Mno-miR164a and MnNAC100 regulate the resistance of mulberry to Botrytis cinerea.

Although microRNAs (miRNAs) regulate the defense response of a variety of plant species against a variety of pathogenic fungi, the involvement of miRNAs in mulberry's defense against Botrytis cinerea has not yet been documented. In this study, we identified responsive B. cinerea miRNA mno-miR164a in mulberry trees. After infection with B. cinerea, the expression of mno-miR164a was reduced, which was fully correlated with the upregulation of its target gene, MnNAC100, responsible for encoding a transcription factor. By using transient infiltration/VIGS mulberry that overexpressed mno-miR164a or knocked-down MnNAC100, our study revealed a substantial enhancement in mulberry's resistance to B. cinerea when mno-miR164a was overexpressed or MnNAC100 expression was suppressed. This enhancement was accompanied by increased catalase (CAT) activity and reduced malondialdehyde (MDA) content. In addition, mno-miR164a-mediated inhibition of MnNAC100 enhanced the expression of a cluster of defense-related genes in transgenic plants upon exposure to B. cinerea. Meanwhile, MnNAC100 acts as a transcriptional repressor, directly suppressing the expression of MnPDF1.2. Our study indicated that the mno-miR164a-MnNAC100 regulatory module manipulates the defense response of mulberry to B. cinerea infection. This discovery has great potential in breeding of resistant varieties and disease control.

Complete Genome Sequence of Pantoea ananatis Strain LCFJ-001 Isolated from Bacterial Wilt Mulberry.

A Pantoea ananatis strain, named LCFJ-001 (GDMCC: 1.6101), was isolated for the first time from bacterial wilt-diseased roots of mulberry (Morus atropurpurea) in the western part of the Guangxi Zhuang Autonomous Region, China. Moreover, through Koch's postulates, it was proven that LCFJ-001 can cause mulberry wilt, which is one of the pathogens of mulberry bacterial wilt. Here, we report a complete, annotated genome sequence of P. ananatis LCFJ-001. The entire genome sequence of P. ananatis strain LCFJ-001 was a 4,499,350 bp circular chromosome with 53.50% GC content. In total, 3,521 genes were annotated, of which 3,418 were assigned protein-coding genes. In addition, 22 ribosomal RNAs and 81 transfer RNAs were identified. The presented resource will help explore the pathogenetic mechanisms of mulberry wilt disease caused by the genus Pantoea.

Study on the Potential for Stimulating Mulberry Growth and Drought Tolerance of Plant Growth-Promoting Fungi.

Drought stress often leads to heavy losses in mulberry planting, especially for fruits and leaves. Application of plant growth-promoting fungi (PGPF) endows various plant beneficial traits to overcome adverse environmental conditions, but little is known about the effects on mulberry under drought stress. In the present study, we isolated 64 fungi from well-growing mulberry trees surviving periodical drought stress, and Talaromyces sp. GS1, Pseudeurotium sp. GRs12, Penicillium sp. GR19, and Trichoderma sp. GR21 were screened out due to their strong potential in plant growth promotion. Co-cultivation assay revealed that PGPF stimulated mulberry growth, exhibiting increased biomass and length of stems and roots. Exogenous application of PGPF could alter fungal community structures in the rhizosphere soils, wherein Talaromyces was obviously enhanced after inoculation of Talaromyces sp. GS1, and Peziza was increased in the other treatments. Moreover, PGPF could promote iron and phosphorus absorption of mulberry as well. Additionally, the mixed suspensions of PGPF induced the production of catalase, soluble sugar, and chlorophyll, which in turn enhanced the drought tolerance of mulberry and accelerated their growth recovery after drought. Collectively, these findings might provide new insights into improving mulberry drought tolerance and further boosting mulberry fruit yields by exploiting interactions between hosts and PGPF.

Chemical and Biological Review of Endophytic Fungi Associated with Morus sp. (Moraceae) and In Silico Study of Their Antidiabetic Potential.

