Effect of Biostimulants on the Recovery of Warm- and Cool-Season Turfgrass in Central Chile

Abstract

This study evaluated the effect of a combined treatment of biostimulants on the recovery of low-maintenance turfgrass grown in central Chile. During the summer of 2025, a trial was conducted in Melipilla (central Chile) in an area planted with turfgrass sods consisting of a mixture of warm- and cool-season grasses under weekly mowing conditions. Three doses of the biostimulant treatment were applied at 15-day intervals, maintaining a controlled irrigation regime based on daily turfgrass evapotranspiration. The turf quality during the study was evaluated using vegetation indices based on RGB digital image analysis (VARI and TGI). During the trial period, only the treated group showed a significant improvement relative to its initial condition in one of the two evaluated indices (VARI). Comparative analyses across evaluation dates revealed significant differences between the treatment and the procedural control, which represented the non-maintained condition and the deterioration targeted by the trial, whereas no significant differences were detected with the irrigated control. These preliminary findings suggest that biostimulant treatments could represent a feasible tool for improving the appearance and recovery of turfgrass recurrently affected during summer conditions in urban areas of central Chile. However, future studies should include repeated damage–recovery events and incorporate multispectral indices, such as NDVI, to strengthen the robustness of the results.

1. Introduction

Lawn grass and its intensive use in urban green spaces play an important role due to their multiple social and environmental services. Among their social functions is that of providing a highly aesthetic space that is irreplaceable for recreational and sporting activities, which in turn has a positive impact on citizens’ mental health. while in environmental terms, turf has greater potential than other surfaces to mitigate temperatures, reduce runoff, increase infiltration, and purify water of sediments and contaminants, while controlling erosion, improving soil quality, and reducing the risk of fires [1,2]. The maintenance of turfgrass required by the industry relies heavily on chemical applications, including fungicides, insecticides, nematicides, and herbicides, to control pests and promote turf health [3]. However, in recent years, growing concern about the interactions between conventional fertilizers and the effects of these conventional fertilizers on users and the environment has led to the search for organic alternatives that have the same effects on turf maintenance and quality [4,5].

On the other hand, some authors [6] conclude that biostimulants cannot replace nitrogen fertilizers but can enhance their efficiency, improving turf quality and resilience. A comparative analysis between conventional fertilizer treatment and treatment with biostimulants, taking into account their primary function, turf response, and environmental impact, can help to understand the relevance of the use of biostimulants on turfgrass management. Primary function: Conventional nitrogen fertilization supplies essential nutrients for growth, color, and density, while biostimulants stimulate physiological processes and enhance nutrient uptake, stress tolerance, and root development. Turf response: Conventional methods provide immediate and predictable improvement in greenness and biomass, while biostimulants provide a more gradual effect, improving resilience, root vigor, and overall turf quality. Environmental impact: Conventional methods have a risk of nitrate leaching, eutrophication, and greenhouse gas emissions, whereas biostimulants have a lower footprint and support sustainable management by reducing reliance on synthetic inputs.

Considering there has been rigorous ongoing debate over the last decade to determine a biologically meaningful definition for biostimulant, Chilean Law 21.349, which establishes standards for the composition, labeling, and marketing of fertilizers and biostimulants, defines biostimulants as products consisting of substances or microorganisms applicable to seeds, plants, or the rhizosphere, whose function is to stimulate natural plant nutrition processes, improving nutrient use efficiency, abiotic stress tolerance, crop quality, and the availability of nutrients present in the soil [7]. This study provides more information regarding the effect of biostimulants in a crop not well studied in Chile but widely used in sports and recreational areas.

In recent years, the use of biostimulants in agriculture has increased with good results [8,9], also extending to the turf industry with improvements in visual parameters such as color, plant development, and reduction in microbial infestation [9,10]. Calvo et al. (2014) explain how different biostimulant categories (seaweed extracts, humic substances, protein hydrolysates, and microbial inoculants) act through distinct physiological pathways, making combined use beneficial [11]. Several biostimulants are applied together because they act through different mechanisms and produce synergistic effects.

