Biostimulant Products Research Articles

Anthocyanins: Biosynthesis, Distribution, Ecological Role, and Use of Biostimulants to Increase Their Content in Plant Foods—A Review

In the past century, plant biostimulants have been increasingly used in agriculture as innovative and sustainable practice. Plant biostimulants have been mainly investigated as potential agents able to mitigate abiotic stress. However, few information is available about their ability to influence fruit quality or change fruit phytochemical composition. In particular, very little is known about their effects on anthocyanin synthesis and accumulation. Due to the increasing demand of consumers for healthier foods with high nutraceutical values, this review tries to fill the gap between anthocyanin content and biostimulant application. Here, we elucidate the chemical structure, biosynthetic pathway, plant distribution, and physiological role of anthocyanins in plants. Moreover, we discuss the potential implications for human health derived from the consumption of foods rich in these molecules. Finally, we report on literature data concerning the changes in anthocyanin content and profile after the application of biostimulant products on the most common anthocyanin-containing foods.

Evaluating biological thatch control on Zoysia matrella (L.) Merr. golf greens

AbstractThatch is a layer of living and dead plant material (stems and roots) between turfgrass leaf tissue and the soil surface. If excessive, thatch can decrease turf playability, increase scalping and disease pressure, reduce pesticide efficacy and water infiltration, and harbor insects. In golf putting greens, disruptive mechanical practices such as vertical mowing, core cultivation, grooming, and topdressing are traditional agronomic methods for managing thatch/organic matter. Non‐disruptive biological thatch reducers have been used as an alternative. Field studies were conducted to evaluate two commercial biostimulant products, a liquid vermicompost extract marketed as Worm Power® Turf and a liquid humate derivative marketed as EarthMAX, and their ability to reduce thatch on a Zoysia matrella (L.) Merr. ‘Diamond’ putting green; rooting depth and weight were also evaluated. EarthMAX® was applied at two rates/frequencies, EarthMAX (1) and EarthMAX (2). In addition, two industry standards were included: blackstrap molasses and sand topdressing. In Year 2, untreated resulted in ≥42% thicker thatch than EarthMAX (2) and blackstrap molasses, no other treatment was statistically different to untreated for both years. All treatments provided lower (≥48%) thatch thickness than the industry standard sand topdressing for both years. No treatment performed differently to the untreated for thatch weight. Blackstrap molasses provided greatest root weight for both years (≥133% and ≥184% than all other treatments); however, it also provided amongst the lowest root length. Inconsistent results make it difficult to recommend biostimulants for thatch reduction. Improvements amongst some treatments over untreated and sand topdressing indicate potential benefits of their use.

Targeting Ascophyllum nodosum (L.) Le Jol. extract application at five growth stages of winter wheat

Seaweed extracts are a category of biostimulant products that have the potential to promote plant growth and thus, have given a promise to develop more resilient systems of crop production that make efficient use of water and nutrients. This field study evaluated the effect of application timing of an Ascophyllum nodosum extract on nutrient uptake and grain yield of winter wheat when the extract was applied singly in five growth stages from 2017 to 2019. Analysis by orthogonal polynomial contrasts indicated that extract application significantly increased yield or biomass in all three site-years. Application at the tillering stage increased average yield by 17%, grain nutrient accumulation (N, P, K) by 21–24%, and N-use efficiency by 11% over the three site-year period, but extract effects were not consistent between site-years both in terms of optimal growth stages of application and magnitude of crop responses. While grain yield increased by 39% with extract application at tillering in 2017, yield gains ranging from 16 to 22% were obtained at the stem elongation and booting stages in 2018. These differences were likely the result of site-year variations in weather patterns and soil properties. Among macro-nutrients, only grain N accumulation was linearly correlated to grain yield across site-years indicating that increased soil N uptake and/or remobilization to the reproductive organs was a key process of the A. nodosum mode of action. An economic analysis indicated that the average return over extract cost was higher than that of the zero-extract control by €79-100 ha-1 when the extract was applied at the tillering or stem elongation stage and assuming higher dilution rates recommended by the industry. Even greater economic benefits can result if N rates are substantially reduced without an adverse effect on yield gains.

