Genetic Diversity and Growth-Promoting Functions of Endophytic Nitrogen-Fixing Bacteria in Apple

Understanding the dominant populations and biological functions of endophytic nitrogen-fixing bacteria in apple plants is of great significance for the healthy growth management and sustainable development of apple cultivation. In this study, we investigated the community diversity and potential plant growth-promoting abilities of endophytic nitrogen-fixing bacteria in different tissues of apple trees by combining high-throughput sequencing of the <i>nifH</i> gene with traditional isolation and cultivation techniques. Sequencing results revealed that the endophytic bacteria were affiliated with 10 phyla, 14 classes, 30 orders, 42 families, and 72 genera. <i>Rhizobium</i> was the dominant genus in the roots and twigs, while <i>Desulfovibrio</i> dominated the leaf tissues. The diversity and richness of endophytic bacteria in the roots were significantly higher than those in the leaves. Using four types of nitrogen-free media, a total of 138 presumptive endophytic nitrogen-fixing bacterial strains were isolated from roots, leaves, and twigs. These isolates belonged to 32 taxonomic groups spanning 5 phyla, 8 classes, 11 orders, 13 families, and 18 genera. The <i>nifH</i> gene was successfully amplified from the representative strains of all 32 groups using specific primers. Nitrogenase activity among the isolates ranged from 26.86 to 982.28 nmol/(h·mL). Some strains also exhibited the ability to secrete indole-3-acetic acid (IAA), solubilize phosphate and potassium, and produce siderophores. Six individual strains and three microbial consortia were tested for their plant growth-promoting effects on apple tissue culture seedlings. All treatments showed growth-promoting effects to varying degrees, with the RD01+RC16 consortium showing the most significant results: plant height, number of leaves, and chlorophyll content were 2.4, 3.3, and 4.2 times higher than those of the control, respectively. These findings demonstrate the rich diversity of endophytic nitrogen-fixing bacteria in apple plants and their promising potential for application in promoting host plant growth.