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1 Acta Microbiologica Sinica 2018, 58(6): DOI: /j.cnki.wsxb Insect Microbes and Environmental Management 昆虫微生物与环境治理黑翅土白蚁共生真菌 BYSTW3 的抗菌活性代谢产物王滢 1#, 徐晓 1#, 石靖晶 1, 尹彩萍 1,2, 翁垆霞 1, 郭威 1* 张应烙 1,2* 摘要 : BYSTW3 BYSTW3 ITS BYSTW3 Pleosporales ( ) 100 μg/ml (Valsa mali) (Fusarium oxysporum f. sp. mornordicae) 54.5% 53.4% 3 Physcion (1) FK-17-p2a (2) Citrinin (3) μg/ 3 (Staphylococcus aureus) (Bacillus subtilis) (Escherichia coli) mm BYSTW3 关键词 : [1 2] [3] [4] Paraphaeosphaeria sp. QTYC11 lunatoic acid C [5] [6] 基金项目 : ( ) (LY17C010002) (2017R404021) * 通信作者 Tel weiguo817@zjnu.cn Tel zhangyl@ahau.edu.cn # 收稿日期 : 修回日期 : 网络出版日期 :

2 1142 Ying Wang et al. Acta Microbiologica Sinica, 2018, 58(6) White Strongman 2 [7] Harrington 4 [8] BYSTW3 1 材料和方法 (29 00'17.37"N, '54.84"E) (1) (Staphylococcus aureus ATCC 6538) (Bacillus subtilis, ATCC 6633) (Escherichia coli, ATCC 8739) (2) (Dothiorella gregaria) (Alternaria solani) (Gibberella zeae) (Fusarium oxysporum f. sp. cucumerinum) (F. oxysporum f. sp. mornordicae) (Curvularia lunata) (Valsa mali) (Verticillium dahilae) (3) (Echinochoia crusgalli L.) M7 (MEA ) (NA ) (PDA ) PCR ( BBI ) SW-CJ-1D ( ) DK-8D ( ) DYY-8 ( ) YXJ-2 ( ) H6-1 ( ) ( BBI ) ( ) ( ) ( ) h μl M7 28 C MEA 4 C 1.3 BYSTW BYSTW3 MEA 28 C 3 d d DNA DNA DNA [9] DNA ITS1 (forward): 5 -TCCGTAGGTGAACCTGCGG-3 ITS4 (reverse): 5 -TCCTCCGCTTATTGATATGC-3 actamicro@im.ac.cn

3 , 2018, 58(6) 1143 ITS-rDNA PCR PCR ITS BLAST MEGA 5.0 (Neighbor-joining NJ) 1000 (Bootstrap) Bootstrap 1.4 BYSTW3 BYSTW3 150 ml ME 250 ml 30 C 150 r/min 7 d BYSTW3 ( ) 6 mg/ml 0.22 μm 5 μl 5 mm 28 C 1.6 BTSTW3 1 mg/ml 1 ml 9 ml MEA 100 μg/ml 5 mm d 20 mm (1) =[( )/ ( 5 mm)] 100% (1) 1.7 ( ) ( ) HPLC ( 1 H-NMR 13 C-NMR HR-ESI-MS ) 2 结果和分析 2.1 BYSTW3 1 MEA BYSTW3 25 d mm [10] ITS 521 bp ( MG820090) NCBI BLAST Pleosporales sp. ( HQ914849) 92.6% 20 MEGA 5.0 ( 2) 2012 Zhang

做了比较详细的系统发育分析[10] 我枯草芽孢杆菌大肠杆菌 3 种致病细菌的生长抑制们的目标菌株与已知的孢腔目真菌

粗提物对大肠杆菌和金黄色葡萄球菌均表现出一 BYSTW3 为格孢腔目真菌的潜在新种定的抑菌活性

2 粗提物的抗细菌活性果(抑菌圈直径均>20 mm)相比抑制程度相对较菌株 BYSTW3 的粗提物对金黄色葡萄球菌

BYSTW3 菌株的菌落(A)和分生孢子(400 ) (B) Colony of strain BYSTW3 (A)

