Valorization of Strawberry Juice Production Wastewater: Possibilities for Polyphenols Recovery and Plant Biostimulant Production.

Fruit juice production is one of the most important branches of the food and beverage industry, considering both the market size and demand. It is also one of the largest generators of industrial wastewater, considering the large consumption of fresh water during fruit processing. Hence, the appropriate treatment strategies are of the utmost importance to minimize the environmental footprint of food industry effluents. This study aimed to investigate the valorization routes for strawberry juice production wastewater (SJPW), both in terms of nutrient recovery and a circular approach to its utilization as a medium for plant biostimulant production. The results show a low antioxidant capacity and low content of polyphenols in SJPW; however, promising results were obtained for the in vitro seed germination and tomato growth promotion when investigating a biostimulant based on Bacillus sp. BioSol021, which was cultivated using SJPW in a lab-scale bioreactor, with root and shoot length improvements of approximately 30% and 25%, respectively, compared to the control samples. The plant growth promotion (PGP) traits indicated the ability of IAA production, in a concentration of 8.55 ± 0.05 mg/L, and the enzymatic activity was evaluated as through the enzymatic activity index (EAI), achieving the following: 2.26 ± 0.04 for cellulolytic activity, 2.49 ± 0.08 for hemicellulolytic activity, 2.91 ± 0.16 for pectinolytic activity, and 1.05 ± 0.00 for proteolytic activity. This study opens a new chapter of possibilities for the development of techno-economically viable circular bioprocess solutions aimed at obtaining value-added microbial products for sustainable agriculture based on the valorization of food industry effluents thus contributing to more sustainable food production at both the agricultural and industrial levels.

The Application of a Foliar Spray Containing Methylobacterium symbioticum Had a Limited Effect on Crop Yield and Nitrogen Recovery in Field and Pot-Grown Maize.

In this study, the effectiveness of an inoculant containing a nitrogen (N)-fixing microorganism (Methylobacterium symbioticum) was evaluated on maize (Zea mays L.) grown both in the field (silage maize) and in pots over two years (2021 and 2022). The field trial included the following two treatments: with (Yes) and without (No) the inoculant. The pot experiment was designed as a factorial arrangement with two factors: the application of the inoculant (Yes and No) and N applied to the soil (0, 0.4, 0.8, and 1.6 g pot-1). In the field, total dry matter yield (DMY) did not differ significantly between treatments, although the average DMY was higher in the inoculant treatment. In pots, the total DMY varied significantly across all N rates but was only significantly affected by the inoculant application in 2022. N fixation estimates in the field were 58.8 and 14.5 kg ha-1 for 2021 and 2022, respectively, representing 23.7% and 9.1% of the N recovered in the aboveground plant parts. In pots, the estimated fixed N values were -49.2 and 199.2 mg pot-1 in 2021 and 2022, respectively, which corresponded to -5.2% and 18.5% of the N found in the aboveground plant parts. Considering the average values obtained across the four cultivation conditions, there was a positive outcome for the treated plants. However, these values cannot be considered significant when compared to nitrogen removal in maize crops. A commercial product should provide an unequivocal and quantitatively relevant contribution to plant nutrition, which did not appear to be the case. Thus, for this inoculant to provide reliable guarantees of positive outcomes for farmers and become a useful tool in promoting more sustainable agriculture, further studies appear necessary. These studies should aim to determine in which crops and under what cultivation conditions the application of the inoculant is truly effective in enhancing N fixation and improving crop productivity.

Phenotypical and biochemical characterization of tomato plants treated with triacontanol.

