Next Article in Journal
Targeting the P10 Peptide in Maturing Dendritic Cells via the DEC205 Receptor In Vivo: A New Therapeutic Strategy against Paracoccidioidomycosis
Previous Article in Journal
Biosynthetic Potential of Hypogymnia Holobionts: Insights into Secondary Metabolite Pathways
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JoF
Volume 9
Issue 5
10.3390/jof9050547
jof-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Qianqian Dong
1,2,
Qingxiang Liu
1,2,
Paul H. Goodwin
3,
Xiaoxu Deng
2,
Wen Xu
2,
Mingcong Xia
2,
Jie Zhang
2,
Runhong Sun
2,
Chao Wu
2,
Qi Wang
4,
Kun Wu
1,* and
Lirong Yang
2,*
1
College of Life Sciences, Henan Agricultural University, Zhengzhou 450046, China
2
Institute of Plant Protection Research, Henan Academy of Agricultural Sciences, Henan Agricultural Microbiology Innovation Center, Zhengzhou 450002, China
3
School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada
4
College of Plant Protection, China Agricultural University, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2023, 9(5), 547; https://doi.org/10.3390/jof9050547
Submission received: 5 February 2023 / Revised: 18 April 2023 / Accepted: 3 May 2023 / Published: 9 May 2023
Browse Figures
Review Reports Versions Notes

Abstract

:
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.

1. 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.

2. Materials and Methods

2.1. 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 × 106 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 12h, 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 × 108 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.

2.2. 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 108 bacterial cells/mL YB-1631 or sdH2O 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 sdH2O treatment − DSI of YB-1631 treatment)/DSI of sdH2O 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.

2.3. Wheat Disease Resistance Enzyme Activities

At 20 days post-sowing [20], 0.1 g of stem tissue from YB-1631 treated or non-treated wheat seedlings grown in soil containing or not containing F. pseudograminearum were taken and stored at −80 °C. Activities of peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), polyphenol oxidase (PPO, EC 1.10.3.1), lipoxygenase (LOX), phenylalanine ammonia lyase (PAL, EC 4.315), and malondialdehyde (MDA) content were determined by their respective detection kits (Cat. Nos. BC0095, BC0175, BC0205, BC0195, BC0325, BC0215, and BC0025, Beijing Solarbio Science Technology Co., Beijing, China).

2.4. Plant Growth Promotion and Biocontrol Traits of YB-1631

Amylase, protease, β-glucanase [30], siderophore, cellulase, and phosphorus solubilization activities were determined from zone diameters on corresponding agar plates as previously reported [20]. IAA production was determined in NB containing L-tryptophan and treated with Salkowski reagent [20,31].

2.5. 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 neighbor-joining 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].

2.6. Genome Sequencing, Assembly, and Annotation

YB-1631 was grown in LB broth for 12 h at 37 °C and 180 rpm, and genomic DNA was extracted as described above. Sequencing of 250 bp paired-end reads was conducted at Frasergen (Wuhan, Hubei, China) using MGIseq2000 (Mgi Tech Co., Shenzhen, China) and 900 bp single end reads with PacBio Sequel II (Pacific Biosciences, Menlo Park, CA, USA). The genome was assembled using pb_assembly_microbial in Smrtlink (10.1.0; Pacific Biosciences) and Pilon (1.24) [40]. The coverage depth was analyzed with Minimap (2.15-r905) [41] to align three sets of HiFi reads to the assembled genome. Coding regions were identified with Glimmer (3.02) [42] and annotated using blastp in Diamond (2.0.9.147) [42] against the Genbank nr, COG/KOG, GO, SwissProt, KEGG basic databases (cut-off E value 1 × 10−5). Genes for the synthesis of secondary metabolites were identified with antiSMASH (https://antismash.secondarymetabolites.org, accessed on 6 December 2022). Genes for extracellular hydrolases were identified using blastp with queries obtained from extracellular hydrolase predicted proteins from B. velezensis FZB42 [43]. Genes annotated for γ-PGA synthesis and other plant growth promotion traits were selected based on the literature [20,43,44,45]. tRNAs were predicted with tRNAscan-SE (2.0.9) [46], rRNAs were predicted with RNAmmer (1.2) [47], and sRNAs were predicted from the Rfam database using Cmscan in Infernal (1.1.4) [48]. Clustered regularly interspaced short palindromic repeat sequences (CRISRRs), genomic islands, and prophages were predicted with MinCED (0.4.2) [49], IslandPath-1.0.6 [50], and PhiSpy-4.2.19 [51], respectively. A genome circle map was generated with Circos (0.69–9) [52].

2.7. Statistical Analysis

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

3. Results

3.1. 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).

3.2. 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.

3.3. 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).
YB-1631 treatment significantly increased the activities of all the defense enzyme activities tested and significantly decreased MDA content compared to control non-inoculated 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.

3.4. 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.
The genome of YB-1631 was predicted to contain 4357 protein-coding genes, 86 tRNAs, 27 rRNAs and 89 other potential RNAs, 6 CRISPR sequences, 10 gene islands, and 9 prophages (Tables S1–S3). Using the NCBI nr, COG, GO, SwissProt, and KEGG databases, annotations were obtained for 96.26%, 82.81%, 72.85%, 51.16%, and 71.47% of the predicted protein sequences, respectively, giving a total annotation of 96.33% (Table S4).

3.5. 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 Table 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.
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).
The YB-1631 genome also contains many putative plant growth promotion-related genes (Table S7). There were 4 indole-3-acetic acid synthesis genes, 2 cytokinin synthesis genes, 5 acetoin and butanediol synthesis genes, 4 nitrogen-related genes, 10 phosphorus-related genes, 3 potassium assimilation and transport genes, and 41 iron assimilation and transport genes, which included 22 siderophore genes. Indole-3-acetic acid production, phosphorus solubilization, and siderophore activity were also detected in vitro (Figure 7A,C,D).
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).

