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从进境加拿大油菜籽中分离到一株疑似十字花科细菌性疫病菌（Pseudomonas cannabina pv. alisalensis,Pca）的分离物，对其进行检测和鉴定。通过形态学观察、PCR检测、16S rRNA及多位点序列分析、系统发育树构建、致病性测试对分离物1121-H1进行鉴定，结果表明，分离物在MT培养基上菌落呈无色至浅白色，培养3 d后直径不超过1.5 mm，形态为圆形至椭圆形，扁平，边缘略有不规则，且不产生水解圈，培养5 d后产生红褐色色素；Pca特异引物hrpK＿fw1/ALS＿rv1和hrpK＿fw2/ALS＿rv2能扩增分离物1121-H1的DNA，得到预期特异条带；分离物1121-H1的16S rRNA序列与GenBank中Pca菌株ES4326和MAFF301419的序列一致，相似性为100.00%；选择gltA、gap1、ropD、pgi、gyrB和acnB等6个看家基因进行多位点序列分析，每段看家基因序列均与Pca菌株NCPPB1820的序列一致，相似性为100.00%；系统发育树显示分离物1121-H1与Pca相关菌株处于同一分支，与假单胞菌属其他菌株处于不同分支；致病性测试显示，涂抹接种后叶片出现褐色坏死斑伴黄绿色晕圈，针刺接种后病斑扩展并伴随组织塌陷，符合Pca典型症状。本研究是国内首次对Pca进行分离和鉴定，为口岸截获Pca提供了科学依据。
Abstract：A suspected isolate of Pseudomonas cannabina pv. alisalensis(Pca),was obtained from imported Canadian rape seeds and subjected to detection and identification. The isolate 1121-H1 was identified through morphological observation,PCR detection,16S r RNA and multi-locus sequence analysis,phylogenic tree construction,and pathogenicity test. The colony of the isolate on MT medium was colorless to light white,with the diameter of no more than 1.5 mm after cultivating for 3 days,and was circular to elliptical,flat,with slightly irregular edges and no hydrolytic zone. After 5 days of culture,it produced reddish-brown pigment. The specific primers hrp K＿fw1/ALS＿rv1 and hrp K＿fw2/ALS＿rv2 for Pca could amplify the DNA of isolate 1121-H1,and the expected specific bands were obtained. The 16S r RNA sequence of isolate1121-H1 was consistent with the sequences of Pca strains ES4326 and MAFF 301419 in Gen Bank,with a similarity of 100.00%. Six housekeeping genes,glt A,gap1,rop D,pgi,gyr B and acn B,were selected for multi-locus sequence analysis,each housekeeping gene sequence was consistent with the sequences of Pca strains NCPPB1820,with a similarity of 100.00%. The phylogenetic tree showed that isolate 1121-H1 was in the same branch as Pca-related strains and in a different branch from other Pseudomonas strains. The pathogenicity test showed that brown necrotic spots with yellowish-green halos appeared on the leaves after smear inoculation,and the lesions expanded and accompanied by tissue collapse after needle puncture inoculation,which were consistent with the typical symptoms of Pca. This study is the first isolation and identification of Pca in China,providing a scientific basis for the interception of Pca at the port.
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基本信息:
DOI：10.19662/j.cnki.issn1005-2755.2025.06.005
中图分类号:S41-30
引用信息:
[1]牟桂萍,许然,胡加谊,等.进境加拿大油菜籽中十字花科细菌性疫病菌的首次分离鉴定[J].植物检疫,2025,39(06):28-34.DOI:10.19662/j.cnki.issn1005-2755.2025.06.005.
基金信息:
海关总署科研项目（2025HK046）; 广东省口岸安全智能化检测重点实验室（2023B1212010011）; “十四五”广东省农业科技创新十大主攻方向(2023SDZG06)