*2.4. Study of B. velezensis BZR 86 and B. velezensis BZR 277 Strains under Greenhouse Conditions*

As a result of comprehensive monitoring, it was found that the cause of the formation of the galls on plant roots is the root-knot nematode M. incognita. In 2016–2018, *B. velezensis* BZR 277 and *B. velezensis* BZR 86 strains were tested against the M. incognita on cucumber plants in the greenhouse. In 2016, a single soil treatment under the root during planting with the studied strains led to plant growth retardation, shredding, and necrosis of the leaves, and then plants wilting. The double treatment under the root with Bacillus strains during the entire growing season caused intensive plant development and no visual signs of plant damage by the nematode were observed. Growth and development indicators of plants after treatment with Bacillus strains significantly exceeded the control. The vegetative period of plants lasted for three months. To the end of the period, complete drying of the control plants was noted. Phytopathological analysis of cucumber plant samples showed that no signs of damage by peronosporosis and powdery mildew were discovered on the plants (Figure 3).

**Figure 3.** Effect of *B. velezensis* BZR 86 and *B. velezensis* BZR 277 treatment on the growth and development of cucumber plants in the greenhouse, 2016–2018: (**A**) plant height, cm; (**B**) leaf area, cm2; (**C**) shoot mass, g; (**D**) root mass, g. The results are presented as the mean <sup>±</sup> standard deviation. Different letters in each column indicate significant difference (p ≤ 0.05).

In 2016, the gall index for the *B. velezensis* BZR 86 strain was five times less, and for the *B. velezensis* BZR 277 strain was 14 times less, than in the control. However, yield is provided not only by a decrease in the number of galls, but also by a direct effect on the growth and development of plants. Therefore, the yield of cucumber plants treated with the studied strains was 5.2% higher for *B. velezensis* BZR 277 (a double treatment), and 21.4% higher for *B. velezensis* BZR 86 (a double treatment) compared to the control (Figure 4).

**Figure 4.** Effect of *B. velezensis* BZR 86 and *B. velezensis* BZR 277 treatment on cucumber yield and gall index in a greenhouse, 2016–2018: (**A**) yield, kg/m2; (**B**) number of galls; (**C**) gall index; (**D**) damage score. The results are presented as the mean ± standard deviation. Different letters in each column indicate significant difference (*p* ≤ 0.05).

In 2017, a double treatment of cucumber plants with *B. velezensis* BZR 86 and *B. velezensis* BZR 277 strains was statistically more effective than a single treatment. It was noted that the *B. velezensis* BZR 277 strain had a greater effect on the development of the aerial part of cucumber plants, while the *B. velezensis* BZR 86 strain stimulated the development of the root system (Figures 3 and 5). Due to the increase in the underground part, a higher percentage of nematode damage was noted for *B. velezensis* BZR 86 strain: two times compared to *B. velezensis* BZR 277. Both strains were noted to influence an increase in yield by 17.8–45.8% compared to the control (Figure 4).

**Figure 5.** Effect of *B. velezensis* BZR 86 and *B. velezensis* BZR 277 strains on the degree of gall formation on the roots of cucumber plants: (**A**) *B. velezensis* BZR86, single treatment; (**B**) *B. velezensis* BZR86, double treatment; (**C**) *B. velezensis* BZR277, single treatment; (**D**) *B. velezensis* BZR277, double treatment; (**E**) control. Black scale bars represent 1 cm.

In 2018, a double treatment of cucumber plants with the *B. velezensis* BZR 86 and *B. velezensis* BZR 277 strains also turned out to be more effective than a single treatment. This may indirectly indicate the ability of the studied strains to enhance the induced systemic resistance of plants. The maximum biometric parameters were recorded for plants treated with the *B. velezensis* BZR 277 strain. It also showed greater activity against M. incognita when it was applied to the root system; two times fewer galls were noted compared to the strain B velezensis BZR 86. As a result, the maximum cucumber yield was recorded with double application of the *B. velezensis* BZR 277 strain—32.7% more than in the control, and 12.7% more than in the option with a double application of the *B. velezensis* BZR 86 strain (Table 1).

### **3. Discussion**

In this study, we assessed the disease control efficacy of *B. velezensis* BZR 86 and *B. velezensis* BZR 277 strains against the root-knot nematode *M. incognita* of cucumber plants in the greenhouse.

