Rhizobium is well-documented for its symbiotic relationship with legume plants, where it plays a crucial role in biological nitrogen-(N)-fixation within their root nodules. However, the isolation, identification, and association of Rhizobium as a free-living diazotroph with potato plants remain relatively less explored. The present study reports the isolation and characterization of free-living Rhizobium strain from the rhizosphere of potato plants and its potential for promoting growth and N-fixation. Diazotrophic strain (TN04) was isolated from rhizosphere of potato plants on nitrogen-free media and identified on the basis of 16S rRNA gene sequence (Accession number: LN833444). TN04 strain also contained nifH gene and showed N-fixation potential (151.70 nmolmg/protein/h) through ARA activity, indicating its ability to fix atmospheric nitrogen. TN04 exhibited potential for phosphate solubilization (272.5 µg/mL) and produced indole acetic acid at concentration of 3.50 µg/mL. To assess the N-fixing ability of TN04 diazotroph, a ¹⁵N dilution experiment was conducted in pots using sterilized sand and sterilized soil under various fertilizer doses. The results of pot experiments demonstrated significant improvement in N content and growth parameters of inoculated potato plants compared to un-inoculated controls, suggesting that diazotrophic strain effectively fixed atmospheric N through isotopic dilution. Moreover, Rhizobium sp. TN04 remarkably improved plant growth and agronomic parameters under field conditions. Significant improvements were observed in N uptake, N utilization, and N use efficiency in field trails. In addition, microscopic analysis using transmission electron and confocal laser scanning microscopy provided insights into the colonization patterns of TN04 strain at the junctions between the secondary and primary roots, forming strong association with potato roots. Our study presents novel insights into the presence and interaction of Rhizobium with non-host plants, shedding light on its N-fixing capabilities in non-leguminous crops. These findings pave the way for developing strategies to explore microbiome of non-leguminous crops and exploit the N-fixation of Rhizobium in non-host crops.

根瘤菌与豆科植物的共生关系已有充分记录，在根瘤内的生物固氮（N）中发挥着至关重要的作用。然而，根瘤菌作为一种自由生活的固氮菌与马铃薯植物的分离、鉴定和关联仍然相对较少被探索。本研究报告了从马铃薯植物根际分离和鉴定自由生活的根瘤菌菌株及其促进生长和固氮的潜力。固氮菌株（TN04）是在无氮培养基上从马铃薯植株根际分离出来的，并根据16S rRNA基因序列进行鉴定（登录号：LN833444）。 TN04菌株还含有nif H基因，并通过ARA活性表现出固氮潜力（151.70 nmolmg/蛋白质/h），表明其固定大气氮的能力。 TN04 表现出磷酸盐溶解潜力 (272.5 µg/mL)，并产生浓度为 3.50 µg/mL 的吲哚乙酸。为了评估TN04固氮菌的固氮能力，在盆中使用灭菌沙和灭菌土壤在不同肥料剂量下进行了¹⁵ N稀释实验。盆栽实验的结果表明，与未接种的对照相比，接种的马铃薯植株的氮含量和生长参数显着改善，这表明固氮菌株通过同位素稀释有效地固定了大气中的氮。此外，根瘤菌sp。 TN04 显着改善了田间条件下的植物生长和农艺参数。田间试验观察到氮吸收、氮利用率和氮利用效率显着改善。此外，使用透射电子和共焦激光扫描显微镜进行显微分析，深入了解TN04菌株在次生根和初生根之间的交界处的定植模式，与马铃薯根形成紧密的关联。我们的研究对根瘤菌的存在及其与非寄主植物的相互作用提出了新的见解，揭示了其在非豆科作物中的固氮能力。这些发现为制定探索非豆科作物微生物组和利用非寄主作物中根瘤菌固氮作用的策略铺平了道路。