Efficacy and Its Mechanism of Bacterial Strain HX0037 on the Control of Anthracnose Disease of Trichosanthes kirilowii Maxim

doi:10.13560/j.cnki.biotech.bull.1985.2023-1172

Abstract

Abstract:

【Objective】The biocontrol ability of bacterium HX0037 against anthracnose disease of Trichosanthes kirilowii Maxim and its biocontrol mechanism were studied. 【Method】The antagonistic activities of HX0037 against four pathogens of fruit anthracnose disease were determined in petri dishes experiments. The biocontrol efficiencies of HX0037 against Trichosanthes leaf anthracnose and Trichosanthes fruit anthracnose were evaluated on detached leaves and fruits in vitro. In addition, HX0037 was further identified based on the analyses on morphological features, physiological and biochemical characteristics and molecular biological identification results of the whole genome, and the secondary metabolites of biocontrol bacteria HX0037 were also predicted. The lipopeptide compounds produced by strain HX0037 were extracted by acid precipitation and analyzed by high-performance liquid chromatography tandem mass spectrometry（LC-MS）. Finally, the possible biocontrol mechanism was explored through two experiments, observing the effects of lipopeptides produced by strain HX0037 on the hyphal growth and morphology of Colletotrichum gloeosporioides, and the abilities of strain HX0037 to produce enzyme and siderophore. 【Result】Strain HX0037 showed varied antimicrobial activity against four pathogens of fruit anthracnose disease, especially the best against C. gloeosporioides, a pathogen of Trichosanthes anthracnose disease, and it obviously changed the cell membrane permeability of the pathogenic fungi. Compared with plant tissues inoculated with the pathogen alone, inoculation with strain HX0037 significantly reduced the incidence of leaf and fruit anthracnose diseases of T. kirilowii Maxim, and the biocontrol efficacy was 61.50% and 52.51%, respectively. Strain HX0037 was further identified as Bacillus amyloliquefaciens, and its genome contained 12 secondary metabolite gene clusters, and it decomposed the protein, cellulose and starch, and also produced siderophore. The hyphae of C. gloeosporioides were distorted, intumescent, broken and more transparent after treatment with the antifungal lipopeptide produced by strain HX0037. Surfactin and fengycin were detected from the lipopeptide extracts of strain HX0037 by LC-MS. 【Conclusion】The B. amyloliquefaciens HX0037 has strong antagonistic activity on C. gloeosporioides, and is expected to be further developed as a biological agent for the biocontrol of Trichosanthes anthracnose diseases.

Key words: Bacillus amyloliquefaciens, biological control, Trichosanthes anthracnose disease, antagonism, whole genome, species identification, lipopeptide

XU Wei-fang, LI He-yu, ZHANG Hui, HE Zi-ang, GAO Wen-heng, XIE Zi-yang, WANG Chuan-wen, YIN Deng-ke. Efficacy and Its Mechanism of Bacterial Strain HX0037 on the Control of Anthracnose Disease of Trichosanthes kirilowii Maxim[J]. Biotechnology Bulletin, 2024, 40(4): 228-241.

