) and 40 cm deep soil (N0b), cultivated once (N1a, N1b) and twice (N2a, N2b), were investigated by Illumina MiSeq sequencing. The predominant bacterial phyla at the phylum classification level were Acidobacteria, Chloracidobacteria, Nitrospira, Spartobacteria, Gemmatimonadetes, Acidobacteria-6. Still, only the relative abundance of Chloracidobacteria and Acidobacteriia increased after two years of replanting of basswood G. lingzhi. At the genus level, the dominant genus included many unclassified bacteria. Among the known genera, the best genus was DA101, which showed a decreasing trend after two years of replanting. Network analysis showed that more connections of bacterial communities were observed in soil samples of the group "a," indicating that the replanting of basswood G. lingzhi can affect the relationship between soil bacterial communities at depths. The phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis showed that the gene metabolism function of soil bacteria was quite different after one year of replanting basswood G. lingzhi. The replanting of basswood G. lingzhi changed the composition and function of the soil bacterial community, and also affected the bacterial community diversity in the soil at different depths.

Infundibulicybe trachyspora, a New Species from Northeastern China Based on Morphology and Molecular Phylogeny.

Infundibulicybe trachyspora is described as a new species from northeastern China. The species is characterized by clitocyboid to omphalioid habit, carneous, greyish-yellow to brownish pileus, brown to dark reddish-brown, longitudinally fibrillose-striate stipe, non-amyloid, non-smooth spores, the absence of cystidia and the presence of clamp connections. A comprehensive description of the species is provided together with photo-illustrations and comparisons with phenotypically similar and phylogenetically related species. The nuclear ribosomal internal transcribed spacer (ITS) region and the nuclear, large subunit rDNA (nrLSU) region of the new species was sequenced and analyzed. The phylogenetic analysis supported the novelty of the species and its placement within the genus. Furthermore, a discussion on the proposal to establish a new section is made, and a key is provided for the Infundibulicybe species reported from China.

Additions to Lyophyllaceae s.l. from China.

Four new species, viz. Calocybe coacta, C. fulvipes, C. vinacea and Clitolyophyllum umbilicatum, are described in northern China. Comparisons are made of macro- and micromorphological features among the new species and closely related species within the genus. The new species feature unique morphological characteristics that separate them from the previously described species. Calocybe coacta is characterized by medium- to large-sized basidiocarps, greyish cream, felty pileus and non-cellular epicutis. The key characteristics of C. fulvipes are rose-brown to greyish-brown pileus, stone-brown stipe and non-cellular epicutis. The unique morphological characteristics of C. vinacea that distinguish it from its closely related species are pastel red to dull-red pileus and stipe surface with densely white pruina. The main characteristics of Clitolyophyllum umbilicatum are deeply depressed dark orange to light-brown pileus, central stipe and subglobose-ellipsoid spores. Phylogenetic analyses based on the ITS and 28S regions indicated that the four new species are distinct and monophyletic. Full descriptions, color images, illustrations and a phylogenetic tree that show the placement of the four new species are provided. A key to the Calocybe species reported from China is also given.

Unraveling the Genetic Basis of Fertility Restoration for Cytoplasmic Male Sterile Line WNJ01A Originated From Brassica juncea in Brassica napus.

