茄子是我国第五大茄科类经济作物，茄子黄萎病属于茄子三大病害之一，给我国的茄子产量和质量带来了极为严重的破坏，造成了巨大的经济损失。黄萎病主要病原菌为以微菌核形式存在于土壤中的大丽轮枝菌。在防治黄萎病时，由于物理嫁接抗性品种费时费力，化学药剂对环境污染严重，因此进行安全、高效且绿色的生物防治是目前最有发展前景的防治手段。使用枯草芽孢杆菌作为生物防治剂进行生物防治则是最受关注的方式之一。项目组前期于新疆连作棉田中分离出的枯草芽孢杆菌J-15（Baciilus subtilis J-15，简称J-15），能够分泌拮抗大丽轮枝菌的次生代谢产物，该菌株具有广谱抗真菌、抗逆性强等特点，其次代物的粗提物在对棉花黄萎病的防治上也取得了良好的效果。基于此，本论文对J-15防治茄子黄萎病的可行性进行了探讨。

本论文首先用平板对峙法和牛津杯法在体外检验了J-15及其次级代谢产物对以茄子为宿主的大丽轮枝菌GA（Verticillium Dahliae GA，简称GA）的拮抗效果，证明其能够拮抗GA；采用实时荧光定量PCR技术，建立了基于标准曲线检测土壤中GA生物量的方法，并模拟了土壤环境中J-15对GA的拮抗效果。结果显示，在每克土壤中含100个GA孢子时，建立的方法仍然可靠，与其他同类研究相比，精确度提高了近10000倍。在模拟的土壤环境中，J-15处理能够使土壤中GA数目从2×10⁷（Colony Formine Unit/g，CFU/g）降低至5×10⁶（CFU/g）。因此，J-15不仅能够在培养基上抑制GA的生长，还能杀灭土壤中的GA。

J-15对茄子黄萎病的生物防治实验，采用北京地区广泛种植的京茄黑宝（HB）和京茄21号（N21）作为植物材料，采取伤根侵染法构建茄子黄萎病的患病模型。通过统计发现，N21和HB在10²~10⁸（CFU/mL）GA孢子液侵染时发病程度与孢子液浓度呈正相关；qPCR检测发现，GA在HB和N21的茎中含量最高，根、叶片和土壤中也能够检测到大量的GA；在相同孢子液浓度侵染时，N21对GA更易感，采用N21进行生物防治实验。由于茄子种植普遍都是采用育苗移栽技术，主要是在移栽至田间后由土壤中的病原菌侵染发病，因此针对育苗移栽过程中易伤根染病这一实际情况，本研究据此设计了三部分实验：（1）使用J-15处理被GA侵染的茄子，观察茄子发病情况。首先使用10⁴、10⁶和10⁸（CFU/mL）J-15水悬液灌根处理患病茄子，结果表明此时J-15水悬液防治效率分别为2.60%，6.49%和16.88%；使用J-15发酵液（OD=2.0）处理被GA侵染的茄子，防治效率为17.86%；（2）在GA侵染茄子前4天使用J-15发酵液（OD=2.0）对茄子幼苗灌根处理，防治效率18.18%；（3）使用J-15发酵液（OD=2.0）处理含GA的土壤，24小时后移栽茄子幼苗，防治效率为17.50%。

以上研究结果表明，J-15可以通过直接处理患病植株、预处理幼苗和土壤等方法，达到一定的防治茄子黄萎病的效果，但是它阻止GA侵染茄子和杀死已经定殖在植株体内的GA的作用有限，因而使用J-15来杀灭土壤中的GA是其应用于茄子黄萎病防治的有效途径，且使用J-15杀灭土壤中的GA需要一定的时间。随后探究J-15对茄子的生长影响，实验结果表明J-15水悬液和发酵液对茄子生长无显著影响，但是J-15能够诱导茄子PR1和PR5基因的表达，帮助植株抗病。

