Several authors have found that humic acid is a sustainable alternative to mitigate the effect of salt stress, because it is an organic compound that is formed through the decomposition of organic matter and improves soil aggregation, resulting in greater water retention, in addition to the chelation of several nutrients, increases the activity of the Rubisco enzyme and reduces the toxicity of some uptake ions. However, previous studies have revealed that humic acid increases its salinity tolerance efficiency and increases yield when combined with a beneficial microorganism.
Although there are a large number of microorganisms, recent studies have shown that the use of Trichoderma harzianum has achieved efficient disease control results and produces organic substances that solubilize ions, improving the availability and uptake of nutrients from the fertilizers applied to the plant. Therefore, the associated application of humic acid and Trichoderma harzianum could improve intercellular communication in salt stress signaling, plant homeostasis and plant performance.
The bell bell pepper (Capsicum annum L.) or sweet bell pepper is one of the main horticultural crops with a high commercial demand worldwide, because the fruit has a high nutritional value, good taste, pleasant aroma and beneficial properties for human health. However, the constant productive growth of this vegetable has resulted in increased exposure to various abiotic stresses such salt stress.
Taking into account that horticultural crops such as Capsicum require good nutritional and hormonal management, so in situations of salt stress is detrimental because the accumulation of salts alters the uptake of nutrients and cellular metabolism of the plant, resulting in low yield and if not intervened in time causes the death of the plant. In view of this, farmers carry out management practices such as the use of desalinators and/or washing with heavy irrigation to mitigate the saline effect.
However, these practices are not completely efficient and water resources are limiting in coastal agriculture. In addition, in sandy soils, salinity reduces beneficial soil microbial activity and these practices do not maintain this activity and do not retain soil moisture, as a result, crop nutritional problems have arisen, the soil biota is poor and it has become more difficult to achieve expected yields.
Agronomic Parameters
The mean squares results of the analysis of variance in Table 1 show significant differences (p<0.05) for the main factors humic acid (HA) and Trichoderma harzianum (TH) in the variables plant height (PH), root dry weight (RDW) and plant dry weight (PDW) during the two seasons, while the effect of electrical conductivity (EC) levels was not significant (p>0.05) in both seasons.
Regarding the interaction of factors, the variables PH, RDW and PDW showed significant effects in the interaction between EC x AH and for AH x TH in both seasons, indicating that salt concentrations were affected by increasing doses of HA, as well as HA levels responded to the application of TH, which indicates that increasing doses of HA with TH produces a significant effect on agronomic parameters.
However, the analysis shows that interaction of three factors for the variables PH and RDW in 2023 season had no significant effect (p>0.05), while the variables PH, RDW and PDW in 2024 season reported a significant effect, which confirms that as time passes, salt concentrations are affected by increasing doses of HA with application of TH.
| Source of Variation | MS | ||||||
|---|---|---|---|---|---|---|---|
| DF | PH-2023 | PH-2024 | RDW-2023 | RDW-2024 | PDW-2023 | PDW-2024 | |
| Blocks | 3 | 2.45ns | 1.14ns | 1.27ns | 0.05ns | 4.79ns | 19.66ns |
| Cond Elec (CE) | 1 | 0.16ns | 1.00ns | 1.92* | 0.21ns | 29.17ns | 6.90ns |
| Ac. Hum (AH) | 2 | 2947.65** | 5395.31** | 259.54** | 1943.55** | 29075.96 ** | 43180.58 |
| CE x AH | 2 | 35.27** | 0.24ns | 0.45ns | 0.22ns | 145.22* | 48.99** |
| Trichoderma (T) | 1 | 410.32** | 759.07** | 185.46** | 210.59** | 4495.39** | 13635.02** |
| CE x T | 2 | 0.03ns | 3.59ns | 0.06ns | 0.30ns | 1.02ns | 62.38** |
| AH x T | 2 | 62.69** | 24.67** | 52.49** | 47.63** | 313.62** | 6.32ns |
| CE x AH x T | 2 | 5.05ns | 9.45** | 0.47ns | 1.21* | 117.94* | 146.53** |
| Error | 33 | 4.27 | 1.33 | 0.4 | 0.25 | 27.32 | 7.83 |
| Total | 47 | ||||||
| VC (%) = | 6.82 | 3.35 | 6.21 | 4.35 | 6.32 | 3.01 | |
Since no interaction was found between the three factors in the PH and RDW variables in the 2023 season, the main effects analysis was carried out, and this analysis shows each factor separately, as shown in the following Figures 1A and 1B. With respect to HA levels, the 30 L ha-1 dose was statistically superior to the other doses, producing a 63.36% increase in PH compared to the 0 L ha-1 HA dose. For the RDW variable, it presented an increase of 57.55% in relation to 0 L ha-1 HA in the 2023 season.
Regarding the TH factor, it is observed that the application of this microorganism statistically exceeds the treatment without application of Trichoderma harzianum by 17.60% for PH and 32.35% for RDW, however, the use of this microorganism in the 2024 season increased its values by 13.42% for PH and 10.27% for RDW compared to the previous season (see Figures 1A and 1B).
Yield Parameters
Significance (p<0.05) in the main factors as in the interaction between EC and HA, as well as for HA and TH were fruit length (FL), fruit width (FW), number of fruits per plant (NFP), number of fruits per plant (WFP) and total yield (TY) during the two seasons. This indicates that the application of irrigation with EC water of 0 and 4 dS m-1 was affected by the increase in HA levels with the use of Trichoderma harzianum producing a significant effect on yield parameters. While the effect of the interaction between EC and TH was not significant (p>0.05) in both seasons.
It is also observed that the variables that showed interaction between the three factors were FL, NFP, WFP and TY, indicating that at doses of 30 and 15 L ha-1 of HA with the application of Trichoderma harzianum did not respond to the increase in EC, while the variable FW did not show interaction between factors in the first season, while in the following season it showed significance.
Regarding the effect of the main factors for the variable FW in the 2023 season. The FW with EC levels was not affected in both seasons. When HA levels were evaluated separately, it was observed that for the 30 and 15 L ha-1 HA doses there was an increase of 36.47 and 32.46% in FW in the first season in relation to the 0 ha-1 HA dose. Likewise, the application of TH reached the highest value in FW, producing an increase of 14.15% compared to that without the use of TH (see Figure 1C).
Regarding NFP and WFP for the first season, treatments T5 and T11 reached the highest values with 8.75 and 8.50 fruits and 1.15 and 0.96 g plant-1, exceeding the control by 85.71 and 85.29%, 85.71 and 85.29% for NFP and 91.49 and 91.3% for WFP, respectively. However, in the following season, the values of treatments T5, T11 and T3 increased by 25.53, 26.09 and 31.82% for NFP and by 22.82, 23.29 and 30.94% for WFP in relation to the previous season.
The total yield in the 2023 season showed that T5 and T11 were significantly superior to the other treatments, obtaining values of 31.84 ± 0.11 and 30.93 ± 0.07 t ha-1 exceeding 89.54 and 89. 23% compared to the control, while in the 2024 season it was observed that at doses of 30 and 15 L ha-1 of HA with TH at an EC of 0 and 4 dS m-1 produced an increase of 23.18, 23.76 and 30.73% with respect to previous season. It is observed that increasing doses of HA with TH showed tolerance to the EC of 4 dS m-1.
Ion Concentrations in Leaf
Based on the results, HA, HT and the interaction between two factors obtained significant effect (p ≤ 0.01) on the concentrations of K+, Ca2+, Na+ and Cl– together with the relationship of K+/Na+ on bell pepper leaves. However, the EC and the interaction between three factors only presented significance in Cl– for both seasons. Also, Figure 2A, show that the concentrations of K+ and Ca2+ decreased significantly with increasing salinity level. However, by increasing the doses of HA combined with microorganism the concentration of K+ and Ca2+ showed an increase of 37.88 and 48.71% in ratio of plants subjected to salt stress, for 2023 and 2024 seasons, respectively (Figure 1D and 1E).