The chronic nature of diabetes mellitus motivates the quest for novel agents to improve its management. The scarcity and prior uncontrolled utilization of medicinal plants have encouraged researchers to seek new sources of promising compounds. Recently, endophytes have presented as eco-friendly leading sources for bioactive metabolites. This article reviewed the endophytic fungi associated with Morus species and their isolated compounds, in addition to the biological activities tested on their extracts and chemical constituents. The relevant literature was collected from the years 2008-2022 from PubMed and Web of Science databases. Notably, no antidiabetic activity was reported for any of the Morus-associated endophytic fungal extracts or their twenty-one previously isolated compounds. This encouraged us to perform an in silico study on the previously isolated compounds to explore their possible antidiabetic potential. Furthermore, pharmacokinetic and dynamic stability studies were performed on these compounds. Upon molecular docking, Colletotrichalactone A (14) showed a promising antidiabetic activity due to the inhibition of the α-amylase local target and the human sodium-glucose cotransporter 2 (hSGT2) systemic target with safe pharmacokinetic features. These results provide an in silico interpretation of the possible anti-diabetic potential of Morus endophytic metabolites, yet further study is required.

Analysis of endophytic bacterial flora of mulberry cultivars susceptible and resistant to bacterial wilt using metagenomic sequencing and culture-dependent approach.

Endophytes have a wide range of potential in maintaining plant health and sustainable agricultural environmental conditions. In this study, we analysed the diversity of endophytic bacteria in four mulberry cultivars with different resistance capacity against bacterial wilt using metagenomic sequencing and culture-dependent approaches. We further assessed the role of 11 shared genera in the control of bacterial wilt of mulberry. The results of the present study showed that Actinobacteria, Firmicutes, and Proteobacteria were the three dominant phyla in all communities, with the representative genera Acinetobacter and Pseudomonas. The diversity analysis showed that the communities of the highly and moderately resistant varieties were more diverse compared to those of the weakly resistant and susceptible varieties. The control tests of mulberry bacterial wilt showed that Pantoea, Atlantibacter, Stenotrophomonas, and Acinetobacter were effective, with a control rate of over 80%. Microbacterium and Kosakonia were moderately effective, with a control rate between 50 and 80%. At the same time, Escherichia, Lysinibacillus, Pseudomonas, and Rhizobium were found to be less effective, with a control rate of less than 40%. In conclusion, this study provides a reasonable experimental reference data for the control of bacterial wilt of mulberry.

Microbial community structure, co-occurrence network and fermentation characteristics of woody plant silage.

BACKGROUND: Feed shortage is a factor restricting animal production in the tropics, therefore how to use natural woody plant resources as animal feed is an important strategy. RESULTS: Under the dual stress of an anaerobic and acidic environment, the microbial response during the fermentation of paper mulberry (PM) silage was found to be sensitive. The Gram-negative bacteria and mould died, and the dominant microbial community rapidly shifted to Gram-positive bacteria, resulting in a large reduction in microbial diversity and abundance. Exogenous bran additives interfered with the stress effects of the woody silage environment. Wheat bran (WB) accelerated the response of microorganisms to the anaerobic stress, and lactic acid bacteria became the dominant microbial community, thereby enhancing the lactic acid fermentation of silage, affecting the metabolic pathways of microorganisms, and improving the flavour and quality of the silage. Addition of rice bran made Enterobacter and Clostridium species quickly respond to the stress of the silage environment and become the predominant bacterial groups. In particular, anaerobic and spore-forming Clostridium species showed a strong tolerance to the silage environment, leading to butyric acid fermentation and protein degradation of the silage, and reducing its fermentation quality. CONCLUSION: The PacBio single-molecule real-time (SMRT) sequencing technology accurately revealed the microbial co-occurrence network and fermentation mechanism of silage. Our results indicate that PM can be used in combination with WB to prepare high-quality silage for animal production. © 2021 Society of Chemical Industry.

Genome Sequencing of Ciboria shiraiana Provides Insights into the Pathogenic Mechanisms of Hypertrophy Sorosis Scleroteniosis.

Ciboria shiraiana causes hypertrophy sorosis scleroteniosis in mulberry trees, resulting in huge economic losses, and exploring its pathogenic mechanism at a genomic level is important for developing new control methods. Here, genome sequencing of C. shiraiana based on PacBio RSII and Illumina HiSeq 2500 platform as well as manual gap filling was performed. Synteny analysis with Sclerotinia sclerotiorum revealed 16 putative chromosomes corresponding to 16 chromosomes of C. shiraiana. Screening of rapid-evolution genes revealed that 97 and 2.4% of genes had undergone purifying selection and positive selection, respectively. When compared with S. sclerotiorum, fewer secreted effector proteins were found in C. shiraiana. The number of genes involved in pathogenicity, including secondary metabolites, carbohydrate active enzymes, and P450s, in the C. shiraiana genome was comparable with that of other necrotrophs but higher than that of biotrophs and saprotrophs. The growth-related genes and plant cell-wall-degradation-related genes in C. shiraiana were expressed in different developmental and infection stages, and may be potential targets for prevention and control of this pathogen. These results provide new insights into C. shiraiana pathogenic mechanisms, especially host range and necrotrophy features, and lay the foundation for further study of the underlying molecular mechanisms.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

Bioactive phytocompound mulberroside C and endophytes of Morus alba as potential inhibitors of HIV-1 replication: a mechanistic evaluation.