Many researchers note the ability of *Bacillus* strains bacteria to reduce the number and harmfulness of phytoparasitic nematodes that attack agricultural crops [3,21]. One of the mechanisms of the nematicidal action of Bacillus strains is the synthesis of extracellular enzymes [22–25]. It is likely that extracellular enzymes, such as proteases, lipases, and chitinases, are capable of destroying the physical and physiological integrity of the nematode cuticle and eggs [26]. It was shown that enzymes can damage the egg membrane of nematodes, which consists of a protein matrix and a chitinous layer [27]. In addition, chitin is the main component of the nematode pharynx. Therefore, any disturbance in the synthesis or hydrolysis of chitin can lead to the death of nematode embryos, the laying of defective eggs, or a violation of molting [28]. The enzymatic activity (chitinase and protease) of the *B. megaterium* culture supernatant provided a nematicidal effect in the range of 21–30% against the larvae of *Meloidogyne* sp. and 30–37% against its eggs [29].

Some studies revealed the ability of Bacillus firmus to synthesize toxins that reduced the number of eggs of *Meloidogyne* spp. [30]. It is known that effective lysis of the cell walls of pests is associated with the complex action of various hydrolytic enzymes. Geng et al. [31] found the ability of *B. firmus* to produce peptidase group enzymes capable of destroying intestinal tissue of nematodes. That research served as the basis for the development of biological products Nortica and VOTiVO, which were successfully used in the United States. Other studies revealed 100% mortality of the second age larvae of the root-knot nematodes *M. incognita* and *M. javanica* within 72 h caused by liquid cultures based on *B. thuringiensis* and *P. fluorescens* bacterial strains. Furthermore, these bacteria stimulated plant growth [32].

Our results demonstrated the activity of *Bacillus* strains against *M. incognita* on cucumber plants (up to 76.4%). According to the study, we assumed the ability of Bacillus strains to synthesize proteolytic enzymes that caused the death of nematodes [10]. In Iran, the treatment of tomato plants with the filtrate of the bacterial culture *Bacillus* sp. in vitro caused mortality of *M. incognita* juveniles by 72% [33]. The use of chitinase and protease synthesized by the *B. pumilus* L1 strain in vitro reduced the hatching of *M. arenaria* eggs by 78%, and the mortality of juveniles was 98.6% compared to the control. In this study, structural damage of nematode bodies and eggs was noted [34]. Under in vitro conditions, *B. subtilis* culture filtrates inhibited the hatching of M. incognita eggs by 94.6% and caused mortality of juveniles by 91.3% [35]. Laboratory studies of *B. subtilis* strains exhibiting nematicidal activity against *M. incognita* showed that the use of a cell suspension provided the mortality of juveniles at the level of 39.3%, while the mortality rate when using the supernatant was 82.3–90.7%, which may be caused by the ability of *B. subtilis* to produce lytic enzymes [36]. These data are confirmed by the studies carried out in 2020, during which liquid culture filtrate caused the death of 90% of juveniles of the second stage and reduced egg hatchability by 97% in vitro [37].

Our studies confirmed the ability of *B. velezensis* BZR 86 and *B. velezensis* BZR 277 to produce a number of enzymes. The *B. velezensis* BZR 86 strain discovered chitinolytic activity. As to the *B. velezensis* BZR 277 strain, a high level of synthesis of lipases and proteases was noted, which may be partially involved in the process of suppression of the nematode *M. incognita*. Various factors have a significant effect on the growth of the number of cells and the synthesis of enzymes: temperature, acidity, the composition of the nutrient medium, and the incubation period [38]. Therefore, it is important to optimize the cultivation conditions for improving the nematicidal activity of bacterial strains. For example, Cheba et al. [39] showed that incubation of the *Bacillus* sp. R2 at 30 ◦C led to the highest level of chitinase synthesis. *B. cereus* JK-XZ3 filtrate cultivated at 30 ◦C showed the highest nematicidal activity, resulting in 100% mortality of Bursaphelenchus xylophilus [40]. However, Dukariya and Kumar [41] reported maximum chitinase productivity at 37 ◦C.

In our studies, we improved the cultivation conditions for strains exhibiting nematicidal activity according to a criterion, such as the number of colony-forming units. It is known that *Bacillus* strains are capable of growing in a temperature range of 5.5 ◦C to 55.7 ◦C [42–44]. According to the data obtained for the *B. velezensis* BZR 86 strain, the optimum temperature is 35 ◦C, and for the *B. velezensis* BZR 277 strain—30 ◦C. Our study is fully consistent with the conclusions of Park et al. [45], according to whom, when cultivating the B. velezensis GH1–13 strain, a titer of 7.5 × 109 CFU/mL was obtained at 37 ◦C. A similar pattern was noted in the study by Usanov et al. [46], according to which an increase in the cultivation temperature to 40 ◦C has a positive effect on the growth dynamics of the *B. subtilis*, and the number of colony-forming units exceeds the control by 88.9%.

