Institute of Molecular Biosciences, College of Sciences, Massey University, Private Bag 11222, Palmerston North, 4442, New Zealand.
J Microbiol Methods. 2011 Oct;87(1):32-7. doi: 10.1016/j.mimet.2011.07.004. Epub 2011 Jul 14.
Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.
长喙壳（Dothistroma）是引起松树赤枯病的病原菌。本研究开发了一种基于绿色荧光蛋白（GFP）的筛选方法，用于评估微生物对长喙壳的生物防治潜力。该筛选方法利用 GFP 表达作为病原菌代谢活性的指标，并利用潮霉素抗性选择来确定相互作用是抑菌作用还是杀菌作用。结果表明，在体外测试的 8 个木霉分离物中有 6 个具有通过杀菌作用来控制长喙壳的潜力。由于长喙壳产生广谱毒素——多氧霉素，因此通过与对照相比，测定木霉对长喙壳的抑制作用来确定多氧霉素对木霉的抑制作用。木霉的抑制作用范围从无抑制到 30%的抑制，并且受到所用测定培养基的影响。还评估了 GFP 筛选方法是否适合筛选具有潜在生物防治能力的细菌。尽管必须使用间接接触方法，但在 4 株芽孢杆菌中有 2 株在体外对长喙壳表现出拮抗活性，通过抑菌作用。这 4 株细菌对长喙壳的生长抑制率为 14.0%至 39.8%。该基于 GFP 的方法代表了一种筛选真菌和细菌拮抗活性的新方法。