4. Discussion

The wheat rhizosphere bacterial isolate, YB-1631, was identified as Bacillus siamensis based on gyrB sequences, ANI values, and morphology. Bacillus siamensis is a common rhizosphere soil bacterium, and many isolates have shown biocontrol potential against plant pathogens, such as B. siamensis S3 from sweet potato soil with activity against Pestalotiopsis versicolor [53], B. siamensis GL-02 from maize soil with activity against F. graminearum [54], and B. siamensis H30-3 from Chinese cabbage soil with activity against Pectobacterium carotovorum subsp. carotovorum PCC21 [13]. Additionally, many B. siamensis isolates from rhizosphere soil have shown plant growth promotion activity, such as B. siamensis H30-3 that significantly increased biomass of Chinese cabbage [13], B. siamensis RGM 2529 from forest soil that increased biomass of tomato [55], and B. siamensis Pbbb1 from bean soil that also increased biomass of tomato [12]. In addition to rhizosphere soil, B. siamensis isolates have been reported in fermented and pickled foods [16,56,57], healthy plant roots, leaves, and seeds [9,58,59,60], animal intestines [61], marine sediments [19], sewage [62], and insect hives and nests [63].
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].
Inhibition of F. pseudograminearum by B. siamensis YB-1631 may have been due to secreted antifungal compounds detected in cell-free culture filtrates. Inhibition of mycelial growth with 5–20% B. siamensis YB-1631 LB culture filtrate ranged from 45 to 84%. In comparison, 10–50% NB culture filtrate of B. velezensis YB-185 inhibited F. pseudograminearum by 44 to 79% [25], 10–50% LB culture filtrate of B.amyloliquefaciens Y1 inhibited F. graminearum by 34 to 45% [70], and 1–10% LB culture filtrate of B. velezensis BM21 inhibited F. graminearum by 42 to 76% [71]. Thus, B. siamensis YB-1631 LB culture filtrate has similar levels of inhibitory activity against F. pseudograminearum and F. graminearum as those from other Bacillus spp.
The antifungal compounds identified in cell-free culture filtrates of B. siamensis isolates include the lipopeptides, surfactin [22,72,73,74], fengycin [9,19], and iturin A [22,74,75], bacillomycin F [22,74], bacillomycin D [73], and the catechol-based siderophore, bacillibactin [19]. The YB-1631 cell-free culture filtrate resulted in ruptured, swollen, and vacuolated hyphae of F. pseudograminearum. In comparison, surfactin causes outgrowths, distortions, protrusions, and swelling of hyphae [76], fengycin causes hyphal cell-surface shrinking [77], iturin A causes swelling and reduced mycelial cytoplasmic content [78], and bacillomycin D results in sunken, lumpy, and wrinkled hyphae [7]. Thus, the damage to the hyphae due to B. siamensis YB-1631 cell-free culture filtrate is consistent with the effects of a number of antifungal compounds reported from B. siamensis isolates.
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.
Inhibition of F. pseudograminearum by B. siamensis YB-1631 may also have come from secreted volatile antifungal compounds that can diffuse through the soil and inhibit conidia germination and mycelial growth [70,84] and volatiles from B. siamensis YB-1631 grown on LB agar inhibited mycelial growth of F. pseudograminearum by 68%. In comparison, B. siamensis G-3 grown on NB agar inhibited raspberry post-harvest diseases caused by Botrytis cinerea and Rhizopus stolonifer by 89%, and the main antifungal compounds detected were 2, 6-di-tert-butyl-4-methylphenol and 2,4-di-tert-butylphenol [15]. Volatiles of B. siamensis LZ88 grown on NB agar inhibited the mycelial growth of A. alternata by 78%, and the active compounds were 2-methylbutanoic acid and 3-methylbutanoic acid [8,75]. Volatiles of B. siamensis N-1 grown on NB agar inhibited mycelial growth of C. gloeosporioides, P. microcystis, and F. incarnatum by 87%, 75% and 43%, respectively, and the active compounds were N-11-undecene, 3-methyl-1-butanol, 2-nonanone, 1,3,5,7-cyclooctatetraene, and phenol [72]. Thus, a variety of volatile compounds may be responsible and similar to levels of inhibition of fungal growth by volatiles of other B. saimensis isolates have been higher than that found in this study.
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 transporter [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.

5. 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.

Author Contributions

Conceptualization, L.Y., K.W. and Q.D.; methodology, Q.D., Q.L. and K.W.; validation: Q.L. and W.X.; formal analysis, Q.D., P.H.G. and X.D.; investigation, Q.D., Q.L. and X.D.; resources, M.X., Q.W., J.Z. and K.W.; data curation, Q.D., W.X. and L.Y.; writing—original draft preparation: Q.D.; writing—review and editing, Q.D. and P.H.G.; visualization, Q.D.; supervision, R.S. and C.W.; funding acquisition, L.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Central Plains Science and Technology Innovation Leader Project (234200510010), the Major Public Welfare Scientific Research Project of Henan Province (201300111600), the Henan Provincial Science and Technology Major Project (221100110100), and the Special Project for Science and Technology Innovation Team of Henan Academy of Agricultural Sciences, (2023TD15).

Institutional Review Board Statement

Not applicable.

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).

