Designing of the biological product "Agrobiolog" for mitigating pesticide stress in agricultural plants and stimulating their growth

The productivity of agricultural plants is directly related to the use of methods for their integrated protection from stress effects: diseases, pests, drought, and weeds. Pesticides are an important element in intensive plant cultivation systems. At least 60% of them are herbicides [1]. Their use, in turn, not only leads to environmental pollution, damages soil fertility and microbiota, but also significantly effects crops [2-4]. It is possible to mitigate these stressful effects with the use of bacterial antistress agents.

In crop production, there is a growing interest in the bacterial-based products that have a positive influence on the growth and development of agricultural plants. The plant growth stimulation is usually caused by a complex of beneficial properties of bacteria, such as the ability to nitrogen fixation, phosphate dissolution, synthesis of phytohormones and antimicrobial compounds, competition for nutrients and roots surface with pathogens [5-7]. According to the scientific review, the treatment of crops with growth-stimulating bacteria (plant growth promoting bacteria – PGPB) turns out to be the most effective in overcoming abiotic stresses [8, 9]. Their use can improve the plants growth exposed Для цитирования

to herbicides and drought [3]. Still reports on the mitigation of pesticide stress of plants by bacteria are few [10-11]. In this case, the ability of bacteria to effectively combine with chemical herbicides comes to the fore. It assumes their resistance to herbicides [12], and the ability to have a complex positive effect on the plant, including protection from moisture deficiency [13] and pathogens [14] against the background of herbicides.

Therefore, the development of biological products based on pesticide stress mitigating bacteria (PSMB) for stimulating the growth of agricultural crops becomes relevant.

Materials and Methods

The purpose of the work is to screen new bacteria that are resistant to herbicides of different chemical structures and promote plant growth, to create on its basis an anti-stress biological product with the operational name "AGROBIOLOG" and optimizing the conditions of its industrial production.

Two methods were used for screening: sowing on microbiological media directly from freshly harvested soil and obtaining enrichment cultures from soil samples with addition of herbicides, of which, after a month of incubation at a temperature of 28 °C, inoculation on selective nutrient media

This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License containing herbicides was carried out. The Raymond mineral medium was used as the basis for the media with the addition of, as selective agents, herbicides based on 2,4-dichlorophenoxyacetic acid ususally used on wheat crops (Octapon extra (2,5 g/l), Flo-rax (2,5 g/l), Dicamba (1 g/l), Chistalan (2,5 g/l)) and based on sulfonylureas (Nanomet (1 g/l), Spetsnaz (1 g/l)). The concentrations of herbicides in the medium were selected based on the concentration of their working solution according to the manufacturers recommendations.

Antagonism to phytopathogens was determined by the simultaneous cultivation of bacteria and fungi in Petri dishes. Testing was performed on the following mycelial fungi: Bipolaris sorokiniana (Sacc.) Shoemaker, Fusarium culmorum (W.G. Smith) Sacc. BKM F-844, F. gibbosum Appel et Wollenw BKM F-848, F. graminearum Schwabe BKM F-1668, F. solani (Mart) Sacc. BKM F-142, F. oxysporum Schltdl BKM F-137, F. nivale (Fr.) Ces. Ex Sacc. BKM F-3106, F. semitectum ВКМ F-1938, F. aven-aceum ВКМ F-132, Alternaria alternate (Fr.) Keissl. ВКМ F-3047, Rhizoctonia solani J.G. Kuehn ВКМ F-895. To measure the nitrogenase activity, an acetylene assay was used, according to the methodology [15]. The ability of the strain to synthesize IAA was determined using the method of enzyme immunoassay [16]. The solubilization of phosphates was evaluated by the formation of a clear halo around the bacterial colony after five incubation days at 28 °C in the Pikovskaya agar as described [17].

When optimizing the cultivation conditions, we relied on the experience of obtaining biological products for agricultural purposes based on pseudomonads [4]. Biological reactors of various volumes were used for scaling (LLC firm “Prointech”, Pushchino, Russia).

Results

A scheme for the selection of anti-stress bacterial agents is proposed (fig. 1), the main condition is the resistance of microorganisms to herbicides.

Stage 1. Herbicide resistance

Stage 2. Availability of PGPB properties: production of phytohormones, production of antibiotics, nitrogen fixation, phosphorus mineralization.