Crosses that lead to heterosis have been widely used in the rapeseed (Brassica napus L.) industry. Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) systems represent one of the most useful tools for rapeseed production. Several CMS types and their restorer lines have been identified in rapeseed, but there are few studies on the mechanisms underlying fertility restoration. Here, we performed morphological observation, map-based cloning, and transcriptomic analysis of the F2 population developed by crossing the CMS line WNJ01A with its restorer line Hui01. Paraffin-embedded sections showed that the sporogenous cell stage was the critical pollen degeneration period, with major sporogenous cells displaying loose and irregular arrangement in sterile anthers. Most mitochondrial electron transport chain (mtETC) complex genes were upregulated in fertile compared to sterile buds. Using bulked segregant analysis (BSA)-seq to analyze mixed DNA pools from sterile and fertile F2 buds, respectively, we identified a 6.25 Mb candidate interval where Rfw is located. Using map-based cloning experiments combined with bacterial artificial chromosome (BAC) clone sequencing, the candidate interval was reduced to 99.75 kb and two pentatricopeptide repeat (PPR) genes were found among 28 predicted genes in this interval. Transcriptome sequencing showed that there were 1679 DEGs (1023 upregulated and 656 downregulated) in fertile compared to sterile F2 buds. The upregulated differentially expressed genes (DEGs) were enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) lysine degradation pathway and phenylalanine metabolism, and the downregulated DEGs were enriched in cutin, suberine, and wax biosynthesis. Furthermore, 44 DEGs were involved in pollen and anther development, such as tapetum, microspores, and pollen wall development. All of them were upregulated except a few such as POE1 genes (which encode Pollen Ole e I allergen and extensin family proteins). There were 261 specifically expressed DEGs (9 and 252 in sterile and fertile buds, respectively). Regarding the fertile bud-specific upregulated DEGs, the ubiquitin-proteasome pathway was enriched. The top four hub genes in the protein-protein interaction network (BnaA09g56400D, BnaA10g18210D, BnaA10g18220D, and BnaC09g41740D) encode RAD23d proteins, which deliver ubiquitinated substrates to the 26S proteasome. These findings provide evidence on the pathways regulated by Rfw and improve our understanding of fertility restoration.

Morphology and Phylogeny Reveal Callistosporium subpetaloideum sp. nov. (Callistosporiaceae) from China.

A new species, Callistosporium subpetaloideum is described from Guizhou province, China. The key characteristics of C. subpetaloideum are its depressed, subpetaloid, finely pubescent pileus, pruinose apexed central stipe, large elliptical spores up to 7 µm long and 5 µm wide, lacking cystidia and clamp connection. Phylogenetic analyses based on the internal transcribed spacer region indicated that C. subpetaloideum occupies an isolated position, while morphological characteristics have also been compared with other phenotypically similar species.

First Report of Green Mold Disease Caused by Trichoderma hengshanicum on Ganoderma lingzhi.

Ganoderma lingzhi is a well-known source of natural fungal medicines which has been given for the treatment of several diseases. China is one of the major commercial producers of Ganoderma mushroom worldwide. However, with the expansion of the commercial cultivation, the occurrence of the fungal diseases on G. lingzhi has also been increased. The green mold disease symptoms were observed in the cultivation base of G. lingzhi in Zuojia Town, Jilin City, Jilin Province, China, causing the basidiomes to be rotten and withered, and the green mycelium layer generated gradually. The pathogenicity tests showed the same symptoms as appeared naturally in Zuojia mushroom base. Morphology characters revealed conidia green, ellipsoid, globose, 2.56-4.83 × 2.09-4.22 µm, length-width ratio was 1.1-1.2 (n = 10). Conidiophores trichoderma-like, often asymmetry, branches solitary, paired or in whorls of 3 phialides formed solitary, paired or in whorl, variable in shape, lageniform, sometimes ampulliform or subulate. While using molecular methodology, comparing with the sequences of Trichoderma hengshanicum from GenBank, the analyzed sequence showed 97.32% homology with the RPB2 sequences, 100% with the TEF1-α sequences. A fungus isolated from the diseased tissues was identified based on morphology and molecular studies as T. hengshanicum. This is the first report of T. hengshanicum causing the green mold disease of G. lingzhi in China.

Phylogenetic Analyses of Some Melanoleuca Species (Agaricales, Tricholomataceae) in Northern China, With Descriptions of Two New Species and the Identification of Seven Species as a First Record.

Two new species (Melanoleuca galerina and M. subgrammopodia) and seven new recorded species from northern China are described here using morphological and molecular methods. Melanoleuca galerina is mainly characterized by its hygrophanous pileus, decurrent lamellae, fibrous stipe and spores with round warts. Key characteristics of M. subgrammopodia include its discolored pileus, fibrous stipe and urticiform cystidia. The divergence time of Melanoleuca fungi as well as the phylogenetic relationships within this genus were analyzed using DNA sequences of the internal transcribed spacer (ITS) and the nuclear large subunit rDNA (nrLSU) gene fragments. Analyses revealed that morphological identifications and phylogenetic relationships were consistent with the results of divergence time, thereby confirming that M. galerina and M. subgrammopodia are new species.

