First Report of Colletotrichum fructicola Causing Anthracnose on Water Hyacinth in China

Water hyacinth (Eichhornia crassipes), a worst invasive aquatic weed has been caused the widespread problems for the water bodies and water resources, particularly the case in China. Plant pathogens are a promising alternative as biocontrol agents (Dagno et al. 2011), but success in this strategy will require the selection of some highly virulent pathogen strains. In September 2020, irregular necrotic lesions on leaves, stems, as well as crown and petiole rots symptoms, occurred on water hyacinth, in Minjiang and Xiyuanjiang watershed, in Fuzhou, China. Fragments from symptomatic leaf tissue (5x5mm) were superficially disinfected in 0.1% MgCl2 solution for 30 s, followed by rinsing three times in sterile water, placed on potato dextrose agar (PDA), and then incubated in darkness at 28°C for 5 days. Two fungal isolates (F3 and F11) were recovered and obtained pure cultures from the affected leaves and deposited in the Institute of Oceanography, Minjiang University. The colonies were stale, with felted, dense, pale grey aerial mycelium, scattered dark based acervuli with orange conidial masses near centre; in reverse side pinkish orange with patches of grey pigment near centre. The hyphae were septate, branched, and 2 to 6 µm in width. Appressoria were not observed. Conidiogenous cells were 20-24 × 3.5-4.5 µm, cylindric to flask-shaped, towards margin the conidiophores with a much looser structure, conidiogenous loci at apex and often also at septa. Asci were 60-80 × 15-20 µm, cylindric to subfusoid, 8-spored. Ascospores were 17-23 × 4-6 µm, gently curved, tapering to quite narrow, rounded ends. Perithecia mature after about 15 days, and were dark brown, subglobose, and 50-150 μm in diameter, and with scattered, dark brown setae about 50-80 µm long. Conidia were 15-25 × 4.5-6 μm, unicellular, colorless, and cylindrical to fusiform. Genomic DNA from two isolates was extracted with a modified DNA Midi Kit (TIANGEN, Inc., Beijing, China), and amplified using ITS4/ITS1F, CL1/CL2A, CHS-79F/CHS-345R, T1/T2 and GDF/GDR primers by PCR (Weir et al. 2012; White et al. 1990). Sequences of F3 and F11 were submitted to GenBank (accession no. ITS, MW307302, MW307303; CAL, MW303427, MW303429; CHS-1, MW303428, MW303430; TUB, MW531006, MW531007; GADPH, MW531008, MW531009). A phylogenetic tree using the maximum likelihood methods and including ITS-CHS-CAL-TUB-GADPH concatenated sequences from Colletotrichum gloeosporioides complex was obtained (Cai et al. 2009; Damm et al. 2018; Weir et al. 2012). Phylogenetic analyses revealed that isolate F3 and F11 were grouped into the clade C. fructicola. To test Koch's postulates, conidial suspensions (107 CFU/ml) of the isolate F3 and F11 were micro-injected into 20 water hyacinth seedlings per isolate. Another 20 seedlings were injected with water without conidia as control. Inoculated plants were kept in 50-liter plastic tanks, and maintained in a greenhouse at room temperature (19-24ºC) for two weeks. The Koch's test was conducted twice. After 10 days, typical anthracnose symptoms similar to the field appeared on the inoculated leaves, while the control leaves remained asymptomatic. The C. fructicola was re-isolated and identified by microscopy, PCR and sequencing, but not on non-inoculated controls. Anthracnose disease caused by C. fructicola has been reported affecting numerous plants worldwide, including cotton, coffea, grape, citrus, ect (Guarnaccia et al. 2017). However, to our knowledge, this is the first report of C. fructicola causing anthracnose on water hyacinth in China. Further studies for the efficacy of C. fructicola and/or of the genus Colletotrichum as biocontrol agent for water hyacinth or another aquatic plant are required (Ding et al. 2007; Dagno et al. 2012).