First report: Co-infection of (sweetbox) by and causes a foliar disease of sweetbox in Pennsylvania.

Sweetbox () are high value ornamental shrubs susceptible to disease caused by () and () (Malapi-Wight et al. 2016; Salgado-Salazar et al. 2019). In July 2018, 18-month old sweetbox with leaf spots and defoliation were observed in a residential landscape in Lancaster County, Pennsylvania. Small tan leaf spots grew to cover half of the leaf, developing a concentric banding with dark brown rings and a yellow halo (Sup. Doc. 1: Sup. Fig. 1). The symptoms agreed with those of disease of sweetbox reported from Washington D.C. (Salgado-Salazar et al. 2019). Diseased plants were located ~1.5 m from with boxwood blight. Morphological and genetic characterization of isolated fungi and pathogenicity tests followed Salgado-Salazar et al. (2019) (Sup. Doc. 2). White to salmon pink spore masses developed on the abaxial leaf surface after humid chamber incubation. Two distinct fungal cultures were recovered (JAC 18-61, JAC 18-79) on potato dextrose agar (Fisher Scientific, Pittsburg, PA). JAC 18-61 presented cultural and morphological characteristics as described for (Crous et al. 2002). JAC 18-79 produced flat, filamentous, light salmon colonies with tan centers and white filiform borders containing pale pink sporodochia, verticillate and simple conidiophores (x̄: 61.8 ± 20.12 µm, N = 20) with lateral, cylindrical phialides (x̄ = 18.1 ± 5.83 x 2.4 ± 0.7 µm, N = 20), and ellipsoid, hyaline conidia without septa (x̄ = 15.2 ± 1.9 x 3.3 ± 0.7 µm, N = 20). Sexual structures and chlamydospores were not observed. The characteristics of JAC 18-79 agree with those reported for (Salgado-Salazar et al. 2019). Bidirectional sequencing of the ITS, beta-TUB, and RPB1 and RPB2 regions was performed as described (Salgado-Salazar et al. 2019). BLASTn comparisons against NCBI GenBank revealed JAC 18-61 sequences (MT318150 and MT328399) shared 100% identity with sequences (JX535321 and JX535307 from isolate CB002). Sequences from JAC 18-79 (MT318151, MT341237 to MT341239) were 100% identical to sequences (MH892596, MH936775, MH936703 from isolate JAC 16-20 and JF832909, isolate CBS 128674). The genome of JAC 18-79 was sequenced and yielded an assembly of 26.3 Mb (204 contigs > 1000 bases, N50 = 264.3 kb, 92x coverage, JABAHV0000000000) that contained the MAT1-2 mating-type idiomorph and shared 98.9% similarity with BPI910731. Isolate JAC 18-61 () caused lesions on wounded and unwounded sweetbox and boxwood leaves (Sup. Table 1). In general, JAC 18-79 () infected only wounded leaves of both hosts; however, in one trial, one unwounded sweetbox and two unwounded boxwood plants developed lesions, possibly due to the presence of natural wounds. Control plants did not develop symptoms. These results diverge to some degree from previous reports of infecting unwounded sweetbox and not infecting wounded boxwood (Salgado-Salazar et al. 2019). These results indicate that virulence variation among isolates might occur. Plating of symptomatic tissue and examination of spores fulfilled Koch's postulates for both pathogens. To our knowledge, this is the first report of blight on sweetbox in Pennsylvania, and the second U.S. report of the disease. This is also the first report of co-infection of and on diseased sweetbox foliage. Given the capacity of to infect both sweetbox and boxwood, inspection for on both hosts is advisable.