Pathogen Identification and Screening of Biocontrol Bacteria for Euonymus japonicus Anthracnose

doi:10.15933/j.cnki.1004-3268.2026.05.010

Abstract

Abstract: To clarify the pathogen species of anthracnose on Euonymus japonicus and screen green biocontrol resources in Ji’nan，Shandong Province，the tissue isolation method was used to isolate pathogens from diseased plants.The taxonomic status of the pathogens was identified via morphological observation，multi‐gene sequence analysis，and pathogenicity test，and the biological characteristics were determined.Further，biocontrol bacteria with significant antagonistic effects against E.japonicus anthracnose were screened from the rhizosphere soil of healthy plants，and their antagonistic activity and control efficacy against the pathogens were evaluated via in vitro dual culture assay and leaf antibacterial test.The results showed that the pathogenic strain EJJN05，isolated from diseased plants，was identified as Colletotrichum siamense through morphological observation，pathogenicity test，and multi‐gene sequence analysis.The optimal growth conditions for this strain were PDA medium，30 ℃ and pH 7.A strain designated as EJSF04 with significant antagonistic effect against the pathogen was isolated from the rhizosphere soil of healthy E.japonicus plants.In dual culture assay，the inhibition rate reached 85.12%，and the in vitro leaf test showed that the fermentation broth of EJSF04 had a control efficacy of 78.32% for preventive treatment of anthracnose，which was significantly higher than that of curative treatment（63.18%）.Combined with morphological observation and 16S rDNA and gyrB gene sequences analysis，strain EJSF04 was identified as Bacillus velezensis.This study provided a solid scientific basis for the effective implementation of green prevention and control measures against E.japonicus anthracnose.

Key words: Euonymus japonicus, Anthracnose, Pathogen identification, Biological characteristics, Biocontrol bacteria, Bacillus velezensis, Antifungal activity

摘要： 为明确山东省济南市大叶黄杨炭疽病的病原菌种类并筛选绿色生防菌资源，采用组织分离法对罹病植株进行病原分离，通过形态特征观察、多基因序列分析及致病性测定鉴定其分类地位，测定其生物学特性，并进一步从健康植株根际土壤中筛选对大叶黄杨炭疽病具有显著拮抗效果的生防菌，通过室内对峙培养和离体叶片抑菌试验，评估生防菌株对病原菌的拮抗能力和防治效果。结果表明，从病株上分离获得病原菌株EJJN05，通过形态学观察、致病性、多基因序列分析将其鉴定为暹罗炭疽菌（Colletotrichum siamense）。该菌最适生长条件为PDA培养基、30 ℃、pH值7。从健康大叶黄杨植株根际土壤中分离得到1株对病原菌具有显著拮抗作用的菌株EJSF04，对峙培养抑菌率达85.12%，离体叶片试验表明，其发酵液对炭疽病的预防处理防效达78.32%，显著高于治疗处理防效（63.18%）。结合形态学观察及16S rDNA和gyrB 基因序列分析，将该菌株鉴定为贝莱斯芽孢杆菌（Bacillus velezensis），为有效实施大叶黄杨炭疽病的绿色防控提供了科学依据和生防资源。

关键词: 大叶黄杨, 炭疽病, 病原鉴定, 生物学特性, 生防菌, 贝莱斯芽孢杆菌, 抑菌活性

CLC Number:

S436.8

ZHANG Zongxia, LÜ Juan, HAN Bo, FAN Susu, YAN Fanfeng. Pathogen Identification and Screening of Biocontrol Bacteria for Euonymus japonicus Anthracnose[J]. Journal of Henan Agricultural Sciences, 2026, 55(5): 96-106.

张宗侠, 吕娟, 韩波, 范素素, 闫凡峰. 大叶黄杨炭疽病病原菌鉴定及生防菌筛选[J]. 河南农业科学, 2026, 55(5): 96-106.

