长叶轮钟草根腐病病原菌的分离鉴定及4种芳香中药植物挥发油对其病原菌的抑制作用

Isolation and identification of pathogens causing root rot in Campanumoea lancifolia and inhibitory effects of essential oils from four aromatic medicinal plants on the pathogenic fungi

摘要:

长叶轮钟草[Campanumoea lancifolia(Roxb.) Merr.]是一种具有药食两用价值的新资源，开发利用前景广阔。田间调研发现，随着其种植面积的扩大，根腐病问题日益严重，在根腐病严重的田间，损失可高达40%，占长叶轮钟草所有病害造成损失的75%−90%，直接导致了果实产量及品质的下降，影响了商品价值及农户的收入，开展长叶轮钟草根腐病的防治工作迫在眉睫。【目的】分离和鉴定长叶轮钟草根腐病病原菌，同时提取4种芳香中药植物的挥发油，研究其对长叶轮钟草病原菌生长的抑制作用。【方法】采用常规组织分离法对典型根腐病症状的长叶轮钟草病害植株进行病原菌的分离纯化，结合形态学和分子生物学手段鉴定病原菌种类，并进行科赫氏法则验证。采用水蒸气蒸馏法从芳香中药植物中提取挥发油，通过牛津杯法评估挥发油的抑菌效果，并通过96孔板法研究了其最低抑菌浓度(minimum inhibitory concentrations,MICs)。【结果】从患病的长叶轮钟草植株根系中分离并鉴定到4株病原菌，将分离得到的4株病原菌回接后，植物出现了与大田一致的根腐病症状。经形态学和分子生物学鉴定，4株菌分别为尖孢镰刀菌(Fusarium oxysporum)、茄腐镰刀菌(Fusarium solani)、麦冬炭疽菌(Colletotrichum liriopes)和蔓枯病菌(Stagonosporopsis pogostemonis)。挥发油抑菌实验发现，4种挥发油对尖孢镰刀菌、茄腐镰刀菌、蔓枯病原菌和麦冬炭疽菌均有很强的抑制效果，抑制率在32.94%−95.29%之间。此外，4种挥发油对4株病原菌MICs在0.031−4.000 mg/ml之间。【结论】本研究表明，尖孢镰刀菌、茄腐镰刀菌、蔓枯病原菌和麦冬炭疽菌均为长叶轮钟草的致病菌，且茄腐镰刀菌、蔓枯病原菌和麦冬炭疽菌为首次报道的长叶轮钟草根腐病病原菌。此外，基于中医“芳香避秽”的思想理论，本研究选取的4种芳香植物的挥发油对长叶轮钟草的根腐病病原菌均有较强的抑制作用。本研究为长叶轮钟草根腐病植物源农药的开发和未来该资源的绿色种植奠定了科学基础。

Abstract:

Campanumoea lancifolia (Roxb.) Merr. is a new plant species with both medicinal and edible values, demonstrating broad prospects for development and utilization. However, as its cultivation area expands, root rot has become increasingly severe. In the fields with severe root rot, the losses can reach up to 40%, accounting for 75% to 90% of the losses caused by all diseases affecting C. lancifolia. Root rot directly leads to declines in fruit yield and quality, affecting the commercial value of the fruits and reducing farmers’ incomes. Thus, it is urgent to address the root rot in C. lancifolia. [Objective] To isolate and identify the pathogens causing root rot in C. lancifolia and investigate the inhibitory effects of essential oils extracted from four aromatic medicinal plants on the growth of these pathogens. [Methods] Pathogens were isolated from C. lancifolia plants displaying typical root rot symptoms by the tissue culture method. The pathogens were identified based on morphological and molecular evidence and verified according to Koch’s postulates. Essential oils were extracted from four aromatic medicinal plants by steam distillation. The Oxford cup method was employed to examine the inhibitory effects of the essential oils on the pathogens, and the 96-well plate method was used to determine the minimum inhibitory concentrations (MICs) of the essential oils. [Results] Four pathogenic strains were isolated and identified from the roots of diseased C. lancifolia plants. Re-inoculation of these pathogens induced root rot symptoms consistent with those observed in the field. The pathogens were identified as Fusarium oxysporum, Fusarium solani, Colletotrichum liriopes, and Stagonosporopsis pogostemonis. The essential oils exhibited strong inhibitory effects on these pathogens, with inhibition rates ranging from 32.94% to 95.29%. Additionally, the MICs of the four essential oils against the pathogens ranged from 0.031 mg/mL to 4.000 mg/mL. [Conclusion] This study demonstrates that F. oxysporum, F. solani, C. liriopes, and S. pogostemonis are pathogenic to C. lancifolia. This is the first report of F. solani, C. liriopes, and S. pogostemonis causing root rot in C. lancifolia. Furthermore, the essential oils extracted from the selected four aromatic plants exhibited strong inhibitory effects on the pathogens causing root rot in C. lancifolia, which coincides with the theory of aromatic plants dispelling pathogens in traditional Chinese medicine. The findings lay a scientific foundation for the development of botanical pesticides against root rot in C. lancifolia and the eco-friendly cultivation of this plant.