Obtaining Melanin-Synthesizing Strains of Bacillus thuringiensis and their Use for Biological Preparations

Background : A pivotal objective in crop production and plant protection lies in developing environmentally friendly insecticidal preparations and biostimulants. Methods : We employed Bacillus thuringiensis strains with varied insecticidal spectra and engineered melanogenic mutants. Results : We demonstrated a significant increase in insecticidal activity in the isolated mutants. Meanwhile, there was no observable impact of the enhanced synthesis of water-soluble melanin on the nature and abundance of spore and crystal formation. This heightened efficacy can be attributed to the photoprotective qualities of the synthesized pigment, shielding spores and crystals against the detrimental effects of UV radiation and insolation. We demonstrated the high biological activity of water-soluble bacterial melanin through in vivo experiments involving multiple plant species. Conclusions : Our findings indicate that bacterial melanin is a potent phytostimulant. This preparation accelerates and amplifies plant growth and development processes, leading to a substantial increase in crop yield by 20–40%. The simultaneous synthesis of two biologically active substance, melanin and insecticidal toxins, ensures an elevated level of effectiveness in utilizing melaninogenic strains.


Introduction
The widespread use of mineral fertilizers and pesticides in agriculture has increased, leading to dangerous and alarming environmental pollution.In response to this concern, there is a current trend in agriculture to curtail traditional fertilizers and chemicals, instead favoring the adoption of biological products derived from natural sources.At the forefront of agricultural development, prioritizing research on environmentally friendly and highly effective biological insecticidal formulations specifically designed for targeted pest control is crucial.Concurrently, a concerted effort exists to explore plant growth stimulants that align with sustainable agricultural practices.Bacterial preparations derived from the Gram-positive spore-forming bacterium Bacillus thuringiensis (B.thuringiensis) are preferred in integrated pest management since their selective action distinguishes them from traditional pesticides and ensures the safety of humans, warm-blooded animals, fish, and beneficial entomofauna [1,2].Although B. thuringiensis preparations have emerged as the most successful bioinsecticides worldwide, their widespread use is hindered by certain drawbacks.In particular, they exhibit sensitivity to various environmental factors, such as sunlight, rainfall, dew, soil pH, and temperature [3].Among these, ultraviolet rays of sunlight are particularly detrimental, causing protein crystal degrada-tion and diminishing insecticidal effectiveness [4].Consequently, repeated spraying becomes necessary [5], escalating preparation costs.Thus, developing UV-resistant preparations of B. thuringiensis with heightened insecticidal activity may address this challenge.
The widespread use of well-known melanins is constrained by their water insolubility and high production costs.Due to the expensive starting materials and intricate technological processes, synthetic melanins are also economically burdensome.Moreover, isolating and purifying natural melanins from protein impurities constitutes a complex and costly endeavor [8][9][10].Furthermore, most synthetic and natural melanins are water-insoluble, which presents significant challenges in formulating and applying medications based on them [11].The acquisition of natural melanin that is cost-effective and watersoluble presents a substantial expansion of its potential applications.Subsequently, we have obtained a large collection of melanin-synthesizing mutant strains from various B. thuringiensis subspecies (maintained at the Microbial Depository Center of the Institute of Scientific and Production Center "Armbiotechnology" of National Academy of Sci-ences (NAS), Republic of Armenia (RA)).From this collection, we isolated strains with the highest water-soluble bacterial melanin (BM) synthesis, which remarkably retained insecticidal activity [12].
Based on these strains, a sustainable and profitable production process can be established, where the biologically active substance, BM, isolated from the culture liquid (CL), acts as a plant growth stimulant.Concurrently, the biomass forms the basis for an insecticidal preparation, generating two valuable biological products through a single fermentation process.Comprehensive technology has been developed for producing BM in fermenters and its subsequent isolation and purification from methods in previous studies [13,14].Likewise, the environmental biosafety of BM and melaninogenic strains of B. thuringiensis has previously been shown [15].The study also highlighted the efficient assimilation of water-soluble BM by soil microorganisms under aerobic and anaerobic conditions when introduced into the soil.This finding is significant as it prevents the accumulation of melanin in groundwater.
This research aims to investigate the biological activity of BM and bioinsecticides within the highly active melaninogenic strains of B. thuringiensis obtained by us.Additionally, it seeks to explore the potential applications of these products in agriculture.

Used Strains
We used 85 strains from the collection of museum cultures of Scientific and Production Center (SPC) "Armbiotechnology".

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

Spore-Crystal Formation
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.

Bacterial Fermentation
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 2.5-3.0Cell suspension grown on meat peptone agar (MPA) at 30 °C for 48 hours was used as an inoculum.

