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Ameliorating potential effects of natural biological formulations and biostimulants on plant health and quality attributes in coriander-fenugreek intercropped strawberry (Fragaria × ananassa Duch.)

BMC Plant Biology volume 25, Article number: 164 (2025) Cite this article

Abstract

The present study documented the effect of bio-organics in legume intercropped strawberry cv. Camarosa during the years 2022 and 2023. Bio-organic fertilizer inputs included were Jeevamrit (JV), Ghan-Jeevamrit (GJ) and Azolla. Coriander-Strawberry-Fenugreek as intercropping system was adopted. The treatments comprised were T1: GJ at 100 g/m2 + JV at 10% +Azolla at 200 g/plant, T2: GJ at 150 g/m2 + JV at 10% +Azolla at 200 g/plant, T3: GJ at 100 g/m2 + JV at 20% +Azolla at 200 g/plant, T4: GJ at 150 g/m2 + JV at 20% +Azolla at 200 g/plant, T5: GJ at 100 g/m2 + JV at 10% +Azolla at 250 g/plant, T6: GJ at 150 g/m2 + JV at 10% +Azolla at 250 g/plant, T7: GJ at 100 g/m2 + JV at 20% +Azolla at 250 g/plant, T8: GJ at 150 g/m2 + JV at 20% +Azolla at 250 g/plant, T9: GJ at 150 g/m2 + JV at 20% as per SPNF, T10: Farmyard manure (100% N basis) and T11: Recommended dose of N: P:K (80:40:40 kg/ha) as control. Application of bio-stimulants at 50 g/plant and AM fungi @ 20 g/ plant was applied uniformly in treatments T1–T8. One month after transplanting, T3 showed positive influence on vegetative growth traits of strawberry plantlets. Minimum number of days taken to flower, maximum duration of flowering (142) and number of flowers (51) were also recorded. This treatment application also observed maximum fruit yield (677.93 g/ plant) and yield efficiency (7.91 g/cm2 of leaf area) compared to all other bio-organic combinations applied. Post harvest soil chemical indicators were also significantly influenced except pH and electrical conductivity compared to FYM (100% N equivalence) and Recommended dose of fertilizers (RDF) of NPK (80:40:40 kg/ha). Microbial biomass in terms of total bacteria, soil fungi, actinobacterial count, phosphorous solubilizing bacteria, AM spore population, Azotobacter count and Soil enzymatic activity of phosphatase and dehydrogenases showed a steady rise after application of GJ @ 100 g/m2 + JV @ 20% + Azolla @ 200 g/plant. In addition, overall increase of the yield of coriander and fenugreek compared to FYM (100% N equivalence) and RDF of NPK (80:40:40 kg/ha) was recorded. The positive influence both on leaf and fruit NPK contents were also recorded when plantlets were supplemented with GJ @100 g/m2 + JV@ 20% + Azolla @ 200 g/plant. This study inferred that application of bio-organic inputs sources which can boost up cropping behavior, post harvest soil indicators, native microbial properties and enzymatic activity in rhizosphere, and thus can have the potential to improve crop resilience and soil productivity on sustainable basis.

Clinical trial number

Not applicable.

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Introduction

The cultivated strawberry (Fragaria × annanasa Duch.) is one of the most widespread and acceptable fruits in the World. It is believed to have originated from a cross breeding of two wild American species between F. chilonensis and F. virginiana in Europe. The crop (berry) is the temperate beauty but its cultivation also extends to the tropics and sub-tropics of Indian sub-continent [1]. Strawberries, with their vibrant red color and distinctive sweetness, have captured human fascination as the berries are not only beloved for their taste but also revered for their nutritional richness. The crop is luscious characterized by juicy texture and has emerged as a powerhouse of vitamin C, minerals (P, K, Fe) and antioxidants including, ellagic acid, ellagitannins, and pelargonidine-3-glucoside [2]. In India, the total area under its cultivation is 1000 ha with an annual production of 5000 MT. Major growing states are Maharashtra, Punjab, Haryana, Himachal Pradesh, Jammu and Kashmir, Uttrakhand, Uttar Pradesh, Rajasthan and West Bengal. Currently, total cultivable area in Himachal Pradesh is 54 ha with annual production of 348 MT. Incidentally, its niche lies in Dhaulakuan in Sirmour district which is producing 99% of the total strawberries with production of 342 MT in the State [3]. Fertilizers play a crucial role to meet the nutritional needs of strawberry which mainly depend upon the farming system, soil conditions and specific crop requirements. However, the excessive use of chemical fertilizers has led to degradation of soil, water, environment and human health besides, reduction in biodiversity [4]. Sustainable fertilizer management practices are increasingly important for both economic and environmental reasons in modern strawberry production. Organically produced fruits thus, are of great demand in market and are preferred by consumers for a variety of reasons including no pesticide residues, higher nutritional value, flavor and ecologically friendly agricultural techniques which help to maintain soil health and productivity [5]. Organic production approach excludes the use of synthetic chemicals such as pesticides, herbicides, and fertilizers, focusing instead on natural methods to maintain soil fertility, manage pests and diseases and promote plant health [6]. Among the various factors which contribute towards the growth and yield of strawberry, nutrient management is an important aspect of crop production [7]. Intensification of conventional farming systems has led to extensive usage of agrochemicals resulting in negative impacts on environment.

Bio-organic formulation including biostimulants can drastically reduce the dependence on chemical fertilizers to boosts up yield, quality and crop productivity by providing tolerance under stress conditions. Organic strawberry farms produce higher- quality of fruit, and organic strawberry farms with higher soil quality have greater microbial functional capability and resilience to stress than conventional [8]. Application of vermicompost enhance the soil microflora by promoting beneficial microbes which in turn enhance plant growth through production of plant growth-regulating hormones and enzymes and also aids in controlling plant pathogens, nematodes and other pests thereby enhancing plant health and minimizing the yield loss [9]. Azolla is a water fern that is used as bio-organic input. It contains N2-fixing cyanobacteria has low C: N which releases minerals faster than other organic fertilizers. It provides 60–80% of nitrogen in a couple of weeks when incorporated in soil with logged water [10]. AMF symbiosis is a crucial component to improve biological equilibrium in mycorrhizosphere to enhance nutrient uptake by the plants. AM fungi cause more uptake of P through their expanded network of hyphae and has shown significant increase in P, Zn and protein content of the crops [11].

Natural Farming considered as a cost- effective farming practices as a viable option to address the current farmers’ distress, other related soil and water issues and to sustain farmer’s incomes. The farming practice has proven to be the most suitable paradigm and offers a solution to improve crop yields, increase farmers’ income, ensure better health, environment conservation, reduce water consumption, minimize cost of production, eliminates usage of synthetic chemical inputs and rejuvenates soil health. These practices harness nature cycles and functions in a quite sustainable manner to provide safer and healthier food to consumers as it has in built mechanisms to regenerate soil, reduce water usage, use of non- chemical locally available natural resources and enhance crop diversity for maintaining crop quality and productivity. In addition, natural farm inputs completely eliminate chemical fertilizers usage that could help to reduce acidification of soil [12]. Natural farming offers a suitable perspective on multiple fronts from production to consumption while addressing wholesome aspects of health (ecological, human and economic). Natural farm inputs include Jeevamrit which is a fermented microbial culture that acts as a bio-stimulant which promotes the activity of beneficial microorganisms in the soil and also the activity of phyllospheric microorganisms when sprayed on foliage of the crops. Jeevamrit contain macro and micro nutrients, essential amino acids and growth promoting substances like auxins, gibberellins and cytokinins along with beneficial microorganisms [13]. Another input, Ghan-Jeevamrit is a solid form of Jeevamrit used for increasing soil fertility by microbial consortia and is made from cow dung and urine of desi cow, besan, jaggery and forest soil, Achhadan i.e., mulching which covers the whole soil surface with crop residue or live intercrops to create favorable microclimate in soil and Waphasa (soil aeration) by creating a condition of moisture and air in 50:50 proportion. Other natural bio-formulations viz., Agniaster and Brahmaster can also be used as crop protection measures [14]. In addition, legume intercrops help to maintain soil productivity and health sustainably. Earlier studies carried out on bio-organic inputs confirmed that microbial species in soil are associated with the increase in microorganisms in rhizosphere and improve the overall growth, fruit yield and quality. Keeping in view, the present study was carried out to evaluate the conjoint effect of biostimulants, bio-fertilization and sequential natural farm inputs on the fruiting behavior, chemical and biological properties in legume intercropped strawberry.

