Methodological Approaches Frame Insights into Endophyte Richness and Community Composition.

Area of Science:

• Microbiology
• Ecology
• Botany

Background:

• Microbial isolation is key for microbiome studies, but limitations exist with culturable and unculturable microbes.
• Culture-free methods like next-generation sequencing (NGS) offer greater microbial richness detection than culturing alone.
• Methodological variations in studying foliar endophytes can hinder large-scale ecological understanding.

Purpose of the Study:

• To assess how cultivation media and leaf storage duration impact endophyte community inferences.
• To determine if these methodological effects vary across different plant taxa.
• To evaluate the complementarity of culturing and NGS when applied to the same plant tissue.

Main Methods:

• Fungal endophytes were studied in a temperate plant community.
• Culturing methods were tested across five different media types.
• Next-generation sequencing (NGS) was used as a complementary culture-free approach.
• Leaf samples were stored for 1, 5, and 10 days to assess storage effects.

Main Results:

• Endophyte richness and composition were consistent across tested cultivation media.
• Both culturing and NGS results remained robust despite variations in leaf storage periods.
• Significant differences were observed in endophyte richness, composition, and diversity between culturing and NGS.
• Both methods consistently identified host-structured endophyte communities.

Conclusions:

• Methodological choices in cultivation media or storage duration have minimal impact on comparative endophyte studies.
• Despite differences, culturing and NGS provide complementary insights into microbial ecology.
• Sampling a wider range of host plant species is more crucial for quantifying endophyte diversity than intensive sampling of fewer species.
• Integrating culturing and NGS methods yields the most comprehensive understanding of foliar endophyte ecology.