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Article

Analysis of the Biological Activity and Whole Genome Sequencing of Bacillus cereus CDHWZ7 Isolated from the Rhizosphere of Lycium ruthenicum on the Tibetan Plateau

by
Xue Yang
1,2,
Yongli Xie
1,2,3,*,
Youming Qiao
3,
Lan Chen
1,
Tian Wang
1,
Lingling Wu
1,
Junxi Li
1 and
Ying Gao
1
1
College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China
2
Key Laboratory of Use of Forage Germplasm Resources on Tibetan Plateau of Qinghai Province, Xining 810016, China
3
State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(5), 1041; https://doi.org/10.3390/agriculture13051041
Submission received: 13 April 2023 / Revised: 5 May 2023 / Accepted: 8 May 2023 / Published: 11 May 2023
(This article belongs to the Special Issue Beneficial Microorganisms and Crop Production)
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Abstract

:
This study examined the biological activity and genome of Bacillus cereus CDHWZ7 isolated from the root of Lycium ruthenicum in the Dachaidan saline area, Haixi Prefecture, Qinghai Province, China. The results revealed that B. cereus CDHWZ7 exhibited strong inhibition activity against the pathogenic fungi Fusarium graminearum, F. acuminatum, and F. oxysporum. CDHWZ7 also demonstrated cellulose-degrading activity, nitrogen-fixing activity, and the ability to secrete indole-3-acetic acid (IAA) at 55.00 mg∙L−1. The strain CDHWZ7 can grow at a salt concentration of 3–11%, a pH range of 5–11, and a temperature of 4 °C–18 °C, and shows good salt tolerance, acid and alkaline tolerance, and low-temperature fitness. The genome of strain CDHWZ7 was sequenced using Illumina HiSeq + PacBio, revealing a circular structure of 5,648,783 bp in length, containing two intact plasmids with an average GC content of 35.2%, and a total number of 5672 encoded genes. It contained 106 tRNA genes, 42 rRNA genes, and 134 sRNA genes. A total of 137 genes were annotated as carbohydrases, with a total base length of 3,968,396,297 bp. The numbers of coding sequences assigned to the Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of Proteins, and Gene Ontology Databases were 4038, 4133, and 2160, respectively. Further analysis of the genome identified genes encoding chitinase activity, cellulases, secondary metabolites, phytohormone production, volatile compounds, nitrogen and phosphate metabolism, and resistance responses to biotic stresses (glycine betaine transporter protein, catalase, superoxide dismutase, low-affinity potassium transporter protein, cold-shock protein, heat-shock protein), as well as genes related to proliferation, stress response, and resistance to pathogenic fungi. Therefore, this study determined that strain CDHWZ7 has several excellent biological traits, such as antagonism to pathogenic fungi, nitrogen-fixation ability, cellulose-degradation ability, and IAA-production ability. The genome sequence of strain CDHWZ7 and several biodefense functional genes were also analyzed, revealing the potential use of strain CDHWZ7 in the development of biological agents.

1. Introduction

Haixi Tibetan Autonomous Prefecture is located in the northern part of the Qinghai–Tibetan Plateau, China, which has a complex topography that encompasses several geomorphic environments, such as high mountains, hills, plains, glaciers, and wetlands. Its unique climate type, with low average annual precipitation, large temperature differences, and high UV radiation makes the region extremely dry [1,2], and these extreme environments are also responsible for the rich microbial resources of the region [3]. Among them, populations of the genus Bacillus have super-resistant budding cells that are able to adapt to unfavorable environments, such as cold, high light radiation, and high salinity. Beneficial Bacillus also have good inter-root colonization, bacteriostatic activity, and growth-promoting ability and are environmentally safe and non-pathogenic [4,5]. Therefore, the use of Bacillus in place of chemical fertilizers in agricultural production meets the requirements for sustainable agricultural development. The beneficial effects of Bacillus on plant growth and yield have been demonstrated in several agricultural crops, including wheat, tomato, soybean, potato, oat, and many others [6].
The inhibition of pathogen growth by Bacillus involves mechanisms, such as competition for nutrients and space, production of antibiotics and hydrolytic enzymes, iron carriers, the induction of systemic resistance, and the production of lipopeptides, organic acids, and volatile organic compounds (VOCs) during metabolism [7,8]. Bacillus subtilis C3 has been reported to disrupt the mycelial structure of the pathogenic fungus Fusarium oxysporum, causing the rupture of protoplasts, chromatin condensation, DNA breakage, and ultimately, cell death [9]. In addition, Bacillus is able to colonize plant roots with a dense biofilm, and, through its metabolism, produce indole-3-acetic acid (IAA) to promote cell division, stem elongation, and increase the surface area of the root system, thereby helping the plant to better absorb water and nutrients, thus establishing a good mutualistic relationship with the plant [10]. Bacillus aryabhattai SRB02 has been reported to produce large amounts of IAA in medium and to activate the IAA signal transduction pathway in soybean plants, increasing the content of the endogenous hormone IAA in soybean plants and encouraging root and shoot growth [11]. Bacillus amyloliquefaciens FZB42 has been shown to secrete growth hormones, which not only promotes plant-root growth but also reshapes the expression of the strain’s Pi transporter protein and enhances organic carbon secretion [12].
Nitrogen and phosphorus elements play important roles in plant productivity and ecosystem stability. Nitrogen is essential to the synthesis of vitamins, proteins, and phospholipids in plants, and can regulate plant physiological metabolism and delay ageing, while phosphorus is an important component for the synthesis of nucleic acids, phosphorus-containing organic matter, and energy substances in plants, and can also improve plant resistance [13,14]. In plant production, nitrogen and phosphorus availability is managed through the application of a large number of chemical fertilizers, which often causes environmental pollution. By contrast, Bacillus, as a novel biofertilizer, is able to convert nitrogen and insoluble phosphorus into ammonia-containing complexes and soluble phosphorylates for direct plant uptake and use, thus increasing plant production [15,16]. One study reported that the inoculation of Cicer arietinum with rhizobia, nitrogen-fixing B. subtilis, and phosphate-solubilizing Bacillus megaterium in a large field in the cold highlands of Erzurum, Turkey, significantly promoted C. arietinum growth, and the combination of the three fungicides had the potential to replace nitrogen and phosphorus fertilizers in C. arietinum production [17].
With continuous improvements in high-throughput sequencing, microbial genomics now provides a new theoretical basis for the application of microorganisms in agriculture, animal husbandry, the food industry, and health through the assembly of complete nucleotide sequences to further understand the structure, function, and regulatory network of their genomes [18]. The objectives of this study were to analyze the biological activity of Bacillus isolated from extreme habitats, which was sequenced using second generation + third generation sequencing, i.e., Illumina HiSeq + PacBio. A functional annotation of its coding genes was carried out. Key genes related to antagonism against pathogenic fungi, plant growth promotion, and resistance of the strain were mined to provide a theoretical basis for revealing the mechanism of growth promotion and antibacterial activity of strain CDHWZ7. Overall, our study demonstrated the effectiveness of CDHWZ7 in growth-promoting activity and antagonism against different pathogens, thereby supporting further research and development.

