First Report of Sphingobacterium multivorum Causing Bacterial Rot of Cauliflower mushroom (Sparassis latifolia) in China.

Cauliflower mushroom (Sparassis latifolia), is widely distributed in Australia, North America, Europe, and East Asia (Bashir et al., 2020). It is known for its medicinal significance due to the availability of various pharmacological substances and their use in health supplements (Bashir et al., 2017). In recent years, with the development of artificial cultivation technology, S. latifolia has been industrialized in China, with an annual output value 50 million dollars. In March 2023, approximately 15% of S. latifolia showed obvious bacterial rot in mushroom hothouse (about 0.05 ha), located in Shuangliu county, Sichuan province, China (104°7'51"N, 30°25'2"E). The affected parts appear water-soaked, and become sunken and softened as the disease progresses. In the finally, all the fruiting body tissues turn into paste, with colors pale yellow, and have a foul smell. The pathogen was isolated from the margin of the lesions by dilution and streaking techniques onto Nutrient Agar, and incubated at 28℃ in the dark for 2-3 days. A single colony was re-streak for purification. Eight isolates were obtained from five samples collected randomly. The representative three isolates were selected for further characterization. For pathogenicity testing, ten health fruit bodies of S. latifolia were selected (for per isolate). Bacterial suspensions (1 × 107 CFU/ml) of the three isolates were applied to the fruiting body until wet, sterile water was used as controls. All the S. latifolia were maintained at 19±1℃, 85-100% relative humidity, and 18 h of light in the mushroom hothouse. Three days later, the inoculated fruiting bodies developed yellow color, and appear water-soaked, five days later, fruiting body gradually turn to soft and part turn to rot, seven days later, the fruiting body tissues completely turn into paste with a foul smell. The symptoms exhibited were similar to those of the original diseased fruiting bodies, while the control group remained healthy. The same bacterial were re-isolated from the infected fruiting bodies and subsequently identified by morphological characteristics and DNA sequenced. The pathogenicity test was conducted three times, each yielding similar results. The colonies of the pathogen are gram-negative rods, medium sized, convex, smooth, opaque, turning yellow after several days at a temperature 28℃. For molecular identification, the DNA of the representative three isolates was extracted using a Bacterial Genomic DNA Extraction Kit (Solarbio, Beijing). The 16S rRNA genes were amplified and sequenced with the primer 27F/1492R (Lane et al., 1985). Finally, the sequences were identical. The generated representative sequence was deposited in GenBank with accession number OR399122. BLASTn analysis showed 100% identity (1404/1404 bp) with previously deposited sequence (accession number CP068224) of S. multivorum FDAARGOS in GenBank. Based on the maximum likelihood method, phylogenetic analysis revealed 100% bootstrap support values with S. multivorum. Finally, the bacterium was identified as S. multivorum. This is the first report of S. multivorum causing bacterial rot of mushroom. The fruiting body of S. multivorum consists of multiple folded flat lobes, which are thin and have large surface area, may facilitate the infection of S. multivorum. Sphingobacterium sp. are named for their synthesize sphingolipids, which play an important role in bacterial infection (Kunz et al., 2019). These results will contribute to developing control strategies for this disease.

A Comprehensive Assessment of Ultraviolet-Radiation-Induced Mutations in Flammulina filiformis Using Whole-Genome Resequencing.