OBJECTIVES: Despite considerable advancement in antiretroviral therapy, development of safe, effective, and multi-targeted drugs for HIV still remains a big challenge. Endophytes are untouched and, hence, an important and novel sources in drug discovery endeavours. The present study was conducted to identify the anti-HIV compounds from Morus alba and endophytes isolated from it. METHODS: The extracts of isolated endophytes were screened using high-performance liquid chromatography (HPLC). Further, all samples were analysed for their cytotoxicity using a thiazolyl blue tetrazolium bromide assay. Subsequently, anti-HIV activity was performed using cell-based and cell-free assay. At the end, potential endophytes were identified using gene sequencing. RESULTS: A total of 27 endophytes were isolated from the eight stem bark samples of M. alba. Of the 27 endophytes, extracts of total of four endophytes showed a profile similar to the M. alba plant when analysed by HPLC. Further experimentation with extracts of these four endophytes, along with an extract of M. alba stem bark and its bioactive molecule, mulberroside C, revealed that all these six samples have good inhibitory potential for HIV. Among them, mulberroside C and two endophytic fungal extracts showed very potent anti-HIV activity. Subsequently, mechanistic studies at the molecular level showed that out of six test samples, three acted as protease inhibitors. Further, all four potential endophytes were identified using gene sequencing. CONCLUSIONS: The overall findings of these studies can help in the development of a novel anti-HIV candidate from mulberroside C, an extract of stem bark of M. alba and extracts of these endophytes. However, further validation and clinical studies are required to develop an anti-HIV drug.

The dynamics in rhizosphere microbial communities under bacterial wilt resistance by mulberry genotypes.

The contribution of crops and soil microbial community structure and functional diversity in soil-borne diseases control mulberry plant production is still inadequately understood. In this work, a comparative study was undertaken on the microbial abundance, community structure, and functional diversity in the soil rhizosphere between the resistant (Kangqing 10) and the susceptible (Guisang 12) mulberry genotypes. The study deployed the use of dilution plate method, micro-ecology technology, and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) techniques. The study aimed at developing better crop management methods for mulberry cultivation as well as preventing and controlling the occurrence and impacts of bacterial wilt on mulberry productivity. The results indicated that the soil rhizosphere microorganisms were more abundant in the normal resistant mulberry genotype than in the normal susceptible mulberry genotype. Carbon source utilization was better in the normal susceptible mulberry genotype. These properties were lower in the sickly resistant mulberry genotype than in the susceptible sickly mulberry genotype. Through the PCR-DGGE, it was shown that the bacterial and fungal community structures of the resistant genotypes were more stable than those of the susceptible genotypes. Through correlation regression analysis, it was shown that the mulberry bacterial wilt significantly contributes to the loss of soil nutrients, particularly organic matter and nitrogen, a possible cause to disrupted balance between the soil microbial community and the loss of soil organic matter. Resistant genotype plants displayed more resistance to bacterial wilt. Therefore, this study recommends the need to promote the cultivation of resistant genotype mulberry for increased yield.

Molecular phylogeny, identification and pathogenicity of Rhizopus oryzae associated with root rot of mulberry in India.

AIMS: Root rot caused by a group of fungi is a serious disease in mulberry. This study aims to identify and characterize Rhizopus oryzae and other fungal species associated with root rot of mulberry in India. METHODS AND RESULTS: Rotted root samples were collected from the mulberry gardens from four states of Southern India. The majority of the isolates identified were R. oryzae, and others were saprophytic fungi, less abundant to occasional. Two methods of inoculations were tested to confirm the pathogenicity of the selected isolates and R. oryzae was found to be pathogenic on susceptible mulberry genotypes RC2 and SRDC-1. Multi gene phylogenetic analyses using the internal transcribed spacer region (ITS), actin (ACT) and translation elongation factor 1-α (TEF), identified the isolates as R. oryzae. Additionally, Ovatospora brasiliensis, Amesia nigricolor, Gongronella butleri, Myrmecridium schulzeri, Scedosporium boydii, Graphium euwallacea, Clonostachys rosea andTalaromyces spp. were also identified. CONCLUSION: This study revealed the existence of eleven species of fungi including the first report of R. oryzae and the occurrence of weak pathogens or saprophytes that are associated with the root rot of mulberry in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of R. oryzae causing Rhizopus rot of mulberry in India. Moreover, the occurrence of saprophytes associated with root rot of mulberry was identified. Further studies should focus more on the ability of these species to generate secondary metabolites and extracellular lytic enzymes as they are beneficial for the management of root rot disease.

