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Article

Endophyte Bacillus vallismortis BL01 to Control Fungal and Bacterial Phytopathogens of Tomato (Solanum lycopersicum L.) Plants

by
Vladimir K. Chebotar
1,*,
Maria S. Gancheva
1,2,
Elena P. Chizhevskaya
1,
Anastasia V. Erofeeva
1,
Alexander V. Khiutti
3,
Alexander M. Lazarev
3,
Xiuhai Zhang
4,
Jing Xue
4,
Chunhong Yang
4,5 and
Igor A. Tikhonovich
1,2
1
All-Russian Research Institute for Agricultural Microbiology, Podbelskogo Hwy, 3, Pushkin, 196608 St. Petersburg, Russia
2
Department of Genetics and Biotechnology, Faculty of Biology, Saint Petersburg State University, 7–9 Universitetskaya Embankment, 199034 St. Petersburg, Russia
3
All-Russian Research Institute of Plant Protection, Podbelskogo Hwy, 3, Pushkin, 196608 St. Petersburg, Russia
4
Institute of Grassland, Flowers and Ecology, Beijing Academy of Agriculture and Forestry Sciences, No.9 Shuguang Garden Middle Road, Beijing 100097, China
5
Institute of Botany, Chinese Academy of Sciences, No.20 Nanxincun, Xiangshan, Beijing 100093, China
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(10), 1095; https://doi.org/10.3390/horticulturae10101095
Submission received: 2 September 2024 / Revised: 3 October 2024 / Accepted: 11 October 2024 / Published: 14 October 2024
(This article belongs to the Section Plant Pathology and Disease Management (PPDM))
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Abstract

:
Some strains of Bacillus vallismortis have been reported to be efficient biocontrol agents against tomato pathogens. The aim of our study was to assess the biocontrol ability of the endophytic strain BL01 Bacillus vallismortis through in vitro and field trials, as well as to verify its plant colonization ability and analyze the bacterial genome in order to find genes responsible for the biocontrol activity. We demonstrated in a gnotobiotic system and by confocal laser microscopy that the endophytic strain BL01 was able to colonize the endosphere and rhizosphere of tomato, winter wheat and oilseed rape. In vitro experiments demonstrated the inhibition activity of BL01 against a wide range of phytopathogenic fungi and bacteria. BL01 showed biological efficacy in two-year field experiments with tomato plants against black bacterial spotting by 40–70.8% and against late blight by 47.1% and increased tomato harvest by 24.9% or 10.9 tons per hectare compared to the control. Genome analysis revealed the presence of genes that are responsible for the synthesis of biologically active secondary metabolites, which could be responsible for the biocontrol action. Strain BL01 B. vallismortis can be considered an effective biocontrol agent to control both fungal and bacterial diseases in tomato plants.

1. Introduction

Agricultural strengthening is an important factor in food safety for the rising world population. Extensive usage of chemical fertilizers and pesticides not only affects soil health by depleting soil fertility and diminishing soil microflora but also poses a threat to human health and the ecosystem [1]. By considering all these problems, researchers are attentive to the substitution of chemical fertilizers and pesticides with microbial-based ones [2].
Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in the world, cultivated worldwide on 5.2 million hectares with a total production of 186 million tons in 2022 [3]. Tomato is a source of important nutrients and bioactive compounds with well-established health benefits [4]. It was reported that tomato plants can be infected by more than two hundred different phytopathogenic fungi and bacteria, which affect both the quality and quantity of tomato production [5,6,7]. The use of chemical plant protection means is still an effective method to control tomato pathogens and prevent yield losses and damage [8]. However, the intensive application of chemicals can lead to the pollution of surface and underground water, degradation of soil, a negative impact on the environment and an increase in pathogen resistance [9]. So, taking into account this concern, biological control is one of the most promising alternatives to the chemical control of plant diseases [10].
The use of Bacillus species as biocontrol agents against tomato diseases was recently reviewed by Karačić et al. [11]. The authors mentioned that dynamic and complex soil–plant–microbe interactions, accompanied by biotic and abiotic stress and the effects of climate change, influence the colonization and action of the introduced Bacillus spp. agents. It was pointed out that integrated multi-omics and bioinformatics technologies should be exploited to underline the mechanisms and efficiency issues of Bacillus spp. agents for managing plant diseases in sustainable agricultural production [11].
Microorganisms existing within the plant, including aboveground and underground parts and seeds, that positively affect plant development can be defined as endophytic [12,13]. Endophytic microorganisms were formed as a result of millions of years of evolution and acquired the ability to use the internal environment of the plant (endosphere) as a unique ecological niche that protects them from environmental changes [14,15,16]. These organisms can be transmitted through generations, from ancestor to descendent, as an integral part of the plant organism endosphere. Endophytic bacteria colonize the same ecological niche in the plant as pathogens, and, therefore, they can be used as a promising biological method to control plant pathogens, i.e., they are so-called “biocontrol” agents [17,18,19]. Nearly 300,000 plant species that exist on Earth are thought to be host to one or more endophytes [20]. Thus, endophytes enable their host to have better survival against biotic and abiotic challenges and competition from other plants.
Tomato productivity is impacted by a number of plant diseases caused by the fungi Alternaria solani Sorauer, Septoria lycopersici Spegazzini, Botrytis cinerea, Fusarium oxysporum f. sp. radicis-lycopersici Jarvis and Shoemaker, Verticillium dahliae Klebahn and Phytophthora infestans (Montagne) de Bary [6]; and the bacteria Pseudomonas syringae pv. tomato (Okabe), Clavibacter michiganensis subsp. michiganensis (Smith) and Xanthomonas campestris pv. vesicatoria (Doidge) [21,22]. To counter these phytopathogens, endophytic bacteria are a viable alternative for plant disease management, especially as a component of an integrated disease management program [23,24,25,26]. Biological control agents antagonize pathogens directly by hyperparasitism and predation, as well as by the production of antibiotics and lytic enzymes, and indirectly by competing for space and nutrients, inducing systemic resistance, and promoting plant growth [27,28,29].
It is known that Bacillus vallismortis can produce metabolites with strong growth inhibition activity against phytopathogenic fungi [30,31]. For instance, it was demonstrated that the treatment of pepper seedlings with the strain BS07 B. vallismortis confers disease resistance against infection with Phytophthora capsici and Colletotrichum acutatum, the most destructive pathogens to pepper plants [32,33,34,35]. Some strains of B. vallismortis provided protection against bacterial speck and bacterial spot of tomato [36,37]. However, the biocontrol potential of B. vallismortis used for the protection of tomato plants is not clear, and data are limited.
In our study, we used the endophytic strain BL01 Bacillus vallismortis isolated from the roots of the drought-resistant plant Artemisia lerchiana Web. from the Astrakhan region, Russia. The genome of this strain was sequenced and deposited in NCBI (National Center for Biotechnology Information) under BioProject accession number PRJNA809498 [38]. The aim of our study was to assess the biocontrol ability of the endophytic strain BL01 Bacillus vallismortis against the phytopathogenic fungi and bacteria in vitro and field experiments and verify its plant colonization ability, as well as to analyze the bacterial genome in order to find the genes responsible for the biocontrol activity.

