Tang Zhaoyang, Lou Jun, Mo Jinying, He Luqian, Chen Yu, Wu Hanqing, Zeng Wenrui, Shen Ying, Wu Choufei, Wang Zhan Qi, Zhang Liqin
Huzhou, China;
Huzhou University, 117774, Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, College of Life Sciences, Huzhou, Zhejiang, China;
Plant Dis. 2023 Feb 1. doi: 10.1094/PDIS-07-22-1724-PDN.
During April 2022, leaf spot was observed on strawberry (Fragaria × ananassa Duch.) with a disease incidence of approximately 45% among 100 plants. Strawberry was cultivated in a nursery at Huzhou University (30.87゜N, 120.13゜E), Zhejiang Province, China. In the strawberry greenhouse, the average temperature was 15-18 degrees, 40%-60% humidity. Early symptoms appeared as dark brown or black spotted necrotic lesions, which expanded from 2 to 6 mm in diameter. Dark brown spots with yellow halos occupied half of the leaf area and eventually developed leaf blight with large yellow halos. To isolate the causal agent, 0.5 cm x 0.5 cm fragments were cut from three symptomatic leaves, and were surface sterilized with 75% ethanol for 30 s and then rinsed three times with sterilized water. The airdried leaf fragments were placed on PDA with 50 μg/ml ampicillin and incubated in the dark at 25℃ for two days. Isolates were obtained by transferring hyphal plugs of 1 mm in diameter onto PDA. The colony morphology was circular and dark brown on the upperside and black on the underside, with cottony mycelium and an large amount of gray aerial mycelium. Conidia were large, light olive-brown to dark olive-brown and light olive-black and septate. The typical conidia were oval or rod-shaped, rarely curved, and dark septa defined the basal and apical cells. In the two typical forms of conidia, the average size of oval conidia was approximately 18.77 × 54.92 μm (11.99 to 26.97 × 35.13 to 74.59 μm, n = 20), and the average size of the rod-shaped conidia was approximately 14.80 × 103.24 μm (11.24 to 24.64 × 73.11 to 131.51 μm, n = 20). The morphological characteristics matched well with previous descriptions of Exserohilum rostratum (Sharma et al. 2014; Liu et al. 2021). The identity of C1-L and C1-S from symptomatic tissues was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted from the mycelium using the CTAB (cetyltrimethylammonium bromide) method (Griffith & Shaw 1998). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, and partial beta-tubulin 2 (TUB2), using the primers ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT512F/ACT783R (Carbone and Kohn 1999), T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson, 1995). The obtained sequences of C1-L and C1-S were the same. Moreover, the sequences have been deposited in GenBank under accession numbers ON982516 (ITS), ON996915 (GAPDH), ON996916 (ACT), and ON996917 (TUB2). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, and ACT had 100% identity with the sequences of E. rostratum (GenBank Accession No. LT837834, LT883550, and LT837672, respectively), the TUB2 had 99.61% similarity with BLAST sequences of E. rostratum (LT899391). These morphological characteristics and molecular analyses allowed the identification of the pathogen as E. rostratum. Koch's postulates were performed with five healthy detached strawberry leaves with three inoculations per leaf of the 'Akihime' strawberry variety. Surface-sterilized leaves were wounded with an aseptic needle, and inoculated with 2 mm diameter mycelial plugs from 5-day-old cultures of E. rostratum. Control leaves were also wounded with the aseptic needle, and inoculated with a sterile PDA agar plug. The leaves were incubated at 25℃ in Petri plates with petioles wrapped in moist sterile cotton. The diseased symptoms included black spots on the epidermis of the wounded leaves within 5, 10, and 20 days after inoculation. Mock-inoculated controls remained asymptomatic, and three biological repetitions were conducted. The fungus reisolated from the diseased leaves was confirmed as E. rostratum by sequencing. Abundant reports have shown that E. rostratum can infect many economically important crops such as maize, rice, and pineapple (Sun et al. 2021; Kabore et al. 2022; Luo et al. 2012). To the best of our knowledge, this is the first report of E. rostratum on strawberry in China and worldwide.