Evaluating biological thatch control in turfgrass

AbstractThatch is a layer of living and dead plant material between turfgrass leaf tissue and soil surface; it can decrease playability, increase scalping and disease pressure, reduce pesticide efficacy and water infiltration, plus harbor insects. In golf putting greens, disruptive mechanical practices such as vertical mowing, core cultivation, grooming, and topdressing are traditionally used for managing thatch; however, several biological products have been marketed as non‐disruptive thatch reducers. Greenhouse experiments were conducted to evaluate thatch reduction and rooting depth of two commercial humic acid based biostimulant products, Worm Power (WP) and EarthMAX (EM). Products were applied at labelled rates and timings. EM was applied at two rates, indicated as EM (1) and EM (2). Two industry standards were also included: blackstrap molasses and sand topdressing. For both years, blackstrap molasses, EM (1), and EM (2) reduced thatch thickness compared to untreated by 30, 24, and 18% respectively. However, no decrease in thatch weight by treatment was observed. In Year 1, EM (1), and sand topdressing provided an average of 16% greater root length than untreated. However, in Year 2, WP provided 16% greater root length than untreated. EM (1) provided 117% greater root mass than untreated control in Year 2. No treatment provided greater root mass in Year 1. Thatch depth reduction indicate these products may be beneficial, but, inconsistent results mean further research is required.

Biostimulant Activity of Azotobacter chroococcum and Trichoderma harzianum in Durum Wheat under Water and Nitrogen Deficiency

Biostimulants hold great potential for developing integrated sustainable agriculture systems. The rhizobacteria Azotobacter chroococcum strain 76A and the fungus Trichoderma harzianum strain T22, with demonstrated biostimulant activity in previous systems, were evaluated in Triticum durum cv Creso for their ability to enhance growth and tolerance to drought stress. Growth and drought tolerance were evaluated in conditions of low and high soil nitrogen, with two levels of water stress. T. harzianum increased plant growth (+16%) under control conditions and tolerance to moderate drought stress (+52%) under optimal fertilization, while A. chroococcum conferred a growth penalty (−28%) in well-watered conditions under suboptimal fertilization and increased tolerance only under extreme drought stress (+15%). This growth penalty was ameliorated by nitrogen fertilization. T. harzianum abundance was found to be positively correlated to extreme soil drying, whereas A. chroococcum-induced tolerance was dependent on soil nitrogen availability. These results indicate that while biostimulants may enhance growth and stress tolerance, nutrient availability soil and environmental conditions heavily influence these responses. These interactions should be considered when designing biostimulant products targeted to specific cultural conditions.

Visualization of probiotics via epifluorescence microscopy and fluorescence in situ hybridization (FISH)

Aerobic plate counts, the standard for bacterial enumeration in the probiotic industry, can be biased towards fast-growing organisms that replicate on synthetic media and can significantly underestimate total bacterial abundance. Culture-independent approaches such as fluorescence in situ hybridization (FISH) hold promise as a means to rapidly and accurately enumerate bacteria in probiotic products. In addition, FISH has the potential to more accurately represent bacterial growth dynamics in the environment in which products are applied without imposing additional growth constraints that are required for enumeration via plate counts. In this study, we designed and optimized three new FISH probes to visualize and quantify Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus licheniformis within probiotic products. Microscopy-based estimates were consistent or higher than label claims for Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus in both a direct fed microbial (DFM) product as well as a crop microbial biostimulant (CMB) product. Quantification with FISH after a germination experiment revealed the potential for this approach to be used after application of the product.

Physiological, Nutritional and Metabolomic Responses of Tomato Plants After the Foliar Application of Amino Acids Aspartic Acid, Glutamic Acid and Alanine.

Agriculture is facing a great number of different pressures due to the increase in population and the greater amount of food it demands, the environmental impact due to the excessive use of conventional fertilizers, and climate change, which subjects the crops to extreme environmental conditions. One of the solutions to these problems could be the use of biostimulant products that are rich in amino acids (AAs), which substitute and/or complement conventional fertilizers and help plants adapt to climate change. To formulate these products, it is first necessary to understand the role of the application of AAs (individually or as a mixture) in the physiological and metabolic processes of crops. For this, research was conducted to assess the effects of the application of different amino acids (Aspartic acid (Asp), Glutamic acid (Glu), L-Alanine (Ala) and their mixtures Asp + Glu and Asp + Glu + Ala on tomato seedlings (Solanum lycopersicum L.). To understand the effect of these treatments, morphological, physiological, ionomic and metabolomic studies were performed. The results showed that the application of Asp + Glu increased the growth of the plants, while those plants that received Ala had a decreased dry biomass of the shoots. The greatest increase in the growth of the plants with Asp + Glu was related with the increase in the net CO2 assimilation, the increase of proline, isoleucine and glucose with respect to the rest of the treatments. These data allow us to conclude that there is a synergistic effect between Aspartic acid and Glutamic acid, and the amino acid Alanine produces phytotoxicity when applied at 15 mM. The application of this amino acid altered the synthesis of proline and the pentose-phosphate route, and increased GABA and trigonelline.