2. Materials and Methods

2.1. Determination of the Colonization Ability of B. vallismortis BL01

Tomatoes (Solanum lycopersicum L., cv. Perseus, Russian Vegetable Garden, Russia) and spring wheat (Triticum aestivum L., cv. Veda, selection of the P.P. Lukyanenko National grain center selection, Russia) were used to analyze the plant colonization ability of the endophytic strain BL01 Bacillus vallismortis in a gnotobiotic system. The Bacillus subtilis Ch-13 rhizospheric strain, used for the production of biopreparations, was used as a reference strain [39].
Both strains of the genus Bacillus were grown overnight in liquid LB medium (Sigma Aldrich, St. Louis, MO, USA) at 28 °C on a rotary shaker at 200 rpm to get a final concentration of bacterial suspension of 1.0 × 106 cfu/mL. Before bacterial inoculation, tomato and spring wheat seeds were subjected to surface sterilization for 30 s in 70% alcohol, followed by 3 min in a 3% NaOCl solution, and then washed six times with sterile distilled water. A gnotobiotic system for studying tomato rhizosphere colonization by Bacillus spp. was used [40]. The system is based on sterile seedlings that are inoculated with one strain and subsequently grown in a sterile glass tube containing quartz sand. For inoculation, sterile seedlings of tomatoes and spring wheat were soaked for 30 min in diluted suspensions of bacterial strains Bacillus subtilis Ch-13 and Bacillus vallismortis BL01 with a cell number of 1.0 × 105 cfu/mL. After inoculation, plant seedlings were planted in quartz sand (at depths of 5–6 mm) in gnotobiotic systems, one plant for each system in six replications for each variant. The plants were grown for 5 days at 21 ± 2 °C under artificial lighting (with a 16 h light period). When determining the number of introduced bacteria in the rhizosphere and on the roots (endosphere), gnotobiotic systems were disassembled under sterile conditions. The rhizosphere layer of sand from the roots was washed off with water for 1 h on a rotary shaker with 120 rpm (5 mL H2O per 1 wheat root and 3 mL H2O per 1 tomato root were added). The sand washed from the roots was suspended in water and used as samples of the rhizosphere. A number of serial dilutions were made, and the number of introduced bacteria was taken into account by inoculating Petri dishes with LB medium. Rhizosphere sand that was washed from the roots was dried at t = 105 °C to account for the weight of the plant rhizosphere. To determine the amount of introduced bacteria on the surface and inside the roots (endosphere), plant roots washed of rhizosphere sand were additionally washed by dipping one root in 20 mL of 0.85% NaCl solution. Then, the roots were crushed and suspended in 10 mL of 0.85% NaCl solution, a number of serial dilutions were made and the number of bacteria on the LB medium was taken into account.

2.2. Root Colonization Study by Using Fluorescence In Situ Hybridization and Confocal Laser Scanning Microscopy

Oilseed rape (Brassica napus L.) root colonization by B. vallismortis BL01 was visualized using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oilseed rape (Brassica napus L., cv. Avatar, NPZ, Holtsee, Germany) seeds were subjected to surface sterilization for 5 min in a 2% NaOCl solution and then washed six times with sterile distilled water. For seed treatment (bio-priming), B. vallismortis BL01 was grown on LB plates (Sigma Aldrich, St. Louis, MO, USA). The strain was grown for 72 h, and then cells were scraped from the plates and suspended in sterile distilled water. Bio-priming of seeds was performed as described in [41]. Briefly, seeds were immersed in the cell suspension for 4 h at 20 °C under agitation, and seeds incubated with sterile distilled water for 4 h served as a control. Infiltrated seeds were dried for 1 h at 20 °C. Primed seeds were placed in germination pouches (Mega International, Minneapolis, MN, USA): seven treated seeds with BL01 and seven not-treated seeds (control) were aseptically placed into one germination pouch that had been filled with 20 mL of sterile distilled water. The prepared pouches were placed in sterile plastic boxes for 14 days at 21 ± 2 °C under artificial lighting (with a 16 h light period). After 14 days of the experiment, the roots were separated from the plants and weighed. Roots of BL01-primed seedlings (14 d) were fixated with paraformaldehyde (PFA). FISH was carried out following the protocol from [42], using the FISH probes EUB338 I-III (Cy3-labelled) mix for universal bacterial staining and the LGC354 A-C (Cy5-labelled) mix for Firmicutes staining. CLSM was done on a Leica TCS SPE DM5500Q microscope (Leica microsystems, Wetzlar, Germany). Root samples were examined at three different regions: mature root with root hairs, elongation zone and root tip. Three-dimensional pictures were edited using Imaris 7.30 (Bitplane, Zürich, Switzerland).

2.3. Evaluation of the Inhibitory Effect of B. vallismortis BL01 on the Phytopathogenic Fungi

To test antifungal activity, strains of six different plant pathogenic fungal species were used, i.e., Diaporthe eres 18-001, Plenodomus lindquistii 19-007, Alternaria solani 747151, Fusarium oxysporum 70523, F. sporotrichioides 286093 and F. culmorum 46504. Strains of Alternaria solani 747151, Fusarium oxysporum 70523, F. sporotrichioides 286093, F. culmorum 46504 and F. culmorum 58800 isolated from the seeds of winter wheat grown in the Chechen Republic were kindly provided by Dr. T. Yu. Gagkaeva; Rhizoctonia solani and Sclerotinia sclerotiorum N14, isolated from the potato tubers grown in the Leningrad region, were kindly provided by Dr. Alexander V. Khiutti; and strains of Diaporthe eres 18-001 and Plenodomus lindquistii 19-007, isolated from the sunflower grown in the Voronezh region, were kindly provided by Dr. M.M. Gomzhina of the All-Russian Research Institute of Plant Protection (Saint-Petersburg, Russia). The antifungal activity of the strain BL01 Bacillus vallismortis was determined as described in [43]. Antagonistic strain BL01 and strains of different plant pathogenic fungi (Diaporthe eres 18-001, Plenodomus lindquistii 19-007, Alternaria solani 747151, Fusarium oxysporum 70523, F. sporotrichioides 286093 and F. culmorum 46504) were grown at 25 °C for 7 days on one Petri dish with potato dextrose agar (PDA, Sigma, Burlington, MA, USA). Briefly, BL01 was placed at a distance of 25 mm from the pathogen plug (D = 6 mm), which was placed on the center of the plate. Plates without strain BL01 were used as controls. The experiment was carried out in three independent replications. Antifungal activity was measured and recorded by using the following formula:
Antifungal activity (%) = [(Control group diameter − treatment group diameter)/Control group diameter] × 100%.
In another experiment, a phytopathogen plug was placed in one part of the Petri dish and a line with BL01 was placed on the opposite part to show the influence of distance on the fungal–bacterial interaction. The experiment was carried out in three independent replications.