Acknowledgments

The author is especially grateful to Honglian Li (Henan Agricultural University) for providing the F. pseudograminearum WZ-8A strain.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kazan, K.; Gardiner, D.M. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: Recent progress and future prospects. Mol. Plant Pathol. 2018, 19, 1547–1562. [Google Scholar] [CrossRef] [PubMed]
  2. Minati, M.H.; Mohammed-ameen, M.K. Interaction between Fusarium Head Blight and Crown Rot Disease Incidence and Environmental Factors and Soil Physiochemical Analysis on Wheat in the South of Iraq, Basra Province. Karbala Int. J. Mod. Sci. 2019, 5, 6. [Google Scholar] [CrossRef]
  3. Pecoraro, F.; Giannini, M.; Beccari, G.; Covarelli, L.; Filippini, G.; Pisi, A.; Nipoti, P.; Prodi, A. Comparative studies about fungal colonization and deoxynivalenol translocation in barley plants inoculated at the base with Fusarium graminearum, Fusarium culmorum and Fusarium pseudograminearum. Agric. Food Sci. 2018, 27, 74–83. [Google Scholar] [CrossRef]
  4. Compant, S.; Duffy, B.; Nowak, J.; Clement, C.; Barka, E.A. Use of plant growth-promoting bacteria for biocontrol of plant diseases: Principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 2005, 71, 4951–4959. [Google Scholar] [CrossRef] [PubMed]
  5. Fira, D.; Dimkic, I.; Beric, T.; Lozo, J.; Stankovic, S. Biological control of plant pathogens by Bacillus species. J. Biotechnol. 2018, 285, 44–55. [Google Scholar] [CrossRef]
  6. Sumpavapol, P.; Tongyonk, L.; Tanasupawat, S.; Chokesajjawatee, N.; Luxananil, P.; Visessanguan, W. Bacillus siamensis sp. nov., isolated from salted crab (poo-khem) in Thailand. Int. J. Syst. Evol. Microbiol. 2010, 60, 2364–2370. [Google Scholar] [CrossRef]
  7. Xu, B.H.; Lu, Y.Q.; Ye, Z.W.; Zheng, Q.W.; Wei, T.; Lin, J.F.; Guo, L.Q. Genomics-guided discovery and structure identification of cyclic lipopeptides from the Bacillus siamensis JFL15. PLoS ONE 2018, 13, e0202893. [Google Scholar] [CrossRef]
  8. Xie, Z.; Li, M.; Wang, D.; Wang, F.; Shen, H.; Sun, G.; Feng, C.; Wang, X.; Chen, D.; Sun, X. Biocontrol efficacy of Bacillus siamensis LZ88 against brown spot disease of tobacco caused by Alternaria alternata. Biol. Control. 2021, 154, 104508. [Google Scholar] [CrossRef]
  9. Gorai, P.S.; Ghosh, R.; Mandal, S.; Ghosh, S.; Chatterjee, S.; Gond, S.K.; Mandal, N.C. Bacillus siamensis CNE6- a multifaceted plant growth promoting endophyte of Cicer arietinum L. having broad spectrum antifungal activities and host colonizing potential. Microbiol. Res. 2021, 252, 126859. [Google Scholar] [CrossRef]
  10. Awan, S.A.; Ilyas, N.; Khan, I.; Raza, M.A.; Rehman, A.U.; Rizwan, M.; Rastogi, A.; Tariq, R.; Brestic, M. Bacillus siamensis Reduces Cadmium Accumulation and Improves Growth and Antioxidant Defense System in Two Wheat (Triticum aestivum L.) Varieties. Plants 2020, 9, 878. [Google Scholar] [CrossRef]
  11. Pastor-Bueis, R.; Mulas, R.; Gómez, X.; González-Andrés, F. Innovative liquid formulation of digestates for producing a biofertilizer based on Bacillus siamensis: Field testing on sweet pepper. J. Plant Nutr. Soil Sci. 2017, 180, 748–758. [Google Scholar] [CrossRef]
  12. Islam, A.; Kabir, M.S.; Khair, A. Characterization and evaluation of Bacillus siamensis isolate for its growth promoting potential in tomato. Agric./Pol’nohospodárstvo 2019, 65, 42–50. [Google Scholar] [CrossRef]
  13. Shin, D.J.; Yoo, S.J.; Hong, J.K.; Weon, H.Y.; Song, J.; Sang, M.K. Effect of Bacillus aryabhattai H26-2 and B. siamensis H30-3 on Growth Promotion and Alleviation of Heat and Drought Stresses in Chinese Cabbage. Plant Pathol. J. 2019, 35, 178–187. [Google Scholar] [CrossRef]
  14. Hussain, T.; Khan, A.A. Determining the antifungal activity and characterization of Bacillus siamensis AMU03 against Macrophomina phaseolina (Tassi) Goid. Indian Phytopathol. 2020, 73, 507–516. [Google Scholar] [CrossRef]
  15. Zhang, X.; Gao, Z.; Zhang, X.; Bai, W.; Zhang, L.; Pei, H.; Zhang, Y. Control effects of Bacillus siamensis G-3 volatile compounds on raspberry post-harvest diseases caused by Botrytis cinerea and Rhizopus stolonifer. Biol. Control. 2020, 141, 104135. [Google Scholar] [CrossRef]
  16. Huang, Y.; Zhang, X.; Xu, H.; Zhang, F.; Zhang, X.; Yan, Y.; He, L.; Liu, J. Isolation of lipopeptide antibiotics from Bacillus siamensis: A potential biocontrol agent for Fusarium graminearum. Can. J. Microbiol. 2022, 68, 403–411. [Google Scholar] [CrossRef]
  17. Jiang, Z.; Li, R.; Tang, Y.; Cheng, Z.; Qian, M.; Li, W.; Shao, Y. Transcriptome analysis reveals the inducing effect of Bacillus siamensis on disease resistance in post-harvest mango fruit. Foods 2022, 11, 107. [Google Scholar] [CrossRef]
  18. Poveda, J.; Calvo, J.; Barquero, M.; González-Andrés, F. Activation of sweet pepper defense responses by novel and known biocontrol agents of the genus Bacillus against Botrytis cinerea and Verticillium dahliae. Eur. J. Plant Pathol. 2022, 164, 507–524. [Google Scholar] [CrossRef]
  19. Pan, H.; Tian, X.; Shao, M.; Xie, Y.; Huang, H.; Hu, J.; Ju, J. Genome mining and metabolic profiling illuminate the chemistry driving diverse biological activities of Bacillus siamensis SCSIO 05746. Appl. Microbiol. Biotechnol. 2019, 103, 4153–4165. [Google Scholar] [CrossRef]
  20. Xu, W.; Yang, Q.; Xie, X.; Goodwin, P.H.; Deng, X.; Zhang, J.; Sun, R.; Wang, Q.; Xia, M.; Wu, C.; et al. Genomic and Phenotypic Insights into the Potential of Bacillus subtilis YB-15 Isolated from Rhizosphere to Biocontrol against Crown Rot and Promote Growth of Wheat. Biology 2022, 11, 778. [Google Scholar] [CrossRef]
  21. Wu, L.; Wu, H.; Chen, L.; Yu, X.; Borriss, R.; Gao, X. Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens. Sci. Rep. 2015, 5, 12975. [Google Scholar] [CrossRef] [PubMed]
  22. Xu, B.H.; Ye, Z.W.; Zheng, Q.W.; Wei, T.; Lin, J.F.; Guo, L.Q. Isolation and characterization of cyclic lipopeptides with broad-spectrum antimicrobial activity from Bacillus siamensis JFL15. 3 Biotech 2018, 8, 444. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, W.Y.; Kong, W.L.; Liao, Y.C.; Zhu, L.H. Identification of Bacillus velezensis SBB and Its Antifungal Effects against Verticillium dahliae. J. Fungi 2022, 8, 1021. [Google Scholar] [CrossRef] [PubMed]
  24. Moura, R.D.; de Castro, L.A.M.; Culik, M.P.; Fernandes, A.A.R.; Fernandes, P.M.B.; Ventura, J.A. Culture medium for improved production of conidia for identification and systematic studies of Fusarium pathogens. J. Microbiol. Methods 2020, 173, 105915. [Google Scholar] [CrossRef]
  25. Zhang, J.; Zhu, W.; Goodwin, P.H.; Lin, Q.; Xia, M.; Xu, W.; Sun, R.; Liang, J.; Wu, C.; Li, H.; et al. Response of Fusarium pseudograminearum to Biocontrol Agent Bacillus velezensis YB-185 by Phenotypic and Transcriptome Analysis. J. Fungi 2022, 8, 763. [Google Scholar] [CrossRef]
  26. Zhang, D.; Yu, S.; Yang, Y.; Zhang, J.; Zhao, D.; Pan, Y.; Fan, S.; Yang, Z.; Zhu, J. Antifungal Effects of Volatiles Produced by Bacillus subtilis Against Alternaria solani in Potato. Front. Microbiol. 2020, 11, 1196. [Google Scholar] [CrossRef]
  27. Han, C.; Yang, P. Studies on the molecular mechanisms of seed germination. Proteomics 2015, 15, 1671–1679. [Google Scholar] [CrossRef]
