Comparison of Rhizosphere Soil Microbial Community Structure and Function among Diploid, Triploid and Tetraploid Watermelons

(1) Background: The rhizosphere microbiome, as the “second genome” of plants, plays a crucial role in nutrient acquisition, stress resistance, and soil health maintenance. Polyploidization is a key breeding strategy for watermelon: triploids are valued for seedlessness and high stress resistance, while tetraploids serve as important parental materials for triploid hybridization. However, how ploidy level (diploid, triploid, tetraploid) shapes the micro-ecological environment and microbial community assembly remains unclear. This study aims to elucidate the link between ploidy and rhizosphere microbiota, providing a theoretical basis for optimizing breeding and cultivation practices. (2) Methods: High-throughput sequencing (Illumina MiSeq PE300 for bacteria, PE250 for fungi) targeting the bacterial 16S rRNA gene (primers 338F/806R) and fungal ITS region (primers ITS1F/ITS2R) was used to analyze rhizosphere microbial communities of diploid (HLL40-3), triploid (Guixi No.5), and tetraploid (MT410n-1) watermelons. Alpha diversity (Shannon/Chao1/ACE/Simpson), beta diversity (PCoA/PLS-DA), taxonomic composition (phylum/genus level), and functional prediction (PICRUSt2/Tax4Fun/FUNGuild) were performed to compare differences among groups. (3) Results: Triploid watermelons exhibited significantly higher bacterial and fungal alpha diversity (Shannon/Chao1) than diploid and tetraploid varieties. Beta diversity analysis confirmed distinct separation of microbial communities across ploidy levels ( p  < 0.05). Taxonomically, diploid rhizospheres were enriched in Proteobacteria, Melanconiella, and Hypomyces ; triploids were enriched in Bacteroidota, Microvirga , Mortierellomycota, Cercophora , and Neocosmospora ; tetraploids were specifically enriched in Patescibacteria , Gaiella , Trichoderma , and Papiliotrema . Functional prediction showed diploids were dominated by plant/wood saprotrophs; triploids harbored diverse functional guilds (animal pathogens, endophytes, soil saprotrophs, etc.); tetraploids had the highest relative abundance of undefined saprotrophs. (4) Conclusions: Ploidy level is a key determinant of watermelon rhizosphere microbial structure and function. Triploid watermelons assemble a more diverse and functionally versatile microbial community through selective recruitment, which may contribute to their superior agronomic traits. These findings deepen our standing of plant-microbe interactions and provide new insights for integrating polyploid breeding with rhizospheric microecological regulation in sustainable watermelon production.