Figures/Tables 8

References

［1］CAI H Y，GU X Z，LI Y T，et al. Cold resistance of Euonymus japonicus Beihaidao leaves and its chloroplast genome structure and comparison with Celastraceae species［J］. Plants，2022，11（19）：2449.
［2］朱旭，李海梅，李彦华，等. 8种灌木对大气颗粒物污染的生理响应［J］. 生态环境学报，2022，31（3）：535‐545.
ZHU X，LI H M，LI Y H，et al. Physiological responses of eight shrubs to atmospheric particulate matter pollution［J］. Ecology and Environment Sciences，2022，31（3）：535‐545.
［3］李娟霞，何靖，孙一梅，等. 10种园林植物功能性状对大气污染的生理生态响应［J］. 生态环境学报，2020，29（6）：1205‐1214.
LI J X，HE J，SUN Y M，et al. Physiological and ecological responses of ten garden plant functional traits to air pollution［J］. Ecology and Environment Sciences，2020，29（6）：1205‐1214.
［4］王恩怡. 大叶黄杨对大气SO2-Pb复合污染的抗性响应机制研究［D］. 济南：山东建筑大学，2017.
WANG E Y. Research on the response mechanism of Euonymus japonicus to compound pollution of atmospheric SO2‐Pb［D］. Ji’nan：Shandong Jianzhu University，2017.
［5］LIU Y Y，TAN X Q，ZHAO J，et al. Diversity of Colletotrichum species associated with anthracnose on Euonymus japonicus and their sensitivity to fungicides［J］. Frontiers in Plant Science，2024，15：1411625.
［6］刘兴元. 鄂西南地区大叶黄杨病害现状及防治对策研究［D］. 武汉：华中农业大学，2006.
LIU X Y. Study on the current situation and prevention countermeasure of Euonymus japonicus thunb diseases in southwest Hubei Province［D］. Wuhan：Huazhong Agricultural University，2006.
［7］吴曼颖，熊兴耀. 张家界大叶黄杨炭疽病的病原菌鉴定［J］. 湖南农业大学学报（自然科学版），2020，46（6）：
706‐710.
WU M Y，XIONG X Y. Pathogen identification of anthracnose on Euonymus japonicus in Hunan Province［J］. Journal of Hunan Agricultural University（Natural Sciences），2020，46（6）：706‐710.
［8］TAN X Q，ZHAO J，QIAO G H，et al. Morphological characterization and pathogenicity of Colletotrichum aenigma and C.siamensecausing anthracnose on Euonymus japonicusin Beijing， China ［J］. Plant Pathology，2023，72（3）：430‐441.
［9］LEE H B，PARK J Y，JUNG H S. First report of leaf anthracnose caused by Colletotrichum boninense on spindle trees［J］. Plant Pathology，2005，54（2）：254.
［10］LIN L，PAN M，GAO H，et al. The potential fungal pathogens of Euonymus japonicus in Beijing，China［J］.Journal of Fungi，2023，9（2）：271.
［11］ALIZADEH A，JAVAN‐NIKKHAH M，ZARE R，et al.New records of Colletotrichum species for the mycobiota of Iran ［J］. Mycologia Iranica，2015，2：95‐109.
［12］HUANG L，LI Q C，ZHANG Y，et al. Colletotrichum gloeosporioides sensu stricto is a pathogen of leaf anthracnose on evergreen spindle tree（Euonymus japonicus）［J］. Plant Disease，2016，100（4）：672‐678.
［13］QIN W T，LI X H，HUANG J B，et al. First report of Colletotrichum theobromicola causing anthracnose on Euonymus japonicus in China［J］. Plant Disease，2023，107（5）：1629.