  28. Smiley, R.W.; Gourlie, J.A.; Easley, S.A.; Patterson, L.M. Pathogenicity of Fungi Associated with the Wheat Crown Rot Complex in Oregon and Washington. Plant Dis. 2005, 89, 949–957. [Google Scholar] [CrossRef]
  29. Zhou, H.; He, X.; Wang, S.; Ma, Q.; Sun, B.; Ding, S.; Chen, L.; Zhang, M.; Li, H. Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China. Environ. Microbiol. 2019, 21, 2740–2754. [Google Scholar] [CrossRef]
  30. Teather, R.M.; Wood, P.J. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 1982, 43, 777–780. [Google Scholar] [CrossRef]
  31. Dhouib, H.; Zouari, I.; Ben Abdallah, D.; Belbahri, L.; Taktak, W.; Triki, M.A.; Tounsi, S. Potential of a novel endophytic Bacillus velezensis in tomato growth promotion and protection against Verticillium wilt disease. Biol. Control. 2019, 139, 104092. [Google Scholar] [CrossRef]
  32. Xu, W.; Zhang, L.; Goodwin, P.H.; Xia, M.; Zhang, J.; Wang, Q.; Liang, J.; Sun, R.; Wu, C.; Yang, L. Isolation, Identification, and Complete Genome Assembly of an Endophytic Bacillus velezensis YB-130, Potential Biocontrol Agent Against Fusarium graminearum. Front. Microbiol. 2020, 11, 598285. [Google Scholar] [CrossRef]
  33. Yamamoto, S.; Harayama, S. PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl. Environ. Microbiol. 2015, 61, 1104–1109. [Google Scholar] [CrossRef]
  34. Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406–425. [Google Scholar]
  35. Richter, M.; Rossello-Mora, R.; Oliver Glockner, F.; Peplies, J. JSpeciesWS: A web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 2016, 32, 929–931. [Google Scholar] [CrossRef]
  36. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  37. Ciufo, S.; Kannan, S.; Sharma, S.; Badretdin, A.; Clark, K.; Turner, S.; Brover, S.; Schoch, C.L.; Kimchi, A.; DiCuccio, M. Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI. Int. J. Syst. Evol. Microbiol. 2018, 68, 2386–2392. [Google Scholar] [CrossRef]
  38. Richter, M.; Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. USA 2009, 106, 19126–19131. [Google Scholar] [CrossRef]
  39. Konstantinidis, K.T.; Ramette, A.; Tiedje, J.M. The bacterial species definition in the genomic era. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2006, 361, 1929–1940. [Google Scholar] [CrossRef]
  40. Walker, B.J.; Abeel, T.; Shea, T.; Priest, M.; Abouelliel, A.; Sakthikumar, S.; Cuomo, C.A.; Zeng, Q.; Wortman, J.; Young, S.K.; et al. Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 2014, 9, e112963. [Google Scholar] [CrossRef]
  41. Li, H. Minimap2: Pairwise alignment for nucleotide sequences. Bioinformatics 2018, 34, 3094–3100. [Google Scholar] [CrossRef] [PubMed]
  42. Delcher, A.L.; Bratke, K.A.; Powers, E.C.; Salzberg, S.L. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 2007, 23, 673–679. [Google Scholar] [CrossRef] [PubMed]
  43. Chen, X.H.; Koumoutsi, A.; Scholz, R.; Eisenreich, A.; Schneider, K.; Heinemeyer, I.; Morgenstern, B.; Voss, B.; Hess, W.R.; Reva, O.; et al. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat. Biotechnol. 2007, 25, 1007–1014. [Google Scholar] [CrossRef] [PubMed]
  44. Wang, L.; Chen, S.; Yu, B. Poly-γ-glutamic acid: Recent achievements, diverse applications and future perspectives. Trends Food Sci. Technol. 2022, 119, 1–12. [Google Scholar] [CrossRef]
  45. Saxena, A.K.; Kumar, M.; Chakdar, H.; Anuroopa, N.; Bagyaraj, D.J. Bacillus species in soil as a natural resource for plant health and nutrition. J. Appl. Microbiol. 2020, 128, 1583–1594. [Google Scholar] [CrossRef]
  46. Lowe, T.M.; Eddy, S.R. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25, 955–964. [Google Scholar] [CrossRef]
  47. Lagesen, K.; Hallin, P.; Rødland, E.A.; Stærfeldt, H.-H.; Rognes, T.; Ussery, D.W. RNAmmer: Consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007, 35, 3100–3108. [Google Scholar] [CrossRef]
  48. Nawrocki, E.P.; Eddy, S.R. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 2013, 29, 2933–2935. [Google Scholar] [CrossRef]
  49. Bland, C.; Ramsey, T.L.; Sabree, F.; Lowe, M.; Brown, K.; Kyrpides, N.C.; Hugenholtz, P. CRISPR recognition tool (CRT): A tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinform. 2007, 8, 209. [Google Scholar] [CrossRef]
  50. Hsiao, W.W.; Ung, K.; Aeschliman, D.; Bryan, J.; Finlay, B.B.; Brinkman, F.S. Evidence of a large novel gene pool associated with prokaryotic genomic islands. PLoS Genet. 2005, 1, e62. [Google Scholar] [CrossRef]
  51. Akhter, S.; Aziz, R.K.; Edwards, R.A. PhiSpy: A novel algorithm for finding prophages in bacterial genomes that combines similarity- and composition-based strategies. Nucleic Acids Res. 2012, 40, e126. [Google Scholar] [CrossRef]
  52. Krzywinski, M.; Schein, J.; Birol, I.; Connors, J.; Gascoyne, R.; Horsman, D.; Jones, S.J.; Marra, M.A. Circos: An information aesthetic for comparative genomics. Genome Res. 2009, 19, 1639–1645. [Google Scholar] [CrossRef]
  53. Ali, M.A.; Ren, H.; Ahmed, T.; Luo, J.; An, Q.; Qi, X.; Li, B. Antifungal Effects of Rhizospheric Bacillus Species Against Bayberry Twig Blight Pathogen Pestalotiopsis versicolor. Agronomy 2020, 10, 1811. [Google Scholar] [CrossRef]
  54. Zhang, K.; Wang, L.; Si, H.; Guo, H.; Liu, J.; Jia, J.; Su, Q.; Wang, Y.; Zang, J.; Xing, J.; et al. Maize stalk rot caused by Fusarium graminearum alters soil microbial composition and is directly inhibited by Bacillus siamensis isolated from rhizosphere soil. Front. Microbiol. 2022, 13, 986401. [Google Scholar] [CrossRef]
  55. Altimira, F.; Godoy, S.; Arias-Aravena, M.; Araya, B.; Montes, C.; Castro, J.F.; Dardón, E.; Montenegro, E.; Pineda, W.; Viteri, I. Genomic and Experimental Analysis of the Biostimulant and Antagonistic Properties of Phytopathogens of Bacillus safensis and Bacillus siamensis. Microorganisms 2022, 10, 670. [Google Scholar] [CrossRef]
  56. Khan, M.M.; Kim, Y.K.; Cho, S.S.; Jin, Y.-Y.; Suh, J.-W.; Lee, D.Y.; Yoo, J.C. Response Surface Optimization of Culture Conditions for Cyclic Lipopeptide MS07 from Bacillus siamensis Reveals Diverse Insights Targeting Antimicrobial and Antibiofilm Activity. Processes 2020, 8, 744. [Google Scholar] [CrossRef]
  57. Thakham, N.; Thaweesak, S.; Teerakulkittipong, N.; Traiosot, N.; Kaikaew, A.; Lirio, G.A.; Jangiam, W. Structural Characterization of Functional Ingredient Levan Synthesized by Bacillus siamensis Isolated from Traditional Fermented Food in Thailand. Int. J. Food Sci. 2020, 2020, 7352484. [Google Scholar] [CrossRef]
  58. Guesmi, S.; Chouchane, H.; Neifar, M.; Hosni, F.; Cherif, A.; Sghaier, H. Radiation-inducible radioprotective exopolysaccharides of Bacillus siamensis CV5 from irradiated roots of Cistanche violacea to decrease free radical damage produced by ionizing radiation. Int. J. Radiat. Biol. 2019, 95, 1552–1563. [Google Scholar] [CrossRef]
  59. Hossain, M.T.; Khan, A.; Harun-Or-Rashid, M.; Chung, Y.R. A volatile producing endophytic Bacillus siamensis YC7012 promotes root development independent on auxin or ethylene/jasmonic acid pathway. Plant Soil 2019, 439, 309–324. [Google Scholar] [CrossRef]
  60. Rungsirivanich, P.; Thongwai, N. Antibacterial Activity and Tannin Tolerance of Bacillus spp. Isolated from Leaves of Miang (Camellia sinensis (L.) Kuntze var. assamica (J.W. Mast.) Kitam.). Int. J. Biosci. Biochem. Bioinform. 2020, 10, 26–33. [Google Scholar] [CrossRef]
