Endophytes have been defined as microorganisms living inside plant tissues without causing negative effects on their hosts. Endophytic microbes have been extensively studied for their plant growth-promoting traits. However, analyses of endophytes require complete removal of epiphytic microorganisms. We found that the established tests to evaluate surface sterility, polymerase chain reaction, and leaf imprints, are unreliable. Therefore, we used scanning electron microscopy (SEM) as an additional assessment of epiphyte removal. We used a diverse suite of sterilization protocols to remove epiphytic microorganisms from the leaves of a gymnosperm and an angiosperm tree to test the influence of leaf morphology on the efficacy of these methods. Additionally, leaf tissue damage was also evaluated by SEM, as damaging the leaves might have an impact on endophytes and could lead to inaccurate assessment of endophytic communities. Our study indicates, that complete removal of the leaf cuticle by the sterilization technique assures loss of epiphytic microbes, and that leaves of different tree species may require different sterilization protocols. Furthermore, our study demonstrates the importance of choosing the appropriate sterilization protocol to prevent erroneous interpretation of host-endophyte interactions. Moreover, it shows the utility of SEM for evaluating the effectiveness of surface sterilization methods and their impact on leaf tissue integrity.

中文翻译：

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从叶子表面去除微生物的简单方法

内生菌被定义为生活在植物组织内而不对其宿主造成负面影响的微生物。内生微生物因其促进植物生长的特性而被广泛研究。然而，内生菌的分析需要完全去除附生微生物。我们发现，用于评估表面无菌性、聚合酶链反应和叶印的既定测试是不可靠的。因此，我们使用扫描电子显微镜 (SEM) 作为对附生植物去除的额外评估。我们使用了一套多样化的灭菌方案从裸子植物和被子植物树的叶子中去除附生微生物，以测试叶子形态对这些方法功效的影响。此外，还通过 SEM 评估了叶组织损伤，因为损坏叶子可能会对内生菌产生影响，并可能导致对内生菌群的不准确评估。我们的研究表明，通过灭菌技术完全去除叶表皮可确保附生微生物的损失，并且不同树种的叶子可能需要不同的灭菌方案。此外，我们的研究证明了选择合适的灭菌方案以防止对宿主-内生菌相互作用的错误解释的重要性。此外，它还显示了 SEM 在评估表面灭菌方法的有效性及其对叶组织完整性的影响方面的效用。通过消毒技术完全去除叶表皮可确保附生微生物的损失，并且不同树种的叶子可能需要不同的消毒方案。此外，我们的研究证明了选择合适的灭菌方案以防止对宿主-内生菌相互作用的错误解释的重要性。此外，它还显示了 SEM 在评估表面灭菌方法的有效性及其对叶组织完整性的影响方面的效用。通过消毒技术完全去除叶表皮可确保附生微生物的损失，并且不同树种的叶子可能需要不同的消毒方案。此外，我们的研究证明了选择合适的灭菌方案以防止对宿主-内生菌相互作用的错误解释的重要性。此外，它还显示了 SEM 在评估表面灭菌方法的有效性及其对叶组织完整性的影响方面的效用。