Biostimulants are heterogeneous products designed to support plant development and to improve the yield and quality of crops. Here, we focused on the effects of triacontanol, a promising biostimulant found in cuticle waxes, on tomato growth and productivity. We examined various phenological traits related to vegetative growth, flowering and fruit yield, the metabolic profile of fruits, and the response of triacontanol-treated plants to salt stress. Additionally, a proteomic analysis was conducted to clarify the molecular mechanisms underlying triacontanol action. Triacontanol application induced advanced and increased blooming without affecting plant growth. Biochemical analyses of fruits showed minimal changes in nutritional properties. The treatment also increased the germination rate of seeds by altering hormone homeostasis and reduced salt stress-induced damage. Proteomics analysis of leaves revealed that triacontanol increased the abundance of proteins related to development and abiotic stress, while down-regulating proteins involved in biotic stress resistance. The proteome of the fruits was not significantly affected by triacontanol, confirming that biostimulation did not alter the nutritional properties of fruits. Overall, our findings provide evidence of the effects of triacontanol on growth, development, and stress tolerance, shedding light on its mechanism of action and providing new insights into its potential in agricultural practices.

Beach wrack: Discussing ecological roles, risks, and sustainable bioenergy and agricultural applications.

The equilibrium of the marine ecosystem is currently threatened by several constraints, among which climate change and anthropogenic activities stand out. Indeed, these factors favour the growth of macroalgae, which sometimes end up stranded on the beaches at the end of their life cycle, forming what is known as beach wrack. Despite its undeniable important ecological role on beaches, as it is an important source of organic matter (OM), and provides food and habitat for several invertebrates, reptiles, small mammals, and shorebirds, the overaccumulation of beach wrack is often associated with the release of greenhouse gases, negatively impacting tourist activities, and generating economic expenses for its removal. Although currently beach wrack is mainly treated as a waste, it can be used for numerous potential applications in distinct areas. This review aimed at providing a solid point of view regarding the process of wrack formation, its spatiotemporal location, as well as its importance and risks. It also contains the current advances of the research regarding sustainable alternatives to valorise this organic biomass, that range from bioenergy production to the incorporation of wrack in agricultural soils, considering a circular economy concept. Although there are some concerns regarding wrack utilisation, from its variable availability to a possible soil contamination with salts and other contaminants, this review comprises the overall beneficial effects of the incorporation of this residue particularly in the organic agricultural model, strengthening the conversion of this wasted biomass into a valuable resource.

Synthesis and characterization of chitosan-zinc-salicylic acid nanoparticles: A plant biostimulant.

The vastly expanding global population raised the demand for profuse food grain production. For food security in India, high yield and nutritional quality of grain crops, both are essential. Zinc is a crucial micronutrient generally deficient in food grains grown in India, reflecting their deteriorating nutritional quality. To address these issues, in the present study, a novel tri-component nanoparticle of chitosan­zinc-salicylic acid (CS-Zn-SA NPs) has been synthesized by ionotropic gelation method. The average size of synthesized CS-Zn-SA NPs was recorded 13.5 nm by dynamic light scattering (DLS) spectroscopy. The presence of chitosan, zinc and salicylic acid and crosslinking among these components in synthesized nanoparticles has been demonstrated by Fourier transforms infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). Further, synthesized CS-Zn-SA NPs at various concentrations (50-200 ppm) were evaluated for seed germination via seed priming, yield, grain zinc content and defence enzyme activity through the foliar application. CS-Zn-SA NPs revealed significant seed germination activities, 19.8 % higher grain yield, 45.5 % increased grain zinc content and manyfold defence enzyme activities than the control. The obtained results exposed the potential of CS-Zn-SA NPs as a stimulant for effective seedling development, higher yield, a virtuous micronutrient fortifying agent and defence enzyme promoter.

Influence of chicken feather waste derived protein hydrolysate on the growth of tea plants under different application methods and fertilizer rates.