While the Na+ concentration in leaves of bell pepper plants without HA application in saline soil and subjected to saline irrigation (EC 4 dS m-1), an increase of 39.78 and 50.96% was found in two seasons (Figure 1F) and for Cl– concentration in leaves showed an increase of 59.24 and 63.29% when plants were subjected to salt stress for 2023 and 2024 seasons respectively.
Regressions for plant height (A), root dry weight (B), number of fruits per plant (C), total yield (D), potassium sodium ratio (E) and proline (F) on two levels of electrical conductivity, three levels of humic acids and two levels of Trichoderma harzianum. Note: EC = electric conductivity (dS m-1).
As for K+/Na+ ratio, a significant reduction of 62.61 and 74.93% was observed when plants in saline soil were subjected to saline water irrigation (EC of 4 dS m-1) in both seasons. Thus, the highest K+/Na+ ratio in bell pepper leaves was found in saline soil treatments with water irrigation at EC of 0 and EC of 4 dS m-1 accompanied by 30 L ha-1 of HA combined with TH (Figure 1G).
Proline
The factors EC, HA, TH and their interactions had a significant effect (p≤0.01). It is observed that the proline content significantly increases with increasing salinity level in bell pepper plants applied with high doses of HA combined with TH. However, the proline content in plants without HA application in saline soil and subjected to saline irrigation (EC of 4 dS m-1) decreased significantly by 41.4 and 33.57% in 2023 and 2024 seasons, respectively.
Likewise, the treatments that obtained the highest amount of proline were found in the saline soil treatment with saline water irrigation (EC of 4 dS m-1) accompanied with the application of 30 L ha-1 of HA combined with TH (4.08 ± 0.08 and 4.25 ± 0.12 mg proline g-1 DW) followed by saline soil treatment with water irrigation (EC of 0 dS m-1) accompanied by the application of 30 L ha-1 of HA combined with TH (4.01 ± 0.07 and 4.17 ± 0.06 mg proline g-1 DW) (Figure 1H).
Regresión
The direct relationship between PH, RDW, NFP and TY and increasing doses of HA with and without the use of Trichoderma harzianum under EC levels, which was fitted to a linear function where the highest value was reached at the 30 L ha-1 dose of HA with TH. Likewise, the coefficient indicates that the increase of 1 L of HA combined with the microorganism under saline soil and water irrigation (0 and 4 dS m-1 of EC) produces an increase of 16.64 and 13.23 cm of plant height, 6.76 and 6.41 g plant-1 of root weight, of 4.5 and 3.75 fruits per plant, and the yield increases by 16.64 and 13.23 t ha-1 (Figure 2).
In addition, EC levels did not affect PH, NFP and WFP when 15 and 30 L ha-1 of HA were applied with TH. This model demonstrates that increasing doses of HA combined with the microorganism show tolerance to salinity and increases plant height, fruit number, root weight and yield.
While for K+/Na+ ratio and proline content, Figure 2E, shows that the increase of 1 L of HA combined with the microorganism under saline soil and with water irrigation of 0 and 4 dS m-1 EC, there is the increase of 35.12 and 31.65% in K+/Na+ ratio and the increase of 1.21 and 1.23 mg proline g-1 DW (Figure 2F). In contrast, the application of increasing doses of HA combined with TH showed higher K+ uptake and proline synthesis as a defense mechanism against salt stress.
Dicussion: Humic Acid and Trichoderma Harzianum Enhance Salinity Tolerance and Productivity of Bell Pepper in Saline Soil
The results showed that saline soil accompanied by irrigations of 0 and 4 dS m-1 of electrical conductivity, reduces the growth and productivity of bell pepper, because the plant subjected to saline stress, loses its ability to uptake water and nutrients, presents a hormonal imbalance, low cell division, stomata closure and toxic effect of Na+. However, increasing the dose of humic acid combined with Trichoderma harzianum significantly increases salinity tolerance and produces a significant effect on agronomic parameters (PH, RDW and PRW) of bell pepper during the 2023 season and these values increase slightly in 2024 season.