Stage 3. The presence of positive properties of PGPB in the presence of herbicides

Figure 1. Scheme of selection of potential anti-stress agents for the protection of agricultural plants

Рисунок 1. Схема подбора потенциальных антистрессовых средств для защиты сельскохозяйственных растений

Microbes resistant to herbicides were isolated from 178 soil samples taken on the territory of agricultural lands and industrial zones of the Republic of Bashkortostan, industrial area in the Komi Republic (Russia). After studying their properties, 11 isolates were selected from them (Table 1). The selection criteria were the presence of agro-nomically valuable characteristics, resistance to herbicides and the ability to grow using different herbicides as a source of nutrients. When testing new strains for resistance in tank mixtures, it was found that the most toxic of the tested for bacteria was the herbicide Chistalan (Table 1). Apparently, the toxicity of Chistalan is also due to the lower probability of detecting its destructors.

Table 1.

The ability of new isolates to grow in a medium with herbicides as the sole source of carbon and energy Таблица 1.

Способность новых изолятов к росту в среде с гербицидами в качестве единственного источника углерода и энергии

Isolate	Herbicide
	Dicamba	Octapon	Nanomet	Florax	Chistalan	Spetsnaz
CH.3.1	–	++	–	+	–	+
12N1	++	++	++	++	–	+
СН5% 1	+	++	+	++	+	+
СН5% 2	++	++	++	+	+	+
6СН1	++	++	–	++	+	+
6СН2	++	+	++	+	++	+
5N1	++	++	++	++	–	+
ДД4	+	+	+	–	+	+
DА1.2	++	++	++	++	++	+
NG	++	++	++	++	++	+
NEKR	++	++	+	++	–	++

Note: ++ active growth, + weak growth, – no growth

The isolates DА1.2, 6СН2, СН5% 2, which were identified as Pseudomonas protegens, P. avallanae and P. plecoglossicida, respectively, were the most promising in the scope of the studied problem according to the set of abilities. Their ag-ronomically valuable characteristics of PGPB are

Chetverikov S.P. et al. Proceedings of VSUET, 2023, vol. 85, no. 4, pp. 86-90

presented in Table 2, according to which and increased up to 200% level of antifungal activity (the diameters of the growth inhibition zone of the studied fungi are in the range of 30–40 mm) the strain DА1.2 is the best for creating the biological prod-

uct "AGROBIOLOG".

Table 2.

Properties of potential bacterial antistressants

Таблица 2.

Свойства потенциального бактериального антистрессового средства

Strain	Nitrogenase activity, nmol С 2 Н 4 ⋅h-1ml-1	Synthesis of auxins, ng/ml	Phosphates solubilization
Pseudomonas plecoglossicida СН5% 2	20,7 ± 0,2	323 ± 16	+
P. avallanae 6СН2	19,8 ± 0,2	169 ± 8	+
P. protegens DА1.2	20,8 ± 0,3	870 ± 44	+

The composition of the nutrient medium (g/l) was selected for the production of the biological product "AGROBIOLOG" : peptone-2, yeast extract-2, glycerin-5, NaCl-3, К 2 НРО 4 -2, МgSО 4 ⋅7Н 2 O-1, as the most optimal in terms of the ratio of the cost of components and the final titer of the culture fluid. When cultured in a laboratory fermenter FA-10 with a volume of 10 liters at a temperature of 28 °C, stirrer speed-200 rpm, aeration-0,5 air volume per 1 min per 1 volume of medium for 72 hours, the concentration of viable cells was 2,8⋅10¹⁰ CFU/ml. Cultivation in a reactor with a volume of 1000 liters under the same conditions allows achieving a titer of 6,0⋅10⁹ CFU/ml.

Discussion

Almost all isolates were able to produce auxins. It is known that auxins accumulation in roots

The joint cultivation of the new bacterial strains with fungal mycelium showed their ability to antagonize phytopathogens. Their fungistatic activity was expressed as strongly as in previously isolated cultures of Pseudomonas [20]. However, unlike them, they are stable and able to grow in environments polluted by herbicides based on low-volatile esters of 2,4-D and sulfonylurea.

The optimization of the nutrient medium and cultivation conditions makes it possible to obtain the culture of P. protegens DА1.2 containing at least 5 billion CFU/ml of culture fluid without losing its characteristic properties. This meets the requirements of industrial cultivation of microorganisms and makes the biological product "AGROBIOLOG" promising for industrial production

Conclusion

An     anti-stress biological product

"AGROBIOLOG" based on PSMB has been developed. It can be used as a biofungicide, biofertilizer or safener in classic tank mixtures with herbicides. The useful properties (fixation of molecular nitrogen, immobilization of phosphorus, disease control), in addition to its effect on the mechanisms of stress in plants, increases its practical and commercial value.

Acknowledgments

The study was supported by funding the theme №  075-03-2021-607 от 29.12.2020 по теме

№122031000309-7 by Ministry of Science and Higher Education of the Russian Federation. This study was carried out using the equipment of the Regional Center for Shared Use «Agidel».