Modern agriculture prioritizes eco-friendly and sustainable strategies to enhance crop growth and productivity. The utilization of protein hydrolysate extracted from chicken feather waste as a plant biostimulant paves the path to waste recycling. A greenhouse experiment was performed to evaluate the implications of different doses (0, 1, 2, and 3 g L-1) of chicken feather protein hydrolysate (CFPH), application method (soil and foliar), and fertilizer rate (50% and 100%) on the growth performance of tea nursery plants. The highest dose of CFPH (3 g L-1) increased the shoot and root dry weights by 43% and 70%, respectively over control. However, no significant differences were observed between 2 and 3 g L-1 doses in plant dry weight, biometric, and root morphological parameters. Foliar application of CFPH significantly increased all the growth parameters compared to soil drenching except N, P, and K concentrations in leaves and roots. Plants grown under 100% fertilizer rate showed better growth performance than 50% fertilizer rate. Tea nursery plants treated with foliar 2 g L-1 dose and grown under full fertilizer rate recorded the highest plant dry weight, root length, and root surface area. However, tea plants under 50% fertilizer rate and treated with foliar 2 and 3 g L-1 doses sustained the growth similar to untreated plants under 100% fertilizer rate. The significantly higher N, P, and K concentrations in leaves were observed in plants treated with soil drenching of 2 and 3 g L-1 CFPH doses under 100% fertilizer rate. Our results indicate that the application of CFPH as a foliar spray is highly effective in producing vigorous tea nursery plants suitable for field planting, eventually capable of withstanding stress and higher yield.

Habitat-adapted heterologous symbiont Salinispora arenicola promotes growth and alleviates salt stress in tomato crop plants.

To ensure food security given the current scenario of climate change and the accompanying ecological repercussions, it is essential to search for new technologies and tools for agricultural production. Microorganism-based biostimulants are recognized as sustainable alternatives to traditional agrochemicals to enhance and protect agricultural production. Marine actinobacteria are a well-known source of novel compounds for biotechnological uses. In addition, former studies have suggested that coral symbiont actinobacteria may support co-symbiotic photosynthetic growth and tolerance and increase the probability of corals surviving abiotic stress. We have previously shown that this activity may also hold in terrestrial plants, at least for the actinobacteria Salinispora arenicola during induced heterologous symbiosis with a wild Solanaceae plant Nicotiana attenuata under in vitro conditions. Here, we further explore the heterologous symbiotic association, germination, growth promotion, and stress relieving activity of S. arenicola in tomato plants under agricultural conditions and dig into the possible associated mechanisms. Tomato plants were grown under normal and saline conditions, and germination, bacteria-root system interactions, plant growth, photosynthetic performance, and the expression of salt stress response genes were analyzed. We found an endophytic interaction between S. arenicola and tomato plants, which promotes germination and shoot and root growth under saline or non-saline conditions. Accordingly, photosynthetic and respective photoprotective performance was enhanced in line with the induced increase in photosynthetic pigments. This was further supported by the overexpression of thermal energy dissipation, which fine-tunes energy use efficiency and may prevent the formation of reactive oxygen species in the chloroplast. Furthermore, gene expression analyses suggested that a selective transport channel gene, SlHKT1,2, induced by S. arenicola may assist in relieving salt stress in tomato plants. The fine regulation of photosynthetic and photoprotective responses, as well as the inhibition of the formation of ROS molecules, seems to be related to the induced down-regulation of other salt stress response genes, such as SlDR1A-related genes or SlAOX1b. Our results demonstrate that the marine microbial symbiont S. arenicola establishes heterologous symbiosis in crop plants, promotes growth, and confers saline stress tolerance. Thus, these results open opportunities to further explore the vast array of marine microbes to enhance crop tolerance and food production under the current climate change scenario.

Lysinibacillus xylanilyticus Strain GIC41 as a Potential Plant Biostimulant.