［14］吴文平. 冬青卫茅上的腔孢菌记述Ⅰ. 黑盘孢目［J］.河北省科学院学报，1992，9（1）：65‐74.
WU W P. Note on some coeldmycetes on Euonymus japonicas Ⅰ. Melanoconiales［J］. Journal of the Hebei Academy of Sciences，1992，9（1）：65‐74.
［15］DA SILVA L L，MORENO H L A，CORREIA H L N，et al. Colletotrichum：Species complexes，lifestyle，and peculiarities of some sources of genetic variability［J］.Applied Microbiology and Biotechnology，2020，104（5）：1891‐1904.
［16］侯圣凡，华战迎，刘峻杰，等. 我国草莓胶孢炭疽菌的多基因联合鉴定与致病性分析［J］. 中国农业大学学报，2022，27（4）：82‐94.
HOU S F，HUA Z Y，LIU J J，et al. Multi‐gene joint identification and pathogenicity analysis of Colletotrichum gloeosporioides complex of strawberry in China［J］. Journal of China Agricultural University，2022，27（4）：82‐94.
［17］CANNON P F，DAMM U，JOHNSTON P R，et al.Colletotrichum‐current status and future directions［J］.Studies in Mycology，2012，73：181‐213.
［18］朱晓琴，方树贤，刘冬梅，等. 辣椒炭疽病生防菌株的筛选、鉴定及其抑菌机理［J］. 植物保护学报，2023，50（4）：913‐922.
ZHU X Q，FANG S X，LIU D M，et al. Screening，identification and antifungal mechanism of bacterial biocontrol strains against hot pepper anthracnose［J］.Journal of Plant Protection，2023，50（4）：913‐922.
［19］孙前军. 北京平谷区桃细菌性黑斑病与炭疽病病原鉴定及生防菌初筛［D］. 南阳：南阳师范学院，2024.
SUN Q J. Identification of pathogen of bacterial black spot and anthracnose of peach in Pinggu District of Beijing and preliminary screening of biocontrol bacteria［D］. Nanyang：Nanyang Normal University，2024.
［20］刘辉，冯月玲，周罗娜，等. 茶炭疽病拮抗木霉菌株的分离鉴定及其对茶炭疽病的拮抗作用［J］. 西南农业学报，2025，38（4）：757‐768.
LIU H，FENG Y L，ZHOU L N，et al. Isolation，screening and antagonism of antagonistic Trichoderma strains against tea anthracnose［J］. Southwest China Journal of Agricultural Sciences，2025，38（4）：757‐768.
［21］张宏鑫. 水果炭疽病拮抗酵母菌的筛选鉴定及其机制研究［D］. 南阳：南阳师范学院，2024.
ZHANG H X. Screening，identification and mechanism of antagonistic yeast against fruit anthracnose［D］.Nanyang：Nanyang Normal University，2024.
［22］YIN X T，LI T G，JIANG X L，et al. Suppression of grape white rot caused by Coniella vitis using the potential biocontrol agent Bacillus velezensis GSBZ09［J］. Pathogens，2022，11（2）：248.
［23］桑华春，邱德勋. 大叶黄杨炭疽病的病原初步研究［J］. 四川农业大学学报，1992，10（1）：189‐199.
SANG H C，QIU D X. A preliminiary study of etiology of anthracnose of Euonymus japonicum Thunb［J］.Journal of Sichuan Agricultural University，1992，10（1）：189‐199.
［24］张勇，张震，邱海萍，等. 3种园林植物炭疽病病原的分离与鉴定［C］//中国植物病理学会. 中国植物病理学会2009年学术年会论文集. 昆明：［出版者不详］，2009：129‐135.
ZHANG Y，ZHANG Z，QIU H P，et al. Isolation and identification of the anthracnose pathogens from three landscape plants ［C］//Chinese Society of Plant Pathology. Proceedings of the 2009 Annual Meeting of the Chinese Society of Plant Pathology. Kunming：［s.n.］，2009：129‐135.
