First Report of Botrytis cinerea Causing Leaf Spot on Strawberry in California

On 29 October 2018, a leaf spot disease was observed on strawberry leaves (Fragaria × ananassa Duch., cultivar Portola) on organically grown plants in high plastic tunnels in Santa Maria, California. Symptoms consisted of circular lesions measuring 2 to 3 cm in diameter, concentric rings with a darker brown outer ring and fungal sporulation in the center of the lesion. The diseased leaf sample was surface sterilized in 10% sodium hypochlorite for 1 min, rinsed thrice in sterile water, and placed on potato dextrose agar (PDA) after drying. The resulting culture produced conidia that were hyaline and round to ellipsoid (8 to 14 × 5 to 8 µm, n = 50) and sclerotia that were hard, dark, round to oblong, and measured 2 to 12 × 2 to 9 mm (n = 50). Based on its morphological characteristics in culture, the isolate was putatively identified as Botrytis cinerea (Hong et al. 2001; Jarvis 1977). The isolate produced an average of 133 sclerotia per Petri dish (100 × 15 mm; n = 3) with PDA, an uncommon feature of B. cinerea isolates from strawberry. Koch’s postulates were performed using two inoculation methods: (i) Botrytis agar plug on leaf; and (ii) conidial suspension on leaf. Leaf inoculations used a spore suspension containing 1 × 10⁶ conidia/ml and were replicated three times. Leaf inoculations using an agar plug or spore suspension were allowed to dry, misted with water, and covered with a plastic bag to maintain continuous moisture. Agar-plug inoculated leaves produced symptoms in 4 days, whereas the spore-suspension inoculations produced symptoms in 9 days. Leaf symptoms matched those observed in the field. The pathogen was reisolated from symptomatic tissue, and its morphological features matched those of the original culture. In both inoculation methods, the noninoculated controls did not result in any disease symptoms. Cross pathogenicity to leaves using an isolate of B. cinerea collected from fruit was successful in causing foliar disease, but symptoms lacked concentric rings. To determine the fungal species, total genomic DNA of five subcultures of the original field isolate was extracted using a DNeasy Plant Mini Kit (Qiagen). The heat shock protein (HSP60) (Hsp60for+/Hsp60 rev+ primers), RNA polymerase II-binding (RPB2) (RPB2for+/RPB2 rev+ primers), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (G3PDHfor+/G3PDH rev+ primers) genes were amplified (Staats et al. 2004). The PCR amplicons were purified and sequenced. The sequences were processed using BLAST in the National Center for Biotechnology, and results showed 100% homology with the HSP60 (MH796663), RPB2 (MH479932), and G3PDH (MH479930) sequence for isolates of B. cinerea. The sequences were deposited in GenBank with accession numbers MK919494 (HSP60), MK919495 (RPB2), and MK919496 (G3PDH). B. cinerea is a common pathogen of strawberry fruit and can cause gray mold, postharvest rot, and dry crown rot but has not been reported as causing a leaf spot. This disease is rare and considered of minor significance to strawberry production. However, it could be of greater significance in protected culture where humidity is higher. To our knowledge, this is the first report of B. cinerea causing strawberry leaf spot in California or elsewhere.