Patel Nikhil N, Gaiero Jonathan R, Sulman Muhammad, Moote Paul, Nesbitt Darlene, Svircev Antonet M, Ellouze Walid
Agriculture and Agri-Food Canada, London Research and Development Centre, 4902 Victoria Avenue North, Vineland Station, ON L0R 2E0, Canada.
Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, ON L2S 3A1, Canada.
Microorganisms. 2025 Apr 16;13(4):923. doi: 10.3390/microorganisms13040923.
This study examines the effect of pre-extraction methods, namely, sonication, grinding, and lyophilization, and the use of peptide nucleic acid (PNA) blockers on the DNA recovery, diversity, and taxonomic resolution of bacterial and fungal communities in apple blossoms. Sonication was the most successful in recovering bacterial 16S and fungal ITS reads across all the collection points and plots. Lyophilization and grinding led to a significant reduction in fungal read counts, while PNA enhanced the recovery of bacterial 16S reads. Sonication improved the efficiency of DNA extraction and yielded greater diversity in the recovered microbial community. Sonicated samples showed greater sensitivity to temporal shifts in microbial community composition. Communities in sonicated samples contained a larger number of bacterial genera, such as , , and , and fungal genera, including and In contrast, lyophilization and grinding led to a reduction in detected taxa. The indicator species analysis determined that 35 bacterial and 21 fungal genera were closely related to sonication, whereas no other pre-extraction method had any associated genera. Our findings suggest that sonication is the most appropriate pre-extraction method for analyzing blossom-associated microbiomes, and that the use of PNA blockers can improve the recovery of bacteria and minimize contamination by host DNA.
本研究考察了提取前处理方法（即超声处理、研磨和冻干）以及肽核酸（PNA）阻断剂的使用对苹果花中细菌和真菌群落DNA回收率、多样性及分类分辨率的影响。在所有采集点和样地中，超声处理在回收细菌16S和真菌ITS读数方面最为成功。冻干和研磨导致真菌读数显著减少，而PNA提高了细菌16S读数的回收率。超声处理提高了DNA提取效率，并在回收的微生物群落中产生了更大的多样性。经超声处理的样本对微生物群落组成的时间变化表现出更高的敏感性。经超声处理的样本中的群落包含更多的细菌属，如 、 和 ，以及真菌属，包括 和 。相比之下，冻干和研磨导致检测到的分类单元减少。指示物种分析确定，35个细菌属和21个真菌属与超声处理密切相关，而没有其他提取前处理方法有任何相关属。我们的研究结果表明，超声处理是分析与花相关的微生物群落最合适的提取前处理方法，并且使用PNA阻断剂可以提高细菌回收率并最大限度减少宿主DNA的污染。