To identify Lysinibacillus strains with the potential to function as plant biostimulants, we screened 10 previously isolated Lysinibacillus strains from the rhizosphere and soil for their plant growth-promoting (PGP) effects. In vitro tests showed that all strains produced indole-3-acetic acid. In primary screening, the PGP effects of these strains were assessed on spinach seedlings grown on Jiffy-7 pellets; strains GIC31, GIC41, and GIC51 markedly promoted shoot growth. In secondary screening, the PGP efficacies of these three strains were examined using spinach seedlings grown in pots under controlled conditions. Only GIC41 exerted consistent and significant PGP effects; therefore, it was selected for subsequent experiments. The results of 6-week glasshouse experiments revealed that GIC41 markedly increased shoot dry weight by ca. 12-49% over that of the control. The impact of fertilization levels on the PGP efficacy of GIC41 was investigated using pot experiments. The application of a specific level of fertilizer was required for the induction of sufficient PGP effects by this strain. The phylogenetic ana-lysis based on the 16S rDNA sequence identified GIC41 as L. xylanilyticus. Collectively, these results show the potential of strain GIC41 to function as a plant biostimulant.

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses.

The use of plant biostimulants contributes to more sustainable and environmentally friendly farming techniques and offers a sustainable alternative to mitigate the adverse effects of stress. Protein hydrolysate-based biostimulants have been described to promote plant growth and reduce the negative effect of abiotic stresses in different crops. However, limited information is available about their mechanism of action, how plants perceive their application, and which metabolic pathways are activating. Here we used a multi-trait high-throughput screening approach based on simple RGB imaging and combined with untargeted metabolomics to screen and unravel the mode of action/mechanism of protein hydrolysates in Arabidopsis plants grown in optimal and in salt-stress conditions (0, 75, or 150 mM NaCl). Eleven protein hydrolysates from different protein sources were used as priming agents in Arabidopsis seeds in three different concentrations (0.001, 0.01, or 0.1 µl ml-1). Growth and development-related traits as early seedling establishment, growth response under stress and photosynthetic performance of the plants were dynamically scored throughout and at the end of the growth period. To effectively classify the functional properties of the 11 products a Plant Biostimulant Characterization (PBC) index was used, which helped to characterize the activity of a protein hydrolysate based on its ability to promote plant growth and mitigate stress, and to categorize the products as plant growth promoters, growth inhibitors and/or stress alleviator. Out of 11 products, two were identified as highly effective growth regulators and stress alleviators because they showed a PBC index always above 0.51. Using the untargeted metabolomics approach, we showed that plants primed with these best performing biostimulants had reduced contents of stress-related molecules (such as flavonoids and terpenoids, and some degradation/conjugation compounds of phytohormones such as cytokinins, auxins, gibberellins, etc.), which alleviated the salt stress response-related growth inhibition.

Ulva lactuca Extract and Fractions as Seed Priming Agents Mitigate Salinity Stress in Tomato Seedlings.

The present study investigates the effect of Ulva lactuca extract as seed-priming agent for tomato plants under optimal and salinity stress conditions. The aims of this experiment were to assess the effect of seed priming using Ulva lactuca extract in alleviating the salinity stress tomato plants were subjected to, and to find out the possible mechanism of actions behind such a positive effect via means of fractionation of the crude extract and characterization. Salinity application decreased the plant biomass and altered different physiological traits of tomato. However, the application of Ulva lactuca methanol extract (ME) and its fractions (residual fraction (RF), chloroform fraction (CF), butanol fraction (BF), and hexane fraction (HF)) at 1 mg·mL-1 as seed priming substances attenuated the negative effects of salinity on tomato seedlings. Under salinity stress conditions, RF application increased the tomato fresh weight; while ME, RF, and HF treatments significantly decreased the hydrogen peroxide (H2O2) concentration and antioxidant activity in tomato plants. The biochemical analyses of Ulva lactuca extract and fractions showed that the RF recorded the highest concentration of glycine betaine, while the ME was the part with the highest concentrations of total phenols and soluble sugars. This suggests that these compounds might play a key role in the mechanism by which seaweed extracts mitigate salinity stress on plants.

Effects of phosphite as a plant biostimulant on metabolism and stress response for better plant performance in Solanum tuberosum.

