Improved real-time PCR protocol for the accurate detection and quantification of Rosellinia necatrix in avocado orchards

[Aims] This study aims to develop and validate a new molecular method of detection and quantification of Rosellinia necatrix fungus in soil samples and compare it with conventional methods.[Methods] We collected 40 soil and root samples (one as negative control) from the soil around avocado trees. The root samples were checked for typical symptoms of R. necatrix and the pathogen was identified using the conventional method of plate culture. These results were then corroborated using a new molecular method of detection and quantification of R. necatrix in soil samples, and a duplex TaqMan qPCR protocol was designed that included an internal positive control to avoid the detection of false negatives.[Results] The molecular detection and quantification method was effective, sensitive and reliable for all 40 soil samples analysed, whereas, with traditional methods, the fungus was isolated in only 24 out of the 26 symptomatic roots from 40 avocado trees sampled. This improved methodology reduces the sample preparation time compared with previous studies, and provides a molecular tool for the reliable and accurate detection and quantification of R. necatrix in naturally infested avocado soils.[Conclusions] This technique could be applied for the rapid assessment of R. necatrix in soils at the pre-planting stage and evaluation of the efficacy of physical, chemical or biological control treatments.This study was partly supported by the Spanish Plan Nacional I + D + I Ministerio de Economía y Competitividad (AGL 2014-52518-C2-2-R). The research was also co-financed by FEDER funds (EU).Peer reviewe