Plant Biostimulants Research Articles

Plant Biostimulants: Mechanisms and Applications for Enhancing Plant Resilience to Abiotic Stresses

Plant Biostimulants: Mechanisms and Applications for Enhancing Plant Resilience to Abiotic Stresses

The use of mineral dynamised high dilutions for natural plant biostimulation; effects on plant growth, crop production, fruit quality, pest and disease incidence in agroecological strawberry cultivation

ABSTRACT Strawberries (Fragaria x ananassa) are one of the most important fruit crops worldwide. Most of the strawberries that are produced today rely on the use of pesticides and fungicides, leading the crop to be listed as one of the most contaminated by contentious inputs. Plant biostimulants are used as a plant health strategy focused on stimulating plant growth and development with a low residual impact. The objective of this study was to assess the plant biostimulation effects of strawberry plants treated with mineral dynamised high dilutions (DHDs): Sulphur 12CH, Phosphorus 12CH, Kali carbonicun 12CH, Calcarea carbonica 12CH, Silicea terra 12CH, Natrum muriaticum 12CH and Mercurius solubilis 12 CH, distilled water 12CH and distilled water (control). The experiment was undertaken under greenhouse conditions at the University of Santa Catarina State (UDESC), in Lages, Santa Catarina (SC), Brazil in 2019 and was repeated in 2021 at the Federal University of Santa Catarina (UFSC), in Curitibanos, SC. The experiment used a randomised block design, following double-blind treatment application. Data were analysed by ANOVA and when significant (p ≤ 0.05) by Dunnett’s test. The results showed that plants treated with DHDs of Sulphur 12CH, Phosphorus 12CH and Kali carbonicum 12CH increased plant growth and crop yield and were less affected by leaf spot disease (Mycosphaerella fragariae). Plants treated with Silicea terra 12CH, or with Calcarea carbonica 12CH showed increased development of the root system. The results obtained suggested that mineral DHDs could be used as plant biostimulants to support agroecological strawberry production.

The effects of humic substances application on the phytohormone profile in Lactuca sativa L.

AbstractHumic substances (HS) are commonly employed as plant biostimulants to enhance crop yields. However, the HS mechanisms of action, as well as the differences between radicular and foliar modes of application, remain unclear. Here, we explored the changes in phytohormonal balance as possible mechanism of HS to enhance lettuce (Lactuca sativa L.) growth, and the difference between both modes of application. For this purpose, BLACKJAK®, a HS‐based product was applied as radicular (R) and foliar (F) at the concentrations (mL/L): 0.20 (R1), 0.40 (R2), 0.60 (R3), 0.80 (R4), 5.00 (F1), 7.50 (F2), 10.00 (F3), and 12.50 (F4). The experiment was performed in pots filled with vermiculite:perlite (3:1) and HS were applied three times with a periodicity of 10 days. Shoot and root growth parameters were measured. In addition, the phythormones indole‐3‐acetic acid (IAA), gibberellins (GAs), trans‐zeatine (tZ), isopentenyl adenine (iP), 1‐aminocyclopropane‐1‐carboxylic acid (ACC), abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA), were analysed by U‐HPLC‐MS. BLACKJAK® application resulted in higher shoot growth at doses R1, R2, R3, F2, and F3, whereas root biomass was increased at R2, R3, F2, F3, and F4, showing radicular better plant growth than foliar applications. Furthermore, HS changed phytohormonal balance in shoots and roots. However, it was with radicular applications, especially at R2, where phytohormonal profile was best associated with plant growth due to the increases observed in IAA, GAs, JA, SA, tZ, and decreased ABA. However, further research is needed to clarify the involvement of hormones in the growth‐promoting action of HS.