2. Materials and Methods

2.1. Experimental Materials

The test strain CDHWZ7 was isolated from the rhizosphere of L. ruthenicum in the arenosols of Dachaidan, Haixi Prefecture, Qinghai Province, China (3400 m above sea level, 95°21′ E, 37°88′ N).

2.2. Determination of Antagonistic Fungal Activity against Pathogens

The activity of strain CDHWZ7 against three pathogenic fungi was determined using the plate confrontation method. F. graminearum, F. acuminatum, and F. oxysporum were reactivated at 4 °C in refrigerated storage, and the plates were punched using a punch. Four sterilized filter-paper sheets of 5 mm in diameter were placed at equidistant symmetrical points, and 5 μL of CDHWZ7 bacterial solution was pipetted onto the center of the filter paper sheets at 25 °C, and this was repeated three times. After 5 d of incubation, the diameter of the inhibition circle was measured and recorded [19].

2.3. Determination of Degraded Cellulose Activity

The cellulose-degradation activity of strain CDHWZ7 was determined by the critical micellar concentration (CMC) method. Four sterilized filter-paper pieces of 5 mm in diameter were placed at equidistant symmetrical points on a CMC-Na medium plate, and 5 μL of the activated bacterial solution to be tested was placed in the center of the filter-paper slices. This process was repeated three times at 37 °C. After 3 d of incubation, Gram’s iodine staining solution was added to the plate for 5–10 min, and the presence of transparent circles was observed. The ratio of the diameter of the hyaline circle D to the diameter of the colony d was measured and calculated, and the cellulose degradation activity was determined according to the size of the ratio A [20].

2.4. Determination of Nitrogen-Fixation Capacity

2.4.1. Solid Plate Method

Four 5-mm-diameter filter-paper plates were placed at equidistant symmetrical points on a modified Ashby nitrogen-free solid medium plate (Shenggong, Shanghai, China). Five microliters of activated CDHWZ7 were drawn onto the center of the filter-paper plate and incubated three times at 37 °C for 3 d. If the strain was able to grow on the plate, it was initially determined to have nitrogen-fixation capacity [21].

2.4.2. Liquid Culture Method

After 100 μL of the activated CDHWZ7 bacterial solution was pipetted into 10 mL of modified Ashby’s nitrogen-free liquid medium (Shenggong, Shanghai, China) three times, the strain was incubated in a shaker (37 °C, 200 r∙min−1) for 3 d. If the strain was able to take nitrogen from the air and fix in the media, it would be reflected by the growth in turbidity or presence of colonies in agar; therefore, this method is a qualitative expression of nitrogen-fixation ability [21].

2.5. Determination of IAA-Producing Activity

Standard solutions of IAA at concentrations of 0, 10, 20, 30, 40, 50, and 60 μg∙mL−1 were prepared, and IAA standard curves were plotted. After activation, the strain CDHWZ7 was inoculated in 20 mL of LB liquid medium at 37 °C and 200 r∙min−1 for 7 d. The suspension was centrifuged (4 °C, 10,000 r∙min−1, 10 min) and 5 mL of supernatant was aspirated with Salkowski’s colorimetric solution in equal amounts in the colorimetric plate and left in the dark for 30 min. The color change of the supernatant was observed, and if all three replicate groups turned red, the strain was considered able to secrete IAA. Its OD530 value was determined, and the concentration of IAA produced by the strain was measured through the standard curve [22].

