BtNEB17 was grown in King’s medium B as previously described (Gray et al., 2006). Cells for the initial broth inoculum were taken from plated material and cultivated in 250 mL flasks with 50 mL medium. For 48 h, the bacterium was cultivated at 28 ± 2 °C on an orbital shaker rotating at 150 rev min ^-1. For the initial culture, 5 mL of subculture was inoculated into 2000 mL of broth, and the culture was grown under the same conditions. Bacterial populations were assessed by an Ultrospec 4050 Pro UV/Visible Spectrophotometer at 600 nm 96 h after initiation. A cell-free supernatant (CFS) containing BtNEB17 was obtained through centrifuging the bacterial culture at 13,000 g for 10 min, followed by analytical-HPLC identification. For partial purification, 800 mL of butanol was added to 2000 mL of bacterial culture for 12 h then the collected upper layer was extracted under vacuum in rotary evaporator. The viscose extract was collected with 12 mL of 30% Acetonitrile and centrifuged at 13,000 g for 13 min, and the supernatant was collected for chromatography (Gray et al., 2006). Th17 was selected based on peaks and retention times. After being lyophilized, the samples were kept at 20 °C for dilution to the required concentrations. For all experiments, 10^-7 (Thu1), 10^-9 (Thu2) and 10^-11 (Thu3) M of Th17 were prepared based on the molecular weight of Th17.

Canola seeds (cultivar L233P) were disinfected with 20% sodium hypochlorite and rinsed with distilled water before applying treatments. Twenty-five sterilized seeds were placed on petri plates (100 x 15 mm) lined with filter paper (QualitativeP8). The plates were randomly treated with 10 mL of Thu1, Thu2, Thu3 solution and distilled water. Petri dishes were then sealed with parafilm to prevent water loss and arranged in a completely randomized design, with four replications for each treatment, in growth chambers. Growth chambers were set to low (5 °C), optimal (20 °C), high (30 °C) temperatures, 70% humidity, and zero illumination. The total number of germinated seeds were counted at 24 h intervals, for 72 h at optimum and high temperature and 7 days for low temperature. Radical and shoot length were measured for all experiments after 7 days, and seedlings were weighted. Each experiment was repeated twice, and the data was pooled for analysis.

Th17 was applied as a pre-planting seed treatment, seeds were soaked in 10 mL Thu1, Thu2, Thu3 solution and distilled water, and root irrigation after the stress induction time. Canola seeds (5 seeds per pot) were placed in 10 cm pots filled with AGRO MIX^® G10 media, which was a mix of peat, limestone, balanced nutrient materials, with micronutrients, gypsum and wetting agent. This growth medium was chosen based on root application of Th17 and the ability of peat to gradually release water and/or the compound. The pots were placed in a growth chamber (Conviron R, Canada) at 22/18 °C day/night, with a photoperiod of 14/10 h light/darkness cycle, 60-70% relative humidity and photosynthetic irradiance of 350–400 µmol m⁻² s ⁻¹ ( Figure 1A ). After a week, the seedlings were counted, and the plants were thinned to one seedling per pot. Plants were regularly watered with half strength Hoagland’s solution and grown in the growth chamber until the end of the third week ( Figure 1B ). Then, 3-week-old seedlings ( Figure 2 ) were randomly selected to transfer to the low (10/5 °C), optimum (22/18 °C) and high temperature (30/25 °C) chambers to determine the effect of stressful temperatures on the vegetative growth of canola. Plants were watered semi-weekly with 250 mL of either water (for controls) or Th17 (for Th17-treated plants). Following the start of the temperature treatment, the plants in each case were allowed to develop for 4 weeks before being sampled for data collection. A LI-COR 6400 portable photosynthesis metre (LI-COR Lincoln, NE), was used to measure the photosynthetic rate, transpiration rate, stomatal conductance and CO₂ concentration inside the leaves. Readings were taken on a weekly basis (4, 5, 6, 7 weeks) after the 3-week-old seedling stage. An upper-most fully expanded leaf was used for determination of physiological variables between 10:00 and 14:00 h ( Figure 1C ). Plants were sampled at the end of the experiment for the following variables: plant height, leaf area, fresh weight, dry weight and root variables. After harvesting plants and measuring morphological traits, roots were coarsely shaken off to remove soil (destructive methods). Roots were then washed with water and adhering soil particles removed with brushes ( Figure 1D ). Total roots were scanned using EPSON-Expression 11000XL then root length, root volume, total root surface and root diameter were analyzed by WinRHIZO^TM Pro software. Each experiment described above was repeated twice with four replicates, and data were pooled for analysis.

