First Report of Magnaporthe oryzae Causing Gray Leaf Spot on Tough Lovegrass (Eragrostis plana) in Brazil

Abstract

Tough lovegrass (Eragrostis plana Nees) is a weed grass in several native pasture areas in South America. This weed has affected the germination and growth of forage species owing to allelopathic compounds released (Medeiros and Focht 2007). In spring and summer of 2017, leaves and stems of E. plana showing gray leaf spots were observed under field and greenhouse conditions in the City of Capao do Leao, Rio Grande do Sul state, Brazil. The incidence of the disease (symptomatic leaves and stems) was up to 45%, and the severity ranged from 30 to 40%. Disease symptoms began as small dark lesions that evolved to elliptical spots, grayish to tan, sometimes presenting olive-brown borders, with a yellow halo. The symptomatic leaves were surface sterilized with a solution of 1% NaOCl for 60 s and plated on water agar medium. Plates were incubated at 25°C for 2 days. Fungal mycelia were observed and transferred to oat dextrose agar medium and incubated at 25°C for 5 days. The isolated pathogen had colonies with gray coloration and velvety texture. Conidiophores, obtained from the cultured colonies, measured 141.3 μm (60.0 to 250.0 μm) × 3.8 μm (3.2 to 4.0 μm) and were observed as singles or grouped, with light brown coloration. Conidia measured 30.0 μm (25.0 to 34.0 μm) × 12.0 μm (8.0 to 13.0 μm) and were hyaline, septate (two to three), with protruding hilum, pyriform-shaped oblate, rounded at the base, and with narrow apex (Muni and Nadarajah 2014). To confirm pathogen identity, molecular analysis was performed using specific primers for internaltranscribed spacer (ITS) (ITS1 and ITS2), actin (ACT) (ACT512F and ACT783R), calmodulin (CAL) (CAL228F and CAL783R), and β-tubulin (TUB 2) (BT1a and BT1) genes as described by Klaubauf et al. (2014). The DNA sequences were deposited in GenBank (accession nos.: ITS, MH745133; ACT, MH748530; CAL, MH748531; and TUB2, MH748532). BLAST searches showed 99 to 100% identity with the existing sequences of Magnaporthe oryzae B.C. Couch (ITS, KM484893; ACT, DQ240884; CAL, DQ240900; and TUB2, DQ240916). Phylogenetic analysis was conducted using maximum likelihood and Bayesian inference using multilocus alignment (ITS, ACT, CAL, and TUB2). Isolates of E. plana were placed along with the reference isolates of M. oryzae in a clade with 99% bootstrap support. To determine the pathogenicity of the M. oryzae isolate, five plants of E. plana at the flowering stage were inoculated with a suspension of 1 × 10³ conidial/ml. A set of five noninoculated plants was considered as a control, with only distilled water applied. All plants were enclosed in plastic bags and incubated in a growth chamber at 25 ± 1°C for 24 h with a 12-h photoperiod. At the end of the 24-h period, plastic bags were removed. At 7 to 8 days after the inoculation, all inoculated plants showed the same leaf symptoms initially observed in the field plants. No symptoms were observed in control plants. M. oryzae was successful reisolated from inoculated plants, and the fungus was morphologically and molecularly confirmed as M. oryzae. M. oryzae infects several species worldwide, including Oryza sativa, which has an enormous economic importance in the Rio Grande do Sul (Fang et al. 2017). Leaves of rice plants (cultivars BRS Querencia and Inov CL) were also inoculated with an isolate of M. oryzae from E. plana using the same methods as described above. Gray leaf spot symptoms were on rice, indicating that this isolate was pathogenic on that crop. This is the first report of M. oryzae infecting E. plana in Brazil. This discovery has considerable importance to the farmers because tough lovegrass could be an alternative host of M. oryzae during the off-season period for rice.