  61. Yang, H.L.; Liu, Z.Y.; Jian, J.T.; Ye, J.D.; Sun, Y.Z. Host-associated Bacillus siamensis and Lactococcus petauri improved growth performance, innate immunity, antioxidant activity and ammonia tolerance in juvenile Japanese seabass (Lateolabrax japonicus). Aquac. Nutr. 2021, 27, 2739–2748. [Google Scholar] [CrossRef]
  62. Chopra, S.; Kumar, D. Characterization and biodegradation of ibuprofen by Bacillus siamensis strain DSI-1 isolated from wastewater. Rend. Lincei. Sci. Fis. E Nat. 2022, 33, 643–652. [Google Scholar] [CrossRef]
  63. Fitri, L.; Yasmin, Y.; Fauziah, F.; Septiani, D.A.; Suhartono, S. Characterization of BSL6 isolates isolated from honeybee hive and to determine its antibacterial activity. Biodivers. J. Biol. Divers. 2020, 21. [Google Scholar] [CrossRef]
  64. Li, S.; Xu, J.; Fu, L.; Xu, G.; Lin, X.; Qiao, J.; Xia, Y. Biocontrol of Wheat Crown Rot Using Bacillus halotolerans QTH8. Pathogens 2022, 11, 595. [Google Scholar] [CrossRef]
  65. Morales, M.; Munne-Bosch, S. Malondialdehyde: Facts and Artifacts. Plant Physiol. 2019, 180, 1246–1250. [Google Scholar] [CrossRef]
  66. Choudhary, D.K.; Johri, B.N. Interactions of Bacillus spp. and plants--with special reference to induced systemic resistance (ISR). Microbiol. Res. 2009, 164, 493–513. [Google Scholar] [CrossRef]
  67. Kildea, S.; Ransbotyn, V.; Khan, M.R.; Fagan, B.; Leonard, G.; Mullins, E.; Doohan, F.M. Bacillus megaterium shows potential for the biocontrol of septoria tritici blotch of wheat. Biol. Control. 2008, 47, 37–45. [Google Scholar] [CrossRef]
  68. Xie, D.; Cai, X.; Yang, C.; Xie, L.; Qin, G.; Zhang, M.; Huang, Y.; Gong, G.; Chang, X.; Chen, H. Studies on the control effect of Bacillus subtilis on wheat powdery mildew. Pest Manag. Sci. 2021, 77, 4375–4382. [Google Scholar] [CrossRef]
  69. Chen, L.; Heng, J.; Qin, S.; Bian, K. A comprehensive understanding of the biocontrol potential of Bacillus velezensis LM2303 against Fusarium head blight. PLoS ONE 2018, 13, e0198560. [Google Scholar] [CrossRef]
  70. Jamal, Q.; Monkhung, S.; Munir, S.; Cho, J.; Moon, J.; Khattak, B.; Malik, M.; Younis, F.; Kim, K. Identification of cyclo (L-Pro-D-Tyr) from Bacillus amyloliquefaciens Y1 exhibiting antifungal activity against Fusarium graminearum to control crown rot in wheat. Appl. Ecol. Environ. Res. 2019, 17, 6299–6314. [Google Scholar] [CrossRef]
  71. Wang, S.; Sun, L.; Zhang, W.; Chi, F.; Hao, X.; Bian, J.; Li, Y. Bacillus velezensis BM21, a potential and efficient biocontrol agent in control of corn stalk rot caused by Fusarium graminearum. Egypt. J. Biol. Pest Control. 2020, 30, 9. [Google Scholar] [CrossRef]
  72. You, W.; Ge, C.; Jiang, Z.; Chen, M.; Li, W.; Shao, Y. Screening of a broad-spectrum antagonist-Bacillus siamensis, and its possible mechanisms to control post-harvest disease in tropical fruits. Biol. Control. 2021, 157, 104584. [Google Scholar] [CrossRef]
  73. Sharma, A.; Kaushik, N.; Sharma, A.; Bajaj, A.; Rasane, M.; Shouche, Y.S.; Marzouk, T.; Djebali, N. Screening of Tomato Seed Bacterial Endophytes for Antifungal Activity Reveals Lipopeptide Producing Bacillus siamensis Strain NKIT9 as a Potential Bio-Control Agent. Front. Microbiol. 2021, 12, 609482. [Google Scholar] [CrossRef]
  74. Santoso, I.; Fadhilah, Q.G.; Safitri, S.D.; Handayani, S.; Maryanto, A.E.; Yasman, Y. Inhibition of the phytopathogenic fungi Curvularia lunata BM and Ganoderma sp. TB4 by antifungal compounds produced by Bacillus siamensis LDR grown on hanjeli (Coix lacryma-jobi L.) starch. Pharmacia 2022, 69, 203–210. [Google Scholar] [CrossRef]
  75. Wang, D.; Li, Y.; Yuan, Y.; Chu, D.; Cao, J.; Sun, G.; Ai, Y.; Cui, Z.; Zhang, Y.; Wang, F.; et al. Identification of non-volatile and volatile organic compounds produced by Bacillus siamensis LZ88 and their antifungal activity against Alternaria alternata. Biol. Control. 2022, 169, 104901. [Google Scholar] [CrossRef]
  76. Park, G.; Nam, J.; Kim, J.; Song, J.; Kim, P.I.; Min, H.J.; Lee, C.W. Structure and Mechanism of Surfactin Peptide from Bacillus velezensis Antagonistic to Fungi Plant Pathogens. Bull. Korean Chem. Soc. 2019, 40, 704–709. [Google Scholar] [CrossRef]
  77. Kim, P.I.; Bai, H.; Bai, D.; Chae, H.; Chung, S.; Kim, Y.; Park, R.; Chi, Y.T. Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26. J. Appl. Microbiol. 2004, 97, 942–949. [Google Scholar] [CrossRef]
  78. Wang, J.; Qiu, J.; Yang, X.; Yang, J.; Zhao, S.; Zhou, Q.; Chen, L. Identification of Lipopeptide Iturin A Produced by Bacillus amyloliquefaciens NCPSJ7 and Its Antifungal Activities against Fusarium oxysporum f. sp. niveum. Foods 2022, 11, 2996. [Google Scholar] [CrossRef]
  79. Zhou, L.; Wang, J.; Wu, F.; Yin, C.; Kim, K.H.; Zhang, Y. Termite Nest Associated Bacillus siamensis YC-9 Mediated Biocontrol of Fusarium oxysporum f. sp. cucumerinum. Front. Microbiol. 2022, 13, 893393. [Google Scholar] [CrossRef]
  80. Feng, X.; Xu, R.; Zhao, N.; Wang, D.; Cun, M.; Yang, B. Isolation, Identification, and Characterization of Endophytic Bacillus from Walnut (Juglans sigillata) Root and Its Biocontrol Effects on Walnut Anthracnose. Agriculture 2022, 12, 2102. [Google Scholar] [CrossRef]
  81. Manikandan, A.; Jaivel, N.; Johnson, I.; Krishnamoorthy, R.; Senthilkumar, M.; Raghu, R.; Gopal, N.O.; Mukherjee, P.K.; Anandham, R. Suppression of Macrophomina root rot, Fusarium wilt and growth promotion of some pulses by antagonistic rhizobacteria. Physiol. Mol. Plant Pathol. 2022, 121, 101876. [Google Scholar] [CrossRef]
  82. Fonseca, R.; Silva, A.; De França, F.; Cardoso, V.; Sérvulo, E. Optimizing carbon/nitrogen ratio for biosurfactant production by a Bacillus subtilis strain. Appl. Biochem. Biotecnol. 2007, 137, 471–486. [Google Scholar] [CrossRef]
  83. Zhao, X.; Han, Y.; Tan, X.Q.; Wang, J.; Zhou, Z.J. Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology. J. Microbiol. 2014, 52, 324–332. [Google Scholar] [CrossRef] [PubMed]
  84. Zou, C.-S.; Mo, M.-H.; Gu, Y.-Q.; Zhou, J.-P.; Zhang, K.-Q. Possible contributions of volatile-producing bacteria to soil fungistasis. Soil Biol. Biochem. 2007, 39, 2371–2379. [Google Scholar] [CrossRef]
  85. Jeong, H.; Jeong, D.E.; Kim, S.H.; Song, G.C.; Park, S.Y.; Ryu, C.M.; Park, S.H.; Choi, S.K. Draft genome sequence of the plant growth-promoting bacterium Bacillus siamensis KCTC 13613T. J. Bacteriol. 2012, 194, 4148–4149. [Google Scholar] [CrossRef]
  86. Poudel, P.; Khanal, S.; Panta, O.P.; Dhakal, S.; Shah, R.; Maharjan, R.; Gautam, A.; Khadka, S.; Sapkota, S. Screening and Optimization of Thermo-Tolerant Bacillus Sp. For Amylase Production and Antifungal Activity. J. Inst. Sci. Technol. 2019, 24, 47–56. [Google Scholar] [CrossRef]
  87. Zhao, M.; Liu, D.; Liang, Z.; Huang, K.; Wu, X. Antagonistic activity of Bacillus subtilis CW14 and its β-glucanase against Aspergillus ochraceus. Food Control. 2021, 131, 108475. [Google Scholar] [CrossRef]
  88. Tomita, M.; Kikuchi, A.; Kobayashi, M.; Yamaguchi, M.; Ifuku, S.; Yamashoji, S.; Ando, A.; Saito, A. Characterization of antifungal activity of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1. Antonie Van Leeuwenhoek 2013, 104, 737–748. [Google Scholar] [CrossRef]
