Development and validation of a Loop-Mediated Isothermal Amplification technique (LAMP) for the detection of Spiroplasma citri, the causal agent of citrus stubborn disease

Authors: DRAIS, MOUNIRA - Mediterranean Agronomic Institue Of Bari (MAI-BARI); MAHESHWARI, YOGITA - Foreign Agricultural Service (FAS, USDA); SELVARAJ, VIJAY - Foreign Agricultural Service (FAS, USDA); VARVARO, LEONARDO - University Of Tuscia; Yokomi, Raymond - Ray; DJELOUAH, KHALED - Mediterranean Agronomic Institue Of Bari (MAI-BARI)

Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2019
Publication Date: 5/7/2019
Citation: Drais, M.D., Maheshwari, Y., Selvaraj, V., Varvaro, L., Yokomi, R.K., Djelouah, K. 2019. Development and validation of a Loop-Mediated Isothermal Amplification technique (LAMP) for the detection of Spiroplasma citri, the causal agent of citrus stubborn disease. European Journal of Plant Pathology. 155(1):125-134. https://doi.org/10.1007/s10658-019-01755-6.
DOI: https://doi.org/10.1007/s10658-019-01755-6

Interpretive Summary: Citrus stubborn disease (CSD) is a bacterial disease caused by Spiroplasma citri. CSD-affected trees are stunted, and low yielding with reduced fruit quality. However, S. citri spread by leafhopper vectors is slow such that good fertilization and horticultural care may offset CSD yield losses. A rapid and simple test to accurately detect S. citri-infected trees is needed to initiate mitigation actions and to distinguish CSD from Huanglongbing (HLB), a devastating citrus disease. To this end, a Loop-Mediated Isothermal Amplification technique (LAMP) was developed to detect S. citri, targeting the spiralin gene. LAMP is a simple, cost-effective technique for detection of specific DNA sequences without thermal cycling and can be carried out using a small portable instrument. The protocol for S. citri LAMP detection was conducted using the BioRanger device and was optimized to use crude plant extracts samples to facilitate on-site field testing. The LAMP assay detected DNA from S. citri to a level of 100 fg/µl with no inhibition by crude plant extract. The LAMP assay was validated with field samples with detection efficiency and percentage yes/no calls comparable to that obtained by real time PCR conducted with DNA extracted and purified from the same sample. The LAMP procedure allows growers, pest control, or diagnostic services to rapidly test for S. citri in the field.

Technical Abstract: Citrus stubborn disease (CSD) is caused by a Gram-positive bacterium, Spiroplasma citri, and is an endemic, but manageable, citrus disease in California. CSD-affected trees are low yielding but the current problem is that its symptoms are similar to and easily mistaken for Huanglongbing (HLB), a devastating citrus disease controlled by quarantines and eradication. Therefore, a rapid and simple test for S. citri is needed to readily distinguish CSD from HLB. To this end, a Loop-Mediated Isothermal Amplification technique (LAMP) was developed to detect S. citri, targeting the spiralin gene. The protocol was optimized for crude plant extracts from infected trees to allow on-site field testing. The LAMP assay showed high specificity to S. citri and detected 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, it performed well in field validations using a portable BioRanger device with crude citrus extracts. The LAMP assay showed detection efficiency and percentage yes/no calls similar to that obtained by real time PCR conducted with DNA extracted and purified from the same sample. The LAMP procedure allows growers, pest control, or diagnostic services to rapidly test for S. citri in the field without a laboratory or DNA purification.