2.6. Determination of Resistance to Stress

2.6.1. Salt Tolerance

CDHWZ7 was inoculated in LB liquid medium for 12 h (37 °C, 200 r∙min−1) to produce a bacterial suspension. One-hundred microliters of the test suspension was incubated in 10 mL of LB liquid medium with NaCl concentrations of 0, 3, 5, 7, 9, 11, and 13% for 1 d (37 °C, 180 r∙min−1). Each treatment was repeated three times, and 50 μL of the bacterial solution was aspirated onto solid LB plates containing NaCl concentrations of 3, 5, 7, 9, 11, 13, and 15% and incubated at 37 °C for 12 h. The incubation was repeated three times for 3 d. Its OD600 value was determined, and the growth of the strains was observed and recorded daily to determine the salt tolerance of the strains [23].

2.6.2. Acid and Base Tolerance

CDHWZ7 was made into a bacterial suspension, of which 100 μL was added to 10 mL of LB liquid medium at pH 3.0, 5.0, 7.0, 9.0, 11.0, and 13.0 for 3 d (37 °C, 180 r∙min−1), and each treatment was repeated three times. Fifty microliters of bacterial suspension were applied to the medium at pH 3.0, 5.0, 7.0, 9.0, 11.0, and 13.0 and incubated at 37 °C for 12 h. Each treatment was repeated three times for 3 d. The OD530 value was determined, and the growth of the strains was observed and recorded daily to determine the salt tolerance of the strains [24].

2.6.3. Low Temperature Tolerance

CDHWZ7 was inoculated into a low-temperature (1 °C) liquid medium and incubated overnight at 37 °C and 200 r∙min−1 for 12 h until the log phase. Ten microliters of the bacteria to be tested were aspirated onto 1 °C solid medium and incubated in a constant temperature incubator (SHENGKE, Shanghai, China) at 4 °C, 10 °C, 14 °C, and 18 °C for 7 d, with each treatment repeated three times. The growth of the strains at different temperatures was recorded, and the low-temperature tolerance activity of the three strains was determined [25].

2.7. Whole-Genome Sequencing of Strain CDHWZ7

2.7.1. Sample Preparation

After activation, CDHWZ7 was inoculated in a triangular flask containing 20 mL LB liquid medium (Shenggong, Shanghai, China) and incubated at 37 °C and 200 rpm for 12 h. After centrifugation for 10 min (10,000 rpm), the bacteriophage was extracted, snap-frozen in liquid nitrogen, and sent to Shanghai Meiji Biomedical Technology Co. (Shanghai, China) for sequencing.

2.7.2. Whole-Genome Sequencing

The purified CDHWZ7 genomic DNA was collected, fragmented using a Covaris (Covaris, MA, USA) instrument to construct a qualified genomic sequencing library, sequenced by Illumina HiSeq (Illumina, CA, USA) after bridge PCR, and the data obtained offline for biological analysis.

2.7.3. Genome Annotation Function and Prediction

The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Groups of Proteins (COG) Databases were used to quickly annotate the bacterial genome and obtain the corresponding annotation information. Prodigal, RNAmmer, and other tools were used to annotate and predict the coding sequences and ribosomal RNA genes, and CGView 1.0 software was used to map the genome loops of strain CDHWZ7 [26].

3. Results

3.1. Antagonistic Activity against Pathogenic Fungi

CDHWZ7 had an inhibitory effect on all three of the tested pathogens(Figure 1). The diameter of the inhibition circle was greater than 22 mm, and the diameter of the inhibition circle of strain CDHWZ7 against F. oxysporum was 26.8 mm (Table 1), showing the best inhibition activity.

3.2. Cellulose-Degrading Activity

The cellulose-degrading activity of strain CDHWZ7 was determined by the CMC method. The results showed that strain CDHWZ7 produced degrading hyaline circles on the CMC-Na medium, indicating that the strain was able to secrete cellulase. Our results showed that the diameter of the hyaline circles was 18 mm, and the diameter of the colony was 6.9 cm. By measuring the size of the ratio A between the diameter of the hyaline circles D and the diameter of the colony d on the plate, the degrading activity was measured based on the ratio of the diameter of the hyaline circle D to the diameter of the colony A. The strain CDHWZ7 had an A value of 2.61 and was a strong cellulose degreaser (Figure 2).

3.3. Nitrogen-Fixation Activity

The nitrogen-fixation activity of strain CDHWZ7 was tested, and the results showed that after 3 d of the inoculation of strain CDHWZ7 in the modified Ashby nitrogen-free liquid medium, the fermentation broth (B) showed obvious turbidity, indicating that strain CDHWZ7 was able to fix nitrogen in the air via nitrogen-fixation enzymes and could grow and multiply in the nitrogen-free medium. Moreover, strain CDHWZ7 was inoculated in the modified Ashby nitrogen-free solid medium, and after 3 d of incubation, white endospores grew. It was tentatively assumed that strain CDHWZ7 exhibited some nitrogen-fixation ability based on these two methods.

3.4. IAA-Producing Activity

The IAA-producing activity of strain CDHWZ7 was determined. The colorimetric solution turned red, indicating that strain CDHWZ7 has the ability to produce IAA. The standard equation for IAA was determined as: y = 61.952x – 0.6997, and the amount of IAA synthesized by strain CDHWZ7 was calculated as 55.00 mg∙L−1 according to the standard equation (Figure 3).