  89. Aktuganov, G.E.; Galimzyanova, N.F.; Melent’ev, A.I.; Kuz’mina, L.Y. Extracellular hydrolases of strain Bacillus sp. 739 and their involvement in the lysis of micromycete cell walls. Microbiology 2007, 76, 413–420. [Google Scholar] [CrossRef]
  90. Chang, W.-T.; Chen, M.-L.; Wang, S.-L. An antifungal chitinase produced by Bacillus subtilis using chitin waste as a carbon source. World J. Microbiol. Biotechnol. 2009, 26, 945–950. [Google Scholar] [CrossRef]
  91. Xu, W.; Yang, Q.; Yang, F.; Xie, X.; Goodwin, P.H.; Deng, X.; Tian, B.; Yang, L. Evaluation and Genome Analysis of Bacillus subtilis YB-04 as a Potential Biocontrol Agent Against Fusarium Wilt and Growth Promotion Agent of Cucumber. Front. Microbiol. 2022, 13, 885430. [Google Scholar] [CrossRef]
  92. Spaepen, S.; Vanderleyden, J.; Remans, R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol. Rev. 2007, 31, 425–448. [Google Scholar] [CrossRef]
  93. Wybouw, B.; De Rybel, B. Cytokinin—A Developing Story. Trends Plant Sci. 2019, 24, 177–185. [Google Scholar] [CrossRef]
  94. Fan, B.; Wang, C.; Song, X.; Ding, X.; Wu, L.; Wu, H.; Gao, X.; Borriss, R. Bacillus velezensis FZB42 in 2018: The gram-positive model strain for plant growth promotion and biocontrol. Front. Microbiol. 2018, 9, 2491. [Google Scholar] [CrossRef]
  95. Suthar, K.; Patel, R.; Singh, D. Polyamine Modulation by antagonistic bacteria Bacillus subtilis in chickpea during Fusarium oxysporum f. sp. ciceri interaction. Legume Res. Int. J. 2021, 44, 109–114. [Google Scholar] [CrossRef]
  96. Ryu, C.M.; Farag, M.A.; Paré, P.W.; Kloepper, J.W. Invisible Signals from the Underground: Bacterial Volatiles Elicit Plant GrowthPromotion and Induce Systemic Resistance. Plant Pathol. J. 2005, 21, 7–12. [Google Scholar] [CrossRef]
  97. Wang, L.; Ning, W.; Mi, D.; Luo, Y.; Guo, J. Poly-γ-glutamic acid productivity of Bacillus subtilis BsE1 has positive function in motility and biocontrol against Fusarium graminearum. J. Microbiol. 2017, 55, 554–560. [Google Scholar] [CrossRef]
Jof 09 00547 g001 550
Figure 1. The in vitro antifungal activity of YB-1631 against F. pseudograminearum. (A) Colonies of F. pseudograminearum on PDA with 5%, 10%, 15%, and 20% of cell-free culture filtrates of YB-1631 grown in LB, Landy, or MSM agar. (B) Inhibitory rate of F. pseudograminearum growth with YB-1631 culture filtrates. (C) Inhibitory rate of F. pseudograminearum growth with YB-1631 volatile substances and inhibitory rate of F. pseudograminearum conidial germination with 66% cell-free culture filtrates of YB-1631 grown in LB. The error bar represents standard deviation (SD), and different letters on the columns in Figure 1B indicate significant differences (p < 0.05) among the concentrations of each culture medium using one-way ANOVA with Tukey’s test.
Figure 1. The in vitro antifungal activity of YB-1631 against F. pseudograminearum. (A) Colonies of F. pseudograminearum on PDA with 5%, 10%, 15%, and 20% of cell-free culture filtrates of YB-1631 grown in LB, Landy, or MSM agar. (B) Inhibitory rate of F. pseudograminearum growth with YB-1631 culture filtrates. (C) Inhibitory rate of F. pseudograminearum growth with YB-1631 volatile substances and inhibitory rate of F. pseudograminearum conidial germination with 66% cell-free culture filtrates of YB-1631 grown in LB. The error bar represents standard deviation (SD), and different letters on the columns in Figure 1B indicate significant differences (p < 0.05) among the concentrations of each culture medium using one-way ANOVA with Tukey’s test.
Jof 09 00547 g001
Jof 09 00547 g002 550
Figure 2. Effect of culture filtrate of YB-1631 on conidial germination of F. pseudograminearum. (A) Morphology of conidia germinating for 12 h in the control treatment. (B) Morphology of conidia germinating for 12 h in 66% LB culture filtrate of YB-1631. Arrows indicate morphological changes in the fungus. Conidia showed lysis (a) and deformation (b), germ tubes showed swelling (c), protoplast condensation (d), shortening of septum intervals (e), wrinkling (f), and lysis (g). The photos were taken with 400× magnifications.
Figure 2. Effect of culture filtrate of YB-1631 on conidial germination of F. pseudograminearum. (A) Morphology of conidia germinating for 12 h in the control treatment. (B) Morphology of conidia germinating for 12 h in 66% LB culture filtrate of YB-1631. Arrows indicate morphological changes in the fungus. Conidia showed lysis (a) and deformation (b), germ tubes showed swelling (c), protoplast condensation (d), shortening of septum intervals (e), wrinkling (f), and lysis (g). The photos were taken with 400× magnifications.
Jof 09 00547 g002
Jof 09 00547 g003 550
Figure 3. Morphology of strain YB-1631 in culture. (A) Colony morphology of YB-1631 on LB agar at 12 h, (B) Gram stain, (C) morphology under scanning electron microscope.
Figure 3. Morphology of strain YB-1631 in culture. (A) Colony morphology of YB-1631 on LB agar at 12 h, (B) Gram stain, (C) morphology under scanning electron microscope.
Jof 09 00547 g003
Jof 09 00547 g004 550
Figure 4. Identification of YB-1631 (A) NJ tree of gyrB sequences of YB-1631 and strains of B. siamensis, B. velezensis, B. amyloliquefaciens, B. subtilis, B. pumilus, B. aerophilus, B. cereus, and B. thuringiensis, (B) heatmap of ANI values based on complete genome sequences of YB-1631 and strains of B. siamensis, B. velezensis, B. amyloliquefaciens, B. licheniformis, and B. subtilis.
Figure 4. Identification of YB-1631 (A) NJ tree of gyrB sequences of YB-1631 and strains of B. siamensis, B. velezensis, B. amyloliquefaciens, B. subtilis, B. pumilus, B. aerophilus, B. cereus, and B. thuringiensis, (B) heatmap of ANI values based on complete genome sequences of YB-1631 and strains of B. siamensis, B. velezensis, B. amyloliquefaciens, B. licheniformis, and B. subtilis.
Jof 09 00547 g004
Jof 09 00547 g005 550
Figure 5. Effect of strain YB-1631 on 21-day-old wheat seedlings with and without F. pseudograminearum inoculation. (A) Potted seedlings at 21 days after sowing. (B) Seedlings with soil removed. CK = sowing seeds with sdH2O treatment into sterile soil treatment, YB-1631 = sowing seeds with 108 cells/mL YB-1631 treatment into sterile soil treatment, YB-1631 + Fp = sowing seeds with 108 cells/mL YB-1631 treatment into sterile soil with 2% F. pseudograminearum soil inoculum treatment, and Fp = sowing seeds with sdH2O treatment into sterile soil with 2% F. pseudograminearum soil inoculum treatment.
Figure 5. Effect of strain YB-1631 on 21-day-old wheat seedlings with and without F. pseudograminearum inoculation. (A) Potted seedlings at 21 days after sowing. (B) Seedlings with soil removed. CK = sowing seeds with sdH2O treatment into sterile soil treatment, YB-1631 = sowing seeds with 108 cells/mL YB-1631 treatment into sterile soil treatment, YB-1631 + Fp = sowing seeds with 108 cells/mL YB-1631 treatment into sterile soil with 2% F. pseudograminearum soil inoculum treatment, and Fp = sowing seeds with sdH2O treatment into sterile soil with 2% F. pseudograminearum soil inoculum treatment.
Jof 09 00547 g005
Jof 09 00547 g006 550
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.
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.
Jof 09 00547 g006
Jof 09 00547 g007 550
