First Report of Lasiodiplodia pseudotheobromae Causing Canker and Shoot Dieback on Apple in China

Abstract

In July 2017, apple trees (Malus pumila Mill.) with canker and shoot dieback symptoms on side branches were observed in several young orchards in Yantai, Shandong Province, China. Symptoms included shoot dieback, discoloration of xylem tissues, cankers, and sunken necrotic bark lesions, which surrounded the branches and resulted in the death of the branches. The disease is symptomatically different from canker diseases of apple earlier described. Small pieces (4 to 5 mm) of necrotic tissues from the shoot cankers were disinfected for 1 min in 75% ethanol, rinsed thrice with sterile distilled water, and plated onto potato dextrose agar (PDA) with 0.05% lactic acid. White colonies developed from symptomatic tissues but became dark gray mycelia with age. Pycnidia were produced on sterilized apple twigs on 2% water agar after incubation for 3 weeks at 25°C. Conidia were unicellular, ellipsoidal, initially hyaline, and became dark brown and developed a central septum with age. Conidia measured 21.3 to 31.9 × 12.4 to 16.9 µm (n = 50). On the basis of morphological characteristics, the isolates were identified as Lasiodiplodia sp., a member of the Botryosphaeriaceae family (Alves et al. 2008; Phillips et al. 2013). For further identification, total DNA was extracted from the mycelium of three monoconidial isolates (La1, La2, and La3). The rDNA internal transcribed spacer (ITS), a partial sequence of β-tubulin gene (TUB), and translation elongation factor 1-α gene (TEF1) were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, and EF1-688F/EF1-1251R, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). The sequences (GenBank accession nos. MK408640 to MK408642 for ITS, MK446722 to MK446724 for TUB, and MH829861 to MH829863 for TEF1) were aligned using BLASTn in GenBank, showing 99 to 100% homology with the sequences of ITS (EF622077), TUB (EU673111), and TEF1 (EF622057) from L. pseudotheobromae isolate CBS 116459 (Alves et al. 2008). The pathogenicity testing of three isolates was conducted using the wound inoculation method both on living apple trees and detached twigs. Thirty 2-year-old twigs were wounded with a scalpel on the phloem tissues and inoculated by placing 5-mm² agar plugs containing 2-day-old cultured hyphae or 10 μl of an inoculum suspension (10⁵ conidia/ml), which were then sealed with Parafilm. The same number of wounded twigs was inoculated with uncolonized PDA plugs or sterile water as controls. After 7 days, necrotic lesions (35 to 60 mm in diameter) developed around the inoculation points, which resembled the symptoms on infected apple shoots in the field. No symptoms were observed on the control twigs, and the pathogenicity test was conducted thrice. The pathogen was reisolated from symptomatic twigs and was reidentified as L. pseudotheobromae on morphological and molecular analysis. Although L. pseudotheobromae was previously reported on several forest and fruit trees (Alves et al. 2008; Awan et al. 2016), to our knowledge, this is the first report of the pathogen causing canker and shoot dieback on apple in China. This report is important for the new research aiming at management of apple canker disease.