Data from the experiments were analyzed, based on a completely randomized factorial design, using the SAS Statistical Package 9.3 (SAS Institute Inc., Cary, NC, USA). For germination data, observed values were analysed similarly to other variables and a non-linear regression model was fitted to predict the probability of germination, in Figure 3 (Piegorsch and Bailer, 2005; Schwinghamer et al., 2015). Duncan’s multiple comparison test was used when there was a significant difference at the 95% confidence level.

There were no significant differences in germination rate under optimal conditions (20 °C) ( Figure 3B ). The control had the highest germination rate, which did not differ significantly from Th17-treated seeds. The lowest germination rate under optimal conditions was for seeds treated with the lowest concentration, Thu3. With increasing temperature to 30 °C Figure 3C , germination decreased although applying Th17 helped alleviate the negative effect of high temperature on germination; in particular Thu2 treatment increased germination significantly. Cold stress strongly affected germination ( Figure 3A ); about 100 h were required to start germination, and it increased slowly after that. In marked contrast, the first germination was recorded approximately 12 h after onset of the experiment at 20 °C. Under cold stress conditions, the interaction of temperature and the compound application was significant (p < 0.002), and application of Th17 significantly influenced germination, mainly mid-range concentration (Thu2) maximized germination rate.

Seed germination is a very important stage in the life cycle of a plant and one that is very sensitive to both the intrinsic and extrinsic factors (Rifna et al., 2019). Among them, temperature is a major environmental factor determining seed germination (Finch‐Savage and Leubner‐Metzger, 2006). Early germination is an essential element that impacts the production of canola (B. napus [L.]). The optimal temperature for canola germination is 22°C (Nykiforuk and Johnson‐Flanagan, 1994; Tasseva et al., 2004). Any temperature either below or above optimal induces uneven germination and, in the field, poor stand establishment, restricting the crop potential to produce yield. In this study, we found that germination and seedling growth indices were negatively affected by both low and high temperatures. Germination was delayed under low temperature conditions, which could have arisen from slowed water uptake, slower enzyme kinetics, higher levels of late embryogenesis-abundant proteins, induction of nuclear stress-responsive proteins and imbalanced amount of inhibitor and promoter phytohormones (Achard et al., 2008; Copeland and McDonald, 2012; Abdul Aziz et al., 2021). However, faster germination occurred at a higher temperature than optimal conditions, which could be due to a thermal effect, related to number of heat units required for germination. These results are consistent with previous studies that found stressfully high and low temperatures hindered final germination percentage of five cultivars of canola between 10 to 20 percent (Schwinghamer et al., 2015). Seedling growth was also impeded by both high and low temperatures, resulting in lower fresh weight and seedling vigor indices than optimum conditions (Zhang et al., 2015). Hence, discovery of technologies that improve seed germination and seedling establishment are of importance in agriculture. Here, the application of a bacteriocin could enhance canola germination percentage, radical and shoot length, seedling fresh weight and seed vigor compared to the controls under both low and high temperature stress conditions. To be more precise, among three levels of the compound, Thu2 considerably ameliorated negative effects of stress and showed high stimulatory impacts on both seeds and seedlings. This result is in line with findings of Gautam et al. (2016), which highlighted the response of soybean to 10^-9 M and 10^-11 M Th17 during germination and seedling development under low temperature. A similar conclusion was reached by He (2009) for Th17 treated corn seeds under a combination of stresses, including low temperature, salinity and drought stress under both controlled environment conditions and a cool spring climate in the field. In another study, the effect of LCO, a compound produced by B. japonicum 532C, Th17 and chitopentaose were tested on high oil content cultivars of canola. Treatment with 10 ^-6 M LCO accelerated germination at 10 °C while seeds treated with 10^-9 M Th17 had more even and uniform germination (Schwinghamer et al., 2015). LCO has been shown to be a stimulant for seed germination and seedling establishment as illustrated in a large body of published research, whereas Th17 has not been well studied (Kidaj et al., 2012; Suganya et al., 2016; Nandhini et al., 2018). The underlying mechanism of LCO and Th17 in soybean germination under stressful conditions was studied through proteomic approaches. The results indicated that energy, carbon and nitrogen metabolic pathways were influenced by signal compounds under stressful environments. Some notable stress-related proteins, including PEP carboxylase, rubisco oxygenase large subunit, pyruvate kinase and other proteins are up regulated in response to LCO and Th17 treatments under stressful conditions (Subramanian et al., 2016). Similar results were observed where over 600 genes, mostly related to stress and defense, were up regulated in LCO treated soybean leaf tissue under stressful conditions (Wang et al., 2012). These findings highlight that signal molecules can alter metabolism and physiology to assist in alleviating sub-optimal conditions and improve germination and early growth.

