ABSTRACT
Strawberry production in California represents over 38,000 acres with an annual farm value of $1.99 billion. Strawberry dieback was observed in February of 2021 in the Salinas Valley in central California. Disease symptoms included dead and dying 'Maverick' strawberry plants with necrotic lesions and black discoloration of the crown, root cortex, epidermis, and vascular tissues. Disease incidence was estimated to be 60% of a 20-acre field. The causal agent was isolated from five randomly selected symptomatic plants by surface disinfesting symptomatic crowns and roots in 1% sodium hypochlorite for 30 s, rinsed twice in sterile water for 30 s then placed on 3.7% potato dextrose agar (PDA) Petri dishes amended with 100 mg/L streptomycin, and then incubated for 7 days at 24°C under a 12-h photoperiod. Consistent white cottony fungal colonies were hyphal tip transferred to fresh PDA dishes and incubated as above for morphological and genetic comparisons. Black acervuli developed 7 to 9 days after incubation. Conidia were ellipsoidal, measuring 25 to 30 × 7.5 to 10 µm (average 26.8 × 9.2 µm, n = 30), with five cells. Apical and basal cells were hyaline, and the three median cells were versicolorous brown, with a single, straight, centric basal appendage and 3 to 4 flexuous apical appendages. Colony diameter averaged 90 mm in 7 days. Based on colony and conidial characters, the fungus was tentatively identified as a species of Neopestalotiopsis (Maharachchikumbura et al. 2014). Total genomic DNA was extracted from three axenic cultures using the Invitrogen Easy-DNA kit. Three genetic loci were PCR amplified and sequenced: internal transcribed spacer (ITS), beta-tubulin (BT), and translation elongation factor 1-alpha (TEF) utilizing the primer pairs ITS1/ITS4, T1/Bt2a, and EF1-688F/EF1-1251R, respectively (White et al. 1990, O'Donnell and Cigelnik 1997, Glass and Donaldson 1995, and Alves et al. 2008). A BLASTn search of NCBI showed 99.6% identity (495/497 bases; OM942910-OM942912) with the type specimen of Neopestalotiopsis rosae CBS 101057 for the ITS locus. Both BT (765/765 bases, OM964802-OM964804) and TEF (475/475 bases; OM964799-OM964801) sequences were 100% identical to CBS 101057. Conidia of isolate PAR027 were scraped from the surface of 14-day-old PDA Petri dishes and inoculated (1 × 106 spores/mL: 2 mL/plant) to four apparently healthy strawberry transplant roots of the cultivar 'Monterey' in 'sunshine mix' potting soil. Two control plants were inoculated with sterile water. The experiment was conducted twice. Strawberry plants were maintained in a hoop house for four weeks, after which dieback and wilt symptoms resembled the symptoms observed in the field. Control plants remained asymptomatic and no pathogens were isolated. Fungal recovery from inoculated plants morphologically matched the original inoculum; thus, Koch's postulates was satisfied. To our knowledge, this is the first report of N. rosae causing crown and root rot disease of strawberry in California. Previously, N. rosae has been reported to cause serious decline of strawberry plants in Florida and several countries (Baggio et al. 2021, Rebollar-Alviter et al. 2020, Wu et al. 2021, Sun et al. 2021). Correct identification of the causal agent provides a proper foundation to identify control strategies for this emerging disease, which has the potential to become a significant problem for strawberry growers in the Salinas Valley of California.
