地黄(又称毛地黄)是一种多年生双子叶草本植物,在中医药中发挥着重要作用。其活性成分对血液系统具有广泛的药理作用,内分泌系统,免疫系统,心血管系统,和神经系统(Zhang等人。2008).2022年5月,在焦作市(35°01\'44.20″N,113°05\'30.63英寸),河南省,中国的发病率约为54%(约1,300种植物)。不规则的棕色病变最初出现在基叶的尖端,然后进展到整个叶子,导致叶子干燥(柔软。Fig.1-A,B,C).从植物的基部到顶部的叶子中相继出现了相同的症状,最终导致整株植物死亡。为了识别病原体,从八株植物中随机收集了八株有症状的叶子,在病灶边界处切成小块(5×5mm)。这些碎片在75%的乙醇中表面消毒15秒,然后用1%NaClO持续1分钟,在无菌水中冲洗三次,置于25℃黑暗条件下的马铃薯葡萄糖琼脂(PDA)培养基上3天。最后,采用单孢子法获得12株纯化分离物(DHY1-DHY12)。用分生孢子悬浮液(106分生孢子/ml)接种R.glinosa幼苗的叶子,每个分离株接种三株植物。用无菌水处理对照。将所有接种和对照植物在25℃、80±10%湿度和8-h/16-h黑暗/光照循环下孵育。该实验重复三次。5天后,接种DHY4-DHY10的叶片出现与幼苗苗圃中病叶相似的症状,而接种DHY1-DHY3,DHY11-DHY12的植物则无症状(柔软。图1-D,E).从病叶中重新分离出相同的真菌,履行科赫的假设。病原体DHY4到DHY10,显示相似的形态,在形态上被鉴定为曲霉属。(Visagie等人。2014).选择分离物DHY5用于进一步研究。在PDA车牌上,殖民地被白色的绒毛菌丝体覆盖(柔软。图1-F)。分生孢子为o石黄色,向外的同心圆。囊泡呈球形,直径约为20.1-26.6μm(柔软。图1-G)。分生孢子柱壁光滑透明,分生孢子头辐射。分生孢子为浅黄色至橙色,渗出液清澈到橙色的小滴。分生孢子直径为(2.53-3.25)μm×(2.58-3.47)μm(n=50)(柔软。图1-H)。为了进一步的分子鉴定,用引物对ITS1/ITS4和BT2a/BT2b(Glass和Donaldson。1995),分别。ITS(PP355445)和TUB(PP382788)序列的BLASTn搜索显示与A.westerdijkiae(OP237108和OP700424)的相似性为100%和98.42%,分别。基于ITS和TUB的串联序列的系统发育分析证实该真菌是A.westerdijkiae,(柔软。图2)。A.westerdijkiae主要报道其次生代谢产物OchratoxinA污染农产品,水果,和各种食品,如咖啡豆(Alvindiaetal2016),葡萄(Díaz等人。2009),橙子和果汁(Marino等人2009),等。据我们所知,这是中国首例引起赤霉病的赤霉病。
Rehmannia glutinosa (also known as Chinese foxglove) is a perennial dicotyledonous herb, which plays an important role in traditional Chinese medicine. Its active ingredients have a wide range of pharmacological effects on the blood system, endocrine system, immune system, cardiovascular system, and nervous system (Zhang et al. 2008). In May 2022, leaf blight was observed on 45-day-old R. glutinosa in a seedling nursery in Jiaozuo City (35°01\'44.20″N, 113°05\'30.63″E), Henan Province, China with an approximate disease incidence up to 54% (~1,300 plants). Irregular brown lesion initially appeared on the tips of basal leaves, then progressed to the entire leaf causing leaf drying out (Supple. Fig. 1-A, B, C). The same symptoms appeared successively in the leaves from the base to the top of the plant, which eventually caused the whole plant to die. To identify the pathogen, eight symptomatic leaves were randomly collected from eight individual plants, and cut into small pieces (5 × 5 mm) at the border of lesions. The pieces were surface disinfected in 75% ethanol for 15 s, followed by 1% NaClO for 1 min, rinsed in sterile water three times, and placed on potato dextrose agar (PDA) medium in the dark for 3 days at 25℃. Finally, 12 purified isolates (DHY1-DHY12) were obtained by using single spore method. Leaves of R. glutinosa seedlings were inoculated with conidial suspension (106 conidia/ml), three plants were inoculated per isolate. Controls were treated with sterilized water. All inoculated and control plants were incubated in a greenhouse at 25℃ under 80 ± 10% humidity and a 8-h/16-h dark/light cycle. This experiment was repeated three times. After 5 days, similar symptoms to those of diseased leaves in the seedling nursery appeared on leaves inoculated with DHY4-DHY10, while plants inoculated with DHY1-DHY3, DHY11-DHY12, and the controls remained asymptomatic (Supple. Fig.1-D, E). The same fungi were re-isolated from diseased leaves, fulfilling Koch\'s postulates. The causal agents DHY4 to DHY10, showed similar morphology, which were morphologically identified as Aspergillus sp. (Visagie et al. 2014). Isolate DHY5 was selected for further study. On PDA plates, the colonies were covered with white velutinous mycelia (Supple. Fig.1-F). Conidia were ochre yellow and outwards concentric circles. Vesicles were globose, and about 20.1-26.6 μm in diameter (Supple. Fig.1-G). Conidiophore stipes were smooth walled and hyaline, with conidial heads radiating. The conidia were light yellow to orange, exudate clear to orange droplets. The conidia were (2.53-3.25) μm × (2.58-3.47) μm in diameter (n=50) (Supple. Fig.1-H). For further molecular identification, the ITS and TUB gene sequences were amplified with primer pairs ITS1/ITS4 and BT2a/BT2b (Glass and Donaldson. 1995), respectively. BLASTn searches of the ITS (PP355445) and TUB (PP382788) sequences showed 100% and 98.42% similarity to those of A. westerdijkiae (OP237108 and OP700424), respectively. Phylogenetic analysis based on the concatenated sequences of ITS and TUB confirmed that the fungus was A. westerdijkiae, (Supple. Fig.2). A. westerdijkiae was mainly reported on its secondary metabolite ochratoxin A contamination of agricultural products, fruits, and various food products, such as coffee beans (Alvindia et al 2016), grapes (Díaz et al. 2009), oranges and fruit juice (Marino et al. 2009), etc. To our knowledge, this is the first report of A. westerdijkiae causing leaf blight on R. glutinosa in China.