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Detection of Xanthomonas citri pv. viticola on grapevine by real-time PCR and BIO-PCR using primers designed from pathogenicity and xanthomonadin gene sequences

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Abstract

Grapevine bacterial canker caused by Xanthomonas citri pv. viticola (X. campestris pv. viticola) (Xcv), was detected in Brazil in 1998 and is currently regarded as a quarantine disease with limited distribution in the country. To improve sensitivity and speed in the detection of Xcv in asymptomatic grapevines, two pairs of primers were designed, targeting sequences of a pathogenicity gene (hrpB) and the xanthomonadin coding cluster. Both pairs were tested in conventional PCR (cPCR) and real-time PCR (qPCR) formats. Primers targeting the hrpB gene showed cross reactions with other Xanthomonas spp. but were effective for use in both cPCR and qPCR, whereas primers for the xanthomonadin gene were highly specific for Xcv but showed low efficiency in qPCR. Enrichment of plant extracts in semi-selective medium before qPCR allowed a significant increase in sensitivity when compared to total DNA extraction, making it possible to detect as low as 101 CFU ml-1. Under natural infection conditions, symptomatic and asymptomatic grapevines were tested by qPCR with hrpB primers and cPCR with xanthomonadin primers. In both cases, plant extracts were enriched for 36-72h. Xcv was detected in all symptomatic samples by qPCR and the result was confirmed by cPCR. For the asymptomatic samples, Xcv was detected in 93.4% with qPCR and in 89.5% with cPCR. These two methods offer advantages in terms of sensitivity and specificity, and they could be useful in quarantine programs, certification of grapevine propagating material and detection of inoculum sources in alternative hosts, contributing to the prevention of pathogen spread to disease-free areas.

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Acknowledgments

We thank the Brazilian National Council for Scientific and Technological Development (CNPq) and CAPES- MEC for doctoral scholarships awarded to JGA Villela and CNPq for a research fellowship to MASV Ferreira (Grant #310860/2016-4). The work was financed by Sistema Embrapa de Gestão (SEG), Projects: 12.13.06.011.00.00 - Management for bacterial canker and decline of grapevine in the São Francisco Valley and 02.13.03.006.00.00 - Developing of new cultivars to the viability and maintenance of Brazilian viticulture - Stage III.

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Authors and Affiliations

  1. Departamento de Fitopatologia, Universidade de Brasília, Brasília, DF, Brazil

    João Gilberto A. Villela, Mauricio Rossato & Marisa A. S. V. Ferreira

  2. Embrapa Uva e Vinho, Bento Gonçalves, RS, Brazil

    Patricia Ritschel & Kétini M. S. Baccin

  3. Embrapa Semiárido, Petrolina, PE, Brazil

    Maria Angélica G. Barbosa

  4. Universidade de Caxias do Sul, Caxias do Sul, RS, Brazil

    Kétini M. S. Baccin

  5. Embrapa Uva e Vinho, EVT, Jales, SP, Brazil

    João D. G. Maia

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  1. João Gilberto A. Villela
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  3. Maria Angélica G. Barbosa
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Correspondence to Marisa A. S. V. Ferreira.

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Table S1

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Fig. S1

Localization of Xpig primers in an alignment of the xanthomonadin coding cluster of Xanthomonas citri pv. viticola (accession CBZT010000000) and Xanthomonas campestris pv. mangiferaeindicae (KF991092). (A) General view of the Xpig forward primer position in an intergenic region of X. citri pv. viticola, with a transposase insertion in X. campestris pv. mangiferaeindicae. (B) Zoomed view of Xpig primer location showing no identity with X. campestris pv. mangiferaeindicae. (PNG 760 kb)

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Fig. S2

Real-time PCR (SYBR® Green) amplification curves with primers Xcv18F/19R for detection of (a) genomic DNA (1.5 ng μl-1 to 150 fg μl-1) and (b) cell suspension (108 to 1 CFU ml -1) from Xanthomonas citri pv. viticola (UnB 1188). Legend (a): A - water negative control; B - 1,5 ng μl-1; C - 150 pg μl-1; D - 15 pg μl-1; E- 1,5 pg μl-1; F- 150 fg μl-1. Legend (b): A - water negative control; B- 108 UFC ml-1; C- 107 UFC ml-1; D- 106 UFC ml-1; E- 105 UFC ml-1; F- 104 UFC ml-1; G- 103 UFC ml-1; H- 102 UFC ml-1 (PNG 1378 kb)

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Villela, J.G.A., Ritschel, P., Barbosa, M.A.G. et al. Detection of Xanthomonas citri pv. viticola on grapevine by real-time PCR and BIO-PCR using primers designed from pathogenicity and xanthomonadin gene sequences. Eur J Plant Pathol 155, 445–459 (2019). https://doi.org/10.1007/s10658-019-01779-y

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