The non-significant response between fertilizer supplemented wastewater and commercial hydroponic fertilizer mix could be caused by the trade-offs in nutrients between wastewater and CHFM.Ligularia fischeriTurcz is a perennial herbaceous plant which belongs to Ligularia, a class of composite family and distributed widely in South Korea, Japan, China, and Eastern Siberia. It usually grows on wood, water, or rocks at an elevation of 1 000 to 1 400 m. The roots and rhizomes of L. fischeri contain pharmaceutically important sesquiterpene compounds used in the treatment of cough, promoting blood circulation, relieving pain, etc. . Further, the medicinal extracts prepared from the plant can be used to cure cough, sputum, sore throat, pulmonary tuberculosis, traumatic injury, etc. Owing to its numerous medicinal properties, the wild L. fischeri plant has been widely threatened by over exploitation by humans . However, the conventional cultivation of L. fischeri by seed propagation is difficult due to dormancy issues whereas the production of artificial planting of L. fischeri is low. In order to develop and utilize the medical value of L. fischeri and to improve the yield of artificial cultivation, the application of hydroponic technology to the artificial cultivation has been widely utilized. Hydroponics is a convenient means for studying plants in the laboratory, for growing commercial crops and easily manipulating the plant’s secondary metabolism for increased pharmacological activities of plant materials. The advantages of hydroponics include the cultivation of identical plant material around the year in a control environment, the possibility forFor phytochemical and antioxidant assays, the leaf, petiole, and root were harvested after a 4-week culture and extracted with methanol according to Gracia-Perez et al. with slight modifications. Briefly, one gram of fresh plant tissue was homogenized using liquid nitrogen and extracted with 10 mL of 80% methanol upon continuous shaking for 5 h at 200 r·min−1. The extract was subsequently centrifuged at 10 000 r·min−1 for 10 min and the supernatant was used for analysis right after centrifugation or saved at 4 °C for afterword analysis. The total phenol content of the extract was estimated by the Folin-Ciocalteu principle according to Kumaran and Karuna Karan . The aliquot of the extracts made up to 1 mL with ddH2O was mixed with 0.5 mL of Folin-Ciocalteu reagent and 2.5 mL of sodium carbonate solution .

The reaction mixture was incubated in the dark for 40 min and then absorbance was recorded at 725 nm. The total phenol content was estimated using standard gallic acid calibration curve. The total flavonoid content was determined by aluminum chloride method outlined by Gracia-Perez et al. . Samples made up to 1 mL with 80% methanol and were used for analysis by adding 1 mL of 2% aluminum chloride solution. The absorbance of the reaction mixture was measured at 415 nm after 30 min incubation and the total flavonoids were determined from the standard quercetin calibration curve.Morphological traits of L. fischeri were significantly varied with culture medium type . Notably, all the growth traits were significantly increased in the dutch bucket hydroponic growth system and this could be due to the continuous recirculation of nutrients in the hydroponics. Shoot length in the hydroponic nutrient solution treatment system increased by 115.2% and 20.0% compared with the Tosilee medium and soil treatment, respectively. Meanwhile, the root length in the hydroponic system was increased by 108.3% and 51.2% compared with the Tosilee and soil treated roots, respectively. To be specific, the leaf length and width grown in the hydroponic nutrient solution treatment were the greatest among the treatments, and chlorophyll content in Tosilee medium was higher than other treatments. Shoot fresh weight in the recirculated hydroponic system increased by 73.0% and 54.9% compared with the Tosilee medium and soil treatment, respectively; root fresh weight in the recirculated hydroponic system treatment increased by 91.5% and 50.3% compared with the Tosilee medium and soil treatment, respectively. These results thus suggest that the recirculated hydroponic system improved the growth traits of L. fischeri and this could be due to the continued circulation of nutrient solution and ambient aeration of the roots. Since L. fischeri is a medicinal plant, the contents of phytochemicals are important indicators in evaluating its quality. In the experiment, the recirculated hydroponic system not only affected the plant growth but also increased the content of L. fischeri’s phytochemicals. Higher total phenol content and total antioxidant capacity were observed in the leaf, petiole, and root extracts of L. fischeri in the recirculated hydroponic system treatment and soil treatment . However, plants grown in Tosilee consisted of the least total phenol content and lowest total antioxidant capacity . The greatest total phenol content was observed in the whole plant in the recirculated hydroponic system . The recirculated hydroponic system significantly increased the total phenol contents of the leaf, petiole, and root extracts by 17.6%, 30.6%, and 20.9% more compared to the soil treatment. The recirculated hydroponic system treatment significantly increased the total antioxidant capacity of root extracts by 55.9% more compared to the soil treatment . But the recirculated hydroponic system treatment did not lead to the significant enhancement of total antioxidant capacity in the leaf and petiole extracts.

