Isolation and Genome-Based Characterization of Biocontrol Potential of Bacillus siamensis YB-1631 against Wheat Crown Rot Caused by Fusarium pseudograminearum

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum is one of the most serious soil-borne diseases of wheat. Among 58 bacterial isolates from the rhizosphere soil of winter wheat seedlings, strain YB-1631 was found to have the highest in vitro antagonism to F. pseudograminearum growth. LB cell-free culture filtrates inhibited mycelial growth and conidia germination of F. pseudograminearum by 84.14% and 92.23%, respectively. The culture filtrate caused distortion and disruption of the cells. Using a face-to-face plate assay, volatile substances produced by YB-1631 inhibited F. pseudograminearum growth by 68.16%. In the greenhouse, YB-1631 reduced the incidence of FCR on wheat seedlings by 84.02% and increased root and shoot fresh weights by 20.94% and 9.63%, respectively. YB-1631 was identified as Bacillus siamensis based on the gyrB sequence and average nucleotide identity of the complete genome. The complete genome was 4,090,312 bp with 4357 genes and 45.92% GC content. In the genome, genes were identified for root colonization, including those for chemotaxis and biofilm production, genes for plant growth promotion, including those for phytohormones and nutrient assimilation, and genes for biocontrol activity, including those for siderophores, extracellular hydrolase, volatiles, nonribosomal peptides, polyketide antibiotics, and elicitors of induced systemic resistance. In vitro production of siderophore, β-1, 3-glucanase, amylase, protease, cellulase, phosphorus solubilization, and indole acetic acid were detected. Bacillus siamensis YB-1631 appears to have significant potential in promoting wheat growth and controlling wheat FCR caused by F. pseudograminearum.


Introduction
Fusarium crown rot (FCR) of wheat, caused by Fusarium pseudograminearum, can result in the rotting of seeds, seedlings, roots, crowns, subcrowns, and lower stems, inhibiting the flow of water and nutrients [1]. It has become one of the most important diseases of wheat, primarily in arid and semi-arid regions of the world. Yield losses caused by FCR are approximately 10% but can be over 30% when conditions favor the disease, such as drought or high humidity, temperature, and soil organic matter [2]. Although F. pseudograminearum only colonizes wheat roots up to the second stem node, the mycotoxin deoxynivalenol (DON) also can be detected in the head, creating a serious threat to the health of humans and animals [3].
Biocontrol agents (BCAs) are a very promising alternative to chemical pesticides for plant disease control. Many plant-growth-promoting bacteria can produce abundant antifungal secondary metabolites and hydrolytic enzymes with strong antagonistic activities against plant pathogenic fungi, as well as trigger-induced systemic resistance (ISR) [4]. Among bacterial BCAs, Bacillus species are considered to be one of the most valuable for field application, producing a wide range of different antimicrobial compounds. They utilize 5-8% of their genomes for secondary metabolite synthesis, and the major antimicrobial metabolites are lipopeptides, non-ribosomally synthesized peptides, polyketides, bacteriocins, and siderophores [5].
Bacillus siamensis was first described in salted crab in Thailand in 2010 [6]. It is widely distributed in the environment, such as in the beltfish gastrointestinal tract [7], rhizosphere soil [8], fermented foods, and the interior of plant tissues [9]. It has been shown to have plant growth promotion activity for a wide range of plants, including wheat [10], sweet pepper [11], tomato [12], and Chinese cabbage [13]. It has also been shown to have the potential to control a range of pathogens by direct antimicrobial activity, such as against charcoal rot fungus [14], post-harvest fruit rot fungi [15], and Fusarium head blight fungus [16]. Additionally, it has the potential to control diseases by inducing host resistance, such as against post-harvest anthracnose of mango [17], fruit rot and wilt of sweet pepper [18], and brown spot of tobacco [8].
Sequencing of the genome of B. siamensis KCTC13613 revealed that it has genes for promoting root colonization, including those for chemotaxis (methyl-accepting chemotaxis proteins and CheV system), biofilm formation (polyglutamic acid, biofilm-surface layer protein, extracellular polysaccharides, biofilm matrix protein), and motility (flagellar and swarming motility proteins). There were also genes for plant growth promotion, including those for phytohormone synthesis (auxin and gibberellic acid) and nutritional assimilation (iron, nitrogen, phosphorus, and potassium). Genes were also found for production of antimicrobial volatile organic compounds (VOCs) (acetoin and 2,3-butanediol), nonribosomal peptide (surfactin and fengycin), polyketides (bacillaene and difficidin), and a hybrid nonribosomal peptide/polyketide (iturin). In addition, there were genes for the production of compounds related to induced systemic resistance (ISR), such as VOCs (acetoin and 2,3-butanediol) and microbe-associated molecular patterns (MAMPs) (peptidoglycan, flagellin, and elongation factor Tu). Many of these genes have also been found in the genomes of other B. siamensis strains [7,19]. Thus, B. siamensis strains can have a wide range of plant growth promotion and biocontrol traits.
In this study, strain YB-1631 was isolated from winter wheat rhizosphere soil and was selected from 58 isolates based on its strongest in vitro antagonistic ability against the F. pseudograminearum. It was identified as B. siamensis, and its antifungal activity in vitro and in vivo and growth promotion ability on wheat seedlings were tested. The genome of strain YB-1631 was sequenced and assembled, and genes related to root colonization, plant growth promotion, antimicrobial activity, and ability to induce host disease resistance were analyzed. This is the first report on the biocontrol of wheat FCR with B. siamensis.