Pararhizobium sp. strains enhancing chilling stress tolerance and yield in tomato plants

In temperate climates, low temperatures represent a significant stressor that adversely affects crop yield and production. Tomato (Solanum lycopersicum L.) is a subtropical crop cultivated in temperate regions. However, most tomato cultivars are sensitive to chilling temperatures, which limit their cultivation in colder regions. Some microorganism-based plant biostimulants have been reported to enhance abiotic stress tolerance in crops. In this study, we isolated two Pararhizobium sp. strains (44 and 128) and tested their potential to trigger chilling stress tolerance in tomato. Through transcriptional, metabolic and biochemical analyses we demonstrate that inoculation with strains 44 and 128 enhance chilling stress tolerance by stimulating the ICE1-CBF-COR cold stress signaling pathway at transcriptional level, improving reactive oxygen species (ROS) detoxifying capacity and boosting the biosynthesis of stress-protective metabolites, such as polyamines and reduced glutathione (GSH). Treatment of tomato plants with these strains under non-stress conditions also increased tomato fruit weight and quality attributes. These findings suggest that Pararhizobium strains 44 and 128 could be valuable biostimulants for improving chilling stress tolerance and crop yield.

Unlocking the potential of biostimulants in sustainable agriculture: Effect of wood distillate on the nutritional profiling of apples

In this work, we report the investigation of the effect of exposure of apple trees to the bioeffector wood distillate (WD), a plant biostimulant used for improving the nutritional profiling of crop plants. We measured the effect by evaluating the biochemical and nutritional profile of both pulps and skin of fruits. WD (0.2 %, v/v) was applied once a week by foliar application, from May 2023 until September 2023. The results indicate that the WD-treated apples have a significant increase in several analyzed parameters (i.e., phenols, flavonoids, tannins, total antioxidant power, sugars, pectin, free amino acids, and mineral element content), especially in the pulp. These data were also confirmed by NMR and LC-ESI-MS techniques. This study pointed out that WD could be a handy tool for the cultivation of fruit trees.

A Plant biostimulant prepared from Ascophyllum nodosum Induces Flowering by Regulating the MIR156-mediated Age Pathway in Arabidopsis.

Flowering, the change from vegetative development to the reproductive phase, represents a crucial and intricate stage in the life cycle of plants, which is tightly controlled by both internal and external factors. In this study, we investigated the effect of Ascophyllum nodosum extract (ANE) on the flowering time of Arabidopsis. We found that a 0.1% concentration of ANE induced flowering in Arabidopsis, accompanied by the upregulation of key flowering time genes: FT (FLOWERING LOCUS T), SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1), and LFY (LEAFY). Further investigation showed that ANE specifically promotes flowering through the MIR156-mediated age pathway. ANE treatment resulted in the repression of negative regulator genes, MIR156, while simultaneously enhancing the expression of positive regulator genes, including SPLs and MIR172. This, in turn, led to the downregulation of AP2-like genes, which are known as floral repressors. It is worth noting that ANE did not alleviate the late flowering phenotype of MIR156-overexpressing plants and spl mutants. Furthermore, ANE-derived fucoidan mimics the function of sugars in regulating MIR156, closely mirroring the effects induced by ANE treatments. It suppresses the transcript levels of MIR156 and AP2-like genes while inducing those of SPLs and MIR172, thereby reinforcing the involvement of fucoidan in the control of flowering by ANE. In summary, our results demonstrate that ANE induces flowering by modulating the MIR156-SPL module within the age pathway, and this effect is mediated by fucoidan.

Reduced Effect of Commercial Leonardite and Seaweed Extract on Lettuce Growth under Mineral, Organic, and No Fertilization Regimes

In this study, two commercial products based on the main groups of contemporary biostimulants—a commercial leonardite and a seaweed extract—were tested with the objective of assessing the conditions under which they can enhance lettuce (Lactuca sativa L.) performance, particularly to determine if synergies with conventional fertilization methods can be observed. The experimental protocol was arranged as a factorial design with two factors: organic or mineral fertilization × plant biostimulant. The organic or mineral fertilization factor included five levels: two rates of a nitrogen (N) fertilizer (40 (Nmin40) and 80 (Nmin80) kg ha−1 of N), the same N rates applied as an organic amendment (Norg40 and Norg80), and an unfertilized control (N0). The plant biostimulants used were a commercial leonardite (leonardite) for soil application before planting, a commercial seaweed extract (algae) for foliar application during the growing season, and a control without plant biostimulant. Leonardite significantly increased lettuce dry matter yield (DMY) compared to the control only in the first growing cycle (11.5 and 13.5 g plant−1) and showed no significant interaction with conventional fertilization. It also consistently increased phosphorus (P) levels in the plant tissues. The seaweed extract did not show any effect on the plant, nor did it have any interactions with conventional fertilization regarding DMY. In contrast, with mineral fertilization, lettuce DMY increased from 8.0 and 4.0 g plant−1 (N0) to 22.2 and 12.0 g plant−1 (Nmin80) in the first and second growing cycles, respectively. The response to organic fertilization was lower, yet DMY still increased from 4.0 to 8.1 g plant−1 in the second growing cycle. Generally, this type of plant biostimulant is tested under some form of environmental stress, where it often yields positive results. In this study, the optimal cultivation conditions maintained for the lettuce in the pots likely explain the limited response to the biostimulants. This study suggests that the product labels should more clearly indicate whether they are recommended for general cultivation conditions or specifically for situations where a particular environmental stress can be anticipated.

