First Report of Colletotrichum fructicola Causing Anthracnose on Paeonia lactiflora in China.

Abstract

Paeonia lactiflora Pall is a traditional famous flower with long cultivated history in China, and has important medical and ornamental functions (Duan et al. 2022). In the middle of June 2022, anthracnose disease was observed nearly 25% (n=90) on P. lactiflora in Poyang County, Shangrao City, Jiangxi Province (29.00° N, 116.67° E) (Figure 1 E). The symptoms of the disease were small, round, light brown spots then grew bigger to round or irregular dark brown lesions (5 to 7 mm diameter) progressively on the leaves with disease spread (Figure 1 A). Subsequently, necrotic tissue was formed in the center and caused fade and wilt on the leaves ultimately, which reduced the medicinal and aesthetic value severely. Small pieces of diseased tissue (5 × 5 mm) were cut from the diseased junction, disinfected with 75% ethanol for 30 to 45 seconds, then 1% NaClO for 1 to 2 minutes, rinsed three times with sterile water. To identify the pathogen, tissues were placed on PDA and incubated for 3 days at 28°C. Single spore isolates were cultured on PDA, the colonies of one representative strain (SY4) were originally white with a lot of aerial mycelium after 5 to 7 days at 28°C in the incubator. The center of the colony turned greyish-white, released tiny orange-yellow particles (conidia) (Figure 1 F and 1 G), which were single, colorless, elongated ovals with rounded ends and measured 11.29 to 23.24 × 3.94 to 5.60 μm (av=15.89 μm × 4.74 μm, n=50) (Figure 1 H and 1 I). The isolate SY4 was identified to Colletotrichum fructicola based on morphological characteristics (Yang et al. 2021; Li et al. 2022b). For further molecular identification, the rDNA-ITS, actin gene (ACT), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), chitin synthase (CHS) and calmodulin gene (CAL) genes were amplified and sequenced with primers of ITS1/ITS4 (Gardes et al. 1993), ACT-512F/ACT-783R, GDF/GDR (Templeton et al. 1992), CHS-79F/CHS-345R (Carbone et al. 1999) and CL1C/ CL2C (Weir et al. 2012) respectively. The accession numbers in GenBank were OP523977 (ITS-rDNA), OP547618 (ACT), OP605733 (GAPDH), OP605732 (CHS), and OP605731 (CAL). The BLAST analysis revealed that these sequences were identical more than 99% with those of C. fructicola (GenBank accession Nos. MZ437948.1, MN525803.1, MN525860.1, MZ13360.1 and ON188684.1) (Figure 2). To confirm pathogenicity, the leaves were cleaned with 75% ethanol, rinsed with sterile water. After the leaf surface was dried naturally, 20 leaves were pricked at two symmetrical places on either side of the main veins of the leaf with a sterilized inoculum needle (2.0 mm in diameter), half of the wounded leaves were inoculated with 20 μL spore suspension (1.0 × 106 spores/mL) (Figure 1 C and 1 D), while the other half were inoculated with sterile water as controls (Figure 1 B). Inoculated leaves were grown for 5 days in an incubator at 28 °C and above 90% relative humidity, repeated three times. The results demonstrated that the wounded leaves with C. fructicola showed the same signs of wilting with the original disease leaves, while control leaves remained healthy. The same fungus was reisolated from the diseased leaves which confirmed with Koch's postulates. The same fungus was re-isolated from the diseased leaves while it was not isolated from control leaves, confirmed with Koch's postulates. In China, it had been reported that C. fructicola caused anthracnose on Persea americana (Li et al. 2022a) and Myrica rubra (Li et al. 2022b). To the best of our knowledge, this is the first report of anthracnose on P. lactiflora caused by C. fructicola in China. The results will help to develop effective control strategies for anthracnose on P. lactiflora.