Effective microorganisms (EMs), categorized as biostimulants, are consortia of beneficial organisms that can be inoculated into the soil, thereby improving its ecology and creating a favorable environment for plant growth and health [9,12]. A study conducted on Lolium perenne L. demonstrated the effectiveness of a commercial product composed of EMs in promoting root extension and increasing tear resistance [13]. Furthermore, an interesting review of the literature on biostimulants for agriculture reported that the additive and/or synergistic effects of combinations of microbial biostimulants with humic acids, plant extracts, or protein hydrolysates could provide more reproducible benefits for plant growth and production [8,9]. In turfgrass, this is consistent with the findings of Bosi et al. (2023), in a study conducted on Agrostis stoloniferous L., where the characteristics of the rhizosphere and the plant were collectively improved by combining a microbial inoculum with humic acid and mycorrhizal fungi compared to treatment with the inoculum alone [9].

Aerial image analysis is an interesting tool to assess crop health and can provide valuable information on the condition of vegetation. Conventional RGB digital images provide a cost-effective alternative for analyzing the color and green coverage of grass, offering information related to its physiological condition and overall health. These images are used to obtain indices such as the visible atmospherically resistant index (VARI), which is used to estimate the fraction of vegetation with minimal sensitivity to atmospheric effects [14], and the triangular greenness index (TGI), which quantifies the intensity of greenness and associates it with plant stress [15]. The visible atmospherically resistant index (VARI) and the triangular greenness index (TGI) are increasingly used in turfgrass quality assessment because they rely only on RGB imagery, making them cost-effective tools for precision turf management. VARI is particularly effective for monitoring canopy greenness under variable light conditions, while TGI correlates strongly with chlorophyll content and nitrogen status. Together, they provide a practical alternative to NDVI when multispectral sensors are unavailable [16]. RGB-based indices (such as VARI and TGI) can complement multispectral indices for objective prediction of turfgrass quality, reducing reliance on subjective visual ratings [17].

This study was conducted in central Chile, one of the country’s most densely populated regions, where turf is widely used for sports and recreational purposes. This area is characterized by a Mediterranean climate, which allows for the coexistence of a mix of warm- and cool-season grass species [18]. Under these conditions, mixed-grass lawns often suffer visual deterioration during the summer season due to intensive use, climatic conditions, and management challenges. In this context, this study evaluated the effect of a combined treatment using biological biostimulants and commercial organic extracts on the recovery of a mixed-grass lawn under previously unmaintained conditions in central Chile. Our hypothesis was that the treatment would improve the quality and visual appearance of the turf between the initial and final phases of the trial, while also representing a potential complementary strategy to reduce dependence on conventional fertilizers in the face of damage or deterioration.

2. Materials and Methods

2.1. Site Description

The experiment was carried out during a 45-day period from January to March of the summer of 2025 in Melipilla (−33.741418, −71.310037), located in central Chile, Melipilla Province, Metropolitan Region, Chile. Based on the Köppen–Geiger climate classification system [19], the study area is characterized by a hot-summer Mediterranean climate (Csa), defined by warm, dry summers. The site has a mean annual temperature of 15.4 °C, with a significant temperature contrast, reaching a maximum of 35 °C in summer, and the average annual precipitation is 278.2 mm, mainly concentrated during the winter season (Chilean Meteorological Directorate-Climate Services, Melipilla-Chocalán Station (national code: 330176), 2025). Figure 1 shows agroclimatic parameters recorded during the trial period, obtained daily from the INIA Agrometeorological Platform using data from the two nearest meteorological stations to the experimental site: San Pedro de Melipilla and Cuncumén San Antonio (INIA).

Based on the USDA soil classification system, the experimental site was characterized as a sandy loam soil, composed of 56% sand, 34% silt, and 10% clay. Soil chemical properties included a pH of 7.22, an organic matter concentration of 5.58%, and macronutrient levels of nitrogen (N), phosphorus (P), and potassium (K) of 95, 64, and 1135 mg kg⁻¹, respectively. Soil samples were collected at depths ranging from 0 to 10 cm using a zigzag sampling method [20]. The experiment was set up in an agricultural field where, during the previous season (January 2024), a 24 m² plot (3 × 8 m²) had been prepared and marked out. Common bermudagrass (Cynodon dactylon (L.) Pers.), the most widely used warm-season turfgrass species in Chile, was growing naturally and was manually removed. Subsequently, 18 turf sod patches, each 0.5 m², corresponding to the standard size of commercial sod sold in Chile, were installed. The sod, marketed as “Stadium Mix”, consisted mainly of cool-season grasses (Lolium perenne L. 84%, Schedonorus arundinaceus (Schreb.) Dumort. 14%, and Cynodon dactylon (L.) Pers. 2%) and was produced locally in Isla de Maipo, Metropolitan Region, Chile. Therefore, for this study, the grass sod had been in place at the site for one year.