Figures/Tables 15

References 57

[1]	韩慧莹, 刘桂荣. 张志远临证运用瓜蒌经验[J]. 中医杂志, 2021, 62(1): 16-18.
	Han HY, Liu GR. ZHANG Zhiyuan's experience in using Gualou(fructus trichosanthis)[J]. China J Chin Mater Med, 2021, 62(1): 16-18.
[2]	张琪, 彭向前. 栝楼的活性成分及其药理作用的研究进展[J]. 山东化工, 2021, 50(14): 98-100.
	Zhang Q, Peng XQ. Research progress on active components and pharmacological effects of Trichosanthes kirilowii maxim[J]. Shandong Chem Ind, 2021, 50(14): 98-100.
[3]	葛江洪. 潜山市瓜蒌产业发展的优势与建议[J]. 安徽农业科学, 2019, 47(16): 238-240.
	Ge JH. Advantages and suggestions on the development of Trichosanthes kirilowii maxim industry in Qianshan city[J]. J Anhui Agric Sci, 2019, 47(16): 238-240.
[4]	Zhang LX, Lin YF, Zhang L, et al. First report of anthracnose caused by Colletotrichum liaoningense on Trichosanthes kirilowii in China[J]. Plant Dis, 2022: PDIS07211363PDN.
[5]	Zhang LX, Song JH, Tan GJ, et al. Characterization of Colletotrichum gloeosporioides responsible for anthracnose disease of Trichosanthes kirilowii Maxim in central China[J]. Phytoparasitica, 2014, 42(4): 549-558. doi: 10.1007/s12600-014-0393-6 URL
[6]	汪霞, 林一帆, 张立新. 栝楼主要病害及综合防治技术[J]. 特种经济动植物, 2021, 24(8): 40-41.
	Wang X, Lin YF, Zhang LX. Main diseases of Trichosanthes kirilowii and their integrated control techniques[J]. Spec Econ Anim Plants, 2021, 24(8): 40-41.
[7]	林一帆, 李相汉, 李华, 等. 栝楼新品种‘皖蒌15号’[J]. 园艺学报, 2020, 47(S2): 3146-3147.
	Lin YF, Li XH, Li H, et al. A new Trichosanthes kirilowii variety ‘Wanlou No.15’[J]. Acta Hortic Sin, 2020, 47(S2): 3146-3147.
[8]	李萍, 吴成方, 刘冬, 等. 安庆市瓜蒌用药现状及瓜蒌炭疽病菌室内药剂筛选试验[J]. 红河学院学报, 2019, 17(5): 127-129.
	Li P, Wu CF, Liu D, et al. Current status of fungicides use for Trichosanthes kirilowii in Anqing city and toxicity test of fungicides to colletootrichum orbiculare[J]. J Honghe Univ, 2019, 17(5): 127-129.
[9]	胡显海, 周晓飞. 栝楼炭疽病的发生规律及防治方法[J]. 现代农业科技, 2006(11): 84-85.
	Hu XH, Zhou XF. Occurrence regularity and control methods of Trichosanthes kirilowii anthracnose[J]. Mod Agric Sci Technol, 2006(11): 84-85.
[10]	徐劲峰, 檀根甲, 韩翔. 瓜蒌炭疽病发生危害及综合控制技术研究[J]. 安徽农业科学, 2006, 34(15): 3741, 3784.
	Xu JF, Tan GJ, Han X. Study on the occurrence and harm of Trichosanthes kirilowii anthracnose and its comprehensive control technology[J]. J Anhui Agric Sci, 2006, 34(15): 3741, 3784.
[11]	韩翔. 瓜蒌炭疽菌的生理生态及病害防治的研究[D]. 合肥: 安徽农业大学, 2004.
	Han X. Studies on physiological and ecological characteristics of Mongolian snakegourd anthracnose pathogen and disease control[D]. Hefei: Anhui Agricultural University, 2004.
[12]	王世伟, 王卿惠. 解淀粉芽孢杆菌相关功能机制研究进展[J]. 生物技术通报, 2020, 36(1): 150-159. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0786
	Wang SW, Wang QH. Research advances in functional mechanisms of Bacillus amyloliquefacien[J]. Biotechnol Bull, 2020, 36(1): 150-159.
[13]	Zouari I, Jlaiel L, Tounsi S, et al. Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds[J]. Biol Contr, 2016, 100: 54-62. doi: 10.1016/j.biocontrol.2016.05.012 URL
[14]	姚佳明, 田亚平. 解淀粉芽孢杆菌抑菌肽的分离鉴定及其抑菌谱表征[J]. 食品科学, 2020, 41(16): 126-131.
	Yao JM, Tian YP. Isolation and identification of antimicrobial peptides produced by Bacillus amyloliquefaciens and characterization of their antibacterial spectra[J]. Food Sci, 2020, 41(16): 126-131.
[15]	Arrebola E, Jacobs R, Korsten L. Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens[J]. J Appl Microbiol, 2010, 108(2): 386-395. doi: 10.1111/j.1365-2672.2009.04438.x pmid: 19674188
