First Report of <i>Colletotrichum henanense</i> Causing Fruit Rot in <i>Ilex verticillata</i> in Yunnan Province, China

Ilex verticillata (L.) A. Gray (Winter Red) is a deciduous shrub and ornamental tree species native to North America with a long fruiting period in the autumn and winter (Qin et al. 2023). In November 2023, disease symptoms of fruit rot were observed in approximately 30% of I. verticillata plantings in Chuxiong City, Yunnan Province, China (25° 31′ 22.27″ N, 102° 08′ 55.47″ E). The symptoms of fruit browning, necrosis, and rot were observed. The symptomatic fruits were collected to isolate and identify the pathogen. Symptomatic fruits were disinfected with 75% ethanol for 1 min, followed by rinsing three times with sterilized distilled water. The infected fruits were cut into 3 × 3 mm squares and transferred to potato dextrose agar (PDA) medium containing 10 mg/L rifampicin and ampicillin to avoid bacterial growth. The plates were incubated at 28 °C in the dark for 3-5 days and hyphal tip of suspected colonies were purified onto PDA medium. Eight isolates were isolated from 10 infected fruits, and three species were identified based on the morphological characteristics of the colonies, including Alternaria sp. (numbered SRF1), Aspergillus sp. (numbered SRF2), and 6 isolates (numbered SRF3-SRF8) of an unknown species exhibiting the highest frequency of isolation and identical colonies. To further confirm the pathogenicity of all the isolates, 10 μL spore suspension (1 × 106 spores/mL) of respective isolates was injected into healthy fruits and an equal volume of sterile water served as a control. The fruits were incubated in an incubator at 28°C for 7 days, in humid conditions. Each treatment consisted of 30 healthy fruits, and the inoculation was repeated three times. After the incubation, isolates SRF3-SRF8 showed pathogenicity towards the fruit, displaying typical brown dry rot symptoms, and the lesions were consistent with field symptomatic samples. Finally, multiple re-isolates obtained from the inoculated fruits exhibited morphological characteristics identical to the original isolates SRF3-SRF8, thereby confirming Koch’s postulates. In contrast, no similar strains were found in the uninoculated control fruits. After comparison, isolates SRF3-SRF8 had the same colony morphology on PDA. The colony morphology on PDA was fluffy, and the colony was initially yellowish-white in color, and later turned grayish-green with a circular whorl on the back. The vegetative hyphae were septate and extensively branched, the conidiophore expanded into an ovoid or clavate shape, conidia were hyaline cylindrical with blunt ends and light green with size of 11.8 ~ 16.5 × 3.2 ~ 4.6 µm (average: 14.2 × 3.9 µm, n = 30). These morphological characteristics were similar to the species of Colletotrichum recorded previously (Liu et al. 2022). The genomic DNA of the isolates SRF3-SRF8 and re-isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) method. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), and actin (ACT) of isolates SRF3-SRF8 and re-isolates were amplified using primer pairs ITS-1/ITS-4, GDF/GDR, CL1C/CL2C and ACT-512F/ACT-783R, respectively (Feng et al. 2023). The ITS region, GAPDH, CAL, and ACT genes of isolates were sequenced and compared to the NCBI database. We confirmed that ITS region sequence showed 99.81% identity with C. siamense (OQ652493.1), GAPDH sequence showed 100.00% identity with C. henanense (OP781534.1), CAL sequence showed 99.01% identity with C. gloeosporioides (OP622633.1), ACT sequence showed to 99.65% identity with C. henanense (OP750621.1). Multilocus phylogenetic analysis based on the above-mentioned molecular markers suggested that SRF5 clustered on the same evolutionary branch with C. henanense. The sequence information was deposited in GenBank under accession numbers PP474958.1, PP492689.1, PP492690.1, and PP492691.1. Additionally, sequences of other isolates and the re-isolates matched those of the SRF5 isolate, further confirming their identity. To our knowledge, this is the first report of C. henanense causing fruit rot in I. verticillata. Colletotrichum spp. is an important pathogen causing Holly fruit rot (Lin et al. 2018). This emerging pathogen has caused serious economic losses to I. verticillata and may pose a potential risk to other plant species therefore appropriate management measures should be taken to avoid further losses in future.