毛桃种子表面根癌土壤杆菌的荧光定量PCR检测

Quantitative detection of Agrobacterium tumefaciens on peach pits by realtime PCR

【目的】明确不同来源毛桃种子携带根癌病菌情况,为切断病原菌的初侵染途径奠定基础。【方法】采用根癌土壤杆菌Agrobacterium tumefaciens的特异性引物virD2.For1/virD2.Rev1,建立了毛桃种子表面A.tumefaciens的real-time PCR检测方法,并利用该方法对不同来源毛桃种子的表面带菌量进行检测。【结果】实时荧光定量PCR法对毛桃种子表面根癌土壤杆菌数量的检测下限为1.60×102cfu·g-1,种子带菌量在1.60×102~1.45×107cfu·g-1范围内时,目的基因拷贝数与病原菌数量线性关系良好。对不同来源的毛桃种子进行表面带菌量检测结果表明,未接触土壤的毛桃种子表面病原菌含量低于检测下限,而接触地面土壤的毛桃种子表面带菌量的数量级达102cfu·g-1。沙藏可使带菌种子的表面带菌量升高2.7~5.0倍,而表面消毒可显著降低种子表面病原菌的含量。【结论】商品毛桃种子表面携带根癌病菌,病原菌来源于果园或其他地面土壤,桃种子表面带菌可能是桃根癌病的传播途径之一。

【Objective】 Clarifying the presence and the population of Agrobacterium tumefaciens on peach-pits will help us to prevent the spread of A. tumefaciens. 【Method】 Primer pair virD2.For1/virD2.Rev1 was used to develop a sensitive real-time PCR method for quantifying A. tumefaciens on peach pits.Using this assay, we detected A. tumefaciens on peach pits collected from different sources. 【Result】 The results showed that a good linear relationship between copy number of target DNA and A. tumefaciens population on peach pits, ranging from 1.60×102to 1.45×107cfu·g-1, was obtained. A. tumefaciens on peach-pits was detected at levels of 102cfu·g-1from the samples touched with the ground of seed-producing orchards and those peach pits purchased in Hebei and Shandong provinces. The A. tumefaciens population on peach pits increased by 2.7 to 5.0 times after storing in clean sand for about 3 months. No realtime PCR fluorescent signal was obtained from peach pits which were pulp- removed cleanly from directpicking peaches from trees. 【Conclusion】 A. tumefaciens could be assayed from commercial peach pits,and its presence on peach pits comes from touching with contaminated orchard soil, and this might be partly responsible for the spread of crown gall disease.