Isolation and Characterization of Serratiopeptidase Producing Bacteria from Mulberry Phyllosphere.

Serratiopeptidase (EC 3.4.24.40), a proteolytic enzyme, is one of the most promising enzymes being used in biopharmaceutical industry. Mulberry phyllosphere, being an unexplored niche for exploration of protease production, was chosen for the present study. Protease producing bacteria were isolated from the tissues of mulberry plant as well as its rhizospheric soil. Two protease producing bacteria belonging to Serratia genus were found to be potential serratiopeptidase producers. Among them, the endophyte, i.e., Serratia marcescens MES-4 presented 95 Units/mL activity, while the soil isolate i.e., Serratia marcescens MRS-11 presented 156 Units/mL activity.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 (MuMLPL329) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 (Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants.

Integrated Transcriptomic and Un-Targeted Metabolomics Analysis Reveals Mulberry Fruit (Morus atropurpurea) in Response to Sclerotiniose Pathogen Ciboria shiraiana Infection.

Mulberry sclerotiniose caused by Ciboria shiraiana is a devastating disease of mulberry (Morus alba L.) fruit in Northwest China. At present, no disease-resistant varieties are used in production, as the molecular mechanisms of this disease are not well understood. In this study, to explore new prevention methods and provide direction for molecular breeding, transcriptomic sequencing and un-targeted metabolomics were performed on healthy (CK), early-stage diseased (HB1), and middle-stage diseased (HB2) mulberry fruits. Functional annotation, gene ontology, a Kyoto encyclopedia of genes and genomes (KEGG) analysis, and a Mapman analysis of the differentially expressed genes revealed differential regulation of genes related to plant hormone signal transduction, transcription factors, and phenylpropanoid biosynthesis. A correspondence between the transcript pattern and metabolite profile was observed in the phenylpropanoid biosynthesis pathway. It should be noted that the log2 ratio of eugenol (isoeugenol) in HB1 and HB2 are 85 times and 23 times higher than CK, respectively. Our study shows that phenylpropanoid biosynthesis may play an essential role in response to sclerotiniose pathogen infection and eugenol(isoeugenol) enrichment in mulberry fruit, which may provide a novel method for mulberry sclerotiniose control.

Enhancing laccase production by white-rot fungus trametes hirsuta SSM-3 in co-culture with yeast sporidiobolus pararoseus SSM-8.

Due to wide application of laccase, many researchers have shown great interest in over production of white-rot fungi laccase by co-culture. In this study, a white-rot fungus Trametes hirsuta SSM-3, and a yeast Sporidiobolus pararoseus SSM-8 were isolated and identified from Mulberry fruit. The capacity of S. pararoseus to enhance laccase production was remarkable in T. hirsuta, yielding 31777 ± 742 U/L, about 9.9 times higher than the result from the monoculture. The stimulatory factor in the S. pararoseus cells might be temperature-sensitive. The laccase production was enhanced by oil-extract of S. pararoseus and ß-carotene induction. The amylase activity was decreased rapidly when strain S. pararoseus SSM-8 was inoculated. The glucose deprivation was occurred both in the mono-culture and co-culture process, and S. pararoseus propagated slowly in co-culture all the time. Native-PAGE revealed an increase of laccase-1(lac-1) level and a laccase-3 (lac-3) in the co-culture. Therefore, it was concluded that competition for resources between the co-cultured microbes leaded to amylase decreasing and the enhanced production of laccase. This conclusion was helpful for the development of laccase fermentation industry because it provided an effective, simple and economic method to improve the yield of laccase.

Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose.

Bacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0 × 106 and 1.0 × 105 CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.

Characterization, Phylogenetic Analyses, and Pathogenicity of Colletotrichum Species on Morus alba in Sichuan Province, China.

