We used 85 strains from the collection of museum cultures of Scientific and Production Center (SPC) “Armbiotechnology”.

Chemical mutagenesis of cultures was conducted according to the method described by Miller [16].

To assess spore–crystal formation, the obtained Cultural Liquids (CLs) were studied by microscopy using a light phase-contrast microscope (LOMO, MIKMED 6, Petersburg, Russia). The spore titer was counted in Goryaev’s counting chamber.

Fermentation was performed in a “Biostat-S” laboratory fermenter with a 7.0 L working capacity (“Brawn”, Sartorius Company, Goettingen, Germany). Fermentation was carried out at the oxygen dissolution rate of 2.5–3.0 g O${}_2$ • L${}^{-1}$ • h${}^{-1}$ (1.2 volume of air to 1 volume of culture medium per minute), temperature 30–32 ℃, pH 7.2–7.8, pressure of 0.03–0.04 MPa in the fermenter, stirrer rate 700–750 rpm.‬ The medium of the following composition was used, %: hydrolyzate of fishmeal –12; peptone –1; CuSO${}_4$ $\times$ 7H${}_2$O –0.005; MnSO${}_4$ and ZnSO${}_4$ $\times$ 7H${}_2$O –0.0005; chalk –3%.

Cell suspension grown on meat peptone agar (MPA) at 30 °C for 48 hours was used as an inoculum.

The level of synthesized melanin was determined on a Perkin Elmer 550S UV-VIS spectrophotometer (Kenilworth, NJ, USA) at a wavelength ($\lambda$) of 315 nm. The resulting OD value was compared with the control OD. Synthetic melanin (Sigma, USA) was used as an aqueous solution (pH 9.0) as the control. Synthetic melanin and the obtained purified preparations of water-soluble melanin were used to prepare standard solutions.

To determine the Infrared (IR) spectra of bacterial melanin, we used a Nexus Nicolet FTIR spectrometer (Catalog number: 912A0760, Thermo Fisher Scientific, Waltham, MA, USA). ZnSe Prism (4000–650 cm${}^{-1}$) with a single reflection.

The biomass obtained after centrifugation (5000 rpm) was used as an insecticidal preparation, and the supernatant was used to obtain water-soluble melanin.

Melanin was sorbted from the supernatant under dynamic conditions at a linear flow rate of 0.034 cm/s on an IA-1r anion exchanger (Waterhim, Moscow, Russia). Melanin was eluted from the resin with a 3.5% ammonia solution at an eluent flow rate of 0.026 cm${}^3$/s.

The biological activity of BM was investigated across various plants. Optimal concentrations of BM were identified for treating the seeds of experimental plants and subsequently irrigating the soil.

Plants were treated with BM in various ways: By immersing the roots of seedlings and the bases of cuttings in melanin solutions of different concentrations, watering the soil after planting, soaking the seeds, and combining both methods. The optimal concentrations and processing modes of BM were selected for each species.

The qualitative reactions were conducted using the isolated melanins [8, 17].

Qualitative reactions with oxidizing agents (H${}_2$O${}_2$, KMnO${}_4$, FeCl${}_3$) were used to identify the resulting pigment for melanins.

The following solutions were added to the melanin:

10% H${}_2$O${}_2$ in a ratio of 1:1 (v/v): The reaction was positive; discoloration of the solution was observed, which is typical for melanin.

0.5 M KMnO${}_4$: The reaction was positive; the solution changed color from brown to green, followed by precipitation and discoloration.

1% FeCl${}_3$: The reaction was positive; a precipitate formed, which was dissolved by adding 3–5 volumes of salt solution.

Anatomical studies of plants, dry matter content, and the determination of the amount of protein were performed according to the methods described by Tretyakov [18].

The amino acid composition of the hydrolyzate was evaluated using a Shimadzu NexeraX2 analyzer (SHIMADZU CORPORATION, Kyoto, Japan).

The High-performance liquid chromatography (HPLC) method (Waters Alliance e2695 separation module, HPLC, Water Corporation, Milford, MA, USA) was used to determine the amount of vitamin C and flavonoids in the treated plants, and the number of essential oils was determined using a gas chromatograph (GC) (thermoscopic lane 1300, Catalog number: CL2GAS000001, Thermo Scientific™ TRACE™, Waltham, MA, USA).

The insecticidal activity of the studied strains was determined in laboratory conditions on silkworm caterpillars (Bombyx mori) of various ages. Mulberry leaves treated with the cultural liquid obtained after fermentation of the studied strains were fed to the caterpillars. Then, the number of living and dead caterpillars was recorded for 3–12 days. The dead caterpillars were examined according to the methods described by Minaeva and Pantene [19, 20]. The insecticidal activity of the studied strains was also tested on golden-tail (Euproctis chrysorrhoea) caterpillars at various developmental stages. The biological effectiveness was assessed based on the number of deceased caterpillars, with counts conducted on the 5th and 8th days following treatment with bacterial preparation.

