Ramularia mali associated with symptoms of dry lenticel rot, an emerging postharvest disease on apples in Italy.

Abstract

Ramularia mali Videira & Crous is an emerging postharvest pathogen on apple (Malus × domestica Borkh.) in Italy and other apple producing countries (Prencipe et al. 2023). After 3 to 6 months of cold storage at 1 - 2 °C and low oxygen levels of 0.5 - 2 %, lenticels show black-brown speckled dry rot of 1 mm - 5 mm in diameter, without colonizing underlying tissue. The most affected cultivar (cv.) in South Tyrol (northern Italy) is Golden Delicious and postharvest losses due to characteristic lenticel spots range from 10 % to above 50 %. Four symptomatic fruits, originating from two orchards (Latsch/Laces and Bozen/Bolzano; South Tyrol, Italy), respectively, were sampled after cold storage (= ultra-low oxygen; 0.5 % O2 and 1 °C). After surface disinfection with 70 % EtOH for 1 min, sixteen explants from lenticel spots were cultivated on potato dextrose agar (PDA) at 25 °C. Two isolates, morphologically identified as Ramularia sp., were sequenced and showed high identities to R. mali type culture CBS 129581: 100 % and 99.31 % identity for ITS region (MH865432); 94.66 % and 91.41 % for TEF-1α (KJ504693); 97.22% and 97.40% for RpbII (KJ504649). Isolates were cultivated at 25 °C for 2 weeks and conidia were harvested with 3.0 mL 0.05 % Tween®20. Inoculation was performed in triplicate on 5-month cold stored fruits cv. Golden Delicious. After surface disinfection for 1 min with cotton swabs, which were immersed in 70 % EtOH, 10 µL spore suspension of each isolate (8.50 × 107 spores mL-1 in 0.05 % Tween®20) were injected horizontally beneath the epidermis with a syringe (Hamilton® model 710N). Also, a mixture of both isolates was used. Controls were carried out with 0.05 % Tween®20 only. Apples were stored either at 9 °C in the dark or at 1°C and 0.5 % oxygen for 4 months. First symptoms were observed for both spore concentrations after 2 weeks at 9 °C. The injection pathway changed to a brownish color, whereas the control did not show any change (Fig. 1). Final evaluation was carried out after 4 months, but the fruits did not show further symptom development. Fruits stored at 1°C for 5 months were simultaneously evaluated, confirming that the pathogen invaded the tissue surrounding the injection site, without penetrating deeper into the fruit flesh. (Fig. 2). Reisolation from artificially infected apples was successfully achieved, and sequence analysis was performed on the DNA extracts from the obtained isolates. Concatenated sequences of ITS (deposited to GenBank under the accession numbers: PP439643 -PP439647), TEF-1α (PP480231-PP480235), and RbpII (PP480226-PP480230) were subjected to multi-locus sequence analysis. References sequences of R. nyssicola CBS 127665, R. collo-cygni CBS 101181, R. vizellae CBS 115981, R. eucalypti CBS 120726, R. hydrangeae-macrophyllae CBS 122272, R. glennii CBS 129441 and R. mali CBS 129581 included and aligned by the CLUSTALW algorithm within the software Geneious® 11.1.5 (Biomatters Inc., New Zealand). Phylogeny was reconstructed with MEGAX (Version 10.2.6) (Kumar et al. 2018) based on the Maximum Likelihood (ML) algorithm (Fig. 3). Isolates from artificially infected fruit clustered with the R. mali type culture. Although Gianetti et al. (2012) and Lindner (2013), respectively, first described Ramularia sp. as a postharvest pathogen on apple, the present study demonstrated the reproduction of lenticel dry rot symptoms by R. mali.