First Report of Diaporthe eres and D. unshiuensis causing Leaf Spots on Sapindus mukorossi in China.

Abstract

Sapindus mukorossi Gaertn., commonly known as soapberry, is widely cultivated as a landscaping tree in Southern China. In June 2019, a foliar disease with an incidence of ∼60% occurred on trees was observed in the soapberry germplasm repository, Jianning, Sanming, Fujian, China. The symptoms initially appeared as irregular small yellow spots, while the center of the lesions became dark brown with time. Fragments (size 3 to 4 mm2) taken from lesion margins were sterilized and cultured based on Wang et al. Two isolates (FJ1 and FJ21) were obtained with the following morphological characteristics on PDA, (1) FJ1: Conidiogenous cells were 9.7 to 25.0 × 1.5 to 2.2 μm (n=20). Alpha conidia were 6.1 to 8.3 × 2.2 to 3.0 μm (n=30), aseptate, hyaline, smooth, ellipsoidal. Beta conidia were 28.3 to 38.2 × 1.3 to 1.7 μm (n=30), hyaline, smooth, curved to hooked. Conidial drops were milky colored; (2) FJ21: Pycnidia were dark brown, 280 to 843 μm (n=30) in diam., globose, or irregular on alfalfa stems. Conidiophores were hyaline, cylindrical, smooth, and slightly tapered to the apex, 17.4 to 35.4 × 1.5 to 2.6 μm (n=20). Conidiogenous cells were 14.7 to 29.7 × 1.4 to 2.6 μm (n=20). Alpha conidia were 5.6 to 7.1 × 2.4 to 3.4 μm (n= 30), hyaline, smooth, ellipsoidal, or clavate, aseptate, biguttulate. Beta conidia not observed. Conidial drops were yellow. The morphological characteristics of FJ1 and FJ21 were similar to those of Diaporthe spp.. DNA of two isolates was extracted, and the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), β-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b, and CYLH3F/H3-1b, respectively. The sequences were deposited in GenBank (accession nos. MW585608 and MW768905 to MW768908 for FJ1; MT755625 and MT776728 to MT776731 for FJ21). The BLASTn results showed that the ITS, TEF1-α, TUB, HIS, and CAL sequences of FJ1 were 100, 99, 98, 98, and 99% identical to those of D. eres (NR144923, KJ210550, KJ420799, KJ420850, and KJ434999, respectively). For FJ21, BLASTing with the same loci showed 100, 100, 100, 99, and 100% similarity with those of D. unshiuensis (MH121530, MH121572, MH121607 MH121488, and MH121448, respectively). Phylogenetic analyses with the concatenated sequences placed FJ1 and FJ21 in the clades of D. eres and D. unshiuensis, respectively. Pathogenicity tests were performed by wounding leaves of 2-year-old soapberry seedlings with a sterile needle. The leaves were inoculated with D. eres and D. unshiuensis isolates, respectively, with 10 µl of conidial suspensions (106 conidia/ml). Three plants were used for each treatment, and the leaves of each plant were inoculated. The control was treated with 10 µl of sterile water. The plants were kept in a greenhouse (RH > 80%, 25 ± 2°C). In 5 days, all inoculated leaves showed lesions similar to the field symptoms. Controls were asymptomatic. Diaporthe eres and D. unshiuensis were reisolated from the diseased leaves. No fungus was isolated from the control. Previously, D. biconispora and D. sapindicola were reported as the causal agents of soapberry, but this is the first report of D. eres and D. unshiuensis causing leaf spots on S. mukorossi in China. These data will help develop effective strategies for managing this disease.