摘要
【目的】旨在明确江西省玉兰炭疽病病原菌种类和生物学特性,对采集的具有典型发病症状的玉兰叶片进行分离鉴定并筛选高效化学防治药剂,为玉兰炭疽病防控提供理论依据。【方法】采用组织块分离法对玉兰炭疽病发病叶片进行病原菌分离纯化,并根据柯赫式法则对分离得到的5株代表菌株进行致病性测定后,对最后分离所得的病原菌BTJ3进行形态学鉴定;以ITS、CHS-1、ACT、TUB2等引物对病原菌进行PCR扩增与测序,在NCBI数据库中进行BLAST同源性比对分析,使用MEGA 7.0软件构建多基因序列系统发育树,以对病原菌进行分子鉴定。利用菌丝生长速率法测定病原菌的生物学特性,并采用含药平板法开展病原菌室内毒力测定。【结果】(1)玉兰炭疽病病斑呈圆形或不规则形,中间深褐色,后期病斑扩大呈深灰色,边缘黑色并带有黄绿色晕圈;从采集的玉兰病叶样本中共分离获得5株炭疽菌菌株,遵循柯赫式法则确定菌株BTJ3为玉兰炭疽病病原,该病原菌形成的菌落初期为灰白色,气生菌丝发达,后期出现灰绿色同心圆环;分生孢子呈长椭圆形,大小为(8.7~19.5)μm×(3.45~6.36)μm,单胞,两端钝圆,透明无隔膜;分生孢子附着胞不规则形,卵圆形或球形,浅棕色至灰黑色,大小为(7~10.8)μm×(4.2~7.3)μm。(2)将菌株BTJ3的ITS、CHS-1、TUB2和ACT基因序列提交至GenBank中,获得序列登录号为PQ225996、PQ243288、PQ243289、PQ233290;基于多基因序列构建的系统发育树结果显示,菌株BTJ3与暹罗炭疽菌聚为同一个进化支,且支持率为93%,通过形态学结合多基因(ITS,TUB2,ACT,CHS-1)联合的系统发育分析,鉴定引起江西省玉兰炭疽病的病原菌为暹罗炭疽菌(Colletotrichum siamense)。(3)适宜该病原菌生长的温度为20~30℃;最适的pH范围为7~9,甘露醇和蛋白胨为最佳碳、氮源,光暗交替环境有利于其生长。(4)室内药剂筛选结果表明,80%多菌灵可湿性粉剂对暹罗炭疽菌有较好的抑制作用,其EC50值为0.28μg/mL。【结论】首次报道引起江西省玉兰炭疽病的病原菌为暹罗炭疽菌,明确了病原菌生物学特性和最佳化学防治药剂,为玉兰炭疽病科学防治提供依据。
[Objective]In order to clarify the species and biological characteristics of Magnolia denudata anthracnose in the campus of Jiangxi Agricultural University,the leaves of Yulania denudata anthracnose with typical symptoms were isolated and identified,and the efficient chemical control agents were screened to provide theoretical basis for the prevention and control of anthracnose in Yulania denudata.[Method]The pathogen was isolated and purified from the diseased leaves of Magnolia denudata by tissue block separation method,and the pathogenicity of the five representative strains was determined according to Koch's rule,and the morphological identification of the pathogen BTJ3 wasconducted.ITS,CHS-1,ACT,TUB2 and other primers were used for PCR amplification and sequencing of pathogenic bacteria.BLAST homology comparison analysis was performed in the NCBI database,and MEGA 7.0 software was used to construct a multi-gene sequence phylogenetic tree for molecular identification of pathogenic bacteria.The biological characteristics of the pathogen were determined by mycelial growth rate method,and the indoor virulence of the pathogen was determined by drug-containing plate method.[Result]The lesions of anthracnose on Magnolia are round or irregular.They are dark brown in the center.In the later stage,the lesions expand and turn dark gray,with black edges and a yellowish-green halo.A total of 5 strains of Colletotrichum were isolated from the collected Magnolia leaf samples.According to Koch's rule,the strain BTJ3 was identified as the pathogen of Magnolia anthracnose.The colony formed by the pathogen was grayish white in the early stage,with developed aerial hyphae,and grayish green concentric rings appeared in the later stage.The conidia were oblong,measuring 8.7-19.5 μm in length and 3.45-6.36 μm in width,unicellular,with bluntly rounded ends,transparent and without septa.The appressoria of the conidia were irregular,ovoid or spherical,light brown to grayish-black,and measure 7-10.8 μm in length and 4.2-7.3 μm in width.The ITS,CHS-1,TUB2 and ACT gene sequences of strain BTJ3 were submitted to GenBank,and the obtained sequence accession numbers were PQ225996,PQ243288,PQ243289 and PQ233290.The phylogenetic tree constructed based on multiple gene sequences showed that that strain BTJ3 clustered into the same evolutionary clade as Colletotrichum siamense,with a bootstrap support value of 93%.The pathogen of Magnolia anthracnose in Jiangxi Province was identified as Colletotrichum siamense by morphology combined with phylogenetic analysis of multiple genes(ITS,TUB2,ACT,CHS-1).The suitable temperature for the growth of the pathogen was 20-30 ℃.The optimum pH range was 7-9,mannitol and peptone were the best carbon and nitrogen sources,and the alternating light and dark environment was beneficial to its growth.The results of indoor fungicide screening showed that 80%carbendazim WP had a good inhibitory effect on Colletotrichum siamensis,and its EC50 value was 0.28 μg/mL.[Conclusion]The pathogen causing anthracnose of Magnolia denudata in Jiangxi Province was first reported to be Colletotrichum siamensis.The biological characteristics of the pathogen and the best chemical control agent were clarified,which laid a foundation for the scientific control of Magnolia anthracnose.
作者
罗佳钰
罗倩影
曾永春
文旺先
张林平
杨滢
操佳
黄绍华
黄佳慧
樊鹏敏
LUO Jiayu;LUO Qianying;ZENG Yongchun;WEN Wangxian;ZHANG Linping;YANG ying;CAO Jia;HUANG Shaohua;HUANG Jiahui;FAN Pengmin(College of Forestry/Jiangxi Provincial Key Laboratory of Conservation Biology,Jiangxi Agricultural University,Nanchang 330045,China;Guixi Academy of Forestry,Guixi,Jiangxi 335400,China;Jiangxi Academy of Forestry,Nanchang,330013,China)
出处
《江西农业大学学报》
北大核心
2025年第1期105-116,共12页
Acta Agriculturae Universitatis Jiangxiensis
基金
国家自然科学基金项目(32260377)
江西省林业科技创新专项(创新专项[2023]19号、创新专项[2024]6号)。
关键词
玉兰
暹罗炭疽菌
生物学特性
多基因联合分析
药剂筛选
Magnolia denudata
Colletotrichum siamense
biological characteristics
multigene joint analysis
fungicide screening
