School of Life Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian, China.
Curr Microbiol. 2021 Aug;78(8):2916-2925. doi: 10.1007/s00284-021-02529-2. Epub 2021 May 28.
Botryosphaeria dothidea is one of the most important diseases which can cause poplar canker. In our previous study, the endophytic Bacillus subtilis N6-34 screened from poplar tissue was found to be an antagonistic strain against B. dothidea. In order to ascertain the colonization rule of B. subtilis N6-34 in poplar plants, colonization of B. subtilis N6-34 labeled with a green fluorescent protein (GFP) was investigated in poplar plants and the rhizosphere soil. To confirm the inhibitory effect of the strain N6-34 on pathogenic fungi, real-time fluorescent quantitative PCR experiment with Fusarium oxysporum as the target strain was carried out. Firstly, a plasmid (pHT01-P43GFPmut3a) containing gfp gene was successfully transformed into wild B. subtilis N6-34, which has the similar characteristics with the strain N6-34 in cell growth and antifungal activity. The poplar pot experiments were carried out to examine the colonization rules and colonization quantity in poplar plants and rhizosphere soil. Observation with a confocal laser scanning microscope showed that GFP-labeled B. subtilis N6-34 (N6-34-GFP) could colonize in primary root, lateral root and adventitious root. With the extension of inoculation time, the colonization quantity of N6-34-GFP in the rhizosphere soil and poplar plants showed a trend of first increasing, then stabilizing for a period of time and then decreasing. The real-time fluorescent quantitative PCR result showed a gradual decrease in the number of F. oxysporum with increasing inoculation time. Therefore, N6-34-GFP exhibited colonization in the rhizosphere soil and different parts of poplar plants. In addition, the strain N6-34 could inhibit the growth of pathogenic fungi. The ability of B. subtilis N6-34 to colonize in the rhizosphere soil and poplar plants and to inhibit fungal growth in vitro suggest a potential application of this strain as a biological control agent.
层出镰刀菌是引起杨树溃疡病的最重要的病害之一。在我们之前的研究中，从杨树组织中筛选出的内生枯草芽孢杆菌 N6-34 被发现是一种拮抗层出镰刀菌的菌株。为了确定枯草芽孢杆菌 N6-34 在杨树植物中的定殖规律，研究了用绿色荧光蛋白（GFP）标记的枯草芽孢杆菌 N6-34 在杨树植物和根际土壤中的定殖情况。为了确认该菌株 N6-34 对病原菌的抑制作用，以尖孢镰刀菌为靶标菌株进行了实时荧光定量 PCR 实验。首先，成功地将含有 gfp 基因的质粒（pHT01-P43GFPmut3a）转化到野生枯草芽孢杆菌 N6-34 中，该质粒与菌株 N6-34 在细胞生长和抗真菌活性方面具有相似的特性。通过杨树盆栽实验，研究了枯草芽孢杆菌 N6-34 在杨树植物和根际土壤中的定殖规律和定殖数量。共聚焦激光扫描显微镜观察表明，GFP 标记的枯草芽孢杆菌 N6-34（N6-34-GFP）可以定殖在初生根、侧根和不定根中。随着接种时间的延长，N6-34-GFP 在根际土壤和杨树植物中的定殖数量呈现出先增加、然后稳定一段时间、然后减少的趋势。实时荧光定量 PCR 结果表明，随着接种时间的增加，尖孢镰刀菌的数量逐渐减少。因此，N6-34-GFP 在根际土壤和不同部位的杨树植物中均有定殖。此外，该菌株 N6-34 可以抑制病原菌的生长。枯草芽孢杆菌 N6-34 在根际土壤和杨树植物中的定殖能力以及体外抑制真菌生长的能力表明，该菌株作为生物防治剂具有潜在的应用价值。
