蒲公英是菊科的多年生草本植物,具有很高的食用和药用价值,在中国广泛种植。2022年8月,在天家寨镇的蒙古毛上发现了叶斑,西宁市,青海省,中国(北纬36°27\'17.65英寸,101°47\'19.65E,海拔:2,408米)。植物表现出圆形或不规则的棕色斑点,和一些斑点的中心是灰色的(图。S1A).在一公顷的土地上进行了调查,叶斑病的发生率达到15%-30%,严重影响了蒙古草的品质和产量。收集了11个蒙古赤霉叶斑病样品。为了分离致病真菌,使用无菌剪刀从感染和健康组织的交界处获得约0.5cm×0.5cm的组织。将有症状的叶子用3%NaClO表面消毒1.5分钟,并用无菌水洗涤三次。将消毒后的片干燥,并在25°C的培养箱中放置在水琼脂板上2天。随后,叶片表面表现出分生孢子和分生孢子。通过单孢子分离获得了11个分离株。在马铃薯葡萄糖琼脂(PDA)上,稀疏的气生菌丝体的颜色为深灰色至黑褐色(图S2A),并产生黑暗,多间隔分生孢子,具有7-11个横向间隔和1-2个纵向间隔(图。S2C).有一个或两个喙的分生孢子是长卵形的,平均长度和宽度为103.4×21.2μm,和80.7×3.9μm的喙。测量了一百一十个分生孢子。通过核糖体DNA内部转录间隔区(rDNAITS)的多位点序列分析证实了11个分离株的鉴定(White等人。1990),和甘油醛-3-磷酸脱氢酶(GAPDH)(Xu等人。2022),肌动蛋白(ACT)(Yang等人。2020),组蛋白3(HIS3)(郑等人。2015),翻译延伸因子1-α(TEF1-α)(Carbone.1999),和RNA聚合酶II的第二大亚基(RPB2)(Liu等人。1999)基因。所有分离株的序列都保存在Genbank(NCBI登录号ITS:OR105029-OR105039,ACT:OR135220-OR135230,GAPDH:OR135231-OR135241,HIS3:OR122992-OR123002,TEF1-α:PP055972-PP055982,andRPB2:PP055983-PP055993),和ITS的序列相似性,ACT,GAPDH,HIS3、TEF1-α和RPB2均为100%,98%,100%,99%,100%,99%的链格孢菌序列,分别。ITS的组合序列,GAPDH,TEF1-α,和RPB2基因连接,并用PAUP*v.4.0α构建最大简约树。结果表明,11个分离株与A.solani聚集在一起(图。S2D)。因此,根据其形态和分子特征,将11个分离株鉴定为A.solani。将11个分离株接种在其宿主上,以执行Koch的假设。分离物在PDA上生长6天。将健康的一个月大的T.mongolicum幼苗种植在10厘米的花盆中(图。S1B)或将幼苗移至培养皿(图。S1C),用涂片法接种5mL菌丝悬液。此外,用无菌水处理相同年龄的幼苗作为对照。将接种的幼苗移入25℃的人工气候箱内,相对湿度为70%,12小时光照/12小时黑暗条件。共接种80株幼苗,15株作为对照。7天后,在接种分离株的植物上观察到类似的症状,而对照植物没有产生症状。该测定进行三次。此外,从有症状的叶子中重新分离出分离株,并且菌落形态与原始分离株相同(图S2A和B)。通过扩增和测序HIS3基因的一部分,将回收的分离株鉴定为A.solani。以前曾有报道称,马铃薯Langterariasolani会导致马铃薯和其他茄属作物的早期疫病(vanderWaals等人。2004;郑等人。2015).据我们所知,这是中国首次报道的solani叶斑病。在管理实践中必须考虑这种疾病,我们的发现为疾病的预防和管理提供了依据。
Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27\'17.65″N, 101°47\'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 μm, and 80.7 × 3.9 μm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3,TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch\'s postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.