First Report of Alternaria crassa Causing Yellow Spots on Datura stramonium in China

Datura stramonium L. (jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in Zhanjiang (21.17°N, 110.18°E), Guangdong Province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned necrotic with a clear yellow halo and a white center. The disease incidence in the field was 85% (n = 50, about 1 ha). Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 × 2-mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed two times with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28°C. After 2 days of incubation, grayish fungal colonies appeared on the PDA medium, and then pure cultures were produced by transferring hyphal tips to new PDA plates. The single-spore isolation method was used to recover pure cultures for three isolates (DSAC-1, DSAC-2, and DSAC-3). The isolates were morphologically identical. The colonies were gray to brownish black. Conidiophores were branched and brown. Conidia were brown and long ellipsoid, had 4 to 12 transverse and 0 to –3 longitudinal septa, and measured 67.5 to 127.8 (average = 105.6) × 12.5 to 27.8 (average = 20.4) μm (n = 30). The apical beak was longer than the conidia body, measuring 40.5 to 423.5 (average = 365.2) × 2.5 to 5.8 (average = 3.2) μm (n = 30). Based on the morphological characteristics, the three isolates were identified as Alternaria crassa (Sacc.) Rands (Simmons 2007). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2), and translation elongation factor (TEF1) with the primers of ITS1/ITS4, GDF1/GDR1, RPB2-5F2/fRPB2-7cR, and EF-1α-F/EF-1α-R, respectively (Nishikawa and Nakashima 2020; Walther et al. 2013; Woudenberg et al. 2015). Amplicons of the isolates were sequenced and submitted to GenBank (ITS, ON430524 to ON430526; GAPDH, ON500656 to ON500658; RPB2, ON500659 to ON500661; TEF1, ON500662 to ON500664). The BLAST analysis showed that the sequences were 100% identical with those of the A. crassa strain CBS 116647. The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. crassa (CBS 116647, CBS 116648, CBS CBS-110.38, and CBS_103.18). Thus, the fungus associated with leaf yellow spot on D. stramonium was identified as A. crassa. Pathogenicity tests were conducted in a greenhouse at 24 to 30°C with 80% relative humidity using the three isolates. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated with the three isolates (DSAC-1, DSAC-2, and DSAC-3). The fungal mycelia on 5-mm-diameter PDA plugs were placed faced down to the leaves. Sterile PDA was used for mock inoculated controls. The test was performed three times. Disease symptoms were observed on the leaves after 7 days, whereas the controls remained healthy. The pathogen was reisolated from the infected leaves and was morphologically identical to the original isolates, thus fulfilling Koch’s postulates. A. crassa was reported causing leaf spot on D. stramonium in Algeria (Nabahat et al. 2020). To our knowledge, this report is the first report of A. crassa causing yellow leaf spot on D. stramonium in China. This pathogen has potential biocontrol properties on the invasive weed, and this study also provides an important reference for the control of the disease on the medicinal plant.