Hydrolysates from cauliflower and artichoke industrial wastes as biostimulants on seed germination and seedling growth: a chemical and biological characterization.

Cauliflower (Brassica oleracea L.) and globe artichoke (Cynara scolymus L.) are vegetables with a high waste index mainly related to stems and leaves. In this study, enzymatic hydrolysates obtained from these wastes were proposed to be used as plant biostimulants. Life cycle assessment methodology was also applied to evaluate environmental performances related to cauliflower and artichoke byproducts. Hydrolysates (HYs) were chemically and biologically characterized. Amino acids, organic acids, amines, polyols, mineral elements, phenols, tannins, flavonoids and sulfur compounds were identified and quantified by means of NMR, inductively coupled plasma mass spectrometry and UV-visible analyses. Cauliflower leaf and flower HYs showed the highest concentration of free amino acids, whereas stems showed the highest concentration of Ca. Regarding artichoke, asparagine, glutamine and aspartic acid were exclusively detected in stems, whereas artichoke leaves showed the highest Mg and Mn levels together with the highest antioxidant activity. The HYs diluted in water were tested as biostimulants. The impacts of five concentrations of HYs (0.00, 0.28, 0.84, 2.52 and 7.56 g L-1) on seed germination and early seedling growth of crimson clover, alfalfa, durum wheat and corn were investigated. The application of artichoke biostimulant (0.28 g L-1) positively influenced the coefficient of velocity of germination in alfalfa, crimson clover and durum wheat, whereas cauliflower biostimulant significantly improved corn germination speed. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of extraction methods on the plant biostimulant activity of the soil microalga Chlorella vulgaris

Effects of extraction methods on the plant biostimulant activity of the soil microalga Chlorella vulgaris

Compost tea and bioslurry as plant biostimulants. Part 1: characterization and phytotoxicity testing

In the present study, the quality of aerated (A), non-aerated (NA) and bioslurry (B) as potential bio-fertilizers were evaluated. Aerated and non-aerated compost teas were brewed from two different compost: exhausted grape marc (G); and a mixture of residues (M): goat manure, grape marc, garden raking leaves and alfalfa cutting hay. Bioslurry was prepared from fresh organic material in anaerobic digestion, according to FAO. Aeration, with shorter extraction time, resulted in higher pH (8.25 and 7.44 regarding 7.32 and 6.98, respectively, for AM, NAM, AG and NAG), but in general with lower nutrient concentration than non-aerated. B presented higher nitrogen content (7.86 mg L−1 N-NO3 − and 40.77 mg L−1 N-NH4 +). No pathogens or phytotoxic effects were detected. To our knowledge, this is the first time that B has been evaluated, and our results suggest that the use of fresh organic matter may generate a high-quality product to be used as a biostimulant. Agricultural bio-inputs, elaborated from organic residues, represent a cheaper and safer alternative to traditional fertilizers, while residues are reduced and reused.

Improvement in Plant Growth, Yield, and Fruit Quality with Biostimulant Treatment in Organic Strawberry Cultivation

The excessive use of chemical fertilizers in agriculture not only causes a decrease in soil fertility but also has negative effects on the environment, natural resources, and human health. Therefore, environmentally friendly practices, such as the use of organic fertilizers (OFs) and plant biostimulants that increase yield and fruit quality can be effective in solving these problems. In the present research study, we investigated the impact of using an OF alone and in combination with as a biostimulant different doses of humic acid (HA) on plant growth parameters, yield, fruit characteristics, and leaf mineral nutrient concentrations in plants of the Monterey and Albion strawberry varieties. As a result of this study, we determined that the combined application of the OF and HA increased the yield, fruit quality, plant growth, and nutritional elements in the crop compared with using the OF alone. In addition, the Monterey variety plants treated with OF and HA (5.0 L·ha−1) in T3 offered the best results among the different treatment groups and varieties. With this treatment, we obtained the highest total yield (262.42 g/plant), fruit weight, total soluble solids (TSS), and TSS/acid ratio, as well as increased growth parameters, and mineral nutrient concentrations in leaves. These results are hopeful for enhancing organic strawberry production.