3.5. Resilience

The results showed that after 3 days of culture, strain CDHWZ7 could grow normally on plates containing NaCl at 3, 5, 7, 9, and 11% and pH 3.0, 5.0, 7.0, 9.0, and 11.0 at 12 °C and 14 °C. After 24 h of culture at 12 °C and 14 °C, strain CDHWZ7 grew normally, with rings growing after 24 h of incubation. At 8 °C, endospores after three days of incubation, and at 4 °C, faint rings grew after seven days. This indicated that Bacillus screened from extreme climatic environments, such as drought, strong UV radiation, and anoxia, were more responsive to acid–base stress and more stable than bacteria from normal habitats, and also had greater potential for development as biocides.

3.6. Sequencing the Genome of Strain CDHWZ7

Basic Characteristics of the Genome

The whole genome of B. cereus CDHWZ7 was sequenced using a sequencing technique combining the second-generation Illumina HiSeq platform with the third generation PacBio platform. The chromosome of CDHWZ7 is a circular structure of 5,648,783 bp in length, containing two intact plasmids with an average GC content of 35.2%, and a total number of 5672 encoded genes. The CDHWZ7 genome-sequencing data were submitted to GenBank with the accession number CP095767, and the CDHWZ7 genome map is shown in Figure 4.

3.7. Functional Annotation of the Genome

3.7.1. COG Annotation Results

The genomic functional annotation of strain CDHWZ7 with the COG database revealed that there were 4038 protein-coding genes annotated in 22 categories, accounting for 71.19% of the total number of annotated genes. Among them, amino acid transport and metabolism had the highest number of annotated genes, with 358 genes, accounting for 8.87% of the total number of annotated genes, followed by inorganic ion transport and metabolism, with 257 genes, accounting for 6.36% of the total number of genes annotated. In addition, replication, recombination, and repair (221 genes, 5.47%), cell wall/membrane/envelope biogenesis (227 genes, 5.62%), carbohydrate transport and metabolism (206 genes, 5.10%), and energy production and conversion (195 genes, 4.83%) were also annotated. There were also 1247 (30.88%) genes of unknown functions (Figure 5).

3.7.2. GO Annotation Results

The protein sequences of B. cereus CDHWZ7 were annotated with the GO database for functional annotation of the genome. A total of 4133 genes in three categories were annotated, accounting for 72.87% of the total genes. The number of genes related to the cellular component was 2069, and these were mainly related to integral component of the membrane, cytoplasm, plasma membrane and membrane formation, with 1380, 343, 205, and 88 genes each, respectively. The number of genes related to the molecular function was 3182, and these were mainly related to gene binding, hydrolase activity, metal ion binding, transcription factor activity, and sequence-specific DNA binding processes, with 419, 274, 194, and 156 genes each, respectively. The number of genes associated with biological processes was 1792, and these were mainly related to the regulation of transcription, DNA-templated methylation, transmembrane transport, and translation, with 123, 87, 82 and 64 each, respectively (Figure 6).

3.7.3. KEGG Annotation Results

The protein sequences of B. cereus CDHWZ7 were annotated with the KEGG database for the functional annotation of the genome. A total of 2160 genes were functionally annotated, with 196 pathways involved in cellular life processes, mainly cell motility, cell growth and death, and community-prokaryotes; 299 pathways involved in environmental signal transduction, signal transduction, and membrane transport; 775 pathways involved in material metabolism, mainly in carbohydrate metabolism, amino acid metabolism, and metabolism of cofactors and vitamins; and 220 pathways involved in the processing of genetic information, mainly in translation, replication and repair, folding, sorting, and degradation (Figure 7).

3.7.4. CAZy Annotation Results

The genome sequence of CDHWZ7 was compared with the CAZy database, and a total of 137 genes were annotated as carbohydrases in the genome of strain CDHWZ7. These were divided into four families, namely 16 auxiliary activities, 43 carbohydrate esterases, 31 glycoside hydrolases, and 47 glycosyl transferases (Figure 8).

4. Analysis of Relevant Functional Genes

4.1. Analysis of Genes Related to Antagonistic Activity

Bacillus produce a large number of antimicrobial metabolites with different chemical structures and diversity, such as biopeptides and hydrolytic enzymes, which can effectively inhibit plant-pathogenic bacteria and promote plant growth [27]. The plate standoff experiments herein revealed that strain CDHWZ7 could significantly inhibit the growth of F. graminearum, F. acuminatum, and F. oxysporum on PDA plates. Genome sequence analysis revealed that strain CDHWZ7 possesses genes encoding for chitinase chiA and chiD. Chitinase plays an important role in plant defense, and Bacillus can also effectively degrade the cell walls of pathogenic fungi and bacteria by secreting degrading cellulases, chitinases, glucanases, and proteases, thereby inhibiting the growth of pathogens [28]. CDHWZ7 was detected to have cellulose-degrading activity by CMC-Na plates, and the genomic analysis revealed that strain CDHWZ7 possesses the cellulase-encoding genes bcsA and celC, the gene celf encoding endocellulase, and the gene malZ encoding α-glucosidase. The antagonistic activity of Bacillus is also usually associated with the production of secondary metabolites with antibiotic properties, and secondary metabolite analysis of the strain CDHWZ7 genome using anti-SMASH predicted the presence of genes encoding the antimicrobial active substances Fengycin, Bacillibactin, and chejuenolide A~B. It has been reported that Bacillus amyloliquefaciens FZB42 can produce the lipopeptide Fengycin that acts directly on fungal cell membranes, causing the efflux of cell contents and ultimately leading to the death of pathogenic microorganisms [29]. One study found that chejuenolide A~B has strong antibacterial activity against Candida albicans and Staphylococcus aureus [30]. Therefore, it is predicted that strain CDHWZ7 is able to increase the fungal inhibitory activity of the strain through several pathways, including the synthesis of bacillibactin, fengycin, chejuenolide A–B, cellulase, and chitinase, via the non-ribosomal pathway.

