First report of soybean damping off caused by Pythium aphanidermatum and P. coloratum in Japan

In Japan more than 80% of soybean crops (Glycine max) are cultivated in fields converted from rice paddies. However, the soil moisture levels of these fields are generally high due to poor drainage which promotes the growth of soil-borne pathogens, such as Pythium spp. (Kato et al., 2013). In June 2016, severe damping-off was found on soybean (cv. Ezomidori) grown in a field converted from a rice paddy in Osaka, Japan. Approximately 10% of all soybean plants were destroyed by the disease. 

A total of ten Pythium isolates were obtained from twenty diseased soybean seedlings (Fig. 1) and identified as Pythium aphanidermatum, P. coloratum and P. ultimum, based on their morphological characteristics (van der Plaats-Niterink, 1981). Pythium ultimum has been reported as a pathogen of soybean in Japan (Kodama et al., 2010).  Pythium aphanidermatum and P. coloratum have been reported as pathogens of soybean in Iowa and North Dakota, USA (Rizvi & Yang, 1996; Zitnick-Anderson & Nelson 2015), but have never been reported in Japan. Detailed descriptions are provided here on representative isolates of P. aphanidermatum (D1) and P. coloratum (D2). Their morphological features corresponded with the descriptions of van der Plaats-Niterink (1981) (Table 1, Figs. 2-3). The ITS regions of isolates D1 and D2 were amplified and sequenced with primers ITS4 and ITS5 (White et al., 1990). The sequence analysis determined that isolates D1 (GenBank Accession No. MF769579) and D2 (MF769577) showed 100% identity with P. aphanidermatum isolate CBS118.80 (HQ665084) and 99.0% identity with P. coloratum isolate CBS154.64 (AY598633), respectively. 

Pathogenicity was studied using isolates D1 and D2 in pot experiments. Pythium species were cultured on autoclaved bentgrass seeds at 25°C for one week. One gramme of the culture was thoroughly mixed with 100 g of commercial nursery soil (Aisai-1, Katakura Chikkarin Co. Ltd., Tsuchiura, Japan) using a mortar and pestle. The infested soil was put in a plastic pot (inner diameter 7 cm, inner depth 6 cm). Five seeds of soybean (cv. Ezomidori) were sown per pot and incubated in a growth chamber at 25°C (12h day) / 20°C (12h night) with 73 mol/m2/s (measured at plant levels) of light intensity and irrigated daily with tap water. Non-infested soil was used as a control. The percentage of seedling emergence was determined seven days after sowing. The experiments were repeated five times using one pot per repetition. Both isolates D1 and D2 caused severe damping-off on soybean. In the non-infested soil 92% of the seedlings emerged (Fig. 4A), whereas only 0 and 24% of the seedlings emerged in the soil infested with D1 and D2, respectively (Fig. 4B and C). Pythium aphanidermatum and P. coloratum were re-isolated from the damaged plants grown in the soil infested with isolates D1 and D2, respectively. 

To our knowledge, this is the first report of P. aphanidermatum and P. coloratum causing damping-off on soybean in Japan.