In Aug 2019, approximately 10% of mung bean plants at the experimental farm of the Jiangsu Academy of Agricultural Science (32.03 N; 118.88 E) showed symptoms of stunting and wilting. Brown and water-soaked stem lesions were often observed at the base of the diseased plants. In severe cases, the plants collapsed and cumulous aerial mycelia were visible on the basal stem surface (Figure S1 A). To identify the causal agent, a total of 20 tissue fragments (5 mm long) were excised from roots and basal stems of five symptomatic plants. The fragments were surface sterilized in 2% sodium hypochlorite solution then plated on 2.5% potato dextrose agar (PDA) plates containing 10 μg/mL pimaricin, 100 μg/mL ampicillin, 10 μg/mL rifampicin, and 10 μg/mL pentachloronitrobenzene (PARP; Beckerman et al. 2017). After 3-4 days incubation at 25oC in dark, 14 colonies with white and cumulous mycelia grew from the tissue pieces (named as JS19-1 to JS19-14). JS19-1 and JS19-2 were purified by hyphal tipping, then grown on PDA medium for 7 days for morphological observation using a compound microscope (Figure S1 B, C). Width of coenocytic hyphae ranged from 3.7 to 8.9 (avg. 6.1, n=20) μm. Terminal oogonia were globose and with a diameter of 13.8 to 25.8 (avg. 22, n=20) μm. Antheridia were barrel-shaped, and mostly intercalary, sometimes terminal. Most of antheridia were diclinous, with 6.2 to 12.5 (avg. 9.3, n=20) μm in width and 7.6 to 15.3 (avg. 12.8, n=20) μm in length. Oogonia were fertilized with one or two (rare) antheridia. Oospores were aplerotic, 10.1 to 23.5 (avg. 20.4, n=20) μm in diameter. Sporangia had terminal inflated hyphal branches (Figure S1 D, E). The two isolates were preliminary identified as Pythium aphanidermatum. For molecular identification, the sequences of internal transcribed spacer (ITS) rDNA, cytochrome oxidase subunit I (CoxI) (Robideau et al. 2011), and β-tubulin (Kroon et al. 2004) of JS19-1 were detected, and deposited in GenBank (MT949538, MT949539 and MT949540). The ITS and CoxI sequences were identical with P. aphanidermatum CBS28779 ITS (759/759 bp, HQ643439.1) and PYT01 CoxI (640/640 bp, MH760243.1) respectively, the β-tubulin sequence showed 99% (830/840 bp) similarity of P. aphanidermatum P2 (AY564048.1). Thus, JS19-1 was confirmed as P. aphanidermatum. To fulfill Koch's postulates, the pathogenicity of JS19-1 was tested using the procedure of Kiyoshi et al. (2021) with some modifications. Barley grains infested with JS19-1 were as inoculum and thoroughly mixed with potting mixture at a rate of 10% in volume. Six mung bean seeds were sown per pot and then grown in a greenhouse. Potting mixture with no inoculum was used as control. Three pots of replicate plants used for inoculation and control. After 3 weeks, emergence in the inoculated pots was 33% and symptoms of stunting and root rot similar to those in field were observed, while control plants were asymptomatic (FigureS1 F, G). P. aphanidermatum was successfully reisolated from symptomatic plants of both methods. The pathogenicity tests were repeated twice. P. aphanidermatum causes seed rot, pre- and postemergence damping-off, or stem/root rot of a wide range of industrial crops and vegetables (Liu et al, 2018). To our knowledge, this is the first report of P. aphanidermatum causing disease on mung bean in China. Since Phytophthora vignae (Sun et al, 2020) and P. myriotylum (Yan et al, 2021) have been reported causing mung bean root rot, integrated disease management should be adopted to reduce damage.
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