［25］KIM Y H，CHO S E，SUNG H J，et al. First report of anthracnose on Euonymus japonicus caused by a provisionally novel species of Colletotrichum in the magnum complex in South Korea［J］. Plant Disease，2024，108（7）：2221.
［26］王成，赵丹阳，扈丽丽，等. 苹婆炭疽病病原菌生物学特性及室内毒力测定［J］. 林业与环境科学，2023，39（4）：21‐26.
WANG C，ZHAO D Y，HU L L，et al. Studies on biological characteristics and lab toxicity test of leaf pathogen（Colletotrichum siamense） on Sterculia monosperma［J］. Guangdong Forestry Science and Technology，2023，39（4）：21‐26.
［27］LI H，LIAO Y C Z，WAN Y，et al. Colletotrichum siamense， a novel causal agent of Viburnum odoratissimum leaf blotch and its sensitivity to fungicides［J］. Journal of Fungi，2023，9（9）：882.
［28］MOMOTAZ R，ALAM K M，ARIFUNNAHAR M，et al.Colletotrichum siamense leaf blight/spot：Characterization of a newly identified disease on Cinnamomum tamala［J］. Journal of Basic Microbiology，2023，63（8）：915‐929.
［29］孔会会，姜昱，何鹏搏，等. 儿茶酚铁载体对贝莱斯芽孢杆菌B9601-Y2的生物膜形成及抑菌活性的影响［J］. 微生物学通报，2025，52（9）：4013‐4024.
KONG H H，JIANG Y，HE P B，et al. Effects of bacillibactin on the biofilm formation and biocontrol activity of Bacillus velezensis B9601‐Y2［J］.Microbiology China，2025，52（9）：4013‐4024.
［30］范田田，王傲雪，刘禹彤，等. 芽孢杆菌抗菌物质研究进展［J］. 生物工程学报，2025，41（10）：3667‐3682.
FAN T T，WANG A X，LIU Y T，et al. Advances in the antimicrobial substances in Bacillus［J］. Chinese Journal of Biotechnology，2025，41（10）：3667‐3682.
［31］朱玉涛，薛冰怡，王豫蓝，等. 生防菌防治棉花黄萎病的研究进展［J］. 山西农业科学，2026，54（1）：29‐38.
ZHU Y T，XUE B Y，WANG Y L，et al. Research progress on biocontrol bacteria for cotton Verticillium wilt［J］. Journal of Shanxi Agricultural Sciences，2026，54（1）：29‐38.
［32］郭翱翔，李志洋，祝春华，等. 贝莱斯芽孢杆菌MD4抑制番茄灰霉病的生物防治潜力［J/OL］. 河南农业大学学报，2025：1‐15（2025‐05‐08）［2026‐04‐09］. https：//doi. org/10. 16445/j. cnki. 1000‐2340. 20250507. 001.
GUO A X，LI Z Y，ZHU C H，et al. Biocontrol potential of Bacillus velezensis MD4 in inhibiting tomato gray mold ［J/OL］. Journal of Henan Agricultural University， 2025： 1‐15 （2025‐05‐08）［2026‐04‐09］. https：//doi. org/10. 16445/j. cnki. 1000‐2340.20250507. 001.
［33］王飞，李雪梦，杨瑾，等. 贝莱斯芽孢杆菌Bv1-4对3种药材的防病促生效果评价及其全基因组分析［J］. 河南农业科学，2025，54（11）：79‐90.
WANG F，LI X M，YANG J，et al. Evaluation of the disease prevention and growth‐promoting effects of Bacillus velezensis Bv1‐4 on three medicinal plants and its whole genome analysis［J］. Journal of Henan Agricultural Sciences，2025，54（11）：79‐90.
［34］梁松，孟祥坤，于新，等. 草莓叶枯病生防芽孢杆菌的分离鉴定及防效研究［J］. 河南农业科学，2023，52（10）：92‐99.
LIANG S，MENG X K，YU X，et al. Isolation and identification of Bacillus for biocontrol of leaf blight of strawberry and study on its biocontrol effect［J］.Journal of Henan Agricultural Sciences，2023，52（10）：92‐99.

