Biocontrol of Diplodia bulgarica, the causal agent of apple canker, using Trichoderma zelobreve.

Apple (Malus domestica Borkh) is one of the most consumed and nutritious fruits. Iran is one of the main producers of the apple in the world. Diplodia bulgarica is the major causal agent of apple tree decline in Iran. Biological control is a nature-friendly approach to plant disease management. Trichoderma zelobreve was isolated from apple trees infected with Diplodia bulgarica in West Azarbaijan province of Iran. The results showed that T. zelobreve strongly inhibited the colony growth of D. bulgarica. In vivo assay on detached branches of apple tree cv. Golden Delicious using T. zelobreve mycelial plug showed that canker length/stem length (CL/SL) and canker perimeter/stem perimeter (CP/SP) indices decreased by 76 and 69%, respectively, 21 days after inoculation. Additionally, wettable powder formulation (WPF) containing the antagonistic fungus "T. zelobreve" decreased CL and CP/SP by 75 and 67%, respectively, 6 months after inoculation. Moreover, canker progress curves and the area under the disease progress curve (AUDPC) supported these findings. The growth temperatures of the antagonist and pathogen were similar, indicating the adaptation of T. zelobreve for biocontrol of apple canker caused by D. bulgarica. The results also showed that T. zelobreve-based WPF stored at 25 °C assure excellent shelf life at least 4 months, allowing the bioproduct to be stored at room temperature, which is a great advantage and cost-effective option.

Serendipita indica Drives Sulfur-Related Microbiota in Enhancing Growth of Hyperaccumulator Sedum alfredii and Facilitating Soil Cadmium Remediation.

Endophytic fungus Serendipita indica can bolster plant growth and confer protection against various biotic and abiotic stresses. However, S. indica-reshaped rhizosphere microecology interactions and root-soil interface processes in situ at the submicrometer scale remain poorly understood. We combined amplicon sequencing and high-resolution nano X-ray fluorescence (nano-XRF) imaging of the root-soil interface to reveal cadmium (Cd) rhizosphere processes. S. indica can successfully colonize the roots of Sedum alfredii Hance, which induces a remarkable increase in shoot biomass by 211.32% and Cd accumulation by 235.72%. Nano-XRF images showed that S. indica colonization altered the Cd distribution in the rhizosphere and facilitated the proximity of more Cd and sulfur (S) to enter the roots and transport to the shoot. Furthermore, the rhizosphere-enriched microbiota demonstrated a more stable network structure after the S. indica inoculation. Keystone species were strongly associated with growth promotion and Cd absorption. For example, Comamonadaceae are closely related to the organic acid cycle and S bioavailability, which could facilitate Cd and S accumulation in plants. Meanwhile, Sphingomonadaceae could release auxin and boost plant biomass. In summary, we construct a mutualism system for beneficial fungi and hyperaccumulation plants, which facilitates high-efficient remediation of Cd-contaminated soils by restructuring the rhizosphere microbiota.

Aspertaichamide B, a new anti-tumor prenylated indole alkaloid from the fungus Aspergillus japonicus TE-739D.

Prenylated indole alkaloids, which are mainly produced by genera Aspergillus and Penicillium, are a class of structurally intriguing specialized metabolites with remarkable biomedical interests. In this study, chemically guided isolation of the Nicotiana tabacum-derived endophytic fungus Aspergillus japonicus TE-739D yielded eight structurally diverse prenylated indole alkaloids, including an undescribed compound, namely aspertaichamide B (ATB, 1), together with seven previously discovered derivatives (compounds 2 - 8). Their chemical structures as well as the stereochemical features were determined by integrated spectroscopic analyses, including HRESIMS, NMR, NMR calculations with DP4 + probability analysis, and a comparison of the experimental ECD data with computed DFT-based quantum chemical calculations. In vitro cytotoxic effects against the gastric cancer MFC cells revealed that the new compound ATB demonstrated considerable activity. Further studies found that ATB suppressed the viability, colony formation, and migration ability of MFC cells, and induced MFC cells apoptosis in a concentration-dependent way. Moreover, ATB stimulated ROS production in MFC cells and inhibited the tumor growth in the MFC-sourced subcutaneous tumor model while not significantly reducing the weight of mice. The pharmacological results suggested that the newly discovered ATB may be a promising anti-tumor lead compound. KEY POINTS: • Eight structurally diverse prenylated indole alkaloids including a new aspertaichamide B (ATB) were isolated from the fungus Aspergillus japonicus TE-739D. • The structure of ATB was elucidated by HRESIMS, NMR, NMR calculations with DP4 + probability analysis, and ECD calculations. • ATB inhibited cell proliferation, promoted apoptosis, and increased ROS production in gastric cancer cells, and exhibited inhibitory effects on tumor growth in vivo.