Materials and methods

Study area

The present study was conducted at the Experimental Research Farm of the Department of Fruit Science at Dr. YS Parmar University of Horticulture and Forestry, situated in Nauni, Solan, Himachal Pradesh, India (Geographical coordinates: 30°52’ North latitude and 77°11’ longitude, with an elevation of 1272 m above sea level). The climate of this location is typically sub-temperate, characteristic of the mid-hill zone, with a maximum mean air temperature of 31.8 °C and a minimum mean air temperature recorded at 12.3 °C. Soil temperatures exhibited variations, with maximum and minimum mean values of 22.9 °C and 15.2 °C, respectively.

Planting material

Uniform plantlets (runners) of the strawberry cultivar Camarosa were utilized in this research. The planting material was supplied by the Department of Fruit Science at Dr. YS Parmar University of Horticulture and Forestry located in Nauni, Solan, Himachal Pradesh, India. The transplantation of the runners took place on elevated nursery beds measuring 2 × 1 m, with a spacing of 0.5 m, during the timeframe from September to early October. Only healthy plants, free from diseases, damage, and any insect or pest infestations, were selected for the study. Throughout the duration of the research, all plantlets were subjected to uniform cultural practices aimed at fostering optimal growth and survival rates. Observations were made regarding vegetative traits, flowering characteristics, fruit yield and the quality attributes of the fruits. Furthermore, the chemical and microbiological properties of the rhizosphere soil, as well as the nutrient content of the leaves, were measured accordingly.

The edaphic conditions

The experimental soil at 15–30 cm depth was characterized as sandy clay loam, demonstrating a neutral reaction with a pH level of 7.73 and an electrical conductivity of 0.10 dS/m. It contained 0.95% organic carbon at a depth of 0–15 cm. The initial measurements for available N (alkaline KMnO4 extractable), Olsen P (0.5 M NaHCO3 extractable), and available potassium (NH4OAC-K) were found to be 313.6, 22.4 and 220.5 kg/ha, respectively. Additionally, the initial microbial counts revealed the presence of bacteria at 10.1 × 106 cfu/g, soil fungi at 8.6 × 103 cfu/g, actinobacteria at 7.3 × 104 cfu/g, and an AM fungal spore population of 112 per 50 g of soil.

Preparation of bioformulations

Jeevamrit

Jeevamrit is a liquid bioformulation prepared by mixing 10 kg cow dung, 10 L cow urine, 2 kg jaggery, 2 kg pulse flour and a handful of soil (100 g) as microbial inoculum. Total volume of this bioformulation is adjusted to 200 L by the addition of water. The mixture is stirred continuously in a clockwise direction using a wooden stick, both in the morning and evening for duration of 7 days. Following an incubation period of 15 days, the preparation is deemed ready for application. The final Jeevamrit solution is characterized by a bacterial count of 21.6 × 106 cfu/ml, soil fungal count of 1.8 × 103 cfu/ml, and an actinobacterial count of 6.4 × 104 cfu/ml. Jeevamrit is utilized as a foliar spray twice a week, commencing one month after transplanting and continuing through the stages of canopy formation and fruit development. The required amount of Jeevamrit (treatment-wise) was weighed and the final volume was made to one liter. Jeevamrit combinations were prepared as per treatment applications. For example, JV at 10%, the solution was prepared using concentrated 10 L of Jeevamrit diluted in 90 L of water. The finally prepared solution of JV @ 1 L/m2 of bed size was used in each stage of spray as foliar application without using any surfactant.

Ghan-Jeevamrit

Ghan-Jeevamrit is a solid form of Jeevamrit utilized to enhance soil fertility through microbial consortia. It is composed of cow dung, cow urine, gram flour, (gur), and forest soil. This bioformulation is applied as powder directly to the rhizosphere of plantlets. The preparation process begins with 200 kg of fresh cow dung, which is dried under direct sunlight. Once dried, the cow dung is crushed into a fine powder using a wooden log and then sifted through a fine mesh. The resulting fine dried dung powder is evenly spread as a layer on a tarpaulin sheet. Subsequently, cow urine is added along with 1 kg of jaggery (raw sugar), 1 kg of pulse flour, and a handful of forest soil and the mixture is kept in the shade for 48 h. After this period, it is dried again for an additional 48 h under direct sunlight. Once fully dried, the mixture is ground into powder and can be stored for duration of 6 to 8 months. Ghan-Jeevamrit is characterized by a bacterial count of 23.6 × 106 cfu/ml, soil fungi at 1.5 × 103 cfu/ml, and an actinobacterial count of 7.2 × 104 cfu/ml.

Agniaster

Agniaster is prepared by combining 5 kg of crushed darek leaves with 0.5 kg of tobacco leaves, 0.5 kg of chilies and 0.5 kg of garlic paste. This mixture is then incorporated into 10 L of cow urine and allowed to cool for approximately 24 h. Following this period, the solution is filtered for use. Prior to application in the field, the solution is diluted with 15 L of water for every half liter of concentrated Agniaster.

Brahmaster

Brahmaster is prepared from five varieties of bitter leaves of darek, guava, mango, papaya and Duranta varigeta, each weighing 200 g. To prepare it, 4 L of cow urine was utilized and boiled for approximately 2 to 3 h. Following this, the mixture is allowed to cool for around 12 h before being filtered through fine cloths. The resulting solution is then diluted with approximately 15 L of water for every 1 L of concentrated Brahmaster. After a period of 48 h, the solution should be stored in a container and can be used for up to 3 months.

Azolla

The genus Azolla includes water ferns that serve as nitrogen-fixing agents and can be used as green manure. The bio-product of Azolla pinnata was collected from aquatic environments, specifically ponds. This bio-product was then supplemented into strawberry plantlets based on fresh weight and contained a nitrogen content of 2.5%.

Arbuscular mycorrhizal fungi

In the month of October, the arbuscular mycorrhizal fungi (AMF) namely, Glomus fasciculatum was inoculated in all treatments in strawberry plantlets. AMF included roasted bentonite clay, exhibited a moisture content ranging from 15 to 20 per cent and contained 100 viable propagules per gram of the product. The microbial formulation was applied at a depth of 15 cm in conjunction with farmyard manure (FYM) within the rhizosphere of each plantlet, followed by a light irrigation to facilitate the proliferation of the propagules.

Biostimulants

The brown alga, Ascophyllum nodosum, blackish brown coloured biostimulants formulation was used. The formulation is common cold-water seaweed belongs to family Fucaceae. The extract possesses a marine scent and contains total solids exceeding 20%, which comprise hydrolyzed proteins, amino acids, and organic acids. Additionally, it consists of 80% aqueous diluents, stabilizers, and preservatives, with a pH greater than 4 and a specific gravity exceeding 1.0 g/cc. Furthermore, the formulation is enriched with various macro- and micro-nutrient cations, including sulfur (20 mg/kg), magnesium (50 mg/kg), calcium (60 mg/kg), boron (0.5 mg/kg), manganese (0.6 mg/kg), and zinc (0.2 mg/kg).

