Biostimulants in horticulture: evaluation of their mode of action on crops using a platform for high-throughput automated phenotyping

In the last decades, with the constant increase in world population, the fast reduction of fertile arable land and the deteriorating environmental conditions, optimization of agriculture has become a priority. The main focus is on increasing the final yield and protecting the crops from unfavourable growing conditions in a sustainable way. A possible solution to this problem is represented by biostimulants, bioactive substances of diverse origins. A very large number of new biostimulants enter the market every year. However, a thorough knowledge of the mode of action of the substances in different crops and in different environmental conditions is still lacking. Traditional testing methods are time-consuming, expensive and, in most cases, destructive. Therefore, in the last years high-throughput automated phenotyping platforms started to be considered an interesting alternative to traditional characterization assays, drawing the attention of biostimulant producers. Different cameras and sensors can be implemented into high-throughput phenotyping platforms, allowing to screen the effects of different substances on a large number of morpho-physiological plant traits in a fast, efficient, cost-effective and non-destructive manner. In our work, we developed a precise methodology to test the effects of a large set of protein hydrolysates on multiple plant species (wheat, Arabidopsis, lettuce and tomato) subjected to abiotic stresses (drought and salinity) at all phenological phases, from seed up to the crop maturity. A large number of morpho-physiological traits of the plants were analysed throughout their life cycle, before and after the application of the PHs substances. The original set of PHs has been subjected to an initial in vitro screening on Arabidopsis plantlets; the substances were applied as seed priming in three different concentrations. The best-performing PHs in control and salt stress conditions have then been used for trials in planta, where they were applied as foliar spray. With the use of a Plant Biostimulant Characterization Index (PBC), we were able to categorize the substances into functional classes according to their mode of action, classifying them as Growth Promoters and /or Stress Alleviators. Leaves of the plants treated with the best- and worst-performing substances were collected and subjected to untargeted metabolomic analysis to elucidate the biochemical pathways activated by the PHs applications. It was clear that the effects of the biostimulants on plants can vary depending on the mode and time of application, the growing conditions, the dose and the plant species they are applied to; therefore, before putting a new biostimulant on the market, it is essential to select the target crop species that could benefit from the treatment. High-throughput automated phenotyping platforms can be an extremely useful tool to speed up the testing process and precisely investigate the effects of the same substance on multiple morpho-physiological traits

Understanding the biostimulant action of vegetal-derived protein hydrolysates by high-throughput plant phenotyping and metabolomics: A case study on tomato

Designing and developing new biostimulants is a crucial process which requires an accurate testing of the product effects on the morpho-physiological traits of plants and a deep understanding of the mechanism of action of selected products. Product screening approaches using omics technologies have been found to be more efficient and cost effective in finding new biostimulant substances. A screening protocol based on the use of high-throughput phenotyping platform for screening new vegetal-derived protein hydrolysates (PHs) for biostimulant activity followed by a metabolomic analysis to elucidate the mechanism of the most active PHs has been applied on tomato crop. Eight PHs (A–G, I) derived from enzymatic hydrolysis of seed proteins of Leguminosae and Brassicaceae species were foliarly sprayed twice during the trial. A non-ionic surfactant Triton X-100 at 0.1% was also added to the solutions before spraying. A control treatment foliarly sprayed with distilled water containing 0.1% Triton X-100 was also included. Untreated and PH-treated tomato plants were monitored regularly using high-throughput non-invasive imaging technologies. The phenotyping approach we used is based on automated integrative analysis of photosynthetic performance, growth analysis, and color index analysis. The digital biomass of the plants sprayed with PH was generally increased. In particular, the relative growth rate and the growth performance were significantly improved by PHs A and I, respectively, compared to the untreated control plants. Kinetic chlorophyll fluorescence imaging did not allow to differentiate the photosynthetic performance of treated and untreated plants. Finally, MS-based untargeted metabolomics analysis was performed in order to characterize the functional mechanisms of selected PHs. The treatment modulated the multi-layer regulation process that involved the ethylene precursor and polyamines and affected the ROS-mediated signaling pathways. Although further investigation is needed to strengthen our findings, metabolomic data suggest that treated plants experienced a metabolic reprogramming following the application of the tested biostimulants. Nonetheless, our experimental data highlight the potential for combined use of high-throughput phenotyping and metabolomics to facilitate the screening of new substances with biostimulant properties and to provide a morpho-physiological and metabolomic gateway to the mechanisms underlying PHs action on plants