4.2. Analysis of Growth-Promoting Functional Genes

The bacteria secreted IAA through metabolism, which acts as a signaling molecule for plant–microbe interactions and can improve the growth of plant roots, thereby promoting the absorption of water and nutrients, leading to increased plant yield [31]. The Salkowski colorimetric method revealed that strain CDHWZ7 has the ability to produce IAA, and the genes trpA (tryptophan synthase α subunit), trpB (tryptophan synthase β subunit), trpC (indole-3-glycerophosphate synthase), trpD (o-aminobenzoic acid phosphate ribosyltransferase) and trpE (o-aminobenzoic acid synthase), trpF (ribose phosphate o-aminobenzoic acid iso), and trpS (tryptophan-tRNA ligase), which are involved in the synthesis of the IAA precursor tryptophan, were detected in the CDHWZ7 genome. The Bacillus CDHWZ7 genome was also found to have the gene dhaS encoding indole-3-pyruvate dehydrogenase, the pyridoxal phosphate-dependent aminotransferase gene encoding patB, the gene encoding indole-3-pyruvate decarboxylase, and a key enzyme of the indole-3-pyruvate pathway, ipdC, which are involved in a variety of biological processes related to IAA biosynthesis. Microbial nitrogen fixation plays a crucial role in the nitrogen cycle, converting nitrogen (N2) in the air to NH4+ for direct plant uptake through nitrogen-fixing enzymes, thereby maintaining and increasing agricultural productivity [32]. Genomic analysis revealed the presence of NifU (the gene encoding nitrogen-fixing enzyme), glutamate synthase gene gltB, nitrate transporter protein gene NarK, nitrite reductase gene nirA, nitrate reductase gene narG, glutamine synthetase gene glnA, and nitrite reductase gene narG in the nitrogen metabolism pathway of strain CDHWZ7. The presence of these genes may have increased the nitrogen-fixation capacity of the strain.
The VOCs produced by Bacillus metabolism can promote the growth of host plants and influence their defense responses [33]. The genome sequence analysis revealed that strain CDHWZ7 possesses genes encoding the volatile compounds alsD (α-acetyl lactate decarboxylase), alsS (acetolactate synthase), and bdhA (D-β-hydroxybutyrate dehydrogenase), which suppress phytopathogenic fungi, promote plant growth, and enhance plant systemic resistance.
In bacteria, the uptake and utilization of inorganic phosphates (Pi) is mainly accomplished by the phosphate-specific transport (Pst) and phosphate transport (Pit) systems [34]. The genomic analysis revealed that strain Pst of CDHWZ7 was found to consist of five proteins: PstS, PstA, PstB, PstC, and PhoU. The bacterial Pst system was reported to be able to increase the phosphorus content in plants by altering the activity of phosphatase, affecting the phosphate solubilization of microorganisms, accelerating the decomposition of phosphorus-containing organic compounds that are not easily absorbed and utilized by plants, and promoting the release of phosphorus [35].

4.3. Analysis of Resistance-Related Functional Genes

Bacillus CDHWZ7 exhibits strong acid and cold tolerance. The genomic analysis revealed that CDHWZ7 has the gene encoding the glycine betaine transporter protein OpuD, the peroxidase katE, the genes encoding superoxide dismutase sodA1, sodA2, and sodF, the genes encoding low-affinity potassium transporter protein mscL and putP, and the genes encoding cold shock protein. The presence of these genes helps strain CDHWZ7 adapt better to different habitats. In addition, the CDHWZ7 genome also contains the genes encoding N1-acetyltransferase, speG, and spermidine synthase, speE. Spermidine is the main polyamine synthesized by bacteria, and it has been reported that spermidine produced by Bacillus can promote plant growth and improve acid and base tolerance in Bacillus [36].

