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The growth and biological decline of alfalfa may be linked to the rhizosphere microbiome. However, plant–microbe interactions in the rhizosphere of alfalfa and associated microbial community variations with stand age remain elusive. This study explored the successional pattern of rhizosphere microbial communities across different aged alfalfa stands and its relationship with alfalfa decline. Rhizosphere soils were collected from 2- and 6-year-old alfalfa stands. Control soils were collected from interspaces between alfalfa plants in the same stands. Soil bacterial and fungal communities were characterized by 16S and ITS rRNA gene sequencing, respectively. Specific microbial taxa colonized the rhizosphere soils, but not the control soils. The rhizosphere-specific taxa mainly included potentially beneficial genera (e.g., Dechloromonas, Verrucomicrobium) in the young stand and harmful genera (e.g., Peziza, Campylocarpon) in the old stand. Alfalfa roots regulated soil microbial communities by selective promotion or inhibition of distinct taxa. The majority of time-enriched taxa were reported as harmful fungi, whose relative abundances were negatively correlated with plant traits. Time-depleted taxa were mostly known as beneficial bacteria, which had relative abundances positively correlated with plant traits. The relative abundances of functional bacterial genes associated with vancomycin biosynthesis, zeatin biosynthesis, and amino acid metabolism trended lower in rhizosphere soils from the old stand. An upward trend was observed for fungal pathogens and wood saprotrophs with increasing stand age. The results suggest that root activity drives the negative succession of rhizosphere microbial communities during alfalfa decline in old stands.

Alfalfa (Medicago sativa L.) is an important forage crop, and its productivity is severely affected by salt stress. Although proline is a compatible osmolyte that plays an important role in regulating plant abiotic stress resistance, the basic mechanism of proline requires further clarification regarding the effect of proline in mitigating the harmful effects of salinity. Here, we investigate the protective effects and regulatory mechanisms of proline on salt tolerance of alfalfa. The results show that exogenous proline obviously promotes seed germination and seedling growth of salt-stressed alfalfa. Salt stress results in stunted plant growth, while proline application alleviates this phenomenon by increasing photosynthetic capacity and antioxidant enzyme activities and decreasing cell membrane damage and reactive oxygen species (ROS) accumulation. Plants with proline treatment maintain a better K+/Na+ ratio by reducing Na+ accumulation and increasing K+ content under salt stress. Additionally, proline induces the expression of genes related to antioxidant biosynthesis (Cu/Zn-SOD and APX) and ion homeostasis (SOS1, HKT1, and NHX1) under salt stress conditions. Proline metabolism is mainly regulated by ornithine-δ-aminotransferase (OAT) and proline dehydrogenase (ProDH) activities and their transcription levels, with the proline-treated plants displaying an increase in proline content under salt stress. In addition, OAT activity in the ornithine (Orn) pathway rather than Δ1-pyrroline-5-carboxylate synthetase (P5CS) activity in the glutamate (Glu) pathway is strongly increased under salt stress, made evident by the sharp increase in the expression level of the OAT gene compared to P5CS1 and P5CS2. Our study provides new insight into how exogenous proline improves salt tolerance in plants and that it might be used as a significant practical strategy for cultivating salt-tolerant alfalfa.