First Report of Pythium irregulare Causing Damping-Off on Soybean in Japan
First Report of Pythium irregulare Causing Damping-Off on Soybean in Japan
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日本首次报告不规则腐霉引起大豆猝倒病
DOI：
10.1094/pdis-12-18-2247-pdn
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发表时间：
2019
期刊：
影响因子：
4.5
通讯作者：
Tojo Motoaki
中科院分区：
文献类型：
--
作者：
You Xiaodong;Zhao Han;Tojo Motoaki
在日本，大豆 (Glycine max [L.] Merr.) 是一种重要的油料和蛋白质作物，深深植根于日本饮食文化中，其产品包括酱油和味噌。最近，许多农民更喜欢大豆移植种植，因为它可以减少鸟类的进食，而且比直接播种种植可以更早收获作物（Motoki 和 Sodeyama 2007）。 2018 年 5 月，日本大阪将大豆幼苗 (cv. Ezomidori) 移栽至稻田后发现严重猝倒病。大约 5% 的移植物被疾病破坏。在有症状的根中发现了丰富的未完成卵孢子。将患病根部的切片用无菌蒸馏水冲洗两次，用无菌滤纸干燥，并在25℃下在腐霉选择性NARM培养基(Morita和Tojo 2007)上铺板3天。分离了两种类腐霉生物（KN1 和 KN2），并使用草叶培养物检查了它们的形态特征（van der Plaats-Niterink 1981）。使用马铃薯胡萝卜琼脂测定不同温度下的菌丝生长速率。两株菌株均具有以下形态特征：主菌丝宽达6μm，卵原细胞为顶生或居间，直径为14～20μm，每个卵原细胞有1～2个花药，卵孢子为非完整的，直径为10～20μm。菌丝膨大呈顶生或居间状，直径9～24μm。生长的基本温度最低为 4°C，最适温度为 28°C，最高为 37°C，25°C 时日径向生长率为 29.5 毫米。形态特征与 van der Plaats-Niterink (1981) 中对不规则腐霉的描述一致。使用引物 ITS4 和 ITS5 对分离株的核糖体内部转录间隔区进行扩增和测序（White 等人，1990）。两个分离株 KN1（GenBank 登录号 MK123416）和 KN2（MK123417）的序列分析显示与不规则假单胞菌分离株 CBS 250.28（AY598702）有 99.0% 的同一性。基于这些特征，分离株 KN1 和 KN2 被鉴定为不规则假单胞菌，并保藏于日本茨城县 NIAS 基因库，分离株号分别为 MAFF 246895 和 MAFF 246896。将分离株 KN1 和 KN2 在高压灭菌的翦股颖种子上于 25°C 黑暗条件下培养 1 周。使用研钵和杵将1克定植的种子与100克育苗介质（Takii，京都，日本）充分混合，并将混合物放入陶瓷盆中。将在育苗介质上生长的15个7日龄的大豆幼苗移植到陶瓷盆中，每盆一株。将盆置于生长室中，温度为 12 或 25°C（白天 12 小时/晚上 12 小时），并每天浇水。使用未接种的盆作为对照。接种KN1和KN2菌株后14天，所有接种的大豆幼苗在25℃时出现轻微根腐病，在12℃时出现严重立枯病。未接种的大豆幼苗在任一温度下均未观察到发病情况。从形态学上证实了患病植物中一致的不规则假单胞菌的重新分离，从而满足了科赫的假设。温度对不规则倒霉的影响与Wei等人(2011)的观点一致，表明冷胁迫增加了大豆对不规则倒霉引起的立枯病的敏感性。据了解，这是日本首次报道不规则疫霉引起大豆猝倒病。结果表明，移植和凉爽的温度可能会增加病原体猝倒的风险。
In Japan, soybean (Glycine max [L.] Merr.) is an important oil and protein crop deeply rooted in Japanese food culture in products such as soy sauce and miso. Recently, many farmers prefer transplantation cultivation of soybean because it decreases feeding by birds and because crops can be harvested earlier than with direct seeding cultivation (Motoki and Sodeyama 2007). In May 2018, severe damping-off was found on soybean seedlings (cv. Ezomidori) after transplanting into a converted rice paddy in Osaka, Japan. Approximately 5% of the transplants were destroyed by the disease. Abundant aplerotic oospores were found in symptomatic roots. Sections of diseased roots were rinsed twice with sterile distilled water, dried with sterilized filter paper, and plated on Pythium-selective NARM medium (Morita and Tojo 2007) at 25 C for 3 days. Two Pythium-like organisms were isolated (KN1 and KN2), and their morphological features were examined using a grass blade culture (van der Plaats-Niterink 1981). Hyphal growth rates at different temperatures were determined using potato carrot agar. Both isolates had morphological features as follows: main hyphae were up to 6 μm wide, oogonia were terminal or intercalary and 14 to 20 μm in diameter, antheridia were one to two per oogonium, and oospores were aplerotic and 10 to 20 μm in diameter. Hyphal swellings were terminal or intercalary and 9 to 24 μm in diameter. Cardinal temperatures for growth were 4 C minimum, 28 C optimum, and 37 C maximum, with a daily radial growth rate of 29.5 mm at 25 C. The morphological features corresponded with the descriptions of Pythium irregulare in van der Plaats-Niterink (1981). The ribosomal internal transcribed spacer regions of the isolates were amplified and sequenced with primers ITS4 and ITS5 (White et al. 1990). Sequence analysis of both isolates KN1 (GenBank accession no. MK123416) and KN2 (MK123417) showed 99.0% identity with P. irregulare isolate CBS 250.28 (AY598702). Based on these features, isolates KN1 and KN2 were identified as P. irregulare and were deposited in the NIAS Genebank, Ibaraki Prefecture, Japan, as isolate numbers MAFF 246895 and MAFF 246896, respectively. Isolates KN1 and KN2 were cultured on autoclaved bentgrass seeds at 25 C in darkness for 1 week. One gram of colonized seeds was thoroughly mixed with 100 g of seedling raising medium (Takii, Kyoto, Japan) using a mortar and pestle, and the mixture was put in a ceramic pot. Fifteen 7-day-old soybean seedlings grown on the seedling raising medium were transplanted into ceramic pots, one plant per pot. Pots were kept at 12 or 25 C in a growth chamber (12 h day/12 h night) and watered daily. Noninoculated pots were used as controls. At 14 days after inoculation of KN1 and KN2 isolates, slight root rot appeared on all inoculated plants at 25 C, and severe damping-off appeared at 12 C. No incidence of disease was observed on the noninoculated soybean seedlings at either temperature. Consistent reisolation of P. irregulare was morphologically confirmed from the diseased plants, thus fulfilling Koch’s postulates. The effect of temperature on P. irregulare agrees with Wei et al.(2011), showing cold stress increases soybean susceptibility to damping-off caused by P. irregulare. To our knowledge, this is the first report of P. irregulare causing damping-off on soybean in Japan. The results suggest that transplanting and cool temperatures might increase risk of damping-off by the pathogen.