Field trials

The experimental unit was comprised of 2 × 1 m plot size with each bed 0.5 m apart. The plantlets were transplanted on raised beds spaced at 30 × 45 cm apart and accommodated 55,000 plants per hectare [15]. Coriander-fenugreek crop sequencing was carried between two rows of strawberry plantlets in the same plot (treatment-wise) during the cropping cycle. The experiment was laid in Randomized Block Design. All the treatments were replicated thrice. The treatments comprised were T1: GJ at 100 g/m2 + JV at 10% +Azolla at 200 g/plant, T2: GJ at 150 g/m2 + JV at 10% +Azolla at 200 g/plant, T3: GJ at 100 g/m2 + JV at 20% +Azolla at 200 g/plant, T4: GJ at 150 g/m2 + JV at 20% +Azolla at 200 g/plant, T5: GJ at 100 g/m2 + JV at 10% +Azolla at 250 g/plant, T6: GJ at 150 g/m2 + JV at 10% +Azolla at 250 g/plant, T7: GJ at 100 g/m2 + JV at 20% +Azolla at 250 g/plant, T8: GJ at 150 g/m2 + JV at 20% +Azolla at 250 g/plant, T9: GJ at 150 g/m2 + JV at 20% as per SPNF, T10: Farmyard manure (100% N basis) and T11: Recommended dose of N: P:K (80:40:40 kg/ha) as control. Different concentrations of Jeevamrit were applied as foliar sprays according to treatments during cropping cycle. Application of bio-stimulants at 50 g/plant and consortium of AM fungi at 20 g/plant was applied uniformly in treatments T1–T8. In addition, foliar application of Agniaster and Bramhaster at 3% as plant protection measures was alternatively applied at weekly interval (T1 -T9). The plantlets were dipped in Jeevamrit solution for 10 min before transplanting, to prevent any disease infestation during cropping period. The plantlets received regular horticultural care with established scientific practices for commercial strawberry production including, weed management and optimal irrigation procedures. NPK fertilizers used were urea (46% N), single superphosphate (16% P2O5), and muriate of potash (60% K2O) to fulfill the complete recommended dosage of N: P:K (80:40:40 kg/ha) for the plantlets. Nitrogen was applied in two split doses, with the first application occurring at the time of transplanting and the second 15 days post-flowering. During the transplanting process, the full dosage of phosphatic and potassic fertilizers, along with farmyard manure (FYM), was added to the rhizosphere of the plantlets.

Sampling protocol and chemical analysis

Composite soil samples weighing 1 kg were obtained from a depth of 0–15 cm using an auger, with four soil cores collected from each treatment. Care was taken during collection to prevent contamination from the applied microbial inoculants. The samples were air-dried and then sieved to a size of 2 mm. They were subsequently stored in a refrigerator at 4 °C, while observations of microbial indicators were documented. The chemical properties of the soil were analyzed using standard methodologies. Soil pH and electrical conductivity (EC) were measured at a ratio of 1:2 soil to water. Soil organic carbon (OC) was assessed through the wet oxidation method [16], while available nitrogen was estimated using the alkaline potassium permanganate method [17]. Olsen phosphorus, extractable with 0.5 M NaHCO3 [18] and potassium, extractable with 1 N neutral ammonium acetate, were measured using a flame photometer [19]. For leaf analysis, each sample consisted of 25 leaves collected from the middle of the shoot in June [20]. Fresh, mature leaf samples were gathered from the shoots. The nitrogen content in the leaves was determined using the Kjeldahl method, while phosphorus content was assessed through the phosphovanadate molybdate method. Leaf potassium content was also measured using a flame photometer.

Assessment of culturable microorganisms in soil

Culture media were used for the enumeration of the total microbial population densities of soil bacteria, fungi and actinobacteria in rhizosphere of saplings. Nutrient agar and coal-vitamin agar medium contained peptone-5 g, beef extract-3 g, NaCl-5 g, agar-agar-15 g and distilled water-1000 ml at 50 ◦C for10 min have been used for the cultivation of bacteria. Potato-dextrose agar (potato-200 g, dextrose-20 g, Agar-15 g and Distilled water, 1000 ml, pH 7.0) [21], supplemented with Bengal rose to prevent excessive fungal growth) and streptomycin as bacteriostatic agents [22], used for the determination of total fungal count in soil. Total actinobacterial colonies were isolated using Kenknight and Munaires media (K2HPO4 − 1 g, NaNO3-0.1 g, KCl-0.1 g, MgSO4-0.1 g, Glucose-1 g, Agar-15 g and distilled water-1000 ml [23]. Nutrient medium for cultivation of phosphorus solubilizing bacteria contained yeast extract-0.5 g, dextrose- 10 g, calcium phosphate-5 g, ammonium sulphate- 0.5 g, potassium chloride- 0.2 g, magnesium sulphate- 0.1 g, manganese sulphate- 0.0001 g, ferrous sulphate-0.0001 g and distilled water-1000 ml [24]. Total Azotobacter count was determined using Jensen’s media (K2HPO4-1 g, NaCl-0.5 g, FeSO4-0.1 g, MgSO4.7H2O-0.5 g, CaCO3- 2 g, Sucrose-20 g, agar-15 g and distilled water-1000 ml [25]. For each dilution, 1 ml was transferred and spread on to Petri dish for incubation at 28 ± 2 ◦C. Colonies of the bacteria, fungi and actinobacteria were counted under a biological microscope after incubation for 7–10 days. The observed inoculums is expressed as the colony forming units (cfu)/g of soil using formula, Colony forming units (cfu) = Average number of colonies appeared in culture plate x100) x dilution factor divided by fresh weight of soil sample taken.

Growth indexes

The height of the saplings was recorded at intervals of 60, 90 and 120 days and then averages calculated. Multiple samples were taken from the plants during the flowering stage until the conclusion of the growth cycle and final harvest. At the flowering stage, 10 plants were randomly chosen from each experimental sub-unit, and the data were collected on the number of leaves per plant during the study. Leaf area of newly expanded leaves was measured at the end of the growth cycle using a LI-COR-3100 benchtop leaf area meter, with results expressed in square centimeters (cm²). Newly developed runners were cut off from the mother plant, and plantlets with 2 to 3 unfolded leaves were excised and placed on the growth medium. Total number of runners was counted weekly throughout the duration of the study.

Yield, yield efficiency and fruit analyses

The fruits were harvested by hand twice weekly, resulting in a total of 8 to 10 harvests throughout the growing season. Data were collected on the total marketable berries, expressed in grams per plant. Yield efficiency (YE) was calculated based on leaf area, measured in grams per square centimeter of leaf area. During the cropping cycle, ripe berries were sampled and promptly transported to the laboratory for physico-biochemical analyses following standard protocols [26]. Total of thirty fruit samples from each treatment combination were randomly selected to assess fresh berry weight using an electronic digital balance with an accuracy of ± 0.01 g, with results expressed in grams per fruit. The dimensions of the berries, including length and breadth, as well as shape index, were measured using a digital Vernier scale with an accuracy of ± 0.05 mm. Berry firmness was evaluated using the Effegi Penetrometer model FT (Effegi, Milan, Italy). Biochemical quality characteristics, such as total soluble solids (TSS) measured with a hand refractometer (°Brix), sugars, and ripening index, were assessed at a temperature of 25 ± 2 °C at the consumer maturity stage. Titratable acidity (TA) was determined through neutralization of a diluted juice solution, titrating to a pH of 8.2 with 0.1 N NaOH. To assess the ascorbic acid content in the fruits, 25 g of fruit pulp was homogenized in a volumetric flask containing 100 ml of meta-phosphoric acid extraction solution. Subsequently, 10 ml of this mixture was titrated with 2, 6-dichlorophenol indo-phenol dye, with the endpoint indicated by the development of a light pink hue. The concentration of ascorbic acid in the fruit juice was calculated using the formula: Ascorbic acid (mg/100 g) = [(Dye factor × Titre value × Volume made up) / (Weight of sample taken × Volume used for estimation)] × 100. The presence of anthocyanins contributes to the color and visual appeal of fruit samples. The quantification of anthocyanins was measured using the absorbance change at two distinct pH levels. A 10 ml juice sample was extracted and combined with 50 ml of ethanol hydrochloric acid, then stored at 4 °C overnight in a refrigerator. The absorbance of the resulting colored solution was measured using a spectrophotometer, and the pigmentation of anthocyanins was documented using the horticultural color chart from the Royal Horticultural Society in London, United Kingdom.

