Wang Xuefeng, Doddapaneni Harsha, Chen Jianchi, Yokomi Raymond K
National Citrus Engineering Research Center, Citrus Research Institute, Southwest University, Chongqing 400712, P. R. China.
Department of Biology, University of Iowa, Iowa City 52242.
Plant Dis. 2015 Jan;99(1):149-154. doi: 10.1094/PDIS-06-14-0572-RE.
Spiroplasma citri is a phloem-limited bacterium causing citrus stubborn disease (CSD). Isolation and culturing of S. citri is technically demanding and time consuming. S. citri is typically low in titer and unevenly distributed in citrus, making reliable detection challenging. The current preferred detection method is polymerase chain reaction (PCR) assays with primers developed from sequences of S. citri housekeeping genes. Recent genome sequencing of S. citri revealed that the bacterium harbors multiple copies of prophage genes. Therefore, targeting multicopy prophage genes was hypothesized to improve sensitivity of PCR detection. Two primer sets, Php-orf1 and Php-orf3, were developed from conserved prophage sequences in the S. citri genome. These primer sets were used to evaluate detection sensitivity in SYBR Green-based quantitative PCR (qPCR) assays with 18 S. citri in cultures isolated from different hosts and locations. Prophage primer set Php-orf1 increased detection sensitivity by 4.91 and 3.65 cycle threshold (Cq) units compared with housekeeping gene primers for spiralin and P58 putative adhesin gene, respectively. Detection was slightly less sensitive for the Php-orf3 primer set at 3.02 and 1.76 Cq units, respectively, over the same housekeeping gene primers. The prophage primer sets were validated for qPCR detection with field samples from three citrus orchards in California's San Joaquin Valley collected from 2007 to 2013. The data showed that S. citri prophage sequences improved sensitivity for qPCR detection of S. citri-infected trees at least 10-fold and reduced the number of false-negative results. No false-positive samples were detected with any of the primer sets. The enhanced sensitivity resulted from the higher copy number of prophage genes in the S. citri genome and, thus, improved CSD diagnosis from field samples.
柑桔螺原体是一种局限于韧皮部的细菌，可引起柑桔顽固病（CSD）。柑桔螺原体的分离和培养在技术上要求很高且耗时。柑桔螺原体的滴度通常较低，并且在柑桔中分布不均，这使得可靠检测具有挑战性。当前首选的检测方法是聚合酶链反应（PCR）检测，使用从柑桔螺原体管家基因序列开发的引物。柑桔螺原体最近的基因组测序表明，该细菌含有多个噬菌体基因拷贝。因此，推测靶向多拷贝噬菌体基因可提高PCR检测的灵敏度。从柑桔螺原体基因组中的保守噬菌体序列开发了两组引物，即Php-orf1和Php-orf3。这些引物组用于评估基于SYBR Green的定量PCR（qPCR）检测中的检测灵敏度，该检测使用从不同宿主和地点分离的18株柑桔螺原体培养物。与用于螺旋蛋白和P58假定粘附素基因的管家基因引物相比，噬菌体引物组Php-orf1的检测灵敏度分别提高了4.91和3.65个循环阈值（Cq）单位。与相同的管家基因引物相比，Php-orf3引物组的检测灵敏度分别略低，为3.02和1.76个Cq单位。使用2007年至2013年从加利福尼亚州圣华金河谷的三个柑桔果园采集的田间样本对噬菌体引物组进行了qPCR检测验证。数据表明，柑桔螺原体噬菌体序列将qPCR检测柑桔螺原体感染树木的灵敏度提高了至少10倍，并减少了假阴性结果的数量。使用任何一组引物均未检测到假阳性样本。灵敏度的提高源于柑桔螺原体基因组中噬菌体基因的拷贝数更高，从而改善了田间样本中柑桔顽固病的诊断。
