First report of leaf brown spot caused by Diaporthe phoenicicola on Lithocarpus polystachyus in China.

IF 4.4 2区 农林科学 Q1 PLANT SCIENCES
Yunpeng Jiang, Huiting Chen, Yue Sun, Wenting Long, Shengxiang Xiao, Dingjie Li, Keke Li, Jianglan Chen, Hao Chen
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The disease initially presented as small yellow lesions that eventually developed into large brown patches with dark brown edges. More than 80% of the area was covered by leaf lesions, which eventually turned into leaf necrosis. To ascertain the pathogenic species responsible for this disease, pathogen isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected by immersing in 75% ethanol followed by 0.1% HgCl2. Small pieces (0.5 × 0.5 cm) were then excised and placed onto PDA medium, and incubated at 28°C for 6-9 days. Sterilized dissecting needles were used to pick mycelia from the edge of the colonies and placed onto PDA for strains purification. On the PDA, the colony color of upper side initially appeared white (Rayner 1A1), and then turned grey (Rayner 11C1), while the reverse side turnd faint yellow (Rayner 4A3). Black pycnidia were induced on PDA at 28°C under a 12 h/12 h light/dark cycle for 12 days. Alpha conidia were 5.37 _8.84 × 1.53 _3.19 μm (average: 6.77 × 2.37 μm, n = 50), hyaline, fusiform or ellipsoidal. Beta conidia were 13.61 _23.45 μm × 0.94 _1.47 μm (average: 18.78×1.18 μm, n = 50), hyaline, aseptate, filiform, straight or hamate. Morphologically, the fungi were identified as Diaporthe sp. (Guarnaccia and Crous 2017). To further affirm the identification of the pathogen, the genomic DNA was extracted from representative isolate, referred to as Dip, for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), β-tubulin (TUB2) and histone H3 (HIS) genes were amplified from genomic DNA using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and CYLH3F/H3-1b, respectively, to sequence for BLAST (Huang et al. 2015). 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引用次数: 0

Abstract

The leaves of Lithocarpus polystachyus (Wall. ex A. DC.), an economically significant tree species in China, are commonly referred to as 'sweat tea' due to their high dihydrochalcone content, which holds biomedical importance, particularly in the treatment of diabetes (Hou et al. 2011). In January 2024, brown spots on L. polystachyus leaves were widely observed in Ningxiang (28°23'N, 112°59'E), Hunan Province, China. According to the investigation, the incidence rate of this disease was about 74% (222/300 plants surveyed). On each infected plant, nearly 60% leaves had symptoms. The disease initially presented as small yellow lesions that eventually developed into large brown patches with dark brown edges. More than 80% of the area was covered by leaf lesions, which eventually turned into leaf necrosis. To ascertain the pathogenic species responsible for this disease, pathogen isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected by immersing in 75% ethanol followed by 0.1% HgCl2. Small pieces (0.5 × 0.5 cm) were then excised and placed onto PDA medium, and incubated at 28°C for 6-9 days. Sterilized dissecting needles were used to pick mycelia from the edge of the colonies and placed onto PDA for strains purification. On the PDA, the colony color of upper side initially appeared white (Rayner 1A1), and then turned grey (Rayner 11C1), while the reverse side turnd faint yellow (Rayner 4A3). Black pycnidia were induced on PDA at 28°C under a 12 h/12 h light/dark cycle for 12 days. Alpha conidia were 5.37 _8.84 × 1.53 _3.19 μm (average: 6.77 × 2.37 μm, n = 50), hyaline, fusiform or ellipsoidal. Beta conidia were 13.61 _23.45 μm × 0.94 _1.47 μm (average: 18.78×1.18 μm, n = 50), hyaline, aseptate, filiform, straight or hamate. Morphologically, the fungi were identified as Diaporthe sp. (Guarnaccia and Crous 2017). To further affirm the identification of the pathogen, the genomic DNA was extracted from representative isolate, referred to as Dip, for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), β-tubulin (TUB2) and histone H3 (HIS) genes were amplified from genomic DNA using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and CYLH3F/H3-1b, respectively, to sequence for BLAST (Huang et al. 2015). The results showed that the ITS (GenBank: PP502145), EF1-α (GenBank: PP505773), TUB2 (GenBank: PP505774) and HIS (GenBank: PP505772) sequences of Dip isolate, respectively, showed 100% (469/469 bp), 98.47% (257/261 bp), 97.63% (617/632 bp), and 100% (379/379 bp) identity to their counterparts (GenBank: MW504747, ON049530, MW514138 and ON113058) in Diaporthe phoenicicola. The Maximum Likelihood tree was built based on the ITS, EF1-α, HIS and TUB2 sequences using MEGA11.0. Isolate Dip clustered with D. phoenicicola. The fungus was finally identified as D. phoenicicola by combining morphological and molecular characteristics. To confirm the pathogenicity of the isolated D. phoenicicola to induce brown spot, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspension (1×105 conidia per mL) was inoculated on 12 unwounded leaves collected from six 3-years-old plants, and sterile water was as control. The inoculated leaves were then incubated in chambers at 28℃ and 90% humidity with a 12 h photoperiod. The experiment was repeated three times. The results showed that inoculated leaves other than control developed brown spot symptoms within six days after inoculation. The test proved D. phoeicicola as the causal agent of this brown spot disease on L. polystachyus. The pathogen was exclusively re-isolated from the infected leaves and showed identical morphological characteristics to those of the original pathogens. To our knowledge, this is the first report of leaf brown spot of L. polystachyus caused by D. phoenicicola in China. This disease severely delays the plant development of L. polystachyus and significantly reduces the yield and quality of sweat tea. Our findings will contribute to the control of brown spot of L. polystachyus.