Nucleotide substitutions have played an important role in molecular evolution, and understanding their dynamics would contribute to genetic studies. Related research with defined DNA sequences lasted for decades until whole-genome sequencing arose. UV radiation (UVR) can generate base changes and other genetic variations in a short period of time, so it would be more meaningful to explore mutations caused by UVR from a genomic perspective. The monokaryon enoki strain WT583 was selected as the experimental material in this study because it can spontaneously produce large amounts of oidia on PDA plates, and the monokaryons originating from oidia have the same genotype as their mother monokaryon. After exposure to UV radiation, 100 randomly selected mutants, with WT583 as the reference genome, were sent for genome sequencing. BWA, samtools, and GATK software were employed for SNP calling, and the R package CMplot was used to visualize the distribution of the SNPs on the contigs of the reference genome. Furthermore, a k-mer-based method was used to detect DNA fragment deletion. Moreover, the non-synonymous genes were functionally annotated. A total of 3707 single-base substitutions and 228 tandem mutations were analyzed. The immediate adjacent bases showed different effects on the mutation frequencies of adenine and cytosine. For adenine, the overall effects of the immediate 5'-side and 3'-side bases were T > A > C > G and A > T > G > C, respectively; for cytosine, the overall effects of the immediate 5'-side and 3'-side bases were T > C > A > G and C > T > A > G, respectively. Regarding tandem mutations, the mutation frequencies of double-transition, double-transversion, 3'-side transition, and 5'-side transition were 131, 8, 72, and 17, respectively. Transitions at the 3'-side with a high mutation frequency shared a common feature, where they held transversions at the 5'-side of A→T or T→A without covalent bond changes, suggesting that the sequence context of tandem motifs might be related to their mutation frequency. In total, 3707 mutation sites were non-randomly distributed on the contigs of the reference genome. In addition, pyrimidines at the 3'-side of adenine promoted its transversion frequency, and UVR generated DNA fragment deletions over 200 bp with a low frequency in the enoki genome. The functional annotation of the genes with non-synonymous mutation indicated that UVR could produce abundant mutations in a short period of time.

First Report of Lecanicillium aphanocladii Causing Rot of Sparassis crispa in China.

Sparassis crispa, also known as cauliflower mushroom, is a new popularly edible mushroom in China, also a medicinal mushroom, which possesses various biological activities, such as immunopotentiation, anti-diabetes, anti-cancer, and anti-inflammatory effects. (Han et al., 2018). In recent years, the artificial cultivation of S. crispa has gained considerable public attention in China. In 2023, approximately 20% of S. crispa (about 0.05 ha of the planting area) showed obvious rot with white molds symptoms in mushroom hothouse, located in Shuangliu county, Sichuan province, China (GPS, 104°7'51"N, 30°25'2"E). Infected fruiting bodies were covered by white mycelia that later turned red or fuchsia. In the final stages of infection, the S. crispa fruiting bodies turned dark red or brown before rotting. The pathogen was isolated from the margin of the lesions by plating onto potato dextrose agar (PDA), and incubated at 25℃ in the dark for a week. Five pure culture fungal isolates were obtained. Collected isolates with similar morphology were described as Lecanicillium spp. (Zare et al., 2001). The colonies were raised, covered with white, the reverse side were violet brown, produced diffusing reddish-purple pigment. Conidiogenous cells produced singly, in pairs, verticillate or in dense irregular clusters on prostrate hyphae, at first flask-shaped, tapering into threadlike neck, with a size of 3.0-6.2×0.8-2.2 µm. Conidia were solitary, oval to subglobose, and 2.3-4.0×1.1-2.1 µm in size, similar to L. aphanocladii (Higo et al., 2021). For pathogenicity testing, ten fruiting bodies of S. crispa (planted in the bottles) were selected. Fungal cake of the isolate Bx-Ljb of L. aphanocladii were applied to the fruiting body of S. crispa, whereas pieces of sterile PDA medium were used as controls. All the bottles were incubated at 19±1℃, 85-100% relative humidity, and 18 h of light in the mushroom hothouse. A week later, the inoculated fruiting bodies developed brown spots and gradually expanding, with symptoms similar to the original diseased fruiting bodies. The controls remained healthy. The same fungus was reisolated from the infected fruiting bodies and subsequently identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. For molecular identification, the DNA of the isolates was extracted using a Fungi Genomic DNA Extraction kit (Solarbio, Beijing). The SSU, LSU, and TEF1-α genes were amplified with the primer as previously described (Zhou et al., 2018). The generated sequences were deposited in GenBank with accession numbers OR206377, OR206378, and OR204702, respectively. BLASTn analyses showed >99.2% identity with previously deposited sequences of L. aphanocladii. Based on the maximum likelihood method, phylogenetic analysis revealed 99% bootstrap support values with L. aphanocladii. The fungus was identified as L. aphanocladii based on morphological and multilocus phylogenetic analyses. To our knowledge, there are two reports of L. aphanocladii on fruiting bodies of Tremella fuciformis and Morchella sextelata in China, and this is the first report of this fungus causing rot of S. crispa in China. It may be a reminder that the risk of L. aphanocladii in mushroom production in China is gradually increasing. These results will contribute to developing managemental strategies for this disease in S. crispa.

