Characterization of biostimulants and their effects on soil health, enzymatic activity, and disease incidence in tomato (Solanum lycopersicum L.)

Introduction

Cultivation of high-yielding varieties and hybrids to enhance food production has increased the dependence on synthetic fertilizers. However, their continuous use may adversely affect soil quality and the environment. Therefore, maintaining soil health is essential for key biochemical processes such as organic matter transformation (Miltner et al., 2011) [13] , nutrient release (Wicherna et al., 2007) [20] and degradation of xenobiotics. Hence, there is a growing need for sustainable agronomic technologies that improve soil health while enhancing plant growth, nutrient uptake, stress tolerance and crop productivity. Bio stimulants have recently gained considerable attention as an environmentally friendly approach for improving crop performance without imposing additional stress on soil resources. The European Biostimulants Industry Council (EBIC, 2016) defines bio stimulants as substances or microorganisms that stimulate natural processes in plants or the rhizosphere, thereby improving nutrient uptake, nutrient use efficiency, tolerance to abiotic stress and crop quality, irrespective of their nutrient content. Biostimulants, which may be natural or synthetic organic substances containing hormones or their precursors, promote plant growth and development when applied in small quantities and contribute to improved crop productivity and soil properties. Absolute control T7 75% RDF+ soil drenching with BS1 @ 3.75 kg/ha T2 100% RDF T8 100% RDF+ soil drenching with BS2 @ 2.5 lt/ha T3 75% RDF T9 100% RDF+ soil drenching with BS2 @ 3.75 lt/ha T4 100% RDF+ soil drenching with BS1 @ 2.5 kg/ha T10 75% RDF+ soil drenching with BS2 @ 2.5 lt/ha T5 100% RDF+ soil drenching with BS1 @ 3.75 kg/ha T11 75% RDF+ soil drenching with BS2 @ 3.75 lt/ha T6 75% RDF+ soil drenching with BS1 @ 2.5 kg/ha Note: 1. BS1 -Bio stimulant solid form BS2 -Bio stimulant liquid form 2. RDF 250:250:250 kg N, P2O5, K2O ha -1 along with FYM @ 25t ha-1 (T2-T11) 3. Biostimulants will be applied at the time of transplanting (T4-T11)

Material and methods

The soil at the experimental site was sandy loam in texture with 62.83, 23.86, and 13.29 per cent sand, silt and clay, respectively and bulk density and maximum water holding capacity of soil was 1.39 Mg m -3 and 30.13%. The soil was slightly acidic in reaction (pH 6.26) and low in soluble salts (0.10 dS m -1 ). The soil was medium in organic carbon (0.52%), medium in available nitrogen (308.6 kg ha -1 ), medium in available P2O5 (34.61 kg ha -1 ), medium in available K2O (254.28 kg ha -1 ) and sufficient in sulphur (18.71 mg kg -1 ). The exchangeable calcium and magnesium content of soil was 4.85 and 3.46 c mol kg -1 , respectively. The content of DTPA extractable iron, manganese, zinc, copper and hot water-soluble boron was 15.44, 7.03, 1.59, 0.87 and 0.38 mg kg -1 , respectively.

Characterization of bio stimulants

The analytical results indicated that pH of solid and liquid form of bio stimulant were 6.24 and 6.46, respectively and electrical conductivity of the solid and liquid form of bio stimulant were 0.25 dSm -1 and 0.27 dSm -1 , respectively. The nutrient composition of solid form of bio stimulant was composed of N (18.06%), P (16.09%), K (21.45%), Ca (0.18%), Mg (0.26%), S (3.56%), Fe (0.124 ppm), Mn (0.64 ppm), Cu (0.51 ppm), Zn (0.51ppm) and B (0.016 ppm). With respect to nutrient composition of liquid form of bio stimulant composed of N (22.37%), P (20.55%), K (18.6%), Ca (0.34%), Mg (1.50%), S (3.12%), Fe (0.146 ppm), Mn (0.68 ppm), Cu (0.73 ppm), Zn (0.73 ppm) and B (0.029 ppm). From the analysis of the data suggested that liquid form of bio stimulant contained more N and P than solid form of biostimulants, whereas solid form of biostimulants contained more K than liquid form of bio stimulant. The similar nutrient composition of biostimulants was reported by Keiji et al. (2012) [11] , Colla et al. (2017) [6] and Rouphael et al. (2017) [16] .