Determination and Isolation of Bacterial Melanin
The level of synthesized melanin was determined on a Perkin Elmer 550S UV-VIS spectrophotometer (Kenilworth, NJ, USA) at a wavelength (λ) 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.
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.

Determination of BM Biological Activity
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.

Qualitative Reactions of Melanins
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.

Phytostimulation Effect of BM
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 (SHI-MADZU 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).

Insecticidal Activity of B. thuringiensis Strains
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.
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× 10 8 spores/mL.The virulence of the strains was determined by calculating the number of deceased caterpillars.
To obtain a working suspension with an approximate titer of 4.0-5.0× 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).

The Insecticidal Activity of B. thuringiensis Strains
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).

Obtaining Melanin-Synthesizing Strains of B. thuringiensis with High Insecticidal Activity by Chemical Mutagenesis Method
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.

Identifying the Resultant Pigment as Melanin
The identification of the resulting pigment as melanin was confirmed through qualitative reactions with oxidizing The LD 50 (semi-lethal concentration) on the indicated day is presented for two species of lepidopteran larvae belonging to different families, which also shows the degree of sensitivity of caterpillars to the tested strains.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].

The Insecticidal Activity of Melaninogenic Strains of B. thuringiensis
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.High efficiency of BM was observed in unstratified peach seeds and indoor flower crops (Reo, Saxifraga), conifers (Silver spruce), and tree species (Albizia).

Biological Activity and Phytostimulation Effect of BM
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 con-  trol.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.

Discussion
Melanin synthesis by the studied melaninogenic B. thuringiensis strains noticeably increased their virulence without impacting the nature and level of spore and crystal formation.These findings align with existing literature data, indicating that the pigment shields spores and crystals from the destructive effects of sunlight, thereby prolonging their insecticidal activity [5][6][7].The increased insecticidal activity of melaninogenic B. thuringiensis strains makes them valuable for controlling various agricultural pests.
Summarizing the data obtained from studying the impact of BM on the growth, development, and fruiting of agricultural, ornamental, and medicinal plants, it can be concluded that it exerts a significant stimulating effect.The preparation demonstrates an auxin-like effect on plants, particularly evident in the pronounced stimulation of meristematic activity.This leads to increased stem growth in both length and width, as well as the overgrowth of the leaf blade.The intensification of the growth of the aboveground parts of plants is associated with the formation of a powerful root system.
BM surpasses other known phytostimulants due to their solubility in water, rapid decomposition in soil, effectiveness at low concentrations, and low cost.These properties enable melaninogenic strains to replace chemical insecticidal preparations with environmentally friendly, highly effective alternatives with a broad range of actions.A biostimulant preparation containing BM contributes to developing new plant cultivation methods.
Using minerals, especially nitrogen-containing fertilizers, as growth stimulants can lead to the accumulation of nitrates in plant foods and environmental pollution.Therefore, replacing them with environmentally friendly bacterial melanin in various crop production sectors will significantly reduce the use of chemicals.
An evident advantage of the highly active obtained melanin-synthesizing insecticidal strains is that they can serve as a basis for profitable waste-free production.The biomass from the fermentation of these strains can form the foundation for an environmentally friendly insecticidal preparation to combat various agricultural pests.At the same time, the culture medium can be a source for isolating the biologically active BM.Ultimately, the environmental safety and affordable price of these preparations broaden their applications in agriculture.

Conclusions
Bioinsecticidal preparations and BM have been obtained based on melaninogenic strains of B. thuringiensis.The inherent photoprotective property of melanins plays a crucial role in protecting the spores and crystals from UV damage and the harmful effects of sunlight, which contributes to the long-term insecticidal activity of these strains.This will make it possible to replace chemical insecticides with environmentally friendly, highly effective drugs.BM is a natural effective phytostimulant.The use of environmentally friendly water-soluble BM in various sectors of crop production will significantly reduce the use of mineral and especially nitrogen-containing fertilizers, which can lead to the accumulation of nitrates in plant foods and environmental pollution.

Fig. 2 .
Fig. 2. Effect of BM on the development of the root system of young grape cuttings.Root systems of young vine plants: 1, without treatment of cuttings with BM; 2, after treatment of cuttings by the immersion of their bases in BM solution (0.20%).

Table 4 . BM influence on some anatomic characteristics of sunflower stems (p < 0.05; n = 3).
± 0.08 20.3 ± 0.84 Similar to previous crops, corn seeds exhibited rapid and robust germination under the influence of BM treatment, subsequently surpassing the control in all measured parameters.