2.4. Evaluation of the Inhibitory Effect of B. vallismortis BL01 on Phytopathogenic Bacteria

Antibacterial properties were tested on six strains of phytopathogenic bacteria: Erwinia carotovora 3304, Erwinia carotovora pv. atroseptica 822, Xanthomonas campestris pv. vesicatoria 7767, Pseudomonas syringae pv. tomato 8949, Pseudomonas syringae pv. atrofaciens P-88 and Pseudomonas syringae 213 (kindly provided by Dr. A.M. Lazarev, All-Russian Research Institute of Plant Protection, Saint-Petersburg, Russia) with the use of agar blocks [44] on 2% potato agar. Bacterial pathogens were spread on the surface of poor potato agar (2%) as a “lawn”. Agar blocks with the strain BL01 (D = 6 mm) were placed on the surface of poor agar with pathogens. Petri dishes without blocks of the strain BL01 served as a control. Petri dishes with agar blocks were cultivated for 3 days at a temperature of 28 °C, after which the presence or absence of phytopathogen lysis zones was recorded, and the diameter of the zones was measured with a ruler. The experiment was carried out in three independent replications.

2.5. Field Tests with Tomato Plants under Natural Infection with Pathogens

Field experiments to study the biocontrol activity of the strain BL01 B. vallismortis were conducted on tomato (Solanum lycopersicum L.) cv Novichok pink in LLC Nadezhda-2, Kamyzyakskyi district, Astrakhan region, Russian Federation, during 2015–2016. The soil of the experimental site was characterized as alluvial meadow loamy with a humus content of 2.2%, and the predecessor was barley. Tillage was carried out as follows: plowing to a depth of 22–25 cm; cultivation with harrowing; and milling of the soil. Fertilizers were not applied. The rate of planting seedlings was 57,000 plantlets per hectare. BL01 was cultured in liquid potato-dextrose broth (PDB, Sigma, Burlington, MA, USA) for 2 days at 28 °C on a rotary shaker at 200 rpm to obtain a final concentration of bacterial suspension of 1.35 × 108 cfu/mL. The treatment period for BL01 in 2015 was as follows: 13 May (soaking of seeds); 15 June (watering); 29 June, 13 and 23 July (spraying). The treatment dates for BL01 in 2016 were as follows: 15 May (soaking of seeds); 10 June (watering); 30 June, 12 and 25 July (spraying).
The experimental design was as follows:
  • Control (background without treatments)
  • B. vallismortis BL01-2 mL/kg of seeds (soaking) + 0.1% concentration (watering seedlings) + 3.0 L/ha (spraying three times)
  • Biological standard Phytolavin—a streptotricin antibiotic complex (BA-120,000 EA/mL, 32 g/L), water-soluble concentrate, 2.0 L/ha (watering seedlings) + 2.0 L/ha (spraying three times)
Spraying at 3 L/ha was performed with the 0.1% solution with a spraying rate of 400 L/ha. During the soaking at the rate of 2 mL/kg of seeds, the cell suspension was centrifuged at 10,000× g for 5 min at 4 °C, and the pellet was resuspended in 1.0 L of water. Soaking was performed for 20 min. Consumption of the working solution was as follows: 1.0 L of water (soaking seeds); 1000 L/ha (watering under the root); and 400 L/ha (spraying). Untreated plants, as well as plants treated with the biological standard Phytolavin (a streptotricin antibiotic complex), served as a control. In field experiments, we evaluated the incidence of tomatoes with black bacterial spotting (Xanthomonas campestris pv. vesicatoria (Doide) Dy) or late blight (Phytophthora infestans (Mont.) de Bary), as well as their yield. In field experiments, plant diseases depended on the natural infectious background. Therefore, due to the current phytosanitary situation, both in the first and the second year of the study, late blight on tomato plants was noted at the end of the growing season before the second harvest. By the time the fruits were ripe, black bacterial spots began to appear. The dates of appearance of diseases in 2015 were 22 August (black bacterial spotting) and 12 September (late blight); in 2016, the dates were 23 August (black bacterial spotting) and 3 September (late blight). The methodology of accounting for the incidence of tomatoes with black bacterial spotting (Xanthomonas campestris pv. vesicatoria (Doide) Dy) and late blight (Phytophthora infestans (Mont.) de Bary.) was carried out according to Dolzhenko [45]. Crop accounting was carried out manually from each accounting plot as the fruits ripened in 2015 on the following dates: 25 August and 5 and 16 September; and in 2016, the dates were 26 August and 6 and 16 September. Climate conditions in 2015–2016 are presented in Tables S1 and S2.

2.6. Genome Analysis of B. vallismortis BL01

Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP-6.1) [46]. A map of the B. vallismortis BL01 genome was constructed by Proksee [47]. AntiSMASH 6.1.1 [48] was used to predict the biosynthetic clusters in the B. vallismortis BL01 genome. OrthoVenn 2018 [49] was used to identify the orthologous proteins between B. vallismortis BL01, B. spizizenii str. W23 (GCA_000146565.1), B. amyloliquefaciens UMAF6639 (GCA_001593765.1), B. subtilis HJ5 (GCA_000973605.1) and B. velezensis FZB42 (GCA_000015785.2).

2.7. Statistical Analysis

Tukey’s test was used to compare the means of two or three groups, respectively. Significance was computed at p < 0.05. The data in the field tests were assessed by Fisher’s least significant difference (LSD) method (ANOVA).