2.2. Experimental Design and Biostimulant Applications

A one-factor experiment with repeated measures was conducted to evaluate the effects of the application of a combined biostimulant treatment, with two levels: treated (hereinafter “treatment”) and untreated (hereinafter “control”). Additionally, three experimental units were designated as “procedural control.” Each group consisted of 3 experimental units (replicates), which, once the trial began, were mowed weekly to maintain a height of 4 cm, to observe the effect of the treatment on turf maintained short for recreational use. The experimental units in the control and treatment groups were irrigated three times a week to fully replenish the crops’ accumulated daily evapotranspiration. The procedural controls were maintained under “no irrigation” conditions, which was the state of all grass sod prior to the start of the trial. The experimental units were not randomized to avoid potential cross-contamination of the soil due to biostimulant treatment.

The treatment consisted of a combination of biological products (microorganisms), seaweed extract, macro- and micronutrients from organic sources produced by two Chilean companies (Agri Marine Terra S.A., Codegua, Chile and Australis Ecoscience S.A., Codegua, Chile). This combination of products is commonly applied in turfgrass management in the country. Three applications were made during the trial period, with a 15-day interval between each one. The products and quantities used in each application are described in

Table 1. Description of treatment with biostimulants, applications, and dosages.

Application Number	Trial Date	Product	Bioestimulant Category	Composition	Concentration Recommended
1, 2, 3		BioLife Rhizo ® (Codegua, Chile)	Microorganisms	Trichoderma rifai (cepa AMTtr02) 1 × 108 CFU/g, T. harzianum (cepa AMTtr03) 1 × 108 CFU/g, T.virens (cepa AMTtr12) 1 × 108 CFU/g, Bacillus amyloliquefaciens (cepa AMTba21) 2.5 × 109 CFU/g, B.subtilis (cepa AMTbsR06) 2.5 × 109 CFU/g.	5 g/L
	Day 1, 15, 30	MicroLife Bio Potasio ® (Codegua, Chile)	Seaweed Extract	Lessonia trabeculata, L. nigrescens, Durvillaea antarctica, Macrocystis integrifolia, Organic potassium 5% w/v.	5 mL/L
		Koru Multimix ® (Codegua, Chile)	Seaweed Extract	Durvillaea antarctica, Lessonia trabeculate, Macro and micronutrients: Nitrogen 12% w/v, Phosphorus 8% w/v, Potassium 6% w/v, Calcium 0.08% w/v, 0.05% w/v, Boron 0.005% w/v, Zinc 0.005% w/v, Iron 0.002% w/v, and Copper 0.001% w/v.	5 mL/L

.

The experimental plots had an area of 0.5 m², so, following the manufacturer’s instructions, 150 cc of the treatment was applied each time. In all three instances, the treatment was applied at approximately 9:00 a.m. using a sprayer on the corresponding experimental plots, which had been irrigated the previous night. Twelve hours later, the plots were irrigated again to ensure the products were incorporated into the soil. Both irrigation treatments were applied to the control and experimental plots in the same manner to maintain consistent conditions.

2.3. Irrigation Determination and Implementation

Both the treatment and control plots received irrigation three times per week, with water applications calculated to replace the cumulative daily evapotranspiration recorded since the previous irrigation event. Reference evapotranspiration (ET_o) data were obtained daily from the INIA Agrometeorological Platform using data from the two meteorological stations nearest to the experimental site, namely San Pedro de Melipilla and Cuncumén San Antonio (INIA), and mean values were calculated. The San Pedro de Melipilla station is situated 20.8 km from the study area at an elevation of 145 m above sea level (masl), whereas the Cuncumén San Antonio station is located 10.3 km away at 215 masl. Actual evapotranspiration (ET_r) was estimated by applying corrections based on the Penman–Monteith equation [21]. Accordingly, daily turf evapotranspiration was calculated using the following equation:

