小麦赤霉病拮抗菌JB7的拮抗活性及鉴定

Antagonistic activity and identification of antagonistic bacteria JB7 against Fusarium head blight

摘要:

由禾谷镰刀菌(Fusarium graminearum, Fg)引起的赤霉病是限制小麦生产的主要病害之一。生物防治是一种高效且可持续的防治方法。【目的】从小麦种子内筛选具有抑制禾谷镰刀菌的菌株并对其生防潜力进行评估，为小麦赤霉病生防制剂的开发与利用提供菌种资源及理论支撑。【方法】采用平板对峙、孢子萌发法和无菌上清液抑菌试验筛选小麦种子内对禾谷镰刀菌具有拮抗活性的内生菌株；利用扫描电镜(scanning electron microscope, SEM)和共聚焦扫描电镜(confocal laser scanning microscope, CLSM)观察并分析无菌上清液对Fg的分生孢子形态、膜完整性以及胞内活性氧的影响；通过盆栽试验验证内生菌对小麦赤霉病的生防效果；应用二代Illumina HiSeq测序平台进行全基因组测序。【结果】从小麦种子中分离出一株高效抑制Fg生长的内生菌株JB7，其衰亡期无菌上清液对Fg孢子萌发抑制率高达85.23%。菌株JB7的无菌上清液使Fg孢子表面凹陷，破坏其细胞膜，造成核酸和蛋白质的渗漏，诱导Fg菌丝活性氧的累积，引起Fg菌丝可溶性蛋白和丙二醛含量的显著升高。该菌株具有分泌蛋白酶、纤维素酶、葡聚糖酶和产铁载体的能力。盆栽试验表明菌株JB7能显著降低小麦赤霉病的病情指数(P<0.05)。经全基因组学鉴定为甲基营养型芽孢杆菌(Bacillus methylotrophicus) JB7，该菌株基因组中含有12个抑菌功能的次级代谢产物合成基因簇。【结论】菌株JB7能抑制禾谷镰刀菌的生长，对小麦赤霉病有较强的防效，可作为生物防治小麦赤霉病的候选菌株。

关键词:小麦赤霉病;禾谷镰刀菌;甲基营养型芽孢杆菌;生物防治;活性氧积累

Abstract:

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the major diseases limiting wheat production. Biocontrol has been considered an effective and sustainable method for the control of crop diseases. [Objective] To screen out endophytic strains with inhibitory effects on F. graminearum from wheat grains and evaluate their biocontrol potential, providing strain resources and theoretical support for the development and utilization of biocontrol agents against FHB in wheat. [Methods] Plate confrontation, spore germination, and cell-free supernatant (CFS) inhibition experiments were carried out to screen out the endophytic strains with antagonistic activity against F. graminearum from wheat grains. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were employed to observe the conidial morphology, membrane integrity, and mycelial reactive oxygen species of F. graminearum treated with the CFS. Pot experiments were performed to verify the biocontrol effects of the endophytic bacteria on FHB. Illumina HiSeq was used for whole genome sequencing. [Results] A highly efficient endophytic strain JB7 inhibiting the growth of F. graminearum was isolated from wheat grains. The CFS of strain JB7 in the decline stage showed the inhibition rate of 85.23% on the spore germination of F. graminearum. Moreover, it led to concavity on spore surface, cell membrane damage, leakage of nucleic acids and proteins, and accumulation of reactive oxygen species in the mycelia of F. graminearum. The CFS of strain JB7 significantly increased the content of soluble protein and malondialdehyde. Strain JB7 could produce protease, cellulase, glucanase, and siderophores. Pot experiments showed that strain JB7 decreased the disease index of FHB (P<0.05) in wheat. The strain was identified as Bacillus methylotrophic JB7 by whole genome sequencing, and it carried 12 gene clusters for the synthesis of secondary metabolites with antimicrobial functions. [Conclusion] Strain JB7 could inhibit the growth of F. graminearum and demonstrated a strong control effect on FHB, serving as a candidate strain for the biocontrol of FHB in wheat.

Key words:Fusarium head blight;Fusarium graminearum;Bacillus methylotrophicus;biocontrol;accumulation of reactive oxygen species