| Summary: | Endophytes have the ability to improve plant nutrition alongside their agronomic performance, among which arbuscular mycorrhizal fungi provide the most benefits to their host. Previously, we reported for the first time that an arbuscular mycorrhizal-like fungus <i>Piriformospora indica</i> had the ability to colonize roots of trifoliate orange (<i>Poncirus trifoliata</i>) and conferred positive effects on nutrient acquisition. Present study showed the changes in fatty acids and sugars to unravel the physiological and symbiotic association of trifoliate orange with <i>P</i>. <i>indica</i> and an arbuscular mycorrhizal fungus, <i>Funneliformis mosseae</i> singly or in combination. All the endophytic fungi collectively increased fructose, glucose, and sucrose content in leaves and roots, along with a relatively higher increase with <i>P</i>. <i>indica</i> inoculation than with <i>F</i>. <i>mosseae</i> alone or dual inoculation. Treatment with <i>P</i>. <i>indica</i> increased the concentration of part unsaturated fatty acids such as C18:3N6, C20:2, C20:3N6, C20:4N6, C20:3N3, C20:5N3, C22:1N9, and C24:1. Additionally, <i>P</i>. <i>indica</i> induced the increase in the concentration of part saturated fatty acids such as C6:0, C8:0, C13:0, C14:0, and C24:0. <i>F</i>. <i>mosseae</i> hardly changed the content of fatty acids, except for increase in C14:0 and C20:5N3. Double inoculation only reduced the C21:0, C10:0, C12:0, C18:3N3, and C18:1 content and increased the C20:5N3 content. These endophytic fungi up-regulated the root <i>PtFAD2, PtFAD6, PtΔ9</i>, and <i>PtΔ15</i> gene expression level, coupled with a higher expression of <i>PtFAD2</i> and <i>PtΔ9</i> by <i>P</i>. <i>indica</i> than by <i>F</i>. <i>mosseae</i>. It was concluded that <i>P</i>. <i>indica</i> exhibited a stronger response, for sugars and fatty acids, than <i>F</i>. <i>mosseae</i> on trifoliate orange. Such results also reveal the <i>Pi</i> (an <i>in vitro</i> culturable fungus) as a bio-stimulator applying to citriculture.
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