First Report of Phytophthora occultans Causing Dieback of Buxus sempervirens in the Czech Republic.

Abstract

In Nov 2011, and then recurrently since Sep 2020, an extensive decline has been recorded in boxwood (Buxus sempervirens), sometimes with several dozens of damaged individuals planted in private gardens and public areas and purchased in amateur markets in the Czech Republic. The leaves of the plants first showed orange-bronze discoloration, then dried and fell off, and the affected plants died. The roots, collars and stems of these plants had dark brown to black necrotic lesions. Phytophthora occultans Man in 't Veld & K. Rosend. was consistently isolated on selective medium PARPNH (Jung et al. 1996) directly from segments of symptomatic collar tissues and from rhododendron leaf pieces used to bait excised roots. On 20% V8 agar (V8A) and on carrot agar (CA), colonies had a stellate pattern. Radial growth at 25°C was 9.4 mm/day on V8A and 5.3 mm/day on CA. The cardinal growth temperatures were min. 7°C, optimum 25 to 27°C, and max. 32°C. The isolates were homothallic and produced on CA colorless globose oogonia ranging from 25.4 to 36.4 µm (n = 40) in diam. Oospores were slightly aplerotic and measured (n = 40) 22.5 to 31.9 µm in diam., with a 0.9 to 1.5 µm thick wall. The antheridia were predominantly paragynous and averaged 11.5 × 9.9 µm (height × width, n = 40). Noncaducous sporangia were obpyriform, ovoid, elongated to irregular and semipapillate, sometimes bipapillate and measured (n = 40) 31.4 to 73.4 × 17.8 to 32.1 µm, and the L:B ratio was 1.9 to 2.0. Chlamydospores and hyphal swellings were not observed. The morphological characteristics resembled those described for P. occultans (Man in't Veld et al. 2015). The isolates were deposited in the Czech Collection of Phytopathogenic Oomycetes (CCPO) under accession nos. 551.11, 1158.20, 1176.21, 1201.21, 1218.21, 1236.21 and 1261.22. For molecular identification, the internal transcribed spacer (ITS) region, cytochrome oxidase subunit 1 gene (COX1), and translation elongation factor-1α (EF) gene from all isolates were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), COXF-CIT/COXR-CIT (Man in't Veld et al. 2015), and ELONGF1/ELONGR1 (Kroon et al. 2004), respectively. The resulting sequences of representative isolates P1158.20 and P1176.21 were deposited in GenBank (accession nos. MW750576 and OP326036 for ITS, ON862131 and OP313505 for COX1 and MW762616 and ON862132 for EF). BLASTn searches of GenBank, using the partial ITS, COX1, and EF sequences, revealed 100, 100, and 99% sequence identity, respectively, to P. occultans ex-holotype culture CBS101557 accessions JX978155, JX978156 and KF650770 (Man in't Veld et al. 2015). Concatenated sequences of the three genes were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11 (Tamura et al. 2021). The isolates were identified as P. occultans based on morphology and a multigene phylogenetic analysis. Koch´s postulates were confirmed by a soil infestation test. Healthy 2-year-old B. sempervirens plants were inoculated (9 plants per isolate and control, isolates no. 1158.20, 1176.21, 1261.22) with three 5-mm-diam. V8A mycelial plugs by inserting into the substrate near the collar. Control plants were treated with sterile agar plugs. All plants were kept in a greenhouse at 25°C and exposed to 24 h of flooding up to collar once a week. All inoculated plants showed wilting, collar lesions and root rot occurred after 21 days, while control plants remained healthy. The pathogen was reisolated from infected plants and confirmed by molecular identification. P. occultans was found for the first time in 1998 on Buxus sempervirens in the Netherlands and later in Belgium, the United Kingdom, Germany and Romania (Man in´t Veld et al. 2015, Nechwatal et al. 2014), as well as in the USA (Reeser et al. 2015, Gitto et al. 2018). This is the first report of P. occultans in the Czech Republic. This pathogen likely poses another significant threat to boxwood cultivation in addition to the previously invaded Cydalima perspectalis and Calonectria pseudonaviculata.