Molecular Microbiology Research 2024, Vol.14, No.2, 99-108 http://microbescipublisher.com/index.php/mmr 101 MiSeq Illumina technology allowed for the identification of endophytic bacteria in different parts of wheat species, providing insights into the stability and changeability of the core microbiome (Kuźniar et al., 2019). 3.2 Geographic and environmental factors Geographic and environmental factors play a significant role in shaping the diversity and composition of endophytic communities in wild wheat. Environmental heterogeneity, including variations in temperature, humidity, and precipitation, can influence the abundance and diversity of endophytes. For instance, a study on the fungal endophytic microbiome of wheat demonstrated that location-dependent weather conditions largely explained differences in the phyllosphere microbiome, while root communities were less affected by abiotic factors. Geographic location also contributes to the differentiation of endophytic communities. Research on cereal crops-related wild grasses revealed substantial differences in community composition across host species and locations, with both stochastic and deterministic processes affecting fungal endophyte community (FEC) assembly (Sun et al., 2020). Additionally, the diversity of endophytic fungi in wild and domesticated wheat showed that wild plants from specific sites had greater richness and diversity compared to domesticated wheat from corresponding fields (Sun et al., 2020). Moreover, the mode of transmission of endophytes, whether vertical (from progenitors) or horizontal (from the environment), influences the composition of endophytic communities. A study on seed fungal endophyte communities in wheat and its wild relatives found that external infection of seeds is the main source for specific taxa, although vertical transmission also plays a role (Sharon et al., 2023). This highlights the importance of both geographic and environmental factors in shaping the endophytic diversity in wild wheat. 4 Molecular Characterization of Endophytes 4.1 DNA sequencing techniques The molecular characterization of endophytes in wild wheat and its relatives has been significantly advanced by the application of various DNA sequencing techniques. One of the most commonly used methods is the sequencing of the internal transcribed spacer (ITS) regions and 16S ribosomal RNA (rRNA) genes. These regions are highly informative for identifying and classifying fungal and bacterial endophytes, respectively. For instance, in the study of endophytes in Triticum dicoccoides and Aegilops sharonensis, both cultivation-dependent and cultivation-independent methods were employed. The ITS region sequences from single cultures were analyzed, resulting in the identification of 67 operational taxonomic units (OTUs) at 97% sequence similarity, and found in total more than half of the cOTUs (36 out of 67) were only detected in stems (Figure 1) (Ofek-Lalzar et al., 2016). Similarly, in another study, next-generation sequencing (NGS) technologies, such as MiSeq Illumina, were utilized to identify the endophytic microbiome in Triticum aestivumand Triticum spelta. This approach allowed for a comprehensive analysis of the endophytic communities across different plant organs (Kuźniar et al., 2019). The use of 16S rRNA gene sequencing has also been pivotal in characterizing bacterial endophytes. For example, in the analysis of endophytic bacteria in Zea mays, isolates were identified using 16S rRNA gene sequencing, which revealed a diverse community of endophytes belonging to major groups such as α-Proteobacteria, γ-Proteobacteria, and Actinobacteria (Pereira and Castro, 2014). Additionally, a meta-analysis of 148 scientific papers highlighted the importance of 16S rRNA gene sequencing in providing a broad overview of culturable plant endophytic bacteria across various plant species and geographical regions (Riva et al., 2022).
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