First Report of Leaf blight Caused by Colletotrichum karstii on Banana Shrub (Michelia figo) in Sichuan, China.

Banana Shrub (Michelia figo (Lour.) Spreng.) is widely cultivated in most of southern China (Wu et al, 2008). It can be used to make essential oil and flower tea（Ma et al, 2012; Li et al, 2010）.The first symptoms were observed in Sept. 2020 at a grower's field in Banana shrub seedlings (0.6 ha), Ya'an city (29°30'N, 102°38'E), Hanyuan county. The symptoms re-occurred in May-June of 2021 and became prevalent from August to September. the incidence rate and the disease index were 40% and 22%, respectively. Initially, purplish-brown necrotic lesions appeared at the leaf tip with dark-brown edges. Progressively, necrosis spread, to the middle of the leaves, and the older area turned gray-white. Dark sunken lesions appeared in the necrotic areas and orange conidial masses were visible under humid conditions. Ten isolates were obtained on potato dextrose agar (PDA) from 10 leaf samples using previously described tissue isolation method (Fang et al. 1998). All the 10 isolates exhibited similar morphological characteristics. Grey to white aerial mycelium at the center and in dispersed tufts, with numerous dark conidiomata scattered over the surface, reverse was pale orange with numerous dark flecks corresponding to the ascomata, orange conidial masses were formed from mature conidiomata. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, apex round, the contents appearing granular 14.8 to 17.2 × 4.2 to 6.4 µm (average: 16.26 × 4.84 µm, n=30) as Colletotrichum spp. (Damm et al. 2012). For molecular identification, DNA was extracted from a representative isolate HXcjA using a plant genomic DNA extraction kit (Solarbio, Beijing). and the partial sequences of internal transcribed spacer region (ITS, OQ641677), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, OL614009), actin (ACT, OL614007), beta-tubulin (TUB2, OL614011), histone3 (HIS3, OL614010), and calmodulin (CAL, OL614008) were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT-512F/ACT-783R, CAL 228F/CAL 737R (Carbone et al. 1999), TUB1F/Bt2bR, CYLH3F/CYLH3R (Crous et al. 2004), respectively. BLASTn analysis for ITS, GAPDH, CAL, ACT, TUB2 and HIS3 sequences showed ≥99.7% identity to C. Karstii, namely, NR_144790 (532/532 bp), MK963048 (252/252 bp), MK390726 (431/431 bp), MG602039 (761/763 bp), (KJ954424, 294/294 bp), (KJ813519, 389/389 bp), respectively. The fungus was identified as C. karstii based on morphology and a multigene phylogeny. The conidial suspension (1 × 107 conidia/mL) with 0.05% Tween 80 buffer was used for pathogenicity test, by spraying 2-year-old Banana Shrub plants. Ten plants were inoculated with spore suspensions (approximately 2ml per plant). An equal number of plants were sprayed with 0.05% Tween 80 buffer to serve as a control. Fifteen days later, the inoculated plants showed similar symptoms as the original diseased plants but the controls remained asymptomatic. C. karstii was re-isolated from the infected leaves and identified by morphology and a multigene phylogeny. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of Banana Shrub leaf blight caused by C. karstii in China. This disease reduces the ornamental and economic value of Banana Shrub, and this work will provide a basis for the prevention and treatment of the disease in the future.

Comparative Proteomics Study on the Postharvest Senescence of Volvariella volvacea.

