**1. Introduction**

In greenhouses, the diversity of harmful species is less than in the field, however, the specific microclimate in greenhouses, as well as the absence of natural enemies as regulatory factors, lead to pest accumulation and increase its harmfulness to cultivated plants. Parasitic phytonematodes are among the most serious pests that negatively affect the quality of vegetables in a greenhouse [1,2]. They are obligate parasites that feed mostly on plant roots with common aboveground symptoms of stunting, yellowing, wilting, and yield losses and belowground root malformation due to direct feeding damage. These factors lead to yield losses [3]. Parasitic phytonematodes feed on many crops worldwide and they can cause enormous yield losses with an estimation of 100 billion dollars a year [4].

**Citation:** Asaturova, A.M.; Bugaeva, L.N.; Homyak, A.I.; Slobodyanyuk, G.A.; Kashutina, E.V.; Yasyuk, L.V.; Sidorov, N.M.; Nadykta, V.D.; Garkovenko, A.V. *Bacillus velezensis* Strains for Protecting Cucumber Plants from Root-Knot Nematode *Meloidogyne incognita* in a Greenhouse. *Plants* **2022**, *11*, 275. https://doi.org/10.3390/plants 11030275

Academic Editors: Carlos Agustí-Brisach and Eugenio Llorens

Received: 29 December 2021 Accepted: 17 January 2022 Published: 20 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Root-knot nematodes are one of the most harmful groups of phytophagous organisms in greenhouses. About 5% of world crop production is annually destroyed by *Meloidogyne* spp. Gall nematodes induce overgrowth of root cells, which leads to the formation of galls on plant roots. The nematode damages the vascular tissue of the roots, thereby interfering with the normal movement of water and nutrients. Infected plants show signs of nutrient deficiency: slow growth, yellowing of leaves, wilting, and dying off of the plant. In such a condition, the yield of plants can drop by 50–80%, and in some cases, there is an entire yield loss [5]. Control of nematodes in greenhouses is expensive and time-consuming, and none of the existing methods completely relieves the plant from its presence. To reduce the number of nematodes in greenhouses, agricultural producers use resistant varieties and steam the soil. In addition, soil fumigation with highly toxic substances prohibited in greenhouses is often used. [6].

In recent years, there have been numerous studies worldwide concerning the possibility of reducing the harmfulness of phytopathogenic nematodes using antagonistic microorganisms [7–9]. Active strains of antagonistic bacteria with high nematicidal activity in combination with high biological and economic efficiency have been identified. Special attention of the experts is paid to *Bacillus*, *Pseudomonas*, and *Pasteuria* bacterial strains [10–13]. In addition, many researchers have found the nematicidal activity of the metabolites of *Trichoderma*, *Paecilomyces*, *Arthrobotrys*, *Beauveria*, *Pochonia*, *Fusarium*, and *Myrothecium* fungi [14–16].

Currently, successful research has been carried out and technologies have been developed for the development of biological products, such as Bioact WG (*Purpureocillium lilacinus*), KlamiC (*Pochonia chlamydosporia*), Econem (*Pasteuria penetrans*), Deny, Blue Circle (*Burkholderia cepacia*), DiTera (*Myrothecium Verruotia*), and Nortica VOTiVO (*Bacillus firmus*). Giant corporations, such as BASF and Bayer CropScience, which are key suppliers of plant protection products in the world market, became interested in the production of these preparations. It should be noted that some products are produced only for use in the country of manufacture [17]. However, none of these biological products are registered for use in Russia.

Currently, only one formulation, Phytoverm, based on *Streptomyces avermectilis* metabolites, is registered against phytonematodes in Russia. However, this group of products belongs to the category of agrochemicals and is prohibited for use in organic farming technologies. So far, there is no registered biological product based on microorganisms against phytonematodes in Russia [18]. The reasons for such a poor assortment of biological products are the insufficient knowledge of the biological characteristics of bacterial and fungal strains as the basis of nematicidal biological products, as well as the study of their trophic needs and resistance to various factors.

