Use of LAMP for Assessing Botrytis cinerea Colonization of Bunch Trash and Latent Infection of Berries in Grapevines.

Melissa Si Ammour,Giorgia Fedele,Vittorio Rossi,Eleonora Castaldo

doi:10.3390/plants9111538

Abstract

A real-time loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Botrytis cinerea in grapevine bunch trash, immature berries, and ripening berries. A simple method for the preparation of crude extracts of grape tissue was also developed for on-site LAMP analysis. When tested with 14 other fungal species frequently found in grapevines, the LAMP assay was specific and sensitive to a B. cinerea DNA quantity of 0.1 ng/µL. The sensitivity was further tested using bunch trash samples with B. cinerea colonization levels between 6 and 100% and with bulk-berry samples composed of 4 pathogen-free berries or 4 berries among which 25 to 100% had been inoculated with B. cinerea. The LAMP assay detected the lowest B. cinerea colonization level tested in bunch trash and in immature and mature berries in less than 20 min. In single-berry experiments, LAMP amplified B. cinerea DNA from all artificially inoculated individual immature and mature berries. No amplification occurred in B. cinerea-free material. The real-time LAMP assay has the potential to be used as a rapid on-site diagnostic tool for assessing B. cinerea colonization in bunch trash and B. cinerea latent infections in berries, which represent critical stages for decision-making about disease management.

Highlights

loop-mediated isothermal amplification (LAMP) assay assay was was evaluated evaluated in interms termsof ofspecificity specificityand andsensitivity sensitivity
Cinerea plus other fungi commonly present on grape bunches tested, When B. cinerea plus 14 other fungi commonly present on grape vinevine bunches werewere tested, the the assay was found to be specific for cinerea and was found to be sensitive to a LAMP assay was found to be specific for B. cinerea and was found to be sensitive to a B. cinerea
Primers to which a template DNA was added (3 μL); the LAMP mix and primers were provided with the kit

Summary

Introduction

Botrytis cinerea infects grapevines via several pathways [4], with one infection window between flowering to young cluster (corresponding to growth stages 53 and 73 [5], respectively), and a second infection window between veraison to berry maturity (growth stages 79 and 89, respectively). Conidia infect flowers through the styles, ovules, stamens, or petals, and infect young berries via the pedicel [4]. These infections cause blossom blight and the saprophytic colonization of “bunch trash” consisting of calyptras, tendrils, dead stamens, aborted flowers and berries [4,6]

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