First Report of Fusarium verticillioides causing Stem Blight of Schizonepeta tenuifolia in China

Abstract

Schizonepeta tenuifolia is an important medicinal plant in China. Over 10000 ha of S. tenuifolia is cultivated in the country annually. However, fungal diseases are a major limiting factor in S. tenuifolia production. In 2022, 50 ha in several S. tenuifolia fields in Hebei province were observed to be severely affected by a disease causing a yield loss of 30%. Results from field surveys suggested an epidemic during seedlings stages that affected S. tenuifolia stems, causing irregularly watery brown lesions. Lesions ranged from 1.5 to 2 × 2.5 to 3 cm. To isolate the causal agent, tissue was removed from the border of lesions and surface sterilized in 75% ethanol for 30 sec and 0.1% HgCl2 for 1 min, then rinsed three times with steriled distilled water(SDW), plated on potato dextrose agar(PDA) at 25℃, and incubated in the dark for 7 days. Five putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar(SNA) with a ~ 1 × 2-cm strip of sterile filter paper on the agar surface(Nirenberg 1976). Cultures were incubated for 7 to 10 days at 20℃ in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined at 400×. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Microconidia produced on polyphialides and aggregating in heads were unicellular, ovoidal or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 μm. Macroconidia were abundant, falcate to straight, three to five septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 μm. Based on morphological characteristics, isolates were tentatively identified as F. verticillioides(A1-Hatmi et al. 2016; Guarro 2013). Pathogenicity tests were performed by injection inoculation of 0.1 mL of conidial suspensions(1×106 conidia/mL) into three S. tenuifolia stems using a disposable needle and syringe. Distilled water was injected into three mock controls. Inoculated plants were placed in a greenhouse at 32 to 34℃ and 95% relative humidity. Typical lesions were observed 7 days after inoculation, except in the control samples. Each treatment was replicated three times. The suspected pathogen was consistently reisolated from diseased tissue according to Koch’s postulates, and was found to be morphologically similar to F. verticillioides. Preliminary morphological identification of the pathogen was further confirmed by using genomic DNA extracted from the mycelia of a 7-day-old culture grown on PDA at 25℃. The translation elongation factor 1-α gene(TEF1) was amplified(O'Donnell et al. 1998) and the TEF region(Genbank Accession No. OR105502) was sequenced by Sangon Biotech Co., Ltd.(Shanghai, China) and displayed 100% nucleotide similarity with rDNA-TEF of F. verticillioides(JF740717) separately after a BLASTn search in Genbank. Based on the symptoms, fungal morphology, TEF sequence, and pathogenicity testing, this fungus was identified as F. verticillioides. to our knowledge, this is the first report of F. verticillioides infecting S. tenuifolia in China. This report will promote further research of F. verticillioides on this host and lead to better understanding of disease prevalence, extent of damage, and possible management options.