Revista Facultad Nacional de Agronomía Medellín

Study site and experimental design

The field experiment was conducted from January to August 2023 at the Cultivation and Conservation Field for Amazonian Medicinal Plants, located at the Facultad de Agronomía, Universidad Nacional de la Amazonía Peruana (UNAP), along Zungarococha Highway, San Juan Bautista District, Loreto, Peru (03°46'13.2"S, 73°22'10.4"W) at 126 meters above sea level (masl). Climatic conditions were obtained from the Servicio Nacional de Meteorología e Hidrología del Perú (SENAMHI), with average temperatures ranging from 26.8 to 29.1 °C, total rainfall of 1,437 mm (January-May) and 387.6 mm (June-August), and relative humidity of 83-91%. The experimental design, including treatments, timeline, and evaluated parameters, is illustrated in Figure 1.

Figure 1: Experimental design of turmeric (Curcuma longa L.) field trial showing treatments (T₀-T₃), application timeline, and evaluated parameters.

The soil was classified as sandy loam (70% sand, 17% silt, and 13% clay), with a pH of 6.55, high phosphorus (76.9 ppm), low potassium (78 ppm), and medium-low organic matter content (2.34%). Soil properties were determined following standard analytical methods.

A randomized complete block design was employed, comprising four treatments and five replications. Each experimental unit (5 m² per plot) included two rows with 18 plants each. Treatments consisted of increasing doses of T. harzianum conidia: T₀ (control): 0 g; T₁: 2 g (6.4×10⁹ conidia); T₂: 4 g (12.8×10⁹ conidia); and T₃: 6 g (19.2×10⁹ conidia). Applications were performed at transplanting and repeated every 60 days, for a total of four applications throughout the crop cycle.

Trichoderma harzianum preparation

Spores of T. harzianum were provided by the Servicio Nacional de Sanidad Agraria del Perú (SENASA), Lima. Replication was conducted at the Microbiology Laboratory of the Instituto de Medicina Tradicional (IMET), Iquitos. Initial spore viability was assessed using the plate count method, showing 3.2x10⁹ viable conidia per gram. For mass propagation, the protocol was modified from Naeimi et al (2020), where 50 g of T. harzianum conidia were inoculated into 500 g of sterilized rice (autoclaved at 121 °C for 20 minutes) supplemented with 60 mL of sterile distilled water in sealed Ziploc bags. The moisture content was adjusted to approximately 40% to optimize fungal growth. The bags were manually agitated daily for 30 seconds to ensure uniform distribution and prevent substrate compaction, then incubated under controlled conditions with a 12-hour light/dark photoperiod at 25±2 °C for 5 days in a laminar flow cabinet. After incubation, the colonized substrate showed characteristic green coloration, indicating successful Trichoderma sporulation. The inoculated substrate was subsequently dried under sterile conditions in a laminar flow cabinet to reduce moisture content to <10% and stored at 16 °C in airtight glass containers with silica gel desiccant until use. Prior to field application, spore concentration was determined by serial dilution and plating on potato dextrose agar, confirming viable spore counts of 3.2x10⁹ conidia per gram of dried substrate.

Curcuma longa crop management

The samples were taxonomically identified by preparing flowering voucher samples, which were deposited at the Herbarium Amazonense (AMAZ), Iquitos-Perú. under accession code AMAZ 42787. Rhizomes containing 3-5 viable buds were planted in 20 cm-deep holes, spaced 0.60 m between rows and 0.50 m between plants. Agronomic practices included hilling every 30 days, manual weeding every 15 days, limited irrigation during peak heat, and fertilization three months after planting. Harvesting was conducted eight months after transplanting.

Data collection

A zigzag sampling pattern was used to select 10 representative plants per plot, excluding border plants. Selection criteria included plant height ≥30 cm, higher leaf number, and shoot vigor. Measured variables included the number of leaves, plant height (collar to apex), number of shoots, and rhizome fresh and dry weights using a Dival Model DS digital balance. All measurements were performed at the end of the experiment, 240 days after planting.