4 1144 Ying Wang et al. Acta Microbiologica Sinica, 2018, 58(6) Pleosporales 做了比较详细的系统发育分析[10] 我枯草芽孢杆菌大肠杆菌 3 种致病细菌的生长抑制们的目标菌株与已知的孢腔目真菌 ITS 序列相似活性如图 3 所示当供试浓度为 30 μg/滤纸片时度低因此结合形态学鉴定可初步确定菌株粗提物对大肠杆菌和金黄色葡萄球菌均表现出一 BYSTW3 为格孢腔目真菌的潜在新种定的抑菌活性但与阳性对照硫酸庆大霉素活性效 2.2 粗提物的抗细菌活性果(抑菌圈直径均>20 mm)相比抑制程度相对较菌株 BYSTW3 的粗提物对金黄色葡萄球菌弱粗提物对枯草芽孢杆菌未表现出抑菌活性图 1. Figure 1. Figure 2. BYSTW3 菌株的菌落(A)和分生孢子(400 ) (B) Colony of strain BYSTW3 (A) and The conidium of BYSTW3 (400 ) (B). 图 2. 基于 BYSTW3 菌株 ITS-rDNA 基因序列所构建的系统发育树 System development tree constructed based on BYSTW3 strain ITS-rDNA gene sequence. actamicro@im.ac.cn

5 , 2018, 58(6) BYSTW μg/ml 54.5% 53.4% 4 15% 40% 2.4 BYSTW3 图 3. BYSTW3 粗提物对 3 种致病细菌的生长抑制活性 (30 μg/ 滤纸片 ) Figure 3. Inhibitory activity of crude extracts against three pathogenic bacteria (30 μg/disc). Results are means±sd of three parallel measurements. 3 3 Physcion (1) FK-17-p2a (2) Citrinin (3) ( 4) Phytopathogens 表 1. BYSTW3 粗提物对 8 种植物致病真菌的生长抑制作用 Table 1. Inhibitory activity of extracts against plant pathogens Crude extract Cycloheximide a b a b V. mali 20.0± ± ± ± ±3.2 D. gregaria 28.5± ± ± ± ±2.7 G. zeae 28.3± ± ± ± ±1.6 A. solani 16.8± ± ± ± ±3.1 F. oxysporum f. sp. mornordicae 19.5± ± ± ± ±2.4 F. oxysporum f. sp. cucumerinum 24.2± ± ± ± ±3.1 C. lunata 27.0± ± ± ± ±2.9 V. dahliae 20.2± ± ± ± ±4.6 a means colony average diameter/mm; b means inhibiting rate/%; CK means blank control; the concentration of compounds is 100 µg/ml. CK 图 4. 化合物 1 3 的结构图 Figure 4. Structures of compounds