Effect of plant biostimulants, harvesting dates, and storage periods on Sesame (Sesamum indicum L.) seed quality in newly reclaimed soils

Effect of plant biostimulants, harvesting dates, and storage periods on Sesame (Sesamum indicum L.) seed quality in newly reclaimed soils

Novel biostimulant bacterial exopolysaccharides production via solid-state fermentation as a valorisation strategy for agri-food waste.

Bacterial exopolysaccharides (EPS) are extracellular polymer-based substances recently defined as potential plant biostimulants, as they can increase nutrient uptake, water retention, and resistance to abiotic stress. As sugar-based substances, the bacteria producing them need to grow in a sugar-rich substrate. Hence, some agri-food by-products could be used as suitable carbon sources for EPS production as a cost-effective and more sustainable alternative to conventional substrates. Thus, this study aimed to produce EPS from specific bacterial strains through solid-state fermentation (SSF) using agri-food waste as a low-cost substrate. Six residues and five bacterial strains were tested in a lab-scale SSF system. From the assessed substrate-strain combinations, Burkholderia cepacia with ginger juice waste (GJW) resulted in the most promising considering several process parameters (EPS production, cumulative oxygen consumption, biomass growth, reducing sugars consumption). Also, dynamic monitoring of the system allowed for establishing 5days as a suitable fermentation time. Then, using response surface methodology (Box-Behnken design), the process was optimised based on airflow rate (AF), inoculum size (IS), and micronutrient concentration (MN). In this stage, the best conditions found were at 0.049 (± 0.014) L h-1 per gram of dry matter (DM) for AF, 8.4 (± 0.9) E + 09CFUg-1 DM for IS, and 0.07 (± 0.01) mL g-1 DM for MN, reaching up to 71.1 (± 3.2) mg crude EPS g-1 DM. Results show the potential of this approach to provide a new perspective on the value chain for the agri-food industry by introducing it to a circular economy framework.

Application of Protein Hydrolysate Improved the Productivity of Soybean under Greenhouse Cultivation

Protein hydrolysates are plant biostimulants containing amino acids, oligopeptides, and peptides in their composition. When supplied to plants, protein hydrolysates (HPs) have been identified to improve nitrogen metabolism, enhance the activity of antioxidant enzymes, boost plant defense response to stresses, and positively impact the quantity and quality of products. Soybean is a crucial global commodity, with nitrogen being the primary nutrient for crop development as it directly affects productivity. This study aimed to evaluate the effect of an HP-based biostimulant on the N metabolism in nodulated soybean plants and their productivity. A greenhouse experiment was conducted to test two modes of application of the 0.20% HP-based biostimulant. Soybean plants, growing in pots, were treated with 0.20% HP either via seed treatment or foliar application (at growth stages V3 and V5). Activities of enzymes and compounds related to N metabolism, gene expression, and productivity components were analyzed. It was observed that the mode of application did not significantly influence the results. The application of HPs increased the concentration of nitrate, amino acids, and ureides in soybean leaves. It also positively altered the expression of genes such as nitrate reductase, urease, and asparagine. Additionally, it enhanced productivity, resulting in plants with a greater number and weight of pods and grains. Therefore, it is possible to consider HPs as a stimulator for increasing soybean productivity, even under non-stressing conditions.

Chitosan-strigolactone mimics with synergistic effect: A new concept for plant biostimulants

The paper reports new multifunctional plant biostimulant formulations obtained via in situ hydrogelation of chitosan with salicylaldehyde in the presence of a mimetic naphthalimide-based strigolactone, in specific conditions. Various analytical techniques (FTIR, 1H NMR, SEM, POM, TGA, WRXD) were employed to understand the particularities of the hydrogelation mechanism and its consequences on the formulations' properties. Further, in order to evaluate their potential for the targeted application, the swelling in media of pH characteristic for different soils, water holding capacity, soil biodegradability, in vitro release of the strigolactone mimic and impact on tomatoes plant growth in laboratory conditions were investigated and discussed. It was found that the strigolactone mimic has the ability to bond to the chitosan matrix via physical forces, favoring a prolonged release. Moreover, the combination of chitosan with the strigolactone mimic in an optimal mass ratio triggered a synergistic effect on the plant growth, up to 4 times higher compared to the neat control soil.

Comparative Effects of Root and Foliar Leonardite-Suspension Concentrate Application on Plant Growth and Photosynthetic Efficiency of Lettuce Plants (Lactuca sativa L.)

Humic substances (HS) have been defined as a potential plant biostimulant to improve crop yield in a sustainable and environmentally friendly way. Leonardite-suspension concentrate (SC) is a type of HS extracted from lignite that is currently employed to enhance various physiological aspects of plants. However, the different effects between both modes of SC application (root and foliar) are poorly understood, especially on photosynthesis performance. Therefore, this study aimed to investigate the influence of a leonardite-SC-based product (BLACKJAK®), on lettuce growth and photosynthesis efficiency, while comparing both methods of application. For this purpose, four root (R): R1 (0.20 mL/L), R2 (0.40 mL/L), R3 (0.60 mL/L), and R4 (0.80 mL/L), and four foliar: F1 (5.00 mL/L), F2 (7.50 mL/L), F3 (10.00 mL/L), and F4 (12.50 mL/L) BLACKJAK® doses were applied to lettuce plants. Related shoot and root growth parameters, photosynthetic efficiency, and sugar and starch content were assessed in lettuce plants. The results showed that BLACKJAK® improved shoot and root biomass, foliar area, and root length, especially at intermediate doses (R2, R3, F2, and F3), with R3 demonstrating the greatest growth increases. Similarly, the main photosynthetic parameters analyzed (net photosynthetic rate and Rubisco carboxylation efficiency), and the soluble sugars and starch content were improved by the same doses, with R3 showing the best photosynthetic performance. Hence, our study suggests that BLACKJAK® improves lettuce yield and photosynthetic efficiency, particularly with radicular application at R3.

Next generation plant biostimulants & genome sequencing strategies for sustainable agriculture development.

The best environment for plant growth and development contains certain essential metabolites. A broad category of metabolites known as "plant biostimulants" (PBs) includes biomolecules such as proteins, carbohydrates, lipids, and other secondary metabolites related to groups of terpenes, specific nitrogen-containing compounds, and benzene ring-conjugated compounds. The formation of biomolecules depends on both biotic and abiotic factors, such as the release of PB by plants, animals, and microorganisms, or it can result from the control of temperature, humidity, and pressure in the atmosphere, in the case of humic substances (HSs). Understanding the genomic outputs of the concerned organism (may be plants or others than them) becomes crucial for identifying the underlying behaviors that lead to the synthesis of these complex compounds. For the purposes of achieving the objectives of sustainable agriculture, detailed research on PBs is essential because they aid in increasing yield and other growth patterns of agro-economic crops. The regulation of homeostasis in the plant-soil-microbe system for the survival of humans and other animals is mediated by the action of plant biostimulants, as considered essential for the growth of plants. The genomic size and gene operons for functional and regulation control have so far been revealed through technological implementations, but important gene annotations are still lacking, causing a delay in revealing the information. Next-generation sequencing techniques, such as nanopore, nanoball, and Illumina, are essential in troubleshooting the information gaps. These technical advancements have greatly expanded the candidate gene openings. The secondary metabolites being important precursors need to be studied in a much wider scale for accurate calculations of biochemical reactions, taking place inside and outside the synthesized living cell. The present review highlights the sequencing techniques to provide a foundation of opportunity generation for agricultural sustainability.