Food availability represents a major worldwide concern due to population growth, increased demand, and climate change. Therefore, it is imperative to identify compounds that can improve crop performance. Plant biostimulants have gained prominence because of their potentials to increase germination, productivity and quality of a wide range of horticultural and agronomic crops. Phosphite (Phi), an analog of orthophosphate, is an emerging biostimulant used in horticulture and agronomy. The aim of this study was to uncover the molecular mechanisms through which Phi acts as a biostimulant with potential effects of overall plant growth. Field and greenhouse experiments, using 4 potato cultivars, showed that following Phi applications, plant performance, including several physio-biochemical traits, crop productivity, and quality traits, were significantly improved. RNA sequencing of control and Phi-treated plants of cultivar Xingjia No. 2, at 0 h, 6 h, 24 h, 48 h, 72 h and 96 h after the Phi application for 24 h revealed extensive changes in the gene expression profiles. A total of 2856 differentially expressed genes were identified, suggesting that multiple pathways of primary and secondary metabolism, such as flavonoids biosynthesis, starch and sucrose metabolism, and phenylpropanoid biosynthesis, were strongly influenced by foliar applications of Phi. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses associated with defense responses revealed significant effects of Phi on a plethora of defense mechanisms. These results suggest that Phi acted as a biostimulant by priming the plants, that was, by triggering dynamic changes in gene expression and modulating metabolic fluxes in a way that allowed plants to perform better. Therefore, Phi usage has the potential to improve crop yield and health, alleviating the challenges posed by the need of feeding a growing world population, while minimizing the agricultural impact on human health and environment.

Co-application of Se and a biostimulant at different wheat growth stages: Influence on grain development.

An appropriate selenium intake can be beneficial for human health. Se-biofortified food in Se-deficient regions is becoming an increasingly common practice but there are still issues to be addressed regarding the observed Se-induced toxicity to the plant. In this respect, plant biostimulants are used to enhance nutrition efficiency, abiotic stress tolerance and crop quality. In this work, the efficacy of a plant biostimulant to counteract the Se-induced stress in wheat plants is experimentally assessed. The co-application of different Se-biofortification treatments and the biostimulant at different growth stages (tillering or heading stage) was investigated. The use of micro focused X-ray spectroscopy allows us to confirm organic Se species to be the main Se species found in wheat grain and that the proportion of organic Se species is only slightly affected by the Se application stage. Our study proves that the biostimulant had a key role in the enhancement of both the amount of grains produced per spike and their dry biomass without hindering Se enrichment process, neither diminishing the Se concentration nor massively disrupting the Se species present. This information will be useful to minimize both plant toxicity and economic cost towards a more effective and plant healthy selenium supplementation.

Transcriptional and Physiological Analyses to Assess the Effects of a Novel Biostimulant in Tomato.

This work aimed to study the effects in tomato (Solanum lycopersicum L.) of foliar applications of a novel calcium-based biostimulant (SOB01) using an omics approach involving transcriptomics and physiological profiling. A calcium-chloride fertilizer (SOB02) was used as a product reference standard. Plants were grown under well-watered (WW) and water stress (WS) conditions in a growth chamber. We firstly compared the transcriptome profile of treated and untreated tomato plants using the software RStudio. Totally, 968 and 1,657 differentially expressed genes (DEGs) (adj-p-value < 0.1 and |log2(fold change)| ≥ 1) were identified after SOB01 and SOB02 leaf treatments, respectively. Expression patterns of 9 DEGs involved in nutrient metabolism and osmotic stress tolerance were validated by real-time quantitative reverse transcription PCR (RT-qPCR) analysis. Principal component analysis (PCA) on RT-qPCR results highlighted that the gene expression profiles after SOB01 treatment in different water regimes were clustering together, suggesting that the expression pattern of the analyzed genes in well water and water stress plants was similar in the presence of SOB01 treatment. Physiological analyses demonstrated that the biostimulant application increased the photosynthetic rate and the chlorophyll content under water deficiency compared to the standard fertilizer and led to a higher yield in terms of fruit dry matter and a reduction in the number of cracked fruits. In conclusion, transcriptome and physiological profiling provided comprehensive information on the biostimulant effects highlighting that SOB01 applications improved the ability of the tomato plants to mitigate the negative effects of water stress.