6 1146 Ying Wang et al. Acta Microbiologica Sinica, 2018, 58(6) 1 HR-ESI-MS [M+H] + m/z C 16 H 13 O C 16 H 12 O H NMR (CDCl 3, 400 MHz) 7.63 (1H, br, s, 4-H), 7.37 (1H, d, J=2.5, 5-H), 7.09 (1H, br, s, 2-H), 6.69 (1H, d, J=2.6, 7-H), 3.94 (3H, s, 16-H), 2.45 (3H, s, 15-H); 13 C NMR (CDCl 3, 150 MHz) (C-9), (C-10), (C-6), (C-8), (C-1), (C-3), (C-14), (C-11), (C-2), (C-4), (C-12), (C-13), (C-5), (C-7), 56.3 (C-(16-OCH 3 ), 22.3 (C-(15-CH 3 )) [11] Physcion 2 HR-ESI-MS [M+H] + m/z C 13 H 17 O 5 m/z C 13 H 16 O H NMR (Actone-d 6, 600 MHz) (1H, s, 1-OH), 9.90 (3H, s, 12-H), 6.42 (1H, s, 4-H), 4.31 (1H, s, 10-H), 4.21 (2H, s, 7-H), 2.72 (2H, m, 9-H), 1.22 (3H, d, J=9.3, 11-H); 13 C NMR (Actone-d 6, 150 MHz) (C-8), (C-12), (C-1), (C-5), (C-3), (C-6), (C-4), (C-2), 64.8 (C-10), 52.2 (C-9), 47.1 (C-7), 23.9 (C-11), 7.3 (C-13) [12] FK-17-p2a 3 HR-ESI-MS [M+H] + m/z C 13 H 15 O 5 m/z C 13 H 14 O H NMR (CDCl 3, 400 MHz) (1H, s, 8-OH), (1H, s, 12-OH), 8.75 (1H, s, 1-H), 4.79 (1H, q, J=6.7, 3-H), 3.00 (1H, q, J=7.1, 4-H), 2.04 (3H, s, 11-H), 1.37 (3H, q, J=6.7, 9-H), 1.25 (3H, q, J=7.2, 10-H); 13 C NMR (CDCl 3, 150 MHz) (C-6), (C-8), (C-12), (C-1), (C-4a), (C-5), (C-8a), (C-7), 81.8 (C-3), 34.8 (C-4), 18.7 (C-9), 18.4 (C-10), 9.6 (C-11) [13] Citrinin mm 24.7 mm 20.6 mm 表 2. BYSTW3 化合物对三种致病细菌的生长抑制活性 (30 μg/ 滤纸片 ) Table 2. Inhibitory activity of compounds against three pathogenic bacteria (30 μg/disc) Compounds Inhibition zone/mm E. coli B. subtilis S. aureus 1 7.3± ± ± ± ± ± ± ±0.7 Gentamycin sulfate 20.7± ± ±0.5 means no inhibition. 3 总结和讨论 BYSTW3 actamicro@im.ac.cn

7 , 2018, 58(6) % 53.4% 3 1 [14] 3 ( ) [15] [1] Czaja K, Góralczyk K, Struciński P, Hernik A, Korcz W, Minorczyk M, Łyczewska M, Ludwicki JK. Biopesticides - towards increased consumer safety in the European Union. Pest Management Science, 2015, 71(1): 3 6. [2] Hunter P. Fight fire with fire: Can biopesticides fill the void left by banning chemical pesticides and herbicides? EMBO Reports, 2009, 10(5): [3] Chen C, Liu W, Shi HP, Wang YY, Xiao R, Yu J, Liu JY. Present status and application perspectives of microbial pesticide in orchards. Chinese Agricultural Science Bulletin, 2015, 31(19): (in Chinese),,,,,,.., 2015, 31(19): [4] Bode HB. Insect-associated microorganisms as a source for novel secondary metabolites with therapeutic potential// Vilcinskas A. Insect Biotechnology. Biologically-Inspired Systems, vol. 2. Dordrecht: Springer, 2011: [5] Shao MW, Kong LC, Jiang DH, Zhang YL. Phytotoxic and antimicrobial metabolites from Paraphaeosphaeria sp. QTYC11 isolated from the gut of Pantala flavescens larvae. Records of Natural Products, 2016, 10(3): [6] Nirma C, Eparvier V, Stien D. Antibacterial ilicicolinic acids C and D and ilicicolinal from Neonectria discophora SNB-CN63 isolated from a termite nest. Journal of Natural Products, 2015, 78(1): [7] White MM, Strongman DB. New species of Spartiella and Legeriosimilis from mayflies and other arthropod-associated trichomycetes from Nova Scotia, Canada. Botany, 2012, 90(11): [8] Harrington TC, Aghayeva DN, Fraedrich SW. New combinations in Raffaelea, Ambrosiella, and Hyalorhinocladiella, and four new species from the redbay ambrosia beetle, Xyleborus glabratus. Mycotaxon, 2010, 111(1): [9] Li S, Shao MW, Lu YH, Kong LC, Jiang DH, Zhang YL. Phytotoxic and antibacterial metabolites from Fusarium proliferatum ZS07 isolated from the gut of long-horned grasshoppers. Journal of Agricultural and Food Chemistry, 2014, 62(36): [10] Zhang Y, Crous PW, Schoch CL, Hyde KD. Pleosporales. Fungal Diversity, 2012, 53(1): [11] Smetanina OF, Kalinovskii AI, Khudyakova YV, Slinkina NN, Pivkin MV, Kuznetsova TA. Metabolites from the marine fungus Eurotium repens. Chemistry of Natural Compounds, 2007, 43(4): [12] Yamaguchi Y, Masuma R, Kim YP, Uchida R, Tomoda H, Omura S. Taxonomy and secondary metabolites of Pseudobotrytis sp. FKA-25. Mycoscience, 2004, 45(1): [13] Marinho AMD, Rodrigues-Fo E. Dicitrinol, a citrinin dimer, produced by Penicillium janthinellum. Helvetica Chimica Acta, 2011, 42(36): [14] Tong Y, Jin Z. Research progress of pharmacological effect of physcion. Chinese Archives of Traditional Chinese Medicine, 2015, 33(4): (in Chinese),.., 2015, 33(4): [15] Reddy CS, Reddy RV, Hayes AW. High-performance liquid chromatographic analysis of the mycotoxin secalonic acid d and its application to biological fluids. Journal of Chromatography A, 1981, 208(1):