Effects of flowering and production inducers in the Mexican lime (Citrus aurantifolia Swingle)

Objective: To evaluate new hormonal and biostimulant products to induce flowering of Mexican lime (Citrus aurantifolia Swingle) for winter production at the state of Guerrero, Mexico. Design/methodology/approach: Prohexadione calcium (PHD-Ca), cytokinins, paclobutrazol, biuretic urea, granulated urea (46%) and a control were evaluated. Results: There are differences between treatments for the number of flowers and number of fruits (Pr>0.0411 and 0.048); regard the “number of flowers” variable, paclobutrazol, prohexadione calcium and granulated urea (46%) stood out; while, prohexadione calcium, paclobutrazol and cytokinins favor the “number of fruits” variable. The yields show statistical differences (Pr>0.0332) and the cytokinins and paclobutrazol stood out. Findings/conclusions: The usage of cytokinins for induction and winter production of Mexican lime is suggested.

Selection of plant growth promoting rhizobacteria sharing suitable features to be commercially developed as biostimulant products

Plant biostimulants (PBs) are an eco-friendly alternative to chemical fertilisers because of their minimal or null impact on human health and environment, while ensuring optimal nutrient uptake and increase of crop yield, quality and tolerance to abiotic stress. Although there is an increasing interest on microbial biostimulants, the optimal procedure to select and develop them as commercial products is still not well defined. This work proposes and validates a procedure to select the best plant growth promoting rhizobacteria (PGPR) as potential active ingredients of commercial PBs. The stepwise screening strategy was designed based on literature analysis and consists of six steps: (i) determination of the target crop and commercial strategy, (ii) selection of growth media for the isolation of microbial candidates, (iii) screening for traits giving major agronomical advantages, (iv) screening for traits related to product development, (v) characterisation of the mode of action of PGPR and (vi) assessment of plant growth efficacy. The strategy was validated using a case study: PGPR combined with humic acids to be applied on tomato plants. Among 200 bacterial strains isolated from tomato rhizosphere, 39 % were able to grow in presence of humic acids and shared the ability to solubilise phosphate. After the screening for traits related to product development, only 6 % of initial bacterial strains were sharing traits suitable for the further development as potential PBs. In fact, the selected bacterial strains were able to produce high cell mass and tolerated drought, aspects important for the mass production and formulation. These bacterial strains were not able to produce antibiotics, establish pathogenic interaction with plants and did not belong to bacterial species associated to human, animal and plant diseases. Most importantly, five of the selected bacterial strains were able to promote tomato seedling vigour in experiments carried out in vitro. These bacterial strains were furtherly characterised for their ability to colonize effectively tomato plant roots, produce phytohormones and solubilise soil minerals. This characterisation led to the selection of two candidates that showed the ability to promote tomato plant growth in experiments carried out in greenhouse conditions. Overall, this work provides a flow diagram for the selection of PGPR candidates to be successfully developed and commercialized as PBs. The validation of the flow diagram led to the selection of two bacterial strains belonging to Pantoea and Pseudomonas genera, potential active ingredients of new commercial PBs.

Application of Biostimulants Containing Amino Acids to Tomatoes Could Favor Sustainable Cultivation: Implications for Tyrosine, Lysine, and Methionine

Tomato (Solanum lycopersicum L.) is one of the most important crops worldwide as per its production and the surface cultivated. The use of biostimulant products plays a fundamental role in mitigating the negative effects of climate change and reducing the use of conventional fertilizers. Many of these products are formulated with amino acids (AAs). This study was conducted to elucidate the effects of the foliar application of tyrosine (Tyr) (15 mM), lysine (Lys) (15 mM), methionine (Met) (15 mM), and a Tyr + Lys + Met (15 mM + 15 mM + 15 mM) mixture on the physiological and metabolic processes, vegetative growth, and nutritional state of Optima variety tomato plants. The results showed that application of the AAs, individually and combined, was beneficial for the growth of the aerial part, net assimilation of CO2, and water use efficiency (WUE). Application of Tyr resulted in the best WUE. The metabolomics study revealed that AA treatments increased the concentration of proline, fructose, and glucose, whose role was to stimulate glycolysis and the Krebs cycle. Thus, the plants could have greater reduction power and energy, as well as more carbon molecules for their growth processes.

