Mixed seeding with sheep manure amendment alters soil nematode network complexity in a sandy grassland

Soil nematodes are key bioindicators of terrestrial ecosystem functions, yet how restoration practices affect their community and interactions remains unclear.

A one-year field experiment in an alpine grassland tested three treatments: bare sandy soil, mixed seeding of Avena sativa L. and Festuca arundinacea, and mixed seeding with sheep manure amendment at 1.75 kg m^-². Notably, the experimental design did not include a sheep-manure-only treatment; therefore, the specific contribution of manure independent of vegetation reconstruction cannot be disentangled.

High-throughput sequencing and co-occurrence network analysis revealed that mixed seeding with manure significantly enhanced plant biomass, soil organic carbon, electrical conductivity, and nematode richness (Chao1 index) by 26.36-31.58% (p < 0.05). Mixed seeding alone increased the relative abundance of predator/omnivore nematodes by 17.46%, whereas mixed seeding with sheep manure shifted the community toward opportunistic bacterivores such as Acrobeloides and reduced predators/omnivores by 15.74% (p < 0.05). Available nitrogen showed the strongest marginal association with community composition (Monte Carlo test, p = 0.065). Network analysis indicated that mixed seeding promoted the key genus Aporcelaimellus, while adding manure shifted the network architecture toward bacterivores dominance.

Both the mixed seeding treatment and the mixed seeding combined with manure treatment enhanced network complexity but increased competition over cooperation, reflecting altered interaction patterns. Whether this structural complexity translates into functional stability requires long-term monitoring. In conclusion, mixed seeding alone fosters a more structured soil food web with higher trophic-level representation, whereas the combination of mixed seeding with organic amendment-though boosting productivity and richness-induces salinization risks (elevated EC) and functional simplification toward bacterivore-dominated channels. The absence of a manure-only control limits our ability to attribute these effects solely to organic fertilization; future studies should include this treatment to isolate independent and interactive effects.