Soil enzymatic activity

Representative samples (1 g) of moist soil stored at -4 °C were weighed into polypropylene bottles in duplicate (5 replicate sub-samples). The activity of acid phosphatase was determined with p-nitrophenol phosphate by adding 4 ml of p-nitrophenol phosphate buffer with a pH of 6.5 and 1 ml of 0.1 M disodium phenylphosphate as substrate. Both enzymes hydrolyze p-nitrophenol phosphate to p-nitrophenyl and perform a bioassay of phosphatase activity [27]. The mixture from the polypropylene vial was incubated at 37 °C for 1 h. Subsequently, at the end of the incubation, 1 ml of 0.5 M CaCl2 and 4 ml of 0.5 M NaOH were added and the mixture was filtered through Whatman filter paper. The intensity of the yellow color of p-NP (supernatant) was analyzed by UV spectrophotometer at a wavelength of 420 nm. The absorbance of the filtrate was compared with that of a p-nitrophenol standard to calculate the result. Dehydrogenase enzyme activity was assessed by reducing 2,3,5-triphenyl tetrazolium chloride (TTC) to triphenyl formazan (TPF) by following calorimetric method [28]. 1 g of moist soil sample was placed in a screw-cap tube along with calcium carbonate (0.01 g), 3% TTC solution (0.5 mL), and 1% glucose solution (0.5 mL). To eliminate the trapped oxygen, bottom of the tube was gently tapped before the incubation at 37 °C for 24 h. Following it, 10 mL of methanol was added to the solution and was shaken for 1 min. Subsequently, the mixture was kept in the dark for an additional 6-hour period. The red colour of the sample was measured using a Nukes UV-VIS spectrophotometer at an absorbance of 485 nm. The standard curve was generated from various concentrations of TPF (10 to 60 ppm) and the dehydrogenase activity was expressed as amount of TPF produced per hour per gram of the soil (µg TPF/g/h soil).

Statistics

Statistical analysis of the data was carried out according to the general linear model to assess the standard errors of the mean. The data obtained through Randomized Block Design (RBD) for each parameter were analyzed using OPSTAT. Statistically significant differences among treatments were examined and treatment means were compared using the critical difference (CD) at a 5% level of probability (confidence) when the results were significant [29]. The computed F-values were compared against the tabulated F-value. In instances where the F-test yielded significant results, the CD was subsequently calculated to determine the comparative effectiveness of the various treatments.

Results

The observations on vegetative growth indices namely, plant height, number of leaves, leaf area, number of crowns, number of runners was recorded. All vegetative growth parameters were significantly improved with the application of bio-organic inputs in legume intercropped strawberry.

Vegetative growth traits

Among various bio-organic combinations, T3 [Ghan-Jeevamrit (GJ) @ 100 g/m2 + Jeevamrit (JV) @ 20% + Azolla @ 200 g/plant)] recorded the highest plant height of strawberry plantlets (13.60 cm), followed by T2 (GJ @ 150 g/m2 + JV @10%+ Azolla @ 200 g/plant), T6 (GJ @ 150 g/m2 + JV @ 10%+ Azolla @ 250 g/plant), T8 (GJ @ 150 g/m2 + JV @ 20%+ Azolla @ 250 g/plant), T5 (GJ @ 100 g/m2 + JV @10%+ Azolla @ 250 g/plant) with corresponding values of 12.66, 13.33, 13.06 and 13.00 (Table 1). The height of plantlets with different quantities of applied bio-organic inputs was followed in the order T3 > T2 > T6 > T8 > T5> T4. The average plant height of strawberry ranges from 13.66 cm to 12.43 cm, respectively. Treatment combination T3 recorded maximum number of leaves (17.66) which is recorded highest among the other treatments. The treatment T9 however, recorded the lowest value and was statistically similar to T11. The plantlets of strawberry in terms of number of leaves presented in the order T3 > T2 > T6 > T8 > T5> T4 with corresponding values of 17.66, 17.33, 17.00, 16.33, 16.00, 15.66, respectively. Similarly, leaf area of strawberry plantlets exhibited a positive impact when supplemented with various bio-organic inputs during the cropping cycle. The average leaf area of plantlets ranges between 85.84 cm2 to 74.61cm2. Among, various treatments applied to the strawberry plantlets, T3 recorded the maximum leaf area of 85.84 cm2 which however, was least in T9 (74.61 cm2) followed by T3 > T2 > T6 > T8 > T5> T4. The average number of crowns ranged between 8.33 and 5.00. Among these various treatments, T3 showed best results with a steady increase in number of crowns which were statistically at par with T2, T6, T8 with presented values of 8.00, 7.66 and 7.33, respectively. Application of bio-organic inputs showed a steady significant increase in number or runners during the cropping cycle. Number or runners found maximum in T3 (233.33) whereas, minimum in T9 (193.33). The statistically similar results were also obtained in treatments T3 and T2 with corresponding values of 233.33 and 226.66.

Table 1 Vegetative growth traits of strawberry cv. Camarosa influenced by bio-organic inputs under coriander and fenugreek intercrop sequencing
Full size table

Flowering and fruiting traits

Among different bio-organics combinations, the treatment T3 exhibited a significant increase in the number of flowers per plant (61.66) which is highest among all the treatments applied (Table 2). The treatment combination however, was also statistically similar to T2 (60.33) in terms of number of flowers per plant during the cropping season. It is clearly evident from the data that application of bio-organic inputs resulted in a significant increase in the duration of flowering in strawberry plantlets. Maximum duration of flowering was observed in T3 (142.0). The data recorded showed a significant increase in cumulative fruit number in treatment T3 (43.33) which was at par with treatment T8 (39.00) with minimum values observed in T9 treatment combination. The treatments arranged in descending order of T3 > T2 > T6 > T8 > T5 > T4 with corresponding values of 43.33 42, 40, 39, 38.33, 37.33, respectively. When compared to T10 treatment combination, T3 application indicated the highest percent increase of 22.64. Fruit set varied from 70.26 to 61.42%. Among different treatments, fruit set recorded in order of T3 > T2 > T6 > T8 > T5> T4 with corresponding values of 70.26, 69.22, 68.63, 67.52, 67.32, 66.30. It is evident from the data that the application of various bio-organic inputs significantly increased fruit yield in legume intercropped strawberry. In treatment T3, fruit yield (677.93 g/ plant) was statistically similar to T2 (674.22 g/ plant), T6 (666.03 g/ plant) which was followed by T8 (644.64 g/ plant), T5 (618.13 g/ plant) and T4 (612.52 g/ plant).

Table 2 Flowering and fruiting of strawberry affected by bio-organic inputs in coriander and fenugreek intercrop sequencing
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It was however, lowest in treatment T9 (510.40 g/ plant). Also, application of bio-organic sources in strawberry plantlets ranked in term of fruit yield as T2 > T6 > T8 > T5 > T4 (Fig. 1) Among different bio-organic sources, yield efficiency in terms of leaf area ranged between 6.83 and 7.91 g/cm2 of leaf area (LA). Maximum yield efficiency of 7.91 g/cm2 of LA were recorded in treatment T3 which was followed in the order of T3 > T2 > T6> T8 > T5 > T4 with corresponding values of 7.91, 7.90, 7.80, 7.78, 7.60, 7.59 g/cm2 of leaf area (Fig. 2).

