First report of Cercospora cf. malloti causing brown eyespot on strawberry in Brazil.

Strawberry (Fragaria x ananassa Duch.) is one of the most consumed fruits from the Rosaceae family in Brazil. It is cultivated on ~4,500 ha in all regions of the country, with a wide range of fungi causing leaf diseases. Strawberry leaves (cv. San Andreas) with symptoms of eyespot usually related to Mycosphaerella fragariae (Ramularia tulasnei) (Mazaro et al. 2006) were observed in April 2019, in a field located in Santa Maria, Rio Grande do Sul state, Brazil (29°43'19.6"S / 53°43'04.6"W). However, the symptoms slightly differed from Mycospherella blight as well as the fungi structures observed under an optical microscope. The symptomatic leaves had small circular spots, initially reddish-purple to dark brown color, which later increased in size. The center of the lesion was grayish pale, where dark brown conidiophores were found over it. The symptom occurred in 50% of the leaves of all plants during the crop season. To identify the causal agent, symptomatic leaves were collected and superficially disinfected with 70% ethanol for 30 s followed by 0.5% bleach for 30 s and then washed in sterile water. The leaves were placed in germination boxes with a moistened paper filter and incubated at 25 ºC and 12 h photoperiod for four days. After the formation of the fungal structures, the fungi was isolated in potato dextrose agar (PDA) medium and kept for 20 days at 25 ºC. Colonies were dark brown with pale pink aerial mycelium. The conidia were hyaline, single- to multi-septated with a filiform shape, straight or slightly curved. The fasciculate conidiophores on the stroma measured 20 to 70 x 2.5 to 3.5 µm. Conidia average measurement was 10.2 to 82.4 x 4. to 8.8 µm, similar to morphological characteristics of the genus Cercospora. The isolate named M6 was deposited in the Brazilian National System of Genetic Registration (SISGEN) no A57AE19. The DNA was extracted from a monosporic culture of M6 in PDA plates, which presented all typical aspects previously described. To confirm the identity, the PCR amplification was carried out for internal transcribed spacer (ITS) region ITS 1-5.8S - ITS 2 and Calmodulin region (CAL) genes (Acqua et al, 2011). The ITS (No. MZ713249) and CAL (No. MZ713254) sequences were deposited in the Genbank database. BLASTn analysis revealed the isolate was 99% similar to Cercospora cf. malloti which access codes in GenBank are KT 193689 and KT 193738 (Nguanhom et al. 2015). A pathogenicity test was performed to fulfill Koch's postulates. Strawberry plants with healthy leaves (60 days old), were inoculated by spraying a conidial suspension (1 × 105 conidia/ml). Control plants were sprayed with sterile water. The plants were incubated in a greenhouse at 28 ºC for 12 h photoperiod at 80% relative humidity under drip irrigation keeping at least 60% of field capacity. Six plants were used as replicates. Twenty days after inoculation, small circular reddish-purple spots were found, which later increased in size. Ten days later, the spots presented dark brown conidiophores over the center of the lesion. The symptoms described above were observed in all inoculated leaves. The control plants remained symptom-free. The pathogen was then re-isolated from the lesions and identified as C. cf. malloti based on the morphological aspects described above on PDA. In Thailand C. cf. malloti is reported on crops like Brassica alboglabra (cabbage) (Nguanhom et al. 2015), but not in strawberries. To our knowledge, this is the first report of C. cf. malloti pathogenic to strawberries in Brazil.