First Report of Anthracnose on Pecan (Carya illinoensis) Caused by Colletotrichum nymphaeae in China

From 2015 to 2017, anthracnose symptoms on pecan were found in Zhejiang province. Field investigations conducted from July to November in 2017 found anthracnose symptoms on at least 50% leaves in a Jiande pecan orchard (N 29°25′17.7″, E 119°18′10.7″). More leaves located at the bottom were infected than in higher parts, and the top leaves hardly showed infection. At first, light yellow spots appeared and then enlarged and, finally, developed into characteristic anthracnose symptoms including irregular-shaped lesion with a chlorotic halo. From September to November, anthracnose symptoms were also observed on the fruits. Infected fruits became dark, rotten, mummified, and finally dropped with a rate of 30 to 50%. Spores were isolated from the fresh infected tissues following the methods of Velho et al. (2014) and were examined under a microscope. Vegetative mycelium was hyaline, smooth-walled, septate, and branched. Conidia were smooth-walled, aseptate, hyaline, guttulate, straight, fusiform to cylindric with one end round and one end round to acute, with a size of 10.1 to 15.8 µm long (mean = 12.3 µm, n = 60) and 3.4 to 4.9 µm wide (mean = 4.1 µm, n = 60). Appressoria were single, medium brown, smooth-walled, elliptical, clavate or irregular in outline, entire, undulate to lobate margin, with a size of 5.4 to 10.5 µm long (mean = 8.1 µm, n = 30) and 4.0 to 6.5 µm wide (mean = 5.2 µm, n = 30). Genomic DNA of 15 isolates was extracted, and six gene sequences (ITS, GAPDH, CHS-1, CAL, ACT, and TUB2) were amplified and sequenced as described in Fu et al. (2019) and were submitted to GenBank with accession numbers of MH231421 (ITS), MH793690 (GAPDH), MH793689 (CHS-1), MH793688 (CAL), MH891493 (ACT), and MH796660 (TUB2). BLAST analysis showed that all sequences shared high identity with Colletotrichum nymphaeae (100% with ITS, ACT, and TUB2, and 99% with the other three genes). Phylogenetic analysis showed that the isolates and C. nymphaeae were clustered in the same clade. Based on the results of morphological and molecular analyses, the isolates were identified as C. nymphaeae. Pathogenicity tests were carried out three times on living trees. Healthy leaves and fruits were surface cleaned with sterile soaked cotton, air-dried, and then wounded with a sterilized needle. An aliquot of 10 μl of spore suspension (1.0 × 10⁶ conidia/ml) of C. nymphaeae was dropped on each wound. Each treated leaf or fruit was enveloped by a plastic zipper bag with a sterile soaked cotton in it to keep moisture. Sterile distilled water drops served as controls. After 24 h, cotton pieces and plastic bags were removed. Lesions began to appear on the 7th day after inoculation. The symptoms of inoculated treatment developed and exhibited similarly to those observed in pecan orchards, and no symptom was observed on the control. C. nymphaeae was reisolated from the symptomatic pecan tissues and was reidentified with both morphological and molecular methods. According to Koch’s postulates, C. nymphaeae was confirmed as the cause of the disease. To our knowledge, this is the first report of anthracnose on pecan caused by C. nymphaeae in China.