Isolation of Strain YB-1631 and In Vitro Antifungal Assays
Two rhizosphere soil samples from 50 wheat seedlings each were collected from two fields of winter wheat in Maqiao, Henan, China. A total of 1 g of soil was dilution plated on LB agar and incubated at 37 • C. Pure cultures from bacterial colonies with different colony characteristics were maintained on Luria-Bertani (LB) agar.
For bacterial colony mycelial growth inhibition, one 5 mm agar plug of F. pseudograminearum WZ-8A was inoculated onto the center of a PDA plate, and then the bacterial isolates were inoculated 2.5 cm from the fungal plug and incubated at 28 • C. The distance of the zone between the fungal and bacterial colonies was measured after 5 days [20].
For bacterial cell-free culture filtrate mycelial growth inhibition, strain YB-1631 was grown in LB broth overnight at 37 • C and 180 rpm and then inoculated into LB, Landy [21], or mineral salt medium (MSM) [22] broth. After incubation at 37 • C and 180 rpm for 72 h, the broths were centrifuged at 17,226× g for 20 min, and the supernatant passed through a 0.22 µm sterile filter. The filter-sterilized extracts were mixed into potato dextrose agar (PDA) at 50 • C to make 5%, 10%, 15%, and 20% PDA + LB, Landy, or MSM extract. The control was PDA alone. Each plate was inoculated with a 5 mm F. pseudograminearum agar plug and then incubated at 28 • C. The growth diameter of F. pseudograminearum was measured at 5 days. The percent inhibition rate of mycelial growth was calculated: (colony diameter on PDA alone-colony diameter on extract + PDA)/colony diameter on PDA alone × 100 [23].
For bacterial cell-free culture filtrate conidial germination inhibition, F. pseudograminearum was grown in 100 mL sodium carboxymethyl cellulose medium (CMC) broth [24] at 28 • C and 180 rpm for 5 days. The broth was filtered through Miracloth (475855-1R, Calbiochem, Darmstadt, Germany), centrifuged at 1914× g for 10 min, and then the conidia were manually diluted to 1 × 10 6 spores/mL in 66% LB bacterial extract + yeast extract peptone dextrose medium (YEPD) broth. The control was YEPD broth alone. Cultures were incubated at 28 • C and 180 rpm for 12 h, and 100 conidia were examined with three replicates (observed at 400 times magnification under a ZEISS Imager A2 microscope). Conidia were considered to have germinated if the germ tube exceeded half the length of the conidia. The percent inhibition rate of conidial germination was calculated: (conidial germination rate with YEPD alone − conidial germination rate with LB extract + YEPD)/conidial germination rate with YEPD alone × 100 [25].
For bacterial volatile mycelial growth inhibition, YB-1631 was grown overnight in LB broth as described above, and then 80 µL of 1 × 10 8 bacterial cells/mL were spread onto LB agar. A 5 mm F. pseudograminearum agar plug was inoculated onto the center of the PDA, then the LB agar and PDA plates without lids were attached face-to-face with parafilm. The control was face-to-face attached plates of non-inoculated LB agar and PDA with F. pseudograminearum. Plates were incubated at 28 • C for 5 days. The mycelial growth diameter of F. pseudograminearum was measured, and the percent inhibition rate of mycelial growth was calculated as described above [26]. Each experiment was repeated three times.

Biocontrol of FCR and Growth Promotion of Wheat by YB-1631
Surface sterilized wheat seeds (Zhengmai 1860) were germinated for 2 days to obtain primary root emergence [27] and then treated with 10 8 bacterial cells/mL YB-1631 or sdH 2 O for 4 h at 28 • C. F. pseudograminearum was grown in wheat grain sand culture for 1 week at 28 • C to produce soil inoculum [20]. Germinated seeds (21 per pot) were planted in 300 g of sterile soil with or without 6 g F. pseudograminearum soil inoculum (2%, W/W), and the pots were placed in a greenhouse with 12 h light/12 h dark at 25 • C. At 21 days after sowing, the seedlings were evaluated using a 0 to 7 Fusarium crown rot (FCR) symptom rating scale, with class 0 having no symptoms, classes 1 to 6 having different ranges of necrotic lesion areas, and with plant death as class 7 [28]. Fusarium crown rot incidence (FCRI) was calculated: FCRI (%) = (total number of seedlings − number of non-infected seedlings class value)/total number of seedlings × 100 [29]. Disease severity index (DSI) was calculated: DSI (%) = [Σ(class value × number of seedlings in each class value)/(total number of seedlings × 7) × 100 [29]. Relative control effect (RCE) was calculated: RCE (%) = (DSI of sdH 2 O treatment − DSI of YB-1631 treatment)/DSI of sdH 2 O treatment × 100 [20]. Shoot height, root length and fresh weight, and total fresh weight were also determined at 21 days post-sowing. The experiment was repeated five times.