Hormopriming to Mitigate Abiotic Stress Effects: A Case Study of N 9-Substituted Cytokinin Derivatives With a Fluorinated Carbohydrate Moiety.

Drought and salinity reduce seed germination, seedling emergence, and early seedling establishment, affect plant metabolism, and hence, reduce crop yield. Development of technologies that can increase plant tolerance of these challenging growth conditions is a major current interest among plant scientists and breeders. Seed priming has become established as one of the practical approaches that can alleviate the negative impact of many environmental stresses and improve the germination and overall performance of crops. Hormopriming using different plant growth regulators has been widely demonstrated as effective, but information about using cytokinins (CKs) as priming agents is limited to only a few studies using kinetin or 6-benzylaminopurine (BAP). Moreover, the mode of action of these compounds in improving seed and plant fitness through priming has not yet been studied. For many years, BAP has been one of the CKs most commonly applied exogenously to plants to delay senescence and reduce the impact of stress. However, rapid endogenous N 9-glucosylation of BAP can result in negative effects. This can be suppressed by hydroxylation of the benzyl ring or by appropriate N 9 purine substitution. Replacement of the 2' or 3' hydroxyl groups of a nucleoside with a fluorine atom has shown promising results in drug research and biochemistry as a means of enhancing biological activity and increasing chemical or metabolic stability. Here, we show that the application of this chemical modification in four new N 9-substituted CK derivatives with a fluorinated carbohydrate moiety improved the antisenescence properties of CKs. Besides, detailed phenotypical analysis of the growth and development of Arabidopsis plants primed with the new CK analogs over a broad concentration range and under various environmental conditions revealed that they improve growth regulation and antistress activity. Seed priming with, for example, 6-(3-hydroxybenzylamino)-2'-deoxy-2'-fluoro-9-(ß)-D-arabinofuranosylpurine promoted plant growth under control conditions and alleviated the negative effects of the salt and osmotic stress. The mode of action of this hormopriming and its effect on plant metabolism were further analyzed through quantification of the endogenous levels of phytohormones such as CKs, auxins and abscisic acid, and the results are discussed.

Comparison of Soaking Corms with Moringa Leaf Extract Alone or in Combination with Synthetic Plant Growth Regulators on the Growth, Physiology and Vase Life of Sword Lily.

Gladiolus is in demand worldwide as a cut-flower or landscaping plant, because of its superior commercial and ornamental value. Application of plant-based biostimulants has gained interest in the horticulture industry as an innovative and promising approach to ensure enhanced and sustainable yields along with better product quality. The influence of pre-plant corm soaks supplemented to 5% (v/v) with an aqueous extract from Moringa oleifera leaves (MLE) either alone or in combination with 50 mg/L salicylic acid (SA) or 50 mg/L gibberellic acid (GA) on the vegetative, physiological, and ornamental characteristics of potted gladiolus (Gladiolus grandiflorus) was investigated. In general, the treatment order for greatest horticultural value for all the parameters examined was: MLE + SA + GA > MLE + GA or SA individually > MLE alone > water-only control. Compared to other treatments, corms soaked in MLE + SA + GA had the earliest sprout time (3.7 days earlier than control), shortest production time (11 days earlier than control), tallest plant (159.5 cm), greatest number of leaves per plant (8.85 leaves), greatest maximum leaf area (66 cm2), highest SPAD reading (112) and photosynthetic activity (6.7 mmol m-2 s-1), longest spike length (91 cm), greatest number of florets per spike (20 florets), longest vase life (14.8 days), greatest N (1.53%), P (0.28%), and K (0.64%) concentrations, and largest corm diameter (4.68 cm) and mass (22.25 g). The highest total protein and proline concentrations were observed with the combined application of MLE + GA + SA. Our findings suggested that MLE either alone or in combination with other plant growth regulators not only increased the yield and quality of cut spikes, but also prolonged the vase life of cut gladiolus.

