First report of Calonectria henricotiae causing box blight in Spain

Abstract

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 Cpsand Chespecific 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 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.