First Report of Pestalotiopsis trachicarpicola, a Causal Agent of Leaf Spot of Eucommia ulmoides in Southwest China

Eucommia ulmoides Oliver is a native species mostly located in the mountainous regions in southwest China. Its timber is widely used for ecological, medical, and agricultural potential, especially in manufacturing (e.g., fuel, fiber, and rubber). Since April 2017, 50% of E. ulmoides individuals in the cultivating areas in Sichuan Province exhibited symptoms similar to leaf spot. Symptoms gradually appeared from leaf edges to middle tissues; the color of symptomatic leaves gradually changed from light brown to yellow. Some leaves showed circular or irregular spots later; these spots exhibited a gray center and a dark brown edge, which were fruiting bodies of the pathogen. Reddish-brown or purple-brown dots appeared on leaves in the initial stage and developed into round or amorphous dark spots. Yellow lesions haloed around the radial edges before falling off or perforating with an irregular edge margin. In the late period, small scattered black particles were found on lesions, which were the pathogen’s conidia. Infected tissues generally resulted in severe defoliation of leaves and branches. To isolate the responsible fungus, 300 infested leaf fragments were taken from the diseased tissues. Infected tissues were systematically sampled by cutting into 5 × 5 mm pieces. Samples were surface sterilized for 30 s in 3% NaClO and 30 s in 75% ethanol, followed by rinsing three times in sterile water before placing on potato dextrose agar (PDA, Solarbio, Beijing) (25°C for 3 to 8 days). Thirty isolates were obtained, of which 22 strains exhibited the morphology described for P. trachicarpicola (Maharachchikumbura et al. 2012), and the isolation rate was 73.3%. On PDA the fungus initially formed a circular colony with a diameter of ∼60 mm in 7 days at 25°C, with edge fimbriate, whitish to pale yellow, with dense aerial mycelium on surface, fruiting bodies black, developing in concentric circles; reverse of culture yellow. The conidiomata of suspicious fungus was 120 to 410 μm in diameter, acervuli, globose, black, semi-immersed on PDA black conidia in a slimy and glistening mass; its conidiogenous cells were fusiform, hyaline, short, and thin walled (Jayawardena et al. 2015). Conidia contained five cells (per size 21.5 to 25.8 × 5.5 to 7.5 µm). Basal cell was conic to acute, hyaline, thin-walled verruculose, 2.8 to 6.8 µm. The apical cell was conic to subcylindrical, hyaline, 2.7 to 6.4 µm. The middle three cells were all olive color; from the top the second cell was 2.9 to 7.4 µm, the third was 3.4 to 6.8 µm, the fourth was 3 to 5.8 µm, two to three apical appendages, length 5 to 17 µm, and a basal appendage of 2.2 to 8.3 µm. A pathogenicity test showed that those leaf spots of E. ulmoides were mainly caused by P. trachicarpicola. DNA was extracted from 22 fungal colonies using a Plant Genomic DNA Extraction Kit (Solarbio). Polymerase chain reaction (PCR) was performed with primers ITS4/ITS5 for internal transcribed spacer (ITS) region (White et al. 1990), BT2A/BT2B for β-tubulin gene (TUB) (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997), and EF1-526F/EF1-1567R for translation elongation factor 1-alpha (TEF-1α) (Bensch et al. 2012). PCR was amplified and its products sequenced and blasted. The BLAST comparison of the sequenced data (MG827240, MG833724, and MG833725) showed a 99% similarity with sequences of P. trachicarpicola of their ITS region, TUB, and TEF-1α (KX609685.1, JX399035.1, and JQ845946.1, respectively). To confirm pathogenicity and fulfill Koch’s postulates, 1-year-old potted E. ulmoides young leaves were inoculated with the same pathogens by spraying 50 µl of a conidial suspension (1 × 10⁶ conidia/ml) on both sides of leaves. Axenic culture medium (10 plants) and sterile water (10 plants) were used as negative controls. Fifteen days later, the inoculated plants showed the same symptoms observed in diseased plants; controls remained asymptomatic. The pathogen was reisolated from symptomatic leaves and identified by morphological characteristics and molecular method. This is the first report of P. trachicarpicola causing leaf spots on E. ulmoides in southwest China.