Physiological, Ecological, and Biochemical Implications in Tomato Plants of Two Plant Biostimulants: Arbuscular Mycorrhizal Fungi and Seaweed Extract.

The worldwide use of plant biostimulants (PBs) represents an environmentally friendly tool to increase crop yield and productivity. PBs include different substances, compounds, and growth-promoting microorganism formulations, such as those derived from arbuscular mycorrhizal fungi (AMF) or seaweed extracts (SEs), which are used to regulate or enhance physiological processes in plants. This study analyzed the physiological, ecological, and biochemical implications of the addition of two PBs, AMF or SE (both alone and in combination), on tomato plants (Solanum lycopersicum L. cv. "Rio Fuego"). The physiological responses evaluated were related to plant growth and photosynthetic performance. The ecological benefits were assessed based on the success of AMF colonization, flowering, resistance capacity, nonphotochemical quenching (NPQ), and polyphenol content. Biochemical effects were evaluated via protein, lipid, carbohydrate, nitrogen, and phosphorous content. Each PB was found to benefit tomato plants in a different but complementary manner. AMF resulted in an energetically expensive (high ETRMAX but low growth) but protective (high NPQ and polyphenol content) response. AMF + nutritive solution (NS) induced early floration but resulted in low protein, carbohydrate, and lipid content. Both AMF and AMF + NS favored foliar instead of root development. In contrast, SE and SE + NS favored protein content and root development and did not promote flowering. However, the combination of both PBs (AMF + SE) resulted in an additive effect, reflected in an increase in both foliar and root growth as well as protein and carbohydrate content. Moreover, a synergistic effect was also found, which was expressed in accelerated flowering and AMF colonization. We present evidence of benefits to plant performance (additive and synergistic) due to the interactive effects between microbial (AMF) and nonmicrobial (SEs) PBs and propose that the complementary modes of action of both PBs may be responsible for the observed positive effects due to the new and emerging properties of their components instead of exclusively being the result of known constituents. These results will be an important contribution to biostimulant research and to the development of a second generation of PBs in which combined and complementary mechanisms may be functionally designed.

Stand-Alone and Combinatorial Effects of Plant-based Biostimulants on the Production and Leaf Quality of Perennial Wall Rocket.

Modern agriculture is facing many difficulties due to a rapidly changing climate, and environmental damage from agricultural production. The commitment of scientists and farmers to increase environmentally sustainable agricultural practices is one way to help mitigate environmental impacts. Among these practices, the use of biostimulants could be beneficial for increasing fertilizer efficiency and reducing excessive use in agriculture, and as plant growth regulators capable of increasing both production volume and quality of crops. In our study, rocket plants were grown in a greenhouse and treated with two biostimulants (protein hydrolysates or tropical plant extract), either individually or combined, to assess the effect on yield, dry biomass, mineral content, qualitative parameters as well as on economic profitability of foliar biostimulant applications. Total yield and dry biomass of the plants treated with the three biostimulant combinations on average increased by 48.1% and 37.2% respectively compared to untreated plants, without significant differences between treatments. Biostimulant application increased the content of chlorophyll, K, Mg and Ca, compared to the untreated plants. Furthermore, a biostimulant synergistic effect was detected concerning the content of total ascorbic acid. Our results confirmed that the biostimulants are eco-friendly products, able to boost plant growth and product quality and thus increase growers' profitability.

Regulatory risks associated with bacteria as biostimulants and biofertilizers in the frame of the European Regulation (EU) 2019/1009.

