First report of Calonectria henricotiae causing box blight in Spain

Box blight is caused by two closely related fungi, Calonectria pseudonaviculata (Cps) and/or C. henricotiae (Che) (Gehesquière et al., 2016). Cps is widespread globally (Daughtrey, 2019) while Che is limited to eight central European countries (Hong, 2023). To determine whether Che is present in southern Europe, we visited ten large boxwood gardens and street plantings in Madrid and Toledo, Spain and in Bordeaux, Lyon and Sainte-Foy-la-Grande, France. Boxwood (Buxus spp.) twigs with black streaks (Figure 1) were only observed in Madrid, Toledo, and Sainte-Foy-la-Grande. Diseased twigs were collected in sealed triple bags and brought to the laboratory under the USDA permit (P526P-21-05960). After surface sterilization with 10% bleach for two minutes and three rinses in deionised water, twigs were placed on a mesh with wet paper towels underneath in closed plastic containers at 23°C and wetted with sterile distilled water every two days until white mould appeared on leaves (Figure 2). The mould was suspended in 15 µL sterile distilled water then transferred onto a glass slide for microscopy. Following observations of rod-shaped conidia, typical of Calonectria spp., 1-µL drops of the suspensions were pipetted from slides onto acidified potato dextrose agar (PDA) for isolation (Kong et al., 2023). Clean colonies were only obtained from the Madrid and Sainte-Foy-la-Grande samples (Figure 3) and were subcultured on regular PDA. Colony PCR (Kong et al., 2005) was performed from a total of 159 colonies grown from three-day PDA cultures, using Cps- and Che-specific primers (Guo & Pooler, 2021). Also included in the PCR were three French isolates from the ANSES collection: LSVM0960 collected by Department Île-de-France in 2013, LSVM1007 by Department Meurthe-et-Moselle in 2014, and LSVM1235 by Department Finistère in 2015. All 99 isolates from Spain were identified as Che while 63 isolates from France were Cps. Seven Spanish and nine French isolates were subcultured in potato dextrose broth for DNA extraction. Their identities were confirmed by PCR of the DNA (Figure 2) and PCR product sequencing (GenBank Accession Nos. PP632113-19 for Che isolates and PP632120-28 for Cps isolates) with the same primer pairs. The same isolates were tested for temperature tolerance. The Spanish isolates had an average colony diameter of 1.6 cm in PDA at 28.5°C for 10 days, while the French isolates did not grow under the same conditions (Figure 4), further confirming their identities (Gehesquière et al., 2016). To our knowledge, this is the first report of Calonectria henricotiae causing box blight in Spain. Knowing the actual distribution of the fungus is critical to preventing its further spreading to other countries and areas currently not affected by this pathogen, promoting safe plant trade. The work was funded by the USDA National Institute of Food and Agriculture - Specialty Crop Research Initiative (2020-51181-32135).