醉酒马草(Achnatheruminebrians)属于禾本科,主要分布在中国北部和西北部的天然草地上。Ergot是一种疾病,不仅可以影响草的生长,但也会导致牲畜中毒(Coufal-Majewski等人。2016)。2018年9月,在兴海县大面积(约15公顷)观察到麦角,青海省,中国(北纬35°47,东经99°53,海拔3559米)。约有65%的念珠菌植物受到影响。症状最初在醉酒的马草的耳朵上显示出水滴状的蜜露,后来观察到棕色到深棕色的菌核。这些是直的到稍微弯曲的,测量为6.7至13.5×1.5至2.1毫米,大约是健康种子大小的1到4倍。从八个不同的领域收集了16个具有典型症状的尖峰。通过浸入75%乙醇30s和1%NaClO90s对菌核进行消毒,在无菌水中冲洗三次,在马铃薯葡萄糖琼脂(PDA)培养基上,在黑暗中在24°C下孵育,并通过从单个孢子培养纯化分离物。最后,从这些菌核中获得了16种具有相似表型的单孢子培养物。24℃在PDA上产生15天的菌落呈灰白色,有蓬松的气生菌丝,直径约60毫米。分生孢子是透明的,卵形至圆柱形,5.42至7.69×2.85至3.75μm(平均5.67×3.2;n=50)。这些形态特征与一般的Claviceps物种的描述一致(Píchovaetal.2018)。为了进一步鉴定克拉维塞斯类。,选择分离物NSZJ(=MHLZU-AI20201012)作为分子表征的代表。通过T2/T12(O\'Donnell和Cigelnik1997)和CARCA-F/M456-5R(Rehner和Buckley2005)扩增了两个核蛋白编码基因TUB2和MCM7,分别,并测序。序列存放在GenBank中(登录号:用于TUB2的MW115640和用于MCM7的MW115641)。对这两个片段的BLAST分析显示与紫菜的分离株W3的那些序列具有>99%的同一性(Pazoutovaetal.2014).为了确认醉酒马草的致病性,在牧场农业科学技术学院的试验田中生长的20种健康植物(2岁),中国兰州大学榆中校区(东经104°39,35°89'N,海拔1653m)在开花期用分生孢子悬浮液(1×106分生孢子/ml)喷雾接种。用灭菌的蒸馏水喷洒田间的另外20株植物作为对照。所有植物分别用透明聚乙烯袋覆盖24小时以保持高相对湿度。7到10天后,在一些小花中观察到黄白色的蜜露小滴。接种后22天,所有接种的圆锥花序每个头都有三到六个菌核,尺寸范围从6.7到13.5毫米,而对照植物保持健康。从接种的穗中一致地重新分离出相同的病原体,并通过如上所述的形态学和分子表征来证实。据报道,紫癜与爱达荷州的A.lemmonii和蒙大拿州的A.robustum中的麦角有关(Alderman等人。2004).据我们所知,这是在中国的A.inebrians中引起麦角的C.purpurea的第一份报告。
Drunken horse grass (Achnatherum inebrians) belongs to the family Poaceae: it is mainly distributed in the natural grasslands of northern and northwestern in
China.
Ergot is a disease that can not only affect the growth of the grass, but also cause livestock poisoning (Coufal-Majewski et al. 2016). In September 2018,
ergot was observed in a large area (about 15 ha) in Xinghai county, Qinghai province,
China (35° 47\' N, 99° 53\' E, Altitude 3559 m). Around 65% of the plants of Achnatherum inebrians were affected. Symptoms initially showed drop-like honeydew on the ears of drunken horse grass, and later brown to dark brown sclerotia were observed. These were straight to slightly curved, measured 6.7 to 13.5 × 1.5 to 2.1 mm, which was approximately 1 to 4 times the size of healthy seeds. Sixteen spikes with typical symptoms were collected from eight different fields. Sclerotia were disinfested by immersion in 75% ethanol for 30 s and 1% NaClO for 90 s, rinsed three times in sterilized water, plated on potato dextrose agar (PDA) medium, incubated at 24°C in the dark and isolates purified by culturing from single spores. Finally, 16 single-spore cultures with similar phenotypes were obtained from these sclerotia. Colonies produced on PDA for 15 days at 24 ℃ were grayish white with fluffy aerial mycelium, about 60 mm in diameter. Conidia were hyaline, ovoid to cylindrical, 5.42 to 7.69 × 2.85 to 3.75 μm (avg. 5.67 × 3.2; n = 50). These morphological characteristics were consistent with the descriptions of Claviceps species in general (PíchovÁ et al. 2018). To further identify the Claviceps spp., isolate NSZJ (=MHLZU-AI20201012) was selected as a representative for molecular characterization. Two nuclear protein-coding genes TUB2 and MCM7 were amplified by T2/T12 (O\'Donnell and Cigelnik 1997) and CARCA-F/M456-5R (Rehner and Buckley 2005), respectively, and sequenced. Sequences were deposited in GenBank (accession nos. MW115640 for TUB2 and MW115641 for MCM7). A BLAST analysis of these two segments showed >99% identity with those sequences of isolate W3 of C. purpurea (Pazoutová et al. 2014). To confirm the pathogenicity on drunken horse grass, 20 healthy plants (2-year-old) grown in an experimental field at the College of Pasture Agriculture Science and Technology, Yuzhong Campus of Lanzhou University in China (104° 39\' E, 35° 89\' N, altitude1653m) were spray-inoculated with conidial suspension (1 × 106 conidia/ml) during the flowering period. Another 20 plants in the field were sprayed with sterilized distilled water as controls. All plants were individually covered with transparent polyethylene bags for 24 h to maintain high relative humidity. After 7 to 10 days, small yellowish-white drops of honeydew were observed in some florets. At 22 days post inoculation, all the inoculated panicles developed three to six sclerotia per head, ranging in size from 6.7 to 13.5 mm, while control plants remained healthy. The same pathogen was consistently re-isolated from inoculated spikes and confirmed by morphological and molecular characterization as described above. Claviceps purpurea was reported to be associated with ergot in A. lemmonii in Idaho and A. robustum in Montana (Alderman et al. 2004). To our knowledge, this is the first report of C. purpurea causing
ergot in A. inebrians in
China.