Volvariella volvacea is difficult to store after harvest, which restricts the production and circulation of V. volvacea fruiting bodies. Low-temperature storage is the traditional storage method used for most edible fungi. However, V. volvacea undergoes autolysis at low temperatures. When fruiting bodies are stored at 15 °C (suitable temperature), V. volvacea achieves the best fresh-keeping effect. However, the molecular mechanism underlying the postharvest senescence of V. volvacea remains unclear. Based on this information, we stored V. volvacea fruiting bodies at 15 °C after harvest and then analyzed the texture and phenotype combined with the results of previous physiological research. Four time points (0, 24, 60, and 96 h) were selected for the comparative proteomics study of V. volvacea during storage at 15 °C. A variety of proteins showed differential expressions in postharvest V. volvacea at 15 °C. Further comparison of the gene ontology (GO) enrichment analysis and KEGG pathways performed at different sampling points revealed proteins that were significantly enriched at several time points. At the same time, we also analyzed differentially expressed proteins (DEPs) related to the RNA transport, fatty acid biosynthesis and metabolism, and amino acid biosynthesis and metabolism pathways, and discussed the molecular functions of the PAB1, RPG1, ACC1, ADH3, ADH2, ALD5, and SDH2 proteins in postharvest V. volvacea senescence. Our results showed that many biological processes of the postharvest senescence of V. volvacea changed. Most importantly, we found that most RNA transport-related proteins were down-regulated, which may lead to a decrease in related gene regulation. Our results also showed that the expression of other important proteins, such as the fatty acid metabolism related proteins increased; and changes in fatty acid composition affected the cell membrane, which may accelerate the ripening and perception of V. volvacea fruiting bodies. Therefore, our research provides a reference for further studies on the aging mechanism of V. volvacea.

Reactive Oxygen Species Distribution Involved in Stipe Gradient Elongation in the Mushroom Flammulina filiformis.

The mushroom stipe raises the pileus above the substrate into a suitable position for dispersing spores. The stipe elongates at different speeds along its length, with the rate of elongation decreasing in a gradient from the top to the base. However, the molecular mechanisms underlying stipe gradient elongation are largely unknown. Here, we used the model basidiomycete mushroom Flammulina filiformis to investigate the mechanism of mushroom stipe elongation and the role of reactive oxygen species (ROS) signaling in this process. Our results show that O2- and H2O2 exhibit opposite gradient distributions in the stipe, with higher O2- levels in the elongation region (ER), and higher H2O2 levels in the stable region (SR). Moreover, NADPH-oxidase-encoding genes are up-regulated in the ER, have a function in producing O2-, and positively regulate stipe elongation. Genes encoding manganese superoxide dismutase (MnSOD) are up-regulated in the SR, have a function in producing H2O2, and negatively regulate stipe elongation. Altogether, our data demonstrate that ROS (O2-/H2O2) redistribution mediated by NADPH oxidase and MnSODs is linked to the gradient elongation of the F. filiformis stipe.

Transcriptome Profiling Reveals Candidate Genes Related to Stipe Gradient Elongation of Flammulina filiformis.

Stipe gradient elongation is an important and remarkable feature in the development of most mushroom fruiting bodies. However, its molecular mechanism has rarely been described. Here, the decreasing trend of stipe elongation and increasing trend of cell length in a gradient from the top to the base of the stipe were determined in a model basidiomycete mushroom: Flammulina filiformis. According to RNA-seq results, 1409 differentially expressed genes (DEGs) were identified among elongation region (ER), transition region (TR), and stable region (SR) samples, including 26 transcription factors (TFs). Based on Short Time-series Expression Miner (STEM) clustering of DEGs, clusters 1 and 3, with obvious expression trends that were consistent with or in contrast to the elongation rate, were screened. The cluster 1 DEGs were mainly involved in the GO cellular component category and KEGG genetic information processing class; however, the cluster 3 DEGs were mainly involved in metabolic processes. Furthermore, qRT-PCR confirmed that key genes of the long-chain fatty acid synthesis pathway were involved in stipe gradient elongation and regulated by NADPH oxidase-derived ROS signaling molecules. These findings provide an essential basis for understanding the molecular mechanism of stipe gradient elongation.

