Mentha longifolia L. (horsemint) is one of the most famous wild perennial herbs of the Lamiaceae family, which has broad usage in therapeutic and pharmaceutical services. In this research, screening of twenty Mentha longifolia L. accessions was assessed according to the physio-chemical variability under low water irrigation. The investigation was a split plot arranged in a completed randomized block design in three replications with two water treatments of full irrigation (100% field capacity as control) and deficit irrigation (60% FC). The interaction effects of water deficit and accessions indicated that drought stress meaningfully (P ≤ 0.01) altered physiological parameters such as CGR, NAR, RGR, LAR, SLA electrolyte leakage, element content, stomatal conductivity, essential oil content, antioxidant activities, proline, relative water content, chlorophyll, and carotenoid content. The main volatile components as 1,8-cineole, (Z)-β-ocimene, menthon, menthol, pulegone, trans-piperitone epoxide, piperitenone, and piperitone oxide were analyzed. The HPLC analysis disclosed the existence of rosmarinic acid, caffecic acid, 2,3-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, ferulic acid, chlorogenic acid, vanillic acid, p-coumaric acid, and rutin as predominant phenolic and flavonoid compounds in horsemint accessions. Results showed that the drought stress not only enhanced the values of rosmarinic acid, ferulic acid, caffeic acid, rutin, 2,3-dihydroxybenzoic acid, and 3,4-dihydroxybenzoic acid but also triggered p-coumaric acid that did not exist in some accessions under full irrigation conditions. However, the findings were affected by the accession type. Cluster analysis based on all the traits categorized the accessions into two main groups, in which the comparisons of the dendrograms between normal and water deficit conditions showed 0.23 entanglement. The analysis showed that ML13, ML17, and ML2 accessions could be offered as tolerant accessions. Regarding the various reactions to drought stress and the variability among the accessions,the results of the current research could be a primary stage in recognizing and obtaining high-yield, drought-tolerance, and elite accessions and can promote horsemint breeding programs and domestication.

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