This study sampled multiple plant species with different functional pathways and leaf architectures to identify the impact of different preservation method on plant microbiome, and evaluated the validity using plant specific primer pair 799F/1193R on soil microbiome approach.

The microbial communities associated with plants (the plant microbiome) play critical roles in regulating plant health and productivity. Because of this, in recent years, there have been significant increase in studies targeting the plant microbiome. Amplicon sequencing is widely used to investigate the plant microbiome and to develop sustainable microbial agricultural tools. However, performing large microbiome surveys at the regional and global scales pose several logistic challenges. One of these challenges is related with the preservation of plant materials for sequencing aiming to maintain the integrity of the original diversity and community composition of the plant microbiome. Another significant challenge involves the existence of multiple primer sets used in amplicon sequencing that, especially for bacterial communities, hampers the comparability of datasets across studies. Here, we aimed to examine the effect of different preservation approaches (snap freezing, fresh and kept on ice, and air drying) on the bacterial and fungal diversity and community composition on plant leaves, stems and roots from seven plant species from contrasting functional groups. Another major challenge comes when comparing plant to soil microbiomes, as different primers sets are often used for plant vs. soil microbiomes. Thus, we also investigated if widely used 16S rRNA primer set (779F/1193R) for plant microbiome studies provides comparable data to those often used for soil microbiomes (341F/805R) using 86 soil samples. We found that the community composition and diversity of bacteria or fungi were robust to contrasting preservation methods. The primer sets often used for plants provided similar results to those often used for soil studies suggesting that simultaneous studies on plant and soil microbiomes are possible. Our findings provide novel evidence that preservation approaches do not significantly impact plant microbiome data interpretation and primer differences do not impact the treatment effect, which has significant implication for future large-scale and global surveys of plant microbiomes.

与植物关联的微生物群落（植物微生物组，plant microbiome）在调控植物健康与生产力方面发挥关键作用。正因如此，近年来针对植物微生物组的研究数量出现了显著增长。扩增子测序（amplicon sequencing）被广泛应用于植物微生物组的探究，以及可持续微生物农业工具的开发。然而，在区域乃至全球尺度开展大规模微生物组调研，会面临多项实操挑战。其中一项挑战与植物样本的保存相关——需维持植物微生物组原始多样性与群落组成的完整性。另一项重大挑战则在于，扩增子测序中存在多种引物套装，尤其针对细菌群落时，会阻碍不同研究间数据集的可比性。本研究旨在评估不同保存方式（快速冷冻、新鲜样本置于冰上、空气干燥）对7种不同功能群植物的叶片、茎秆及根系样本中细菌与真菌多样性及群落组成的影响。在比较植物与土壤微生物组时，另一项主要挑战在于，植物与土壤微生物组通常会使用不同的引物套装。因此，我们还利用86份土壤样本，探究了植物微生物组研究中常用的16S rRNA引物套装（779F/1193R），与土壤微生物组常用的（341F/805R）能否产出具有可比性的数据。研究结果显示，细菌或真菌的群落组成与多样性对不同保存方式均具有鲁棒性。植物研究常用的引物套装所得结果与土壤研究常用的引物套装相近，这表明同时开展植物与土壤微生物组研究具备可行性。本研究的发现为以下结论提供了新的实证依据：保存方式不会对植物微生物组数据的解读造成显著影响，引物差异也不会影响处理效应，这一结论对未来开展大规模乃至全球尺度的植物微生物组调研具有重要意义。