3. Results

3.1. Colonization Ability of B. vallismortis BL01

The colonization ability of the strain BL01 B. vallismortis was studied in a gnotobiotic system using tomato and spring wheat plants in comparison with Bacillus subtilis Ch-13, the reference rhizospheric strain used in the formulation of a commercial biofungicide. The Bacillus subtilis Ch-13 rhizospheric strain was used as a reference strain [39]. Previously, in model biocontrol experiments on tomatoes, this strain induced a 2.5-fold reduction in plants suffering from the phytopathogenic fungus Fusarium oxysporum. In these experiments, strain Ch-13 colonized the tomato rhizosphere and, at the same time, inhibited fungal development, the amount of which on roots was 6.9-fold less in the case of bacterial inoculation compared to an infective background [39].
As a result of the conducted research, it was found that the strain BL01 B. vallismortis was able to actively colonize the rhizosphere and internal tissues of agricultural crops such as tomatoes and spring wheat. The number of BL01 colonies was comparable with the number of colonies of the reference strain Ch-13 Bacillus subtilis (Figure 1). Thus, the rhizospheric strain Ch-13 more actively colonized the rhizosphere of tomatoes—with 17.0 ± 1.5 × 106 CFU/g compared to strain BL01 with 14.3 ± 1.2 × 106 CFU/g—but the endophyte BL01 more actively colonized the endosphere of tomatoes, with 7.1 ± 0.5 × 106 CFU/root compared to strain Ch-13 with 5.6 ± 0.4 × 106 CFU/root (Figure 1a). During the colonization of the rhizosphere and endosphere of spring wheat plants, the endophyte BL01 demonstrated better colonization ability compared with the rhizospheric strain Ch-13, with 6.7 ± 0.5 × 106 CFU/g and 5.5 ± 0.3 × 106 CFU/root, respectively (Figure 1b).
The visualization of rapeseed colonization using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) showed a clear colonization pattern for the strain BL01 B. vallismortis (Figure 2a–c).
BL01 formed either small and scattered or big and elongated colonies that are typically stretched in the growth direction, mainly found on the surface of matured roots in the root hair zone. We also observed endophytic cells inside the root, as indicated by the arrows in Figure 2b,c. BL01 colonies (red) covered root and root hairs (green), as demonstrated in Figure 2a. These results indicate that strain BL01 effectively colonizes plant roots, which is important for efficient plant–bacteria interaction.

3.2. Antifungal Activity of B. vallismortis BL01

B. vallismortis BL01 showed inhibitory activity in vitro against phytopathogenic fungi Diaporthe eres 18-001, Alternaria solani 747151, Plenodomus lindquistii 19-007, Fusarium oxysporum 70523, F. sporotrichioides 286093 and F. culmorum 46504 (Figure 3 and Figure 4), which are the causal agents of rootstock death, early blight, fusarium ear blight, seedling blight and so on.
It was demonstrated in vitro that B. vallismortis BL01 showed a strong inhibitory effect against Fusarium culmorum 58800, Rhizoctonia solani and Sclerotinia sclerotiorum N149 in co-cultivation in one Petri dish when they were placed near each other (Figure 5).

3.3. Antibacterial Activity of B. vallismortis BL01

B. vallismortis BL01 demonstrated inhibitory activity in vitro against a wide range of phytopathogenic bacteria, causing such diseases as bacterial stem rot, soft rot, bacterial leaf spot on peppers and tomatoes, fruit spot and foliage blight in tomatoes (Figure 6).
For instance, the lysis zones of strain BL01 B. vallismortis varied from 7.0 ± 0.5 mm on strain P-88 Pseudomonas syringae pv. atrofaciens to 13.1 ± 0.9 mm on strain 3304 Erwinia carotovora.

3.4. Field Tests with Tomato Plants and B. vallismortis BL01

Due to the fact that strain BL01 showed high antifungal and antibacterial activity in the laboratory, we tested its properties in two-year field experiments on tomato plants. The assessment of diseases was carried out as their symptoms manifested on plants (Figure 7 and Figure 8, Tables S3–S6).
In the first year of the study, the biological efficacy of B. vallismortis BL01 against black bacterial spotting in the 102 days after sowing (das) compared to the control was 69.0%. Later, during the fruit ripening period, the above-mentioned situation persisted and amounted to 66.7–46.4% in the variant with BL01 compared to 61.2–43.2% in the Phytolavin standard, with the development of the disease in the control being 8.4–19.2% (Figure 7, Table S3).
It was not possible to objectively assess the effectiveness of the tested strain BL01 against late blight due to the late appearance of the disease (before the last harvest) due to the prevailing weather conditions. However, against this background, the biological efficiency of B. vallismortis BL01 was 41.8%, which exceeded the efficiency of the standard Phytolavin, which was 34.3% (Figure 7, Table S5).
The tomato yield with B. vallismortis BL01 increased by 22.1% or 9.7 tons per hectare compared to the control, which was higher than that with the Phytolavin standard, for which the yield increased by 17.2% or 7.5 t/ha (Table S7). Taking into account that spraying by B. vallismortis BL01 at 3 L/ha was done using a 0.1% solution, but the control was treated with water, part of the activity may be due to the culture medium.
In the second year of the field tests, the biological efficacy of B. vallismortis BL01 against black bacterial spotting in the 102 days after sowing compared to the control was 72.6%, and the efficacy of Phytolavin was 56.2%. Later, during the second and third harvest, the biological efficacy of B. vallismortis BL01 was 68.8 and 33.5%, respectively, and also exceeded the Phytolavin standard, the effectiveness of which was 54.1 and 24.1%, respectively, with the development of the disease in the control being 10.9–17.0% (Table S4). Against late blight, the biological efficacy of B. vallismortis BL01 was 52.3% and exceeded the efficacy of Phytolavin, which was 43.8%, with the development of the disease in the control being 17.6% (Figure 8, Table S6). However, the difference between both products was not significant. Tomato harvest when using B. vallismortis BL01 increased by 27.7% or 12.1 tons per hectare compared to the control, which was higher than that with the Phytolavin standard, where the yield increased by 21.0% or 9.2 t/ha (Table S7). However, the difference between both products was not statistically significant. Thus, on average, over two years of field tests, B. vallismortis BL01 showed biological efficacy against black bacterial spotting on day 32 after the last treatment of 70.8%, during fruit ripening of 67.8 and 40%, and against late blight of 47.1% compared with 39.1% for the Phytolavin standard. The tomato harvest when using B. vallismortis BL01 significantly increased by 24.9% or 10.9 tons per hectare on average over the two years of field tests compared to the control (Table S7).

3.5. Genome Features and Phylogeny of B. vallismortis BL01

The genome of B. vallismortis BL01 was previously sequenced using Illumina paired sequencing technology and collected by SPAdes [38]. The complete genome of BL01 (Accession no. CP092751) contained one chromosome with a size of 4,115,091 bp. The functional annotation revealed 3900 protein-coding genes, 94 RNA genes and 129 pseudogenes (Table 1, Figure 9).