Figure 7. Plant growth promotion and biocontrol traits of strain YB-1631. (A) IAA, the upper plate is the non-inoculated broth control, the lower plate is the YB-1631 culture broth, where the broth was placed in a hole in the center of the plate, and the pink color of the broth indicated the presence of IAA. Activities of (B) β-1, 3-glucanase, (C) siderophores, (D) phosphorus solubilization, (E) amylase, (F) cellulase, and (G) protease were visible as transparent circles or light-colored areas around the YB-1631 colony in the center of each plate.
Figure 7. Plant growth promotion and biocontrol traits of strain YB-1631. (A) IAA, the upper plate is the non-inoculated broth control, the lower plate is the YB-1631 culture broth, where the broth was placed in a hole in the center of the plate, and the pink color of the broth indicated the presence of IAA. Activities of (B) β-1, 3-glucanase, (C) siderophores, (D) phosphorus solubilization, (E) amylase, (F) cellulase, and (G) protease were visible as transparent circles or light-colored areas around the YB-1631 colony in the center of each plate.
Jof 09 00547 g007
Table
Table 1. The width of no fungal growth between the F. pseudograminearum and soil bacterial colonies on PDA.
Table 1. The width of no fungal growth between the F. pseudograminearum and soil bacterial colonies on PDA.
StrainWidthStrainWidth
Mq202003-12.33 ± 0.29 lmMq202003-304.60 ± 0.17 abcde
Mq202003-21.17 ± 0.29 noMq202003-313.40 ± 0.17 hi
Mq202003-34.17 ± 0.29 cdefgMq202003-324.07 ± 0.12 cdefg
Mq202003-42.83 ± 0.29 jkMq202003-333.87 ± 0.12 fgh
Mq202003-50.67 ± 0.29 opqMq202003-342.07 ± 0.12 m
Mq202003-80.40 ± 0.17 pqMq202003-364.40 ± 0.17 bcdef
Mq202003-94.83 ± 0.29 abMq202003-374.07 ± 0.12 defg
Mq202003-104.67 ± 0.29 abcMq202003-381.23 ± 0.25 no
Mq202003-114.00 ± 1.00 fgMq202003-392.40 ± 0.17 lm
Mq202003-124.17 ± 0.29 cdefgMq202003-403.67 ± 0.29 ghi
Mq202003-130.83 ± 0.29 nopMq202003-413.93 ± 0.12 fg
Mq202003-141.00 ± 0.00 noMq202003-423.13 ± 0.12 ijk
Mq202003-152.50 ± 0.50 lmMq202003-432.33 ± 0.29 lm
Mq202003-165.07 ± 0.12 aMq202003-444.17 ± 0.29 cdefg
Mq202003-174.23 ± 0.25 cdefgMq202003-454.67 ± 0.29 ab
Mq202003-184.17 ± 0.29 cdefgMq202003-474.40 ± 0.17 bcdef
Mq202003-193.17 ± 0.29 ijkMq202003-481.33 ± 0.29 n
Mq202003-202.50 ± 0.50 lmMq202003-493.33 ± 0.29 ij
Mq202003-214.17 ± 0.29 cdefgMq202003-502.17 ± 0.29 lm
Mq202003-220.83 ± 0.29 nopMq202003-512.67 ± 0.29 kl
Mq202003-234.67 ± 0.29 abcMq202003-530.83 ± 0.29 nop
Mq202003-243.17 ± 0.29 ijkMq202003-540.67 ± 0.29 opq
Mq202003-252.50 ± 0.50 lmMq202003-561.33 ± 0.29 n
Mq202003-270.23 ± 0.25 qMq202003-571.40 ± 0.17 n
Mq202003-280.67 ± 0.29 opqMq202003-584.83 ± 0.29 ab
Data in the table are mean ± standard deviation (SD). Means with a different letter (a–q) after the antifungal width indicates significant difference (p < 0.05) using one-way ANOVA with Tukey’s test.
Table
Table 2. Effect of YB-1631 on wheat seedling growth and FCR levels. CK indicates dsH2O-treated seeds and no F. pseudograminearum inoculation, YB-1631 indicates YB-1631-treated seeds and no F. pseudograminearum inoculation, Fp indicates dsH2O-treated seeds and F. pseudograminearum inoculation, YB-1631 + Fp indicates YB-1631-treated seeds and F. pseudograminearum inoculation.
Table 2. Effect of YB-1631 on wheat seedling growth and FCR levels. CK indicates dsH2O-treated seeds and no F. pseudograminearum inoculation, YB-1631 indicates YB-1631-treated seeds and no F. pseudograminearum inoculation, Fp indicates dsH2O-treated seeds and F. pseudograminearum inoculation, YB-1631 + Fp indicates YB-1631-treated seeds and F. pseudograminearum inoculation.
TreatmentRoot Length (cm)Plant Height (cm)Root Fresh Weight (mg)Total Fresh Weight (mg)FCR Incidence (FCRI %)Disease Severity Index (DSI %)Relative Control Effect (RCE %)
CK8.89 ± 0.15 b25.29 ± 0.58 b26.36 ± 1.13 c407.40 ± 6.34 c
YB-163112.19 ± 0.52 a28.90 ± 0.73 a31.88 ± 0.58 a449.60 ± 10.48 a
Fp6.41 ± 0.19 c18.15 ± 1.63 c13.80 ± 2.56 d225.40 ± 15.87 d90.75 ± 3.67 a48.69 ± 1.33 a
YB-1631 + Fp12.07 ± 0.18 a27.98 ± 0.40 b29.80 ± 0.84 b426.20 ± 5.84 b14.50 ± 1.60 b8.17 ± 0.93 b83.23 ± 1.75
Data in the table are mean ± standard deviation (SD). Means with a different letter (a–d) in each column indicate significant difference (p < 0.05) between treatments using one-way ANOVA with Tukey’s test.
Table
Table 3. Activities of polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), peroxidase (POD), lipoxygenase (LOX), and MDA content in treated wheat seedlings. Treatments were water (CK), bacteria (YB-1631), F. pseudograminearum inoculation (Fp), YB-1631, and F. pseudograminearum inoculation (YB-1631 + Fp).
Table 3. Activities of polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD), peroxidase (POD), lipoxygenase (LOX), and MDA content in treated wheat seedlings. Treatments were water (CK), bacteria (YB-1631), F. pseudograminearum inoculation (Fp), YB-1631, and F. pseudograminearum inoculation (YB-1631 + Fp).
TreatmentPPO (U/g)CAT (U/g)PAL (U/g)SOD (U/g)POD (U/g)LOX (U/g)MDA (nmol/g)
CK22.38 ± 1.04 d143.56 ± 3.11 d24.86 ± 2.38 c9.61 ± 1.54 c15,175.45 ± 249.40 d752.40 ± 13.14 d21.36 ± 0.79 b
YB-163125.5 ± 0.67 c156.03 ± 5.19 c34.17 ± 2.38 b24.64 ± 1.70 b16,674.29 ± 384.66 c3261.24 ± 94.44 c16.66 ± 2.15 c
Fp38.06 ± 0.46 b238.68 ± 3.48 b37.20 ± 1.37 b31.66 ± 1.64 a34,486.16 ± 544.57 b1008.90 ± 22.57 b37.88 ± 1.52 a
YB-1631 + Fp45.89 ± 1.28 a567.73 ± 10.22 a51.76 ± 0.84 a34.53 ± 0.70 a42,529.88 ± 651.12 a4022.64 ± 77.42 a20.93 ± 2.44 c
Data in the table are mean ± standard deviation (SD). Means with a different letter (a–d) in each column indicate significant difference (p < 0.05) between treatments using one-way ANOVA with Tukey’s test.
Table
Table 4. Putative gene clusters for synthesis of secondary metabolites in B. siamensis YB-1631 genome analyzed by antiSMASH.
Table 4. Putative gene clusters for synthesis of secondary metabolites in B. siamensis YB-1631 genome analyzed by antiSMASH.
ClustersTypesFromToMost Similar Known ClustersSimilarity
Cluster 1NRPS337,277401,867Surfactin biosynthetic gene cluster from B. velezensis FZB4282%
Cluster 2NRPS1,865,3261,973,480Fengycin biosynthetic gene cluster from B. velezensis FZB4293%
Cluster 3NRPS3,131,5643,183,345Bacillibactin biosynthetic gene cluster from B. subtilis subsp. subtilis str. 168100%
Cluster 4TransAT-PKS1,709,5981,809,529Bacillaene biosynthetic gene cluster from B. velezensis FZB4292%
Cluster 5TransAT-PKS2,383,5112,477,284Difficidin biosynthetic gene cluster from B. velezensis FZB42100%
Cluster 6PKS-like1,011,8001,053,044Butirosin A biosynthetic gene cluster from B. circulans7%
Cluster 7terpene1,137,5291,154,562None
Cluster 8terpene2,002,9462,023,869None
Cluster 9NRP + Polyketide1,882,0311,926,708Bacillomycin D biosynthetic gene cluster from B. velezensis FZB4284%
Cluster 10NRP + Polyketide199,769267,925Locillomycin biosynthetic gene cluster from B. subtilis21%
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Dong, Q.; Liu, Q.; Goodwin, P.H.; Deng, X.; Xu, W.; Xia, M.; Zhang, J.; Sun, R.; Wu, C.; Wang, Q.; et al. Isolation and Genome-Based Characterization of Biocontrol Potential of Bacillus siamensis YB-1631 against Wheat Crown Rot Caused by Fusarium pseudograminearum. J. Fungi 2023, 9, 547. https://doi.org/10.3390/jof9050547