Identification of YB-1631
Gram staining was performed as per Xu et al. [32]. For SEM, YB-1631 was scraped off LB agar at 16 h at 37 • C, fixed with 2.5% glutaraldehyde, and examined with a Hitachi SU8100 SEM microscope (Hitachi, Tokyo, Japan). Genomic DNA of YB-1631 was extracted using the MiniBEST Bacterial Genomic DNA Extraction Kit Ver. 3.0 (Takara, Beijing, China). A 1200 bp gyrB sequence was amplified from the YB-1631 genomic DNA using primers UP-1S and UP-2Sr [33] and sequenced (Tsingke Biotechnology, Beijing, China). The gyrB sequence was used as a query with blastn against the NCBI nr database. In addition, 16 other gyrB sequences were selected for the construction of a tree using the neighborjoining method with 1000 bootstrap iterations [20,34].
The genome of YB-1631 was sequenced and assembled as described below. The complete genomes of 10 strains of Bacillus were downloaded from the EzBioCloud (http://www.ezbiocloud.net, accessed on 12 December 2022), and pairwise comparisons of Average Nucleotide Identity (ANI) of the genomes were calculated using the JSpeciesWS Online Service [35]. The ANI values were used to draw a heatmap using TBtools software [36]. Strains with ANI values higher than 95-96% are defined as the same bacterial species [37][38][39].

Statistical Analysis
Statistical analyses were performed using IBM SPSS Statistics 21 using one-way ANOVA with Tukey's test (p < 0.05).

Isolation and In Vitro Antifungal Activity of YB-1631
Of 58 bacterial strains isolated from wheat rhizosphere soil, 50 showed some in vitro antifungal activity against F. pseudograminearum on PDA plates ( Table 1). The largest zone of growth inhibition between the pathogen and soil bacteria was with strain Mq202003-16 at 5.07 mm, which was significantly greater than the other bacterial strains, except for Mq202003-10, Mq202003-23, Mq202003-30, Mq202003-45, and Mq202003-58. Mq202003-16 was deposited in the Henan Academy of Agricultural Sciences soil microbial culture collection as YB-1631. YB-1631 cell-free culture filtrate from LB, Landy, and MSM media all inhibited the mycelial growth of F. pseudograminearum ( Figure 1A). The 20% LB culture filtrate resulted in the highest antifungal activity at 84.14% ( Figure 1B). Conidial germination of F. pseudograminearum was inhibited with 66% LB culture filtrate of YB-1631 by 92.23% compared to the control ( Figure 1C). The cell walls of conidia and germ tubes with control treatment appeared intact and without deformation (Figure 2A), while conidia treated with cell-free culture filtrates showed lysis and deformations, and the germ tubes showed swelling, protoplast condensation, shortening of septum intervals, crumpling, and lysis ( Figure 2B, arrows a to g). Antifungal volatiles of YB-1631 were detected with a face-to-face plate assay showing inhibition of F. pseudograminearum growth by 68.16% ( Figure 1C). pseudograminearum was inhibited with 66% LB culture filtrate of YB-1631 by 92.23% compared to the control ( Figure 1C). The cell walls of conidia and germ tubes with control treatment appeared intact and without deformation (Figure 2A), while conidia treated with cell-free culture filtrates showed lysis and deformations, and the germ tubes showed swelling, protoplast condensation, shortening of septum intervals, crumpling, and lysis ( Figure 2B, arrows a to g). Antifungal volatiles of YB-1631 were detected with a face-toface plate assay showing inhibition of F. pseudograminearum growth by 68.16% ( Figure 1C).  in the highest antifungal activity at 84.14% ( Figure 1B). Conidial germination of F. pseudograminearum was inhibited with 66% LB culture filtrate of YB-1631 by 92.23% compared to the control ( Figure 1C). The cell walls of conidia and germ tubes with control treatment appeared intact and without deformation (Figure 2A), while conidia treated with cell-free culture filtrates showed lysis and deformations, and the germ tubes showed swelling, protoplast condensation, shortening of septum intervals, crumpling, and lysis ( Figure 2B, arrows a to g). Antifungal volatiles of YB-1631 were detected with a face-toface plate assay showing inhibition of F. pseudograminearum growth by 68.16% ( Figure 1C).

Identification of YB-1631
YB-1631 colonies were slimy, translucent, convex, smooth, and pale yellow on LB agar at 12 h at 37 • C ( Figure 3A). Under light microscopy, YB-1631 appeared rod-shaped and was Gram-positive ( Figure 3B). Under SEM, the rod-shaped cells had an average size of 3.22 µm × 0.72 µm and were surrounded by an extracellular matrix ( Figure 3C).

Identification of YB-1631
YB-1631 colonies were slimy, translucent, convex, smooth, and pale yellow on LB agar at 12 h at 37 °C ( Figure 3A). Under light microscopy, YB-1631 appeared rod-shaped and was Gram-positive ( Figure 3B). Under SEM, the rod-shaped cells had an average size of 3.22 μm × 0.72 μm and were surrounded by an extracellular matrix ( Figure 3C). Based on gyrB sequences of eight Bacillus species, YB-1631 clustered with Bacillus siamensis B28, B. siamensis SRCM100169, and B. siamensis KCTC13613 ( Figure 4A). Based on genome comparisons with five other Bacillus species, YB-1631 also had the highest ANI value of 98.69% with B siamensis SRCM100169, followed by 98.47% with B. siamensis KCTC13613. Both ANI values are higher than the 95-96% identity threshold for defining bacterial species. In contrast, ANI values with other Bacillus species ranged from 72.02% to 93.91% ( Figure 4B). Thus, YB-1631 was identified as a strain of B. siamensis.

B. siamensis YB-1631 In Vivo Biocontrol and Plant Growth Promotion Activities
Wheat seedlings treated or not treated (control) with YB-1631 showed no symptoms of FCR, whereas wheat seedlings inoculated with F. pseudograminearum showed FCR symptoms of stunting, black or brown leaf sheaths, and root rot ( Figure 5A,B). Seedlings with YB-1631 treatment plus F. pseudograminearum inoculation appeared similar to the control and YB-1631 treatment alone. For YB-1631 treatment plus F. pseudograminearum inoculation, DI and DSI for FCR were significantly less than with F. pseudograminearum

B. siamensis YB-1631 In Vivo Biocontrol and Plant Growth Promotion Activities
Wheat seedlings treated or not treated (control) with YB-1631 showed no symptoms of FCR, whereas wheat seedlings inoculated with F. pseudograminearum showed FCR symptoms of stunting, black or brown leaf sheaths, and root rot ( Figure 5A,B). Seedlings with YB-1631 treatment plus F. pseudograminearum inoculation appeared similar to the control and YB-1631 treatment alone. For YB-1631 treatment plus F. pseudograminearum inoculation, DI and DSI for FCR were significantly less than with F. pseudograminearum alone, with an RCE of 83.23% (Table 2).
J. Fungi 2023, 9, x FOR PEER REVIEW 9 of 20 control. YB-1631 treatment plus F. pseudograminearum inoculation significantly increased the activities of all the defense enzymes relative to the control and other treatments, except for SOD relative to F. pseudograminearum alone. MDA content with YB-1631 treatment plus F. pseudograminearum inoculation was significantly reduced relative to all other treatments except for YB-1631 alone. At 21 days post-sowing with YB-1631 treatment, wheat root length, plant height, root fresh weight, and total fresh weight were significantly higher than the non-treated control ( Table 2). In contrast, inoculation of F. pseudograminerum significantly decreased all those parameters compared to the control. YB-1631 treatment plus F. pseudograminearum inoculation resulted in root length, root fresh weight, and total fresh weight significantly greater than the control, while plant height was not significantly different from the control. All those parameters, however, were significantly greater than F. pseudograminearum inoculation alone.    YB-1631 treatment significantly increased the activities of all the defense enzyme activities tested and significantly decreased MDA content compared to control noninoculated plants (Table 3). Inoculation with F. pseudograminearum also significantly increased the defense enzyme activities, but it decreased MDA content compared to the control. YB-1631 treatment plus F. pseudograminearum inoculation significantly increased the activities of all the defense enzymes relative to the control and other treatments, except for SOD relative to F. pseudograminearum alone. MDA content with YB-1631 treatment plus F. pseudograminearum inoculation was significantly reduced relative to all other treatments except for YB-1631 alone.   At 21 days post-sowing with YB-1631 treatment, wheat root length, plant height, root fresh weight, and total fresh weight were significantly higher than the non-treated control ( Table 2). In contrast, inoculation of F. pseudograminerum significantly decreased all those parameters compared to the control. YB-1631 treatment plus F. pseudograminearum inoculation resulted in root length, root fresh weight, and total fresh weight significantly greater than the control, while plant height was not significantly different from the control. All those parameters, however, were significantly greater than F. pseudograminearum inoculation alone.