Nutrient and mycoremediation of a global menace 'arsenic': exploring the prospects of phosphorus and Serendipita indica-based mitigation strategies in rice and other crops.

KEY MESSAGE: Serendipita indica induced metabolic reprogramming in colonized plants complements phosphorus-management in improving their tolerance to arsenic stress on multifaceted biological fronts. Restoration of the anthropic damage done to our environment is inextricably linked to devising strategies that are not only economically sound but are self-renewing and ecologically conscious. The dilemma of heavy metal (HM) dietary ingestion, especially arsenic (As), faced by humans and animals alike, necessitates the exploitation of such technologies and the cultivation of healthy and abundant crops. The remarkable symbiotic alliance between plants and 'mycorrhizas' has evolved across eons, benefiting growth/yield aspects as well as imparting abiotic/biotic stress tolerance. The intricate interdependence of Serendipita indica (S. indica) and rice plant reportedly reduce As accumulation, accentuating the interest of microbiologists, agriculturists, and ecotoxicological scientists apropos of the remediation mechanisms of As in the soil-AMF-rice system. Nutrient management, particularly of phosphorus (P), is also praised for mitigating As phytotoxicity by deterring the uptake of As molecules due to the rhizospheric cationic competition. Taking into consideration the reasonable prospects of success in minimizing As acquisition by rice plants, this review focuses on the physiological, metabolic, and transcriptional alterations underlying S. indica symbiosis, recuperation of As stress together with nutritional management of P by gathering case studies and presenting successful paradigms. Weaving together a volume of literature, we assess the chemical forms of As and related transport pathways, discuss As-P-rice interaction and the significance of fungi in As toxicity mitigation, predominantly the role of mycorrhiza, as well as survey of the multifaceted impacts of S. indica on plants. A potential strategy for simultaneous S. indica + P administration in paddy fields is proposed, followed by future research orientation to expand theoretic comprehension and encourage field-based implementation.

New Naphthalene Derivatives from the Mangrove Endophytic Fungus Daldinia eschscholzii MCZ-18.

Five new naphthalene derivatives dalesconosides A-D, F (1-4, 6), a known synthetic analogue named dalesconoside E (5), and eighteen known compounds (7-24) were isolated from Daldinia eschscholzii MCZ-18, which is an endophytic fungus obtained from the Chinese mangrove plant Ceriops tagal. Differing from previously reported naphthalenes, compounds 1 and 2 were bearing a rare ribofuranoside substituted at C-1 and the 5-methyltetrahydrofuran-2,3-diol moiety, respectively. Their structures were determined by detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses, while the absolute configurations were established by theoretical electronic circular dichroism (ECD) calculation. Compounds 1, 3, 13-17 and 19 showed broad ranges of antimicrobial spectrum against five indicator test microorganisms (Enterococcus faecalis, Methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans); especially, 1, 16 and 17 were most potent. The variations in structure and attendant biological activities provided fresh insights concerning structure-activity relationships for the naphthalene derivatives.

Diisoprenyl Cyclohexene-Type Meroterpenoids with Cytotoxic Activity from a Mangrove Endophytic Fungus Aspergillus sp. GXNU-Y85.

