|DRAIS, MOUNIRA INAS - University Of Tuscia|
|MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA)|
|SELVARAJ, YIJAYANANDRAJ - Foreign Agricultural Service (FAS, USDA)|
|VARVARO, LEONARDO - University Of Tuscia|
|Yokomi, Raymond - Ray|
|DJELOUAH, KHALED - Mediterranean Agronomic Institue Of Bari (MAI-BARI)|
Submitted to: Conference of International Organization of Citrus Virologists
Publication Type: Abstract Only
Publication Acceptance Date: 2/8/2019
Publication Date: 3/10/2019
Citation: Drais, M., Maheshwari, Y., Selvaraj, Y., Varvaro, L., Yokomi, R.K., Djelouah, K. 2019. Progress on the development of a field-use optical sensor for screening of citrus pathogens in California. Conference of International Organization of Citrus Virologists. p. 7-24.
Technical Abstract: Citrus stubborn disease (CSD) is caused by the bacterium, Spiroplasma citri. CDS-affected trees are stunted, low yielding with reduced fruit quality. This research developed innovative serological and molecular tools for on-site detection of S. citri to facilitate large-scale pathogen detection surveys. Firstly, a Direct Tissue Blot ImmunoAssay (DTBIA) that uses a polyclonal antiserum to S. citri was developed and validated in a field survey of 112 citrus trees sampled from the Mitidja region, the main citrus-growing region of Algeria. This survey detected two S. citri-infected trees in two varietal collections and results were confirmed by molecular assays of a spiralin gene sequence from DNA extracted from infected field trees. The resulting sequence shared 99% identity with the Iranian Fasa I strain (GenBank Accession No.FJ755921.1). Secondly, a Loop-Mediated Isothermal Amplification technique (LAMP) was developed. The LAMP assay targeted the spiralin gene and was optimized for crude plant extracts. The LAMP assay showed high specificity and detected S. citri DNA to a level of 100 fg/µl with no inhibition by crude plant extracts. Although the LAMP assay was 9 times less sensitive than qPCR with purified DNA templates in dilution series lab tests, the Android-based portable isothermal RNA/DNA amplification system yielded comparable yes/no calls for S. citri infection with that of qPCR with purified DNA from the same trees in field validations. These methods allow growers, pest control or diagnostic services to rapidly test for S. citri in the field without a laboratory or DNA purification.