Pseudocercospora exilis Causing Leaf Spot Disease on Brassica rapa subsp. parachinensis in China

Abstract

Chinese flowering cabbage (Brassica rapa L. subsp. parachinensis) is an important vegetable crop of China and widely cultivated in the southern provinces. During February of 2017 to 2019, a severe leaf spot disease was seen on Chinese flowering cabbage plants in different agricultural fields around Lianzhou and Guangzhou cities of Guangdong province, China. Disease incidence reached up to 70% in severely infested fields. Early disease symptoms observed were small circular or irregular spots of brown color, 3 to 11 mm in diameter and often coalesced. Ten symptomatic leaves were cut into small pieces (5 × 5 mm), surface sterilized with 1% NaOCl, plated onto potato dextrose agar (PDA) medium, and incubated at room temperature for 7 days under continuous fluorescent light. A total of 11 pathogen isolates were purified by successively subculturing on new PDA medium plates using the single-spore technique. A cercosporoid fungus was consistently isolated from diseased leaves. The colonies were raised and greenish black in color. Conidiophores were brown, subcylindrical, multiseptate, straight or slightly curved, unbranched, and smooth. Conidiogenous cells were pale brown, smooth, and arranged terminally. Conidia were pale brown, smooth, obclavate or fusoid, straight to slightly curved, with obtuse apex, one- to seven-septate, and 44.3 to 69.12 × 4.8 to 6.2 μm (n = 20). The morphological and cultural characteristics of all isolates were consistent with Pseudocercospora exilis Hern.-Gut. & Dianese (Hernandez-Gutierrez and Dianese 2009; Silva et al. 2016). One monosporic isolate (PE-1) was obtained by plating individual spores on V8 agar medium. To further confirm the species, genomic DNA was extracted from the hyphal mass of the isolate PE-1 using the DZup fungal DNA extraction kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) and translational elongation factor (EF-1α) regions of the isolate PE-1 were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) and sequenced by Sangon Biotech. The obtained DNA sequences were deposited in GenBank (accessions MN971630 for the ITS sequence and MN991198 for the EF-1α). BLAST analysis showed that the ITS and EF-1α sequences shared 99% nucleotide identity with the previously submitted P. exilis sequences (GenBank accessions NR147302 and KT290193 for ITS and EF-1α, respectively). Pathogenicity testing was performed by spraying conidial suspension (1 × 10⁶ conidia/ml) of isolate PE-1 on the leaves of 40-day-old Chinese flowering cabbage plants. Five plants were used for inoculation. Another five plants were sprayed with distilled sterilized water to serve as a control. Plants were kept in transparent boxes for 2 days to retain moisture and subsequently incubated in a growth room at 28 ± 2°C with a 12-h photoperiod. Similar leaf spots were observed on the leaves of all inoculated plants after 1 week of inoculation. No symptoms were seen on control plants. The same pathogen was reisolated consistently from inoculated leaves. The species identity was confirmed by both morphological and molecular characteristics, thus fulfilling Koch’s postulates. Previously, P. exilis has been reported to cause leaf spot disease on Chamaecrista orbiculata in Brazil (Hernandez-Gutierrez and Dianese 2009). To our knowledge, this is the first report of leaf spot on Chinese flowering cabbage caused by P. exilis. This disease can cause severe economic losses, because Chinese flowering cabbage is mainly sold in the market and used in salads or cooked dishes. Timely measures must be taken to manage this disease.