All the determined chemical parameters of the PA and SL increased as their concentrations, increased except for pH, which showed a slight decline in PA and a slight increase in SL (Table 1 and Table 2). The pH levels had a significant (p < 0.001) decrease with higher concentrations of PA liquid. Salinity increased significantly (p < 0.001) with increasing concentrations of PA liquid and SL liquid. The electrical conductivity (EC) of 2% liquid showed significant (p < 0.001) differences from other concentrations, particularly for SL liquid.

The nitrogen content (N, HNO₃, NO₂⁻) of PA liquid was markedly higher than that of SL liquid (Table 3). However, SL liquid contained higher levels of Mg, Ca, K, Mn, Fe, and B than PA liquid. The content of cobalt (Co), molybdenum (Mo), selenium (Se), and Barium (Ba) were present at levels below the reported limits in both liquids. This composition of PA is consistent with the findings of previous studies [31]. These differences in chemical properties can be attributed to the feedstock and pyrolysis temperature [9].

Germination occurred in all treatments after the pouches were incubated in a dark environment for two days, except for the 1% and 2% PA-treated seeds, which did not germinate. PA had a significant (p ≤ 0.05) inhibitory effect on the germination of kale seeds, especially at 1% and 2% PA. The germination rate of kale seeds showed a distinct (p < 0.05) decrease with increasing PA concentration, and 0.5% PA reduced germination rate by ca. 43% compared to the control. (Figure 1A). This may be due to the high PA concentrations, which have high electrical conductivity and low pH. Changes in the pH and EC of acidic solutions affect biological activity, and plants are more sensitive to salinity during germination and seedling growth [32]. Most plants grow and perform well at an optimal pH between 5 and 8. The 1% and 2% PA had a complete inhibition on the germination of kale seeds, and all other concentrations of PA delayed the germination of seeds (Figure 1A). However, the 0.01% and 0.1% PA-treated kale seeds had similar germination rates, but the germination rate of 0.1% PA was slightly higher than that of 0.01% PA at a later stage (Figure 1A). Several studies have demonstrated that low PA concentration can facilitate metabolic activities and enhance plant germination [19,33]. The biostimulatory ability of PA has been attributed to the presence of butanolide, a biologically active compound belonging to the Karrikin family of phytohormones and other phenolic compounds which are known to play a critical role in regulating seed photomorphogenesis [33,34].

There was no obvious difference (p > 0.05) in the germination rate of kale seeds between different concentrations of SL on day 9 (Figure 1B). The germination rate of kale seeds treated with 0.01% SL was less than the germination rate of seeds treated with all other concentrations (Figure 1B). The germination rate of the control was greater than all other SL concentrations at a later stage (after seven days) (Figure 1B). On day 7, the germination rate of the control was 4.8% higher than that of 0.01% SL (Figure 1B). High salinity inhibits seeds due to the high EC in the SL liquid, which could negatively affect seed germination. Similar results were found in previous studies, where the germination rate of lettuce (Lactuca sativa) and corn (Zea mays) seeds decreased with an increasing concentration of the process water generated in the HTC of vinasse and sugarcane bagasse [35]. This study used the same HTC process water as the SL liquid. The diluted hydrothermal charring filtrate had a significant (p < 0.05) inhibitory effect on the germination and growth of maize [36].

It is obvious from Figure 2 that the accumulative rate of root elongation of kale treated with the PA and SL liquids was faster than that of the control. The kale seeds maintained a rapid growth rate in the early stage (first four days), which began to slow down and level off in the later stage (after day 4). The root elongation of 0.1% PA- and 0.01% PA-treated plants was significantly (p < 0.05) greater than that of the control, while that of 0.5% PA was inhibited significantly (p < 0.05) and lowered by ca. 82.8% compared to the control (Figure 2A). The inhibition effect of PA on kale was reduced after seed germination, and low concentrations of PA promoted the growth of kale roots. Infiltration of seeds with 600-fold-diluted PA promotes wheat seed germination and growth and significantly increases yield. This is attributed to the high bioactivity of acids and phenolics in PA, which can promote seedling growth and nitrogen uptake at low concentrations [37]. The 1% and 2% PA had no growth data because they completely inhibited the germination of treated seeds (Figure 2A).