In general, the occurrences of higher phenolic contents enhance the antioxidant property of the plant because phenols possess ideal structural properties for scavenging free radicals and thus they prevent oxidative damages . The antioxidant ability assessment by DPPH radical scavenging revealed the greatest DPPH scavenging ability of the root among leaf, petiole, and root.However, there is no significant difference among growing medium treatments. In conclusion,the growth traits,total phenol content, and total antioxidant capacity of L. fischeri can be enhanced in hydroponics cultivation compared to Tosilee medium. From the element contents and phytochemical analysis, we found that hydroponics is the optimal medium for the higher growth and development of L. fischeri.The nutrient-rich rhizosphere is a hotspot for microbial activity, containing up to 1011 bacteria per gram root and housing more than 30,000 prokaryotic species . Among those are beneficial bacteria which are actively recruited by the plant through root exudate secretion and subsequently colonize the root from where they benefit the plant through various mechanisms . One well-known plant growth-promoting rhizobacterium is the spore-forming B. subtilis that has been isolated from various plant species . Its plant-beneficial traits and promising role as a biocontrol agent have fueled the interest in studying B. subtilis–plant interactions and led to the elucidation of mechanisms involved in the establishment of B. subtilis on the root and behind its plant beneficial properties. An obvious prerequisite for successful root colonization is the ability of the bacterium to reach the plant root. Chemotaxis toward root exudates was shown to be important for the early colonization of A. thaliana roots by B. subtilis under hydroponic conditions , whereas solid surface motility has been suggested to play a role during tomato root colonization in vermiculites . After reaching the plant root, B. subtilis initiates biofilm formation . Similar to in vitro conditions, the formation of plant root-associated biofilms depends on the production of the matrix components EPS and TasA . The expression of the operons involved in matrix production, epsA-O and tapA-sipW-tasA, is controlled by the biofilm repressor SinR . In response to environmental cues, one or more of the five histidine kinases, KinA-E, are activated resulting in phosphorylation of the master regulator Spo0A through a phosphorelay . At threshold concentrations of Spo0A P, SinI is produced , which binds to and inhibits SinR , resulting in matrix gene expression.

As an environmental cue for root colonization, root exudates from tomatoes were shown totrigger biofilm formation in B. subtilis in a KinD-dependent manner , whereas another study reported biofilm induction by plant polysaccharides via KinC and KinD . B. subtilis benefits from such microbe–plant interactions by acquiring carbon source from the plants. In turn, B. subtilis protects the plant against disease directly by producing antimicrobials and indirectly through niche competition and elicitation of induced systemic resistance in the plant . Moreover, B. subtilis promotes plant growth by improving nutrient availability and producing growth-promoting phytohormones . Mutualistic bacteria-plant interactions are a result of a long-term co-evolution of bacteria and plants that started with the colonization of land by ancestral plants 450 million years ago . Here, we were interested in studying how B. subtilis adapts to plant roots on a much shorter evolutionary timescale. Experimental evolution provides a powerful tool to study microbial adaptation to different environments in real-time . We recently studied EE of B. subtilis on A. thaliana plants roots, which revealed diversification of B. subtilis into three distinct morphotypes. A mix of the three morphotypes displayed increased root colonization compared with the sum of the three morphotypes in monocultures weighted by their initial relative abundance in the mix,dutch buckets system which was demonstrated to be caused by complementarity effects . Such morphological diversification has also been observed in EE of B. subtilis biofilm pellicles formed at the air-liquid interface as well as during EE of Burkholderia cenocepacia biofilms on polystyrene beads . In this study, we performed EE of B. subtilis on one-week-old A. thaliana roots under axenic conditions with the initial hypothesis that B. subtilis would adapt to the plant root environment by acquiring mutations that would provide the bacteria with a fitness advantage over the ancestor during root colonization. We found that B. subtilis rapidly adapted to the plant root as observed by improved root colonizers already after 12 consecutive transfers. In addition, two selected evolved isolates from independent populations from the final transfer out competed the ancestor during root colonization. Furthermore, re-sequencing of single evolved isolates from independent populations and different time points as well as of the endpoint populations revealed mutations within genes related to different bacterial traits. To further elucidate which bacterial traits were altered during the adaptation to plant roots, evolved isolates from the final transfer were subjected to additional phenotypic characterization. We found that evolved isolates from independent populations displayed robust biofilm formation in response to plant polysaccharides, impaired motility, and altered growth on plant compounds.

Finally, we demonstrate that adaptation of B. subtilis to A. thaliana roots is accompanied by an evolutionary cost, and report an evolved isolate displaying increased root colonization also in the presence of resident soil microbes.To explore the evolutionary adaptation of B. subtilis to plant roots, we employed an experimental evolution setup previously established for another Bacillus species . In short, B. subtilis DK1042 was inoculated onto A. thaliana seedlings under hydroponic conditions in seven parallel populations. The MSNg medium used in the EE is a minimal medium supplemented with a very low concentration of glycerol , and the bacteria thereby move toward the plant root to access a carbon source. Every 48 h for a total of 64 days, the newly colonized seedling was transferred to a fresh medium containing a new sterile seedling, thereby enabling re-colonization . We hypothesized, that during the successive transfers B. subtilis would adapt to the plant roots by acquiring mutations that would confer a fitness advantage over the ancestor during root colonization, resulting in these mutations being selected. In this setup, we specifically selected for a regular cycle of dispersal from the root-associated biofilm, chemotaxis toward the new root, and biofilm formation on the root surface. To follow potential changes in root colonization of the evolving populations during the ongoing EE, the productivity, i.e. colony-forming unit per root, was quantified at different time points. All seven independent populations showed a rapid increase in root colonization within the first seven transfers, after which the productivity of the populations remained rather stable with slight increases and drops dependent on the certain population . While this rapid increase in productivity could be owing to genetic adaptation to the plant root, the initial rise could also be caused by physiological adaptation to the experimental conditions. Interestingly, such a rapid increase in productivity of populations evolving on plant roots is consistent with our recent study where B. subtilis evolved on older A. thaliana roots .