Rhizome quality analysis

Rhizome quality analyses were performed on a dry weight basis at the Laboratorio de Compuestos Bioactivos - Instituto Tecnológico de la Producción (ITP), Lima. Protein content was determined using the Kjeldahl method; ash content by incineration in a Barnstead Thermolyne 48000 muffle furnace; fat content using Soxhlet extraction (Buchi E-800). Curcuminoid content was determined by UV-Visible spectrophotometry following the validated method of Hazra et al. (2015), in wich a 500 µg mL-1 curcumin standard was prepared in methanol and diluted to 5-20 µg mL-1 for calibration curve construction, with absorbance measurements read at 421 nm using a Thermo Scientific Genesys 180 spectrophotometer and results expressed as curcumin equivalents per gram of dry sample.

Statistical analysis

Data were analyzed using parametric methods within a randomized complete block design consisting of four treatments and five replications. Analysis of variance (ANOVA) was performed to assess treatment effects, followed by Tukey's HSD test for mean separation (α=0.05).

The treatment sum of squares was partitioned into linear, quadratic, and cubic polynomial components using orthogonal contrasts for deeper interpretation of dose-response relationships. Regression analysis was also performed to model variable responses and identify the optimal conidial dose. All statistical analyses were conducted using SISVAR and RStudio (v4.4.1).

Agronomic characteristics of Curcuma longa

The agronomic variables of C. longa were significantly affected by the application of T. harzianum (Table 1). Leaf number and plant height showed highly significant differences (P≤0.01), while shoot number varied significantly at P≤0.05. Quadratic models were fitted for leaf number and plant height, and a cubic model for shoot number (Figure 2).

Table 1: Analysis of variance of the regression of the agronomic characteristics, yield and quality of C. longa by T. harzianum action.

Figure 2: Quadratic regression models showing the effects of T. harzianum doses on A. leaf number, B. plant height, and C. shoot number.

According to treatment means (Table 2), plants inoculated with 4 g developed more vigorous vegetative growth than the control, with averages of 47.25 leaves, 61.50 cm in height, and 1.33 shoots per plant. Regression analysis estimated the maximum responses at intermediate doses: 49.11 leaves at 4.30 g (Figure 2A), 63.28 cm in height at 3.88 g (Figure 2B), and 1.38 shoots per plant at 4.30 g (Figure 2C). At higher doses, the models predicted reductions in all three traits. Visual differences among treatments at harvest are shown in Figure 3.

Table 2: Effects of Trichoderma harzianum doses on agronomic traits and rhizome yield in Curcuma longa, with mean separation by Tukey&apos;s test.

Figure 3: Growth performance of turmeric (Curcuma longa L.) under Trichoderma harzianum application: control (T₀) and three inoculation doses (T₁-T₃).

The enhanced vegetative development observed with T. harzianum application aligns with findings reported by Verma et al. (2019), who documented significant improvements in turmeric growth parameters under organic practices involving T. harzianum seed treatment at 5 g kg-1 seed rhizome plus soil application. The beneficial effects can be attributed to the biocontrol and growth-promoting capabilities of Trichoderma species documented by Tripura et al. (2018), who reported that the combined application of the recommended dose of fertilizer with T. harzianum resulted in higher growth parameters, including plant height, pseudostem diameter, leaf area, and leaf-area index in turmeric.

The quadratic response patterns observed for leaf number and plant height suggest optimal bioagent concentrations exist, beyond which diminishing returns occur. This dose-dependent response has been documented in integrated management systems by several researchers. Altaf et al. (2022) found that T. harzianum effectiveness in sustainable management approaches varied with application methods, while Nanda et al. (2024) demonstrated that moderate concentrations of biocontrol agents, including T. harzianum provided optimal results in sesame cultivation compared to higher doses. The cubic model for shoot number indicates more complex interactions between T. harzianum and plant developmental processes, possibly related to hormonal balance modifications.

Yield of Curcuma longa rhizomes

Rhizome production also varied significantly among treatments (P≤0.01; Table 1). Fresh and dry yields fitted quadratic regression models (Figure 4).

Figure 4: Quadratic regression models of rhizome yield response to fertilizer doses. A. Fresh and B. Dry.

The observed data (Table 2) indicated increases from 6.52 kg per plot in the control to 8.92 kg at 4 g for fresh yield, and from 0.87 to 1.18 kg per plot for dry yield. The models estimated maxima slightly above these values, with 9.14 kg per plot (10.83 t ha-1) at 5.27 g for fresh yield (Figure 4A) and 1.23 kg per plot (1.44 t ha-1) at ~5 g for dry yield (Figure 4B). At doses greater than 5 g, yields decreased. Tukey's test (Table 2) confirmed significant differences between the control and all inoculated treatments, but no differences among the treated groups.

The yield improvements obtained with T. harzianum application demonstrate substantial economic benefits for turmeric cultivation. The 36.8% increase in fresh yield observed at optimal doses is notable because Tripura et al. (2018) reported the highest benefit-cost ratio of 4.87 with RDF + VAM + T. harzianum treatment combinations. Studies evaluating biocontrol efficacy show that T. harzianum can significantly improve plant performance through multiple mechanisms, including disease suppression and enhanced nutrient uptake (Jakatimath et al. 2017).

The quadratic response pattern for both fresh and dry yields indicate an optimal inoculum density of around 5 g per application, beyond which competitive effects may limit benefits. Wagh et al. (2017) demonstrated that bioagents, including T. harzianum exhibited optimal mycelial growth inhibition at specific concentrations, with effectiveness declining at higher doses. Similar patterns have been observed by Gaur et al. (2011), who found that T. harzianum paint formulations showed optimal effectiveness at intermediate concentrations for disease management in citrus. The strong correlation between fresh and dry yield responses suggests that T. harzianum influences both water accumulation and dry matter partitioning to rhizomes.

Rhizome quality evaluation

Chemical composition of the rhizomes was also influenced by T. harzianum (Table 1). Protein, fat, and ash contents followed cubic models, while curcuminoids adjusted to a quadratic response (Figure 5).

Figure 5: Polynomial regression models showing the relationship between T. harzianum doses and rhizome quality attributes: A. Protein content, B. Fat content, C. Ash content, and D. Curcuminoid content.

Mean comparisons (Table 2) showed that protein increased from 7.20% in the control to 8.45% at 4 g, while curcuminoids rose from 6.59% in the control to 6.98% at 2 g. The regression models estimated higher values than those observed in the treatments, with 8.72% protein at 5.31 g (Figure 5A) and 7.13% curcuminoids at 2.30 g (Figure 5D). Fat and ash contents fluctuated across doses without a defined optimum (Figures 5B-C).

The enhancement of protein content (17.4% increase) and curcuminoids (5.9% increase) represents improvements in rhizome quality. Research on C. longa chemical composition has shown substantial variations depending on cultivation practices and environmental conditions. Setzer et al. (2021) documented major variations in essential oil components among five different C. longa varieties cultivated in North Alabama, with ar-turmerone ranging from 6.8-32.5%, α-turmerone from 13.6-31.5%, and β-turmerone from 4.8-18.4%, indicating that cultivation practices strongly influence bioactive compound profiles.

The mechanisms underlying quality improvements may involve enhanced secondary metabolite biosynthesis pathways. Gururani et al. (2022) demonstrated that cultivation practices significantly affect phytochemical composition and biological activities in C. longa accessions from different altitudes, with variations in major compounds including turmerone, germacrone, eucalyptol, caryophyllene, and α-curcumene. Environmental and biological factors can modulate the production of these bioactive compounds through enhanced antioxidant activities and improved nutrient metabolism.

The quadratic response pattern for curcuminoids, with an optimum at 2.30 g, suggests that lower bioagent concentrations may be more effective for enhancing secondary metabolite production compared with vegetative growth parameters. Marchant et al. (2022) reported that cultivation conditions significantly influence phytochemical profiles in C. longa, with specific treatments increasing polyphenols, flavonoids, and curcumin synthesis while boosting radical scavenging activity. The differential optima observed (2.30 g for curcuminoids vs. 5.31 g for protein) indicate that T. harzianum applications can be tailored according to production goals.

The observed improvements in curcuminoid content are particularly significant given their commercial and therapeutic value. Fernández-Marín et al. (2021) demonstrated that cultivation methods influence the final properties of C. longa oil, including total phenolic content and antioxidant properties, with optimized conditions yielding 10.32% extraction efficiency and improved bioactive. Similarly, Baka (2023) reported that C. longa rhizome extract showed enhanced phenolic content and bioactive properties when plants were subjected to specific cultivation treatments, with curcumin content reaching 3,220.8 μg g-1 dry weight. The integrated approach of combining biological treatments with optimized cultivation practices offers potential for sustainable enhancement of both yield and quality parameters in medicinal plant production systems.