First Report of Leaf Spot Disease Caused by Colletotrichum gloeosporioides on Chaenomeles sinensis in China

Abstract

Chaenomeles sinensis is a shrub or small arbor of the genus Chaenomeles in Rosaceae, which is widely planted in China. It is a kind of garden ornamental plant and has high economic value. Since 2020, a leaf disease occurred on the foliage of C. sinensis at the campus of Nanjing Forestry University, Nanjing, China. After investigating, C. sinensis was found with leaf spot disease at a 100% infection rate, which causing gigantic ornamental loss. Leaf spots are round to irregular distributing on the leaves, in addition, the color of spots is brown. There are yellow halos on the edge of the lesion. Small leaf tissues (3 to 4 mm2) from lesion margins were surface sterilized with 75% ethanol for 30s and then rinsed with sterile dH2O for three times. Afterwards, placed on potato dextrose agar (PDA) at 25°C. Pure cultures were obtained by monosporic isolation, and a representative isolate (NJTJ.1) was obtained. When cultured on PDA, the colony of NJTJ.1 was white and cottony. On the reverse side, the color of colony nearly light yellow. The colony were placed in the liquid Carboxymethyl cellulose (CMC) medium. After culturing for 24h in a shaker at 25℃ and 150rmp/min, the spore liquid was taken by us. The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and measured 15.1 to 23.6× 5.4 to 7.9 µm (n =30). Appressoria were one-celled, brown, thick-walled, ellipsoidal, and measured 7.7 to 13.8 × 6.4 to 10.3 µm (n =30). The morphological characteristics of NJTJ.1 fitted with the description of the Colletotrichhum gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS-1) were amplified with primers ITS1/ITS4, GDF/GDR, ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al, 2019), respectively. The sequences were deposited in GenBank [Accession Nos.MT984264, MW030495 and MW030496 to MW030497 for NJTJ.1]. A Blast search of GenBank showed that ITS, GAPDH, ACT and CHS-1 sequences of NJTJ.1 were 99%, 99%, 100% and 100% identical to those of C. gloeosporioides (MH571757.1 ,KY995355.1 , MN058143.1 and MN313581.1). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate NJTJ.1 clustered in the C. gloeosporioides clade with 99% bootstrap support. The pathogenicity of the NJTJ.1 was verified both on detached and living leaves. The detached leaves were inoculated with 5-mm mycelial plugs cut from the edge of 6-day old cultures on PDA and 20 μL of spore suspension (106 conidia/mL) and each treatment had 5 replicates. Controls were treated with sterile dH2O. The inocula were placed at a distance of 2 to 3 cm on the leaves which were wounded with a sterile needle. All of them were placed in 20-cm dishes on wet filter paper at 25°C. After 5 days, all the inoculated points showed lesions which were similar to those outdoor observed. Whereas, controls were asymptomatic.At the same time, the plugs of C. gloeosporioides were inoculated on living leaves.After 7 days, the leaves which were inoculated also appeared lesions. This is the first report of C. gloeosporioides causing leaf blotch on Chaenomeles sinensis in China. These data will help develop effective strategies for managing this disease.