First report of strawberry root rot caused by Monosporascus eutypoides in China

Strawberry (Fragaria × ananassa) is an important economic crop in Hebei, China. In May 2023, root rot was observed in strawberry plantations (cultivar ‘Benihoppe’) in Shijiazhuang (37°57′23″N, 115°16′34″E), Hebei, China. The incidence of the disease reached up to 30% in the field. The infected plants displayed symptoms of reduced vigor and stunted growth, yellowing leaves, blackened roots, and eventual rotting. Ten diseased strawberry plants were collected for pathogen isolation. Root tissues of symptomatic plants were surface sterilized sequentially in 70% ethanol (1 min), 3.125% NaOCl (6 min), and 70% ethanol (30 s), then they were rinsed in sterile water three times (Sahu et al. 2022). The sterilized roots were then cut into 3.0 × 3.0 mm pieces and placed on potato dextrose agar (PDA) medium supplemented with chloramphenicol (100 μg/mL). After 2 to 3 days incubated at 25℃ in darkness, single colonies from the initial isolates were streaked onto PDA agar plates to obtain pure cultures. The fungal colonies appeared milky white on PDA medium and exhibited only hyphae growth. Globose perithecia were observed on V8-juice medium after 30 days of incubation, with black, smooth, spherical ascospores 13.9 to 19.1 μm in diameter (n=50). For molecular analysis, the representative isolates ME10-2 and ME10-3 from different strawberry plants were selected for the amplification of internal transcribed spacer (ITS) and elongation factor 1-alpha (EF-1α) sequences using the primer sets ITS1/ITS4 (Bellemain et al. 2010) and EF1-688F/EF1-1251R (Alves et al. 2008). The sequences were deposited in GenBank (accession no. PP990210 and PP990211 for ITS, and PQ001579 and PQ001580 for EF-1α). BLASTn analysis revealed that the sequences of ITS had above 99% (504/511 bp and 503/506 bp) similarity to Monosporascus eutypoides (MK183805.1), the sequences of EF-1α had 94% (625/661 bp and 619/656 bp) similarity to Monosporascus eutypoides (JQ958959.1). Phylogenetic analysis based on concatenated sequences of ITS and EF-1α using the Maximum Likelihood method with IQ-TREE version 2.2.3 (Nguyen et al. 2015) also indicated that the two isolates clustered with M. eutypoides. To fulfill Koch’s postulates, pathogenicity tests were performed (Chen et al. 2024). Due to challenges in ascospore production, an improved wheat grain medium (wheat grain with 2% dextrose) was used for fungal culture (Sainos et al. 2006). After 14 days of inoculation at 25℃, the medium was crushed and mixed into sterilized soil with a 10% ratio by weight. Four-week-old strawberry plants were transplanted into the inoculated soil and grown in a growth chamber at 25℃ (16 h light/8 h dark). Strawberry plants transplanted into sterilized uninoculated soil served as a negative control. Three weeks post-inoculation, the inoculated strawberry plants exhibited symptoms similar to those observed in field-infected, whereas no symptoms were observed on the control plants. The pathogen was reisolated from the symptomatic plants and identified as M. eutypoides based on colony morphology on a PDA plate and ITS sequences information. M. eutypoides has been found to cause Monosporascus root rot and vine decline on muskmelon, watermelon and cucumber (Martyn and Miller 1996; Salem I. B. et al. 2013). To our knowledge, this is the first report of M. eutypoides causing root rot on strawberry. This finding suggests a potential risk of strawberry root rot when planting subsequent to cucurbit crops and assists strawberry growers in the region in adjusting their planting and disease management decisions.