[16]	Wong JH, Hao J, Cao Z, et al. An antifungal protein from Bacillus amyloliquefaciens[J]. J Appl Microbiol, 2008, 105(6): 1888-1898. doi: 10.1111/j.1365-2672.2008.03917.x pmid: 19120637
[17]	An JY, Zhu WJ, Liu Y, et al. Purification and characterization of a novel bacteriocin CAMT2 produced by Bacillus amyloliquefaciens isolated from marine fish Epinephelus areolatus[J]. Food Contr, 2015, 51: 278-282. doi: 10.1016/j.foodcont.2014.11.038 URL
[18]	刘锋, 何群, 陈兴帮, 等. 贝莱斯芽孢杆菌ES2-4的生防潜力及全基因组分析[J]. 应用与环境生物学报, 2023, 29(6): 1459-1466.
	Liu F, He Q, Chen XB, et al. Biocontrol potential and complete genome analysis of Bacillus velezensis ES2-4[J]. Chinese Journal of Applied and Environmental Biology, 2023, 29(6): 1459-1466.
[19]	Xu WF, Sun T, Du JH, et al. Structure and ecological function of the soil microbiome associated with ‘Sanghuang’ mushrooms suffering from fungal diseases[J]. BMC Microbiol, 2023, 23(1): 218.
[20]	张紊玮, 王艳玲, 毕阳, 等. 一株马铃薯干腐病拮抗菌的筛选、鉴定及其生物防效[J]. 微生物学通报, 2018, 45(8): 1726-1736.
	Zhang WW, Wang YL, Bi Y, et al. Screening and identification of an antagonistic strain against potato dry rot[J]. Microbiol China, 2018, 45(8): 1726-1736.
[21]	魏丽梅, 邹小文, 徐婷璐, 等. 农抗211对水稻纹枯病菌细胞膜和抗氧化酶活性的影响[J]. 核农学报, 2021, 35(5): 1084-1090. doi: 10.11869/j.issn.100-8551.2021.05.1084
	Wei LM, Zou XW, Xu TL, et al. Effects of antifungalmycin 211 on cell membrane and antioxidant enzyme activity of Rhizoctonia solani[J]. J Nucl Agric Sci, 2021, 35(5): 1084-1090.
[22]	王若琳, 徐伟芳, 王飞, 等. 桑树内生拮抗菌的分离鉴定及其对桑断枝烂叶病的生防初探[J]. 微生物学报, 2019, 59(11): 2130-2143.
	Wang RL, Xu WF, Wang F, et al. Isolation and identification of an antagonistic endophytic bacterium from mulberry for biocontrol against Boeremia exigua[J]. Acta Microbiol Sin, 2019, 59(11): 2130-2143.
[23]	陈帅康, 王彩霞, 施万斌, 等. 嗜果刀孢菌寄主范围的测定及PCR快速检测[J]. 林业科学研究, 2023, 36(6): 162-171.
	Chen SK, Wang CX, Shi WB, et al. Host determination of the Wil-sonomyces carpophilus of wild apricot forest of Tianshan and establishment of a rapid detection system[J]. For Res, 2023, 36(6): 162-171.
[24]	章乐乐, 王冠, 柳凤, 等. 芒果炭疽病拮抗菌分离、鉴定及生防机制研究[J]. 生物技术通报, 2023, 39(4): 277-287. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0908
	Zhang LL, Wang G, Liu F, et al. Isolation, identification and biocontrol mechanism of antagonistic bacterium against anthracnose on mango caused by Colletotrichum gloeosporioides[J]. Biotechnol Bull, 2023, 39(4): 277-287.
[25]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001: 296.
	Dong XZ, Cai MY. Handbook of identification of common bacterial systems[M]. Beijing: Science Press, 2001: 296.
[26]	Bergey DH, Holt JG. Bergey's Manual of determinative bacteriology[M]. 9th ed. Baltimore: Williams & Wilkins, 1994.
[27]	Xu WF, Ren HS, Ou T, et al. Genomic and functional characterization of the endophytic Bacillus subtilis 7PJ-16 strain, a potential biocontrol agent of mulberry fruit sclerotiniose[J]. Microb Ecol, 2019, 77(3): 651-663. doi: 10.1007/s00248-018-1247-4
[28]	Medema MH, Blin K, Cimermancic P, et al. antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences[J]. Nucleic Acids Res, 2011, 39(Web Server issue): W339-W346.
[29]	刘龙, 荣华, 郑童童, 等. 莫海威芽孢杆菌对梨腐烂病的抑菌防病效果[J]. 中国农学通报, 2022, 38(18): 140-146. doi: 10.11924/j.issn.1000-6850.casb2021-1050
	Liu L, Rong H, Zheng TT, et al. Antifungal and control effect of Bacillus mojavensis on pear Valsa canker[J]. Chin Agric Sci Bull, 2022, 38(18): 140-146.
