First Report of Cercospora rodmanii Causing Leaf and Petiole Lesions on Water Hyacinth in China.

Abstract

The rapid spread and colonization of water hyacinth (Eichhornia crassipes) leads to a series of serious environmental problems for water bodies, prompting microbiologists to develop effective mycoherbicides to alleviate the water hyacinth population (Julien et al. 2001). In September 2020, numerous leaftip diebacks and petiole rots of water hyacinth, with 40 to 50% incidence, were observed within an area of 2 ha (ca. 2 km) mat on Jinjiang River tributary, Fujian, China. Ten infected leaf samples were collected and symptomatic tissues were cut into small pieces, surface disinfected in 75% ethanol followed by 0.1% MgCl2 solution and placed on potato dextrose agar (PDA). Pure cultures (Isolates J1 and J5) were obtained and their colonies on PDA appeared as white villi with wrinkled surfaces and dense colorless mycelium on the upper surface, while they were dark olivaceous-gray at the bottom. Internal mycelium consisted of septate, branched, smooth hyphae. There lacked stromata. Conidiophores were solitary to 2 to 5 in loose fascicles, thick-walled, conically truncated, 40 to 80 × 5 to 8 µm. Conidiogenous cells integrated, terminal, 30 to 150 × 4 to 6 µm, slightly protuberant, apical and lateral. Conidia were solitary, hyaline, acicular to obclavate, slightly curved, acute at the apex, 50 to 80 ×3 to 6 µm, indistinctly 1 to 3 septate. Genomic DNA from the two isolates was extracted for PCR, and the internal transcribed spacers (ITS), calmodulin (CAL), translation elongation factor 1-alpha (TEF), actin (ACT), histone H3 (H3) and chitin synthase (CHS) were amplified and sequenced using the primer pairs ITS1/ITS4, CL1/CL2A, EF1Fd/EF1Rd, ACT1Fd/ACT1Rd, CYLH3F/CYLH3R and CHS-79F/CHS-345R (Weir et al. 2012; White et al. 1990), respectively. The ITS (MZ436974-MZ436975), CAL (MZ519385-MZ519386), TEF (OK340826-OK340827), ACT (OK340824-OK340825), H3 (OK340828-OK340829) and CHS (MZ519387-MZ519388) sequences were deposited in GenBank. A phylogenetic tree based on concatenated sequences of ITS-CAL-TEF-ACT-H3-CHS from the genus Cercospora was constructed using a maximum likelihood method, showing that the present isolates and Cercospora rodmanii formed a monophyletic group with 99% bootstrap support. Therefore, the fungus was identified as C. rodmanii (Groenewald et al. 2013; Nguanhom et al. 2015). To test Koch's postulates, petioles of a set of 20 water hyacinth seedlings of 30 to 40 days old were wounded using a sterile needle and then spray-inoculated with 20 μl of the spore suspension of each isolate at 106 CFU/ml. Another set of 20 seedlings inoculated with sterile distilled water served as the controls. Inoculated plants were kept in 50-liter plastic tanks and maintained in a greenhouse at room temperature 22 to 28°C with a relative humidity of 80 to 85%. After 7 to 20 days, lesions were observed on the inoculated leaves but not on the control leaves. The same fungus was reisolated and identified by microscopy and PCR-sequencing. The pathogenicity test was conducted twice. Cercospora rodmanii and C. piaropi have been reported on water hyacinth in America, Brazil, México, and Zambia (Charudattan et al. 1985; Montenegro-Calderón, 2011; Moran, 2015). To our knowledge, this is the first report of C. rodmanii causing leaf and petiole lesions on water hyacinth in China. This report will help identify indigenous plant pathogens in China and develop a novel bioherbicide strategy for control of water hyacinth.