The kale seedling root growth of SL-treated kale seeds was significantly (p < 0.05) promoted, especially at low concentrations of SL. The length of the kale roots at 0.5% SL was 1.8 times longer than the length of the roots in the control. This may be related to the dilution concentration and that the SL liquid was rich in nutrients that promote plant root growth. Similarly, SL contains phenolic substances that promote plant growth and improve plant stress tolerance [15]. The seedling growth of SL-treated plants was longer than the roots of PA treatments, and all concentrations of SL were much better than the control. Also, 0.5% SL had the highest root elongation and the fastest growth rate among all concentrations of SL, and a trend of rapid growth on the eighth day (Figure 2B).

All SL treatments seemed to have similar effects on the germination and seedling growth of kale seeds and had a pronounced growth promotion effect compared to the control. PA liquid had a significant (p < 0.05) reduction effect on all the seedlings in the present study. As clearly shown in Figure 3, PA-treated seeds had a significant (p < 0.05) inhibitory effect on the germination and growth of kale seeds, which increased with an increasing concentration of PA.

There were significant differences (p < 0.05) in the total length, total surface area, and root volume of kale seedlings grown at different concentrations of PA (Table 4). Low concentrations of PA promoted the growth of kale seeds compared to the control. However, high concentrations of PA (≥0.5% PA) had a significant (p < 0.05) negative effect on seedling growth (Table 4). In addition, the 0.01% PA-treated kale seeds had the highest total length, which was higher than that of the control by ca. 18.5% (Table 4). It is suggested that the effectiveness of PA is highly dependent on the concentration used [9]. This indeed confirms that lower levels of PA may contain a tight proportion of bioactive compounds to stimulate kale seedling growth.

There were no significant (p > 0.05) differences in the total length, total surface area, and root volume of kale seedling growth with the different concentrations of SL. However, it was observed that the growth of kale seeds treated with 0.5% SL was relatively higher compared with that of control, followed by 0.1% SL (Table 4). The total length of kale seeds treated with all concentrations of SL liquid was non-significantly (p > 0.05) higher than the control (Table 4).

All treatments were photographed to compare the growth and color of the kale (Figure 4). The application of biostimulants had a significant effect (p < 0.05) on the color of the kale. Different concentrations of PA and SL had little effect on the color of the kale, but all treatments were significantly (p < 0.05) higher than the control. Comparing the yellow-blue value b* and red-green value a*, it was obvious that the control leaves were yellowish in color and the treatment leaves were greenish (Table 5). The 0.25% PA and 0.5% SL-treated plants had the greenest leaf color, while the 0.5% PA- and 0.5% SL-treated plants had the highest chlorophyll content (Table 5). The brightness L* of both PA- and SL-treated plants was significantly (p < 0.05) higher than that of the control. Specifically, the 0.25% PA- and 0.5% SL-treated plants recorded the highest brightness compared to the control (Table 5). Taken together, 0.25% PA and 0.5% SL were greener and brighter in terms of kale leaves.

The physiological response of kale to different concentrations of PA treatment showed a positive trend. The stomatal conductance and transpiration rate of 1% PA-treated plants were significantly higher (p < 0.05) than those of the control group, by ca. 204% and 100%, respectively (Table 6). The intercellular CO₂ concentration of the control plants was significantly (p < 0.05) higher than that of all PA-treated group plants, while the transpiration rate of the control was lower compared to other concentrations of PA (Table 6). In the study of Ofoe et al. [38], the application of low concentrations of PA was less toxic to the root system and could improve plant nutrient uptake and promote root growth. The results showed that 0.25% PA had the best growth promotion effect on kale with the highest photosynthetic rate and biomass yield. However, the 1% PA exhibited some biological toxicity and a relatively low growth-promoting effect. The application of PA increased the leaf area index and dry matter accumulation of rapeseed and improved plant resistance to low temperatures and diseases. Pyroligneous acid can also be mixed with other plant hormones or liquid fertilizers with significantly greater effect than PA alone [37].

