Abstract:

[Objective] Biological nitrogen fixation (BNF), mediated by diazotrophic microorganisms via the nitrogenase enzyme complex, represents a critical natural source of reactive nitrogen in terrestrial ecosystems and thus underpins vegetation productivity and long-term ecosystem nutrient balance. In arid environments, where soil fertility is inherently low and nitrogen availability frequently limits plant growth, understanding the ecology of soil diazotrophs is of particular importance. Among nitrogenase structural genes, nifH encodes the iron protein subunit and is widely used as a molecular marker for studying diazotrophic abundance and diversity due to its functional conservation and broad phylogenetic distribution. Increasing evidence has demonstrated that non-symbiotic nitrogen fixation—performed by free-living or associative diazotrophs—constitutes a substantial component of ecosystem nitrogen input, suggesting that changes in environmental conditions or land use may directly influence nitrogen fixation potential by altering diazotrophic communities. In northwestern China, oases serve as major socio-economic centers. However, rapid population growth and increasing agricultural demand have driven extensive land conversion from native desert to irrigated cropland. Reclamation practices—typically involving tillage, irrigation, and fertilization—modify soil moisture regimes, nutrient status, carbon availability, salinity, and microhabitats, thereby exerting strong effects on soil microbial communities. Previous studies have shown that cultivation enhances soil organic matter and nutrient accumulation, while long-term fertilization can restructure bacterial community networks. Nevertheless, despite the recognized importance of free-living nitrogen fixation in arid ecosystems, the combined impacts of desert reclamation and prolonged fertilization on nifH-harboring diazotrophs remain poorly understood, particularly in grey desert soils (Calci-Orthic Aridosols) distributed across oasis-desert ecotones.[Methods] In this context, the present study investigated how land reclamation and long-term fertilization influence soil physicochemical properties and the diversity and composition of nifH-bearing diazotrophic communities in a representative grey desert soil. The research was conducted at the Fukang Station of Desert Ecosystem Observation and Research, where a long-term fertilization experiment has been maintained since 1990. Nine fertilization treatments were examined: an unfertilized control (CK); chemical fertilization treatments with different combinations of nitrogen (N), phosphorus (P), and potassium (K) (PK, NK, NP, NPK, N2P2, N2P2K); and integrated organic-inorganic treatments (NPKR with wheat straw returning and NPKM with farmyard manure). Adjacent native desert soils under three vegetation types—bare land, herbaceous cover, and shrub cover—served as reference baselines representing natural desert conditions. Soil samples were collected from 0-20 cm depth in June 2020 following winter wheat harvest. Soil physicochemical properties, including pH, electrical conductivity (EC), organic carbon (SOC), total nitrogen (TN), and total phosphorus (TP), were quantified using standard protocols. Soil DNA was extracted, nifH fragments were PCR-amplified, sequenced, and processed through a bioinformatic pipeline for quality control, OTU clustering (97% similarity), taxonomic annotation, and alpha diversity estimation (Shannon and Chao1 indices). Redundancy Analysis (RDA) and distance-based Redundancy Analysis (db-RDA) were employed to intuitively illustrate the relationships between nifH-harboring diazotrophic communities and environmental factors, while simultaneously accounting for non-linearity issues in the data. [Results] The results revealed that reclamation and fertilization markedly altered soil edaphic properties. Compared with native desert soils, cultivated soils exhibited substantially lower EC, reflecting reduced salinity due to irrigation and drainage. SOC and TN increased significantly under fertilization treatments, with particularly strong enhancement under N2P2K and NPKM. Soil pH showed treatment-specific changes, with chemical fertilization generally acidifying soils relative to desert baselines, while organic amendments promoted SOC accumulation and moderated pH changes. These findings collectively indicate that agricultural management improves soil quality by increasing nutrient availability, enhancing carbon inputs, and alleviating salinity stress. High-throughput sequencing showed that nifH-harboring diazotrophs in grey desert soils were dominated by taxa belonging to Proteobacteria, Cyanobacteria, and Actinobacteria. Native desert soils were characterized by high relative abundance of Alphaproteobacteria, particularly the genus Azospirillum, which dominated the diazotrophic community. Following cultivation, substantial community shifts occurred. The relative abundance of Proteobacteria declined significantly across all cultivated treatments, with the sharpest reductions in NPKR and NPKM, while Cyanobacteria increased markedly—especially under P-containing fertilization regimes (NP, NPK, N2P2, N2P2K). At the genus level, Azospirillum dominance decreased sharply after reclamation, whereas Azotobacter, Azoarcus, Klebsiella, Leptolyngbya, and Nostoc increased significantly under fertilized conditions. These changes suggest ecological restructuring driven by increased resource availability, reduced salinity, and altered soil microhabitats. Alpha diversity analysis showed that reclamation and fertilization significantly enhanced diazotrophic Shannon diversity, although Chao1 richness remained relatively unchanged across treatments, indicating increased community evenness rather than expansion of taxonomic richness. Beta diversity analyses revealed distinct clustering patterns among desert, unfertilized, and fertilized soils, with CK positioned between desert and fertilized groups. Notably, shrub-covered desert soils exhibited microbial compositions closer to fertilized soils than to bare or herbaceous soils, suggesting that natural shrub-soil interactions may partially mimic the effects of cultivation on microbial nitrogen fixation. RDA identified EC and SOC as the primary environmental drivers shaping diazotrophic community composition, followed by pH, TN, and TP. Fertilized soils were associated with higher SOC and nutrient levels, whereas desert soils clustered with higher pH and EC. These relationships highlight mechanistic pathways through which NP fertilization and organic inputs enhance SOC storage, relieve salinity constraints, and create favorable niches for nitrogen-fixing microorganisms. Given that nitrogenase activity is energetically demanding and highly sensitive to osmotic stress, the combined effects of carbon enrichment and salinity reduction are likely central to explaining the observed shifts in diazotrophic assemblages. [Conclusion] Collectively, this study demonstrates that desert reclamation and long-term fertilization substantially modify soil diazotrophic communities in grey desert soils. Long-term fertilization, particularly treatments combining N, P, and organic amendments, improved soil quality, enhanced diazotrophic diversity, and increased the potential for microbial nitrogen fixation. The shift from oligotrophic Alphaproteobacteria dominance toward more diverse Cyanobacteria- and heterotroph-enriched assemblages illustrates the sensitivity of diazotrophs to land-use change and nutrient inputs. These findings provide novel insights into microbial nitrogen cycling in desert-oasis transition zones and offer theoretical support for optimizing fertilization strategies to promote soil fertility and sustainable oasis agriculture.

