Li Yanxin, Liu Xin, Ding Chunyan, Rui Lu, Shi Rujie
Chongqing, China;
Chongqing Three Gorges University, College of biology and food engineering, Chongqing, Chongqing, China;
Plant Dis. 2025 Aug 11. doi: 10.1094/PDIS-04-25-0749-PDN.
Lonicera japonica Thunb., belonging to Caprifoliaceae, is a widely cultivated traditional Chinese medicinal plant with high economic value. In May of 2024, symptoms of a root rot disease were observed on L. japonica in commercial plantings in Longju Town, Baitu Town, and Gaoliang Town in Wanzhou District (30°23'50″N, 107°52'22″E), Chongqing City (southwestern China). Aboveground parts of infected plants were entirely necrotic and displayed weakened lignification of roots with incidence rate of 45 to 70%. To identify the pathogen, root tissues from 17 diseased plants were collected, surface disinfected in 75% ethanol for 30 seconds, immersed in 5% NaClO for 1 minute, rinsed three times with sterile water, dried on sterile absorbent paper, placed on potato dextrose agar (PDA), and incubated at 28°C in the dark. After 5 days, 95% of the samples developed similar-appearing colonies. A single isolate, B1-V1, was selected for morphological and molecular characterization. After 15 days of incubation at 28℃ on PDA, colonies of B1-V1 reached a diameter of 80 mm; white mycelium grew slightly above the surface of the plate, was velvety in texture, and produced a yellow pigment. Hyaline, septate hyphae produced verticillate conidia that were branched like broomsticks, and conidiophores were transparent and elliptical with a single conidiophore measuring (2.31 - 4.75) × (5.33 - 8.75) μm (n = 32). The morphology was consistent with a previous description of Clonostachys rosea (Wang et al. 2024). The internal transcribed spacer region (ITS), β-tubulin gene (TUB2) and second subunit of RNA polymerase II gene (RPB2) were amplified by primers ITS1/ITS4 (Wang et al. 2024), T1/βt2b (Wang et al. 2024). and RPB2F/RPB2R (Long et al. 2021), respectively. The corresponding sequences from B1-V1 were submitted to GenBank under accession numbers PV368674 (ITS), PV390661 (TUB2), and PV390662 (RPB2). According to a BLAST search, the pathogen displayed the highest sequence similarity to strains GY12 (ITS: OR789466.1, identities: 545/545 bp), JABY1 (TUB2: OP868694.1, identities: 601/617bp) and HZL-6-8 (RPB2: PP486409.1, identities: 896/900bp). A phylogenetic tree was constructed based on concatenated ITS-TUB2-RPB2 sequences using Maximum Parsimony methods by MEGA7 software, revealing that the isolate was most closely related to C. rosea strain TMB (ITS: 491/498bp, TUB2: 311/455bp, RPB2:753/753bp). To confirm pathogenicity, both cuttings derived from branches of two-year-old plants of L. japonica Thunb. and one-year-old plants (n = 6 per group) were surface-sterilized with 75% ethanol. Roots were wounded with a sterile needle and soaked in a 4 × 10⁷ conidia/mL suspension of isolate B1-V1 for 20 days. After inoculation, symptoms including leaf wilting, yellowing, and rot browning and rot were observed, and the number of new roots significantly decreased. Colonies re-isolated from symptomatic roots were morphologically consistent with the original inoculum, thus fulfilling Koch's postulates (Wang et al, 2024). To our knowledge, this is the first report of C. rosea causing root rot in L. japonica Thunb. and provides an essential basis for root rot disease management on this crop in the future.
忍冬属忍冬科植物，是一种广泛种植的具有高经济价值的传统中药材。2024年5月，在中国西南部重庆市万州区龙驹镇、白羊镇和高粱镇（北纬30°23′50″，东经107°52′22″）的商业种植忍冬中观察到根腐病症状。受感染植株的地上部分完全坏死，根部木质化减弱，发病率为45%至70%。为鉴定病原菌，采集了17株患病植株的根组织，在75%乙醇中表面消毒30秒，浸入5%次氯酸钠中1分钟，用无菌水冲洗三次，在无菌吸水纸上干燥，置于马铃薯葡萄糖琼脂（PDA）上，于28℃黑暗中培养。5天后，95%的样本长出外观相似的菌落。挑选出一个单一分离株B1-V1进行形态学和分子特征分析。在PDA上于28℃培养15天后，B1-V1的菌落直径达到80毫米；白色菌丝体在平板表面略向上生长，质地天鹅绒状，并产生黄色色素。透明、有隔膜的菌丝产生轮状分生孢子，分生孢子像扫帚一样分枝，分生孢子梗透明且呈椭圆形，单个分生孢子梗大小为（2.31 - 4.75）×（5.33 - 8.75）μm（n = 32）。其形态与先前对粉红粘帚霉的描述一致（Wang等人，2024年）。使用引物ITS1/ITS4（Wang等人，2024年）、T1/βt2b（Wang等人，2024年）和RPB2F/RPB2R（Long等人，2021年）分别扩增内部转录间隔区（ITS）、β-微管蛋白基因（TUB2）和RNA聚合酶II基因的第二亚基（RPB2）。来自B1-V1的相应序列已提交至GenBank，登录号分别为PV368674（ITS）、PV390661（TUB2）和PV390662（RPB2）。根据BLAST搜索，该病原菌与菌株GY12（ITS：OR789466.1，一致性：545/545 bp）、JABY1（TUB2：OP868694.1，一致性：601/617 bp）和HZL-6-8（RPB2：PP486409.1，一致性：896/900 bp）的序列相似性最高。使用MEGA7软件通过最大简约法基于拼接的ITS-TUB2-RPB2序列构建系统发育树，表明该分离株与粉红粘帚霉菌株TMB关系最为密切（ITS：491/498 bp，TUB2：311/455 bp，RPB2：753/753 bp）。为确认致病性，对忍冬两年生植株和一年生植株的枝条扦插苗（每组n = 6）均用75%乙醇进行表面消毒。用无菌针损伤根部，然后将其浸泡在分离株B1-V1的4×10⁷个分生孢子/毫升悬浮液中20天。接种后，观察到叶片萎蔫、变黄、腐烂褐变等症状，新根数量显著减少。从有症状的根部重新分离得到的菌落形态与原始接种物一致，从而满足了科赫法则（Wang等人，2024年）。据我们所知，这是首次报道粉红粘帚霉引起忍冬根腐病，为今后该作物根腐病的防治提供了重要依据。