3.6. Secondary Metabolic Potential of Strain BL01

Eight putative gene clusters that function to produce bioactive secondary metabolites were identified, including genes for the biosynthesis of non-ribosomal lipopeptides, polyketides and ribosomally synthesized and post-translationally modified peptides (Table 2). These gene clusters are involved in synthesizing sporulation killing factor, surfactin, macrolactin H, bacillaene, fengycin, bacillibactin, subtilosin A and bacilysin. Altogether, more than 500 kb of the B. vallismortis BL01 genome is involved in the synthesis of antimicrobial molecules.
According to the analysis using OrthoVenn, it was found that there are 2919 clusters shared among the Bacillus species included in this study (Figure 10). B. vallismortis BL01 showed four unique clusters involved in the phosphorelay signal transduction system (GO:0000160), phosphoprotein phosphatase activity (GO:0004721) and the antibiotic biosynthetic process (GO:0017000) (Figure 10).

4. Discussion

Bacteria from the genus Bacillus are the most well-known candidates for the development of bioinoculants to combat biotic and abiotic stresses, as well as to stimulate plant growth due to their ability to form spores [50]. Currently, most commercial biofertilizers contain different strains of bacilli, such as Alinit (Bacillus subtilis), Kodiak (Bacillus subtilis GB03), Quantum-400 (B. subtilis GB03), Rhizovital (B. amyloliquefaciens FZB42), Serenade (Bacillus subtilis QST 713) and YIB (Bacillus sp.), and are focused on the biocontrol of phytopathogenic fungi and the stimulation of plant growth [51]. However, most of them are aimed at the biocontrol of phytopathogenic fungi but not bacteria. Therefore, the development of bioinoculants that are able to control both phytopathogenic fungi and bacteria seems to be a very promising scientific direction.
We isolated the endophytic strain BL01 Bacillus vallismortis from the roots of the drought-resistant plant Artemisia lerchiana Web. from the Astrakhan region, Russia. B. vallismortis is one of the Bacillus species that has not been extensively studied since it was first identified in 1996 [52]. However, since 1996, the isolation of endophytic Bacillus vallismortis was reported from various plants. Thus, Bacillus vallismortis was isolated from healthy stems of the Ilex latifolia Thunb [30], Kobreasia capillifolia in China [53], Citrus sinensis L. leaves in China [54], Carica papaya stem in the USA [55], stems of medicinal herb Coleus forskohlii [56] and the rhizosphere of Juniperus sabina L. from Spain [31]. Khan et al. [57] reported on the isolation of Bacillus vallismortis as an endophytic contaminant from in vitro cultures of the medicinally important plant Fagonia indica. So, it can be concluded that strains of Bacillus vallismortis are ubiquitous inhabitants in the plant endosphere and rhizosphere.
The attachment or adhesion of bacterial cells to the plant surface is considered the first and most important step of the colonization process. Bacteria in the rhizosphere can move towards the roots by using chemotactic affinities for root exudates. This is followed by attachment to the root surface, which is likely important in getting access to potential entry sites at lateral root emergence areas or other openings caused by wounds or mechanical injuries [58]. Endophytes penetrate into plants mainly through roots but also through leaves, flowers and stems [16]. Indeed, the vast majority of endophytes are soil inhabitants, and plant colonization seems to occur mainly from the rhizosphere. Evidence supporting this possibility has been obtained using microbiological and microscopic research methods. Thus, it has been shown that some endophytes systematically spread from the initial site of penetration and are found in distant tissues and organs of plants [59]. Consequently, the population density of endophytes is usually higher in the roots than in any other plant organ. That is why it is important to know the colonization ability of the isolated endophytes. We demonstrated in a gnotobiotic system and using confocal laser microscopy that the endophytic strain BL01 B. vallismortis was able to colonize the roots and rhizosphere of tomato, winter wheat and oilseed rape. Our results were supported by other researchers. Thus, it was reported that strain EXTN-1 Bacillus vallismortis (a commercial biocontrol agent, EXTN, Dongbu HiTech Co., Seoul, Korea) was able to effectively colonize pepper roots one and seven days after treatment at 4.40–4.51 log cfu/cm root [60]. It was also reported that endophytic bacilli were able to effectively colonize the tomato roots, stems and leaves at 3.5–6.8 log CFU/g fresh weight fifteen days after inoculation [61], which coincides with our results.
We demonstrated in in vitro experiments the inhibitory activity of B. vallismortis BL01 against phytopathogenic fungi such as Diaporthe eres, Alternaria solani, Plenodomus lindquistii, Fusarium oxysporum, F. sporotrichioides and F. culmorum, which are the causal agents of rootstock death, early blight, fusarium ear blight, seedling blight and so on. The same effect has been reported for other strains of B. vallismortis. Thus, endophyte B. vallismortis 263XY1 isolated from Kobreasia capillifolia grown in alpine grasslands on the Tibetan Plateau, China, demonstrated the best in vitro inhibition activity among 42 tested endophytes against three phytopathogenic fungi: Fusarium avenaceum, Colletotrichum coccodes and Phoma foveata [53]. Endophytic B. vallismortis Ps isolated from the papaya stem in the USA demonstrated strong inhibition activity in vitro against Phytophthora capsici, Fusarium solani, Peyronellaea pinodella, Macrophomina phaseolina and Glomerella guttata [55,62]. Some strains of B. vallismortis have been proven to have strong antagonistic activity against the anthracnose disease of Cymbidium sp. caused by the pathogen Colletotrichum gloeosprioides and cotton wilt caused by Verticillium dahlia [63]. The strain R2 B. vallismortis supported more than 50% inhibition of different phytopathogenic fungi (Alternaria alternata, Rhizoctonia oryzae, Fusarium oxysporum, Fusarium moniliforme, Colletotrichum sp., Helminthosporium sp. and Magnaporthe grisea) in in vitro experiments [64]. Strong growth inhibition activity in vitro against phytopathogenic fungi, such as Fusarium graminearum, Alternaria alternata, Rhizoctonia solani, Cryphonectria parasitica and Phytophthora capsica, has been observed for the strain ZZ185 B. vallismortis [65]. The antagonistic effect of B. vallismortis against a number of fungal pathogens was reported, including Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani, F. graminearum, Rhizoctonia solani, Athelia rolfsii and Thanatephorus cucumeris [63,66].
We also demonstrated in in vitro experiments the inhibition activity of B. vallismortis BL01 against a wide range of phytopathogenic bacteria. Usually, the biocontrol effect of endophytic bacteria is focused on phytopathogenic fungi, and there were only a few reports of their effect on phytopathogenic bacteria. However, the inhibition effect of endophytic Bacillus vallismortis CFM3 isolated from the stems of medicinal herb Coleus forskohlii and cultivated in the experimental fields of CSIR-IIIM, Jammu, India, was reported against pathogenic bacteria such as Escherichia coli (MTCC 730), Klebsiella pneumoniae (ATCC 75388) and Staphylococcus aureus [56]. Disease resistance in Arabidopsis thaliana L. was reported against Pseudomonas syringae infection by the strain BS07 Bacillus vallismortis due to the production of cyclic dipeptides [67].
Through in vitro experiments, we demonstrated the inhibitory activity of the strain BL01 B. vallismortis to control black bacterial spotting (Xanthomonas campestris pv. vesicatoria (Doide) Dy) and late blight (Phytophthora infestans (Mont.) de Bary.) in two-year field experiments with tomato plants. Thus, on average, over two years of research, B. vallismortis BL01 showed biological efficacy against black bacterial spotting on day 32 after the last treatment of 70.8%, during fruit ripening of 67.8 and 40%, and against late blight of 47.1% compared with 39.1% for the Phytolavin standard. Tomato harvest when using B. vallismortis BL01 increased by 24.9% or 10.9 tons per hectare on average over the two years of research compared to the control. Taking into account that spraying by B. vallismortis BL01 at 3 L/ha was performed using a 0.1% solution, but the control was treated with water, part of the activity may be due to the culture medium. A similar effect of B. vallismortis was observed by other researchers. For instance, some strains of B. vallismortis provided protection against bacterial speck and bacterial spot of tomato [36,37]. Moreover, the strain BS07 B. vallismortis had the same effect on the bacterial phytopathogen Pectobacterium carotovorum SCC1, which causes soft rot infection of chili peppers, as the chemical preparation benzothiadiazole (BTH) [34]. In the field experiment, the chili pepper plants treated with BS07 and BTH significantly reduced the percent disease incidence of soft rot to 20 and 16.6%, respectively, compared with 71.6% in the untreated control.
Some strains of B. vallismortis have been proven to have strong antagonistic activity against the anthracnose disease of Cymbidium sp. caused by Colletotrichum gloeosprioides and cotton wilt caused by Verticillium dahlia [68]. In our study, we demonstrated a strong effect of B. vallismortis BL01 against Phytophthora infestans, which causes tomato late blight.
The cyclic lipopeptides (CLPs), namely, iturins, fengycins and surfactins, belong to the most important bioactive substances produced by Bacillus spp., and they exhibit excellent properties, such as broad-spectrum antibiotic activity, good stability, low toxicity and high biodegradability [69,70,71,72]. Among them, the iturins exhibit strong antifungal activity but have limited antibacterial activity; the fengycins have strong antifungal activity; and the surfactins demonstrate bactericidal activity against Bacillus spp. strains, providing protection against various phytopathogens for both bacteria and fungi [73,74]. For instance, in in vitro experiments, surfactin produced by B. velezensis inhibited the growth of bacterial pathogens Xanthomonas campestris and X. vesicatoria [75]. Surfactins are able to solubilize the phospholipid bilayer and create pores and ionic channels, causing cell death [65,76]. Fengycins produced by B. amyloliquefaciens cause alterations in inhibition in in vitro Xanthomonas axonopodis pv. vesicatoria cell topography, which results in cell death by intracellular content filtration [74].
An analysis of the B. vallismortis BL01 genome revealed eight putative gene clusters that function to produce bioactive secondary metabolites, including gene clusters involved in synthesizing sporulation killing factor, surfactin, macrolactin H, bacillaene, fengycin, bacillibactin, subtilosin A and bacilysin. Some researchers have isolated antifungal compounds as a mixture of bacillomycin D (n-C14) and bacillomycin D (iso-C15) from the n-butanol extract of strain B. vallismortis ZZ185 [30]. Other researchers revealed the presence of surfactin, iturin A and fengycin-like compounds in acid-precipitated biomolecules [64]. Biosurfactants produced by strain TU–Orga21 B. vallismortis were identified as surfactin and iturin by mass spectrometry [77].
These results demonstrated that strains of B. vallismortis have great potential for studying their ability to produce antifungal and antibacterial compounds.
For future research on this strain, we suggest performing a chemical analysis of extracts of B. vallismortis BL01 to check secondary metabolites. So, assuming these data are obtained, we can expect that strain B. vallismortis BL01 has the ability to produce antifungal and antimicrobial substances, such as surfactin, macrolactin H, bacillaene, fengycin, bacillibactin, subtilosin A and bacilysin, as was demonstrated by the analysis of the B. vallismortis BL01 genome. However, this should be demonstrated by mass spectrometry analysis in future research.