Key words: gray desert soil, desert reclamation, long-term fertilization, nitrogen-fixing microorganisms, nifH

摘要：

【目的】旨在研究灰漠土开垦与长期施肥模式下土壤化学性质和固氮微生物nifH基因群落多样性的响应特征。【方法】选择中国科学院阜康荒漠生态站长期施肥试验地（自1990年至今）的9个处理：不施肥（CK）、化肥（PK、NK、NP、N2P2、NPK、N2P2K）、化肥配施有机肥（NPKR（R：150kg秸秆）、NPKM（M：农家肥）），与同一毗邻荒漠（包含裸地、草本与灌木覆被），探究荒漠开垦过程中土壤固氮微生物nifH基因群落的多样性变化。【结果】与荒漠土壤相比，开垦后的所有处理中，土壤变形菌门的相对丰度显著减少，尤其是α变形菌纲，CK、NPKR和NPKM处理的降幅最大（79.40%－81.93%）。蓝细菌门相对丰度显著增加，NP、N2P2、NPK、N2P2K和NPKR增幅最大（233.18%－354.81%）。开垦与长期施肥显著增加了土壤固氮微生物nifH基因群落多样性指数（Shannon）；土壤电导率和有机碳是固氮微生物nifH基因群落分异的最大影响因子。【结论】荒漠开垦和长期施肥显著增加了nifH基因多样性，开垦与施肥后的固氮微生物nifH基因群落与荒漠中裸地、草本覆被具有最大微生物群落分异，与灌木覆被的微生物群落结构相近。氮磷肥施用对于加强土壤有机碳固持、提升固氮微生物nifH基因群落多样性及其贡献率具有正向作用。

关键词: 灰漠土, 荒漠开垦, 长期施肥, 固氮微生物, nifH