8 1148 Ying Wang et al. Acta Microbiologica Sinica, 2018, 58(6) Antimicrobial metabolites of BYSTW3, a symbiotic fungus from Odontotermes formosanus Ying Wang 1#, Xiao Xu 1#, Jingjing Shi 1, Caiping Yin 1,2, Luxia Weng 1, Wei Guo 1*, Yinglao Zhang 1,2* 1 College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua , Zhejiang Province, China 2 School of Life Sciences, Anhui Agricultural University, Hefei , Anhui Province, China Abstract: [Objective] We identified fungus BYSTW3, from Odontotermes formosanus, and studied its active secondary metabolites. [Methods] BYSTW3 was identified by morphological observation and ITS (Internal Transcribed Spacer) sequence analysis. The antibacterial and antifungal activities of crude extracts were tested by filter paper method and growth rate method respectively. The crude extracts were separated and purified by a variety of chromatographic column separation methods, and the structures of the compounds were analyzed with a variety of spectral data. [Results] Strain BYSTW3 was identified as a potential new species of Pleosporales sp. The crude extract of BYSTW3 inhibited radical growth of Valsa mali and Fusarium oxysporum f. sp. mornordicae with the inhibition rates of 54.5% and 53.4% at the concentration of 100 μg/ml. Bioactivity-guided fractionation lead to the isolation of three fungal metabolites 1 3, including physcion (1), FK-17-p2a (2) and citrinin (3). Both compounds 1 and 3 were firstly isolated from the fungus of Pleosporales sp.. At the concentration of 30 μg/disc, compound 3 showed potent antibacterial activity against Staphyloccocus aureus, Bacillus subtilis and Escherichia coli with the mean halo diameter of 23.0 mm, 24.2 mm and 20.7 mm, respectively. [Conclusion] BYSTW3 is a potential new strain with good prospects as a new antimicrobial agents from microbial resources. Keywords: Odontotermes formosanus, symbiotic fungus, Pleosporales sp., metabolites, antimicrobial activity ( 本文责编 : 张晓丽 ) Supported by the National Natural Science Foundation of China ( ), by the Zhejiang Provincial Natural Science Foundation of China (LY17C010002) and by the Projects Funded by Scientific and Technological Innovation Activities for Zhejiang Undergraduate (2017R404021) * Corresponding author. Wei Guo, Tel: , weiguo817@zjnu.cn; Yinglao Zhang, Tel: , zhangyl@ahau.edu.cn #These authors contributed equally to this work. Received: 9 March 2018; Revised: 23 April 2018; Published online: 27 April 2018 张应烙,1976 年 2 月生, 博士, 安徽农业大学皖江学者特聘教授, 博士生导师, 中国昆虫学会昆虫微生物组专业委员会委员主要从事昆虫共生菌及其活性代谢产物研究, 先后主持国家自然科学基金 3 项, 省部级项目 8 项 ; 以第一或通讯作者在国内外刊物上发表论文 40 余篇, 其中 SCI 论文 18 篇, 主要论文发表在 Journal of the American Chemical Society Angewandte Chemie International Edition Bioresource Technology Food Chemistry Journal of Agricultural and Food Chemistry 等主流杂志上 ; 获授权发明专利 9 件 actamicro@im.ac.cn

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