Total daily replenishment (L) =
ET_o (L m⁻² day⁻¹) × 0.975 × 1 × 1.5 × 1.5 × 0.5 (m²) − (r (mm day⁻¹) × 0.5 (m²))

(1)

where the first term corresponds to the average daily evapotranspiration obtained from the meteorological stations, and the second term is the average of the crop coefficients (Kc) for the set of species (1.15 for Lolium perenne L. and 0.8 for Schedonorus arundinaceus (Schreb.) Dumort.) [22]. The third, fourth, and fifth terms are correction factors: shading (with or without), orchard age (first year of establishment or older), and irrigation type (hose, drip, or sprinklers), respectively. In this case, the study was conducted without shading, with orchards more than one year old, and a hose was used for irrigation. The sixth term in Equation (1) is the area of the experimental units, and finally, the terms in parentheses, which include daily precipitation, are subtracted from the final calculation of the liters of water that fell daily per experimental unit due to precipitation (during the trial, there were no precipitation events). Using the evapotranspiration rate, the daily water volume required for replenishment in each experimental unit was estimated and accumulated according to the number of days since the previous irrigation event. The resulting value was then adjusted based on the irrigation system flow rate (1035 L h⁻¹) to calculate the total irrigation time required. Irrigation was ultimately applied three times per week.

2.4. Variable Measurement

Digital RGB aerial images were acquired using a DJI MAVIC MINI 3 PRO drone (Da-Jiang Innovations Science and Technology Co., Shenzhen, China) equipped with a 12-megapixel RGB camera (4000 × 3000 px). Images were captured weekly (seven days apart) from a zenithal perspective, capturing the entire test area within the image at approximately 3:00 p.m. Using these images, the visible atmospherically resistant index (VARI) and triangular greenness index (TGI) (

Table 2. RGB vegetation index and equation.

Vegetation Index	Equation	Reference
Visible atmospherically resistant index (VARI)	(Green − Red)/(Green + Red − Blue)	(Gitelson et al., 2002) [14]
Triangular greenness index (TGI)	(Green − (0.39 × Red) − (0.61 × Blue))	(Hunt et al., 2011) [23]

) were calculated for each experimental plot on the different measurement dates using the Raster Calculator tool in QGIS.

For each experimental unit, the RGB index was obtained on each measurement date (7 measurements during the trial period). These values were plotted as averages by treatment, showing their normalized temporal variation and a comparison of the index between the start and end dates of the experiment within each group.

2.5. Statistical Analysis

To evaluate differences in turf appearance based on the VARI and TGI indices obtained from the treatments on different measurement dates (between groups) and to compare the initial and final measurements (within each group), a two-way repeated-measures ANOVA was conducted; subsequently, the Bonferroni test was used for multiple comparisons. Statistical analyses and graphical outputs were achieved using GraphPad Prism version 9.3.1.

3. Results

A positive trend in normalized VARI and TGI was observed for both the control and treatment groups, whereas the procedural control, which maintained the initial condition of all experimental units without maintenance, saw its quality deteriorate over the course of the trial (Figure 2). Starting on the third measurement date (day 15 of the trial), prior to the second application of the biostimulant (arrow 2), the treatment group began to show improvements in the VARI that were statistically significant compared to the procedural control (p < 0.05, p < 0.01, and p < 0.001), while the control group showed no significant differences compared to either of the other two groups (p > 0.05) (Figure 2A). A similar pattern was observed for TGI, where an improvement in treatment outcomes compared to the procedural control group was also noted, becoming statistically significant starting from measurement date 3 (p < 0.05), and a significant difference (p < 0.05) was observed between the control group and the procedure group on measurement date 3 (Figure 2B).

Because all experimental units were initially unmaintained and exhibited varying degrees of deterioration in visual quality, the VARI and TGI were compared between the start and end dates of the trial within each group (Figure 3). According to the VARI, three scenarios were observed: the control group showed no significant changes between the initial and final measurements (p > 0.05); the treatment group showed a significant improvement (p < 0.05); and the procedural control group showed a highly significant deterioration (p < 0.01) (Figure 3A). This differs from the results for TGI (Figure 3B), where a significant change (deterioration) was observed only between the initial and final measurements of the procedural control group.