Management of Abiotic Stress in Horticultural Crops: Spotlight on Biostimulants

Horticultural crops are currently exposed to multiple abiotic stresses because of ongoing climate change. Abiotic stresses such as drought, extreme temperatures, salinity, and nutrient deficiencies are causing increasing losses in terms of yield and product quality. The horticultural sector is therefore searching for innovative and sustainable agronomic tools to enhance crop tolerance towards these unfavorable conditions. In a recent review published in Agronomy, “Biostimulants Application in Horticultural Crops under Abiotic Stress Conditions”, Bulgari and colleagues discussed the main pieces of evidence of the use of biostimulants to manage abiotic stresses in vegetable crops. The intent of this editorial was to focus the attention on aspects related to the stress development in plants (i.e., timing and occurrence of multiple stress factors), in combination with the application of biostimulants. The large number of factors potentially involved in the enhancement of crop tolerance toward stress calls for an intensification of research activities, especially when conducted in field conditions and with well-defined protocols. This must be seen as a mandatory task for a successful implementation of biostimulant products among the available agronomic tools for the management of abiotic stresses in horticultural crops.

Field-Scale Evaluation of Botanical Extracts Effect on the Yield, Chemical Composition and Antioxidant Activity of Celeriac (Apium graveolens L. Var. rapaceum).

The use of higher plants for the production of plant growth biostimulants is receiving increased attention among scientists, farmers, investors, consumers and regulators. The aim of the present study was to examine the possibility of converting plants commonly occurring in Europe (St. John’s wort, giant goldenrod, common dandelion, red clover, nettle, valerian) into valuable and easy to use bio-products. The biostimulating activity of botanical extracts and their effect on the chemical composition of celeriac were identified. Plant-based extracts, obtained by ultrasound-assisted extraction and mechanical homogenisation, were tested in field trials. It was found that the obtained formulations increased the total yield of leaves rosettes and roots, the dry weight of leaves rosettes and roots, the content of chlorophyll a + b and carotenoids, the greenness index of leaves, the content of vitamin C in leaves and roots. They mostly decreased the content of polyphenols and antioxidant activities in leaves but increased them in roots and conversely affected the nitrates content. Extracts showed a varied impact on the content of micro and macroelements, as well as the composition of volatile compounds and fatty acids in the celeriac biomass. Due to the modulatory properties of the tested products, they may be used successfully in sustainable horticulture.

Microalgae: New Source of Plant Biostimulants

Biostimulant manufacturers have developed innovative products targeting specific agronomic needs, hence attracting the attention of the scientific community, extension specialists, and industry stakeholders including policymakers and crop producers. Microalgae acquire a broad economic value in the production of nutrient dense food and supplementary diet produce, in addition to their high importance in biofuel production and wastewater bioremediation. Recently, microalgae, which comprise blue-green algae (eukaryotic and prokaryotic cyanobacteria), have gained prominence as biostimulant products due to their potential to increase germination, seedling growth, plant growth, productivity, nutrient use efficiency, as well as tolerance to a wide range of abiotic stresses (salinity, drought, sub- and supra-optimal temperatures, and heavy metals contamination). Although it is well established that green and blue-green algae produce several bioactive and signaling molecules active on horticultural and agronomic crops, their targeted applications in plant science are still in their infancy stage. The aim of this editorial paper is to provide an updated overview of this far-reaching new category of plant biostimulants and the possible physiological and molecular mechanisms behind the biostimulatory action based on the recent scientific literature. Finally, this editorial paper identifies the main bottlenecks that hamper market introduction and farmers from reaping the full benefit of microalgae-based biostimulants; it also pinpoints the future relevant areas of microalgae research to enhance the biostimulant action of microalgal extracts in agriculture.

Combining Molecular Weight Fractionation and Metabolomics to Elucidate the Bioactivity of Vegetal Protein Hydrolysates in Tomato Plants.

The comprehension of the bioactive fractions involved in the biostimulant activity of plant derived protein hydrolysates (PH) is a complex task, but it can also lead to significant improvements in the production of more effective plant biostimulants. The aim of this work is to shed light onto the bioactivity of different PH dialysis fractions (PH1 < 0.5–1 kDa; PH2 > 0.5–1 kDa; PH3 < 8–10 kDa; PH4 > 8–10 kDa) of a commercial PH-based biostimulant through a combined in vivo bioassay and metabolomics approach. A first tomato rooting bioassay investigated the auxin-like activity of PH and its fractions, each of them at three nitrogen levels (3, 30, and 300 mg L−1 of N) in comparison with a negative control (water) and a positive control (indole-3-butyric acid, IBA). Thereafter, a second experiment was carried out where metabolomics was applied to elucidate the biochemical changes imposed by the PH and its best performing fraction (both at 300 mg L−1 of N) in comparison to water and IBA. Overall, both the PH and its fractions increased the root length of tomato cuttings, compared to negative control. Moreover, the highest root length was obtained in the treatment PH1 following foliar application. Metabolomics allowed highlighting a response to PH1 that involved changes at phytohormones and secondary metabolite level. Notably, such metabolic reprogramming supported the effect on rooting of tomato cuttings, being shared with the response induced by the positive control IBA. Taken together, the outcome of in vivo assays and metabolomics indicate an auxin-like activity of the selected PH1 fraction.

Isolation of Rhizosphere Bacteria That Improve Quality and Water Stress Tolerance in Greenhouse Ornamentals.

Water deficit stress is a major contributor to the loss of ornamental crop value due to its negative effects on plant growth and flowering. In addition, post-production water stress can reduce the photosynthetic capacity of plants, negatively impacting crop quality at retail and in the consumer’s home and garden. While the application of microbe-containing biostimulant products can increase stress tolerance and crop quality, the success of most commercially available biostimulants in greenhouse production systems is inconsistent. To identify beneficial bacteria with consistent biostimulant activity in greenhouse ornamentals, our study isolated bacteria from the rhizosphere of water stressed greenhouse ornamentals. Five species of popular ornamentals were obtained from 15 different commercial greenhouse facilities and then subjected to three cycles of water stress. Over 1,000 bacterial isolates were collected from the rhizosphere and screened in vitro for osmoadaptability and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Eighty selected isolates were evaluated in a high-throughput greenhouse trial for their ability to increase plant size and flower number of water-stressed Petunia × hybrida. Ten bacterial strains selected from the high-throughput trial were then evaluated in a greenhouse validation trial for their ability to increase plant growth and to mitigate the reduction in photosynthetic parameters of water stressed P. hybrida and Pelargonium × hortorum. Application of certain bacteria increased plant size in both species and flower number of P. hybrida after recovery from water stress when compared to untreated water stressed plants. In addition, bacteria application increased the chlorophyll fluorescence parameters including quantum yield and efficiency of photosystem II (PSII) and electron transport rate (ETR), while decreasing the extent of electrolyte leakage during water stress and recovery. Overall, this study identified bacterial strains that can increase tolerance to and recovery from water stress in two commercially important ornamental crop species.

Strategies for production, characterization and application of protein-based biostimulants in agriculture: A review

Strategies for production, characterization and application of protein-based biostimulants in agriculture: A review

Integrated valorization of Sargassum muticum in biorefineries

Marine macroalgae represent an excellent raw material for the production of bioactives, adsorbents, plant biostimulants, soil fertilizers and biogas. The success in the exploitation of seaweeds depends on their characteristics, and the approach used to separate their specific active components. In the context of circular economy, invasive species are a good candidate for exploitation, and biorefinery a key valorization technique. Here we investigate a novel biorefinery scheme for an integral valorization of Sargassum muticum. An initial pressing stage allowed the production of a Sap fraction, which showed potential as a plant biostimulant, increasing both root development and shoot/root ratio, especially when used at a dose of 0.1 g/L lyophilized Sap. The solids after pressing were processed by non isothermal autohydrolysis, using pressurized hot water (up to 120-210 °C), a process previously optimized to solubilize the fucoidan and phlorotannin fractions. The residual solids remaining after pressing and autohydrolysis stages were evaluated for the production of biogas. The obtained value (150 mL CH4/g residual solids at 150 °C) was significantly higher than that found for the raw seaweed. The optimal autohydrolysis temperature (150 °C) is compatible with the production of the fucoidan fraction, although the phenolic content is favoured under more severe operation conditions. We also discuss the possibility of preparing adsorbents for pollutant removal and mineral amendments from the autohydrolysis waste solids.