Five new diisoprenyl cyclohexene-type meroterpenoids, aspergienynes J-N (1-5), along with three known analogues (6-8), were obtained from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y85. The chemical structures, including their absolute configurations, were established via spectroscopic data and comparison of experimental and calculated ECD spectra. Cytotoxicity assay results indicated that compound 8 had strong cytotoxicity against HeLa cancer cells, and its IC50 value was 11.8 µM. In addition, flow cytometry analysis revealed that the cytotoxicity of 8 was due to the induction of G1 cell cycle arrest and apoptosis in HeLa cells.

An Investigation of Quorum Sensing Inhibitors against Bacillus cereus in The Endophytic Fungus Pithomyces sacchari of the Laurencia sp.

Bacillus cereus, a common food-borne pathogen, forms biofilms and generates virulence factors through a quorum sensing (QS) mechanism. In this study, six compounds (dankasterone A, demethylincisterol A3, zinnimidine, cyclo-(L-Val-L-Pro), cyclo-(L-Ile-L-Pro), and cyclo-(L-Leu-L-Pro)) were isolated from the endophytic fungus Pithomyces sacchari of the Laurencia sp. in the South China Sea. Among them, demethylincisterol A3, a sterol derivative, exhibited strong QS inhibitory activity against B. cereus. The QS inhibitory activity of demethylincisterol A3 was evaluated through experiments. The minimum inhibitory concentration (MIC) of demethylincisterol A3 against B. cereus was 6.25 µg/mL. At sub-MIC concentrations, it significantly decreased biofilm formation, hindered mobility, and diminished the production of protease and hemolysin activity. Moreover, RT-qPCR results demonstrated that demethylincisterol A3 markedly inhibited the expression of QS-related genes (plcR and papR) in B. cereus. The exposure to demethylincisterol A3 resulted in the downregulation of genes (comER, tasA, rpoN, sinR, codY, nheA, hblD, and cytK) associated with biofilm formation, mobility, and virulence factors. Hence, demethylincisterol A3 is a potentially effective compound in the pipeline of innovative antimicrobial therapies.

Dicerandrol C Suppresses Proliferation and Induces Apoptosis of HepG2 and Hela Cancer Cells by Inhibiting Wnt/ß-Catenin Signaling Pathway.

Overwhelming evidence points to an aberrant Wnt/ß-catenin signaling as a critical factor in hepatocellular carcinoma (HCC) and cervical cancer (CC) pathogenesis. Dicerandrol C (DD-9), a dimeric tetrahydroxanthenone isolated from the endophytic fungus Phomopsis asparagi DHS-48 obtained from mangrove plant Rhizophora mangle via chemical epigenetic manipulation of the culture, has demonstrated effective anti-tumor properties, with an obscure action mechanism. The objective of the current study was to explore the efficacy of DD-9 on HepG2 and HeLa cancer cells and its functional mechanism amid the Wnt/ß catenin signaling cascade. Isolation of DD-9 was carried out using various column chromatographic methods, and its structure was elucidated with 1D NMR. The cytotoxicity of DD-9 on HepG2 and HeLa cells was observed with respect to the proliferation, clonality, migration, invasion, apoptosis, cell cycle, and Wnt/ß-catenin signaling cascade. We found that DD-9 treatment significantly reduced tumor cell proliferation in dose- and time-dependent manners in HepG2 and HeLa cells. The subsequent experiments in vitro implied that DD-63 could significantly suppress the tumor clonality, metastases, and induced apoptosis, and that it arrested the cell cycle at the G0/G1 phase of HepG2 and HeLa cells. Dual luciferase assay, Western blot, and immunofluorescence assay showed that DD-9 could dose-dependently attenuate the Wnt/ß-catenin signaling by inhibiting ß-catenin transcriptional activity and abrogating ß-catenin translocated to the nucleus; down-regulating the transcription level of ß-catenin-stimulated Wnt target gene and the expression of related proteins including p-GSK3-ß, ß-catenin, LEF1, Axin1, c-Myc, and CyclinD1; and up-regulating GSK3-ß expression, which indicates that DD-9 stabilized the ß-catenin degradation complex, thereby inducing ß-catenin degradation and inactivation of the Wnt/ß-catenin pathway. The possible interaction between DD-9 and ß-catenin and GSK3-ß protein was further confirmed by molecular docking studies. Collectively, DD-9 may suppress proliferation and induce apoptosis of liver and cervical cancer cells, possibly at least in part via GSK3-ß-mediated crosstalk with the Wnt/ß-catenin signaling axis, providing insights into the mechanism for the potency of DD-9 on hepatocellular and cervical cancer.