SL liquid had a beneficial effect on the growth of kale leaves. There was no obvious difference in the potential photosynthetic capacity and the maximum quantum yield of kale leaves with the different SL concentrations. However, the intercellular CO₂ concentration of the control plants was significantly (p < 0.05) higher than that of all SL-treated group plants (Table 6). Transpiration rate and stomatal conductance were highest in kale plants treated with 1% SL, and were 103% and 75% higher than the control, respectively. (Table 6). The 0.25% SL significantly (p < 0.05) increased the photosynthetic rate of kale, which was 14 times higher than that of the control (Table 6). SL liquid had a beneficial effect on the growth of kale, and 1% SL exhibited the best performance compared to the other SL treatments. SL liquid contains N, Mg, and K, which are essential nutrients for plant growth and can enhance the water uptake of plants [39]. The rich K content increases the disease resistance of the plant, helps to regulate the water status of the plant, and controls the opening and closing of stomata [32], which explains the significantly (p < 0.05) enhanced photosynthetic rate of SL-treated kale. The SL liquid obtained by the HTC process has a higher carbon content compared to raw sea lettuce, and it contains phenolic substances that contribute to plant growth [15].

Two liquids had a great growth-promoting effect on kale. The effects of the different concentrations of the liquids on leaf growth did not differ much; all treatments were relatively similar in the first period (first nine days), and the promotion effect was more pronounced in the later period (Figure 5A). In the 0.25% PA-treated plants compared to other PA treatments, there was a significant (p < 0.05) difference in the elongation and growth of the leaves (Figure 5A). Moreover, the leaf elongation rate of SL-treated plants changed from slow to fast with time; in the first stage (first 9 days), both 0.25% and 0.5% SL-treated plants had shorter leaves than the control. However, in the later stage (after 9 days), all SL-treated plants were clearly longer than the control, especially 0.25% SL-treated plants (Figure 5B). Overall, 0.25% PA and 0.5% SL performed the best in leaf elongation (Figure 5).

The number of kale leaves was not significantly (p < 0.05) different in the PA and SL treatments. The control treatment had the highest number of kale leaves compared to PA and SL treatments (Figure 6A). The number of leaves of PA- and SL-treated kale decreased with 0.25% and 0.5% applications of the liquids (Figure 6A). Aside from the control, 1% PA had the next highest number of leaves compared to the other treatments (Figure 6A). However, the addition of the solution to the soil did not have an obvious promotional effect on leaf numbers.

There was no difference between the two liquids at different concentrations for the total height of the whole kale plant. Different concentrations of PA and SL liquids have similar promotion effects on height with kale. The average height of kale in the control was the lowest compared to the other treatments, and the data proved that PA liquid and SL liquid still had some promotional effect on the total plant height of kale (Figure 6B). The 0.5% PA and 0.25% SL had the highest effect on the height of kale plants (Figure 6B).

The stem diameter of kale had significant (p < 0.05) effects with PA and SL treatment. The stem diameter of the control was the lowest among all treatments, proving that the two liquids treatments had a significant (p < 0.05) increase in the growth of kale. The results of SL and PA treatments for stem diameter were very similar; the stem diameters of 0.25% PA and SL fluids were ca. 17.1% and 14.6% wider than the control, respectively, and the diameter of treated kale stems decreased with increasing PA or SL concentrations (Figure 6C). The 0.25% PA and SL liquids showed higher performances among the treatments (Figure 6C). There was a significant (p < 0.05) difference between different concentrations of PA on the growth of kale stem diameter (Figure 6C). The growth of kale main stems in 0.25% PA and SL liquids was higher than that of controls.

The fresh weight of kale performed best under the treatment of the liquid, with significant differences (p < 0.05) between the different concentrations. Although there was no significant (p < 0.05) difference in the fresh weight of kale treated with different concentrations of PA, an increase of 25.7% in total fresh weight was observed with 0.5% PA compared to the control (Figure 6D). There was a significant (p < 0.05) difference between different concentrations of SL liquid in the growth of kale. The 1% SL treatment recorded the highest fresh weight of kale compared to the control (Figure 6D). Among all treatments, the control group had the lowest fresh weight, and the data indicated that these two liquids could effectively promote fresh weight and increase the yield of kale (Figure 6D).