5. Conclusions

The endophytic strain BL01 Bacillus vallismortis, isolated from the roots of the drought-resistant plant Artemisia lerchiana Web. from the Astrakhan region, Russia, has been studied. We demonstrated that strain BL01 B. vallismortis was able to colonize the endosphere and rhizosphere of various agricultural crops, such as tomatoes, spring wheat and oilseed rape, at the rate of 5.5–7.1 × 106 CFU/root. Cells of BL01 were mainly found on the surface of matured oilseed rape roots and in the root hair zone, and endophytic cells were found as well. The inhibition activity of BL01 against a wide range of phytopathogenic fungi and bacteria was demonstrated in in vitro experiments. The ability of the strain BL01 to control black bacterial spotting and late blight was demonstrated in two-year field experiments with tomato plants. Strain BL01 showed biological efficacy against black bacterial spotting of 40–70.8% and against late blight of 47.1%, and it increased tomato harvest by 24.9% or 10.9 tons per hectare compared to the control. Analysis of the B. vallismortis BL01 genome revealed gene clusters that function to produce bioactive secondary metabolites, which can be responsible for antifungal and antimicrobial activity. Strain BL01 Bacillus vallismortis can be considered a potential biocontrol agent to control both fungal and bacterial diseases in tomato plants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10101095/s1, Table S1: Meteorological data for the 2015 growing season (according to the Kamyzyaksky weather station of the Kamyzyaksky district of the Astrakhan region), Table S2: Meteorological data for the 2016 growing season (according to the Kamyzyaksky weather station of the Kamyzyaksky district of the Astrakhan region), Table S3: Bactericidal activity of B. vallismortis BL01 against black bacterial spotting in 2015, Table S4: Bactericidal activity of B. vallismortis BL01 against black bacterial spotting in 2016, Table S5: The effect of B. vallismortis BL01 on late blight disease in 2015, Table S6: The effect of B. vallismortis BL01 on late blight disease in 2016, Table S7: The effect of B. vallismortis BL01 on tomato yield.