AMA Style

Dong Q, Liu Q, Goodwin PH, Deng X, Xu W, Xia M, Zhang J, Sun R, Wu C, Wang Q, et al. Isolation and Genome-Based Characterization of Biocontrol Potential of Bacillus siamensis YB-1631 against Wheat Crown Rot Caused by Fusarium pseudograminearum. Journal of Fungi. 2023; 9(5):547. https://doi.org/10.3390/jof9050547

Chicago/Turabian Style

Dong, Qianqian, Qingxiang Liu, Paul H. Goodwin, Xiaoxu Deng, Wen Xu, Mingcong Xia, Jie Zhang, Runhong Sun, Chao Wu, Qi Wang, and et al. 2023. "Isolation and Genome-Based Characterization of Biocontrol Potential of Bacillus siamensis YB-1631 against Wheat Crown Rot Caused by Fusarium pseudograminearum" Journal of Fungi 9, no. 5: 547. https://doi.org/10.3390/jof9050547

APA Style

Dong, Q., Liu, Q., Goodwin, P. H., Deng, X., Xu, W., Xia, M., Zhang, J., Sun, R., Wu, C., Wang, Q., Wu, K., & Yang, L. (2023). Isolation and Genome-Based Characterization of Biocontrol Potential of Bacillus siamensis YB-1631 against Wheat Crown Rot Caused by Fusarium pseudograminearum. Journal of Fungi, 9(5), 547. https://doi.org/10.3390/jof9050547

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop