$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
The experimental workflow for evaluating the efficacy of microbial inoculants in enhancing water-stress tolerance in Romaine lettuce (Lactuca sativa L. var. Paspartu) is summarized in Figure 1. The protocol integrated continuous monitoring of precise irrigation regimes with periodic acquisition of physiological and thermal data. Two distinct microbial products were utilized: (I) microbial consortium, a complex consortium of 67 strains comprising PGPR, actinomycetes, and beneficial fungi, designed to act as a physiological buffer through root colonization and exopolysaccharide (EPS) secretion; and (II) Trichoderma harzianum, a single-strain inoculant of Trichoderma harzianum aimed at inducing systemic resistance and modifying root architecture. The reagents, equipment, and software used are listed in the Table of Materials.
1. Microbial inoculation
A standardized inoculation regime was implemented for both microbial treatments, consisting of six soil-drenched applications targeting the rhizosphere using high-precision micropipettes to simulate field-scale dosages. The first phase occurred during the seedling stage at 5 and 15 days after sowing (DAS). At 30 DAS, seedlings were transplanted into larger cavity trays, with the second phase of inoculations performed 1 day after transplanting (DAT), followed by successive applications at 7, 15, and 20 DAT.
For the microbial consortium, dosages were adjusted according to plant phenology: the initial two applications were applied at 500 g/ha (3 mL/plant), followed by a high-concentration dose of 2 kg/ha at transplanting, and concluding with three maintenance doses of 300 g/ha. Similarly, T. harzianum was applied at 1 L/ha during the first two stages, 3 L/ha at transplanting, and 1 L/ha for the final three applications. This phased approach was designed to ensure robust root colonization and to maintain the microbial population's role as a physiological buffer throughout the critical growth and stress-induction periods.
2. Crop preparation
Certified Romaine lettuce seeds (Lactuca sativa L. var. Paspartu) were germinated in 311-cavity polystyrene trays (11 mL per cavity) using a professional-grade peat moss-based substrate at a depth of 1 cm. Germination was conducted in a controlled bioclimatic chamber maintained at a temperature range of 15 °C to 22 °C and a relative humidity of 60%–70%. Upon reaching 90% germination (approximately 3–5 days after sowing), seedlings were transplanted into larger trays with a 55 mL root-ball capacity to allow for unrestricted root development (Figure 2 and Figure 3). The trial was organized in a completely randomized design (CRD), with 9 experimental units (trays), and 20 seedlings per tray. This setup was structured to accommodate a 3 × 3 factorial arrangement (3 microbial treatments × 3 irrigation regimes). Detailed substrate physical-chemical properties and specific bioclimatic chamber programming14are provided in Figure 3, Supplementary Table 1, Supplementary Table 2, and Supplementary Table 3.
Experimental trays were distributed using a completely randomized layout within the bioclimatic chamber to eliminate edge effects and ensure uniform exposure. The chamber was programmed to simulate dynamic field-like conditions, utilizing a high-temperature regime to replicate open-field thermal stress. During the vegetative stage, environmental parameters were maintained within a variable relative humidity range, and a 9-h photoperiod provided by high-intensity LED lighting (set above 50% of maximum capacity to ensure adequate photosynthetically active radiation)15. Following the establishment phase, the environmental program was adjusted to reflect the intensifying requirements of the water-deficit treatments, as illustrated in the stepwise atmospheric transitions15 (Figure 4 and Figure 5). Specific diurnal/nocturnal setpoints for temperature, humidity, and light intensity are detailed in Supplementary Table 1 and Supplementary Table 2 to streamline the main protocol.
The fertilization strategy was designed to meet the specific phenological requirements of Romaine lettuce, maintaining a balanced nutrient flux throughout the vegetative cycle. During the seedling stage (12 to 24 DAS), nutrients were supplied via targeted applications of Potassium Nitrate (KNO3), Monopotassium Phosphate (KH2PO4), and Calcium Nitrate (Ca(NO3)2). To enhance early establishment, a commercial biostimulant was supplemented during the first 24 days16.
From 26 DAS through the final evaluation at 76 DAS, the fertigation program transitioned to a maintenance regime where the biostimulant was eliminated to isolate the specific effects of the microbial inoculants. All applications were calibrated to maintain optimal electrical conductivity (EC) and pH within the rhizosphere. The complete chronological schedule of mineral concentrations and specific dosing volumes16is detailed in Supplementary Table 3.
3. Treatment with microorganisms
To evaluate the efficacy of biological interventions against hydric stress, three distinct treatments were established: (I) microbial consortium, a high-diversity microbial consortium comprising 67 beneficial strains, including Plant Growth-Promoting Rhizobacteria (PGPR), Actinomycetes, and fungi; (II) T. harzianum, a specialized single-strain formulation of Trichoderma harzianum; and (III) a non-inoculated Control. These treatments were selected based on their potential for Induced Systemic Tolerance (IST), primarily through mechanisms such as root architecture modification, ACC-deaminase production, and exopolysaccharide (EPS) secretion.
Inoculation followed a phased strategic approach. The initial application (3 mL per plant) was performed via high-precision micropipette directly into the rhizosphere at the second true leaf stage to ensure early root colonization. Subsequent applications were calibrated to field-scale equivalents (simulating a density of 75,000 plants/ha). For T. harzianum, a concentration of 2 L/ha was applied, while the microbial consortium was administered at 1 kg/ha. These formulations were dissolved in the irrigation water and applied every 15 days to maintain a stable microbial population as a physiological buffer throughout the vegetative cycle17.
4. Different irrigation treatments
To evaluate the physiological and agronomic plasticity of L. sativa under varying hydric conditions, three irrigation regimes were implemented based on the replacement of crop evapotranspiration (ETc). The volumetric water content was calibrated against the substrate’s field capacity (FC) to ensure precise stress induction. The regimes were defined as follows: (I) Control/Well-watered (CC-100% ETc), where full evapotranspiration was replenished; (II) Moderate Water Deficit (75% ETc), designed to induce intermediate hydraulic limitation; and (III) Severe Water Deficit (50% ETc), aimed at generating critical water stress. Irrigation was administered daily after capturing thermal images using graduated pipettes to ensure accurate replacement of the volume of water lost through evapotranspiration18.
5. Data and image recording
To evaluate the plants' thermal distribution and water status, infrared thermal images were captured daily from Monday to Friday throughout the experimental period. All photographic recordings were conducted between 12:00 PM and 2:00 PM to standardize measurements during the period of maximum transpiration and solar intensity. It is critical to take these images immediately before the daily irrigation treatments to record the cumulative water stress experienced by the plants over the previous 24-h cycle. For image acquisition, the infrared thermal camera was connected to a mobile device and maintained a perpendicular orientation relative to the back of the lettuce plants at a consistent distance of 60–70 cm. The software was configured with a multi-color thermal palette in which temperature gradients are represented from lowest to highest: starting with purple (coldest), transitioning through royal blue, turquoise, green (midpoint), yellow, orange, and finally red (hottest). Within the software interface, the specific tray area containing the plant canopy was manually selected to capture the thermal data. This color scale serves as a physiological indicator: well-hydrated plants exhibit cooler canopy temperatures (purple/blue) due to active transpiration, while dehydrated plants display higher canopy temperatures (orange/red) due to stomatal closure and reduced evaporative cooling. Subsequently, these high-resolution images were processed using the three-point measurement function over specific regions of interest (ROI) to extract precise canopy temperature (Tc) values for the calculation of the Crop Water Stress Index (CWSI).
If CWSI is:
0, then the crop is not under water stress
1 then the crop is under severe water stress
Where:
Tc is the crop canopy temperature, measured with infrared imaging. Tw is the canopy temperature under non-water stress conditions. Td is the canopy temperature under maximum water stress conditions19.
Indicators of plant growth, development, and physiological condition, such as height, leaf number, and leaf temperature, assessed using infrared thermography, are shown in Supplementary Table 4 along with the study variables and conceptual and operational definitions used in the experiment. The combination of these variables allowed for a comprehensive assessment of the impact of the experimental treatments on the physiological performance and development of each plant.
6. Data analysis
Physiological and morphological data were analyzed using a One-Way ANOVA for each irrigation regime to determine the effect of the different microbial treatments. All statistical procedures were performed using statistical software. Before conducting the inferential analysis, the fundamental requirements for parametric statistics were verified to ensure the reliability of the results. The normality of the residuals was confirmed using the Anderson-Darling test (p > 0.100), indicating normality. Additionally, the homogeneity of variances was validated using Levene’s test (p = 0.989), confirming high homoscedasticity across groups. Since both assumptions were met, the Tukey HSD (Honestly Significant Difference) post-hoc test was used to identify significant differences (p < 0.05) among the Treatment means. The individual plant was defined as the experimental unit for all statistical evaluations.