These results are consistent with the findings tested applications of beneficial microorganisms with different doses of HA and found a significant effect on agronomic parameters of bell pepper when increasing the doses of HA with the microorganism under salt stress conditions.
In study reported when applying HA combined with Trichoderma, plant height ranged from 44.55 to 55.93cm without salinity conditions, while in the study it was lower because saline soil conditions and saline irrigation of more than 2 dS m-1 restricts the movement of water to the leaf tissue, showing a reduction in plant dry matter.
It was observed in this study that saline soil with water irrigation of 0 and 4 dS m-1 EC caused significant reductions in root dry weight and leaf biomass of bell pepper. This is corroborated that revealed that plant growth retardation under salt stress is due to Na+ accumulation in plant tissue, which triggers electrolyte leakage, leading to low root and leaf growth. Specifically, the accumulation of salts in rhizosphere of plant reduces and hinders the entry of water into the vascular bundles of the roots, causing the cells to lose turgor and a physiological imbalance to occur, hindering the uptake of nutrients.
However, in our research, increasing HA doses combined with Trichoderma for two seasons significantly reduced the negative effect of salinity and increased root development (root dry weight), improving nutrient and water uptake to plant. This effect is due to the double action of HA and Trichoderma. Recent studies have shown significant effects of the combined use of HA and beneficial microorganisms on root and leaf growth of barley.
Regarding HA, the effect is due to soil aggregation, ionic imbalance and moisture retention. Specifically, HA produces soil aggregates by holding moisture longer, which causes nutrients to be mobilized by mass flow to the roots. It has been reported that soil aggregation is a dynamic process in which plant and microorganisms interact and influence the increase of soil fertility and this is accompanied by an improvement in the availability of nutrients.
While the action of Trichoderma, someone indicated that the application of this microorganism proliferates in the rhizosphere of the plant and through it synthesizes organic compounds that solubilize ions, improving the availability of these and takes advantage of the water retained by the soil aggregates, resulting in increased root growth of the plant.
In addition, the Trichoderma applied from the transplant and in early stages of the plant of bell pepper, tends to increase its proliferation, helping to root the seedlings and stimulates in it, the synthesis of endogenous hormones (gibberellins and auxins), producing greater root development, which results in the increase of the uptake of nutrients. On the other hand, someone indicated that Trichoderma produces fungal elicitors in the rhizosphere of the plant, which solubilizes nutrients and produces mycoparasitism in pathogenic fungi of the soil, fulfilling a double function as biofertilizers and biofungicides.
Regarding fruit characteristics, the supply of saline irrigation of 4 dS m-1 of EC in saline soils reduced 42.47% in fruit diameter and 54.26% in fruit length. Soil salinity causes osmotic stress in the plant, which significantly reduces the movement of water from soil to plant tissue, leading to metabolic alterations in the cells and fruit filling of the bell pepper fruit. On the other hand, the application of increasing doses of HA was favored when using Trichoderma to obtain a fruit of better caliber, because it increases the tolerance to salinity and implies that the conduction of water is continuous, which results in a continuous flow of carbohydrates and water to the fruit achieving a better caliber.
Likewise, results show that salt stress increases flower drop and reduces fruit set and increasing doses of HA combined with Trichoderma produce a significant effect on NFP and WFP. These results confirm that the beneficial microorganism improves intercellular communication in salt stress signaling and plant homeostasis.
In addition, this microorganism in the rhizosphere produces organic acids which generates acidic conditions involving nutrient solubilization, improves soil aggregation and allows increased water and nutrient transmission to growing fruits. After one year, the combination of HA and Trichoderma, improves the uptake of water and nutrients, as well as a continuous flow of photosynthates and water to the fruit, achieving a higher number of fruit set and higher fruit weight, as evidenced by significant increases in these characteristics.
With respect to yield, this study showed that the application of high doses of AH combined with TH significantly reduces salt stress on bell pepper yield. In this regard, someone have revealed that the application of high doses of HA the yield of bell pepper ranged between 30 to 40 t ha-1, while the yield was higher when combining HA with a beneficial microorganism.
