First Report of Leaf Spot on Cucumber Caused by Fusarium incarnatum in China

Abstract

Cucumber (Cucumis sativus L.) is a globally cultivated vegetable crop with great economic and nutritional importance. In May 2019, a new leaf spot disease was observed on cucumber in Jiyang, Shandong Province, China. Disease incidence was estimated at approximately 15% across the survey area. Foliar symptoms began with small pale-yellow lesions, circular to irregular in shape. Subsequently, they developed into large irregular spots, yellow to dark brown, with a gray central area. Finally, the lesions were densely distributed on the leaves. To isolate the pathogen, leaf tissues (5 × 5 mm) were cut from the junction of diseased tissue and healthy tissue, surface disinfected in 75% alcohol for 15 s, soaked in 0.1% mercuric chloride for 1 min, washed with sterile distilled water three times, and cultured on potato dextrose agar (PDA) at 25°C for 3 to 5 days. Seventeen fungal isolates with similar morphological characteristics were obtained and further purified by a single-spore isolation method. The colony of representative isolate SCS6G on PDA was fluffy, white, and showing beige coloration on the reverse side. Macroconidia had three to five septa (more commonly three septa), slightly curved at the apex, ranging from 16.2 to 33.4 × 3.1 to 4.8 µm (n = 100). Microconidia were single celled to one septum, ranging from 6.8 to 17.3 × 2.6 to 4.1 μm (n = 100). Chlamydospores with thick, roughened walls were abundant, singly, in chains or in clumps, ellipsoidal or subglobose. The morphological characteristics and measurements of this fungal isolate were consistent with the descriptions of Fusarium incarnatum (Leslie and Summerell 2006). To validate the species identification, the rDNA internal transcribed spacer (ITS) region (White et al. 1990), β-tubulin gene (Glass and Donaldson 1995), and partial translation elongation factor 1-α (TEF1-α) gene (Cong et al. 2016) were amplified and sequenced. The ITS, β-tubulin, and TEF1-α sequences of isolate SCS6G were submitted to GenBank (MN493639, MN508785, and MN508784, respectively). BLASTn analysis of the sequences showed 99% identity with F. incarnatum (MN317371, KY509037, and KY509036). Furthermore, phylogenetic analysis based on the alignment of the ITS, β-tubulin, and TEF1-α sequences using MEGA7 revealed that the isolate was grouped in the same clade as F. incarnatum. Pathogenicity tests were performed by spraying a conidial suspension (1 × 10⁵ conidia/ml) on 25 healthy cucumber plants at the five-true-leaf stage. Sterile water served as a control. All samples were placed in a dew chamber for 48 h and then kept in a growth chamber at 25°C. All inoculated leaves showed symptoms similar to those observed in the field after 7 days, but no disease occurred on control plants. The same fungus was successfully reisolated from inoculated leaves and reidentified based on morphology and molecular evidence, thus confirming Koch’s postulates. The pathogenicity test was repeated with similar results. To our knowledge, this is the first report of leaf spot caused by F. incarnatum on cucumber in China. This disease may pose a serious threat to cucumber production in China.