Fig. 1
figure 1

The graph shows the comparative effect of bio-organic inputs on strawberry fruit yield. The bars represent the mean values along with standard errors. The letters above the bars denote significant differences among the different bio-organics according to the DMRT at p ≤ 0.05

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Fig. 2
figure 2

The graph illustrates the quantitative results of yield efficiency in relation to leaf area, with mean values influenced by bio-organic inputs. Statistical analysis was performed to confirm the significance of the observed differences, indicated by * = < 0.05 and ** = < 0.01. ns, non-significant

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Fruit quality characteristics

Physical

It is clearly evident from Fig. 3 that overall effect of bio-organic inputs led to a significant increase in physical parameters of fruit samples with maximum increase in treatment T3 (length: 33.05 mm) and (diameter: 24.59 mm). However, treatment T9 showed minimum results in both length (22.82 mm) and diameter (20.04 mm). Also, bio-organic sources ranked in term of length/ diameter of fruit samples as T3 > T2 > T6 > T8 > T5> T4 with value range of 33.05/24.59, 32.38/24.63, 31.28/24.30, 30.52/23.97, 30.40/23.74, 29.26/23.44. In case of length of fruit samples, treatment T2 is at par with T3, while, in case of diameter treatments of T2, T4, T5, T6 and T8 were at par with T3 treatment. Application of different bio-organic inputs has significant effect on fruit weight of strawberry. Application of T3 showed maximum weight of fruit (18.17 g) followed by treatment T2 (17.66), whereas, it was lowest in T9 (15.07 g). Treatment T3 showed 13.91% increase in weight of fruit samples compared to T10. Treatment T3 also recorded statistically at par with treatments of T2, T6 and T8. Among various bio-organic sources, fruit firmness varied from 7.09 to 8.15 lbs/inch2. Maximum (8.15 lbs/inch2) fruit firmness was registered in T3 which was statistically significant compared to all other applied treatments (Table 3). The lowest values (7.09 lbs/inch2) were, however, noted in the treatment T9. The progression of fruit firmness under bio-organic sources observed in the order of T3 > T2 > T6 > T8 > T5 with corresponding values of 8.15, 7.86, 7.82, 7.58 and 7.44 lbs/inch2. Shape index of strawberry fruit samples showed significant effects among the different bio-organic treatments applied. Treatment T3 recorded the highest (1.34) shape index of fruit samples. Treatment T3 is recorded statistically at par with treatment T2, T4, T5, T6, T8 with respective values of 1.30, 1.24, 1.26, 1.28 and 1.27.

Fig. 3
figure 3

The graph depicts the impact of different bioorganic inputs on dimension of strawberry fruit samples. Statistical analysis was undertaken to confirm that the differences are statistically significant, where p < 0.01= **, p < 0.001= ***

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Bio-chemical

The data depicted in Table 4 revealed a significant effect of bio-organic inputs on TSS content of strawberry fruits. Maximum TSS content (9.96 °B) was recorded in treatment T3, which showed statistically at par effects with treatment with treatments T2 and T6. However, it was minimum (8.20 °B) in treatment T9. The treatment of T3 also recorded an increase of 16.3% TSS compared to T10.Titratable acidity of strawberry fruits varied between 0.61 and 0.74% with statistically non-significant effects among the different bio-organic treatments applied. Lowest titratable acidity (0.61%) was reported in treatment T9, whereas, it was highest (0.74%) in T3 treatment. Among various bio-organic treatments, the titratable acidity of fruits arranged in order of T2 > T6 > T8 =T5 each with corresponding values of 0.73, 0.72, and 0.69%. Application of bio-organic inputs showed up significant change in TSS: acid ratio with steady increase in the value range. Treatment T3 showed maximum TSS: acid ratio of 9.96 which was highest among all the other treatments. However, it was minimum (8.20) in treatment T9.

Table 3 Physical parameters of strawberry fruits influenced by bio-organic input sources in coriander and fenugreek intercrop sequencing
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Table 4 Bio-chemical parameters of strawberry fruits influenced by bio-organic sources in coriander and fenugreek intercrop sequencing
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The treatment of T3 also recorded an increase of 16.3% TSS: acid ratio compared to T10. Among different bio-organic sources applied, maximum reducing sugars content of 6.76% was observed in T3 followed by T2, T6, T8 and T5 with respective values of 6.46, 6.18, 6.10 and 6.01%, whereas, it was attained minimum (5.73%) in T9 treated strawberry plantlets. Moreover, T3 also recorded 17.9% increase in reducing sugars content over T10. Application of varied bio-organic sources exerted a significant influence on non-reducing sugars content of strawberry fruits. The average values for non-reducing sugars ranged from 1.94 to 2.70%. Treatment T3 exhibited maximum non-reducing sugars content which also showed statistically similar effects with T2, T6, T8 and T5. The treatment of T9, however, demonstrated the minimum non-reducing sugars (1.94%). A perusal of the data illustrated a pronounced increase in total sugars content of fruit samples due to the application of bio-organic sources during the cropping cycle. Total sugars content exhibited an average of 7.91 to 9.46%. Treatment T3 recorded the highest total sugars (9.46) among all other treatments followed by treatment T2 (9.08). However, treatment T9 observed the minimum value of 7.91%. Moreover, T3 also exhibited 17.2% increase in non-reducing sugars content over T10 treatment. Application of bio-organic inputs showed up significant increase in ascorbic acid content in fruit samples. Treatment T3 showed the highest ascorbic acid content of 15.07% compared to T10. However, it was minimum (63.57 mg/100 g) in treatment T9. Application of bio-organic inputs recorded a significant increase in anthocyanins content of fruit samples. Among all the other treatments, T3 recorded the highest (10.02 mg/100 g) followed by T2 (9.93 mg/100 g). Treatment T9 showed the lowest content of anthocyanins (8.15 mg/100 g), whereas, it was highest in T3 (10.02 mg/100 g).

Soil chemical indicators

Application of different bio-organic combinations recorded statistically non-significant results for soil pH during the course of study. However, the treatments combinations of T3, T2, T6, T8 and T5 depicted analogous results with corresponding values of 7.81, 7.75, 7.75, 7.73 and 7.69 (Table 5). Non-significant effect on the electrical conductivity (EC) of soil was recorded in different bio-organics combinations applied in legume intercropped strawberry plantlets. It is evident from the data that soil organic carbon content was significantly influenced when application of bio-organics in strawberry plantlets were applied. Maximum organic carbon content (2.96%) was recorded in treatment T3, which was statistically similar to T2 (2.90%). However, minimum organic carbon (1.99%) was exhibited by treatment T9. Moreover, statistically equivalent results were also observed with treatments combinations of T6, T4 and T8 with corresponding values of 2.73, 2.57 and 2.63%. The treatment of T3 also recorded an increment of 34.5% when compared to T10 with a difference is equal to 0.76%. Application of different bio-organics combinations recorded a significant increase in available N content of soil in legume intercrop sequencing.

Available N content was maximum (399.92 kg/ha) in treatment T3, which was statistically superior to all other applied treatment combinations. However, the lowest values were observed in T9 (345.42 kg/ha). Furthermore, an increment of 12.6% was observed in T3 compared to T9. The order followed pertaining to available N content for different bio-organic combinations followed as T2 > T6 > T8 > T5 with corresponding values of 396.78, 386.41, 385.72 and 384.34 kg/ha. Available P content of soil showed a positive effect through the application of bio-organic combinations in strawberry. Treatment combination of T3 recorded maximum available P content (39.52 kg/ha) which was statistically at par at par with T2 and T6. However, treatment of T9 recorded the minimum (31.03 kg/ha). An increment of 11.6% was observed in T3 compared to T9. A perusal of the data indicated the significant influence of application of bio-organics combinations on available K content of soil. The treatment T3 recorded the maximum available K content (261.04 kg/ha) followed by treatment T2 (250.51 kg/ha). However, the lowest value (217.7 kg/ha) was found in treatment T9. Moreover, the available K content in soil through the application of different bio-organics combinations was followed in the order T3 > T2 > T6 > T8 > T5 with corresponding values of 261.04, 250.51, 242.26, 241.91 and 238.36 kg/ha.

Table 5 Chemical properties of soil influenced by bio- organic inputs in coriander and fenugreek intercropped strawberry
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Soil microbiological properties

The data depicted the influence of application of bio-organic sources on the microbiological properties of rhizosphere soils of strawberry plantlets (Table 6). The spider graph illustrating different microbial properties in rhizosphere under various bio-organic inputs is shown in Fig. 4. The data illustrated a pronounced effect of bio-organic sources on total bacterial count in the rhizosphere of strawberry. The treatment of T3 demonstrated maximum total bacterial count (17.60 × 106 cfu/g) which was followed by T2 (15.63 × 106 cfu/g). The lowest value (10.20 × 106 cfu/g) was recorded in treatment T9. The superior treatment also experienced an increase of 52.2% in total bacterial count in rhizosphere soils of strawberry. The total bacterial count (× 106 cfu/g) for the various bio-organics combinations was followed in the order of T3 > T2 > T6 > T8 > T5 with respective values 17.60, 15.63, 14.93, 13.96, 13.96 and 13.90. Application of bio-organic supplements exhibited significant impact on population of phosphorus solubilizing bacteria (PSB) in rhizosphere soils of strawberry plantlets. PSB count varied from 6.80 × 104 cfu/g to 8.83 × 104 cfu/g. Treatment T3 recorded the highest PSB count (8.83 × 104 cfu/g) which was statistically at par with T2 (8.63 × 104 cfu/g). However, it was lowest (6.80 × 104 cfu/g) in treatment T9 (Table 8). Moreover, the results also illustrated an increment of 25.1% in T3 compared to T10. The relationship between available P and phosphorus-solubilizing bacterial count in the strawberry rhizosphere under various bio-organic inputs is presented in Fig. 5. Application of bi-organic sources exerted a noticeable impact on the Azotobacter count of rhizosphere soils of strawberry plantlets. Treatment T3 exhibited the maximum Azotobacter count (4.66 × 105 cfu/g) followed by T2, T6, T8 and T5 corresponding values of 4.23, 3.93, 3.80, 3.66 × 105 cfu/g. It was however, minimum (2.36 × 105 cfu/g) in the treatment T9. Application of bio-organic supplements exhibited significant impact on population of total soil fungal population in rhizosphere soils of strawberry plantlets. Treatment T3 recorded the highest soil fungal count (12.60 × 103 cfu/g). Moreover, the results also illustrated an increment of 51.8% in T3 compared to T2. The results pertaining to actinobacterial count in rhizosphere of strawberry plantlets showed statistically significant difference among different bio-organic treatment combinations. Actinobacterial count was ranged between 7.73 × 104 cfu/g and 10.26 × 104 cfu/g (log10 transformed). Maximum (10.26 × 104 cfu/g) count was recorded in treatment T3. Furthermore, the results indicated 21.3% increment in treatment of T3 compared to T10.

Fig. 4
figure 4

Spider graph illustrating microbial properties in strawberry rhizosphere under different bio-organic inputs

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Fig. 5
figure 5

Relationship between available phosphorus and phosphorus-solubilizing bacterial count in the strawberry rhizosphere under various bio-organic inputs. The letters above the bars indicate significant differences among the treatments based on the DMRT at p ≤ 0.05

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Table 6 Microbial population in strawberry rhizosphere influenced by application of bio-organics in coriander and fenugreek intercrop sequencing
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Actinobacterial count (× 104 cfu/g) for the various bio-organics combinations was followed in the order of T3 > T2 > T6 > T8 > T5 with respective values 10.26, 10.13, 9.86, 9.60, 9.43. Application of bio-organic sources recorded a significant increase in AM fungal (AMF) spore population in rhizosphere soils of strawberry. Maximum AMF spore count (159.00 spores/50 g of soil) was recorded in treatment T3, whereas, it was minimum (121.33 spores/50 g of soil) in T9. The order of AMF spore population in different bio-organic sources recorded in the order of T3 > T2 > T6 > T8 > T5. Moreover, an increment of 19.85% on AMF spore population was also observed in T3 as compared to T10. The data obtained in the current study clearly indicate a treatment-wise variation in AM fungal spore counts, which evidenced the establishment and extent of the symbiotic relationship. The significantly highest AM fungal spore count was recorded under treatment T3 (GJ at 100 g/m2 + JV at 20% +Azolla at 200 g/plant), suggesting that this combination of bio-organic inputs provided optimal conditions for quantified AM fungal sporulation which caused effective colonization in strawberry plantlets. Improved AM fungal spore count under T3 indicated a stronger symbiotic interaction with strawberry roots compared to other treatments.

Soil enzymatic activity

Application of bio-organic supplements exhibited significant impact on phosphatase enzymatic activity in rhizosphere soils of strawberry plantlets. Phosphatase varied from 3.37 to 4.68 µmole p-nitrophenol/g/h. The results also illustrated an increment of 38.1% phosphatase enzymatic activity in T3 compared to T10. The order of phosphatase enzymatic activity in rhizosphere in different bio-organic sources recorded in the order of T3 > T2 > T6 > T8 > T5 with corresponding values of 4.68, 4.51, 4.32, 4.29, 4.15 µmole p-nitrophenol/g/h. There was significant variation in dehydrogenase activity in rhizosphere of strawberry plantlets subjected to different bio-organics combinations (Table 7). The values for soil dehydrogenases varied from 19.21 to 24.01 µg TPF/h/g soil. Treatment combination of T3 recorded the maximum dehydrogenases (24.01 µg TPF/h/g soil), whereas, the treatment combination of T9 recorded the minimum (19.21 µg TPF/h/g soil). Among other treatment combinations, the treatments T2, T6 and T5 were statistically equivalent to each other.

Leaf analysis

A perusal of the data indicated the positive influence of bio-organics supplements on leaf N content in legume intercropped strawberry plantlets (Table 8). The treatment combination of T3 observed maximum leaf N content (2.86%) followed by T2 (2.77%). However, the minimum leaf N (2.12%) content was found in treatment T9. The percent increase of 27.5 was also recorded T3 as compared to T10. Moreover, the leaf N content for various bio-organic combinations was followed in the order of T3 > T2 > T6 > T8. Analysis of data revealed that application of bio-organics exerted a significant influence on leaf P content of strawberry. The average values for leaf P content however, ranged between 0.27 and 0.47%. Treatment T3 exhibited maximum leaf P (0.47%) content and was statistically at par with treatment T2 (0.40%). In addition, the leaf P content for various bio-organic combinations was followed in the order of T3 > T2 > T6 > T8 > T5 with respective values of 0.47, 0.40, 0.39, 0.37 and 0.37%. Leaf K content was positively influenced when supplemented with bio-organics in legume intercropped strawberry. Treatment T3 recorded the highest leaf K content (2.78%) which was statistically at par with to T2 (2.60%). However, it was lowest was recorded in T9. Furthermore, the results indicated 27.5% increment in the treatment combination of T3 as compared to T9.

