Occurrence of Nigrospora osmanthi Causing Leaf Blight on Water Lettuce in China

Water lettuce (Pistia stratiotes L.), is one of the emerging invasive weeds for inland water bodies in Asia and become a major problem for local water ecosystem. Biocontrol of water lettuce by mycobiota is being considered as a promising and sustainable method (Kongjornrak et al. 2019). During July 2021, a leaf blight of water lettuce was observed within about 1.5 ha in Shenxi stream (N25°66', E119°05') in Putian, Fujian, China. The disease severity was about 100% with 80% incidence, early symptoms appeared as small irregularly yellow or brown blight, severely infected leaves turned to be rot, then death and sink. Small pieces (5 × 5 mm) of symptomatic leaves were excised and surface disinfected with 75% ethanol and 0.1% HgCl2 solution, air dried and plated on potato dextrose agar (PDA). 3~5 days after incubation at 28°C, six fungal pure cultures showing similar morphology were obtained from the infected leaves. On PDA, colonies were flat, aerial mycelium grew sparsely, most of it grew inside the agar medium, it reverses white to grey to black with age. Hyphae were branched, septate, smooth and hyaline. Conidiophores mostly reduced to conidiogenous cells and setae were not observed. Conidiogenous cells were monoblastic, discrete and solitary, at first hyaline, subspherical, then turning to pale brown, ampulliform, 4.5-10 × 3.5-6 μm in size. Conidia were solitary, globose or ellipsoidal, black, smooth, some of it formed directly from the mycelia, aseptate, 8-12 μm diam (n=10). Genomic DNA was extracted from one of the representative isolate Z1. ITS1/ITS4 (Mills et al. 1992), Bt-2a/Bt-2b (Glass and Donaldson 1995) and EF1-728F/EF-2 (O'Donnell et al. 1998) primer pairs were used to amplify the isolate's internal transcribed spacer (ITS), the Beta-tubulin fragment (TUB) and the partial translation elongation factor (TEF1), respectively. The isolate's sequences were deposited in the GenBank with accession numbers of OM279539 (ITS), OM296034 (TUB) and OM296035 (TEF1). Phylogenetic analysis using maximum likelihood based on the ITS-TUB-TEF1 concatenated sequences from Nigrospora species revealed that isolate Z1 is closely clustered with N. osmanthi strain LC4487. The fungus was identified as N. osmanthi based on the morphological characteristics and molecular analyses (Hao et al. 2020; Wang et al. 2017). Pathogenicity test were performed using twenty inoculated and control plants, respectively. Conidial suspensions (107 CFU/ml) of Z1 isolate were spray-inoculated on the leaves of healthy water lettuce seedlings, while sterile distilled water was used as control. Inoculated and control plants were kept in the differential 50-liter plastic tanks and maintained in a greenhouse at room temperature (19 to 24°C) for one month. Symptoms appeared 7 days post inoculation, which was similar to what occurs in the field. No symptoms occurred on controls. Pathogen was reisolated and confirmed by morphology and molecular analysis. Koch's postulates were conducted twice. N. osmanthi is a pathogenic fungus of many crop plants, such as buckwheat (Shen et al 2021), Java tea (Ismail et al. 2022) or buffalograss (Mei et al. 2019) in Asia and particularly in China. However, to our knowledge, this is the first report of N. osmanthi causing leaf blight on water lettuce. Further studies on how to apply formulated N. osmanthi will be required so that the strain could be effectively used to control water lettuce, moreover, its environmental safety also need a rigorous experimental evaluation.