The Cytochalasins and Polyketides from a Mangrove Endophytic Fungus Xylaria arbuscula QYF.

Twelve compounds, including four undescribed cytochalasins, xylariachalasins A-D (1-4), four undescribed polyketides (5-8), and four known cytochalasins (9-12), were isolated from the mangrove endophytic fungus Xylaria arbuscula QYF. Their structures and absolute configurations were established by extensive spectroscopic analyses (1D and 2D NMR, HRESIMS), electronic circular dichroism (ECD) calculations, 13C NMR calculation and DP4+ analysis, single-crystal X-ray diffraction, and the modified Mosher ester method. Compounds 1 and 2 are rare cytochalasin hydroperoxides. In bioactivity assays, Compound 2 exhibited moderate antimicrobial activities against Staphylococcus aureus and Candida albicans with MIC values of 12.5 µM for both Compound 10 exhibited significant cytotoxic activity against MDA-MB-435 with an IC50 value of 3.61 ± 1.60 µM.

Diaporchalasins A-E, New Cytochalasins from the Endophytic Fungus Diaporthe sp. BMX12 Isolated from Aquilaria sinensis.

Five new cytochalasins, diaporchalasins A-E (1-5), together with 14 known congeners (6-19) were isolated from the endophytic fungus Diaporthe sp. BMX12, which was isolated from the branches of Aquilaria sinensis. The structures of the new compounds were elucidated by extensive spectroscopic analyses including high-resolution electron spray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR). Their absolute configurations were assigned by theoretical electronic circular dichroism (ECD) calculations. Compounds 11 and 12 featuring a keto carbonyl at C-21 displayed cytotoxicity toward K562, BEL-7402, SGC-7901, A549, and HeLa cell lines with IC50 values ranging from 4.4 to 47.4 µM.

Graphiumisides A-D, Monoterpene Glycosides from an Animal-Derived Endophytic Fungus Graphium sp. GD-11.

Four new monoterpene rhamnosides, graphiumisides A-D (1-4), along with four known steroid compounds (5-8) were isolated from the fermentation extract of animal-derived endophytic fungus, Graphium sp. GD-11. The chemical structures of all compounds were elucidated using 1D and 2D NMR, HRESIMS spectroscopic analyses, and other spectroscopic methods. Compounds 1-4 exhibit a distinctive structure connected by one p-menthane type monoterpene and one L-rhamnose. This is the first report of monoterpene glycosides from Graphium sp. All compounds (1-8) were tested for cytotoxic activities against four cancer cell lines (HepG2, SMMC7721, SW480, and A549), and only compound 1 showed weak anti-tumor activity against SMMC7721 cells.

Anti-inflammatory Polyketides from an Endophytic Fungus Chaetomium sp. UJN-EF006 of Vaccinium bracteatum.

Seven new polyketides including three chromone derivatives (1-3) and four linear ones incorporating a tetrahydrofuran ring (4-7), along with three known compounds (8-10), were obtained from the fermentation of an endophytic fungus (Chaetomium sp. UJN-EF006) isolated from the leaves of Vaccinium bracteatum. The structures of these fungal metabolites have been elucidated by spectroscopic means including MS, NMR and electronic circular dichroism. A preliminary anti-inflammatory screening with the lipopolysaccharide (LPS) induced RAW264.7 cell model revealed moderate NO production inhibitory activity for compounds 1 and 4. In addition, the expression of three LPS-induced inflammatory factors IL-6, iNOS and COX-2 was also blocked by 1 and 4.