中国石竹(Lithocarpus polystachyus)叶片褐斑病报道首例。
石竹(Lithocarpus polystachyus)的叶子。(ex A. DC.)是中国一种具有重要经济意义的树种,由于其高二氢查尔酮含量,通常被称为“汗茶”,具有生物医学意义,特别是在治疗糖尿病方面(Hou et al. 2011)。2024年1月,在湖南宁乡(28°23′n, 112°59′e)广泛观察到羊草叶片上的褐斑。据调查,该病的发病率约为74%(222/300株)。在每棵受感染的植物上,近60%的叶子都有症状。该疾病最初表现为小的黄色病变,最终发展为大的棕色斑块,边缘为深褐色。超过80%的面积被叶片病变覆盖,最终变成叶片坏死。为了确定该疾病的致病种,采用组织分离法进行病原体分离(Xu et al. 2023)。用75%的乙醇和0.1%的HgCl2浸泡对感染的叶片组织进行表面消毒。然后切除小片(0.5 × 0.5 cm),置于PDA培养基上,28℃孵育6-9天。用灭菌的解剖针从菌落边缘采摘菌丝,置于PDA上进行菌株纯化。PDA上菌落颜色最初为白色(Rayner 1A1),后转为灰色(Rayner 11C1),而背面则转为淡黄色(Rayner 4A3)。在28°C的PDA上,在12 h/12 h明暗循环下,培养12 d。α分生孢子大小为5.37 × 8.84 × 1.53 × 3.19 μm(平均为6.77 × 2.37 μm, n = 50),呈透明状、梭形和椭圆形;β分生孢子大小为13.61 _23.45 μm × 0.94 _1.47 μm(平均:18.78×1.18 μm, n = 50),呈透明状、无菌状、丝状、直状或链状。形态学鉴定该真菌为Diaporthe sp. (Guarnaccia and Crous 2017)。为了进一步确认病原体的鉴定,从具有代表性的分离物(称为Dip)中提取基因组DNA进行分子鉴定。利用引物ITS1/ITS4、EF1- 728f /EF1- 986r、Bt2a/Bt2b和CYLH3F/H3-1b分别从基因组DNA中扩增出内部转录间隔区(ITS)、翻译延伸因子1α (EF1-α)、β-微管蛋白(TUB2)和组蛋白H3 (HIS)基因,进行BLAST测序(Huang et al. 2015)。结果表明,Dip分离物ITS序列(GenBank: PP502145)、EF1-α序列(GenBank: PP505773)、TUB2序列(GenBank: PP505774)和HIS序列(GenBank: PP505772)与对应序列(GenBank: MW504747、ON049530、MW514138和ON113058)的同源性分别为100% (469/469 bp)、98.47% (257/261 bp)、97.63% (617/632 bp)和100% (379/379 bp)。基于ITS、EF1-α、HIS和TUB2序列,利用MEGA11.0构建最大似然树。分离沾聚与D. phoenicola。结合形态特征和分子特征,最终鉴定为D. phoenicola。为了确认分离的D. phoenicola诱导褐斑病的致病性,根据Koch的假设评估了致病性测定(Gradmann, 2014)。将分生孢子悬液(1×105分生孢子/ mL)接种于6株3年生植物的12片未伤叶片上,并用无菌水作为对照。将接种后的叶片置于28℃、90%湿度的室内培养,光周期12 h。这个实验重复了三次。结果表明,除对照外,接种叶片在接种后6 d内出现褐斑病症状。结果表明,该病菌是水蛭褐斑病的病原。从被侵染的叶片中完全分离出病原菌,并表现出与原病原菌相同的形态特征。据我们所知,这是中国第一次报道由D. phoenicicola引起的L. polystachyus叶褐斑病。此病严重延缓了毛蕊花的植株发育,显著降低了汗茶的产量和品质。本研究结果将有助于水蛭褐斑病的防治。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Plant disease
Plant disease 农林科学-植物科学
CiteScore
5.10
自引率
13.30%
发文量
1993
审稿时长
2 months
期刊介绍: Plant Disease is the leading international journal for rapid reporting of research on new, emerging, and established plant diseases. The journal publishes papers that describe basic and applied research focusing on practical aspects of disease diagnosis, development, and management.
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