First Report of Botryosphaeria dothidea Causing Stem Canker and Plant Death in Malosma laurina in Southern California

Malosma laurina (Nutt.) Abrams is a deep-rooted shrub growing in coastal foothills and mountains of southern California. M. laurina frequently occurs in mixed chaparral stands and co-dominates the landscape. At such sites, during acute drought, deep-rooted M. laurina remains hydrated and largely unaffected (Venturas et al. 2016). However, in January 2015 after several years of chronic drought, we observed M. laurina dieback in coastal exposures of the Santa Monica Mountains (SMM). Branch dieback was associated with extensive stem-canker formation, including the abrupt appearance of newly dead leaves in terminal regions of large branches distal to cankers, and retention of green leaves proximal to cankers. Whole plant death was higher at 10 low elevation (low rainfall) sites than at 10 high elevation (high rainfall) sites in the SMM. By fall 2016, 62% whole-plant mortality was observed in a low elevation population near Malibu (n = 314). Stems with cankers were collected in May 2015, surface sterilized (EtOH 1 min then 10% bleach 10 min), and ∼1 mm³ of xylem tissue aseptically transferred to potato dextrose agar (Difco) and incubated at 25°C. Black pycnidia with a mean diameter of 182 ± 12.9 µm, 1 SE, n = 12, with conidia 26.9 ± 0.51 µm long and 7.0 ± 0.15 µm wide, n = 24, were observed erupting from the surface of outer bark on recently dead branches in the field (34°02′20″N, 118°42′29″W). Fungal cultures were fast-growing, gray, and fuzzy for all canker samples collected from the field (n = 20). We used an isolate, #PE8, to PCR amplify and sequence the internal transcribed spacer, elongation factor 1α, and beta tubulin 2 using primer pairs ITS1 and ITS4, EF1-728F and EF1-986R, and BT2a and Bt2b, respectively (Slippers et al. 2004). DNA sequence identified Botyrosphaeria dothidea (Moug. ex Fr.) Ces. & De Not. with greater than 99% sequence match to the type isolate for all three genes examined using BLAST searches of the GenBank database. Our GenBank accession numbers are MF597795, MF597793, and MF597794. Koch’s postulates were confirmed by development of stem canker formation with distal leaf death after a 1.5 mm³ PDA plug containing hyphae from colony peripheries of isolate #PE8 were aseptically inoculated into stem xylem of healthy saplings (n = 16). Eight weeks after inoculation, B. dothidea was reisolated from 80% of inoculated saplings but 0% of controls, inoculated only with sterile PDA. B. dothidea is known to be an endophytic opportunistic pathogen and one that can spread undetected until severe host plant stress occurs (Marsberg et al. 2017). In our case, the opportunistic state of the fungal pathogen was expressed during California’s protracted 5-year drought from 2012 to 2016. This is particularly significant because M. laurina is an extremely deep-rooted keystone member of coastal chaparral shrub communities, tapping deep moisture reserves during dry summers, providing slope stability, and displaying unusual resilience to periodic wildfire and acute drought events (Venturas et al. 2016). It now appears that M. laurina may be less resilient in the presence of chronic drought. To our knowledge, this is the first report of B. dothidea infecting M. laurina.