Secondary metabolites from the endophytic fungus Letendraea helminthicola A820 with Anti-inflammatory activity.

Two undescribed letendrones A-B (1-2), along with three known compounds, ZL-6 (3), dankasterone B (4) and minimoidione B (5) were isolated from the Aquilaria-derived fungus Letendraea helminthicola A820. The structures of 1 and 2 were established by analysis of spectroscopes including 1D/2D NMR, IR, UV, and HRESIMS. Among them, the configuration of 1 was further confirmed by single-crystal X-ray diffraction. Letendrones A and B were the new phenalenyl derivatives with radical form that were firstly found in nature. In addition, bioactivity of these compounds was evaluated and compounds 3-5 exhibited inhibitory activity against LPS-induced NO production in macrophages with IC50 values of 4.64, 13.90, and 34.07 µM. Furthermore, potential targets of the new compounds were analyzed by molecular docking in silico. As a result, compound 1 showed high binding with predicted 5-HT2c receptor (∆G=-8.2 kcal/mol) potentially associated with depression disease, and compound 2 showed significant connection with phosphodiesterase 3A (∆G=-9.4 kcal/mol) probably against cardiovascular disorders. Our findings firstly reported the high symmetry phenalenyl compounds from natural products which would provide a basis for further development and utilization of the secondary metabolites from the endophytic fungus Letendraea helminthicola A820.

A new resorcylic acid lactone from the endophytic fungus Chaetosphaeronema sp.

A new 14-membered resorcylic acid lactone (RAL14), chaetolactone A (1), along with three known ones (2-4), was obtained from the fermentation of the soil-derived fungus Chaetosphaeronema sp. SSJZ001. Their structures were established based on extensive spectroscopic data analyses (UV, IR, HRESIMS, 1D, and 2D NMR),13C NMR chemical shifts calculations coupled with the DP4+ probability method, theoretical calculations of ECD spectra, as well as X-ray diffraction analysis. All compounds were evaluated for their cytotoxic effects against A549, HO-8910, and MCF-7 cell lines.

Talaroacids A-D and Talaromarane A, Diterpenoids with Anti-Inflammatory Activities from Mangrove Endophytic Fungus Talaromyces sp. JNQQJ-4.

Five new diterpenes including four diterpenes with 1,2,3,4,4a,5,6,8a-octalin skeleton talaroacids A-D (1-4) and an isopimarane diterpenoid talaromarane A (5) were isolated from the mangrove endophytic fungus Talaromyces sp. JNQQJ-4. Their structures and absolute configurations were determined by analysis of high-resolution electrospray ionization mass spectroscopy (HRESIMS), 1D/2D Nuclear Magnetic Resonance (NMR) spectra, single-crystal X-ray diffraction, quantum chemical calculation, and electronic circular dichroism (ECD). Talaromarane A (5) contains a rare 2-oxabicyclo [3.2.1] octan moiety in isopimarane diterpenoids. In bioassays, compounds 1, 2, 4, and 5 displayed significant anti-inflammatory activities with the IC50 value from 4.59 to 21.60 µM.

In Vitro Analysis of the Antagonistic Biological and Chemical Interactions between the Endophyte Sordaria tomento-alba and the Phytopathogen Botrytis cinerea.

