First Report of Epicoccum nigrum Causing Leaf Spot of Eugenia involucrata in Brazil.

Abstract

Eugenia involucrata (DC) is a native fruit species of forest formations in the Atlantic Complex and in the forests and savannas of the Paraná State, Brazil (Donadio, 2002). In February 2021, in the experimental area at the Universidade Tecnológica Federal do Paraná, in the Dois Vizinhos city, Paraná State, Brazil, a foliar disease was observed on twenty-two12 years old E. involucrata trees, with 20 to 80% of the leaves per tree affected. Symptoms were small, irregularly to circular shaped, reddish-brown lesions with yellow halos. As the disease progressed, the lesions increased in size and showed no distinction between mature and young tissues. Twenty symptomatic leaves (from each tree) from 11 trees grown at different locations in the orchard (50% of the total number of trees) were collected. For fungal isolation, the leaf surfaces were disinfected with 0.5% NaOCl solution for 1 min, rinsed in sterile distilled water (SDW) and dried on sterile filter paper. Five fragments (0.3 cm) of diseased leaf tissue were placed on potato dextrose agar medium. After 7 days of incubation at 25°C, orange colonies appeared, with a reddish pigment on the reverse side. Conidial were brown globular to pear solitary, verrucous and multicellular (average of 21.74 µm x 24.45 µm, n = 30). The morphological characteristics of the colony and conidia of the eight isolates matched the description of the fungal genus Epicoccum (Valenzuela-Lopez et al. 2018). Further identification of eight isolates was confirmed by amplifying and sequencing three phylogenic loci (ITS, β- tubulin and RPB2) using the ITS1/ITS4, Bt2a/Bt2b and 5F2/7cR primer pairs, respectively (White et al., 1991, Glass and Donaldson, 1995, O Donnell et al., 1998). The sequences of one representative isolate (ENcm) were submitted to GenBank (ITS, MZ442338, β-tubulin, MZ447127 and RPP2, MZ447128) respectively. A phylogenetic tree was constructed by the maximum likelihood method with 1,000 replicas of bootstrapping based on concatenated ITS, β-tubulin, and RPB2 sequences of the ENcm and strains of 14 species of the genus Epicoccum. Isolate ENcm grouped with Epicoccum nigrum stains CBS 173.73 (Chen et al., 2017). For the pathogenicity tests four young healthy branches containing 20 leaves were spray inoculated, with 1.5 mL of conidia suspension of ENcm (106 conidia/mL) covered with a punched transparent plastic bag and moistened with distilled water in orchard. The air temperature ranged from 14ºC to 25ºC. SDW was used as control. Three replicates (pathogen and control) on different trees were performed. After 7 days the fungus was re-isolated from the symptomatic lesion, showing morphological characteristics similar to those of ENcm. Control branches did not show fungal growth. The inoculation test was repeated once, confirming the symptoms described above. This is the first report of the leaf spot caused by E. nigrum on E. involucrata in Brazil as well as in the world. E. nigrum on E. involucrata leaves could pose potential threat on productivity, whose impact may affect the fruit tree's ability to perpetuate, its survival in natural conditions or in commercial orchards.