The plant kingdom is of great importance to human life on nutritional, therapeutic and environmental levels. The chemical composition of plants has been considered a complete pharmacy for treating many pathogens throughout history. With the development of science that accompanied the industrial revolution, the use of medicinal plants and the extraction of compounds with therapeutic effects from them has increased (Mohammed et al., 2024). The medicinal components of plants are either fatty, carbohydrate, or volatile oils that evaporate upon exposure to air. They have a distinctive aroma and various properties, some of which are insoluble as food. Most are saturated fatty acids and the majority is used for pharmaceutical purposes, primarily as preventatives and tonics against external infections (Wiart, 2019).
       
Agricultural methods have evolved and attention has shifted towards plant propagation that avoids pollution problems. Experts have sought to reconsider traditional farming systems that rely on chemical additives, aiming to protect the ecosystem from pollution and degradation and to move towards clean farming methods and the use of biofertilizers (Kadhim et al., 2020). Recently, biofertilizers have been relied upon to meet agricultural production requirements by supplying plants with nutrients and evaluating the physical, chemical and biological properties of the soil to enhance plant growth indicators. Furthermore, these organisms have been shown to play a crucial role in the natural cycle of macronutrients and their release from organic compounds. They also possess the ability to produce growth regulators and inhibit the growth of pathogens, in addition to increasing plant tolerance to environmental stresses that have become a threat to agricultural production (Ornales et al., 2025).
       
The importance of sage for medicinal and ornamental purposes and the variety of methods used to cultivate and propagate it such as seed, vegetative and tissue culture. Sage is sensitive to high temperatures and its cultivation conditions are often incompatible with the climatic realities of Iraq. Therefore, the study aimed to study the effect of temperature regimes and biofertilizers on major bioactive compounds of Salvia officinalis.

A factorial experiment was conducted in a plastic shade structure in Hilla city to study the effect of various temperature ranges, controlled by shading and the use of biofertilizers (Bacterial and fungal), on the chemical content of unsaturated fatty acids in three sage rootstocks propagated sexually, vegetatively and tissue culture.
 
Study factors
 
First/rootstock type
 
Three rootstocks of Sage plants were used in this research. The first was sexually propagated from seeds of a Spanish variety imported and sown on November 6, 2024, in Styrofoam trays (stems) filled with peat moss and covered with green plastic film. After reaching the four-true-leaf stage, they were transplanted into 30 cm diameter plastic pots filled with a 1:1 mixture of peat moss and potting soil until the beginning of March 2025, when they were moved to a greenhouse. The second type was obtained from a nursery in Baghdad and consisted of six-month-old plants propagated by cuttings. The third type was obtained from a private scientific laboratory in Baghdad and consisted of tissue-cultured plants aged between six and ten months. These plants were acclimatized and placed in pots containing a 3:1:1 mixture of peat moss, perlite and potting soil.
       
Second, the temperature range: Three temperature ranges were used in the research: partial open-field cultivation, where the sides of the shade structure were covered with plastic sheeting while the top remained uncovered throughout the experiment; semi-shaded cultivation, where the plastic sheeting was removed daily for 12 hours and then closed for the same duration throughout the experiment; and fully protected cultivation, where the plants were placed under the plastic sheeting for the entire duration of the experiment.
       
Third, the biofertilizer: Two beneficial biofertilizers were used in the research: Azotobacter bacteria, whose pure isolates were obtained from a scientific laboratory and Trichoderma fungus, which was propagated in the Bioresistance Laboratory at Al-Musayyib Technical College. The other experimental plants were left untreated as control plants.
 
Treatments and experimental design
 
A factorial experiment (3×3×3) was conducted using a complete random design with three replicates per treatment. Each treatment contained three experimental units, totaling 27 treatments per replicate, randomly distributed. Each treatment contained three pots, with one plant per pot, which served as an experimental unit within the treatment, for a total of 243 pots. Statistical analysis of the studied traits was performed using GenStat 2012 software. The results were tested across all experimental treatments by comparing the means to determine significance using the least significant difference (LSD) at a probability level of 5% (Al-Asadi, 2019).
 