The NADPH oxidase in Volvariella volvacea and its differential expression in response to mycelial ageing and mechanical injury.

NADPH oxidases are enzymes that have been reported to generate reactive oxygen species (ROS) in animals, plants and many multicellular fungi in response to environmental stresses. Six genes of the NADPH oxidase complex components, including vvnoxa, vvnoxb, vvnoxr, vvbema, vvrac1 and vvcdc24, were identified based on the complete genomic sequence of the edible fungus Volvariella volvacea. The number of vvnoxa, vvrac1, vvbema and vvcdc24 transcripts fluctuated with ageing, and the gene expression patterns of vvnoxa, vvrac1 and vvbema were significantly positively correlated. However, the expression of vvnoxb and vvnoxr showed no significant difference during ageing. In hyphae subjected to mechanical injury stress, both O2- and H2O2 concentrations were increased. The expression of vvnoxa, vvrac1, vvbema and vvcdc24 was substantially upregulated, but vvnoxb and vvnoxr showed no response to mechanical injury stress at the transcriptional level. Additionally, the transcription of vvnoxa, vvrac1, vvbema and vvcdc24 could be repressed when the intracellular ROS were eliminated by diphenyleneiodonium (DPI) chloride and reduced glutathione (GSH) treatments. These results indicated a positive feedback loop involving NADPH oxidase and intracellular ROS, which might be the reason for the oxidative burst during injury stress.

Comparative Transcriptomics of Flammulina filiformis Suggests a High CO2 Concentration Inhibits Early Pileus Expansion by Decreasing Cell Division Control Pathways.

Carbon dioxide is commonly used as one of the significant environmental factors to control pileus expansion during mushroom cultivation. However, the pileus expansion mechanism related to CO2 is still unknown. In this study, the young fruiting bodies of a popular commercial mushroom Flammulina filiformis were cultivated under different CO2 concentrations. In comparison to the low CO2 concentration (0.05%), the pileus expansion rates were significantly lower under a high CO2 concentration (5%). Transcriptome data showed that the up-regulated genes enriched in high CO2 concentration treatments mainly associated with metabolism processes indicated that the cell metabolism processes were active under high CO2 conditions. However, the gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with cell division processes contained down-regulated genes at both 12 h and 36 h under a high concentration of CO2. Transcriptome and qRT-PCR analyses demonstrated that a high CO2 concentration had an adverse effect on gene expression of the ubiquitin-proteasome system and cell cycle-yeast pathway, which may decrease the cell division ability and exhibit an inhibitory effect on early pileus expansion. Our research reveals the molecular mechanism of inhibition effects on early pileus expansion by elevated CO2, which could provide a theoretical basis for a CO2 management strategy in mushroom cultivation.

A hydrophobin gene, Hyd9, plays an important role in the formation of aerial hyphae and primordia in Flammulina filiformis.

Flammulina filiformis is an edible fungus that is largely cultivated and widely consumed around the world. The quantity and quality of the primordia, which gives rise to the fruiting body, affects its production efficiency. Hydrophobins are involved in the formation of the fruiting body of macrofungi. However, functional verification of the hydrophobin genes is limited to date. In this study, we used gene silencing and overexpression analyses to investigate the function of one F. filiformis hydrophobin gene (Hyd9) during the development of the fruiting body. The Hyd9-silenced transformants exhibited sparse aerial hyphae, resulting in fewer primordia and fruiting bodies. In contrast, the Hyd9 overexpression strain displayed denser aerial hyphae and more primordia. The phenotypes of these transgenic lines strongly suggested that Hyd9 plays an important role in the formation of aerial hyphal knots (the primary stage of primordia) and primordia in F. filiformis. These results will be beneficial for developing more efficient methods to induce primordia formation in F. filiformis and other commercially valuable mushrooms.