Eggplant is the fifth largest commercial crop of the Solanaceae family in China. And verticillium wilt is one of the three major diseases of eggplant, which brings extremely serious damage to the yield and quality of eggplant in China and causes huge economic losses. Verticillium Dahliae, the main pathogen of eggplant verticillium wilt, is present in the soil in the form of microsclerotia. Regarding the control of verticillium wilt, due to the time-consuming and laborious physical grafting of resistant varieties and the serious environmental pollution caused by chemical agents, the biological control for safe, efficient and environment-friendly is the most promising means of control at present. And the use of Bacillus subtilis as biological control agent is one of the most promising methods. Bacillus subtilis J-15 (J-15), which was isolated from continuous cotton fields in Xinjiang, can secrete the secondary metabolites of antagonistic Verticillium dahliae extracellularly, and has the characteristics of broad-spectrum antifungal and strong resistance.The crude extracts of the secondary metabolites also achieved good results in the control of verticillium wilt of cotton. Based on this, this paper explored the feasibility of J-15 for the control of verticillium wilt of eggplant.

Firstly, we examined the antagonistic effect of J-15 and its secondary metabolites on the eggplant Verticillium dahliae GA (GA) in vitro by the plate standoff method and Oxford cup method, and proved that it could antagonize the GA. A method for detecting GA biomass in soil based on a standard curve was established using Real-time fluorescence quantitative PCR (qPCR). qPCR could detect a minimum of 100 GA per gram of soil, which was 10,000 times more accurate than the common PCR method. In the simulated soil environment, J-15 treatment was able to reduce the number of GA in the soil from 2×10⁷ (Colony Formine Unit/g, CFU/g) to 5×10⁶ (CFU/g).Therefore, J-15 was not only able to inhibit the growth of GA on the medium, but also killed GA in the soil.

Biological control experiments of J-15 on eggplant verticillium wilt were carried out by using the plant material of Jingqie Heibao (HB) and Jingqie N21 (N21), which are widely grown in Beijing, and the disease model of eggplant verticillium wilt was constructed by wounded root infestation. Statistically, it was found that both N21 and HB could be diseased when infested with 10²~10⁸ (CFU/mL) GA spores, and the degree of disease on N21 and HB was positively correlated with spore solution concentration. The qPCR result shows that the relative copy number of GA was highest in the stems of HB and N21, and significant amounts of GA could also be detected in roots, leaves and soil. At the same spore concentration, N21 was more susceptible to GA, and N21 was used for biological control experiments. Given that eggplant cultivation is commonly done by seedling transplanting technology, which is mainly infested by pathogenic bacteria in the soil after transplanting to the field, the thesis designed three parts of experiments based on the actual situation that seedlings are prone to root injury and disease during seedling transplanting: (1) Eggplants were infested with GA and then treated with J-15 to observe the disease index. The results showed that the biocontrol efficiency of 10⁴, 10⁶ and 10⁸ (CFU/mL) J-15 water suspension by root irrigation were 2.60%, 6.49% and 16.88%, respectively. At this time; the biocontrol efficiency was 17.86% when J-15 fermentation broth (OD=2.0) was used to treat the infected eggplant. (2) Treatment of eggplant seedlings by root irrigation with J-15 fermentation solution (OD=2.0) 4 days before GA infestation resulted in 18.18% biocontrol efficiency (3)Soil containing GA was treated with J-15 fermentation solution (OD=2.0) and eggplant seedlings were transplanted 24 hours later with a biocontrol efficiency of 17.50%.

The results of the above study showed that J-15 can achieve some effect in controlling eggplant verticillium wilt by directly treating diseased plants, pretreating seedlings and soil. However, it has limited effect in preventing GA from infesting eggplant and killing GA already colonized in the plants, so using J-15 to kill GA in soil is an effective way to apply it to eggplant verticillium wilt control, and using J-15 to kill GA in soil also takes some time. Subsequently, the effect of J-15 on the growth of eggplant was explored. The result shows that neither the J-15 water suspension nor the fermentation broth had the effect of promoting growth, but J-15 could induce the expression of PR1 and PR5 genes in eggplant and help the plants to resist the disease.