Unfavourable temperature, as a major environmental factor, considerably affects plant growth and development. Some plants adapt themselves by altering their morphology, while others adjust their physiology or exhibit changes in gene expression, which modifies how they grow, survive and tolerate stresses (Raza et al., 2020; Gupta et al., 2020). However, susceptibility or tolerance is directly associated with plant lifecycle stage. We hypothesized that during vegetative growth of canola, different levels of Th17 would assist in tolerating stressful temperatures and enhance growth variables. The exposure of canola to cold conditions in northern climates like western Canada is unavoidable in early spring because early spring sowing is required to ensure that the crop matures without suffering yield losses due to summer heat stress. Canola, as a cold-acclimated crop, has the capacity to develop cold stress tolerance, known as cold acclimation, once exposed to chilling but not freezing temperature, although its growth might be slowed. Furthermore, the likelihood of high temperature occurrence during mid to end of canola vegetative growth is relatively high, as we experienced during May-June of the 2020 and 2021 canola field seasons in eastern Canada (unpublished data). In this study, plant height, leaf area and biomass accumulations were negatively altered, in contrast to optimal temperature, after 3 weeks of growing in the 10/5°C temperature range, but surprisingly application of the signal molecule efficiently acted as an alleviator and effective plant growth regulator. In contrast, canola showed positive responses to 30/25 °C temperature, suggesting acclimation ability to moderately high temperature. This could arise from thermomorphogensis, a group of morphological and architectural alterations caused by higher ambient temperatures, below the heat-stress level (Quint et al., 2016; Casal and Balasubramanian, 2019; Ludwig et al., 2021). Based on this, canola height was significantly enhanced across all treatments at moderately high temperature, which may indicate that the plant shifted from vegetative stage to flowering by stem elongation; leaf area and fresh biomass were also increased while irrigation with Thu2 and Thu3 resulted in higher levels of these variables than the control and Thu1 treatments. In contrast to many previous studies, which indicated heat as a growth inhibitor, in our experiment canola growth and development continued well at higher temperature, implying that heat acclimation ability, with assistance of the signal compound, could be quite high. However, stress-induction stage, duration of stress and combination with other stressors play a significant role in response to heat. A body of research has taken shaped around the effects of high temperature on crops, but only a few studies have examined the interaction of microbial signal molecules and high temperature on plants (Kilasi et al., 2018; Hussain et al., 2019; Anwar and Kim, 2020; Ayub et al., 2021; Lohani et al., 2021; Ahmad et al., 2021). Our findings fit well with previous studies that reported the interaction of 10^-6 M LCO irrigation with relatively high temperature (30/30 °C), resulting in 1 more leaf per canola plant, as apposed to the 25/20 °C water-treated controls, which were unifoliate. Similarly, Th17 treatment increased the dry biomass of canola cultivar 04C111, by approximately 70 mg under saline (0.1 M NaCl) and moderately high (30/30 °C) temperature conditions (Schwinghamer et al., 2016). In this study, we observed that two concentrations, Thu2 and Thu3, had considerable positive impacts whereas the higher concentration level, Thu1, showed no significant stimulatory effect compared to the control. It is not surprising that such results were reported because most of the microbially produced substances are signalling molecules which are similar to phytohormones in that their presence, at very low concentrations, has long been recognised to either promote or inhibit plant growth (Backer et al., 2018; Antar et al., 2021). More than this, there are some other factors that highlight the effect of concentrations, including plant species, stage of life, growth conditions, method of application and so on. While certain chemicals can significantly improve plant growth at extremely low concentrations, others must be used at relatively high levels to have a positive impact.