Table 7 Soil enzymatic activity affected by natural bio-organic inputs in coriander and fenugreek intercropped strawberry
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Table 8 Leaf nutrient content of strawberry affected by bio-organic inputs in coriander and fenugreek intercrop sequencing
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Fruit analysis

The recorded data exhibited variation in N content of strawberry fruit samples subjected to different bio-organic sources, which varied from 2.00 to 2.33% (Table 9). Treatment T3 observed the maximum fruit N content (2.33%) followed by T2 (2.20%), however, it was minimum in T9 (2.00%). The percent increase of 14.9 was also recorded T3 as compared to T10 treatment. The results pertaining to P content of fruit samples showed statistically significant difference among different bio-organic treatments, which ranged between 0.17 and 0.28%. Fruit P content was observed maximum (0.28%) in treatment T3. However, minimum values (0.17%) were exhibited by treatment T9. Fruit P content for various bio-organic combinations was followed in the order of T3 > T2 > T6 > T8 > T5 > with respective values of 0.28, 0.26, 0.25, 0.24 and 0.23%. Data also illustrated a pronounced effect on K content of fruit samples when supplemented with bio-organic sources. The treatment combination of T3 demonstrated the highest fruit K content (2.26%). However, the lowest values (1.56%) were recorded in treatment T9. The superior treatment also experienced an increase of 29.8% in fruit K content over T10 (Table 10).

Table 9 Fruit nutrient content of strawberry cv. Camarosa influenced by natural bio-organic inputs in coriander and fenugreek intercropping system
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Biomass of intercrops within strawberry cropping system

Application of bio-organic inputs in legume intercropped strawberry resulted in overall increase of the yield of coriander. Treatment T3 showed maximum increase in the yield (2312.0 g/plot) followed by treatment T2 (2251.5 g/plot). However, treatment T9 showed minimum yield of 1725.0 g/plot. Treatment T3 showed statistical superior results to T2 and also recorded the highest yield of coriander with 25.8% increase compared to T10. It is also evident from the data that there is significant increase in yield of fenugreek. Followed in order T3 > T2 > T6 > T8 > T5 with corresponding values of 683.3, 646.0, 631.0, 591.6 and 585.0 g/plot. The benefits of legume intercropping include reduce risk, efficient use of resources, increasing the yield of crops by fixing atmospheric nitrogen to made available readily to plants, preventing erosion and ensuring food security. Because intercropping creates a healthy soil cover, the temperature of the soil remains relatively low. This prevents nutrients from being lost and the organic materials in the soil from burning. Moreover, it creates a microclimate that could be advantageous for associated crops.

Decomposing roots and other leftovers from the intercrop’s harvest provide nitrogen and other nutrients to the main crop, facilitating their uptake of the nutrient sources and boosting the production of crops nearby. It was well documented that legume-based intercropping systems are effective in maintaining soil fertility and boost production stability. Because the available pool of nitrogen quickly mineralizes and increases the yield of the main crop, the most available nitrogen was obtained when Jeevamrit + Ghan-Jeevamrit along with legume intercrops is used in combination.

Table 10 Effect of bio-organic inputs on cumulative yield of coriander and fenugreek grown additively in strawberry plantlets
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Discussion

In the present investigation, the vegetative growth parameters of strawberry plantlets showed significant positive results with the application of bio-organic inputs in legume intercropped sequencing. Plant height, number of leaves, leaf area, number of crowns and number of runners was remarkably increased. Application of 100% recommended dose of nitrogen supplemented through Jeevamrit @ 500 ml at crown and fruit development stages significantly increased vegetative growth characteristics of the plantlets [30]. In addition, application of biostimulants to the strawberry plantlets at early growth stages stimulated rapid growth of plantlets in terms of height, number of leaves and leaf size. Exogenous application also boosts up crop productivity by increasing the overall vegetative parameters [31]. Application of bio-organic inputs and FYM has increased the beneficial microbial populations in soil, due to increased production of plant growth hormones and hence, improved leaf area and leaf number. Earlier studies documented an increased leaf area due to increased N availability supplied through bio-organic formulations [14, 32]. Besides, biostimulants and FYM increased the beneficial microbial populations, which enhanced the production of growth regulators including, auxins, gibberellins and cytokinins in strawberries. The study also indicated that these hormones and acids led to improved plant growth viz., leaf area, shoot biomass, number of flowers and runners and yield [33, 34]. Significant increase in plant height and growth due to the availability of small quantities of macronutrients, micronutrients and growth promoting substances aided to huge beneficial microbial population in Jeevamrit treated plantlets, and thus enhanced the plant growth characteristics.

Application of bio-organic input sources to strawberry plantlets increased flowering traits including, number of flowers per plant. This might be due to increased supply of macronutrients required in larger quantities for the growth and development of plantlets. Application of N through bio-organics attributed to the acceleration in development of growth and reproductive phases [35]. Application of natural farm inputs such as Ghan-Jeevamrit and Jeevamrit in strawberry has recorded 50% more floral buds and number of flowers per plant compared to conventional chemical inputs [36]. From the study carried out with these organic input sources, it is observed that flower induction in strawberries exhibited a marked increase after application of these organic fertilizer sources. In addition, biostimulants also promoted growth and flowering of plants during production cycle. Increased photosynthetic rate due to phosphorous content available bio-organic sources and hence, recorded increased flowering traits. They further also concluded that the quantum of nutrients such as NPK and phytohormones provided by organic inputs plays a significant role in increasing these flowering parameters of the strawberry plantlets [37]. The flowering duration also increased to maximum (24.51 days) due to application of vermicompost. The reason ascribed to differences in source and nutrients composition as well as timing of nutrients to become available to the flowering plants.

Application of bio-organic inputs significantly increased the number of fruits per plant in strawberry [38]. Biostimulants applied exogenously to the crop and are considered as substances for plants to boost crop productivity to improve the nutrient use efficiency which led to uptake of nutrients from the soil and thereby, has increased fruit yield [39]. Application of biostimulants positively interferes with growth, productivity and quality traits of fruit crop with observed greater weight of fruits increasing their yield [40]. Application of Jeevamrit produced (50%) more floral buds, yield, and fresh weights of plants when treated with this application [36]. Besides, application of organic inputs increased the photosynthetic activity and other minerals supplied by the integrated nutrient sources resulted in improved level of carbohydrates and other quality parameter of the fruit through the way of enzymatic activity that stimulated the plant growth and substances produced by application of organic manure and other nutrient increased the yield parameter of the following crop [41]. Application of biostimulants led to improvement in plant growth, nutrient use efficiency, and stress tolerance, all of which enhance plant growth, fruit set, crop productivity, and nutrient use efficiency [42]. Recent studies have indicated that biostimulants can improve crop yields and also significantly reduce the need for fertilizers. Biostimulants are a promising and environmentally friendly innovative new tool for sustainable agriculture. Improve yield components such as quantity, weight, size, and quality. Azolla is a water fern and comprises of a bacteria called cyanobacteria which helps in atmospheric nitrogen fixation because of its independence for carbon and nitrogen forms an important N input cultivation. It helps in improvement of soil management and cropping practice along with this bio-fertilizer found so useful to increase the yield [43]. Application of Jeevamrit resulted in 7.98 to 26.20% increase in fruit yield as compared to without Jeevamrit application [44]. Jeevamrit and Ghan- Jeevamrit are liquid organic manure comprise rich sources of beneficial micro-flora who stimulated the plant growth and help in getting better vegetative growth and also good quality yield as they disperse in water rapidly and easily taken up by the plants as compared to solid organic fertilizers.

