A comparison of real-time PCR protocols for the quantitative monitoring of asymptomatic olive infections by Verticillium dahliae pathotypes

Early, specific, and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity, and efficiency of eight real-time quantitative polymerase chain reaction protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems, and leaves. Results showed that some of those protocols were not specific for V. dahliae or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the -tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae. The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D V. dahliae DNA were measured in olive stems, whereas the average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as helpful tools in breeding resistance to V. dahliae in olive. © 2013 The American Phytopathological Society

Emergence of Xylella fastidiosa in Balearic Islands, Spain: Current situation

During official surveys in late autumn 2016 in Mallorca Island, Spain, the bacterium was first detected in a garden center near the locality of Manacor. Since then, as of April 24, 2017, a total of 189 positive samples in 11 different host species have been found in different foci in Mallorca (124), Menorca (16) and Ibiza (49) islands. Sequence analysis of the RNA polymerase sigma 70 factor sequence and MLST typing revealed the presence of X. fastidiosa subsp. fastidiosa ST1 and X. fastidiosa subsp. multiplex ST6*(a new ST with the closest being ST6) and ST7 in Mallorca island, X. fastidiosa subsp. multiplex ST6* in Menorca island and X. fastidiosa subsp. pauca ST80 (a new ST) in Ibiza island. Polygala myrtifolia was found to be infected by all subspecies and ST types. Altogether, these results suggest that the emergence of X. fastidiosa in Balearic Islands is likely due to several introduction events of diverse strains from different subspecies. Eradication measures were taken in the garden center according to the Spanish contingency plan and EU legislation. Following the Commission Decision 2015/789/EU if establishing a radius of 10 km to delimit the buffer zone for each infected foci 80%, 50% and 90% of the territory of Mallorca, Menorca and Ibiza islands, respectively, are considered as demarcated areas. Consequently, the best strategy to control the different outbreaks is under study. Acknowledgment This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N. 635646 “Pest Organisms Threatening Europe POnTE” and grant agreement N. 727987 “Xylella fastidiosa Active Containment Through a multidisciplinary- Oriented Research Strategy XF-ACTORS”. Bibliography Minsavage GV, Thompson CM, Hopkins DL, Leite RMVBC, & Stall RE, 1994. Development of a polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology 84, 456–461. Yuan X, Morano L, Bromley R, Spring-Pearson S, Stouthamer R, & Nunney L, 2010. Multilocus sequence typing of Xylella fastidiosa causing Pierce's disease and oleander leaf scorch in the United States. Phytopathology 100, 601-11