Enumeration of microorganisms for biostimulants

Quantitative analysis of solid form of bio stimulant revealed that, it consists of bacterial count (1×10 7 CFU/g), fungal count (2×10 5 CFU/g), actinomycetes (1×10 5 CFU/g), nitrogen fixing bacterial (5×10 6 CFU/g) and phosphate solubilizing microorganisms (2×10 5 CFU/g). Quantitative analysis of liquid form of bio stimulant revealed that, it consists of bacterial count (1×10 8 CFU/ml), fungal count (5×10 6 CFU/ml), actinomycetes (1×10 6 CFU/ml), nitrogen fixing bacterial (1×10 7 CFU/ml) and phosphate solubilizing microorganisms (5×10 6 CFU/ml) (Table 3).

Results and discussion

Soil physical properties

The data pertaining to bulk density and maximum water holding capacity as influenced by the application of biostimulants and presented in Table 4. Results indicated that bulk density of the post-harvest soil ranged from 1.30 -1.38 Mg m -3 . The data revealed that, there was no significant difference in the bulk density between the treatments. Maximum water holding capacity in soil and the influence of biostimulants on various treatments was found to be statistically significant. However, among the treatments, T9 treatment with application of 100% RDF along with soil drenching of BS2 @ 3.75 L ha -1 exhibited the maximum water holding capacity about 39.03% and found on par with 100% RDF+ soil drenching with BS1 @ 3.75 kg ha -1 (T5) about 38.11% and had significant difference with 100% RDF (T2) about 34.60%. Keen rackzowski method Baruah and Barthakur, 1997) [3] Maximum water holding capacity Keen rackzowski method Baruah and Barthakur, 1997) [3] pH (1:2.5) Potentiometric method Jackson (1973) [9] EC (1:2.5) Conductometric method Jackson (1973) [9] Available nitrogen (kg N ha -1 )

Alkaline potassium permanganate method Subbaiah and Asija (1956) [19] Available phosphorous (kg P2O5 ha -1 ) Bray's extractant using spectrophotometer (Bray and Kurtz, 1945) [5] Available potassium (kg K2O ha -1 ) Ammonium acetate extract using flame photometry Jackson (1973) [9] Exchangeable calcium (c mol (p+) kg -1 )

Complexometric titration (Jackson, 1973) [9] Exchangeable magnesium (c mol (p+) kg -1 )

Complexometric titration (Jackson, 1973) [9] Available sulphur (mg kg -1 ) Turbidometric method (Black, 1965) [4] Soil Chemical properties Soil pH and Electrical Conductivity Application of biostimulants, did not show significant differences in soil pH and electrical conductivity. The plots treated continuously with recommended dose of fertilizers alone recorded lower pH and EC values than those treated in combination with biostimulants. However, the higher pH value (6.63) and electrical conductivity (0.17 dS m -1 ) was observed with the 100% RDF along with soil drenching of liquid form of bio stimulant @ 3.75 L ha -1 (Table 5).

Available Nitrogen

Results revealed that the soil application of 100% RDF along soil drenching with BS2 @ 3.75 L ha-1 (T9) showed higher available nitrogen (301.44 kg ha -1 ) and found on par with treatments T5: 100% RDF along soil drenching with BS1 @ 3.75 kg ha -1 (298.77 kg ha-1), T7 supplied with 75% RDF along with BS1 @ kg ha -1 (294.12 kg ha -1 ) and treatment T11: 75% RDF along soil drenching with BS2 @ 3.75 L ha -1 (296.17 kg ha -1 ) and differ significantly with 100% RDF (286.23 kg ha -1 ). Arjunmend et al. (2015) [1] reported that biostimulants can stimulate the growth and activity of beneficial soil microbes, including nitrogenfixing bacteria. These bacteria convert atmospheric nitrogen into a form that can be used by plants. Biostimulants would also help to improve the mineralization of organic nitrogen into inorganic form in the soil (Table 6).

Available Phosphorous

Plots treated with 100% RDF along with soil drenching with liquid form of bio stimulant @ 3.75L ha -1 (T9) showed higher available phosphorous (50.29 kg ha -1 ) and found on par with 100% RDF along with soil drenching with solid form of bio stimulant @ 3.75 kg ha -1 (48.51 kg ha -1 ), T7 which receives 75% RDF+ soil drenching with BS1 @ 3.75 kg ha -1 (46.55 kg ha -1 ) and treatment T11: 75% RDF+ soil drenching with BS2 @ 3.75 L ha -1 (47.92 kg ha -1 ) and found significantly difference with 100% RDF (37.14 kg ha -1 ). The increase in available phosphorus levels may be attributed to enhanced phosphate solubilization by application of biostimulants, a process facilitated by certain microorganisms that possess the capacity to solubilize inorganic phosphorus through mechanisms such as chelation, exchange reactions, phosphate production and the release of organic acids, thereby transforming insoluble phosphates into soluble forms. Similar results were also reported by Bade et al., (2017) [2] in Chilli and Kumari et al., (2013) [12] in tomato (Table 6).

Available Potassium:

The available potassium content of soil was recorded significantly higher with the 100% RDF along with soil drenching with BS2 @ 3.75 L ha -1 (286.80 kg ha -1 ) and found on par with with treatments T5: 100% RDF along with soil drenching with BS1 @ 3.75 kg ha -1 (284.15 kg ha -1 ), T7 which received 75% RDF+ soil drenching with BS1 @ 3.75 kg ha -1 (274.40 kg ha -1 ) and T11: 75% RDF+ soil drenching with BS2 @ 3.75 L ha -1 (280.92 kg ha -1 ) over 100% RDF (267.07 kg ha -1 ). The increase in potassium content could potentially be attributed to enhanced mineral dissolution rates, leading to the release of potassium, as well as the enzymatic activities of chitinase and cellulases, which contribute to the breakdown of minerals. These results align with previous studies conducted by Bade et al., (2017) [2] in chilli and Kumari et al., (2013) [12] in tomato (Table 6).

Exchangeable Calcium and Magnesium

Exchangeable calcium and magnesium in soil (5.34 and 3.79 c mol (p+) kg -1 ) was significantly recorded higher in treatment T9 with application of 100% RDF along with soil drenching of liquid form of bio stimulant @ 3.75 L ha-1 and found on par with treatments T5 (5.28 and 3.78 c mol (p+) kg -1 ) with application of 100% RDF along with soil drenching of solid form of bio stimulant @ 3.75 kg ha -1 , T7 (5.18 and 3.71 c mol (p+) kg -1 ) which receives 75% RDF along with soil drenching of solid form of bio stimulant @ 3.75 kg ha -1 and treatment T11 (5.28 and 3.76 c mol (p+) kg -

1

) which receives 75% along with soil drenching of liquid form of bio stimulant @ 3.75 L ha -1 and having significantly difference with treatment T2 (4.80 and 3.47 c mol (p+) kg -1 ) receiving100% RDF (Table 6).

Available Sulphur

Plots treated with 100% RDF along with soil drenching of BS2 @ 3.75 L ha-1 (T9) recorded significantly higher available sulphur in soil (28.44 mg kg -1 ) and found on par with the treatment T5 (27.13 mg kg -1 ) receiving 100% RDF along with soil drenching of BS1 @ 3.75 kg ha -1 and having significantly difference with treatment T2 (20.52 mg kg -1 ) receiving 100% RDF (Table 6).

The increase of secondary nutrients (Ca, Mg and S) availability might be due to stimulatory effect of biostimulants and FYM on release of Ca, Mg and S from its mineral sources by releasing acidifying compounds from biostimulants and FYM decomposition. Mindari et al.

(2014) [14] stated that in kharif rice, the acidic compounds by organic substances dissolve the elements from the mineral source. Application of biostimulants and FYM increasing the availability of nutrients by improving soil structure and beneficial microbial activity because of presence of polysaccharides which leads to breakdown of organic matter and release of nutrients to the soil.