Insecticidal activity was assessed by semi-lethal concentration (LD${}_{50}$) using the Kerber method [21].

Silkworm (Bombyx mori) caterpillars in the third instar and golden-tail caterpillars in the third and fourth instars were employed as test subjects. The LD${}_{50}$ calculation was based on the enumeration of deceased caterpillars with counts performed on the 1st, 2nd, and 3rd days for silkworm larvae and on the 3rd, 6th, and 8th days for golden-tail caterpillars.

The selected melaninogenic strains were also tested in the field on golden-tail caterpillars, with a working suspension titer of 4.0–5.3 $\times$ 10${}^8$ spores/mL. The virulence of the strains was determined by calculating the number of deceased caterpillars.

Based on the laboratory results, field tests were carried out on the gypsy moth (Liparis dispar), II–III instar grape moth (Tortrix viridana), and goldenrod (Euproctis chrysorrhoea) caterpillars.

To obtain a working suspension with an approximate titer of 4.0–5.0 $\times$ 10${}^8$ spores/mL from the bacterial biomass of each strain by dilution with tap water. Oak trees 2.5–3.0 meters high were treated with working suspensions of the original and melaninogenic strains. The trees were sprayed with a backpack sprayer of the Baltika brand (16 L, Baltika, Petersburg, Russia). The percentage of insect death was used to assess virulence. Dead caterpillars were counted on 12 linear meters in different parts of the crown after 3, 7, 10, and 12 days.

Statistical parameters (mean, standard deviation) used in the experiments were calculated using analysis of variance (ANOVA).

To obtain melanin-synthesizing strains that retain insecticidal activity, 85 strains of B. thuringiensis with different spectra of insecticidal action were studied.

Initially, 21 strains of B. thuringiensis were selected based on their optimal efficiency in sporulation and crystal formation, where the number of free spores ranged from 80 to 90%, and sporulating vegetative cells with crystals constituted 10–20%. Subsequently, nine strains were further selected from this group as representatives of different subspecies based on their superior efficiency in spore and crystal formation. These included representatives from the ssp kurstaki (1), ssp sotto (1), ssp darmstadiensis (1), ssp caucasicus (2), ssp israelensis (2), and ssp berliner (2) subspecies.

Culture liquid (CL) from new melaninogenic strains of B. thuringiensis was tested on golden-tail caterpillars at various developmental stages. The biological effectiveness was assessed based on the number of deceased caterpillars, with counts conducted on the 5th and 8th days following treatment with the preparation (Table 1).

Chemical mutagenesis was performed on the selected insecticidal strains, resulting in B. thuringiensis mutants that synthesized extracellular water-soluble melanin. This facilitated subsequent isolation and purification processes of melanin. Importantly, these mutants retained their insecticidal activity. The morphology of colonies and cells and the degree of sporulation and crystal formation remained consistent with the original strains. By refining the melanin biosynthesis technology, which included adjustments to the fermentation medium composition and optimization of fermentation conditions, the newly obtained melanin-synthesizing mutants of B. thuringiensis produced up to 11 g/L of melanin (while ensuring a high level of aeration). Based on these test results, mutant strains of B. thuringiensis ssp. kurstaki 1224 (BTKAS1), ssp. caucasicus 837 (BTCc1), and B. thuringiensis S1 ssp. berliner–BTC1 were selected for further research and development.

The identification of the resulting pigment as melanin was confirmed through qualitative reactions with oxidizing agents (H${}_2$O${}_2$, KMnO${}_4$, FeCl${}_3$) [8, 17]. Upon the addition of both KMnO${}_4$ and H${}_2$O${}_2$ to the pigment solution, discoloration occurred. In the presence of a small quantity of FeCl${}_3$, a brown flocculent precipitate formed in the pigment solution, dissipating with increased salt addition. These experimental outcomes suggest the presence of quinoid and phenolic structures in the molecule of the resulting pigment, providing further evidence of its melanin nature.

The analysis to determine the IR spectrum BM revealed the presence of bands characteristic of melanins [22].

The presence of paramagnetic centers in the BM was demonstrated using the electron paramagnetic resonance (EPR) method [13]. The paramagnetism of natural melanins significantly influences various important properties, including electrical conductivity, exchange capacity, chemical reactivity, antioxidant properties, biological activity, etc. [23, 24].

One of the characteristic features of melanin is its photoprotective activity, which protects spores and crystals of insecticide strains from the damaging effect of UV radiation and insolation. This increases the insecticidal activity of melaninogenic B. thuringiensis strains.

Compared to the original strains, the insecticidal activity of melanogenic mutants (pig+) against golden-tail caterpillars increased noticeably, with an increment of more than 10% (Table 2).

When utilizing B. thuringiensis preparations in the field, the primary concern is the potential harm caused by UV radiation and sunlight, which may adversely affect the spores and crystals, leading to a loss of their insecticidal activity. Melanins, being natural photoprotectors, play a crucial role in shielding spores and crystals from UV damage and the deleterious effects of sunlight [5, 6, 7]. This inherent property of melanins contributes to the prolonged insecticidal activity of melaninogenic B. thuringiensis strains.

A BM with high biological activity was obtained using melaninogenic strains of B. thuringiensis, and demonstrated on different plants. Experimental findings underscore the necessity of selecting the optimal BM concentration tailored to each plant species.

The findings from studies conducted on various vegetables, fodder, fruit, and ornamental plants have demonstrated a pronounced stimulating effect of BM. In terms of biometric indicators for vegetable crops such as potatoes, tomatoes, beans, chickpeas, peppers, beets, small-fruited carrots, etc. (Fig. 1), the positive impact of the BM solution was evident at low concentrations ranging from 0.03% to 0.08%. Treated plants exhibited accelerated seed germination, robust growth, increased stem branching, a shift towards intensive and prolonged fruit formation, and the ripening of large, fleshy fruits with many large seeds. These effects collectively contributed to a substantial enhancement in plant productivity, estimated at approximately 20–40%.

In bush beans treated with a 0.03% BM solution, growth in the leaf blade was observable, attributed to marginal meristem activation. Across all experiments, a consistent stimulation of growth and development was observed, with an increased number of beans being established. Furthermore, the number and size of seeds in the treated group significantly exceeded those in the control group (Table 3).

Grapes hold significant agricultural importance, particularly in the food industry, mainly in winemaking. An essential consideration lies in the production and application of biostimulants for grape growth. It was demonstrated that shortened grape cuttings (16 varieties), when treated with a 0.1–0.2% BM solution after 6–7 months, developed a powerful root system, facilitating the growth of the aboveground mass and the ripening of internal tissues. This ultimately ensured a high survival rate of seedlings and resistance to unfavorable abiotic factors and stimulated the growth and ripening of shoots. These favorable outcomes, shown in Fig. 2, highlight the efficacy of pre-soaking seeds in promoting a high survival rate of grape seedlings.

The effect of 0.08–0.12% BM solution is shown on grain and fodder crops such as wheat, sorghum, and sunflower. This effect was achieved through pre-sowing seed soaking and soil watering methods. The treated crops intensified root system growth, providing enhanced access to water and mineral elements. This, in turn, contributed to the plant’s rapid growth in height and thickness and the development of a significant number of elongated leaves (57–63%) characterized by a vibrant bright green. All treated plants exhibited increased growth in the above-ground portion (32–35%) and the root system.

In cultivated plants, longitudinal growth is especially activated through the stimulation of the apical meristem, while the thickening and branching of stems result from the activation of the cambium.

Cambial activity stimulation was evident in the intensified formation of xylem vessels with wider diameters and greater quantities. The diameter of these vessels exceeded that of the control by 29%, leading to a 63.6% increase in xylem square within a vessel bundle compared to the control. Additionally, the overall width of the xylem and cambium zones in the stems of experimental plants surpassed that of the control by 34%, indicating a notable enhancement in cambial activity (Table 4). These changes ultimately resulted in the distinct thickening of the stalk.

BM facilitated an increase in the number and size of leaves and other indicators, such as stem height, stem weight, and average leaf and ear count. The increase in biomass can be attributed to the growth of all studied parameters. Unlike other growth stimulants, stem elongation, in this case, results from the emergence of new leaves rather than internode elongation. An increase in the number and size of leaves, coupled with increased green growth, also contributed to an increase in the number of seeds due to enhanced photosynthesis, evident in the intensified green color of leaves. Consequently, both seed productivity and feed mass growth increased. The enlargement of plant habitus was simultaneous with the enhanced development of lateral roots to those found in sorghum. These roots are crucial in preventing lodging in up to 2-meter tall plants. It has also been demonstrated that a 0.08% BM solution stimulates the growth and development of medicinal plants, such as Matricaria chamomilla, Rhamnus, Crataegus, Thymus, calendula, Panax ginseng, etc. To produce medicines, it is important to monitor biochemical indicators such as vitamin C, flavonoids, and essential oils, the content of which is positively influenced by BM.

Plants treated with BM exhibited increased resistance to abiotic environmental factors.

The results of studies conducted on various plants have indicated that BM has a biostimulating effect.