Endophytic and Molecular Microbiology Laboratory, Division of Biotechnology, ICAR-Indian Institute of Horticultural Research (IIHR), Hessaraghatta Lake, Bengaluru, 560089, India.
Planta. 2017 Nov;246(5):879-898. doi: 10.1007/s00425-017-2733-5. Epub 2017 Jul 11.
Molecular and microscopic analyses reveal enormous non-cultivable endophytic bacteria in grapevine field shoots with functional significance. Diverse bacteria enter tissue cultures through surface-sterilized tissues and survive surreptitiously with varying taxonomic realignments. The study was envisaged to assess the extent of endophytic bacterial association with field shoot tissues of grapevine and the likelihood of introduction of such internally colonizing bacteria in vitro adopting molecular techniques targeting the non-cultivable bacterial community. PowerFood-kit derived DNA from surface-sterilized field shoot tips of grapevine Flame Seedless was employed in a preliminary bacterial class-specific PCR screening proving positive for major prokaryotic taxa including Archaea. Taxonomic and functional diversity were analyzed through whole metagenome profiling (WMG) which revealed predominantly phylum Actinobacteria, Proteobacteria, and minor shares of Firmicutes, Bacteroidetes, and Deinococcus-Thermus with varying functional roles ascribable to the whole bacterial community. Field shoot tip tissues and callus derived from stem segments were further employed in 16S rRNA V3-V4 amplicon taxonomic profiling. This revealed elevated taxonomic diversity in field shoots over WMG, predominantly Proteobacteria succeeded by Actinobacteria, Firmicutes, Bacteroidetes, and 15 other phyla including several candidate phyla (135 families, 179 genera). Callus stocks also displayed broad bacterial diversity (16 phyla; 96 families; 141 genera) bearing resemblance to field tissues with Proteobacterial dominance but a reduction in its share, enrichment of Actinobacteria and Firmicutes, disappearance of some field-associated phyla and detection of a few additional taxonomic groups over field community. Similar results were documented during 16S V3-V4 amplicon taxonomic profiling on Thompson Seedless field shoot tip and callus tissues. Video microscopy on tissue homogenates corroborated enormous endophytic bacteria. This study elucidates a vast diversity of cultivation-recalcitrant endophytic bacteria prevailing in grapevine field shoots, their in vitro introduction, and unsuspecting sustenance with possible silent participation in tissue culture processes.
分子和微观分析揭示了葡萄田间嫩枝中大量具有功能意义的不可培养内生细菌。不同的细菌通过表面消毒的组织进入组织培养，并通过不同的分类重排秘密存活。本研究旨在评估内生细菌与葡萄田间嫩枝组织的关联程度，以及采用靶向不可培养细菌群落的分子技术在体外引入此类内部定殖细菌的可能性。PowerFood-kit 从 Flame Seedless 葡萄田间嫩枝的表面消毒嫩枝尖端中提取的 DNA，采用细菌分类特异性 PCR 筛选，结果为古菌等主要原核生物类群呈阳性。通过全宏基因组分析（WMG）分析了分类和功能多样性，结果表明，主要的细菌门为放线菌门、变形菌门，还有少量的厚壁菌门、拟杆菌门和 Deinococcus-Thermus 门，具有不同的功能作用，归因于整个细菌群落。进一步将田间嫩枝尖端组织和来自茎段的愈伤组织用于 16S rRNA V3-V4 扩增子分类分析。这揭示了田间嫩枝的分类多样性高于 WMG，主要为变形菌门，其次是放线菌门、厚壁菌门、拟杆菌门和其他 15 个门，包括几个候选门（135 个科，179 个属）。愈伤组织也显示出广泛的细菌多样性（16 个门；96 个科；141 个属），与田间组织相似，但具有变形菌门优势的减少，放线菌门和厚壁菌门的富集，一些与田间相关的门的消失，以及在田间群落中检测到一些额外的分类群。在对 Thompson Seedless 田间嫩枝尖端和愈伤组织进行 16S V3-V4 扩增子分类分析时，也记录了类似的结果。对组织匀浆的视频显微镜检查证实了大量的内生细菌。本研究阐明了在葡萄田间嫩枝中普遍存在的大量具有培养抗性的内生细菌的多样性，它们在体外的引入以及在组织培养过程中可能沉默参与的意想不到的维持。