Brown spot disease caused by Colletotrichum species was found on leaves of mulberry (Morus alba L.) in Dujiangyan, Sichuan Province, China. Fungal isolates from leaf lesions were identified as six Colletotrichum species based on morphological characteristics and DNA analysis of the combined sequences ITS, GAPDH, ACT, CHS-1, TUB2, and GS. These included Colletotrichum fioriniae, C. fructicola, C. cliviae, C. karstii, C. kahawae subsp. ciggaro, and C. brevisporum. Results showed that the most important causal agent of mulberry anthracnose was C. fioriniae, causing typical brown necrotic spots or streaks, followed by C. brevisporum, C. karstii, and C. kahawae subsp. ciggaro, whereas the two other species (C. fructicola and C. cliviae) showed no pathogenicity to mulberry. This study is the first report of these species associated with mulberry in China.

Laccase Gene Sh-lac Is Involved in the Growth and Melanin Biosynthesis of Scleromitrula shiraiana.

Scleromitrula shiraiana causes the popcorn disease in mulberry trees resulting in severe economic losses. Previous studies have shown that melanin may play a vital role in establishing the pathogenicity of fungi. In the present study, we identified the melanin produced in S. shiraiana belongs to DHN melanin by gas chromatography-mass spectrometry, and cloned the laccase Sh-lac, a potential DHN melanin biosynthesis gene from S. shiraiana. We obtained two stable Sh-lac silenced transformants using RNAi, ilac-4 and 8 to elucidate the DHN melanin biosynthetic pathway in S. shiraiana. The melanin production of ilac-4 and ilac-8 was significantly reduced, and their vegetative growth was also suppressed. Results such as these led to a proposal that Sh-lac played a key role in DHN melanin formation in S. shiraiana and may function differentially with other melanin biosynthetic genes. The inhibition of melanin was accompanied by the decrease of oxalic acid and the adhesion of hyphae was impaired. Our results indicated that laccase was an important enzyme in the synthesis of melanin and might play a critical role in the pathogenicity of S. shiraiana.

Mulberry leaf extract fermented with Lactobacillus acidophilus A4 ameliorates 5-fluorouracil-induced intestinal mucositis in rats.

The objective of the present study was to evaluate the effect of mulberry leaf extract (ME) fermented with Lactobacillus acidophilus A4 (A4) on intestinal mucositis induced by 5-fluorouracil (5-FU) in a rat model. Male Wistar rats were gavaged with A4, ME, fermented mulberry leaf extract FME) or lafutidine (LAF) for 10 days and injected intraperitoneally with 5-FU (150 mg kg-1 ) or saline (normal control) on day 7 to induce mucositis. After euthanizing the animals, their small and large intestines were removed for evaluation of histopathologic parameters, myeloperoxidase (MPO) activity, mucin content, and mRNA expression of the mucin gene and pro-inflammatory cytokine interleukin (IL)-1ß. 5-FU induced significant weight loss, shortened villi height, and increased histological severity, IL-1ß expression, and MPO activity compared to the normal control group. These pathological changes were markedly ameliorated by treatment with A4, ME and FME. These treatments also stimulated MUC2 and MUC5AC gene expression and mucin production, and reduced IL-1ß expression and MPO level. Interestingly, FME had the greatest protective effect on 5-FU-induced mucositis in rats. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that fermented mulberry leaf extract (ME) may provide synergistic therapeutic benefits of both probiotics and natural plant extracts in prevention of 5-fluorouracil-induced mucositis. These impacts are particularly significant given the induction of MUC2 and MUC5AC gene expressions for production of mucins and the reduction of pro-inflammatory cytokine interleukin-1ß in gut environments. Therefore, we proposed that enhanced functionality of ME by fermentation of Lactobacillus acidophilus A4 can be applied as food-grade adjuncts for mucositis therapy and prevention in food industry.

[Biological and epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, Ciboria shiraiana].

Objective: We studied the biological and the epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, which is a devastating fungal disease of mulberry. Methods: We studied the asexual and sexual reproductive phase stages of C. shiraiana, including the infection ability of hyphal, dormancy of sclerotia, the structures, release, number and germination of ascospores from apothecia, as well as the phenology of sclerotial germination. Results: In C. shiraiana, hyphae had no infection ability toward the female flowers of mulberry. Sclerotia of C. shiraiana must undergo cold treatment above 6 weeks, then the dormancy-breaking sclerotia could germinate to apothecia. One to fifteen apothecia were germinated from one sclerotium, and the number of ascospores in a 1.5 cm diameter apothecia could contain up to (5.6-6.3)×107. Ascospore C. shiraiana had significantly higher germination rates in acid than in neutral and alkaline environments. From late January to middle April, sclerotia germinated to apothecia, and got the highest value in the middle of March. Conclusion: C. shiraiana is a formidable pathogen to cause epidemic disease and damage in mulberry.