5. Discussion

Haixi Prefecture, Qinghai Province, is located in the northeastern part of the Qinghai–Tibet Plateau, where climatic events such as snowstorms, frost damage, high winds, and droughts occur all year round. The natural environment is harsh, and Bacillus originating from here have undergone natural selection that has primed them for their unique environmental adaptability and greater stability [37]. In this study, it was found to have good antagonistic activity against the pathogenic fungi F. graminearum, F. acuminatum, and F. oxysporum. The CMC-Na medium analysis revealed that strain CDHWZ7 has some cellulose-degrading activity, and through genomic analysis, it was found that strain CDHWZ7 possesses the cellulase-encoding genes bcsA and celC, endocytic cellulase-encoding gene celf, and α-glucosidase-encoding gene malZ. This suggests that Bacillus CDHWZ7 may be able to degrade the cell wall of pathogenic fungi, causing rupturing and deformation of the mycelium and thus controlling the growth of pathogenic fungi. The antagonistic activity of Bacillus is also usually associated with the production of secondary metabolites with antibiotic properties. Secondary metabolite analysis of the genome of strain CDHWZ7 using anti-SMASH predicted the presence of compounds encoding the antimicrobial active substances fengycin, bacillibactin, and chejuenolide A~B. Fengycin is a lipopeptide compound synthesized by B. subtilis through the non-ribosomal synthesis pathway, and its compounds are structurally stable and important antibacterial substances produced by B. subtilis. Fengycin produced by B. subtilis Z-14 has been reported to be able to disrupt the internal cell structure by digesting the cytoplasm and organelles of wheat Holothuria, inducing mycelial contraction and deformation [38]. With regard to the role of bacillibactin, it is now widely believed that ferricin chelates Fe3+ around the soil and rhizosphere, making it impossible for the pathogenic bacteria to obtain sufficient iron ions and thus inhibiting their growth and reproduction. The fungicidal activity of the strain against Pseudomonas syringae was significantly enhanced by the secretion of ferricin [39]. The strain CDHWZ7 also possesses the genes chiA and chiD, which encode chitinases, which are a group of glycosyl hydrolases that catalyze the degradation of chitin and are important for the control of pathogenic fungi. and it has been reported that B. subtilis TV-125A produced a chitinase that is effective in inhibiting the activity of Fusarium culmorum [40]. The high adaptability of the enzyme facilitates effective biocontrol in unstable environments [41]. Therefore, strain CDHWZ7 is predicted to be able to inhibit the growth of pathogenic bacteria through multiple pathways such as the synthesis of secondary metabolites via the non-ribosomal pathway, as well as the synthesis of cellulase and chitinase.
In recent years, as the development of eco-friendly agriculture continues to advance, the interest in microbial fertilizers has grown. Bacillus nitrogen-fixing genera have become the best biofertilizer choice due to their easy storage and fast growth and reproduction [42]. Bacillus nitrogen-fixing bacteria are able to increase the above- and below-ground biomass of plants, thereby playing an active role in achieving sustainable agricultural development. In this study, we found that CDHWZ7 was able to grow in nitrogen-free culture, as determined by the Asby nitrogen-free medium method, and it was initially concluded that it exhibits the ability to fix nitrogen. Genomic analysis revealed that CDHWZ7 possesses the nitrogen-fixing enzyme gene NifU, the glutamate synthase-encoding gene gltB, the nitrate transporter protein-encoding gene NarK, the nitrite reductase-encoding gene nirA, the nitrate reductase-encoding gene narG, the glutamine synthetase-encoding gene glnA, and the nitrite reductase-encoding gene nirB. Therefore, strain CDHWZ7 may promote plant growth and increase plant biomass through the nitrogen-metabolism pathway [43].
Indole-3-acetic acid is a growth hormone that can act on the entire plant growth and development process. IAA affects plant cell division, elongation, and differentiation, seed germination, root development, and the process of nutritional growth [44]. In this study, the Salkowski colorimetric method revealed that strain CDHWZ7 was able to secrete IAA, and the amount of IAA synthesized was 55.00 mg∙L−1. Genomic analysis also revealed that CDHWZ7 possessed the genes trpA (tryptophan synthase α subunit), trpB (tryptophan synthase β subunit), trpC (indole-3-glycerophosphate synthase)), trpD (o-aminobenzoic acid phosphoribosyltransferase), trpE (o-aminobenzoic acid synthase), trpF (ribose phosphate o-aminobenzoic acid iso), and trpS (tryptophan-tRNA ligase, the gene encoding indole-3-pyruvate dehydrogenase, dhaS, the gene encoding pyridoxal phosphate-dependent aminotransferase, patB, and the gene encoding indole-3-pyruvate decarboxylase, ipdC (a key enzyme of the indole-3-pyruvate pathway), which are involved in IAA precursor L-tryptophan production, IAA production, and the IPA pathway. It has been shown that plants are able to use IAA synthesized by bacteria to improve plant-root development and increase the surface area and volume of roots, thus promoting better water and nutrient uptake by plants while also promoting the secretion of metabolites by the roots, which in turn can promote bacterial reproduction. This suggests that the interaction between plant- and microbial-synthesized growth factors is important for a beneficial symbiotic relationship between plants and microorganisms [45]. According to a previous report, the IAA-producing Bacillus thuringiensis RZ2MS9 was able to promote growth and alter the root structure of tomato [46].
