Yokomi Raymond K, Mello Alexandre F S, Saponari Maria, Fletcher Jacqueline
United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Parlier, CA 93648.
USDA-ARS, Parlier.
Plant Dis. 2008 Feb;92(2):253-260. doi: 10.1094/PDIS-92-2-0253.
Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using primers based on sequences of the P89 putative adhesin gene and the P58 putative adhesin multigene of Spiroplasma citri. Real-time PCR also was developed with detection limits estimated to be between 10 and 10 ng by serial dilution of a recombinant S. citri plasmid into DNA extracts from healthy Madam Vinous sweet orange. PCR for the detection of S. citri by these new primers was validated by comparing culturing of the pathogen, the traditional method of diagnosis, with PCR assays from samples taken from two citrus plots in Kern County, CA. Fruit columella was collected from 384 and 377 individual trees in each of two fields, respectively; one portion was used for culturing and the other for DNA extraction and PCR. PCR results matched those of culturing 85 to 100% of the time depending on the primers used. More importantly, PCR detected S. citri from culture-negative trees in 5 to 15% of the cases, suggesting that PCR performed as well or better than culturing for detection of S. citri in field samples. Real-time PCR proved to be the best method for detection. Differential reaction of the samples to the P58 primer pairs suggested that two populations of S. citri occur in historical and present-day field isolates. Citrus stubborn disease incidence was estimated to be 58.3 and 3.7% in the two orchards. The results presented here support the use of PCR for reliable detection of S. citri in field trees.
基于聚合酶链反应（PCR）检测柑橘顽固病的方法得到了改进，使用了基于柑橘螺旋体P89假定粘附素基因和P58假定粘附素多基因序列的引物。还开发了实时PCR，通过将重组柑橘螺旋体质粒连续稀释到来自健康的“夫人葡萄”甜橙的DNA提取物中，估计其检测限在10到10 ng之间。通过将病原体培养（传统诊断方法）与从加利福尼亚州克恩县两个柑橘园采集的样本进行PCR检测相比较，验证了这些新引物对柑橘螺旋体的检测效果。分别从两个果园的384棵和377棵单株树上采集果实果心；一部分用于培养，另一部分用于DNA提取和PCR。根据所使用的引物不同，PCR结果与培养结果的匹配率在85%至100%之间。更重要的是，在5%至15%的情况下，PCR检测到了培养结果为阴性的树上的柑橘螺旋体，这表明在检测田间样本中的柑橘螺旋体时，PCR的表现与培养法相当或更好。事实证明，实时PCR是最佳检测方法。样本对P58引物对的差异反应表明，在历史和现代田间分离株中存在两个柑橘螺旋体种群。两个果园中柑橘顽固病的发病率估计分别为58.3%和3.7%。本文给出的结果支持使用PCR可靠检测田间树木中的柑橘螺旋体。
