# Impact of Pre-Extraction Methods on Apple Blossom Microbiome Analysis

## TL;DR

This study compares methods to prepare apple blossom samples for DNA analysis, finding that sonication gives the best results for studying microbial communities.

## Contribution

The study identifies sonication as the optimal pre-extraction method for analyzing apple blossom microbiomes and highlights the benefits of using PNA blockers.

## Key findings

- Sonication outperformed grinding and lyophilization in recovering bacterial and fungal DNA reads.
- PNA blockers improved bacterial DNA recovery and reduced host DNA contamination.
- Sonication revealed higher microbial diversity and temporal sensitivity compared to other methods.

## Abstract

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 Bacillus, Staphylococcus, and Erwinia, and fungal genera, including Didymellaceae and Cladosporium. 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.