This effect is due to the fact that HA reduces salt stress through enzymatic activity and antioxidant compounds (phenols, flavonoids and carboxylic acid) that inhibit oxygen free radicals, while the beneficial microorganism stimulates the plant defense mechanisms and biosynthesis of phytohormones, promoting root proliferation, in addition, it produces organic acids that solubilize nutrients of different mineral forms (precipitates and fertilizers), stabilize soil aggregates and thus improve the uptake of nutrients and water by the plant, so that this combination increases photosynthetic activity, the accumulation of carbohydrates and water in fruit, resulting in a yield similar to bell pepper cultivation in non-saline conditions.
Regarding the concentration of ions in bell pepper leaves, the results indicate that the concentration of K+ and Ca2+ decreases when the plants are subjected to saline soil and saline water irrigation, while the concentration of Na+ and Cl– increase. This fact is due to the fact that the plant in saline soil has difficulty in absorbing water and nutrients, resulting in ionic stress. In addition, Na+ and K+ competition is high, causing the plant to absorb less K+ since the ionic radii of these two ions are similar.
However, absorbed Na+ causes alterations in cell metabolism, such as replacement of Ca2+ ion at cell wall binding sites, resulting in damage to cell permeability and integrity. However, the addition to the soil of HA combined with Trichoderma harzianum significantly increases the concentration of K+ and Ca2+ in bell pepper leaves, since HA produces soil aggregation and the adhesion of ions to the sand particle, allowing greater availability of K+ and Ca2+ in soil solution and through its function as a pseudocytokinin increases the uptake of K+.
In addition, when HA is combined with the microorganism, it produces organic acids that solubilize nutrients, which results in increased K+ uptake and reduces the uptake of Na+ ions and Cl– in saline soil conditions with saline water irrigation and increases the K+/Na+ ratio, confirming that HA is more efficient when combined with a beneficial microorganism.
Likewise, salt stress causes the plant to synthesize proline as a defense mechanism. However, the study showed that plants without HA and TH application, the proline content was lower compared to increasing doses of HA combined with the microorganism, so that osmoregulation was higher in these treatments.
This useful finding may be due to the fact that beneficial microorganisms communicate with and stimulate the plant, accumulating osmolytes at the cellular level that regulate the water potential and solubilize K+ allowing to maintain the Na+ and K+ ratio in plant. Also, the microbes produce exopolysaccharide which is a biofilm that retains water and binds Na+ and Cl– ions, resulting in stabilization of soil aggregates, generating the movement of nutrient and water transmission to the root efficiently.
In addition, Trichoderma produces organic acids that solubilize nutrients from different mineral forms (precipitates and fertilizers) and humic acid, stabilizes soil aggregates and thus, fertilizer nutrients are better utilized, increasing the soil microbial community, photosynthetic activity, carbohydrate accumulation and water conduction to the fruit. Therefore, as the dose of humic acids combined with the beneficial microorganism increases, the plant shows tolerance to soil salinity and saline water irrigation of up to 4 dS m-1, achieving good growth and productivity in bell pepper, similar to plants without salt stress. However, further experiments on the study of the physical, chemical and biological properties of the soil are required for a better understanding.
Conclusions
The combined application of humic acid and Trichoderma harzianum, increases the tolerance to salinity and produces a significant effect on plant height, plant dry weight and increases root development, which improves the uptake of nutrients and water, favoring fruit set, fruit size and fruit weight per plant, resulting in the increase of the total yield of bell pepper in both seasons.
It was also found that the plant subjected to salt stress, increases the uptake of sodium and chlorine, causing harmful effects on it. However, the increase of the doses of humic acids combined with Trichoderma harzianum, significantly increases the concentration of K+ and Ca2+ in leaves, also, the proline content increases, generating defense mechanisms to the saline stress. Therefore, this combination is a sustainable alternative of bell pepper to reduce salt stress in arid and semi-arid areas.
To learn more about the combination of Fulvic acid and Trichoderma harzianum, please contact Dora team. We supply raw materials, and you hold formulations.