DISCUSSION
In order to better understand the phytotoxic potential of C. cardunculus and increase the production of secondary metabolites, the effect of light stress (shading) in field conditions was studied on the STL profile of cultivated cardoon leaf extracts. The influence of light stress on the production of plant STLs has never been studied. Given the importance of these chemical compounds for their numerous biological activities(Chaturvedi 2011), the knowledge of the factors affecting their biosynthesis and production represents an important aspect that should be considered for their future application.
In this study, the yield extract was greater in the April harvest than January, with the stressed treatment showing the highest yield. These yields are similar to those reported by Ramos et al. (Ramos et al. 2013) and Scavo et al. (Scavo et al. 2019f). The yields of extracts were closely correlated to STL concentrations. In fact, extracts belonging to April harvest generally showed higher concentrations of total and single STLs than January, probably due to the higher levels of global radiation and longer photoperiod. A similar trend was reported by Scavo et al. (Scavo et al. 2019f) for STLs on the three C. cardunculus taxa and by Pandino et al. (Pandino, Lombardo, Lo Monaco & Mauromicale 2013) on globe artichoke leaf polyphenols. The leaves of many Asteraceae members, including C. cardunculus , are known for the high cocentrations of STLs, mainly represented by cynaropicrin, which is responsible of its bitter taste(Seaman 1982). Here, in accordance with Ramos et al. (Ramoset al. 2013), cynaropicrin was the most abundant STL detected. However, most of previous studies are focused on the globe artichoke(Rouphael et al. 2016), while cultivated cardoon has been little investigated.
While the influence of light quality (e.g. ionizing or ultraviolet radiation, intensity of visible light, red and far-red light) on the allelopathic expression of plants has been reported in previous researches (del Moral 1972; Kato-Noguchi 1999; Li et al. 2009), only few works investigated the effect of daylength. Zucker et al. (Zucker, Nitsch & Nitsch 1965) stated that chlorogenic acid concentration in Nicotiana spp. species is affected by photoperiodic alteration. Rice (Rice 1984) indicated that long days generally increase the production of inhibitory compounds in donor plants. Similarly, Taylor (Taylor 1965) found that the concentration of different phenolics and cinnamic acid derivatives increase in the leaves of Xanthium strumarium L. with increases in daylength. Here we found an opposite trend to those reported in literature. Indeed, the amount and composition of STLs in cultivated cardoon leaf extracts varied in relation to both light stress and harvest time, with a more pronounced effect of the latter factor, suggesting how the meteorological conditions (e.g. global radiation, photoperiod, air temperature and total rainfall) affect their biosynthesis. However, except for aguerin B, the shading significantly increased the concentration of total and single STLs in the April harvest, compared to control, and this increase was more marked if combined with harvest time. It is known that once a stress factor is recognised at cellular level by a plant, a signal transduction begins leading to gene expression and metabolic responses with the aim of helping the plant survive or minimizing the effectiveness of the stress agent (Reigosa et al. 1999). Eljounaidi et al. (Eljounaidi et al. 2014) elucidated the genes and the enzymes involved in the biosynthetic pathway of STLs in C. cardunculus . However, the alteration of gene expression in this species is still unknown, but appears reasonable how this plant, as commonly happens in other ones, prevents the oxidative damages induced by ROS (reactive oxygen species such as hydrogen peroxide, superoxide, etc.) through the synthesis of secondary metabolites, STLs and polyphenols in this case. However, in nature plants interact with the complex of abiotic and biotic influences of the environment, hence an allelopathic phenomenon depends on the type and intensity of stress, on the genotype and development stage of the plant, as well as on the seasonal changes and, therefore, may vary through time (Scavo, Abbate & Mauromicale 2019a). The influence of genotype and harvest time have been previously studied (Scavo et al. 2019f), while light stress is the first abiotic factor evaluated in C. cardunculus .
With the aim to find a correlation between the STL composition and phytotoxicity, cultivated cardoon leaf extracts were bioassayed on the wheat coleoptile elongation, as well as on germination, root and shoot length of A. retroflexus and P. oleracea . In agreement with previous results with the same bioassay and target species bothC. cardunculus (Scavo et al. 2019e) and other donor plants (da Silva et al. 2017), seed germination percentage was the least affected parameter. Contrariwise, wheat coleoptile elongation and root length of both weeds were significantly inhibited. The phytotoxic activity increase at increasing concentrations, as widely reported in literature (Ambika 2013), and in correspondence of the April harvest, as found also by Scavo et al. (Scavo et al. 2019f) and Pandino et al. (Pandino et al. 2013) for polyphenols. These results are corroborated by STL profile. During April, in fact, the plant accumulated more STLs in the leaves, mainly cynaropicrin. Probably, the combination of high temperatures, low rainfall, high global radiation and long photoperiod stimulates the production of C. cardunculussecondary metabolites (caffeoylquinic acids, flavones and STLs) and increases its inhibitory potential. Nevertheless, the imposition of plant shading in April was found to stimulate the production of STLs in the leaves and to enhance, albeit to a lesser extent, the allelopathic activity.
In conclusion, here we demonstrated, for the first time, that plant shading in field conditions enhances the production of STLs in the leaves of cultivated cardoon, especially in the April harvest. This increase was accompanied by a higher phytotoxicity on wheat coleoptile elongation, germination percentage, root and shoot length of A. retroflexus and P. oleracea . The inhibitory activity of cultivated cardoon extracts caused by shading was weak if taken alone, becoming stronger when combined with the harvest time due to the optimal combination of climatic conditions, development stage of the plant and intensity of light stress. The induction of light stress by plant shading could be a useful tool to stimulate the biosynthesis of STLs for industrial applications and to increase the phytotoxic potential of cultivated cardoon as a sustainable weed control method within an integrated weed management system for the future production of a bioherbicide.