First Report of Fusarium meridionale Causing Stalk Rot of Ryegrass in China.

Abstract

Members of the Fusarium graminearum species complex (FGSC) are the main causing agents of head blight, seedling blight, or stalk rot in wheat and other cereals worldwide. Surveys on species composition and mycotoxin production of FGSC populations have mainly focused on food crops such as wheat, maize, and barley, but little is known about the identity of FGSC pathogens present in pasture grass. In April 2021, a survey of grass diseases in the Hongya County (29.90661 N; 103.37313 E) in Sichuan Province was conducted to understand the etiology of stalk rot in perennial ryegrass (Lolium perenne). It was observed in several pastures that about 10% of yield loss in perennial ryegrass was caused by stalk rot. Affected plant stalks were brown to dark brown in colour and appeared soggy. As infections continued or under conditions of high humidity, some plant stalks also became flattened. Perennial ryegrass samples with symptoms of stalk rot or browning of the stem were collected. Symptomatic tissues were cut into short segments (approximately 5 mm), surface-sterilized in 3% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, air dried, plated onto potato dextrose agar (PDA), and then incubated in the dark at 28 °C. After 3 to 5 days, Fusarium-like fungal colonies with reddish-orange mycelium were collected and transferred to new PDA plates for further purification, and the purified cultures were obtained by single spore isolation. Four uniform isolates were obtained and their colonies on PDA resembled typical FGSC colonies (Leslie and Summerell 2006; O'Donnell et al. 2004). Colonies had an average radial growth rate of 8.5 to 11.0 mm/day at 28 °C in the dark on PDA. Conidial characteristics were studied on Spezieller Nährstoffarmer agar (SNA) as described by Wang et al. (2014). Macroconidia were falcate to almost straight, usually with parallel dorsal and ventral lines, 3- to 5-septate, 20.65 to 55.22 μm in length (average 39.16 μm), and 2.38 to 6.93 μm in width (average 4.42 μm) (n = 200). No microconidia were observed. The pathogenicity of the isolated Fusarium strains was then tested on healthy perennial ryegrass (variety Changjiang 1). Ryegrass plants grown for 2 months were inoculated by punching a hole in the stem using a sterile toothpick, followed by an injection of 20 μL macroconidia suspension at a concentration of 105 spores/mL. Ryegrass stems treated with water served as the control. Twenty plants were included in each treatment. After inoculation, the plants were grown in a growth chamber at 25 °C and 90% humidity for 24 h. Stalk tissues at the wound site turned brown after 3 days and the brown area then extended to regions above and below. No symptoms were observed in the water-treated controls. As well, the same pathogen was reisolated from the infected grass stems, but not from the controls. Thus, the isolated Fusarium spp. are a cause of stalk rot in perennial ryegrass based on the fulfillment of Koch's postulates. To identify the Fusarium spp. to species level, portions of the translation elongation factor 1-α (TEF) gene sequences from all four strains were amplified and sequenced as described by Wang et al. (2015). The obtained sequences were identical, and a sequence of isolate SC1 was submitted to GenBank (accession no. MZ964308). BLASTn searches were conducted on the TEF sequence (607 bp) in two databases, revealing it had 100% similarity to the sequence of Fusarium meridionale strain DS27 (accession no. MN629330) in NCBI and strain NRRL28723 from FUSARIUM-ID (http://isolate.fusariumdb.org/). A concatenated four-gene phylogeny (supplementary figure) resolved SC1 and the type specimens of F. meridionale (NRRL28723, 29010, and 28436) in a monophyletic clade with 100% bootstrap support, confirming that the strain SC1 belongs to F. meridionale. Finally, trichothecene productions of F. meridionale strains were evaluated using rice cultures kept at 28 °C in the dark for two weeks, as described by Desjardins and Proctor (2011). LC-MS/MS analysis indicated that the fungus could produce NIV and 4ANIV in rice cultures with average concentrations of 1400.44 and 3144.10 μg/kg, respectively. To the best of our knowledge, this is the first report of F. meridionale causing disease in perennial ryegrass in China. Further research will be necessary to determine its distribution, aggressiveness, and trichothecene production.