First report of Calonectria canadiana causing peach fruit rot in China.

Peach (Prunus persica (L.) Batsch) is one of the most popular fruits grown in Northern China. In July 2021, a fruit rot outbreak on the peach cultivar "Yonglian Sweet" occurred after unusual rains in Baoding, Hebei Province, China. Sixty peach trees from three orchards were assessed, and a 30% disease incidence was estimated. The disease initiated as a small concave spot on the fruit surface expanding circularly rotting the fruit (3-5 cm deep) with the appearance of grayish-white mycelia (Figure S1A). The infected fruit did not disintegrate but turned light brown. To identify the pathogen, 20 infected fruits were collected, and fruit tissues from lesion margins were inoculated on the potato dextrose agar (PDA) medium. A total of 15 fungal pure cultures with highly similar morphological characteristics were obtained by the hyphal-tipping method. The fungal culture formed smooth-edged colonies of extensive, dense, wooly aerial mycelium, with color changing from sienna to luteous, and to grayish-white along the radius of colonies (Figure S1B) Chlamydospores were extensive and developed micro-sclerotia after 20 d of growth. The conidiophore produced three branches in a "broom" shape, with the primary branch ranging 7.5-25.0 μm in length, the secondary branch 5.5-15.5 μm, and the tertiary branch 10-12.5 μm (N = 30). The top of the tertiary branch tapered and produced conidia. Conidia were colorless and culm-like, 40.0-57.5 μm long and 3.8-6.25 μm wide (N = 30). Hyphae occasionally produced spherical chlamydospores with a diameter of around 7.5 μm (N = 30). Conidia germinated after 12 h in moist conditions, and germ tubes originated from multiple points on the conidia. Based on these morphological features, the isolated fungus was identified as Calonectria spp. (Lombard et al. 2010). Six loci, including ITS, act, cmdA, his3, tef1, and tub2, were amplified and sequenced for molecular identification of an isolate F099 using primers listed in Table S1. The obtained ITS (528 bp, GenBank accession no. OL635556), act (263 bp, OL694221), cmdA (470 bp, OL694222), his3 (432 bp, OL694223), tef1 (487 bp, OL694224), and tub2 (535 bp, OL694225) sequences showed 100% similarity to the ex-type strain of Calonectria canadiana, CMW 23673 (accession nos. MT359667, MT334976, MT335206, MT335446, MT412737, and MT412958, respectively; Figure S1D) (Kang et al. 2001, Lechat et al. 2010, Liu et al. 2020). The isolate F099 of C. canadiana was further subjected to pathogenicity tests. Koch's postulates were performed by placing three mycelial disks (ten-day old, 5 mm) with conidia on the sterile needle-acupunctured surface of healthy fruits of the peach cultivar "Yonglian Sweet" (N= 10). Mock inoculations with sterile PDA disks were served as a control. All the inoculated fruits were kept in a moist chamber (25℃, 16-h light and 8-h dark period). The inoculation assay was repeated twice. Rotting symptoms developed on all the inoculated fruits about 5 days post-inoculation (dpi) and grayish-white mycelia appeared around ten days post inoculation while mock inoculated fruits did not show any rotting. The pathogen of interest was re-isolated from the inoculated fruits and validated as C. canadiana by ITS and tef1 sequences. All above evidence collectively indicates that the fungal pathogen causing the peach fruit rot is C. canadiana. The new host plant and new geographic distribution reported here will inform future management of this fungal species.