First report of causing root rot on Strawberry in China.

In Henan, strawberry cultivation occurs on approximately 10,000 hectares, with annual production approaching 230,000 tons. In April 2022, a root rot disease with a 10% incidence rate was observed on the strawberry cultivars 'Ningyu' and 'Sweet Charlie' grown in plastic greenhouses (0.7 ha) located in Xingyang (113.39°E, 34.79°N), Henan, China. Disease symptoms included yellowing of the outer mature leaves, stunted growth, and subsequent wilting of the entire plant. The roots developed dark brown spots, which gradually turned necrotic (Figures 1a, 1b). To determine the causal agent, four symptomatic plants (two plants per cultivar) were collected. Twelve symptomatic root tissues (three root tissue samples per plant) were surface sterilized with 75% ethanol and 0.1% mercuric chloride, washed thrice in sterile water, air dried, and then placed on PDA at 25°C for 3 days. Eight pure isolates were obtained by hyphal-tip isolation (Fang 2007). Each colony had a dark olivaceous green to brown, cottony appearance with a round margin, and the reverse side was grey-black near the center (Figure 1c). Conidia were ellipsoidal, aseptate, with rounded ends, and 3.1 to 4.8 μm × 1.0 to 2.5 μm in size (Figure 1d). Chlamydospores were ellipsoidal, pale brown, and 7.9 to 11.9 μm × 7.6 to 10.7 μm in size (Figure 1e). A representative fungus isolate, designated as Z5, was selected for further molecular identification. Genomic DNA was extracted from the mycelia of isolate Z5, and four gene partial regions (ITS, , , and LSU) were amplified using the primer pairs ITS1/ITS4, Bt-2a/Bt-2b, RPB2-5F/RPB2-7CR and LROR/LR5, respectively (White et al.1990, O'Donnell et al.1997, Reeb et al. 2004, Rehner and Samuels 1994). PCR products were sequenced and submitted to GenBank with the following accession numbers OQ130480 (ITS), OQ190093 (TUB2), OQ190092 (RPB2), and OQ255570 (LSU). BLASTn search revealed that the ITS, , , and LSU gene sequences of isolate Z5 showed 99.42% (513/516 bp), 99.69% (320/321 bp), 100% (1071/1071 bp), and 100% (857/857 bp) identity with those of ex-type stain ZHKUCC 21-0001 (Dong et al. 2021), respectively. A phylogenetic tree was constructed showing that Z5 clustered with (Figure 2). The isolates in this study were identified as based on morphological and molecular evidence. To confirm pathogenicity, five one-month-old 'Ningyu' cultivar strawberry seedlings were planted in sterilized nursery soil mixed with wheat grains (0.5% w/w) coated with Z5 mycelia (Fang 2007). An equal number of strawberry seedlings were placed in pots filled with non-infected potting mix to serve as controls. The seedlings were kept in a greenhouse under a 12 h light/dark photoperiod at 25°C. After two weeks, the inoculated seedlings displayed symptoms such as leaf wilting and root necrosis, similar to those observed in the greenhouses, while the control seedlings showed no symptoms (Figures 1f, 1g). The experiment was performed thrice. The identical fungus was re-isolated from the symptomatic roots and identified as based on morphological characteristics and molecular analysis, thus fulfilling Koch's postulates. This is the first report of causing root rot on strawberries worldwide. Our findings will contribute to a more comprehensive study on investigating and managing this disease.