Biological properties of soil Dehydrogenase activity

Dehydrogenase activity (DHA) is an index of microbial activity in the soil. Significantly, higher soil dehydrogenase activity was recorded in treatment T9 with application of 100% RDF along soil drenching with liquid form of bio stimulant @ 3.75 L ha-1 (53.68 g TPF g -1 h -1 ) and found on par with treatmenst T5: 100% RDF along soil drenching with solid form of bio stimulant @ 3.75 kg ha -1 (51.58 g TPF g-1 h-1),T7: 75% RDF along soil drenching with solid form of bio stimulant @ 3.75 kg ha-1 (49.58 g TPF g -1 h -1 ) and T11: 75% RDF along soil drenching with liquid form of bio stimulant @ 3.75 L ha -1 (50.67 g TPF g-1 h-1) and found significantly difference with treatment T2: 100% RDF (46.55 g TPF g -1 h -1 ) (Table 7).

The increased dehydrogenase activity in soil following seaweed extract and humic acid and FYM application is likely due to the combined effects of increased nutrient availability, stimulation of microbial activity and improved soil structure (Zhang et al., 2017) [21] .

Acid phosphatase activity

Significantly, higher soil acid phosphatase activity was recorded in treatment T9 with application of 100% RDF along soil drenching with liquid form of bio stimulant @ 3.75 L ha-1 (46.90 µg PNP g -1 h -1 ) and found on par with treatments T5: 100% RDF along soil drenching with solid form of bio stimulant @ 3.75 kg ha -1 (44.21 µg PNP g -1 h -1 ), T7 treatment receiving 75% RDF along soil drenching with solid form of bio stimulant @ 3.75 kg ha-1 (42.84 µg PNP g -1 h -1 ) and T11 treatment receiving 75% RDF along soil drenching with liquid form of bio stimulant @ 3.75 L ha -1 (42.94 µg PNP g -1 h -1 ) and found significantly difference with treatment T2 which receives 100% RDF (38.43 µg PNP g -1 h -1 ) (Table 7). The increased enzymatic activity observed due to the application of bio stimulants and FYM along with full dose of NPK had a positive effect on soil health and microbial communities. This application of bio stimulants in conjunction with FYM likely increased the biomass production and the generation of abundant root exudates in the rhizosphere. The application of biostimulants induces a more robust proliferation of roots, which in turn contributes to the augmented activity of soil enzymes. Similar findings were documented by Sabeetha et al. (2011) [17] .

Urease enzymatic activity

Plots treated with 100% RDF along with soil drenching of liquid form of bio stimulant @ 3.75 L ha -1 (T9) recorded significantly higher soil urease activity T9 (41.93 µg NH4-N g -1 h -1 ) and found on par with treatments T5 (40.71 µg NH4-N g-1 h-1) with application of 100% RDF along soil drenching with solid form of bio stimulant @ 3.75 kg ha -1 which was statistically on par with T7 treatment (39.88 µg NH4-N g -1 h -1 ) receiving 75% RDF along soil drenching with solid form of bio stimulant @ 3.75 kg ha-1 and T11 treatment (40.29 µg NH4-N g -1 h -1 ) supplied with 75% RDF along soil drenching with liquid form of bio stimulant @ 3.75 L ha -1 and found significantly difference with treatment T2 which receives 100% RDF (35.97 µg NH4-N g -1 h -1 ) (Table 8).

The increase in urease activity can be attributed to the ability of biostimulants to improve soil structure and moisture retention, creating a more favourable environment for microbial growth and enzyme function. Biostimulants also stimulate microbial activity directly, providing substrates that boost enzyme production. As a result, the addition of HA enhances the overall metabolic processes in the soil, leading to increased urease activity (Kaur and Reddy, 2015) [10] .

Table 7: Soil dehydrogenase, acid phosphatase and urease enzyme activites in soil after the harvest of tomato as influenced by biostimulants application

Treatment details

Dehydrogenase activity (μg TPF g-1 soil h -1 )

Urease activity (µg NH4-N g

Percent incidence disease Damping off

The data revealed that highest incidence of damping off is observed in the control treatment T1 (7.90%). The application of 100% RDF reduces the incidence to 6.57%, and suggesting that fertilizers alone have some effect in reducing the disease and slightly higher in treatment T3 (6.18%) due to reducing fertilizer application might lead to a slight increase in susceptibility to damping off. The lowest incidence of damping off is observed in T9 (100% RDF + BS2 @ 3.75 L ha -1 ) with 2.10%, indicating that this treatment is most effective in controlling the disease. The trend shows that higher application rates of BS1 and BS2 (T5 -100% RDF+ soil drenching with BS1 @ 3.75 kg ha-1 and T9 -100% RDF+ soil drenching with BS2 @ 3.75 L ha-1 are more effective than lower rates (T4 -100% RDF+ soil drenching with BS1 @ 2.5 kg ha-1 and T8 -100% RDF+ soil drenching with BS2 @ 2.5 L ha-1) (Table 8).

