Effects of endophytic fungus Piriformospora indica on cold tolerance in basil (Ocimum basilicum L.): insight into the expression of cold-responsive genes

Cold stress severely limits the cultivation of chilling-sensitive basil ( Ocimum basilicum L.). This study investigated the potential of the endophytic fungus Piriformospora indica to improve cold tolerance in two Iranian landraces, Tehran (cold-tolerant) and Khash (cold-sensitive). Plants were inoculated with P. indica and subjected to cold stress (3 °C for 18 h). Col​d stress reduced growth traits in non-inoculated plants (all traits in Tehran; shoot height, shoot fresh weight, and root length in Khash). Under cold stress, inoculation restored shoot height to the level of non-stressed plants in both landraces and was associated with higher root length in both landraces and with higher shoot and root fresh and dry weights in Tehran. Inoculation reduced chilling injury (CI) scores from 1.0 to 0 in Tehran and from 3.5 to 0.33 in Khash. Under the same conditions, inoculation also reduced electrolyte leakage (EL) and malondialdehyde (MDA) in both landraces, with larger absolute reductions in Khash, and improved relative water content (RWC) in Khash only, whereas hydrogen peroxide (H₂O₂) was not significantly reduced. Soluble carbohydrates, soluble proteins, phenolics, and DPPH radical scavenging activity were higher in inoculated plants of both landraces, and flavonoids in Khash only. Compared with non-inoculated stressed plants, inoculation increased the activities of catalase (CAT), guaiacol peroxidase (GPX), and superoxide dismutase (SOD) in Tehran, and of CAT, ascorbate peroxidase (APX), and SOD in Khash, whereas APX in Tehran and GPX in Khash did not change significantly; the increases in APX and SOD were largest in Khash. Quantitative real-time PCR showed that, under cold stress, inoculated plants had higher expression of CBF1 (9.48- and 9.54-fold in Tehran and Khash, respectively), CBF3 (19.79- and 9.17-fold), and COR47 (4.64- and 15.90-fold) relative to non-inoculated, non-stressed plants. This research indicates that P. indica inoculation improved cold tolerance in basil, which was associated with higher expression of the cold-responsive genes CBF1 , CBF3 , and COR47 , together with higher antioxidant activity and osmoprotectant accumulation. These findings suggest the potential of P. indica as a biostimulant to improve basil performance under chilling conditions.