In our study, the maximum titer of both strains was observed at pH 3. However, it should be noted that when the strains were cultivated on media with different levels of acidity, no significant jumps in cell density were observed. These data are confirmed by Mohapatra et al. [47] 2015, according to whom, insignificant fluctuations in CFU were observed during cultivation of the *Bacillus* sp. P1on media with pH 6–9 (5.0–8.4 × 105 CFU/mL). *B. velezensis* XT1 strain was able to grow in a wide pH range of 5–10 [48]. A relatively high

cell density at pH 3 was noted during the cultivation of the *B. velezensis* CE 100 strain, the initial pH of the medium of 7.5 decreased to 4.7 over 24 h of cultivation, which may indicate the ability of some *B. velezensis* strains to grow in an acidic medium [49].

Molasses has been shown to optimize the parameters of growing strains-producers of biological products [50,51]. Being a source of not only sugars, but also vitamins, macroand microelements, this ingredient ensures the active growth of microorganism cultures. For strains *B. velezensis* BZR 86 and *B. velezensis* BZR 277, the cell titer in liquid cultures using molasses as media component was 2–3 times higher than on media with sucrose, glucose, and glycerol and amounted up to (1.6 ± 0.03) × <sup>10</sup><sup>9</sup> CFU/mL for the *B. velezensis* BZR 86 strain and (5.8 ± 0.39) × <sup>10</sup><sup>8</sup> CFU/mL for the *B. velezensis* BZR 277 strain. Peptone was the most preferred source of nitrogen nutrition for both strains. It should be noted that the B. velezensis BZR 86 strain turned out to be more sensitive to the source of nitrogen nutrition: when NaNO3 was added to the nutrient medium, the titer was (3.7 ± 0.4) × 106 CFU/mL, and when peptone was added—(1.8 ± 0.07) × <sup>10</sup><sup>8</sup> CFU/mL. These results are consistent with the studies on the optimization of the nutrient medium for the cultivation of the *B. velezensis* NRC-1 strain, according to which the maximum cell growth and mannanase synthesis were achieved on the medium with peptone [52].

The cultivation time is an important parameter for the growth of biocontrol agents. If bacterial cultures are incubated for too long, some metabolites may be converted to other compounds. In contrast, if the incubation period is not long enough, it is possible that important metabolites have not yet been synthesized (for example, enzymes that are formed during the stationary growth phase). This demonstrates the importance of making bacterial growth curves [53]. During our research, it was noted that the phase of initial growth began after the introduction of a parent culture with a titer of (1.4 ± 0.05) × 108 CFU/mL in the *B. velezensis* BZR 277 strain and (8.1 ± 0.01) × 107 CFU/mL in the *B. velezensis* 86 strain and lasted up to 8 h. The phase of the most active growth began 16 h after the introduction of the mother culture and began to decay after 36 h for the *B. velezensis* 277 strain and from 8 to 36 h for the *B. velezensis* 86 strain. The maximum cell titer was observed during this period and amounted to (1.7 ± 0.02) × 109 CFU/mL in the *B. velezensis* BZR 277 strain and (1.3 ± 0.11) × 109 CFU/mL in the B. velezensis BZR 86 strain. The phase of bacterial cell death occurred 36 h after the start of cultivation for both strains, which contradicts the data obtained in the study of the cultivation of the *B. velezensis* IP22 strain, according to which the stationary phase lasted up to 60 h [54].

Three-year vegetation tests on the Courage variety of cucumber plants under the greenhouse conditions showed that plants treated with the B. velezensis BZR 86 and *B. velezensis* BZR 277 strains developed more intensively; the growth and development indicators significantly exceeded the control ones. This tendency was observed in research by Sahebani and Omranzade [55], according to which the introduction of a liquid culture based on *B. megaterium* wr101 into the soil infected with *M. javanica* contributed to an increase in the mass of cucumber shoots twice as compared to the control. Other authors showed that the decrease in the number of M. incognita contributed to the improvement of plant development, which was manifested by an increase in biometric indicators and in cucumbers compared with the control. It was noted that the use of two Bacillus strains contributed to the formation of an additional cucumber yield from 4.6% to 45.8%, which confirms the results of the 2019 studies, in which the treatment of cucumber plants with a liquid culture based on the B. subtilis Bs-1 strain provided an additional yield of up to 53.1% in combination with a high nematicidal effect against *M. incognita* [56].

In our research, we showed that in three consecutive years, application of two Bacillus strains in the greenhouse demonstrated that the number of galls per gram of cucumber root mass decreased by 13 times for the *B. velezensis* BZR 277 strain, and by 7.2 times for the *B. velezensis* BZR 86 strain. In general, the *B. velezensis* BZR 277 strain exhibited a more pronounced nematicidal and plant growth-promoting effect. Probably, such difference is due to the selectivity of the nematicidal action of lipases and proteases synthesized by the *B. velezensis* BZR 277 strain against predominantly adult nematodes. It should be noted that the cuticle of nematodes contains different types of structural proteins and their proportions change throughout their life cycle as other authors have shown [54,57].