The response to abiotic stressors (e.g., drought, salinity, and climate extremes in temperature) induced by inter-root probiotic bacteria is known as induced systemic tolerance, which is essential for mitigating the effects of climatic change on crop yield [47]. The diverse geographical environment and unique natural climatic conditions of the Qinghai Province are conducive to the research and development of extreme microorganisms. In this study, the tolerance of B. cereus strain CDHWZ7 from the plateau to low temperature, acid and alkali, salt was determined. The results showed that B. cereus isolated from extreme climatic environments characterized by drought, strong UV radiation, and hypoxia responded to acid–base stress more stably than B. cereus from normal habitats. CDHWZ7 was able to grow in salt concentrations of 3–11%, showing strong salt tolerance. In the low temperature assay, CDHWZ7 grew coils at temperatures of 12 °C and 14 °C for 24 h, and at a low temperature of 4 °C, after 48 h of incubation, it exhibited good low-temperature fitness. Genomic analysis revealed that CDHWZ7 possesses several resistance genes, including OpuD encoding glycine betaine transporter protein, katE encoding catalase, the genes sodA1, sodA2, and sodF encoding superoxide dismutase, mscL and putP encoding low-affinity potassium transporter protein, cspA encoding cold shock protein, Hsp20 encoding heat shock protein, speG encoding N1-acetyltransferase, and speE encoding spermidine synthase. Spermidine is a polyamine that promotes plant growth, and it has been reported that spermidine produced by B. subtilis OKB105 can inhibit the expression of the ethylene biosynthesis gene ACO1, resulting in a decrease in ethylene content in plant root cells [48]. One study reported that the gene encoding superoxide dismutase sodA1 of B. cereus 0–9 was found to play a major role in oxidative stress and spore formation [49]. In addition, the OpuD-encoded glycine betaine provides a high degree of osmotolerance for B. subtilis [50]. Therefore, it is indicated that these genes can enhance the survival of strain CDHWZ7 under low temperature, high salinity, and other adversity stresses.
Many inter-root microorganisms such as Bacillus velezensis, Streptomyces setonii, and Bacillus subtilis are capable of releasing VOCs during metabolism. The main volatile inhibitors are 2,3-butanediol and 3-hydroxy-2-butanone [51,52]. Compared to other non-volatile bacteriostatic substances, VOCs are readily permeable in air and soil, and are of potential research value [53]. The VOCs released by B. subtilis CL2 inhibited four pathogenic true curly mildew species, including Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. The growth of the mycelium of the strain CL2 was analyzed by scanning electron microscopy, and the VOCs produced by the strain CL2 were able to cause morphological deformation, distortion, folding, and contraction of the mycelium of the pathogenic fungus, thereby inhibiting the growth of the pathogenic fungus [54]. The VOCs produced by microorganisms can also enhance the resistance of plants to disease. For instance, the VOCs produced by Pantoea gDez632 and Pseudomonas Bt851 induced significant upregulation of defense genes related to soft-rot resistance in Beta vulgaris, thereby reducing the incidence of soft rot. The genome sequence analysis revealed that strain CDHWZ7 possessed genes encoding the volatile compounds alsD (α-acetyl lactate decarboxylase), alsS (acetolactate synthase), and bdhA (D-β-hydroxybutyrate dehydrogenase), and it was therefore hypothesized that these volatile compounds could suppress phytopathogenic fungi, promote plant growth, and enhance plant systemic resistance.
Phosphorus (P) is an essential component in the maintenance of plant life and is essential in the processes of carbon metabolism, energy metabolism, and membrane formation, and the synthesis of the key biomolecules ATP, nucleic acids, and phospholipids. In agricultural production, P deficiency is one of the most important factors limiting crop yields. The Pst system is a high-affinity inorganic phosphate transporter found in many bacterial species, The Pst system can accelerate the breakdown of P-containing organic compounds that are not easily absorbed by soil species by altering the phosphatase activity of microorganisms and promoting the release of P, thereby increasing the P content in plants. The genomic analysis revealed that the Pst of strain CDHWZ7 consists of five proteins, namely PstS, PstA, PstB, PstC, and PhoU, and it has been reported that genes from Bacillus subtilis predicted to encode a phosphate-specific transport (Pst) system were shown by mutation to affect high-affinity Pi uptake [55].

