Improved methods for detecting Xylella fastidiosa in pecan and related Carya species

Authors: HILTON, ANGELYN - Texas A&M University; Wang, Xinwang; Zhang, Minling; CERVANTES, KIM - New Mexico State University; FRENCH, JOSHUA - New Mexico State University; RANDALL, JENNIFER - New Mexico State University; Bock, Clive; Grauke, Larry; JO, YOUNG - Texas A&M University

Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2020
Publication Date: 6/30/2020
Citation: Hilton, A.E., Wang, X., Zhang, M., Cervantes, K., French, J., Randall, J.J., Bock, C.H., Grauke, L.J., Jo, Y.K. 2020. Improved methods for detecting Xylella fastidiosa in pecan and related Carya species. European Journal of Plant Pathology. https://doi.org/10.1007/s10658-020-02050-5.
DOI: https://doi.org/10.1007/s10658-020-02050-5

Interpretive Summary: Pecan bacterial leaf scorch (PBLS) is a widespread disease of pecan, caused by Xylella fastidiosa subsp. multiplex. The disease can cause leaves to drop and reduce nut quality. The main symptom is leaf scorch, which can be caused by many things other than this disease. Accurate detection methods are important and were refined in this research. Sap collected from shoots under pressure was better than ground or incubated leaf-parts for enzyme-linked immunosorbent assay (ELISA) tests. Sap must be diluted for polymerase chain reaction (PCR)-based assays. A previously reported PCR primer set (HL5/HL6) was found to bind to pecan genes, creating bands unrelated to the disease. A new primer (NMU3) based on the disease protein (HL) was developed, tested and found specific for X. fastidiosa. Sample preparation was critical for both ELISA and PCR based assays, and crude sap proved the most reliable template.

Technical Abstract: Pecan bacterial leaf scorch (PBLS), caused by Xylella fastidiosa subsp. multiplex, was first documented in Louisiana, and recently reported in Georgia, Arizona, New Mexico, California and Texas. Systemic infections in pecan (Carya illinoinensis) can result in severe defoliation as well as a reduction in nut weight and kernel quality. Accurate diagnostic methods are critical for pathogen detection and subsequent management of PBLS. Symptomatic and asymptomatic shoots were collected from pecan and other Carya relatives in Texas, Indiana and Georgia, and validated by immunological and polymerase chain reaction (PCR)-based assays. Diluted xylem sap and plant tissue samples were used for side-by-side testing. Sample preparation comparisons showed that the use of crude sap provided the optimal template in enzyme-linked immunosorbent assay (ELISA) diagnostics. The use of blocking agents did not significantly impact absorption measurements when determining presence of the bacterium by ELISA. X. fastidiosa-specific genes were amplified with previously published PCR primer sets, and their PCR amplicons were verified as X. fastidiosa. One primer set (HL5/HL6) based on the hypothetical protein (HL) revealed unspecific binding to the genome of pecan. Xylella-specific NMU primers based on the HL were subsequently generated and confirmed non-binding to the pecan genome or other bacterial species, demonstrating high specificity to X. fastidiosa. Pecan crude sap could be directly used as a template in PCR reactions but must be diluted for successful amplification to remediate inhibitory effects by other sap compounds. PCR analysis conducted in different labs confirmed that the protocols outlined in this study can be reproducible and reliable. The real-time PCR assay using a fluorogenic exonuclease (TaqMan) chemistry was tested for the detection of X. fastidiosa, but it could be negatively impacted by inhibitors present in pecan tissue. We evaluated multiple diagnostic methods that can be suitable for detection of X. fastidiosa in pecan and related Carya. Sample preparation was critical for both ELISA and PCR based assays, and crude sap proved the most reliable template. To assure correct diagnosis of PBLS, combined methods of testing multiple samples from an individual tree should be used.