Author Contributions

Conceptualization, V.K.C., I.A.T. and M.S.G.; methodology, E.P.C., A.V.E., A.M.L., X.Z. and M.S.G.; software E.P.C. and M.S.G.; validation, V.K.C., A.V.K., J.X. and M.S.G.; formal analysis, C.Y., A.V.K. and A.M.L.; investigation, E.P.C., A.V.K. and A.V.E.; resources, V.K.C., A.M.L. and A.V.K.; data curation, E.P.C., C.Y. and M.S.G.; writing—original draft preparation, V.K.C. and M.S.G.; writing—review and editing, V.K.C., E.P.C., X.Z. and M.S.G.; visualization, E.P.C. and M.S.G.; supervision, V.K.C. and I.A.T.; project administration, V.K.C.; funding acquisition, I.A.T., V.K.C. and M.S.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Russian Science Foundation project No 23-66-10013.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

The research was performed using equipment of the Core Centrum “Genomic Technologies, Proteomics and Cell Biology” in ARRIAM. We are thankful to T. Yu. Gagkaeva (All-Russian Research Institute of Plant Protection, Saint-Petersburg, Russia) for providing us strains of Alternaria solani 747151, Fusarium oxysporum 70523, F. sporotrichioides 286093, F. culmorum 46504 and F. culmorum 58800 isolated from the seeds of winter wheat grown in the Chechen Republic, M. M. Gomzhina (All-Russian Research Institute of Plant Protection, Saint-Petersburg, Russia) for providing us strains of Diaporthe eres 18-001 and Plenodomus lindquistii 19-007, isolated from the sunflower grown in the Voronezh region, A. V. Khiutti (All-Russian Research Institute of Plant Protection, Saint-Petersburg, Russia) for providing us strains of Rhizoctonia solani and Sclerotinia sclerotiorum N14, isolated from the potato tubers grown in the Leningrad region, and A. M. Lazarev (All-Russian Research Institute of Plant Protection, Saint-Petersburg, Russia) for providing us strains of Erwinia carotovora 3304, Erwinia carotovora pv. atroseptica 822, Xanthomonas campestris pv. vesicatoria 7767, Pseudomonas syringae pv. tomato 8949, Pseudomonas syringae pv. atrofaciens P-88 and Pseudomonas syringae 213.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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Figure 1. The number of bacilli in the rhizosphere and endosphere of tomatoes (106 CFU per 1 root of the tomato plant) (a) and spring wheat (106 CFU per 1 root of the wheat plant) (b). Bars represent the mean ± SD of three replications.
Figure 1. The number of bacilli in the rhizosphere and endosphere of tomatoes (106 CFU per 1 root of the tomato plant) (a) and spring wheat (106 CFU per 1 root of the wheat plant) (b). Bars represent the mean ± SD of three replications.
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Figure 2. (a) IMARIS-edited CLSM picture of BL01 B. vallismortis-primed oilseed rape, old root parts. Rendered root parts (green), Firmicutes (red). (b,c) IMARIS-edited CLSM pictures of matured roots in the root hair zone treated with BL01 B. vallismortis. The view is from various directions, and the arrow points to endophytic cells of BL01 B. vallismortis.
Figure 2. (a) IMARIS-edited CLSM picture of BL01 B. vallismortis-primed oilseed rape, old root parts. Rendered root parts (green), Firmicutes (red). (b,c) IMARIS-edited CLSM pictures of matured roots in the root hair zone treated with BL01 B. vallismortis. The view is from various directions, and the arrow points to endophytic cells of BL01 B. vallismortis.
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Figure 3. Inhibitory effects of B. vallismortis BL01 on Diaporthe eres 18-001 (a), Alternaria solani 747151 (b), Plenodomus lindquistii 19-007 (c), Fusarium oxysporum 70523 (d), F. sporotrichioides 86093 (e) and F. culmorum 46504 (f).
Figure 3. Inhibitory effects of B. vallismortis BL01 on Diaporthe eres 18-001 (a), Alternaria solani 747151 (b), Plenodomus lindquistii 19-007 (c), Fusarium oxysporum 70523 (d), F. sporotrichioides 86093 (e) and F. culmorum 46504 (f).
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Figure 4. Antifungal activity (%) of B. vallismortis BL01 against phytopathogenic fungi Diaporthe eres 18-001, Alternaria solani 747151, Plenodomus lindquistii 19-007, Fusarium oxysporum 70523, F. sporotrichioides 286093 and F. culmorum 46504. Bars represent the mean ± SD of three replications.
Figure 4. Antifungal activity (%) of B. vallismortis BL01 against phytopathogenic fungi Diaporthe eres 18-001, Alternaria solani 747151, Plenodomus lindquistii 19-007, Fusarium oxysporum 70523, F. sporotrichioides 286093 and F. culmorum 46504. Bars represent the mean ± SD of three replications.
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Figure 5. Inhibitory effects of B. vallismortis BL01 on Fusarium culmorum 58800, Rhizoctonia solani and Sclerotinia sclerotiorum N149 in co-cultivation in one Petri dish.
Figure 5. Inhibitory effects of B. vallismortis BL01 on Fusarium culmorum 58800, Rhizoctonia solani and Sclerotinia sclerotiorum N149 in co-cultivation in one Petri dish.
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Figure 6. Antibacterial activity (lysis zones, mm) of strain BL01 B. vallismortis against phytopathogenic bacteria: Erwinia carotovora 3304, Erwinia carotovora pv. atroseptica 822, Xanthomonas campestris pv. vesicatoria 7767, Pseudomonas syringae pv. tomato 8949, Pseudomonas syringae pv. atrofaciens P-88, Pseudomonas syringae 213. Bars represent the mean ± SD of three replications.
Figure 6. Antibacterial activity (lysis zones, mm) of strain BL01 B. vallismortis against phytopathogenic bacteria: Erwinia carotovora 3304, Erwinia carotovora pv. atroseptica 822, Xanthomonas campestris pv. vesicatoria 7767, Pseudomonas syringae pv. tomato 8949, Pseudomonas syringae pv. atrofaciens P-88, Pseudomonas syringae 213. Bars represent the mean ± SD of three replications.
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Figure 7. Bactericidal activity of B. vallismortis BL01 against black bacterial spotting. Box plots were drawn using the R boxplot() function. The box of a boxplot starts in the first quartile (25%) and ends in the third (75%). Hence, the box represents 50% of the central data, with a line inside that representing the median. Standard—Phytolavin (streptotricin antibiotic complex). ***, p-value < 0.001; das—days after sowing.
Figure 7. Bactericidal activity of B. vallismortis BL01 against black bacterial spotting. Box plots were drawn using the R boxplot() function. The box of a boxplot starts in the first quartile (25%) and ends in the third (75%). Hence, the box represents 50% of the central data, with a line inside that representing the median. Standard—Phytolavin (streptotricin antibiotic complex). ***, p-value < 0.001; das—days after sowing.
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Figure 8. The effect of B. vallismortis BL01 on late blight disease (a) and yield (b) of tomato compared with control and standard Phytolavin (streptotricin antibiotic complex). Box plots were drawn using the R boxplot () function. The box of a boxplot starts in the first quartile (25%) and ends in the third (75%). Hence, the box represents 50% of the central data, with a line inside that representing the median. ***, p-value < 0.001.
Figure 8. The effect of B. vallismortis BL01 on late blight disease (a) and yield (b) of tomato compared with control and standard Phytolavin (streptotricin antibiotic complex). Box plots were drawn using the R boxplot () function. The box of a boxplot starts in the first quartile (25%) and ends in the third (75%). Hence, the box represents 50% of the central data, with a line inside that representing the median. ***, p-value < 0.001.
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Figure 9. Map of the B. vallismortis BL01 genome constructed by Proksee (https://proksee.ca/, accessed on 28 August 2024).
Figure 9. Map of the B. vallismortis BL01 genome constructed by Proksee (https://proksee.ca/, accessed on 28 August 2024).
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Figure 10. Protein comparison among B. vallismortis BL01 and related species (B. spizizenii str. W23, B. amyloliquefaciens UMAF6639, B. subtilis HJ5 and B. velezensis FZB42). The Venn diagram illustrates the overlap and unique clusters among Bacillus species. The bar plot displays the total cluster content for each Bacillus species analyzed.
Figure 10. Protein comparison among B. vallismortis BL01 and related species (B. spizizenii str. W23, B. amyloliquefaciens UMAF6639, B. subtilis HJ5 and B. velezensis FZB42). The Venn diagram illustrates the overlap and unique clusters among Bacillus species. The bar plot displays the total cluster content for each Bacillus species analyzed.
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Table 1. Genetic features of B. vallismortis BL01.
Table 1. Genetic features of B. vallismortis BL01.
Accession NumberCP092751
Assembly methodSPAdes v. 3.14.1
Genome coverage43.0×
Sequencing technologyIllumina HiSeq
Total sequence length4,115,091
Annotation pipelineNCBI Prokaryotic Genome Annotation Pipeline (PGAP)
Annotation methodBest-placed reference protein set; GeneMarkS-2+
Annotation software revision6.1
Genes (total)4123
CDSs (total)4029
Genes (coding)3900
Pseudo genes129
Genes (RNA)94
rRNAs9
tRNAs80
ncRNAs5
Table 2. List of the putative gene clusters encoding for secondary metabolites in the BL01 genome. NRP—non-ribosomally produced peptide; NRPS—NRP synthetase cluster; PKS—polyketide synthase; T1PKS—Type I PKS; T3PKS—Type III PKS; CDPS—tRNA-dependent cyclodipeptide synthases; RiPP—ribosomally synthesized and post-translationally modified peptide.
Table 2. List of the putative gene clusters encoding for secondary metabolites in the BL01 genome. NRP—non-ribosomally produced peptide; NRPS—NRP synthetase cluster; PKS—polyketide synthase; T1PKS—Type I PKS; T3PKS—Type III PKS; CDPS—tRNA-dependent cyclodipeptide synthases; RiPP—ribosomally synthesized and post-translationally modified peptide.
RegionTypeGenome LocationsMost Similar Known ClusterSimilarity with
the antiSMASH Database
1Ranthipeptide-
sactipeptide		167692-189797sporulation killing factorRiPP:Head-to-tail cyclized peptide100%
2NRPS321941-386583surfactinNRP:Lipopeptide82%
3NRPS, T1PKS640163-720769zwittermicin ANRP + polyketide18%
4transAT-PKS1501848-1591234macrolactin HPolyketide100%
5transAT-PKS, PKS-
like NRPS		1816614-1921458bacillaenePolyketide + NRP100%
6NRPS, transAT-
PKS, betalactone		1982463-2109472fengycinNRP100%
7terpene2171729-2193627
8T3PKS2240506-2281603
9NRPS3123215-3170351bacillibactinNRP100%
10CDPS3460672-3481418
11sactipeptide3703716-3725326subtilosin ARiPP:Thiopeptide100%
12other3728594-3770012bacilysinOther100%
13epipeptide3982227-4003928thailanstatin ANRP + polyketide10%
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Chebotar, V.K.; Gancheva, M.S.; Chizhevskaya, E.P.; Erofeeva, A.V.; Khiutti, A.V.; Lazarev, A.M.; Zhang, X.; Xue, J.; Yang, C.; Tikhonovich, I.A. Endophyte Bacillus vallismortis BL01 to Control Fungal and Bacterial Phytopathogens of Tomato (Solanum lycopersicum L.) Plants. Horticulturae 2024, 10, 1095. https://doi.org/10.3390/horticulturae10101095