The effect of Hypomyces perniciosus on the mycelia and basidiomes of Agaricus bisporus.

Hypomyces perniciosus has been reported as a destructive pathogen of Agaricus bisporus. Previous research suggested that the pathogenesis may not only be perpetuated by H. perniciosus, but also by bacteria. Clarification of the interaction between A. bisporus and H. perniciosus is a prerequisite for the development of effective control measures against wet bubble disease. Here, the effects of H. perniciosus on A. bisporus mycelia are examined in dual culture on agar media and in open-ended test tubes. During disease development, the putative causal agents and cytology of wet bubble-diseased mushrooms were followed microscopically. The interaction between H. perniciosus and the basidiome of A. bisporus was also studied using dual-cultured H. perniciosus and basidiome tissues. Dual-cultured mycelia from both fungi showed that growth continued even after contact was made, without any observable antagonistic lines or cytoplasmic changes of A. bisporus mycelia. Hypomyces perniciosus could be isolated from diseased basidiomes any time after inoculation, but bacteria were only recovered after the basidiomes of A. bisporus had been killed by H. perniciosus. Dual culture of the basidiome tissue of A. bisporus and H. perniciosus on agar media established that H. perniciosus can independently and rapidly degrade the basidiomes of A. bisporus. We conclude that H. perniciosus has no pathogenic activity on the mycelial stage of A. bisporus, but it can destroy A. bisporus basidiomes in the absence of bacteria. Wet bubble disease is evidently not caused by bacteria, but by the fungus, although bacteria likely participate in the disease after invasion by the fungus.

[The development of Agaricus bisporus wet bubble disease and the nuclear phase of pathogen].

Objective: We studied the dynamic nuclear behavior of Hypomyces perniciosus on axenic culture and its disease progression after infection on different growth stages of Agaricus bisporus. Methods: Infection process was initiated by inoculating different stages of A. bisporus fruit body, and different depths of compost and casing soil with H. perniciosus. Disease progression was studied by observing symptoms on the fruit body using light microscopy and scanning electron microscopy. The nuclear behavior of H. perniciosus was determined by observation using fluorescence light microscopy after binding of DNA specific fluorochrome dye (DAPI:4, 6-Diamidino-2-phenylindole dihydrochloride) to the nuclei. Results: Inoculating H. perniciosus on different depths of compost and casing soil resulted in different disease rate as follows:on the surface of casing soil>in the center of casing soil>between the casing soil and the compost>in the center of compost. H. perniciosus can infect any stage of fruit body development, when young primordial (up to 3 mm) was infected, large, irregular and tumorous fungal masses were formed. H. perniciosus directly penetrated A. bisporus without the formation of appressorium-like structures. The germination of the conidia led to a necrotic brown lesion symptom on A. bisporus at the beginning stages of disease development. The mycelium of A. bisporus plasmolysed, hydropically degenerated, cytoplasmolysed, emptied of mycelium cytosol and eventual death as the disease advanced. H. perniciosus produced two types of conidia. Group I conidia had no septa, colorless and smooth containing one nucleus. Group II didymoconidium had septa, containing two nuclei, separated by septa. The first round of mitosis occurred in conidia with no nucleus in the germinal tube. Another kind of asexual spore for thicker cell wall wart convex chlamydospore, chlamydospore had two cells. The upper cell had two nuclear while the basal cell had one or two nuclear, when germinated, it produced one or two germinal tubes. The number of nuclear in the germinal tube was irregular, usually contained 0 to 2 nuclear. Conclusion: H. perniciosus can infect any part of the A. bisporus fruit body and can cause tremendous cytology changed. If we perform single spore isolation to do genetic analysis, one must isolate conidia with no septa.