First Report of Top Blight of Cunninghamia lanceolata Caused by Diaporthe unshiuensis and Diaporthe hongkongensis in China.

Abstract

Cunninghamia lanceolata (Lamb.) Hook. is an important conifer species widely planted in southern China. A top blight, with an incidence of 20% (40/200 seedlings), occurred on 1-year-old seedlings of C. lanceolata in a nursery, Luzhai, Guangxi, China in August 2021. The disease mainly occurred on shoot tips. The infected needles and shoots appeared brown to brownish red. White conidial tendrils oozed from pycnidia under wet-weather conditions. Lesion margins from fresh samples were cut into small pieces (n=100), which were sterilized according to Mao et al., and placed on potato dextrose agar (PDA) at 25°C. Three isolates (GXJ2, GXJ4, and GXJ6) were obtained and deposited in The China Forestry Culture Collection Center (CFCC 55717, CFCC 55716, and CFCC 55722). The colony of GXJ2 on PDA was white, with sparse aerial mycelia, and became grey with time. The α conidia were fusiform, hyaline, and aseptate, 6.7±0.6 μm × 2.6±0.2 μm (n=30). The β conidia were filiform, hyaline, and curved, 30.4±2.1 μm × 1.4±0.1 μm (n=30). Colonies of GXJ4 and GXJ6 were white, with moderate aerial mycelia, which collapsed at the center, and the collapsed parts were iron-gray. The α conidia were 7.8±0.8 μm × 2.5±0.2 μm (n=30). The β conidia were absent. Morphological characters of 3 isolates matched those of Diaporthe spp.. The partial sequences of ITS, EF1-α, CAL, β-tub, and HIS genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R and CAL228F/CAL737R, βt2a/βt2b and CYLH3F/H3-1b according to Gomes et al. 2013, respectively. The sequences for the five genes of each of 3 isolates were deposited in GenBank (Accession Nos. see Supplementary Table 1). BLAST results showed that the ITS, EF1-α, β-tub, HIS, and CAL sequences of GXJ2 were highly similar (>99%) with sequences of Diaporthe unshiuensis, while sequences of GXJ4 and GXJ6 were highly similar (>99%) to those of D. hongkongensis (Supplementary Table 1). Phylogenetic analyses using concatenated sequences placed GXJ2 in the clade of D. unshiuensis, while GXJ4 and GXJ6 in the clade of D. hongkongensis. Based on the phylogeny and morphology, GXJ2 was identified as D. unshiuensis, GXJ4 and GXJ6 as D. hongkongensis. Pathogenicity tests were performed on nine 1-year-old seedlings of C. lanceolata, and 10 needles at shoot tip per seedling were slightly wounded and inoculated with 5-mm mycelial plugs from one of 3 isolates. Three control seedlings were treated with PDA plugs. Each plant was covered with a plastic bag after inoculation and kept in an air-conditioned nursery at 25°C/16°C (day/night). The symptoms appeared 5-8 days after inoculation and were similar to those observed in the nursery. D. unshiuensis and D. hongkongensis were re-isolated from the inoculated seedlings and were confirmed based on morphology and ITS sequences. The controls were symptomless, and no fungus was isolated from them. D. unshiuensis was first reported as an endophyte on the fruit of Citrus unshiu, and caused peach constriction canker, shoot blight of kiwifruit. D. hongkongensis was first described from fruit of Dichroa febrifuga and caused shoot canker of pear, shoot blight and leaf spot of kiwifruit, and fruit rot of peach. This is the first report of D. unshiuensis and D. hongkongensis causing the top blight of C. lanceolata. This study provides a basis for controlling this newly emerging disease in the nursery.