[30]	姜军坡. 芽孢杆菌源抗肿瘤活性脂肽的分离纯化、结构表征、活性评价与生物合成调控[D]. 保定: 河北大学, 2021.
	Jiang JP. Isolation, purification, structure characterization, activity evaluation and biosynthesis regulation of anti-tumor lipopeptides from bacillus[D]. Baoding: Hebei University, 2021.
[31]	臧威, 何旭, 孙剑秋, 等. 桦木内生真菌的分离与代谢产物的抑菌活性[J]. 生态环境学报, 2012, 21(4): 661-665. doi: 10.16258/j.cnki.1674-5906(2012)04-0661-05
	Zang W, He X, Sun JQ, et al. Antimicrobial activity of metabolites and isolation of endophytic fungi in Betula spp[J]. Ecol Environ Sci, 2012, 21(4): 661-665.
[32]	马欢欢, 林洋, 吕欣然, 等. 96孔板法筛选抗黑曲霉性乳酸菌及抑菌机理研究[J]. 食品工业科技, 2017, 38(12): 171-175.
	Ma HH, Lin Y, Lv XR, et al. Screening and inhibition mechanism of lactic acid bacteria against Aspergillus niger using 96-well microtiter plates[J]. Sci Technol Food Ind, 2017, 38(12): 171-175.
[33]	Zhao YJ, Selvaraj JN, Xing FG, et al. Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum[J]. PLoS One, 2014, 9(3): e92486.
[34]	Stein T. Whole-cell matrix-assisted laser desorption/ionization mass spectrometry for rapid identification of bacteriocin/lantibiotic-producing bacteria[J]. Rapid Commun Mass Spectrom, 2008, 22(8): 1146-1152. doi: 10.1002/rcm.v22:8 URL
[35]	袁玉娟. Bacillus subtilis SQR9的黄瓜促生和枯萎病生防效果及其作用机制研究[D]. 南京: 南京农业大学, 2011.
	Yuan YJ. Cucumber growth promotion and wilt disease suppression of Bacillus subtilis SQR9 and their mechanisms[D]. Nanjing: Nanjing Agricultural University, 2011.
[36]	刘丽萍, 高洁, 李玉. 植物炭疽菌属Colletotrichum真菌研究进展[J]. 菌物研究, 2020, 18(4): 266-281.
	Liu LP, Gao J, Li Y. Advances in knowledge of the fungi referred to the genus Colletotrichum[J]. J Fungal Res, 2020, 18(4): 266-281.
[37]	冯江鹏, 邱莉萍, 梁秀燕, 等. 草莓胶孢炭疽菌拮抗细菌贝莱斯芽孢杆菌JK3的鉴定及其抗菌活性[J]. 浙江农业学报, 2020, 32(5): 831-839. doi: 10.3969/j.issn.1004-1524.2020.05.11
	Feng JP, Qiu LP, Liang XY, et al. Identification of antagonistic bacteria Bacillus velezensis JK3 against anthracnose of straw-berry and its antipathogenic activity[J]. Acta Agric Zhejiangensis, 2020, 32(5): 831-839.
[38]	裴张新, 王志华, 于静亚, 等. 园林植物炭疽病研究进展[J]. 中国森林病虫, 2023, 42(6): 33-38.
	Pei ZX, Wang ZH, Yu JY, et al. Research progress of garden plant anthracnose[J]. For Pest Dis, 2023, 42(6): 33-38.
[39]	Yang LN, He MH, Ouyang HB, et al. Cross-resistance of the pathogenic fungus Alternaria alternata to fungicides with different modes of action[J]. BMC Microbiol, 2019, 19(1): 205.
[40]	Tudi M, Ruan HD, Wang L, et al. Agriculture development, pesticide application and its impact on the environment[J]. Int J Environ Res Public Health, 2021, 18(3): 1112.
[41]	廖思晨, 田露, 王旭阳, 等. 产抗菌脂肽解淀粉芽孢杆菌wxy-b3的抑菌活性及其分离鉴定[J]. 陕西科技大学学报, 2022, 40(6): 55-61.
	Liao SC, Tian L, Wang XY, et al. Bacteriostatic activity, isolation and identification of Bacillus amyloliquefaciens wxy-b3 producing antimicrobial lipopeptide[J]. J Shaanxi Univ Sci Technol, 2022, 40(6): 55-61.
[42]	李洋. 2021年国内新登记的生物农药品种[J]. 世界农药, 2022, 44(2): 1-8.
	Li Y. New biopesticides registered in China in 2021[J]. World Pestic, 2022, 44(2): 1-8.
[43]	袁海峰, 周舒扬, 甄涛, 等. 新型芽孢杆菌在农业领域应用研究进展[J]. 国土与自然资源研究, 2020(2): 92-94.
	Yuan HF, Zhou SY, Zhen T, et al. Research progress on application of new Bacillus in agriculture[J]. Territ Nat Resour Study, 2020(2): 92-94.
[44]	刘超, 刘洪伟, 汪步青, 等. 解淀粉芽孢杆菌BA-26抗菌物质分离及对灰葡萄孢抑菌作用研究[J]. 生物技术通报, 2019, 35(7): 83-89. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0055
