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This study aims to investigate how co-inoculation with arbuscular mycorrhizal fungus Rhizophagus irregularis and Trichoderma harzianum can enhance licorice drought tolerance and secondary metabolite production, providing insights for sustainable agriculture in arid regions. The results demonstrate that inoculation with R. irregularis significantly improved biomass, drought stress tolerance, and increased glycyrrhizin and liquiritin concentrations by 29.9% and 3.3-fold, respectively, particularly under drought conditions. Co-inoculation with T. harzianum further boosted glycyrrhizin yield by 93.7%, indicating a synergistic relationship between the two microbes. The expression of key biosynthetic genes, including squalene synthase (SQS1) for glycyrrhizin and chalcone synthase (CHS) for liquiritin, was significantly upregulated, enhancing water use efficiency and the biosynthesis of secondary metabolites. Nutrient analysis showed improved phosphorus uptake, alongside reduced root carbon and nitrogen concentrations, leading to greater nutrient utilization efficiency. These findings suggest that co-inoculating R. irregularis and T. harzianum is a promising approach to improving licorice growth and medicinal quality under drought stress, with broad applications for sustainable crop management.<\/jats:p>","DOI":"10.3390\/jof11070488","type":"journal-article","created":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T13:06:17Z","timestamp":1751288777000},"page":"488","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Synergistic Effects of Rhizophagus irregularis and Trichoderma harzianum Co-Inoculation on Enhancing Drought Tolerance and Secondary Metabolite Production in Licorice (Glycyrrhiza uralensis)"],"prefix":"10.3390","volume":"11","author":[{"given":"Kangxu","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Regional and Urban Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengyao","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Regional and Urban Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China"},{"name":"School of Earth Sciences and Resources, China University of Geosciences (Beijing), Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiyan","family":"Feng","sequence":"additional","affiliation":[{"name":"School of Earth Sciences and Resources, China University of Geosciences (Beijing), Beijing 100083, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xia","family":"Wei","sequence":"additional","affiliation":[{"name":"Foshan IronMan Environmental Technology Co., Ltd., Foshan 528051, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Xie","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Regional and Urban Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China"},{"name":"China CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Fu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Regional and Urban Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lanping","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Science, Beijing 100700, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8504-5213","authenticated-orcid":false,"given":"Xin","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Regional and Urban Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1211-596X","authenticated-orcid":false,"given":"Zhipeng","family":"Hao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Regional and Urban Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1790-7800","authenticated-orcid":false,"given":"Baodong","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Regional and Urban Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100049, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"931","DOI":"10.3389\/fpls.2017.00931","article-title":"Glomus mosseae inoculation improves the root system architecture, photosynthetic efficiency and flavonoids accumulation of liquorice under nutrient stress","volume":"8","author":"Chen","year":"2017","journal-title":"Front. 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