4. Discussion

The data from this study show a significant improvement when comparing the VARI between the start and end of the trial in the areas treated with biostimulants and mowed weekly. These results are consistent with those of Fidanza et al. (2023), who highlight that mowing frequency and height directly affect turf stress levels, and biostimulants (such as seaweed extracts, humic acids, and amino acids) can mitigate this stress by improving antioxidant activity and recovery [24]. In this context, and considering the mowing frequencies typical of sports and recreational areas, the use of biostimulants is recommended. Furthermore, Mackiewicz-Walec and Olszewska (2023) note that mowing reduces carbohydrate reserves and photosynthetic capacity, while biostimulants improve nutrient uptake and root growth, thereby promoting turf resilience under frequent mowing [6]. VARI has been applied in turfgrass irrigation studies as a practical greenness measure, demonstrating its usefulness for detecting greenness and turf performance under different management regimes [25,26]. The present study confirms this index correlation with turfgrass vigor. TGI is a useful index for stress detection but lacks direct turfgrass irrigation correlation studies. TGI has been used in other crops [15], such as corn, in which it correlated strongly with canopy chlorophyll and nitrogen status. Under reduced irrigation procedural control in this study, TGI values dropped earlier than VARI, making it a sensitive drought indicator, as can be seen in this study (Figure 3).

Although no significant differences were observed in the normalized VARI between the control group (irrigation only) and the treatment group at the various measurement dates, only the latter group showed differences in all measurements taken after the start of the trial compared to the procedural control group, which represented the initial condition of the trial involving the unmaintained lawn (Figure 2A). The same trend was observed for the normalized TGI, except on date 3 (Figure 2B), when the control group also showed a significant improvement. This indirect difference is complemented by the results presented in Figure 3A, where a statistically significant improvement was observed between the initial and final values exclusively in the treated group, a pattern not observed in the control. This result constitutes a relevant finding considering that the study’s objective was to evaluate the recovery of turf quality in the face of deterioration. Its relevance is particularly evident in urban green spaces subject to high maintenance demands and frequent mowing, where preserving turf quality is essential [2], especially under the climatic conditions and management challenges characteristic of summer in central Chile.

Figure 2 and Figure 3 show some differences in the results for the two indices analyzed. Since this study was based on raw RGB images without inter-date correction, from a methodological perspective, the use of VARI, which aims to reduce the influence of atmospheric and lighting variability [14], could represent a more robust response variable than TGI, which does not account for this in its formulation [23]. Furthermore, studies have reported a significant correlation between VARI and NDVI in active vegetation [27] and consistent results across these indices under similar test conditions [28], suggesting that this index serves as a good proxy for vegetation condition when working with more cost-effective alternatives such as RGB imagery. On the other hand, it has been observed that VARI is influenced by leaf area [29], which shows in this case that the improvements observed during the trial period between the initial condition (without irrigation), represented by the procedural control group, and the treatment group, where the experimental units were weekly mowing, are determined exclusively by an improvement in quality and visual appearance.

This study’s preliminary results are presented as a basis for justifying further studies that include longer evaluation periods, with repeated events that cause deterioration (such as accidental water shutoffs, which frequently occur in urban parks due to system failures), to evaluate this type of product, which has a lower environmental impact, in the repeated recovery of turf throughout a season, as may occur in real-world maintenance contexts.

5. Conclusions

The results of this study preliminarily suggest that the evaluated biostimulant combination may support turfgrass recovery under environmental stress conditions, particularly when integrated into active management practices such as weekly mowing. Although statistically significant differences between treated and control groups were not consistently observed across all measurement dates, only the treated group showed a significant improvement between baseline and final values, while maintaining a positive response relative to its initial deteriorated condition throughout the experimental period. Considering that the study objective was focused on turfgrass recovery and that the procedural control represented the initial condition of an unmaintained, non-irrigated lawn, these findings constitute a relevant preliminary indication of a potentially positive response associated with the treatment. However, this interpretation should be treated with caution, and further studies involving a larger number of replicates, repeated deterioration–recovery cycles, and longer evaluation periods are needed to better understand the interaction between these products and turfgrass development. This would allow for a more robust assessment of their potential as an effective and environmentally friendly complementary strategy for turfgrass maintenance.