Impact of Seed Maturation on the Morphology, Nutrition, Microbiome Composition and Germinability of Subterranean Clover (Trifolium subterraneum) Seeds

The annual pasture legume subterranean clover (Trifolium subterraneum L.; sub clover) plays a crucial role in the agricultural systems of southern Australia and other regions with Mediterranean climates by enhancing pasture quality, nitrogen fixation and biodiversity, contributing to sustainable farming. Sub clover, uniquely characterised by its below-ground seed development in burrs, has been insufficiently studied regarding the effects of seed maturation on key physiological factors, despite its agricultural significance. This study aimed to explore the effects of seed maturation on morphology, nutrition, microbiome community composition, and germinability in sub clover seeds. Three cultivars, one each from the three sub clover subspecies, were grown in a paddock using a randomised block design. Seed-bearing burrs were collected at defined developmental and maturity stages, as determined by microscopic analysis and measurements of seed fresh weight and length. Seed nutritional composition was assessed using a macro elemental analyser. Seed microbiome community composition was characterised by sequencing the bacterial 16S rRNA gene and the fungal ITS2 region. Seed germinability was evaluated based on radicle emergence and length following 14 days of incubation. Across all cultivars, seed maturation was characterised by progressive increases in seed length and weight, accompanied by dynamic changes in nutrient composition. While some nutrients showed consistent declines, others exhibited increasing trends as seeds matured. The seed microbiome community compositions were influenced by both developmental stage and cultivar type. Seed germinability decreased from 37 to 43 days post-anthesis, as hardseededness increased. This study provides insights into the dynamic interplay of morphological traits, nutritional composition, microbial community composition, and germinability during the maturation of sub clover seeds.