Arbuscular mycorrhizal fungi and intercropping Vicia villosa mediate plant biomass, soil properties, and rhizosphere metabolite profiles of walnuts

Intercropping is a prevalent soil management strategy within orchards, whereas it is unclear how inoculation with arbuscular mycorrhizal (AM) fungi and intercropping affect tree growth, soil properties, and rhizosphere metabolite profiles. This study investigated the effects of inoculation with Diversispora spurca and intercropping with hairy vetch (Vicia villosa) on biomass production, soil available nutrients, water-stable aggregate (WSA) distribution, phosphatase activity, and secondary metabolite profiles in walnuts (Juglans regia). The intercropping only elevated soil nitrate N levels and WSA distribution at the 0.5–2 mm size, and also triggered 2159 differential metabolites (1378 up-regulated and 781 down-regulated), with armillaramide as the most prominently up-regulated metabolite, followed by the substance diminished upon D. spurca inoculation. Conversely, D. spurca inoculation increased walnut biomass, WSA distribution across the 0.25 − 2 mm size, and acid and neutral phosphatase activities, as well as triggered 2489 differential metabolites (897 up-regulated and 1592 down-regulated), with pteroside D being highest up-regulated differential metabolite, allowing a competitive advantage to AM plants in combating soil pathogens. Despite significantly suppressing root AM fungal colonization and biomass production in AM walnuts, intercropping significantly increased soil ammonium and nitrate N levels in AM walnuts as well as WSAs at the 1–4 mm size, exhibiting a synergistic effect. Flavone and flavonol biosynthesis and pyruvate metabolism were simultaneously involved following AM inoculation or intercropping. Co-application of AM inoculation and intercropping triggered 1006 differential metabolites, with urocanic acid being the most up-regulated metabolite, although it decreased following AM inoculation, suggesting the involvement of mycorrhizal hyphae in soil histidine uptake. Under intercropping, AM inoculation elicited 418 differential metabolites, with the most up-regulated metabolite being implicated in flavonoid pathways. AM inoculation primarily triggered the biosynthesis of unsaturated fatty acids, regardless of intercropping or not, implying a potential increase in unsaturated fatty acid contents of walnut kernels. It concluded that AM inoculation and intercropping interactively affected walnut growth, soil attributes, and soil microenvironment.