2022年4月期间,在草莓(Fragaria × ananassa Duch.)上观察到叶斑病,在100株植株中发病率约为45%。草莓种植于中国浙江省湖州大学(北纬30.87゜,东经120.13゜)的苗圃中。在草莓温室中,平均温度为15 - 18摄氏度,湿度为40% - 60%。早期症状表现为深褐色或黑色斑点坏死病斑,直径从2毫米扩展到6毫米。带有黄色晕圈的深褐色斑点占据叶片面积的一半,最终发展为带有大黄晕圈的叶枯病。为分离致病因子,从三片有症状的叶片上切下0.5厘米×0.5厘米的片段,用75%乙醇进行表面消毒30秒,然后用无菌水冲洗三次。将风干的叶片片段置于添加50μg/ml氨苄青霉素的PDA培养基上,在25℃黑暗条件下培养两天。通过将直径1毫米的菌丝块转移到PDA培养基上获得分离物。菌落形态为圆形,上表面深褐色,下表面黑色,有棉絮状菌丝体和大量灰色气生菌丝体。分生孢子大,浅橄榄褐色至深橄榄褐色以及浅橄榄黑色,有隔膜。典型的分生孢子为椭圆形或棒状,很少弯曲,深色隔膜界定基部和顶端细胞。在两种典型的分生孢子形态中,椭圆形分生孢子的平均大小约为18.77 × 54.92μm(11.99至26.97 × 35.13至74.59μm,n = 20),棒状分生孢子的平均大小约为14.80 × 103.24μm(11.24至24.64 × 73.11至131.51μm(n = 20)。这些形态特征与之前对喙突埃里格孢(Exserohilum rostratum)的描述非常吻合(Sharma等人,2014年;Liu等人,2021年)。通过多位点基因测序确认了来自有症状组织的C1 - L和C1 - S的身份。使用CTAB(十六烷基三甲基溴化铵)法(Griffith & Shaw,1998年)从菌丝体中提取基因组DNA。通过对内部转录间隔区(ITS)rDNA区域、部分甘油醛 - 3 - 磷酸脱氢酶(GAPDH)基因、部分肌动蛋白(ACT)基因和部分β - 微管蛋白2(TUB2)进行测序进行分子鉴定,使用引物ITS1/ITS4(White等人,1990年)、GDF/GDR(Templeton等人,1992年)、ACT512F/ACT783R(Carbone和Kohn,1999年)、T1(O'Donnell和Cigelnik,1997年)以及Bt2b(Glass和Donaldson,1995年)。获得的C1 - L和C1 - S序列相同。此外,这些序列已存入GenBank,登录号分别为ON982516(ITS)、ON996915(GAPDH)、ON996916(ACT)和ON996917(TUB2)。基本局部比对搜索工具(BLAST)分析结果显示,ITS、GAPDH和ACT与喙突埃里格孢的序列具有100%的同一性(GenBank登录号分别为LT837834、LT883550和LT837672),TUB2与喙突埃里格孢的BLAST序列(LT899391)具有99.61%的相似性。这些形态特征和分子分析确定该病原菌为喙突埃里格孢。对‘红颜’草莓品种的五片健康离体叶片进行了柯赫氏法则验证,每片叶接种三次。用无菌针划伤表面消毒的叶片,接种来自5日龄喙突埃里格孢培养物的直径2毫米的菌丝块。对照叶片也用无菌针划伤,接种无菌PDA琼脂块。将叶片在25℃下培养于培养皿中,叶柄用湿润的无菌棉花包裹。接种后5、10和20天,患病症状包括受伤叶片表皮上出现黑斑。模拟接种的对照保持无症状,进行了三次生物学重复。从患病叶片上重新分离的真菌经测序确认为喙突埃里格孢。大量报道表明,喙突埃里格孢可感染许多经济上重要的作物,如玉米、水稻和菠萝(Sun等人,2021年;Kabore等人,2022年;Luo等人,2012年)。据我们所知,这是喙突埃里格孢在中国和全球范围内首次在草莓上的报道。