Diaporthe species are often reported as plant pathogens, endophytes, and saprobes. In this study, three new species: Diaporthe foliicola, D. monospora, and D. nanjingensis on Acer palmatum were described and illustrated based on morphological characteristics and phylogenetic analyses. Phylogenetic relationships of the new species were determined by multilocus phylogenetic analyses based on partial sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF), beta-tubulin (TUB), histone H3 (HIS) and calmodulin (CAL) genes. Genealogical Concordance Phylogenetic Species Recognition (GCPSR) with a pairwise homoplasy index (PHI) test was used to verify the conclusions of the phylogenetic analyses. All species were illustrated and their morphology and phylogenetic relationships with other related Diaporthe species are discussed. In addition, the tests of Koch’s postulates showed that the three new species were pathogens causing leaf blight on A. palmatum.
Liriodendron chinense × tulipifera is widely planted in China as an ornamental plant. In July 2018, a foliar disease, with an incidence of ~23% trees and ~ 45% leaves per individual tree infected, occurred on L. chinense × tulipifera on the campus of Nanjing Forestry University, Jiangsu Province, China. Symptoms were small brown to dark spots surrounded by a yellow halo initially, which enlarged and coalesced into regular or irregular brown necrotic lesions up to 8.5 ± 1.3 mm across. Fungi were isolated from the margins of the lesions. Three representative isolates (E3‐2, E3‐3 and E3‐4) were selected for phylogenetic analyses. One representative isolate E3‐2 was used for pathogenicity tests and morphological identification. The pathogenicity of isolate E3‐2 to L. chinense × tulipifera was confirmed on 1‐year‐old seedlings with lesions after 5 days. The phenotypic characteristics of the isolates were similar to those of Colletotrichum sp. (Glomerellaceae). Phylogenetic analyses using concatenated sequences of ITS, ACT, CHS, GADPH, CAL, SOD2 and TUB2 placed E3‐2, E3‐3 and E3‐4 in the clade of C. fructicola. Based on the morphological and phylogenetic studies, the isolates were identified as C. fructicola. This is the first report of C. fructicola causing L. chinense × tulipifera leaf spots in China.
Viburnum odoratissimum var. awabuki (K. Koch) Zabel ex Rumpl. is an evergreen tree, used as a landscape plant in China. In June 2019, a foliar disease of ~60% incidence was observed on V. odoratissimum var. awabuki at the campus of Nanjing Forestry University, Jiangsu, China. The symptoms were initially irregular small red-brown spots, later enlarged and became brown to black. Small pieces of tissue (3 to 4 mm2) cut from lesion margins were surfaced sterilized in 75% ethanol for 30 s and 1.5% NaClO for 60 s, then rinsed in sterile water and placed on potato dextrose agar (PDA) at 25℃. Pure cultures were obtained from the tip of hyphae. Using the standard phytopathological procedure, two representative isolates (SH161 and SH181) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelium, radiate, and the reverse was white. Black pycnidia developed on the sterilized alfalfa stems at 25°C with a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, straight to sinuous, 9.4 to 26.0 × 1.0 to 2.5 μm (n=30). Conidiogenous cells were 2.1 to 15.1 × 0.9 to 2.5 μm (n=30). Alpha conidia were 7.4 ± 0.6 × 2.0 ± 0.2 μm (n=50), hyaline, ellipsoidal to lanceolate. Beta conidia were 29.5 ± 1.8 × 1.1 ± 0.1 μm (n=30), aseptate, hyaline, smooth, curved to hooked. Morphological features of two isolates matched those of Diaporthe spp.. DNA of two isolates was extracted and the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-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. The sequences were deposited into GenBank (Accession Nos. for isolate SH161: OK326730 for ITS, OK413403 to OK413406 for TUB, CAL, HIS and TEF1-α; and isolate SH181: OK331347 for ITS, OK413407 to OK413410 for TUB, CAL, HIS, and TEF1-α). BLAST search of SH161 showed high similarities with sequences of Diaporthe eres (AR5193) [KJ210529 (ITS), Identities = 438/512, (94%); KJ420850 (HIS), Identities = 466/472, (99%); KJ210550 (TEF1-α), Identities = 345/350, (99%); KJ434999 (CAL), Identities = 344/345, (99%); KJ420799 (TUB), Identities = 508/517, (98%)]. BLAST results of SH181 are listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed SH161 and SH181 in the clade of D. eres. Based on the multi-locus phylogeny and morphology, two isolates were identified as D. eres. The pathogenicity was tested on 1-yr-old cuttings of V. odoratissimum var. awabuki in the greenhouse. Healthy leaves were wounded with a sterile needle, then inoculated with 5-mm plugs from the edge of two isolates cultures. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags bidaily to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. Three days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe eres was reisolated from inoculated leaves. No fungus was isolated from controls. Diaporthe eres was reported from Viburnum lantana in Austria. Also, it was reported from V. odoratissimum and V. tinus in Ukraine. This is the first report of D. eres causing V. odoratissimum var. awabuki leaf spots in China. This finding will provide an effective basis for developing control strategies for the disease.
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