First Report of Colletotrichum brevisporum Causing Anthracnose on Pumpkin in China

Abstract

In the summer of 2015, dark brown lesions were observed on green husk pumpkin cultivar Xiangli No. 23, in Changchun (125.39°E, 43.45°N), Jilin Province, China. Nearly half of the plants in a 0.01-ha field of pumpkin were symptomatic. Symptoms on fruit were initially irregular dark green spots of 2 mm in diameter. As the disease progressed, spots expanded and gradually became round or oval, and the outside and inside of the necrotic areas became black. With higher humidity, abundant white aerial mycelia were produced on the black disease spots and orange conidial cirrhi visible in concentric rings on the margins of the lesions, and fewer acervuli were seen. Finally, the fruit were rotten. To isolate the pathogen, small pieces of symptomatic fruit were surface sterilized in 1.5% NaOCl for 1 min, washed twice with sterile distilled water, and then placed onto potato dextrose agar (PDA). Monoconidial isolate CCCM12 was grown on PDA under a 12-h photoperiod at 25°C. The color of the upper surface of the colony varied from white to off-white with cottony aerial mycelium, and was pale to orange with heavy sporulation on the underside. Conidia (n = 150) were one-celled, straight, hyaline, cylindrical with round ends (98%) or one end with acute apices (2%), smooth-walled, guttulate, and measured 10.8 to 21.4 (16.3 ± 2.1) μm in length and 3.6 to 10.4 (5.7 ± 1.0) μm in width. Appressoria (n = 50) were brown to dark brown, variable in shape including ovoid, clavate, or slightly irregular, and measured 4.9 to 14.5 × 4.7 to 10.8 μm. Based on morphological characteristics, the fungus was preliminarily identified as Colletotrichum brevisporum following descriptions of Noireung et al. (2012). The internal transcribed spacer (ITS), β-tubulin (TUB2), actin (ACT), and chitin synthase (CHS-1) gene regions of isolate CCCM12 were amplified by using ITS4/ITS5, Bt2A/Bt2B, ACTF/ACTR, and CHSF/CHSR primers, respectively (Weir et al. 2012). Their GenBank accession numbers were obtained and were submitted to GenBank. A BLAST search revealed that all sequences (GenBank accession nos. KY797632 for ITS, KY797630 for TUB2, KY797629 for ACT, and KY797631 for CHS-1) matched with 99 to 100% identity to C. brevisporum (KC790943 for ITS, JN050244 for TUB2, JN050217 for ACT, and KF687760 for CHS-1). Molecular phylogenetic trees constructed using MEGA7 confirmed this. To confirm pathogenicity, following Bezerra et al. (2016), 5-mm-diameter hyphal plugs were taken from a 7-day-old culture of CCCM12 and singly placed on each of 15 mature and healthy Xiangli No. 23 pumpkins superficially wounded with a sterilized needle. In control fruits, only PDA discs without fungal growth were used. Fruits were covered with plastic bags and incubated in a growth chamber at 25°C at 95% relative humidity. Symptoms (pale to black sunken lesions) developed on inoculated fruit 6 days after inoculation. No lesions were observed on control fruit. When fungi were reisolated from lesions of inoculated fruit onto PDA, colony and conidial morphology were identical to the original isolate. The experiment was performed three times. The fungus was placed in the Herbarium of Mycology of Jilin Agricultural University. This pathogen has been previously reported on Pandanus pygmaeus and Lycium chinense in Thailand and Korea (Noireung et al. 2012; Paul et al. 2014), on fruits of Carica papaya and Sechium edule in Brazil (Bezerra et al. 2016; Vieira et al. 2013), and on pepper fruits in China (Liu et al. 2016). This is the first report of C. brevisporum causing anthracnose on green husk pumpkins.