Estimation of the medicinally active compounds in leaves using high-performance liquid chromatography (HPLC)
 
Extraction and separation of the active compounds: Plant leaf samples were collected on October 30, 2025, from three replicates and analyzed in the laboratories of the Ministry of Science and Technology using HPLC, according to the separation conditions shown in Table (1). 25 µmol of the oil sample was injected into the aforementioned device to qualitatively detect the fatty acids in the oil sample. This was done by matching the retention time of the fatty acid with the retention time of the standard compound in the standard curve for fatty acids (Fig 1) and Table (2) for unsaturated fatty acids in each oil sample, after calculating the concentration of each according to the following equation (Ebru et al., 2017).

 

Thugone (%)
 
Plants propagated vegetatively by cuttings yielded the highest percentage at 21.16%, while those propagated by tissue culture recorded the lowest at 16.76%. Regarding the covering treatments and temperature ranges during the experiment, plants subjected to a half-cover/uncovered system exhibited the highest thugone percentage at 20.34%, while the open-field treatment yielded the lowest at 17.88%. Biofertilizers also contributed to an increase in the fatty acid percentage. Treating the soil of the experimental plants with Trichoderma fungus resulted in the percentage at 20.13%, surpassing all other treatments, while the control plants yielded the lowest at 18.58% (Table 3).


       
The interaction of the two study factors resulted in significant differences in thugone content. The interaction of the vegetative plant culture with plants grown in semi-open fields recorded 22.52%. The content was recorded in the interaction with tissue-cultured plants grown in open fields, at 16.01%. The interaction of the plant culture with biofertilizer also had a significant difference, as shown in the same table. The interaction with vegetatively cultured plants treated with fungi resulted in the content at 21.95%, surpassing most other interaction treatments. The content was recorded in the interaction with tissue-cultured plants not treated with biofertilizer at 16.23%. The type of cultivation was combined with the addition of a biofertilizer to the soil, it had a significant effect on increasing the active ingredient. The two-way interaction (Semi-open field cultivation with the fungal biofertilizer) yielded the at 21.13%, while the lowest was obtained by plants grown in the open field without any biofertilizer treatment, at 17.22%.
 
Cineole (%)
 
The results recorded significant differences in the percentage of cineole due to variations in plant origin. Vegetative propagated plants yielded the highest average percentage at 15.42%, while tissue-cultured plants recorded the lowest percentage at 13.74%. The type of cultivation also had a significant effect on this trait. The biofertilizer increased the fatty acid content, with the treatment of the experimental plants’ soil with azotobacter bacteria yielding the increase of 15.00%, without a significant difference compared to the soil treatment with Trichoderma fungus, which recorded an average of 14.76%. The lowest decrease was observed in the control treatment, at 14.22% (Table 4).


       
The most significant decrease was recorded in the control treatment. The two-way interaction produced significant differences in the above trait. The interaction of vegetative propagation with plants subjected to the full-coverage system achieved the result of 15.89%, surpassing most other treatments in the experiment. The lowest result, 13.21%, was recorded with the interaction of tissue-cultured plants grown in the open field. The interaction of vegetative propagation with a biofertilizer also produced significant differences. The interaction between the type of cultivation and the biofertilizer had a significant effect on increasing the active ingredient. The interaction (Protected cultivation system with bacterial fertilizer) reaching 15.42%, while lowest 13.68%, was achieved by plants grown in the open field and not treated with biofertilizer.
 
Camphor (%)
 
There are significant differences were found in the percentage of camphor due to variations in plant origin. Vegetatively propagated plants yielded the highest at 11.43%, while tissue-cultured plants recorded the lowest percentage at 10.01%. The cultivation system did not significantly affect this trait. However, biofertilizers increased camphor acid levels. The interaction of vegetative plant material with plants subjected to the semi-exposed cover system achieved 12.02%, while the lowest was recorded with the interaction of tissue-cultured plants grown in the open system, at 9.77%. The interaction of plant material with biofertilizer also showed a significant response (Table 5).


       
The interaction of vegetative plants treated with bacteria recorded 11.86%, while the lowest was recorded with the interaction of tissue-cultured plants not treated with biofertilizer, at 9.36%. The interaction between the type of cultivation and the biofertilizer had a significant effect on increasing the active ingredient. The three-way interaction of experimental factors resulted in a significant increase in camphor acid. The value 12.56%, was achieved in the treatment involving this interaction (vegetatively propagated plants grown in a semi-protected system and treated with a bacterial fertilizer). In contrast, the lowest value, 8.54%, was achieved in tissue-cultured plants grown in open fields without treatment with a biofertilizer.
 
