中国浙江省におけるコイダモの葉面斑紋の初報告

First Report of Leaf Spot of Caused by in Zhejiang province, China.

抄録

コイズカは中国では非常に一般的なハーブで、薬効が高いことでも有名です（Lee et al.、2007）。2019年夏、中国の主要産地である浙江省阜陽県で、総株数約300株のうち74株に黒色葉痕が見られた 。発病率は25％で、高温多湿条件下で増加しました。葉の初期症状は、褐色の縁に囲まれた黒または褐色の斑点として現れ、不規則に拡大した。最終的には直径1.0〜1.5cmの楕円形または不規則な黒褐色の大きな斑点が葉に現れ、その後病変は壊死に転じた。病原菌の分離は、症状のある葉の縁から小片（5×5mm）を取り出し、75%エタノールで30秒、2%次亜塩素酸ナトリウムで2分間表面殺菌した後、滅菌水で5回洗い、ポテトデキストロース寒天培地（PDA）で28℃、暗黒下で培養して行われた。精製コロニーは白色からピンク色で，気生菌糸は密に凝集し，桃色から橙色の色素を帯びていた．マクロコニダは通常3裂し，26.7〜43.3×3.1〜5.3μm（n=50），薄肉でわずかに湾曲し，暗黒下で連続培養すると頂端と基端の細胞が湾曲した状態になった．微小子嚢の大きさは5.9〜14.3×2.3〜3.9μm（n=50）で、ほとんどが無菌の卵形楕円体で、直線からわずかに湾曲していた。菌糸と胞子から球形のクラミドスポアが単数または対で産生された。これらの特徴は、属の記述と一致した（Leslie and Summerell, 2006）。種を同定するために、翻訳伸長因子-1α領域（TEF-1α）およびミトコンドリア小サブユニット（mtSSU）をそれぞれプライマーEF-3／EF-22（Palmore et al,2010およびO'Donnell et al,1998）およびMS3F／MS3R（Stenglein et al,2010）により増幅した。配列はGenBankに寄託されている（MT263720, OM203177, OM203178, OM203179, OM203180, OM203181 and MN853662, MZ028170.1, MZ028171.1, MZ028172.1, MZ028173.1, MZ028174.1).これらの6株はそれぞれ100%および98%の類似度でクレードに分類された。各菌株の病原性を調べるため，8週齢の鉢植え植物5株に傷口接種を行い，直径5 mmの菌糸ディスクを用いた。対照として，5本の植物に5 mmのPDAプラグを接種した。すべての植物を個別にビニール袋で覆い、25±2℃、12時間光周期、相対湿度70～80%の温室内で保管した。5日後、接種した植物にのみ、圃場と同様の典型的な症状が現れた。また，鉢植えの健常株3株の若葉に分生子懸濁液（1×10spores/ml）を散布した。対照として、滅菌蒸留水を散布した3株を用いた。7日後、接種したすべての葉で典型的な症状が観察された。本実験は3回繰り返した。形態学的および分子生物学的同定に基づき，病原体は中国において，(Zhuang, 2005), (Tan et al., 2012), (Sang et al., 2006), (Zhang et al., 2018) の葉斑病の原因物質として報告されている。 我々の知る限り，これは中国で引き起こされた葉斑病についての最初の報告である。損失を減らすために、効果的な防除戦略を確立する必要がある。

Koidz is a very common herbs in China, also famous for its high medicinal value (Lee et al., 2007). In summer of 2019, in Fuyang county of Zhejiang province, the main production area of China, 74 plants of from a total of about 300 plants, showed black leaf spots . The incidence of the disease was 25% and increased under high temperature and humidity conditions. Initial leaf symptoms appeared as black or tan spots surrounded by brown margins and expanded irregularly. Finally, large blackish brown spots appeared on the leaves, elliptical or irregular, 1.0 to 1.5 cm in diameter, and then lesions turned necrotic. To isolate the pathogen, small pieces (5×5 mm) from the margin of symptomatic leaves were surface-sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 2 min, rinsed five times with sterile water, and incubated on potato dextrose agar (PDA) at 28°C in darkness. Purified colonies were white to pink with densely floccose to fluffy aerial mycelium and peach-orange pigmentation. Macroconidia, usually three-septate, were 26.7 to 43.3×3.1 to 5.3μm (n=50), thin-walled, slightly curved, with apical and basal cells curved when cultured in continuous darkness. Microconidia were mostly aseptate, ovate-oblong, straight to slightly curved, and measuring 5.9 to 14.3×2.3 to 3.9μm in size (n=50). Spherical chlamydospores were produced singly or in pairs from mycelium and spores. These characteristics were consistent with the description of spp. (Leslie and Summerell, 2006). To identify the species, the translation elongation factor-1 alpha regions (TEF-1α) and the mitochondrial small subunit (mtSSU) were amplified using primers EF-3/ EF-22 (Palmore et al.,2010 and O'Donnell et al., 1998) and MS3F/ MS3R (Stenglein et al., 2010), respectively. Sequences were deposited in GenBank (MT263720, OM203177, OM203178, OM203179, OM203180, OM203181 and MN853662, MZ028170.1, MZ028171.1, MZ028172.1, MZ028173.1, MZ028174.1). These six isolates clustered in the clade with 100% and 98% similarity, respectively. To test pathogenicity of every isolate, five 8-week-old potted plants were wound-inoculated and mycelial discs of 5-mm diameter were used to inoculate. As a control, five plants were inoculated with 5-mm PDA plugs. All plants were individually covered with a plastic bag and kept in a greenhouse at 25 ± 2°C with a 12-h photoperiod at 70 to 80% relative humidity. Typical symptoms similar to those of the field appeared only in inoculated plants after five days. In addition, a conidial suspension (1×10 spores/ml) was sprayed onto young leaves of three potted healthy plants. Three plants sprayed with sterile distilled water served as controls. After 7 days, typical symptoms were observed on all inoculated leaves. Experiments were replicated three times. was successfully re-isolated from diseased plants. Based on morphological and molecular identification, the pathogen was identified as In China, (Zhuang, 2005), (Tan et al., 2012), (Sang et al., 2006) and (Zhang et al., 2018) were reported as causal agents of the leaf spot disease of . To our knowledge, this is the first report of leaf spot disease on caused by in China. Effective control strategies need to be established to reduce the losses.