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甘蔗赤条病是由燕麦食酸菌燕麦亚种(Acidovorax avenae subsp.avenae,Aaa)引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术,根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物,建立Aaa巢式PCR检测方法。结果表明,建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A.oryzae具有特异性,可扩增出454 bp目的条带,对近缘种德氏食酸菌A.delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L.xyli subsp.xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板,巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL,是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测,阳性检出率分别为100.0%和28.6%,表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。
Sugarcane streak disease is a worldwide cane bacterial disease caused by Acidovorax avenae subsp. Avenae (Aaa). In order to establish a rapid and sensitive detection technique for Aaa, two pairs of specific primers were designed according to the 16S ~ 23S ribosomal gene of the bacterium and ITS of its transcribed spacer region, and a Aa nested PCR assay was established. The results showed that the established nested PCR method was specific for the Aaa standard strain, A. oxysporum A. oryzae, and amplified a 454 bp band. The results showed that A. neoformans A. delafieldii and other families Genus Leifsonia rubra and L. xyli subsp. Xyli did not amplify any bands. The minimum detectable limit of detection of nested PCR was 10 fg / μL, 10 copies / μL and 36 CFU / mL, respectively, based on the total DNA of sugarcane leaves infected with Aaa, the plasmid DNA standard containing ITS target fragment and the standard strain of Aaa. Is 1000 times the sensitivity of conventional PCR. The nested PCR and routine PCR were used to detect the parallel samples of 14 sugarcane leaf samples with the symptom of red blight. The positive detection rates were 100.0% and 28.6%, respectively, indicating that the nested PCR method has higher sensitivity than the conventional PCR method . The nested PCR method established in this study is suitable for molecular detection and identification of red cane disease in the field.