Genome Assembly and Annotation of B. siamensis YB-1631
A total of 276,759,717 bp were obtained with PacBio Sequel II sequencing, and 2,205,483,000 bp were obtained using MGIseq2000 sequencing. This resulted in a 67.38× coverage depth of the assembled genome. The assembled genome consisted of one chromosome of 4,090,312 bp and 45.92% GC content ( Figure 6). The PacBio Sequel II, MGIseq2000, and YB-1631 assembled genome sequences were deposited at NCBI with the accession numbers SRR22106234, SRR22077931, and CP110268, respectively.

Genes and Activities Potentially Related to FCR Biocontrol, Plant Growth Promotion, and the Interaction of B. siamensis YB-1631 with Plants
Ten gene clusters of secondary metabolite synthesis were predicted using the anti-SMASH database (Tables 4 and S5). For nonribosomal peptide synthesis gene clusters, there were 20 genes in cluster 1 for surfactin, 14 genes in cluster 2 for fengycin, and 13 genes in cluster 3 for bacillibactin. For transAT polyketide synthesis, there were 14 genes in cluster 4 for bacillaene and 15 genes in cluster 5 for difficidin. Cluster 6 for polyketide-like synthase had 2 genes for butirosin A, clusters 7 and 8 for terpene synthesis had 4 genes in cluster 7 for squalene/phytoene and 3 genes in cluster 8 for squalene-hopene, and clusters 9 and 10 for nonribosomal peptide + polyketide synthase had 10 genes in cluster 9 for bacillomycin D and 5 genes in cluster 10 for locillomycin.

Genome Assembly and Annotation of B. siamensis YB-1631
A total of 276,759,717 bp were obtained with PacBio Sequel II sequencing, and 2,205,483,000 bp were obtained using MGIseq2000 sequencing. This resulted in a 67.38× coverage depth of the assembled genome. The assembled genome consisted of one chromosome of 4,090,312 bp and 45.92% GC content ( Figure 6). The PacBio Sequel II, MGIseq2000, and YB-1631 assembled genome sequences were deposited at NCBI with the accession numbers SRR22106234, SRR22077931, and CP110268, respectively. Figure 6. Circular map of Bacillus siamensis YB-1631 genome. Circles from the outside to the inside are as follows: ring 1 for genome size (black line), ring 2 for restriction-modification system (RMS), ring 3 for bases modification, forward strand (red), and reverse strand (blue), ring 4 for COG classifications of protein-coding genes on the forward strand and reverse strand, ring 5 for the distribution of tRNAs (brown) and rRNAs (green), ring6 for GC skew, ring 7 for GC content.