Root growth relies on cell production within the root meristem, cell expansion and differentiation in which environmental factors play a vital role as stimuli. The root system possesses spatial architecture by which it can dynamically adapt itself to changes in the environment, such as shifts in temperature (Bardgett et al., 2014; Onwuka and Mang, 2018). It is worth noting that unlike the broad optimum temperature ranges for the development of aerial parts of various species, optimum root temperature (12-20 °C) is quite similar for almost all species except for tundra plants (Bell and Bliss, 1978). When plants are exposed to low and high temperatures, changes in the root system occur (Alsajri et al., 2019; Fonseca de Lima et al., 2021). In this study, root system growth was inhibited by low temperature, which is in line with previous studies (Rymen et al., 2007; Shibasaki et al., 2009; Zhou et al., 2021). This could be explained by the fact that low temperature hinders cell cycle progression and cell division in root meristems. In addition, lower carbohydrate assimilation under chilling conditions impedes nutrient allocation to the underground system. Interestingly, the interaction of temperature and Th17 was significant; root system architecture was positively modified by irrigation with the compound, particularly Thu3-treated roots, which produced the highest length, surface area, volume, and dry weight across all temperatures. This might be associated with the role of Th17 in phytohormones modifications. In this regard, Prudent et al. (2015) reported that abscisic acid (ABA) was the first plant hormone to be considerably increased in soybean leaves and roots after the application of Th17. This phytohormone facilitates root elongation by restricting ethylene production, suggesting that a reason for changes in root architecture could be a shift in the balance of plant hormones (originating at the roots). As such, Th17 treatment could cause plants to modify their ABA content, which may have resulted in maximal root growth. However, to ascertain the mode of action of Th17 in canola roots, precise phytohormone measurements would be required in future studies.

Most physiological functions of plants, including photosynthesis and transpiration are impacted by temperature. Stomatal conductance controls both carbon assimilation, and transpiration, and they mutually influence one another. Hence, temperature affects stomata indirectly by alterations in plant water status and vapor pressure deficit. We determined that photosynthesis increased linearly with time and was maximum under optimal temperature conditions whereas a considerable decline was seen across all physiology variables at low temperature. In accordance with our results, many studies have demonstrated a significant reduction of carbon assimilation rate and stomatal conductance at low temperatures, which is partly explained by disruption of chloroplast structure, thylakoid membrane damage, reductions in enzyme activities, decreased chlorophyll levels, reductions in expression and resulting concentrations of photosynthesis proteins, and reduced electron transport (Liu et al., 2012; Paredes and Quiles, 2015; Li et al., 2018; Khan et al., 2019; Zhao et al., 2020). Regarding high temperature, in line with many studies, carbon assimilation rate decreased with increasing temperature although in our study, the Th17 application could mitigate the reduction. In this regard, Thu2 and Thu3 enhanced photosynthesis rate compared to the control. Increasing conductivity with time under optimal conditions, coupled with more diffusion of CO₂, resulting in greater assimilation rates, whereas it caused higher transpiration rates which could lead to reduced leaf water potential at high temperatures. These results agree with those studies that reported stomata opening and increased transpiration at higher temperatures (Lu et al., 2000; Mott and Peak, 2010; Xu et al., 2016; Urban et al., 2017), while our findings are in contrast with some experiments that observed no significant changes of conductivity and/or closure of stomata (von Caemmerer and Evans, 2015; Abdulmajeed et al., 2017; Hlaváčová et al., 2018). It is worth noting that higher leaf temperature causes reduction in protein translation, chlorophyll breakdown, rubisco inactivation, photosystem II activity, electron transport chain activity and increased damage to photosynthetic apparatus (Kang et al., 2017; Wang et al., 2018a; Balfagón et al., 2019; Hu et al., 2020). As such, lowering leaf temperature by transpiration would be an approach to compensate for assimilation reduction which could be beneficial, even if, to some extent, water loss was not constrained. We observed greater transpiration rates in Th2 and Th3 treated plants which meant enhanced photosynthesis came at the cost of losing more water; however, it would assist in cooling leaves when water is not a limiting factor; therefore, photosynthesis systems could function more efficiently. This might be one of the explanations for continued increase of photosynthesis in Thu2 and Thu3 plants despite lower intercellular CO₂ amounts than the control. Lee et al. (2009) demonstrated that application of 10^-9 and 10^-11 M Th17, either through root irrigation or leaf spray enhanced soybean photosynthetic rate and leaf greenness. Our finding partly ties with a conclusion reached by Prudent et al. (2015); they demonstrated the positive effects of Th17 treatment on improving carbon assimilation in water-stressed soybean plants, but this was associated with an increased level of ABA, resulting in stomatal closure and reduced transpiration which is opposite to our findings.