First Report of Leaf Spot Caused by Paramyrothecium foliicola on Peanut in China.

Abstract

Peanut (Arachis hypogaea) is an important economic and oil crop in China. In September 2022, leaf spots wereobserved on peanut in Luoyang city, Henan province, China (34°49'N, 112°37'E). The disease occurred on about 30% ofthe peanutleaves in only one 0.5-acre field. Symptoms appeared primarily as brown spots, that varied in shape, and appeared round, oval or irregular. In addition, some disease patches exhibited a concentric ring pattern. Small pieces (5×5 mm) of five diseased leaves were surface disinfected in 3% NaClO for 2 minutes, rinsed three times in sterile distilled water, dried on sterilized filter paper, and cultured on potato dextrose agar (PDA) at 25°C for 3 days. Five isolates with uniform characteristics were obtained and subcultured by transferring hyphal tips to fresh PDA. The colonies of the isolates were circular and the margins were clean. The colonies showed white coloration, and after 5-7 days of incubation on PDA plates, concentric rings with dark green sporodochia appeared on the surface of the colonies. The conidiophores branched repeatedly. The conidiophore stipes unbranched, hyaline, 10.0 to 23.2×1.5 to 3.3 μm (n=50). The conidia were rod-shaped or long oval and single-celled, measuring 4.6 to 8.6×1.4 to 3.1 μm (n=100). Based on these characteristics, the five isolates were identified as Paramyrothecium foliicola (Lombard et al 2016). Genomic DNA was extracted from the representative isolates LH-1-1 and LH-1-2. The internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), calmodulin (CmdA), and translation elongation factor 1-alpha (tef1) loci were amplified and sequenced using the following primer pairs: ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7cR (O'Donnell et al. 2007), CAL-228F/CAL-2Rd (Carbone & Kohn 1999), and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. BLASTn analysis revealed that the sequences of ITS (OR352397.1 and OR417392.1), RPB2 (OR413573.1 and OR420678.1), CmdA (OR413572.1 and OR420677.1), and tef1 (OR413574.1 and OR420679.1) had 99 to 100% (553/558 bp, 721/721 bp, 597/598 bp, and 384/389 bp) similarity to P. foliicola (MN593634.1, MN398038.1, OM801785.1, MK335967.1). A phylogenetic tree based on the Maximum Likelihood method also confirmed that the two isolates converge on the same branch as P. foliicola. Pathogenicity tests were performed using leaves of 60-day-old peanut plants (cv. Zhonghua 8). Briefly, uninfected healthy leaves (non-wounded) were inoculated with 30-µl drops containing a spore suspension (5×105 conidia/ml) of LH-1-2, and peanut leaves inoculated with sterile distilled water served as controls. All treatments were incubated in an incubator at 25℃ and high relative humidity with a 12:12 hour light-dark cycle. After 5-7 days, inoculated leaves showed symptoms similar to those observed in the field, while no symptoms were observed on control leaves. The pathogenicity tests were repeated three times. The fungus was reisolated from the infected leaves and identified as P. foliicola based on morphological and molecular characteristics, thus fulfilling Koch's postulates. P. foliicola has previously been reported to cause leaf spot of tomato and mung bean, stem canker of cucumber (Huo et al. 2022; Sun et al.2020; Huo et al. 2021). To our knowledge, this is the first report of P. foliicola causing leaf spot on peanut in the world. Identification of this pathogen will be helpful in monitoring peanut diseases and developing disease control strategies.