Pumpkin seed protein hydrolysate treatment alleviates salt stress effects on Phaseolus vulgaris by elevating antioxidant capacity and recovering ion homeostasis

Pumpkin seed protein hydrolysate (PH) is a biostimulant product that contains high antioxidant activities, peptides and amino acids. This biostimulant is able to improve plant tolerance to abiotic stresses, including salinity. A pot experiment was conducted during summer season of 2018, with the aim of determining growth and productivity, physio-biochemical attributes, and antioxidant system components in salt (3.9 or 7.8 dS m−1 using NaCl)-stressed Phaseolus vulgaris plants treated with PH (1000 or 2000 μL L‒1). Salt stress was applied in irrigation water, and PH was applied three times as foliar application. Under 7.8 dS m−1, Na+ content was significantly increased, however, PH application at 1000 or 2000 μL L‒1 reduced it by 11 or 13%, respectively. The negative impacts of salinity on essential nutrients (e.g., N, P, K+, Ca2+, and Mg2+) contents, leaf photosynthetic pigments contents, and tissue health (in terms of relative water content, membrane stability index, and excised leaf water retention) were considerably mitigated by PH application. Increase in malondialdehyde (MDA) content was associated by increases in proline, soluble sugars, and glutathione contents, and antioxidant enzyme activities under salt stress, however, PH application further improved these parameters along with a worthy reduction in MDA content in salt-stressed plants. PH can maintain plant anatomical features, growth and yield measurements, which were disrupted by salt stress. Results of this study recommend the use of PH as foliar application, especially at 2000 μL L‒1 to obtain satisfactory growth and yield of Phaseolus vulgaris plants grown under salt stress up to 7.8 dS m−1.

Characterization and plant growth promoting potential of microbial groups associated with a Coffea sp. collection

The genus Coffea spp. is the most important of the Rubiaceae family and both Coffea arabica L. and Coffea canephora P. constitute the most economically important species. This research aimed to characterize morphologically, physiologically and biochemically diazotrophic rhizobacteria associated with coffee plants from a living collection, and to study their plant growth promoting potential. Semi-solid nitrogen-free media NFb and JNFb were used to isolate the bacteria. Ten isolates were selected for micro-morphological, physiological-biochemical and plant growth potential characterization. The solubilization of calcium phosphate, the synthesis of indole compounds and the production of siderophores were analyzed. The results showed that high population levels of bacteria, fungi and actinomycetes are present in the rhizosphere of the coffee collection. 95 native isolates were selected, and 8 were classified as Azospirillum and Herbaspirillum. Phosphate solubilizing activity was detected in the ten isolates, highlighting the C8 isolate. Synthesis of indole compounds was also detected by the ten isolates, although in amounts significantly lower than the controls. Only isolates C2, C9 and C10 produced siderophores. The multivariate cluster analysis resulted in the formation of three groups, the C8 grouped with the positive control in one of them. The beneficial potential of this isolate makes it a source of interest to develop a biostimulant product that could be applied as a sustainable alternative for the cultivation of Coffea sp.

Exploratory Study on the Foliar Incorporation and Stability of Isotopically Labeled Amino Acids Applied to Turfgrass

There is increasing interest in the use of amino acid-based biostimulant products due to their reported abilities to improve a number of quality characteristics in a variety of specialty crops. However, when it comes to the foliar application of amino acids to turfgrass, there are still many basic questions about their uptake forms and incorporation into cellular metabolism. In this study, we shed light on the fate of amino acids exogenously applied to turfgrass foliage through a series of time-course, isotopic-labeling studies in creeping bentgrass (Agrostis stolonifera L.) leaves. Using both 15N-labeled and 15N,13C double-labeled L-glutamate applied exogenously to creeping bentgrass foliage, we measured the uptake of glutamate and its integration into γ-aminobutyric acid (GABA) and L-proline, two amino acids with known roles in plant stress adaptation. Our results demonstrate that glutamate is rapidly absorbed into creeping bentgrass foliage and that it is utilized to produce GABA and proline. Based on the labeling patterns observed in the endogenous pools of glutamate/glutamine, GABA, and the proline from applied glutamate-[13C515N1], we can further conclude that glutamate is predominantly taken up intact and that mineralization into other forms of nitrogen is a minor fate. Taken together, the collective findings of this study provide evidence that amino acids exogenously applied to turfgrass foliage can be rapidly absorbed, and serve as stable sources of precursor molecules to be integrated into the metabolism of the plant.