First Report of black spot of Shine Muscat fruit caused by Cladosporium allicinum in China.

Shine muscat is a Vitis vinifera hybrid (Akitsu-21 × Hakunan), that has emerged as a popular table grape cultivar in China. In recent years, shine muscat has been widely cultivated with 66,667 ha being cultivated in 2021. In November 2021, symptoms of fruit spot were observed on shine muscat during the storage at 0~3℃ and 85%~90% RH, while stored at National Agricultural Product Preservation Engineering Technology Research Center, in Tianjin (N 116°20', E 39°09'), China. The incidence of this disease was about 35%. Affected grape berries initially had small brown spots. The spots on the fruit expanded to an ellipse or circular sunken area with a black center. The central peel of the diseased spots were ruptured and collapsed. The diseased fruits eventually fell off the vine. To isolate the pathogen, grape peels with typical symptoms were cut into small pieces, sterilized with 75% ethanol for 45-sec, rinsed with sterilized distilled water three times, and then transferred onto potato dextrose agar (PDA) medium.The plates were incubated at 25°C in the dark. After 10 days, 26 single spore isolates with similar morphology were obtained from 30 symptomatic grape berries. Fungal colonies were grayish brown, with abundant conidia on the obverse-side on PDA. Conidiophores were cylindrical, straight with unbranched, solitary or clustered, elongation at the tip and ranged in size from 3.2-6.8 × 35.6-150.9 µm (n=50). Conidia were grew in chains, ovoid, aseptate, and 2.2-6.0 × 8.3-16.8 µm (n=50). The morphological characteristics were consistent with Cladosporium allicinum (Bensch et al. 2012). Molecular data were also used to support the microscopic identification by extracting genomic DNA from 26 isolates using a Plant Genomic DNA kit (Tiangen, China). Amplicons were generated for the internal transcribed spacer (ITS), translation elongation factor 1-alpha(tef1-α), and actin (act) using the following primers ITS1/ITS4, EF1-728F/ EF1-986R and ACT-512F/ ACT-783R, respectively (Bensch et al. 2012). Blast analysis showed that three amplified fragments of 26 isolates were highly similar to C. allicinum, with 98.96~100% sequence identity with Cladosporium allicinum accessions in GenBank (ITS, OK661041; tef1-α, MF473332; act, LN834537). Three amplified fragments of representative isolate YG03 were deposited in GenBank with accession nos. OP799670 for ITS, OP888001 for tef1-α and OP887999 for act, respectively. Neighbor-joining trees based on concatenated sequences of three genes were constructed using MEGA5.2. The results showed that the strain YG03 from shine muscat was closely related to C. allicinum. Pathogenicity tests of 26 isolates were performed on healthy shine muscat berries using pin pricks and a humidor. In each wound, 5 µL of conidial suspension (1×106 conidia/mL) and sterile distilled water were inoculated on 30 berries, and maintained in a dark incubator at 25°C, 90% relative humidity. Each treatment was repeated twice. After 10 days, the wounded berries inoculated with the spore suspension showed dark brown spots, similar to the original diseased fruits, while no symptoms were observed on the control treament. Pathogen re-isolated from inoculated fruits were identical to the original strains on colony and microscopic morphology, and identified to Cladosporium allicinum based on act gene by molecular method, thereby fulfilling Koch's postulates. C.allicinum has been reported causing leaf spot on 11 host plants around the world (Bensch et al. 2012, 2015; Quaedvlieg et al. 2014; Jurisoo et al. 2019). To our knowledge, this is the first report of C. allicinum causing black spot on fruit of Vitis vinifera worldwide. The identification of this disease could establish a foundation for developing management strategies to reduce losses in storage period.

A Lethal Case of Disseminated Cladosporium allicinum Infection in a Captive African Bullfrog.

Cladosporium infections have a poor prognosis in animals, most likely due to a lack of knowledge about diagnosis and treatment. In this study, we described a case of a lethal Cladosporium allicinum infection in a captive bullfrog (Pyxicephalus adspersus) in Europe. One adult male bullfrog was referred with clinical signs of lethargy and a cutaneous nodule. Fungal infection was suspected on cytology and confirmed by histology and cultural isolation. The mold was identified by molecular methods using partial sequencing of the TEF1α gene and the ITS region of rDNA. Climbazole antifungal treatment was started but the frog died after 30 days, and necropsy was done. Pigmented hyphae and structures consistent with muriform bodies were found on a background of diffuse granulomatous inflammation at cytological and histopathological examinations. Fungal culture revealed the presence of pigmented fungi identified as Cladosporium allicinum only by partial sequencing of the TEF1α gene. A focally extensive granuloma with intralesional hyphae and muriform bodies effacing the architecture of head, liver, kidneys, lungs, and large intestine were retrieved after necropsy. This study is the first Italian report of the occurrence of lethal C. allicinum infection in a frog and highlights the role of this Cladosporium sp. in chromoblastomycosis.