Genes and Activities Potentially Related to FCR Biocontrol, Plant Growth Promotion, and the Interaction of B. siamensis YB-1631 with Plants
Ten gene clusters of secondary metabolite synthesis were predicted using the antiSMASH database (Table 4 and S5). For nonribosomal peptide synthesis gene clusters, there were 20 genes in cluster 1 for surfactin, 14 genes in cluster 2 for fengycin, and 13 genes in cluster 3 for bacillibactin. For transAT polyketide synthesis, there were 14 genes in cluster 4 for bacillaene and 15 genes in cluster 5 for difficidin. Cluster 6 for polyketidelike synthase had 2 genes for butirosin A, clusters 7 and 8 for terpene synthesis had 4 genes in cluster 7 for squalene/phytoene and 3 genes in cluster 8 for squalene-hopene, and clusters 9 and 10 for nonribosomal peptide + polyketide synthase had 10 genes in cluster 9 for bacillomycin D and 5 genes in cluster 10 for locillomycin. Figure 6. Circular map of Bacillus siamensis YB-1631 genome. Circles from the outside to the inside are as follows: ring 1 for genome size (black line), ring 2 for restriction-modification system (RMS), ring 3 for bases modification, forward strand (red), and reverse strand (blue), ring 4 for COG classifications of protein-coding genes on the forward strand and reverse strand, ring 5 for the distribution of tRNAs (brown) and rRNAs (green), ring6 for GC skew, ring 7 for GC content. In addition, YB-1631 was likely to produce some small antimicrobial compounds because 5 genes for amylocyclin and one each gene for uberolysin/carnocyclin family circular bacteriocin, and antimicrobial peptide Lci antibacterial were found in the YB-1631 genome. However, YB-1631 likely cannot produce bacilysin because only one transporter gene for bacilysin was found, and it cannot produce macrolactin, plantazolicin, and aurantinin for the lack of synthetic genes.
The YB-1631 genome also contains 51 extracellular hydrolase genes (Table S6). Corresponding to the two genes for β-glucanase, one gene for amylase, one gene for cellulase, and ten genes for protease, the enzymatic activity of β-glucanase, amylase, cellulase, and protease was detected in vitro from YB-1631 ( Figure 7B,E,F,G). gene for bacilysin was found, and it cannot produce macrolactin, plantazolicin, and aurantinin for the lack of synthetic genes.
The YB-1631 genome also contains 51 extracellular hydrolase genes (Table S6). Corresponding to the two genes for β-glucanase, one gene for amylase, one gene for cellulase, and ten genes for protease, the enzymatic activity of β-glucanase, amylase, cellulase, and protease was detected in vitro from YB-1631 ( Figure 7B,E,F,G).
For genes potentially related to plant-microbe interaction in the YB-1631 genome, there were 11 genes related to bacterial chemotaxis, 28 genes for flagellar assembly and motor proteins, 4 genes for swarming motility, 62 genes for quorum sensing, and 54 genes for biofilm formation, including 5 genes for the synthesis of the secreted polymer, γ-PGA, 4 genes for bacterial target molecules of the general plant immune response, 9 genes for lipopolysaccharide biosynthesis, and 9 genes for teichuronic acid/lipopolysaccharide biosynthesis (Table S8). For genes involved in the breakdown, transport, and utilization of plant-derived substrates, there were 11 genes related to cellulose and hemicellulose, 1 gene related to chitin and chitosan, 2 genes related to protein and peptide, and 20 genes related to opine (Table S9). Finally, in the category of response to environmental stress, there were 2 genes for oxidative stress, 3 genes for nucleic acid-binding protein, and 1 gene for nucleic acid modification (Table S10).
Bacillus siamensis YB-1631 showed the strongest in vitro antagonistic activity against F. pseudograminearum among the isolates tested in this study. It also showed strong in vivo activity against FCR, with a biocontrol efficacy in the greenhouse at 83%, which was higher than that reported for B. velezensis YB-185 at 66% [25], B. subtilis YB-15 at 81% [20], and B. halotolerans QTH8 at 62% [64]. Wheat defense enzyme activities and MDA content were also altered by B. siamensis YB-1631, indicating that treatment with this isolate may have resulted in induced systemic resistance of wheat against F. pseudograminearum and reduced the oxidative damage of wheat seedlings caused by F. pseudograminearum [65]. There are many reports of Bacillus isolates inducing resistance against plant pathogens in a variety of crops [66]. In wheat, some examples of induced systemic resistance due to Bacillus isolates are resistance to Septoria tritici [67], Blumeria graminis f. sp. tritici [68], and Fusarium graminearum [69].
One factor that can affect the production of antimicrobial compounds in cell-free culture filtrates from Bacillus species is the type of culture medium. All the media tested in this study have been reported to result in the production of culture filtrates containing antifungal compounds, such as LB with B. siamensis S3, YC-9, and WB1 against Pestalotiopsis versicolor, Fusarium oxysporum, and Colletotrichum acutatum [53,79,80], Landy with B. siamensis BRBac21-1 against Macrophomina phaseolina and Fusarium oxysporum f. sp. udum [81], and MSM with B. siamensis JFL15 against Magnaporthe grisea, Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium graminearum [7,16,22]. Although previous studies have used different media, this is the first report comparing multiple media, and cell-free culture filtrate of B. siamensis YB-1631 grown in LB showed the highest antifungal activity. LB media may be the best as it had the lowest carbon-to-nitrogen ratio (C/N) among the four media tested. Fonseca et al. [82] reported that the lowest C/N ratio (3 versus 9 or 15) tested in a mineral media resulted in the greatest biomass and highest production of antifungal lipopeptides by B. subtilis YRE207. However, other media nutrients are important, such as phosphorus and metal ions, which were critical for the production of antifungal lipopeptides by Bacillus sp. BH072 [83]. Further research is needed to establish the relationship between specific nutrients and antimicrobial production by B. siamensis YB-1631.
Sequencing the genome of B. siamensis YB-1631 revealed a number of gene clusters potentially related to antibiotic production. Gene clusters were found for the production of lipopeptides, polyketides, dipeptides, cyclic peptides, cationic peptides, and aminoglycosides antibiotics. In comparison, there were similar or identical numbers of genes for such compounds in the genomes of four other B. siamensis isolates reported to have antimicrobial, biocontrol, and/or plant growth promotion activities, B. siamensis KCTC 13613T [85], B. siamensis SCSIO 05746 [19], B. siamensis JFL15 [22], and B. siamensis RGM 2529 [55] (Table S11). However, there were a few exceptions. Among those isolates, there were many fewer genes for difficidin in B. siamensis RGM 2529 and many more genes for bacilysin in B. siamensis SCSIO 05746. However, it appears that except for aurantinin, macrolactin, and plantazolicin, which were limited to the isolates, B. siamensis RGM 2529, B. siamensis SCSIO 05746, and B. siamensis JFL15, respectively, the B. siamensis YB-1631 genome appears to encode all the antibiotics found in those other biocontrol B. siamensis isolates.