	Liu C, Liu HW, Wang BQ, et al. Isolation of antifungal substances from Bacillus amyloliquefaciens BA-26 and its antifungal activity against Botrytis cinerea[J]. Biotechnol Bull, 2019, 35(7): 83-89.
[45]	杜佳慧, 徐伟芳, 杨晓冬, 等. 多花黄精产吲哚乙酸内生菌的分离筛选及其对黄精种子萌发的影响[J]. 生物技术通报, 2022, 38(12): 223-232. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0078
	Du JH, Xu WF, Yang XD, et al. Isolation and screening of endophytes producing indole acetic acid from Polygonatum cyrtonema Hua. and its effect on seed germination of Polygonatum[J]. Biotechnol Bull, 2022, 38(12): 223-232.
[46]	林占军, 汤江武, 陈小龙, 等. 微生物菌剂的茶园抑草效果及对茶叶品质的影响[J]. 浙江农业科学, 2023, 64(10): 2474-2477.
	Lin ZJ, Tang JW, Chen XL, et al. Effect of microbial agents on weed control in tea garden and its influence on tea quality[J]. J Zhejiang Agric Sci, 2023, 64(10): 2474-2477.
[47]	郭世堂, 李齐. 乙醇生产副产物在枯草芽孢杆菌培养中的应用[J]. 当代化工, 2022, 51(6): 1402-1405, 1410.
	Guo ST, Li Q. Application of by-products of ethanol production in Bacillus subtilis culture[J]. Contemp Chem Ind, 2022, 51(6): 1402-1405, 1410.
[48]	汝甲荣, 明立伟, 李志新, 等. 芽孢杆菌防治马铃薯主要病害的研究现状与展望[J]. 中国种业, 2023(9): 22-25, 29.
	Ru JR, Ming LW, Li ZX, et al. Research status and prospects of Bacillus subtilis for controlling major potato diseases[J]. China Seed Ind, 2023(9): 22-25, 29.
[49]	苏正川, 熊仁科, 罗小艳. 解淀粉芽孢杆菌的作用及其产品开发[J]. 农药科学与管理, 2019, 40(6): 21-30.
	Su ZC, Xiong RK, Luo XY. Function and product development of Bacillus amyloliquefaciens[J]. Pestic Sci Adm, 2019, 40(6): 21-30.
[50]	Yuan HB, Shi BK, Wang L, et al. Isolation and characterization of Bacillus velezensis strain P2-1 for biocontrol of apple postharvest decay caused by Botryosphaeria dothidea[J]. Front Microbiol, 2022, 12: 808938. doi: 10.3389/fmicb.2021.808938 URL
[51]	Ahmed W, Zhou GS, Yang J, et al. Bacillus amyloliquefaciens WS-10 as a potential plant growth-promoter and biocontrol agent for bacterial wilt disease of flue-cured tobacco[J]. Egyptian Journal of Biological Pest Control, 32(1): 25.
[52]	喇文军, 贾书娟, 吴小丽, 等. 抑制辣椒炭疽菌的生防芽孢杆菌菌株筛选和鉴定[J]. 深圳职业技术学院学报, 2016, 15(3): 48-52.
	La WJ, Jia SJ, Wu XL, et al. Screening and identification of an antagonistic Bacillus sp. against Colletotrichum capsici[J]. J Shenzhen Polytech, 2016, 15(3): 48-52.
[53]	Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions[J]. Mol Microbiol, 2005, 56(4): 845-857. doi: 10.1111/mmi.2005.56.issue-4 URL
[54]	李苏冉, 李雪, 冯佳霖, 等. 生防菌解淀粉芽孢杆菌SQ-2全基因组测序及生物信息学分析[J]. 微生物学通报, 2023, 50(3): 1073-1097.
	Li SR, Li X, Feng JL, et al. Whole genome sequencing and genomics analysis of Bacillus amyloliquefaciens SQ-2 with biocontrol activity[J]. Microbiol China, 2023, 50(3): 1073-1097.
[55]	何亚芳, 包慧芳, 王宁, 等. 甜瓜镰刀菌果腐病菌拮抗菌筛选及其拮抗性研究[J]. 园艺学报, 2023, 50(10): 2257-2270. doi: 10.16420/j.issn.0513-353x.2022-0741
	He YF, Bao HF, Wang N, et al. Screening of antagonistic bacteria against Fusarium spp. causing melon fruit rot and the antagonistic properties[J]. Acta Hortic Sin, 2023, 50(10): 2257-2270.
[56]	欧婷, 金必堃, 高海英, 等. Bacillus velezensis SWUJ1拮抗物质分离纯化及抑菌机理研究[J]. 西南大学学报: 自然科学版, 2022, 44(1): 75-87.
	Ou T, Jin BK, Gao HY, et al. Purification and research of inhibitory mechanism of antagonist substances from Bacillus velezensis SWUJ1 strain[J]. J Southwest Univ Nat Sci Ed, 2022, 44(1): 75-87.
[57]	李铮, 王金辉, 丁丽丽, 等. 贝莱斯芽孢杆菌菌株NZ-4生防潜能及基因组学分析[J]. 江苏农业科学, 2023, 51(2): 117-125.
	Li Z, Wang JH, Ding LL, et al. Biocontrol potential and genomic analysis of Bacillus Vé lez strain NZ-4[J]. Jiangsu Agric Sci, 2023, 51(2): 117-125.