Camphene (%)
 
The results shows that the plant rootstock used in propagating sage plants significantly affected the percentage of camphene. Plants yielded recorded the highest average value 7.90%, while tissue-cultured plants recorded the lowest, at 6.12%. The significant effect of the cultivation system on the above trait continued, with the semi-open system achieving, 7.20%, while open-field plants recorded the lowest value, 6.67%. The addition of a biofertilizer to the growing soil significantly increased the camphene content. The bacterial treatment yielded 7.12%, without significantly differing from the fungal treatment, which recorded an average of 7.02%. The lowest was recorded in the control treatment, at 6.65% (Table 6).


       
The interaction of semi-open-field cultivation with bacterial and fungal fertilizers yielded 7.33% and 7.32%, respectively. Conversely, the lowest, 6.33%, was achieved by plants grown in open-field cultivation without any biofertilizer treatment. Significant differences were found in the percentage of camphene acid in the leaves of sedge plants due to the three-factor interaction. The 8.86%, was recorded with the three-factor interaction (Vegetatively propagated plants grown in semi-protected soil treated with bacterial fertilizer). Tissue-cultured plants grown in open-field cultivation without biofertilizer treatment achieved the lowest 5.86%.
       
This allowed for the study of their effect on the synthesis and formation of secondary metabolites under varying climatic conditions. The significant superiority of plants propagated vegetatively by cuttings may be due to this method being the optimal propagation method for sage plants. Bagdat et al., (2017) stated that the common propagation method for sage plants is the use of various types of hardwood cuttings, which can produce plants with good growth and high resistance to the various climatic conditions and fluctuations they encounter during their life cycle.
       
The significant differences caused by protected and semi-protected farming systems compared to open-field farming, this may be explained by the fact that plants grown under protected or semi-protected systems were provided with optimal growth conditions, especially the extreme temperatures experienced during the growing season from summer to winter. Since sedges thrive in cold environments, extreme temperatures can be a hindering factor in plant growth and the quality of secondary metabolite formation, including unsaturated fatty acids (Calvo et al., 2014; El-Shanhorey and Sorour, 2019). In contrast, open-field farming exposed plants directly to environmental and climatic variations, including temperature, light and humidity, which can directly affect vital and physiological processes, thus weakening the plant’s resilience, immunity and growth response to the temperature fluctuations observed during the research period (Hosseinzadeh et al., 2021).
       
It plays a role in the formation of bacterial nodules and has the ability to dissolve many poorly soluble nutrients, such as zinc, iron and copper, due to the presence of biochemical compounds produced in large quantities that are responsible for soluble elements (Abena, 2023). This was confirmed by Karpi and Tewari (2010), who found that Trichoderma spp. fungal isolates are efficient at soluble phosphate and increase phosphorus availability through the production of the enzyme phosphatase. Pandya and Saraf (2010) also confirmed that the biofertilizer Trichoderma spp. has the ability to process phosphorus and some micronutrients.
       
Aruna et al., (2025) concluded that the metabolites produced by Trichoderma spp. It is responsible for the chelation of iron, increasing its reduction and converting it into a form readily available for absorption. It also plays a role in the breakdown of chlorinated organic compounds and contributes to increased production of the hormone IAA by oxidizing the amino acid tryptophan secreted by the roots. This, in turn, contributes to an increase in root hairs. This fungus also enhances plant resistance by secreting three types of resistance-inducing compounds: enzymes, protein analogs and oligosaccharides, along with other low molecular weight compounds (Al-Juheishy, 2025). The reason may also be attributed to the combined positive effect of the study’s factors, which were largely linked to increased biological activity of the microorganisms added to the potting soil. These microorganisms, in conjunction with the root system of the experimental plants, positively contributed to improved growth parameters (Vasileva et al., 2022; Botey et al., 2022; Alarkwazi, 2026).

The study concludes that the study factors have a significant impact on improving the study indicators. The study found that the vegetative rootstock of sage propagated by cuttings had the greatest effect on growth and the formation of secondary metabolites, especially when cultivated under protected and semi-protected farming systems. Furthermore, the addition of the fungus Trichoderma spp. and the bacteria Azotobacter spp. was of paramount importance in improving the study indicators. The interaction between all of the above (vegetative rootstock, protected farming system and the addition of bacteria and fungi) yielded the best results for the studied traits under the experimental conditions, thus offering the possibility of replacing the use of chemical fertilizers with their harmful environmental impact.

All authors declare that there is no conflict of interest of this article.