Fusarium Wilt

The data revealed that highest incidence of Fusarium wilt is observed in the control treatment T1 (7.50%). The application of 100% RDF reduces the incidence to 5.91%, suggesting that fertilizers alone have some effect in reducing the disease and slightly higher in treatment T3 (7.12%) due to reducing fertilizer application might lead to a slight increase in susceptibility to damping off. The lowest incidence of damping off is observed in T9 (100% RDF + BS2 @ 3.75 L ha -1 ) with 1.97%, indicating that this treatment is most effective in controlling the disease. The trend shows that higher application rates of BS1 and BS2 (T5: 100% RDF+ soil drenching with BS1 @ 3.75 kg ha-1 and 100% RDF+ soil drenching with BS2 @ 3.75 L ha-1 (T9) are more effective than lower rates (T4: 100% RDF+ soil drenching with BS1 @ 2.5 kg ha-1 and T8: 100% RDF+ soil drenching with BS2 @ 2.5 L ha-1) (Table 8).

Bacterial Wilt

The highest incidence of bacterial wilt was recorded in the control treatment (T1, 7.43%). Application of the recommended dose of fertilizers (T2) reduced the disease incidence to 4.98%, whereas a slightly higher incidence was observed under 75% RDF (T3, 5.11%). Among the treatments, the lowest bacterial wilt incidence (1.54%) was recorded in T9 (100% RDF + soil drenching with BS2 @ 3.75 L ha⁻¹), which was significantly lower than the control.

Treatments receiving the liquid biostimulant (BS2) generally recorded lower bacterial wilt incidence than those receiving the solid biostimulant (BS1). The reduction in disease incidence may be attributed to the beneficial effects of biostimulants on plant vigour, nutrient uptake, microbial activity and induced plant defence mechanisms, which collectively enhance resistance against pathogen infection (Table 8). [15] . Biostimulants improve nutrient uptake, nutrient use efficiency, and tolerance to abiotic stresses, thereby indirectly reducing the impact of pathogens (Hamid et al., 2021) [7] . Biostimulants enhance seedling vigour and root development, leading to stronger and more resilient plants that can better resist pathogen attacks. It also stimulates the plant's immune system, improving its ability to fend off diseases like damping-off and root rot. Additionally, biostimulants improve soil structure and microbial activity, which can suppress the growth and spread of harmful pathogens like Fusarium solani (Hussein and Aljarah., 2023) [8] . Biostimulants enhance the production of key defence-related enzymes like phenylalanine ammonia lyase, chitinase, peroxidase, and polyphenol oxidase, which play crucial roles in pathogen resistance. Additionally, the biostimulants increase phenolic content and alter the expression of defence genes, leading to a stronger and more effective immune response against fungal blast disease. The combined application methods further optimize the plants' resistance to pathogens (Sahana et al., 2022) [18] .

Conclusion

The characterization of both solid and liquid biostimulants revealed the presence of appreciable quantities of nutrients and beneficial microorganisms. The results of the field experiment demonstrated that the application of FYM @ 25 t ha⁻¹ and the recommended dose of fertilizers (250:250:250 kg N:P₂O₅: K₂O ha⁻¹) in combination with biostimulants, particularly the liquid biostimulant (BS2) @ 3.75 L ha⁻¹, improved soil nutrient availability, water-holding capacity and soil enzymatic activities. The integrated application of biostimulants also reduced the incidence of damping-off, Fusarium wilt and bacterial wilt in tomato. Therefore, the use of biostimulants along with recommended nutrient management practices can be considered an effective strategy for improving soil health and promoting sustainable tomato production.

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