培养基名称Name of culture medium	组分Component/（g·L^-1）
马铃薯葡萄糖培养基Potato dextrose medium, PDB	马铃薯200.0，葡萄糖20.0
LB培养基Luria-Bertani medium	胰蛋白胨10.0，酵母粉5.0，氯化钠10.0
PDB改良培养基Modified potato dextrose broth	马铃薯200.0，麦芽糖20.0，蛋白胨10.0，硫酸铵5.0，磷酸氢二钠1.5
纤维素酶检测培养基Cellulase assay medium	羧甲基纤维素钠 2.0，硫酸铵 2.0，磷酸二氢钾 1.0，硫酸镁 0.5，刚果红 0.1
蛋白酶检测培养基Protease assay medium	葡萄糖15.0，脱脂奶粉20.0，氯化钾 0.5，硫酸镁 0.5
淀粉酶检测培养基Amylase assay medium	牛肉膏5.0，蛋白胨10.0，氯化钠 5.0，可溶性淀粉10.0
铁载体检测培养基主要成分Siderophore assay medium	葡萄糖4.0，硝酸钾0.2，硫酸镁 0.1，氯化钠0.1，磷酸氢二钾 0.1，硫酸亚铁 0.002
Landy发酵培养基Landy fermentation medium	葡萄糖20.0，谷氨酸钠 5.0, 酵母粉1.0, 磷酸二氢钾 1.0，硫酸镁 0.5，氯化钾 0.5，硫酸锰 0.000 05，硫酸铜 0.000 16，硫酸亚铁 0.000 15，苯丙氨酸 0.02