Extracellular hydrolases produced with PGPRs have been reported to be involved in antifungal and plant growth-promotion processes, such as glucanases, amylase, protease, and chitosanases to degrade fungal cell walls [86][87][88], and siderophores and phosphatases to promote plant absorption of minerals [87][88][89]. Genes were also identified in the B. siamensis YB-1631 genome for a variety of extracellular hydrolases. These included glucanases, lipases, protease, and chitosanases that have been found in a number of Bacillus isolates, such as Bacillus sp.739, B. subtilis NPU 001, B. circulans MH-K1, B. subtilis CW14, and B. subtilis strain J9 that show antifungal activity against Bipolaris sorokiniana, Fusarium oxysporum, Penicillium expansum, Aspergillus awamori, and Aspergillus ochraceus, Fusarium graminearum, and Fusarium avenaceum [87][88][89][90]. Comparing the B. siamensis YB-1631 genome to the genomes of B. siamensis KCTC 13613T, B. siamensis SCSIO 05746, B. siamensis JFL15, and B. siamensis RGM 2529 revealed that there were similar numbers of genes for extracellular hydrolases with a few exceptions (Table S12). Only the B. siamensis YB-1631 and B. siamensis KCTC 13613T genomes contained the ribonuclease gene yokF, only the genome of B. siamensis KCTC 13613T was missing the ribonuclease gene yhcR, only the genome of B. siamensis RGM 2529 was missing the ribonuclease gene yobL, and only the B. siamensis YB-1631 genome contained the protease gene blaSE. Thus, there were a few differences between the strains in their extracellular hydrolase genes, and further research, such as gene knockouts and gene expression patterns, is needed to determine if that affects their biocontrol activity.
In addition to FCR control, B. siamensis YB-1631 also significantly increased root and shoot growth in healthy wheat seedlings. Root length and root fresh weight without F. pseudograminearum stress was increased by 37% and 21%, respectively, by B. siamensis YB-1631. In comparison, B. siamensis Pbbb1 in healthy tomato increased root length and root fresh weight by 50% and 92%, respectively [12], and B. siamensis MH559649 in healthy wheat cv. NARC-2009 increased root length by 15% [10]. Thus, B. siamensis YB-1631 was within the range of increased root growth produced by other B. siamensis isolates. Plant height and total plant fresh weight was increased by 14% and 10% by B. siamensis YB-1631. Similar results were found for other B. siamensis isolates, such as B. siamensis Pbbb1 in healthy tomato, which increased plant height and total fresh weight by 15% and 65%, respectively [10], and B. siamensis MH559649 in healthy wheat cv. NARC-2009, which increased plant height and total fresh weight by 13% and 15%, respectively [12].
B. siamensis YB-1631 also significantly increased root and shoot growth of wheat seedlings with F. pseudograminearum infection. This could be due to a combination of plant growth promotion and disease biocontrol. Root length and root fresh weight with F. pseudograminearum infection was increased by 88% and 116% by B. siamensis YB-1631, which was much greater than with healthy plants. This was greater than B. subtilis YB-15 in wheat seedlings with F. pseudograminearum infection, which increased root length and root fresh weight by 9% and 68%, respectively [20], and somewhat similar to B. subtilis YB-04 in cucumber seedlings with Fusarium oxysporum f.sp. cucumerinum infection, which increased root length and fresh weight by 46% and 230%. Plant height and total fresh weight with F. pseudograminearum infection were increased by 88% and 89% by B. siamensis YB-1631. In comparison, B. subtilis YB-15 increased plant height and total fresh weight by 26% and 14%, respectively, in wheat seedlings with F. pseudograminearum infection [20], and B. subtilis YB-04 increased plant height and total fresh weight by 190% and 27%, respectively, in cucumber seedlings with F. oxysporum f.sp. cucumerinum infection [91].
The B. siamensis YB-1631 genome contained a number of genes potentially related to plant growth promotion. There were genes for the synthesis of the phytohormones IAA and cytokinin. IAA is the main auxin in plants regulating cell division and differentiation [92], and cytokinin can act synergistically with IAA to promote cell differentiation and growth [93]. There were also genes for nutrient acquisition, such as phosphate, nitrate/nitrite, potassium, and iron assimilation found in many PGPRs [94], and genes for the production of the polyamines putrescine and spermidine. Polyamines increase plant growth, development, and tolerance to abiotic stress by acting as signaling molecules inside plant cells [95]. Finally, genes for the VOCs acetoin and 2,3-butanediol were identified in the B. siamensis YB-1631 and the four other B. siamensis genomes. Acetoin and 2,3-butanediol improve plant growth dependent on cytokinin activation [96]. The same number of those genes were also found in the genomes of B. siamensis KCTC 13613T, B. siamensis SCSIO 05746, B. siamensis JFL15, and B. siamensis RGM 2529, except for the B. siamensis RGM 2529 genome, which lacked the iron assimilation genes efeM and efeU (Table S13). Thus, B. siamensis YB-1631 and these other B. siamensis isolates have a nearly identical array of genes for these potential plant growth promotion traits.
Genes were also found in the B. siamensis YB-1631 genome for different factors related to plant-microbe interactions, utilization of plant-derived substrates, and response to environmental stress. Genes in the B. siamensis YB-1631 genome related to plant-microbe interactions were those for chemotaxis, such as chemotaxis protein and methyl-accepting chemotaxis protein [94], motility, such as flagellar motor protein and swarming motility protein [94], colonization and biofilm, such as levansucrase, surface adhesion protein, γ-PGA, extracellular polysaccharide, and biofilm-surface layer protein [94,97], and ISR elicitors, such as elongation factor, flagellin HAP, flagellin HAG, teichuronic acid, and lipopoly-saccharide biosynthesis [94]. Genes for utilization of plant-derived substrates were also found, such as xylanase, chitosanase, bacillopeptidase F, and ABC sugar trans-porter [94], as were genes potentially important for response to environmental stress, such as cold shock protein and superoxide dismutase [94]. Compared to the genomes of B. siamensis KCTC 13613T, B. siamensis SCSIO 05746, B. siamensis JFL15, and B. siamensis RGM 2529, there were the same number of genes for these traits, except for the B. siamensis RGM 2529 genome, which was missing the colonization and biofilm formation genes lytA, lytB, lytC, and yhcK, and the colonization and biofilm formation gene pznL only found in the B. siamensis JFL15 genome (Table S14). This indicates that B. siamensis YB-1631 has similar traits for plant-microbe interactions, utilization of plant-derived substrates, and response to environmental stress as other B. siamensis isolates, and perhaps B. siamensis JFL15 may be less effective in colonizing plant roots. However, future work would need to compare the strains directly for these traits to determine if the differences in the genomes have a significant effect on its biology.

