|Mello,, Alexandre - OKLAHOMA ST.UV-STILLWATER|
|Fletcher,, Jacqueline - OKLAHOMA ST.UV-STILLWATER|
|Saponari,, Maria - INST. DE VIROL VEG. BARI|
Submitted to: Conference of International Organization of Citrus Virologists
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 17, 2008
Publication Date: May 14, 2011
Citation: Yokomi, R.K., Mello,, A.F., Fletcher,, J., Saponari,, M. 2011. Estimation of Citrus Stubborn Disease Incidence in Citrus Groves by real-time PCR. Conference of International Organization of Citrus Virologists. p.131-141. Interpretive Summary: There is a need for a rapid and accurate method to detect Spiroplasma citri, the causal agent of citrus stubborn disease (CSD). A real-time polymerase chain reaction (PCR) assay was developed to detect the pathogen in infected citrus trees. A molecular analysis was conducted and verified specificity of the real time reaction. The best tissue for detection of S. citri was fruit columella because pathogen titer was highest in this tissue. The real time PCR assay was used to determine the field incidence of CSD in five Navel orange orchards in Kern and Tulare Counties in central California. A systematic sampling plan was used in which 25% of the trees in each orchard were sampled. Results indicated CSD incidence in the plots at 58.9%, 4.2%, 0%, 22.4%, and 28.6%. Infection rates higher than 10% indicates that CSD is present at significant levels. These data are being used to examine if secondary spread of S. citri is occurring and to facilitate identification of the leafhopper vector(s) responsible for this spread.
Technical Abstract: A rapid and sensitive method is needed to detect Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), for epidemiology studies and implementation of CSD management strategies. Real-time polymerase chain reaction (PCR) was developed for detection of S. citri using the DNA binding fluorophore SYBR Green and primer pair P-58-3f/4r which is based on sequences from the P58 putative adhesin multigene of S. citri. Multiple alignment of sequences from cloned amplicons from S. citri shared 100% identity with the nucleotide sequence of the putative adhesin gene of S. citri strain BR3-3X. Assay sensitivity was estimated to be 1.6 x 10-4 to 2.5 x 10-6 ng/mg of tissue collected from field citrus trees. S. citri titer was consistently higher in fruit columella than in leaf midribs making it the best choice for sampling. Real time PCR was used to assay 1,239 trees in five test fields in two central California counties and resulted in estimates of CSD incidence of 58.9%, 4.2%, 0%, 22.4% and 28.6%. This indicated that CSD incidence is at significant levels in some citrus orchards. Further, the infection rates in the high incidence groves (>20%) suggests tree to tree spread is occurring. This data is being used to further analyze spread patterns of CSD and to identify the leafhopper vector responsible for this spread. Our study demonstrated the utility of real time PCR as a new tool to assess CSD incidence. Further, since CSD shares some similar symptomologies with Huanglongbing (HLB) or greening, another phloem-limited prokaryote, the real time assay for CSD will be important to include when suspect samples are tested for HLB in areas where CSD is known to occur.