柑橘花器和种子中黄龙病菌的定量分布及应用  

Quantitative distribution of Candidatus Liberibacter asiaticus in flowers and seeds of Shatian pummelo and its applications

作　　者：娄兵海[1] 宋雅琴[1] 赵小龙[2] 白先进[3] 邓崇岭[1] 

机构地区：[1]广西壮族自治区柑桔研究所,广西柑橘生物学重点实验室,桂林541004 [2]广西农业职业技术学院,南宁530007 [3]广西农业科学院,南宁530007

出　　处：《植物保护学报》2014年第4期447-452,共6页Journal of Plant Protection

基　　金：国家自然科学基金(31301635);广西省自然科学基金(2013GXNSFBA019110);广西科学研究与技术开发计划(桂科合14123001-15)

摘　　要：为准确、快速测定感病柑橘花器和种子中黄龙病菌(Candidatus Liberibacter asiaticus,Las)的含量,比较了SYBR GreenⅠ实时荧光定量PCR(SGI-qPCR)和单管双引物对TaqMan探针qPCR(STDP-qPCR)的检测灵敏度,并用STDP-qPCR法定量检测了感病沙田柚花器和种子中的Las。结果显示,STDP-qPCR检测灵敏度为1×100拷贝/μL,比SGI-qPCR高100倍;花器中的雄蕊、花瓣、雌蕊和花粉等组织,以及种子的种皮和胚乳组织中均可检测到Las,但含量差异较大,其中种皮组织中Las含量最高,达到109842个细胞/μg DNA,花粉中的Las含量最低,为308个细胞/μg DNA,所有种壳中均未检测到Las;基于雄蕊组织的黄龙病分子诊断准确率达93.8%。表明Las在感病柑橘花器和种子中呈不均匀分布,基于雄蕊组织的黄龙病诊断方法可辅助用于该病害的高通量检测。In order to quantify the concentration of Candidatus Liberibacter asiaticus( Las) in flowers and seeds of huanglongbing( HLB)-infected citrus,two real-time fluorescence quantitative PCR( qPCR)methods,SYBR Green I qPCR( SGI-qPCR) and 'single closed tube dual primer'TaqMan qPCR( STDP-qPCR),were compared for their detection sensitivity,and STDP-PCR was used to quantify the concentration of Las in different floral and seed parts collected from HLB-infected Citrus maxima( Burm.) Merr. cv. Shatian T.T.Yu trees. The results showed that the detection sensitivity of STDP-qPCR was 1 × 100 copies /μL DNA,100 times higher than that of SGI-qPCR. Quantitative analysis indicated that Las was distributed in stamens,petals,pistils,pollen,seed coats and endosperms. However,the mean concentration of Las in different tissues was different. The highest concentration of Las was observed in seed coats,up to 109 842 cells /μg DNA,and the lowest was observed in pollen,only 308 cells /μg DNA. No amplicon was obtained from seed hull samples. The incidence of conventional PCR detection of Las based on stamen tissues was 93. 8%. These results indicated that Las was unevenly distributed inflowers and seeds collected from HLB-infected citrus trees,and the new diagnosis system of HLB based on stamen tissues could be used for large-scale detection of Las as an adjective method.

关 键 词：黄龙病 花器 种子 实时荧光定量PCR 雄蕊 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]