Isolating microbes is vital to study microbiomes, but insights into microbial diversity and ecology can be constrained by recalcitrant or unculturable strains. Culture-free methods (e.g., next-generation sequencing, NGS) have become popular in part because they detect greater richness than culturing alone. Both approaches are used widely to characterize microfungi within healthy leaves (foliar endophytes), but methodological differences among studies can constrain large-scale insights into endophyte ecology. We examined endophytes in a temperate plant community to quantify how certain methodological factors, such as the choice of cultivation media for culturing and storage period after leaf collection, affect inferences regarding endophyte communities; how such effects vary among plant taxa; and how complementary culturing and NGS can be when subsets of the same plant tissue are used for each. We found that endophyte richness and composition from culturing were consistent across five media types. Insights from culturing and NGS were largely robust to differences in storage period (1, 5, and 10 days). Although endophyte richness, composition, and taxonomic diversity identified via culturing vs. NGS differed markedly, both methods revealed host-structured communities. Studies differing only in cultivation media or storage period thus can be compared to estimate endophyte richness, composition, and turnover at scales larger than those of individual studies alone. Our data show that it is likely more important to sample more host species, rather than sampling fewer species more intensively, to quantify endophyte diversity in given locations, with the richest insights into endophyte ecology emerging when culturing and NGS are paired.

分离微生物对于研究微生物组至关重要，但对微生物多样性和生态学的了解可能会受到顽固或不可培养的菌株的限制。无培养方法（例如，下一代测序，NGS）之所以流行，部分原因是它们比单独培养检测到更大的丰富度。这两种方法都被广泛用于表征健康叶子（叶内生菌）中的微真菌，但研究之间的方法差异可能会限制对内生菌生态学的大规模洞察。我们检查了温带植物群落中的内生菌，以量化某些方法学因素，例如培养培养基的选择和采叶后的储存期，如何影响对内生菌群落的推断；这种影响在植物分类群中有何不同；以及当同一植物组织的子集被用于每一种时，培养和 NGS 的互补性如何。我们发现来自培养的内生菌丰富度和组成在五种培养基类型中是一致的。来自培养和 NGS 的见解在很大程度上对储存期（1、5 和 10 天）的差异具有很强的鲁棒性。尽管通过培养与 NGS 确定的内生菌丰富度、组成和分类多样性显着不同，但这两种方法都揭示了宿主结构的群落。因此，可以比较仅在栽培介质或储存期方面不同的研究，以在比单独研究更大的规模上估计内生菌丰富度、组成和周转率。我们的数据表明，对更多寄主物种进行采样可能比对更少物种进行更密集的采样更为重要，