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

Abstract

Pecan bacterial leaf scorch (PBLS), caused by Xylella fastidiosa has been reported in Georgia, Louisiana, Texas, New Mexico, Arizona, and California. Accurate methods are critical for the early detection of X. fastidiosa, but the validation of current diagnostic tools for pecan has yet to be investigated. Collected petioles, leaflets, and shoots from pecan and other Carya relatives in Texas, Indiana, and Georgia were used as tissue samples, and to isolate crude xylem sap and gDNA for side-by-side testing using immunological (ELISA) and molecular-based assays [traditional PCR and real-time quantitative PCR (qPCR)]. Isolated crude sap was found to be the most reliable template for ELISA diagnostics. X. fastidiosa-specific genes were amplified with previously published PCR primer sets; however, they revealed non-specific binding. New Xylella-specific primers were subsequently generated and validated using infected tissue from pecan and related Carya species. Two new primer sets (NMU3 and 5) produced expected amplicons specific to X. fastidiosa but did not amplify any non-specific bands of the pecan gDNA. When compared to that of total gDNA as the template in PCR reactions, diluted crude sap was found to be an efficient way to detect X. fastidiosa in pecan petioles. A novel TaqMan qPCR assay was also developed for the detection of X. fastidiosa. The results of the qPCR experiments were equivalent to the traditional PCR amplification when crude sap was used as the template. Comparative PCR analysis confirms that the PCR protocol outlined in this study can be replicated across different laboratories.

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Acknowledgments

This article reports the results of research only. Mention of a trademark or proprietary product is solely for the purpose of providing specific information and does not constitute a guarantee or warranty of the product by the US Department of Agriculture or Texas A&M AgriLife Extension and does not imply its approval to the exclusion of other products that may also be suitable.

Funding

This work was supported by USDA-ARS CRIS 3091–21000-042-00D “Management of the National Collection of Carya Genetic Resources and Associated Information”; USDA-ARS CRIS 6042–21220–012-00-D “Mitigating Alternate Bearing of Pecan”; National Plant Germplasm System Grant 58–3091–6-022 “Screening Xylella fastidiosa in the USDA ARS National Collection of Genetic Resources for Carya”; and the Southern Integrative Pest Management (IPM) Center Program (project #1702922).

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Correspondence to Xinwang Wang or Young-Ki Jo.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. This research did not involve human participants or animals.

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figure11

Supp. Figure 1 Optimization of annealing temperature for PCR amplification using previously designed primers. The three previously designed Xylella fastidiosa-specific primer sets, S-S-X.fas-0838-a-S-21/ S-S-X.fas-1439-a-A-19, RST31/RST33 and HL-5/HL-6 were used to amplify X. fastidiosa target DNA. Three gDNA samples were isolated from three different petioles of a pecan tree (cv. Cape Fear), where the presence of X. fastidiosa was confirmed. Three different annealing temperatures, 55 °C, 58 °C, and 60 °C, are compared for PCR yield and specificity. Amplicons were separated on a 1.5% agarose gel stained with ethidium bromide. Arrows point to the X. fastidiosa amplicon. M = O’GeneRuler Express Ladder, + = positive control (gDNA of X. fastidiosa subsp. multiplex), − = non-template control (nuclease-free dH2O). (PNG 1346 kb)

figure12

Supp. Figure 2 The NMU3 primer set was validated for specificity by screening against the purified DNA of 13 bacterial isolates different from Xylella fastidiosa. A PCR screen using the NMU3 primer set against 13 bacterial isolates did not result in non-specific bands, indicating this primer set has a high specificity to X. fastidiosa. Arrows point to X. fastidiosa amplicon. M = Low Range GeneRuler DNA Ladder, + = positive control (gDNA of X. fastidiosa), − = non-template control (nuclease-free dH2O). (PNG 1138 kb)

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ESM 3

Alignment of HL (hypothetical protein) gene of Xylella fastidiosa isolates used in this study. (XLSX 26 kb)

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Hilton, A., Wang, X., Zhang, M. et al. Improved methods for detecting Xylella fastidiosa in pecan and related Carya species. Eur J Plant Pathol (2020). https://doi.org/10.1007/s10658-020-02050-5

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Keywords

  • Diagnostics
  • Pecan bacterial leaf scorch
  • ELISA
  • PCR
  • qPCR