The content of chlorophyll and carotenoid pigments is closely related to photosynthesis. Biostimulant application significantly (p < 0.05) increased chlorophyll b content, while the effect on chlorophyll a content and carotenoid content were not obvious. Chlorophyll a content was not significantly (p > 0.05) influenced by different concentrations of PA but was significantly (p < 0.05) affected by concentrations of SL treatment. Chlorophyll a content and SL liquid concentrations were positively correlated. The 0.5% SL treatment showed significantly (p < 0.05) decreased chlorophyll a than the control (Figure 7A). The low concentration of biostimulants was not favorable for chlorophyll synthesis. The 0.5% PA and 1% SL treatments had a significant effect (p < 0.05) on chlorophyll b, increasing it by 5.6% and 6.1%, respectively. PA and SL liquids had an inhibitory effect on carotenoids. The 0.5% PA and 1% SL significantly (p < 0.05) decreased carotenoid contents by 17.2% and 18.0%, respectively, compared to the control (Figure 7C).

PA and SL significantly (p < 0.05) elevated the ascorbic acid content of kale, while the dehydroascorbic acid content was not affected. The 1% PA and 0.25% SL treatments significantly (p < 0.05) elevated the ascorbic acid content by ca. 117% and 175% compared to the control (Figure 8A). The 0.25% and 1% PA treatment effectively increased the ascorbic acid and dehydroascorbic acid contents in kale leaves (Figure 8). The concentrations of SL liquid and ascorbic acid and dehydroascorbic acid content were negatively correlated, and the low concentration of SL facilitated the accumulation of ascorbic acid and dehydroascorbic acid content. The contents of ascorbic acid and dehydroascorbic acid contents were increased by 2.75 and 1.39 times, respectively, following the application of 0.25% SL, compared with the control (Figure 8).

PA and SL liquids increased the phytochemical composition of kale leaves, while SL liquid had a more pronounced phytochemical promotion effect. As shown in Figure 9A, the sugar content of both PA and SL treatments showed remarkable changes. The sugar content of 0.25% SL and 1% SL increased by ca. 162% and 148% compared to the control (Figure 9A). There was no great difference in the protein content of PA at different concentrations in the kale leaves. However, SL treatment had a significant (p < 0.05) effect on protein content. The protein content of 0.5% SL significantly (p < 0.05) reduced the protein content compared to the control (Figure 9B). However, 1% SL significantly (p < 0.05) increased the protein content, indicating that a high concentration of SL can effectively increase the protein content of kale leaves (Figure 9B). This suggests that PA and SL could be used to enhance crop quality for human health and nutritional purposes.

PA and SL liquids were effective in increasing the phenolic content in kale leaves, and the phenolic content was more pronounced with lower concentrations of PA and SL. The 0.25% PA and 0.5% SL treatments significantly (p < 0.05) increased phenolic content by ca. 119% and 127%, respectively, compared with the control (Figure 9C). Numerically, the phenolic content of SL treatments was slightly higher than that of PA treatments. PA and SL liquids significantly (p < 0.05) increased flavonoid content, but the flavonoid contents were affected differently by PA and SL treatments. Different concentrations of PA liquid and flavonoid content were inversely proportional, while the flavonoid content of SL treatments increased with increasing concentration. Flavonoid was significantly (p < 0.05) increased with 0.25% PA and 1% SL by ca. 134% and 152%, respectively, compared with the control (Figure 9D). A high concentration of SL liquid or a low concentration of PA liquid can effectively increase the flavonoid content in kale leaves. Kale is considered to be an excellent source of phytochemicals including vitamins, flavonoids, and phenolics, which are crucial for scavenging reactive oxygen species (ROS) radicals [22]. Studies have reported that PA application enhances the antioxidant content of edible parts of leaves and fruits [37,40]. Therefore, the increase in these antioxidant compounds with PA application is expected and can be attributed to the high organic acids and phenolic compounds in PA since previous studies have demonstrated that phenolic compounds in PA exhibited high ROS-scavenging activities, reducing power, and anti-lipid peroxidation capacity [41]. Although the exact mechanism of the biostimulant-mediated increase in antioxidant compound in the leaves has not been examined, it is plausible that the increase in these compounds could be due to the activation of defense responses, improved nutrient absorption and assimilation, and the upregulation of genes and enzymes involved in the phenylpropanoid pathway [42,43].