Conclusions
In this study, a new strain of B. siamensis, YB-1631, was examined with strong in vitro antagonism to F. pseudograminearum, control of FCR, and plant growth promotion. An examination of different media to evaluate in vitro antifungal production by B. siamensis YB-1631 revealed that a low C/N ratio may be a critical factor for production. Future work should use completely defined media where several C/N ratios can be evaluated to choose the optimal one. The complete genome sequence of B. siamensis YB-1631 reveals many genes potentially related to biocontrol, plant growth promotion, and ISR. While similar to the genes found in the genomes of other B. siamensis biocontrol isolates, there were some differences. Sequencing more genomes of B. siamensis biocontrol isolates should help to reveal the range of genes that may be involved. However, future work should examine each potential biocontrol and plant growth promotion gene to assess their contribution to those traits. Although B. siamensis has been shown to be a BCA for a variety of plant diseases, this is the first report on the biocontrol of wheat FCR using a B. siamensis isolate.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof9050547/s1, Table S1: Characteristics of the Bacillus siamensis YB-1631 genome; Table S2: RNA genes in the Bacillus siamensis YB-1631 genome; Table S3: Gene islands, CRISPR and prophage structures in the Bacillus siamensis YB-1631 genome; Table S4: Functional annotation of the Bacillus siamensis YB-1631 genome; Table S5: Genes for antimicrobial compounds in the Bacillus siamensis YB-1631 genome; Table S6: Genes for extracellular hydrolases in the Bacillus siamensis YB-1631 genome; Table S7: Genes for putative plant growth promotion traits in the Bacillus siamensis YB-1631 genome; Table S8: Genes for putative plant-microbe interaction in the Bacillus siamensis YB-1631 genome; Table S9: Genes for breakdown, transport and utilization of plant derived substrates in the Bacillus siamensis YB-1631 genome; Table S10: Genes for putative response to environmental stress in the Bacillus siamensis YB-1631 genome; Table S11: Comparison of genes for antimicrobial compounds in the genomes of five B. siamensis isolates; Table S12: Comparison of genes for extracellular hydrolases in the genomes of five B. siamensis isolates; Table S13: Comparison of genes for putative plant growth promotion traits in the genomes of five B. siamensis isolates; Table S14: Comparison of genes for putative plant-microbe interaction and bacterial survival traits in the genomes of five B. siamensis isolates.

Informed Consent Statement: Not applicable.
Data Availability Statement: The genome sequence data and assemblies of Bacillus siamensis YB-1631 reported in this paper are associated with the NCBI BioProject: PRJNA894992, BioSample: SAMN31484458. Accession Numbers: CP110268.1 in GenBank. The gyrB sequence data are available at https://www.ncbi.nlm.nih.gov/protein/2325799032 (accessed on 6 November 2022).