A Combined Phenotypic and Metabolomic Approach for Elucidating the Biostimulant Action of a Plant-Derived Protein Hydrolysate on Tomato Grown Under Limited Water Availability

Plant-derived protein hydrolysates (PHs) are an important category of biostimulants able to increase plant growth and crop yield especially under environmental stress conditions. PHs can be applied as foliar spray or soil drench. Foliar spray is generally applied to achieve a relatively short-term response, whereas soil drench is used when a long-term effect is desired. The aim of the study was to elucidate the biostimulant action of PH application method (foliar spray or substrate drench) on morpho-physiological traits and metabolic profile of tomato grown under limited water availability. An untreated control was also included. A high-throughput image-based phenotyping (HTP) approach was used to non-destructively monitor the crop response under limited water availability (40% of container capacity) in a controlled environment. Moreover, metabolic profile of leaves was determined at the end of the trial. Dry biomass of shoots at the end of the trial was significantly correlated with number of green pixels (R2 = 0.90) and projected shoot area, respectively. Both drench and foliar treatments had a positive impact on the digital biomass compared to control while the photosynthetic performance of the plants was slightly influenced by treatments. Overall drench application under limited water availability more positively influenced biomass accumulation and metabolic profile than foliar application. Significantly higher transpiration use efficiency was observed with PH-drench applications indicating better stomatal conductance. The mass-spectrometry based metabolomic analysis allowed the identification of distinct biochemical signatures in PH-treated plants. Metabolomic changes involved a wide and organized range of biochemical processes that included, among others, phytohormones (notably a decrease in cytokinins and an accumulation of salicylates) and lipids (including membrane lipids, sterols, and terpenes). From a general perspective, treated tomato plants exhibited an improved tolerance to reactive oxygen species (ROS)-mediated oxidative imbalance. Such capability to cope with oxidative stress might have resulted from a coordinated action of signaling compounds (salicylic acid and hydroxycinnamic amides), radical scavengers such as carotenoids and prenyl quinones, as well as a reduced biosynthesis of tetrapyrrole coproporphyrins

Valorization of bio-refinery by-products in potted ornamental shrub cultivation: effects on growth, water relation and leaf gas exchange

In the coming few years new research trends such as the production of green chemicals (e.g. biorefineries) will continue to gain more importance, in order to minimize the dependence on fossil-based resources. Furthermore, this exponential growth of biorefinery concept will contribute to the development of a circular economy model, where lignocellulosic materials, used to generate bio-based products, are recovered and recycled. The possibility of an agrochemical use of bio-refinery by-products is an important tool of innovation in horticulture in terms of sustainability and safety of the crops. By-products residuals often contain bio-molecules able to stimulate the plant performance. The aim of the current research was to assess the effects of a lignocellulosic by-product of the industrial process of extraction of succinic acid by Arundo donax biomass, on plant growth parameters, water relations, gas exchange and mineral composition of two potted grown ornamental shrubs: Bougainvillea spectabilis Willd and Viburnum lucidum L. The treatments consisted of a control (no residual addition) and the addition to the substrate of 40 or 80 g l-1 of residual. Our results clearly demonstrated that the addition of the residual significantly improved the crop performance of both ornamental shrubs. The addition of 80 g l-1 one time per month increased plant growth parameters such as plant height and shoot length and this was attributed to a better leaf gas exchange status

Reduced Cancer Incidence in Huntington's Disease: Analysis in the Registry Study

Background: People with Huntington's disease (HD) have been observed to have lower rates of cancers. Objective: To investigate the relationship between age of onset of HD, CAG repeat length, and cancer diagnosis. Methods: Data were obtained from the European Huntington's disease network REGISTRY study for 6540 subjects. Population cancer incidence was ascertained from the GLOBOCAN database to obtain standardised incidence ratios of cancers in the REGISTRY subjects. Results: 173/6528 HD REGISTRY subjects had had a cancer diagnosis. The age-standardised incidence rate of all cancers in the REGISTRY HD population was 0.26 (CI 0.22-0.30). Individual cancers showed a lower age-standardised incidence rate compared with the control population with prostate and colorectal cancers showing the lowest rates. There was no effect of CAG length on the likelihood of cancer, but a cancer diagnosis within the last year was associated with a greatly increased rate of HD onset (Hazard Ratio 18.94, p < 0.001). Conclusions: Cancer is less common than expected in the HD population, confirming previous reports. However, this does not appear to be related to CAG length in HTT. A recent diagnosis of cancer increases the risk of HD onset at any age, likely due to increased investigation following a cancer diagnosis