培养基名称Name of culture medium	组分Component/（g·L^-1）
马铃薯葡萄糖培养基Potato dextrose medium, PDB	马铃薯200.0，葡萄糖20.0
LB培养基Luria-Bertani medium	胰蛋白胨10.0，酵母粉5.0，氯化钠10.0
PDB改良培养基Modified potato dextrose broth	马铃薯200.0，麦芽糖20.0，蛋白胨10.0，硫酸铵5.0，磷酸氢二钠1.5
纤维素酶检测培养基Cellulase assay medium	羧甲基纤维素钠 2.0，硫酸铵 2.0，磷酸二氢钾 1.0，硫酸镁 0.5，刚果红 0.1
蛋白酶检测培养基Protease assay medium	葡萄糖15.0，脱脂奶粉20.0，氯化钾 0.5，硫酸镁 0.5
淀粉酶检测培养基Amylase assay medium	牛肉膏5.0，蛋白胨10.0，氯化钠 5.0，可溶性淀粉10.0
铁载体检测培养基主要成分Siderophore assay medium	葡萄糖4.0，硝酸钾0.2，硫酸镁 0.1，氯化钠0.1，磷酸氢二钾 0.1，硫酸亚铁 0.002
Landy发酵培养基Landy fermentation medium	葡萄糖20.0，谷氨酸钠 5.0, 酵母粉1.0, 磷酸二氢钾 1.0，硫酸镁 0.5，氯化钾 0.5，硫酸锰 0.000 05，硫酸铜 0.000 16，硫酸亚铁 0.000 15，苯丙氨酸 0.02

病原菌 Pathogenic bacterium	抑菌率 Inhibition rate/%（x±s, n=3）
栝楼炭疽病菌C. gloeosporioides	57.59±0.00a
梨炭疽病菌C. fructicola	38.97±0.01c
枣炭疽病菌C. coccodes	48.54±0.01b
苹果炭疽病菌C. malicorticis	34.27±0.01d

病原菌 Pathogenic bacterium	抑菌率 Inhibition rate/%（x±s, n=3）
栝楼炭疽病菌C. gloeosporioides	57.59±0.00a
梨炭疽病菌C. fructicola	38.97±0.01c
枣炭疽病菌C. coccodes	48.54±0.01b
苹果炭疽病菌C. malicorticis	34.27±0.01d

检测项目 Test item	检测结果 Test result
葡萄糖（产酸产气）Glucose（Producing acid and gas）	+
乳糖 Lactose	+
麦芽糖 Maltose	-
甘露醇 Mannitol	-
蔗糖 Sucrose	+
蛋白胨水 Peptone water	-
V-P试验Voges-Proskauer reaction	+
西蒙氏柠檬酸盐 Symon's phosphate	+
硫化氢 H₂S production	-
尿素 Urea	-
半固体琼脂Semi-solid agar	-