胡桃木(铁核桃)主要分布在中国西南部,并对木材和坚果有价值(Feng等人。2018)。2020年4月,我们调查了位于白沙镇的核桃园,万源市,(四川,中国),在100种植物中的10种树木的叶子上观察到褐斑症状,当树木严重感染时,这种疾病会导致生长潜力降低。在病叶上观察到带有分生孢子的坏死性和亚圆形病变。收集有症状的叶子,并带回实验室进行进一步分析。使用Chomnunti等人开发的单孢子分离技术。(2014),在马铃薯葡萄糖琼脂培养基(PDA)上从感染的叶子上生长了五个分离株。五个分离株具有相似的菌落形态,最初是白色的,近圆形,逐渐变黄,有黑点,发育蓬松的气生菌丝体。在PDA上使用光学显微镜检查形态特征。分生孢子细胞呈亚圆柱形至圆柱形,或者壶腹状,透明玻璃,很少分枝。大分生孢子是月肉,肾形,透明玻璃,1-3-隔膜,主要是1-隔膜,在隔膜处明显收缩,基底细胞是钝圆的,顶端细胞有一个急性末端,基底细胞等于或大于顶端细胞,测量22至40.5×2.5至8.3μm(平均值=32×6.2μm,n=50)。微分生孢子是肉毒杆菌,或亚梭形,透明玻璃,两端都是圆形的,直的或弯曲的,无菌,测量为10至28.5×1.9至3.7μm(平均值=17.2×2.7μm,n=20)。进行多位点方法以精确鉴定代表性分离株SICAUCC20-0012。内部转录间隔区(ITS),鸟嘌呤核苷酸结合蛋白亚基β基因(MS204),分离株SICAUCC20-0012的翻译延伸因子1-α(tef1-α)被扩增并测序,如Sogonov等人所述。(2008)和Walker等人。(2012a)。GenBank登录号对于ITS,MS204和tef1-α分别是MW250303、MW246773和MW246775。系统发育分析显示,蛇形菌具有100%的支持(Fr。)索戈诺夫,形态与Walker等人记录的无性阶段一致。(2012b)。为了测试科赫的假设,如Desai等人所述,在用小别针受伤后,用分生孢子悬浮液(104分生孢子/mL)接种每株植物5-8叶的西吉拉塔(2至3岁)。(2019),相同数量的健康植物受伤,并喷洒无菌蒸馏水作为对照。每天定期用蒸馏水喷洒植物,并将其置于25℃的生长室中,采用12小时的荧光/黑暗方案。15天后,在接种的叶子上出现典型的褐斑症状,但不是控制。如上所述,将真菌O.leptostyla从病变中重新分离出来,但未从未接种的叶子中分离出来。O.在一些核桃树上报道了leptostyla;例如:J.ailantifolia,J.加州,J.Cinerea,J.印度教,J.少校,J.Mandshurica,J.Nigra,和J.Regia(Farr&Rossman2020)。然而,根据我们的知识,这是O.leptostyla在J.sigillata上引起褐色叶斑病的第一份报告。西吉拉塔是中国西南地区的重要经济树种,和杀真菌剂处理应考虑防止这种真菌传播之前,它变得更广泛。杨春林,YuDeng,王飞虎对这项工作做出了同样的贡献。本研究得到四川省重点研发项目(2021YFYZ0032)的资助。
Juglans sigillata Dode (Iron walnut) is mostly distributed in southwestern
China, and valued for wood and nuts (Feng et al. 2018). In April 2020, we surveyed a walnut garden located in Baisha Town, Wanyuan City, (Sichuan,
China), where brown spot symptoms were observed on leaves of ten trees among of 100 plants, and this disease can result in a reduced growth potential when trees are severely infected. Necrotic and subcircular lesions with conidiamata were observed on diseased leaves. Symptomatic leaves were collected and taken back to the laboratory forfurther analysis. Using the single spore isolation technique developed by Chomnunti et al. (2014), five isolates were grown from the infected leaves on Potato Dextrose Agar medium (PDA). The five isolates had similar colony morphology, which was initially white, suborbicular, gradually turning yellowish with black spots, developing fluffy aerial mycelium. Morphological characteristics were examined using light microscopy on the PDA. Conidiogenous cells were subcylindrical to cylindrical, or ampulliform, hyaline, rarely branched. Macroconidia were lunate, reniform, hyaline, 1-3-septate, mostly 1-septate, distinctly constricted at the septum, the basal cell was bluntly rounded, the apical cell had an acute end, and the basal cell was equal to or larger than the apical cell, measuring 22 to 40.5 × 2.5 to 8.3 μm (mean = 32 × 6.2 μm, n = 50). Microconidia were botuliform, or subfusiform, hyaline, both ends were rounded, straight or curved, aseptate, and measured 10 to 28.5 × 1.9 to 3.7 μm (mean= 17.2 × 2.7 μm, n = 20). A multilocus approach was conducted for precise identification of a representative isolate SICAUCC 20-0012. The internal transcribed spacer regions (ITS), guanine nucleotide-binding protein subunit beta gene (MS204), and translation elongation factor 1-alpha (tef1-α) of isolate SICAUCC 20-0012 were amplified and sequenced as described by Sogonov et al. (2008) and Walker et al. (2012a). GenBank Accession Nos. for ITS, MS204, and tef1-α are MW250303, MW246773, and MW246775, respectively. Phylogenetic analyses showed 100% support with Ophiognomonia leptostyla (Fr.) Sogonov, and the morphology was consistent with the asexual stage of O. leptostyla documented by Walker et al. (2012b). To test Koch\'s postulates, five healthy plants of J. sigillata (2- to 3-year-old) with 5-8 leaves per plant were inoculated with conidial suspensions (104 conidia/mL) after wounded with a small pin as described by Desai et al. (2019), and the same number of healthy plants were wounded and sprayed with sterile distilled water as controls. Plants were sprayed regularly with distilled water every day and placed in a growth chamber at 25℃ with a 12-h fluorescent light/dark regimen. After 15 days, typical brown spot symptoms developed on inoculated leaves, but not on the controls. The fungus O. leptostyla was reisolated from the lesion as described above but not from non-inoculated leaves. O. leptostyla has been reported on some walnut trees; for example: J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. nigra, and J. regia (Farr & Rossman 2020). However, to our knowledge, this is the first report of O. leptostyla causing brown leaf spot on J. sigillata. J. sigillata is an economically important tree in southwest
China, and fungicide treatments should be considered to prevent the spread of this fungus before it becomes more widespread. Chunlin Yang, Yu Deng, and Feihu Wang contributed equally to this work. This research was supported by the Key Research and Development Project of Sichuan Province (2021YFYZ0032).