6. Conclusions

Bacillus strain CDHWZ7, isolated from the extreme habitats of the plateau, has a higher potential for research and development because of its superb resistance, its ability to survive and grow in complex and diverse environments, and its good stability. In summary, strain CDHWZ7 has strong antagonistic activity against phytopathogenic fungi, can provide a nitrogen source for plants through its nitrogen-fixation ability, and has antagonistic, cellulose-degrading, nitrogen fixation, low-temperature tolerance, salinity tolerance, and the ability to secrete Indole-3-acetic acid. The genomic analysis showed that CDHWZ7 possesses genes encoding for chitinase activity, cellulase, secondary metabolites, phytohormone production, VOCs, nitrogen and phosphate metabolism, and resistance responses to biotic stresses (glycine betaine transporter protein, catalase, superoxide dismutase, low-affinity potassium transporter protein, cold-shock protein, and heat-shock protein), thus indicating that this strain has potential applications for complex microbial fertilizer development in agricultural production.

Author Contributions

Conceptualization, software, formal analysis, data curation, writing—original draft preparation, writing—review and editing, X.Y.; visualization, supervision; project administration, funding acquisition, Y.X. and Y.Q.; investigation, L.C., L.W., T.W., J.L. and Y.G.; All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Science Foundation of China, grant number 32160030 and Qinghai Provincial Science and Technology Department Special Projects, grant number 2023-ZJ-709.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The CDHWZ7 genome sequencing data were submitted to GenBank with the accession number CP095767.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Antagonistic activity of Bacillus strains against pathogenic fungi as: (A) antagonistic activity against F. acuminatum; (B) antagonistic activity against F. oxysporum; and (C) antagonistic activity against F. graminearum.
Figure 1. Antagonistic activity of Bacillus strains against pathogenic fungi as: (A) antagonistic activity against F. acuminatum; (B) antagonistic activity against F. oxysporum; and (C) antagonistic activity against F. graminearum.
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Figure 2. Determination of the cellulose-degrading activity of Bacillus.
Figure 2. Determination of the cellulose-degrading activity of Bacillus.
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Figure 3. Strain producing IAA color development reaction. Note: (A) the supernatant of strain DGL1 was spiked with equal amounts of Salkowski colorimetric agent, a color development reaction occurs; and (B) the same volume of the liquid medium is added with an equal amount of Salkowski colorimetric agent as CK group.
Figure 3. Strain producing IAA color development reaction. Note: (A) the supernatant of strain DGL1 was spiked with equal amounts of Salkowski colorimetric agent, a color development reaction occurs; and (B) the same volume of the liquid medium is added with an equal amount of Salkowski colorimetric agent as CK group.
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Figure 4. Circular map of Bacillus cereus strain CDHWZ7. Note: the first and fourth circles from outside to inside are CDS on positive and negative strands, different colors indicate different COG functional classifications; the second and third circles are CDS, tRNA, rRNA on positive and negative strands, respectively; the fifth circle is GC content, the outward part indicates that the GC content of the region is higher than the genome-wide average GC content, the higher the peak indicates the larger the difference with the average GC content. The sixth circle is the GC-Skew value, the specific algorithm is G − C/G + C, which can help to determine the leading strand and lagging strand, generally the leading strand GC skew > 0, the lagging strand GC skew < 0, also can help to determine the replication starting point (cumulative offset minimum) and the end point (cumulative offset maximum). and the end point (cumulative offset maximum), especially for the loop genome; the innermost circle is the genome size marker.
Figure 4. Circular map of Bacillus cereus strain CDHWZ7. Note: the first and fourth circles from outside to inside are CDS on positive and negative strands, different colors indicate different COG functional classifications; the second and third circles are CDS, tRNA, rRNA on positive and negative strands, respectively; the fifth circle is GC content, the outward part indicates that the GC content of the region is higher than the genome-wide average GC content, the higher the peak indicates the larger the difference with the average GC content. The sixth circle is the GC-Skew value, the specific algorithm is G − C/G + C, which can help to determine the leading strand and lagging strand, generally the leading strand GC skew > 0, the lagging strand GC skew < 0, also can help to determine the replication starting point (cumulative offset minimum) and the end point (cumulative offset maximum). and the end point (cumulative offset maximum), especially for the loop genome; the innermost circle is the genome size marker.
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Figure 5. Metabolic pathways annotated using the COG database. Note: The horizontal coordinates represent the different COG types, and the vertical coordinates represent the number of genes. The legend on the right for a functional description of each specific COG type.
Figure 5. Metabolic pathways annotated using the COG database. Note: The horizontal coordinates represent the different COG types, and the vertical coordinates represent the number of genes. The legend on the right for a functional description of each specific COG type.
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Figure 6. Metabolic pathways annotated using the GO database. Note: The horizontal coordinates represent the three major branches of GO, namely BP (biological process), CC (cellular component), MF (molecular function), and a further level 2 classification; the vertical coordinates represent the relative proportions occupied by genes.
Figure 6. Metabolic pathways annotated using the GO database. Note: The horizontal coordinates represent the three major branches of GO, namely BP (biological process), CC (cellular component), MF (molecular function), and a further level 2 classification; the vertical coordinates represent the relative proportions occupied by genes.
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Figure 7. Metabolic pathways annotated using the KEGG database. Note: The vertical coordinate indicates the level 2 hierarchical classification of the KEGG pathway, and the horizontal coordinate indicates the number of genes under the annotated classification.
Figure 7. Metabolic pathways annotated using the KEGG database. Note: The vertical coordinate indicates the level 2 hierarchical classification of the KEGG pathway, and the horizontal coordinate indicates the number of genes under the annotated classification.
Agriculture 13 01041 g007
Agriculture 13 01041 g008 550
Figure 8. Classification statistics of the CAZy annotation.
Figure 8. Classification statistics of the CAZy annotation.
Agriculture 13 01041 g008
Table
Table 1. Antagonistic activity to pathogenetic fungi (unit: mm).
Table 1. Antagonistic activity to pathogenetic fungi (unit: mm).
Strains(F. acuminatum)(F. oxysporum)(F. graminearum)
Diameter of the Inhibition Zone25.926.822.8
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Yang, X.; Xie, Y.; Qiao, Y.; Chen, L.; Wang, T.; Wu, L.; Li, J.; Gao, Y. Analysis of the Biological Activity and Whole Genome Sequencing of Bacillus cereus CDHWZ7 Isolated from the Rhizosphere of Lycium ruthenicum on the Tibetan Plateau. Agriculture 2023, 13, 1041. https://doi.org/10.3390/agriculture13051041

AMA Style

Yang X, Xie Y, Qiao Y, Chen L, Wang T, Wu L, Li J, Gao Y. Analysis of the Biological Activity and Whole Genome Sequencing of Bacillus cereus CDHWZ7 Isolated from the Rhizosphere of Lycium ruthenicum on the Tibetan Plateau. Agriculture. 2023; 13(5):1041. https://doi.org/10.3390/agriculture13051041

Chicago/Turabian Style

Yang, Xue, Yongli Xie, Youming Qiao, Lan Chen, Tian Wang, Lingling Wu, Junxi Li, and Ying Gao. 2023. "Analysis of the Biological Activity and Whole Genome Sequencing of Bacillus cereus CDHWZ7 Isolated from the Rhizosphere of Lycium ruthenicum on the Tibetan Plateau" Agriculture 13, no. 5: 1041. https://doi.org/10.3390/agriculture13051041

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