AMA Style

Chebotar VK, Gancheva MS, Chizhevskaya EP, Erofeeva AV, Khiutti AV, Lazarev AM, Zhang X, Xue J, Yang C, Tikhonovich IA. Endophyte Bacillus vallismortis BL01 to Control Fungal and Bacterial Phytopathogens of Tomato (Solanum lycopersicum L.) Plants. Horticulturae. 2024; 10(10):1095. https://doi.org/10.3390/horticulturae10101095

Chicago/Turabian Style

Chebotar, Vladimir K., Maria S. Gancheva, Elena P. Chizhevskaya, Anastasia V. Erofeeva, Alexander V. Khiutti, Alexander M. Lazarev, Xiuhai Zhang, Jing Xue, Chunhong Yang, and Igor A. Tikhonovich. 2024. "Endophyte Bacillus vallismortis BL01 to Control Fungal and Bacterial Phytopathogens of Tomato (Solanum lycopersicum L.) Plants" Horticulturae 10, no. 10: 1095. https://doi.org/10.3390/horticulturae10101095

APA Style

Chebotar, V. K., Gancheva, M. S., Chizhevskaya, E. P., Erofeeva, A. V., Khiutti, A. V., Lazarev, A. M., Zhang, X., Xue, J., Yang, C., & Tikhonovich, I. A. (2024). Endophyte Bacillus vallismortis BL01 to Control Fungal and Bacterial Phytopathogens of Tomato (Solanum lycopersicum L.) Plants. Horticulturae, 10(10), 1095. https://doi.org/10.3390/horticulturae10101095

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