Thus, one of the promising solutions to the problem of protecting plants from *M. incognita* can be biological control of the pest population based on the use of effective microbiological agents that are currently not available in Russia.

This study focused on the isolation, identification, and characterization of *B. velezensis* BZR 86 and *B. velezensis* BZR 277 isolated from winter wheat roots. They showed high fungicidal activity against *F. graminearum* [19] and high nematicidal activity in vitro against the root-knot nematode *M. incognita* Kofoid et White [20]. The strains were selected from the BRC «State collection of beneficial insects, mites and microorganisms» of the Laboratory for the creation of microbiological plant protection products and the collection of microorganisms (FSBSI FRCBPP, Russia), since they have enzymatic, growth-stimulating, and nematicidal activity against the root-knot nematode *M. incognita*. In addition, the conditions for cultivating *Bacillus* strains were optimized.

#### **2. Results**

#### *2.1. Characteristics and Identification of Bacillus strains*

The cells of the *B. velezensis* BZR 86 strain are rod-shaped with rounded ends; single or paired; cells are motile; and have sizes of 2.8–4.1 × 6.6–9.4 microns. The cells usually contain spores. Coloring indicates positive results in the Gram stain. On the MPA, the shape of the colonies is round with a scalloped edge. The colonies are shiny and colorless. The profile of the colonies is flat, the edge is wavy, the structure is fine-grained, the texture is soft, and the colonies do not stick to the loop. The diameter of the colonies is 2–4 mm. On the PGA, rhizoid colonies with a smooth edge are formed. The colonies are matte and cream-colored. The profile of the colonies is convex, the structure is streaky in the center, fine-grained along the edge, the texture is soft, mucous, and the colonies adhere to the loop. The diameter of the colonies is 1–3 mm.

*B. velezensis* BZR 277 cells are rod-shaped with rounded ends; single or paired; cells are motile; and have sizes of 1.3–1.8 × 4.5–5.4 microns. The cells usually contain spores. Coloring indicates positive results in the Gram stain. On the MPA, the shape of the colonies is round with a scalloped edge. Colonies are opaque and colorless. The profile of the colonies is flat, the structure is fine-grained, the texture is soft, and the mucous colonies adhere to the loop. The diameter of the colonies is 0.5–2 mm. On the PGA, rhizoid colonies with a smooth edge are formed. Colonies are matte and cream-colored or yellowish-brown. The profile of the colonies is curved, the structure is streaky, the consistency is soft, mucous, and the colonies adhere to the loop. The diameter of the colonies is 1–3 mm.

MALDI-TOF MS analysis of bacterial colonies of the studied strains showed score values of ≥0.65 (BactoSCREEN ID) and ≥1.952 (Bruker Autoflex). These data confirm taxonomic identification only up to the genus Bacillus (Figure 1).

**Figure 1.** Identification of *Bacillus* strains BZR 86 and BZR 277: (**A**) Unique spectrum of ribosomal proteins of strain BZR 86 (blue peaks belong to the reference strain *Bacillus subtilis*, the rest of the peaks in the upper part of the profile belong to the studied strain BZR 86 (BactoSCREEN)). Green and yellow peaks coincide with the data of the reference strain *B. subtilis*, red ones do not match. Similar data were obtained by this method for the BZR 277 strain (data not shown); (**B**) Unique ribosomal protein spectra of strains BZR 86 and BZR 277 showing taxonomic identity of strains (Bruker).

A phylogenetic tree based on genome-wide sequencing of 120 conserved marker genes shows that strains BZR 86 and BZR 277 clearly cluster with *B. velezensis* NRRL B-41580 (*B. velezensis* GCF 001461 825.1 at the phylogenetic level); the average nucleotide identity (ANI) is 97.59% (Figure 2). Strains BZR 86 and BZR 277 are closer to *B. siamensis* and B*. amyloliquefaciens* than to *B. subtilis* (as determined using 16S rRNA).

**Figure 2.** Maximum likelihood phylogenetic tree constructed using amino acid sequences of 120 conserved marker genes. The tree was constructed using PhyML v.3.3.