Plant pathogenic infections causing substantial global food losses are a persistent challenge. This study investigates a potential biocontrol strategy against the necrotrophic fungus Botrytis cinerea using the endophytic fungus Sordaria tomento-alba isolated from Gliricidia sepium in Colombia. Today, synthetic fungicides dominate B. cinerea control, raising environmental and health concerns. S. tomento-alba exhibits notable in vitro effects, inhibiting B. cinerea growth by approximately 60% during co-culture and 50% in double disc co-culture. Additionally, it suppresses botryanes production and produces the compound heptacyclosordariolone, which has proven effective in inhibiting B. cinerea mycelial growth and spore germination in vitro. This biocontrol agent could be a potential eco-friendly alternative to replace synthetic fungicides. Our study provides insights into the chemical and biological mechanisms underpinning the antagonistic activity of S. tomento-alba, emphasizing the need for further research to understand its biosynthesis pathways and optimize its biocontrol potential. It also contributes molecular evidence of fungal interactions with implications for advanced forums in molecular studies in biology and chemistry, particularly in addressing plant pathogenic infections and promoting sustainable agriculture.

Bioactive α-Pyrone Analogs from the Endophytic Fungus Diaporthe sp. CB10100: α-Glucosidase Inhibitory Activity, Molecular Docking, and Molecular Dynamics Studies.

Two α-pyrone analogs were isolated from the endophytic fungus Diaporthe sp. CB10100, which is derived from the medicinal plant Sinomenium acutum. These analogs included a new compound, diaporpyrone F (3), and a known compound, diaporpyrone D (4). The structure of 3 was identified by a comprehensive examination of HRESIMS, 1D and 2D NMR spectroscopic data. Bioinformatics analysis revealed that biosynthetic gene clusters for α-pyrone analogs are common in fungi of Diaporthe species. The in vitro α-glucosidase inhibitory activity and antibacterial assay of 4 revealed that it has a 46.40% inhibitory effect on α-glucosidase at 800 µM, while no antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Mycolicibacterium (Mycobacterium) smegmatis or Klebsiella pneumoniae at 64 µg/mL. Molecular docking and molecular dynamics simulations of 4 with α-glucosidase further suggested that the compounds are potential α-glucosidase inhibitors. Therefore, α-pyrone analogs can be used as lead compounds for α-glucosidase inhibitors in more in-depth studies.

Anti-Inflammatory Properties of Oxygenated Isocoumarins and Xanthone from Thai Mangrove-Associated Endophytic Fungus Setosphaeria rostrata.

Chronic inflammation plays a crucial role in the development and progression of numerous chronic diseases. To search for anti-inflammatory metabolites from endophytic fungi isolated from plants growing in Thai mangrove areas, a chemical investigation of those fungi was performed. Five new oxygenated isocoumarins, setosphamarins A-E (1-5) were isolated from the EtOAc extract of an endophytic fungus Setosphaeria rostrata, along with four known isocoumarins and one xanthone. Their structures were determined by extensive spectroscopic analysis. The absolute configurations of the undescribed compounds were established by comparative analysis between experimental and calculated circular dichroism (ECD) spectroscopy. All the compounds were evaluated for their anti-inflammatory activity by monitoring nitric oxide inhibition in lipopolysaccharide-induced macrophage J774A.1 cells. Only a xanthone, ravenelin (9), showed potent activity, with an IC50 value of 6.27 µM, and detailed mechanistic study showed that it suppressed iNOS and COX-2 expression.

The effect of cadmium on soil and plants, and the influence of Serendipita indica (Piriformospora indica) in mitigating cadmium stress.

Due to environmental pollution, the risk of cadmium stress for crops is soaring, so researchers are exploring inexpensive solutions to enhance cultivated crops in contaminated soil. Using microorganisms to reduce cadmium risk has been one of the most effective strategies in recent decades. Serendipita indica (Piriformospora indica) is one of the best endophyte fungi that, in addition to reducing heavy metal stress for crops, can significantly reduce the threat of other abiotic stresses. As part of this research, cadmium in soil has been investigated, as well as its effects on plants' morphophysiological and biochemical characteristics. The present review has also attempted to identify the role of Serendipita indica in improving the growth and performance of crops, as well as its possible effect on reducing the risk of cadmium. The results showed that Serendipita indica enhance the growth and productivity of plants in contaminated environments by improving soil quality, reducing cadmium absorption, improving the activity of antioxidant enzymes and secondary metabolites, raising water and mineral absorption, and altering morphophysiological structures.

Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.