Agriculture represents an important mechanism for the reduction in plant, animal and microbial biodiversity and the alteration of the environment. The high pressure to cope with increasing demands of food for the human population intensifies the environmental impact and requires alternative ways to produce more food and to minimize the decrease in biodiversity. The use of biostimulants and biofertilizers could represent one of such alternatives. However, we need to guarantee that their use does not result in a greater damage for human health and for the environment. The European authorities try to protect humans, plants, animals and the environment by developing the recent European Regulation (EU) 2019/1009 on biostimulants. In the present study, we discuss the taxonomic approach of the EU to limit the use of microorganisms as biostimulants. We also propose an alternative method to evaluate the safety of microorganisms that are used as biostimulants and biofertilizers. That is, we suggest the use of panels of bioassays on model organisms and potential improvements on this system to evaluate the risks associated with the use of microorganisms as biostimulants and biofertilizers.

Effect of cell disruption methods on the extraction of bioactive metabolites from microalgal biomass.

Microalgae synthesize a variety of potentially high-value compounds. Due to their robust cell wall, cell disruption is necessary to improve extraction of these compounds. While cell disruption methods have been optimized for lipid and protein extraction, there are limited studies for other bioactive compounds. The present study investigated the effect of freeze-drying combined with sonication or ball-milling on the extraction of antioxidant and plant biostimulating compounds from Chlorella sp., Chlorella vulgaris and Scenedesmus acutus. Both cell disruption methods resulted in higher extract yields from the biomass compared to freeze-dried biomass using 50% methanol as a solvent. Antioxidant activity of Chlorella extracts was generally higher than freeze-dried extracts based on the diphenylpicrylhydrazyl (DPPH) and ß-carotene linoleic acid assays. However, the effectiveness of each treatment varied between microalgae strains. Sonication resulted in the highest antioxidant activity in Chlorella sp. extracts. Ball-milling gave the best results for C. vulgaris extracts in the DPPH assay. Both cell disruption methods decreased antioxidant activity in S. acutus extracts. Plant biostimulating activity was tested using the mung bean rooting assay. Damaging the membrane by freeze-drying was sufficient to release the active compounds using water extracts. In contrast, both cell disruption methods negatively affected the biological activity of the extracts. These results indicate that bioactive compounds in microalgae are sensitive to post-harvest processes and their biological activity can be negatively affected by cell disruption methods. Care must be taken to not only optimize yield but to also preserve the biological activity of the target compounds.

Effects of Megafol on the Olive Cultivar 'Arbequina' Grown Under Severe Saline Stress in Terms of Physiological Traits, Oxidative Stress, Antioxidant Defenses, and Cytosolic Ca2.

Salinity is one of the most impacting abiotic stresses regarding crop productivity and quality. Among the strategies that are attracting attention in the protection of crops from abiotic stresses, there is the use of plant biostimulants. In this study, Megafol (Meg), a commercial plant biostimulant, was tested on olive plants subjected to severe saline stress. Plants treated with salt alone showed substantial reductions in biomass production, leaf net photosynthesis (Pn), leaf transpiration rate (E), stomatal conductance (gs), and relative water content (RWC). In addition, samples stressed with NaCl showed a higher sodium (Na+) content in the leaves, while those stressed with NaCl and biostimulated with Meg increased the potassium (K+) content in the leaves, thus showing a higher K+/Na+ ratio. Salinity caused the accumulation of significant quantities of hydrogen peroxide (H2O2) and malondialdehyde (MDA) due to decreases in the activity of antioxidant enzymes, namely superoxide dismutase (SOD - EC 1.15.1.1), ascorbate peroxidase (APX - EC 1.11.1.11), guaiacol peroxidase (GPX - EC 1.11.1.9), and catalase (CAT - EC 1.11.1.6). When olive plants under saline stress were biostimulated with Meg, the plants recovered and showed physiological and biochemical traits much improved than salt stressed samples. Finally, Meg exhibited Ca2+-chelating activity in olive pollen grains, which allowed the biostimulant to exert this beneficial effect also by antagonizing the undesirable effects of hydrogen peroxide on Ca2+ metabolism.