[1]	LI Mingzhe, MAO Yuqing, YANG Jieqiong, GUO Runxia, DUAN Zhenwei, BAN Litong, WANG Yuanhong, YU Haili. Isolation and Identification of the Main Trichoderma Pathogens in Shiitake Mushrooms and Study on the Antibacterial Effects of Five Plant Essential Oils [J]. Journal of Henan Agricultural Sciences, 2026, 55(4): 90-99.
[2]	YIN Kun, HUANG Yubo, LIANG Lu, ZHUANG Qiuli, TIAN Kaiyue, XU Bohan, ZHANG Weijie, ZHANG Donghui. Isolation，Identification，Biological Characterization of Diaporthe longicolla and Screening of Antifungal Drugs [J]. Journal of Henan Agricultural Sciences, 2026, 55(3): 97-106.
[3]	HAN Yu, SUN Tianqi, TANG Yuman, XU Lingui, SUN Xiaobo, WANG Lihong, SUI Chun. Root Rot Pathogen Identification and Resistant Germplasm Screening of Isatis indigotica Fort. [J]. Journal of Henan Agricultural Sciences, 2026, 55(3): 118-125.
[4]	WU Shuangshuang, ZHANG Zekun, YE Changwen, YAN Jianying, WANG Qingyue, SUN Zhikang, LI Liqun, HAO Jie, LIU Dandan. Optimization of Fermentation and Application of Amylase‑producing Bacillus velezensis A8‑1 [J]. Journal of Henan Agricultural Sciences, 2026, 55(2): 156-170.
[5]	HAN Jie, WANG Qinghai, ZHENG Yufei, HU Jin, ZHU Wencheng, WANG Xiuguo, LIU Tong. Screening and Residue Characteristics Analysis of Efficient Control Agents for Typical Diseases of Polygonatum sibiricum#br# [J]. Journal of Henan Agricultural Sciences, 2026, 55(1): 101-109.
[6]	ZHANG Changcheng, JIANG Jun, PAN Shouhui, XUE Yuan, ZHANG Quan, LI Zikuo, WANG Hancheng, SHI Caihua, FANG Shouguo, ZHANG Songbai, LI Xiquan. Molecular Identification and Complete Sequence Analysis of Chilli Veinal Mottle Virus GZ‑tobacco Isolate [J]. Journal of Henan Agricultural Sciences, 2025, 54(8): 102-110.
[7]	FENG Lili. Research Progress on Application in Livestock，Poultry and Aquaculture Production of Bacillus subtilis and Its Effect on Intestinal Health of Animals [J]. Journal of Henan Agricultural Sciences, 2025, 54(5): 1-9.
[8]	DU Chenyang, SHEN Fengying, WU Junwei, ZHAO Xinrui, WANG Xue, WU Weigang. Identification，Biological Characterization and Screening of Prevention and Control Agents of the Pathogens Causing the Rot Disease of Malus×robusta（CarriŠre）Rehder [J]. Journal of Henan Agricultural Sciences, 2025, 54(3): 99-109.
[9]	WANG Fei, LI Xuemeng, YANG Jin, GUO Zhichen, SONG Luyang, QIN Yanhong, WEN Yi, GAO Suxia, LU Shuhao, LIU Qi, QI Hongzhi, LI Shaojian. Evaluation of the Disease Prevention and Growth‑promoting Effects of Bacillus velezensis Bv1‑4 on Three Medicinal Plants and Its Whole Genome Analysis [J]. Journal of Henan Agricultural Sciences, 2025, 54(11): 79-90.
[10]	LI Haicheng, WANG Fanzhen, JIANG Yongcheng, SONG Xin, CHEN Zhaorong. Identification and Indoor Fungicide Screening of Pathogen Causing Leaf Spot on Rehmannia glutinosa [J]. Journal of Henan Agricultural Sciences, 2024, 53(4): 92-101.
[11]	WANG Wenxiao, LIU Meiling, QUE Yawei, ZHENG Lei, ZHANG Qiang, YU Dazhao, YANG Lijun, GONG Shuangjun, ZENG Fansong, LI Li. Combination of Bacillus velezensis EA19 and Carbendazim to Control Fusarium Head Blight on Wheat [J]. Journal of Henan Agricultural Sciences, 2024, 53(10): 117-126.
[12]	WANG Peng, BIE Zhaowei, XIA Lele. Pathogen Identification of Leaf Diseases of Louisiana Iris and Screening of Control Agents [J]. Journal of Henan Agricultural Sciences, 2023, 52(12): 107-115.
[13]	YU Shicheng, XIU Zhijun, BAI Songlin, BAN Ruijuan, XU Linbo, WANG Fengwu, WANG Qianjun, LI Yunxia, ZHANG Xiaoyu. Evaluation of Toxicity and Field Control Effect of Different Fungicides against Oat Anthracnose [J]. Journal of Henan Agricultural Sciences, 2023, 52(12): 97-106.
[14]	YANG Xiao, WANG Shuguang, LIANG Shen, LOU Changcheng, WANG Limin, SHI Man, ZHANG Hechen. Isolation and Identification of the Pathogen of Catalpa bungei Stem Rot and Differences in Disease Resistance of Different Catalpa Species [J]. Journal of Henan Agricultural Sciences, 2022, 51(3): 104-111.
[15]	PENG Qichao, HUANG Delong, ZHANG Zhipeng, WEI Hao, WU Shufeng, WU Yan, LI Jun, DENG Zuke, WANG Zongkang. The Biocontrol Effect of Bacillus velezensis DPT‑03 on Sclerotium rolfsii in Peanut [J]. Journal of Henan Agricultural Sciences, 2022, 51(2): 97-103.