Review of the Use of Biostimulant Microalgae to Influence the Growth and Development of Ornamental Plants

The concept of plant biostimulants refers to substances or microorganisms used to improve the efficiency of nutrient uptake of a plant, improve stress tolerance, and improve overall quality. Extracts of microalgae are gaining increasing attention among biostimulants due to their rich bioactive content. Microalgae offer new opportunities in agriculture, wastewater treatment, pharmaceuticals, etc. These tiny organisms are known for their role in carbon dioxide sequestration, bioremediation, and the production of valuable compounds. Biostimulants act on plants through various mechanisms, promoting growth, nutrient mobilisation, and resistance to stress. The abundant amino acids and protein hydrolyzesate in microalgae improve nutrient absorption and act as osmoprotectants against stressors such as heavy metals and salt. The visual appearance of the potted plants plays a crucial role in determining their quality. Although research suggests a consumer preference for organically grown plants, the economic viability of organic ornamental plant production requires careful consideration of production costs and market demand. With the increasing demand for sustainable agricultural practices, the application of plant biostimulants, particularly those derived from microalgae, presents a promising opportunity to enhance productivity, improve soil health, address environmental issues, and overcome challenges posed by new regulations in the European Union related to the use of plant protection products.

Mealworm Larvae Frass Exhibits a Plant Biostimulant Effect on Lettuce, Boosting Productivity beyond Just Nutrient Release or Improved Soil Properties

There is a need for alternatives or complements to synthetic fertilizers to enhance agricultural sustainability. Applying organic amendments can play a significant role in this. Insect droppings show high potential, though studies evaluating their agronomic value have only recently begun to emerge. This study compared black soldier fly (Hermetia illucens L.) and mealworm (Tenebrio molitor L.) larvae frass with another organic amendment (Nutrimais) derived from composting forestry, agro-industrial, and domestic waste. The experiment also included ammonium nitrate at two rates [the same as the organic amendments, 50 kg ha–1 nitrogen (N) (FullR), and half that rate (HalfR)] and an unfertilized control. The study spanned two growth cycles of lettuce (Lactuca sativa L.) grown in pots, followed by unfertilized oats (Avena sativa L.) to assess the residual effects of the fertilizing treatments. Mealworm larvae frass mineralized rapidly, with an apparent N recovery of 37.4% over the two lettuce growth cycles, indicating its high availability to soil heterotrophic microorganisms. The average dry matter yield (DMY) of lettuce was the highest among all treatments (12.8 and 9.8 g plant–1 in the first and second lettuce cycles), even compared to the FullR treatment (12.2 and 7.8 g plant–1), though without significant differences. Although mealworm larvae frass exhibited a high mineralization rate, the DMY cannot be attributed solely to N supply, as plants in the FullR treatment showed better N nutritional status. Mealworm larvae frass provided strong evidence of a plant biostimulant effect, not explained by the variables measured in this study. Black soldier fly larvae frass exhibited typical behavior of a moderately reactive organic amendment, while Nutrimais showed low reactivity, with a near-neutral mineralization/immobilization balance. The results suggest mealworm larvae frass is recommended for early maturing vegetable crops, whereas Nutrimais appears more suitable for perennial crops with low short-term nutrient requirements.

Influence of peat-derived humic acid on the growth of agarwood seedlings

Humic acid (HA) is widely used in agriculture as a biostimulant to enhance plant growth and nutrient uptake. Studies on the early growth promotion of agarwood seedlings using plant biostimulants have not been explored. Therefore, the objective of this study was to extract and characterize peat-derived HA and to determine the optimum rate that would exert biostimulant action on the early growth of agarwood seedlings at the nursery stage. HA was extracted from peat soil using a modified rapid alkaline hydrolysis method. Produced peat humic acid (PHA) was compared with commercial humic acid (CHA) for its chemical and spectroscopic characteristics. One-month-old agarwood seedlings were treated with PHA at different rates (0, 20, 40, 80, 160 and 320 mg kg−1) before the commencement of the experiment. The study was conducted for four months. The C/N ratio of both PHA and CHA were 25.85 and 26.93, respectively. Scanning electron microscope (SEM) and fourier-transform infrared spectroscopy (FTIR) spectra observation reveals that PHA and CHA have similar surface morphology and functional groups respectively. Results revealed that agarwood seedlings treated with 80 mg kg−1 of PHA significantly increased the plant height, stem diameter, the number of leaves, leaf area, dry matter weight, N and P concentrations in plant tissues, and N, P and K uptakes. Based on the findings, incorporation of PHA at the rate of 80 mg kg−1 with planting medium could be used as an optimum rate to enhance the growth of agarwood seedlings in the nursery stage.