Earlier studies stated that influence of bio-organic fertilizers showed positive results in terms of improved breadth and fruit weight with the application of RDF + beejamrit + Jeevamrit + panchagavya [45]. Application of biostimulants increased K oxalate-dimethylsulphoxide soluble pectins mainly composed of galacturonic acid and thereby, increased firmness of fruit samples [46]. Application of biostimulants increased the firmness of fruits and shelf-life along with large sized berries of strawberry when supplemented with bio-organic amendments [40]. The study concluded that application of biostimulants increased total chlorophyll content that helps in the synthesis of total carbohydrates and proteins leading to improving the nutritional status of plant and maintaining a good balance between total carbohydrates and nitrogen and increasing both cell division and enlargement which in turn caused an increase of fruit fresh weight and fruit size [47]. Besides, AM fungi inoculated observed higher equatorial diameter, improved size and weight of fruit samples. Application of Jeevamrit in the current study increased the total soluble solids content due to the mobilization of carbohydrates from the source to the sink. This happened because the microbial formulation of Jeevamrit resulted in the synthesis of plant hormones [48]. The increase in sugars and ascorbic acid content of fruits ascribed due to the conversion of reserved starch and other insoluble carbohydrates into soluble sugars [49]. Application of bio-organic inputs has been attributed to increase rate of translocation of photosynthetic products from leaves to developing fruits and thereby increasing total sugars [31]. Biostimulants are rich in peptides, amino acids and minerals, and if applied as a foliar spray on the leaves at 5% via fertigation made 15–30 days after transplanting led to an increase in the content of proteins, amino acids, reducing sugars, phenolics and flavonoids in the ripe fruits improving the quality of fruit [50]. There is a significant increase in the levels of soluble sugars and the total antioxidant activity of the fruit and lead to increased levels of P content in the fruit ultimately the content of anthocyanin has increased in the fruit peel. AM fungal inoculation could effectively promote growth and nutrient uptake, and improves the fruit yield and fruit nutrient quality, compared with ordinary cultivation. Moreover, AM fungal inoculation mitigate the inhibitory effect of salt stress on the plant growth under drought conditions, made the plant N, P and K contents increased by 7.3, 11.7 and 28.2%, respectively.

Application of Jeevamrit promoted biological activity in the soil and made the nutrients available to the crop, improved soil aeration and enhanced soil biomass. There are conflicting reports on the direct and residual effects of bio-organic inputs to increase in the soil pH [44]. Introduction of legumes in crop sequence and substitution of N through FYM and other supplied bio-organic inputs improved the soil physical and chemical properties. It is stated that organic carbon content of soil has increased significantly through the application of biostimulants [51]. FYM recorded higher values for NPK availability in soil [52]. Soil pH values were also increased by the application of biostimulants and also lead to increase in soil organic matter. The application of organic amendments rich in K which is compost and vermicompost had a positive impact on soil exchangeable K and also showed beneficial effects on soil properties like EC and pH [53]. Application of Jeevamrit increased in activity of microorganisms in rhizosphere such as nitrogen fixer-Azotobacter and phosphorus solubilizing bacteria by solubilization and also enhanced nutrient uptake [54]. Higher bacterial, fungal and actinomycetes population recorded with application of Jeevamrit @ 20% at 2 weeks interval and was also at par with application of Jeevamrit @ 10% at 2 weeks interval and Jeevamrit @ 20% at 3 weeks interval [55]. Increase in the overall fungal concentration in soil when applied biostimulants with high N concentration. AMF and acts as an aggregate binder material, which led to the aggregate stability and stabilize soil macro- aggregation [56]. AMF also recruits mycorrhizal helper bacteria that produce alkaline phosphates to mineralize organic P, also influenced the composition, diversity and activity of microbial communities in the soil [57]. Microbial population of bacteria, fungi and actinomycetes were significantly enhanced with addition of use of bio-organic inputs [58].

Soil enzymatic activity, reflects the tendency of nutrient transformation, and can serve as an indicator for field fertility [59]. Application of biostimulants, Jeevamrit lead to an increase in soil microbes like bacteria and fungi which in return secrete extracellular enzymes such as phosphatases which constitute an important part of the soil matrix as abiotic enzymes. Phosphatase enzymes transform P from non-available, organically bound forms into phosphate ions that can be absorbed by microorganisms and plants [60]. Activity of dehydrogenases in the soil occurs as an integral part of intact cells, and determination of the activity of these enzymes in the soil is an indicator of the metabolism of soil microorganisms. Increase of dehydrogenase activity under the influence of organic fertilizers was observed in the studies [61]. Soil enzyme activities commonly correlate with microbial parameters. Enzyme activities were generally higher in the rhizosphere of the soil and these organic amendments increase the activities of acid phosphatase enzymes significantly [62]. The activity of soil microorganisms is also influenced by application of bio-organic inputs such as Jeevamrit and FYM which leads to increase in the H+ ions concentration is a key factor influencing the microflora of soil. This greatly influences the soil enzymatic activity [63].

It is clear that integration of bio-organic fertilizers had significant effect on leaf macro nutrients content. Higher leaf nutrient content comprising integrated application of vermicompost and organic manure due to higher mineralization coupled with microbial activity in the presence of vermicompost which facilitate better absorption and translocation of mineral nutrients by the plant system [64]. Significant steady increase in macronutrient contents of leaves with the application of vermicompost and cow urine was also observed [65]. The increase in leaf N content might be due to high mobility of N and its efficient translocation under abundant supply from root to the leaves which could have accounted for its enhanced accumulation in the leaves. AMF inoculation increases leaf N, P, K by 8.75, 24.61 and 13.54%, respectively [66]. The highest leaf N, P content was recorded in treatment recommended dose of NPK + 50 kg vermicompost [67]. In another study, AM fungi inoculation increased P, K, Cu, Zn and Mn concentrations of the fruit with a considerable increase in K and B content of fruits [68]. Application of organic manure + RDF + Jeevamrit after 15–20 days of transplanting recorded significantly higher nutrient uptake of N, P and K [69]. FYM + other organic manure with Azotobacter and Azospirillum to the fruits reveals maximum P content in fruits due to production of enzyme complexes which solubilized the unavailable form of P and made it available to the plants [70]. Application of Jeevamrit increased the fruit N content which is directly related with the synthesis of protein through amino acid.

Conclusion

The research findings indicated that the application of Ghan-Jeevamrit @100 g/m2 + Jeevamrit @ 20% + Azolla @ 200 g/plant along with biostimulants at 50 g per plant applied 30 days after transplanting in coriander-strawberry-fenugreek sequencing proved as the most effective treatment. This approach resulted in a notable enhancement of vegetative growth parameters, flowering, yield, quality characteristics, and nutrient profiling in strawberry plantlets. Furthermore, the study suggested that the use of bio-organic input sources along with biostimulants can significantly increase crop load, improve post-harvest soil indicators, enhance microbial properties and boost enzymatic activity in the rhizosphere, thereby holding the potential to elevate soil productivity and crop resilience in a sustainable manner. The findings of this study also underscore the potential of bio-organic inputs to improve soil and crop health, and thus offer a promising pathway for sustainable agricultural practices that can meet the demands of food production while preserving environmental integrity.

Data availability

Data is provided within the manuscript.

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Acknowledgements

We are thankful to all the supporting staff of Dr YS Parmar University of Horticulture & Forestry for their valuable assistance in maintaining all the laboratory and field experiments.

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Authors and Affiliations

  1. Department of Fruit Science, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India

    Priya Thakur, Pramod Kumar, CL Sharma & Tanzin Ladon

  2. Department of Soil Science and Water Management, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India

    Uday Sharma

  3. Department of Biotechnology, Graphic Era (Deemed to be University), Clement Town, Dehradun, Uttrakhand, 248002, India

    Nisha Sharma

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  1. Priya Thakur
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Contributions

Methodology, Software, Writing-original draft preparation-PT, PK, NS, TL; Conceptualization, Visualization, Supervision & Project administration- PK, CLS, US; Writing-review and editing- TL, NS. All authors have read and agreed to the published version of the manuscript.

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Thakur, P., Kumar, P., Sharma, C. et al. Ameliorating potential effects of natural biological formulations and biostimulants on plant health and quality attributes in coriander-fenugreek intercropped strawberry (Fragaria × ananassa Duch.). BMC Plant Biol 25, 164 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-025-06184-8

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