Molecular Microbiology Research 2024, Vol.14, No.5, 226-235 http://microbescipublisher.com/index.php/mmr 227 2 Diversity of Seed-Associated Endophytes inZea 2.1 Types of endophytes found inZea seeds Seed-associated endophytes in Zea, which includes both wild ancestors like teosinte and domesticated maize, exhibit a diverse array of microbial species. Studies have identified a core microbiota conserved across different Zea genotypes, including Clostridiumand Paenibacillus species, which were detected through terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rDNA (Johnston-Monje and Raizada, 2011). Additionally, culturing methods have revealed widespread presence of Enterobacter, Methylobacteria, Pantoea, and Pseudomonas species, with γ-proteobacteria being the most prevalent class. In another study, phylogenetic analysis of culturable bacterial endophytes fromZea mays identified three major groups: α-Proteobacteria, γ -proteobacteria, and Actinobacteria, with genera such as Pseudomonas, Agrobacterium, Variovorax, and Curtobacteriumbeing prominently represented (Geisen et al., 2017). These endophytes are not only diverse but also exhibit various plant growth-promoting traits, such as phosphate solubilization and production of acetoin butanediol, which are crucial for plant health and development. 2.2 Geographic and environmental influences on diversity The diversity and community composition of seed-associated endophytes in Zea are influenced by geographic and environmental factors. For instance, the movement of Zea from its native Mexico to regions as far as Canada has shown that while a core microbiota is conserved, the abundance and specific composition of endophytes can vary. Environmental factors such as soil contamination with heavy metals like Zn and Cd also play a significant role in shaping the endophytic community (Cai et al., 2024). In contaminated soils, endophytes such as Pseudomonas, Agrobacterium, and Curtobacterium have shown high tolerance to metal stress and possess plant growth-promoting traits, indicating an adaptation to harsh environmental conditions. Furthermore, studies on other plant species have demonstrated that factors like urbanization and cone age significantly influence endophyte diversity and composition, suggesting that similar factors could affect Zea endophytes as well (Franić et al., 2020; Philpott et al., 2023). Overall, while host identity remains a primary driver of endophyte diversity, environmental conditions and geographic distribution also contribute to the dynamic and complex nature of seed-associated endophytes in Zea. 3 Methods for Studying Endophyte Diversity 3.1 Culturing techniques and isolation Culturing techniques are fundamental for isolating and studying endophytic bacteria from seeds. Traditional culturing methods involve the use of various growth media to promote the growth of endophytes. For instance, tailored media that mimic the nutritional composition of the seed endosphere have been shown to enhance the diversity of culturable endophytic bacteria. This approach was successfully applied to soybean seeds, revealing a higher richness of unique genera in the embryonic axis compared to cotyledons (Gerna et al., 2022). Additionally, the use of plant extracts in nutrient media has been found to optimize the isolation and cultivation of bacterial endophytes, increasing both the number and diversity of isolates (Eevers et al., 2015). Another effective method is the dilution-to-extinction technique, which has been used to isolate leaf-inhabiting endophytic fungi, demonstrating that different cultivation techniques can significantly influence biodiversity assessments (Langill et al., 2023). These culturing methods are crucial for obtaining pure cultures of endophytes, which are essential for further functional studies and biotechnological applications. 3.2 Molecular identification and genomic approaches Molecular identification and genomic approaches have revolutionized the study of endophyte diversity by allowing the detection of a broader range of microbial taxa, including those that are difficult to culture. Techniques such as next-generation sequencing (NGS) provide comprehensive insights into the phylogenetic diversity of endophytic communities. For example, NGS has been used to profile the seed endophytic and rhizospheric bacterial communities of Noccaea caerulescens, revealing a conserved community structure dominated by Proteobacteria and Actinobacteria (Langill et al., 2023). Similarly, high-throughput Illumina MiSeq
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