摘要
【目的】利用柑橘黄龙病病原亚洲种16S rDNA序列的特异引物检测疑似黄龙病的砂糖橘叶片样品,为砂糖橘黄龙病的综合防控提供理论依据。【方法】选取采自广西9市17个采样点的214份疑似黄龙病及无症状砂糖橘叶片样品,用试剂盒法提取叶脉基因组DNA,采用柑橘黄龙病亚洲种16S rDNA特异引物进行PCR扩增,扩增结果在1%琼脂糖凝胶电泳上检测,统计待检样品柑橘黄龙病病原菌亚洲种的带菌率。【结果】对214份样品的Nested-PCR扩增结果,部分样品可得到1160 bp左右的特异条带,与预期大小相符但条带明暗不同。来自广西9市17个采样点的砂糖橘叶片样品均检测出黄龙病亚洲种病原,平均检出率均在66.0%以上,其中检出率最高的是来自东兴市的样品,检出率为100.0%,检出率最低的是来自南宁市的样品,检出率为66.9%;斑驳、缺素型黄化、叶脉黄化、小叶和无症状叶片样品中均可检测出黄龙病亚洲种。【结论】Nested-PCR技术具有灵敏度高、特异性强的特点,能够快速、准确检测出各种症状样品的黄龙病病原,可用于柑橘黄龙病的早期诊断。
【Objective】To provide theoretical references for HLB synthetic control and prevention, special primers of 16S rDNA genes of Candidatus Liberibacter asiaticus were used to detect Shatangju samples with suspicious Huanglongbing.【Method】214 samples with suspicious Huanglongbing were collected from 17 spots of 9 cities in Guangxi. The leaf vein genomic DNA was extracted by kit and PCR amplification was conducted by special primers of 16S rDNA gene of Candidatus Liberibacter asiaticus. The results of PCR amplification were detected by 1% agarose gel electrophoresis. The carrier rate of Candidatus Liberibacter asiaticus was calculated statistically. 【Result】1160 bp-primers were produced from samples, which accorded with the expected size and varied in brightness. The HLB agent existed in the majority of 214 samples from 17 spots of 9 cities in Guangxi and the rates of the average percentage of HLB suspicious samples was over 66.0%. The detection rate of the samples from Dongxing was the highest （100.0%） and the lowest was from Nanning （66.9%）. The causal agent of HLB was found in the samples with symptoms including blotchy-mottle, nutrient deficiency, yellow veins, leaflets and asymptomatic leaves. 【Conclusion】Nested-PCR characterized with high sensitivity and specificity could be used for fast and accurate detection of Huanglongbing pathogen, hence it’s suitable for early diagnose of citrus Huanglongbing.
出处
《南方农业学报》
CAS
CSCD
北大核心
2013年第12期1997-2000,共4页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31160390)
广西科学研究与技术开发计划项目(桂科攻1123003-2A)
广西自然科学基金回国基金项目(2011GXNSFC018012)
广西农业科学院基本科研业务费专项项目(桂农科2012YZ06
桂农科2012YM21)
