Lipopeptide C17 Fengycin B Exhibits a Novel Antifungal Mechanism by Triggering Metacaspase-Dependent Apoptosis in Fusarium oxysporum.

Fusarium wilt is a worldwide soil-borne fungal disease caused by Fusarium oxysporum that causes serious damage to agricultural products. Therefore, preventing and treating fusarium wilt is of great significance. In this study, we purified ten single lipopeptide fengycin components from Bacillus subtilis FAJT-4 and found that C17 fengycin B inhibited the growth of F. oxysporum FJAT-31362. We observed early apoptosis hallmarks, including reactive oxygen species accumulation, mitochondrial dysfunction, and phosphatidylserine externalization in C17 fengycin B-treated F. oxysporum cells. Further data showed that C17 fengycin B induces cell apoptosis in a metacaspase-dependent manner. Importantly, we found that the expression of autophagy-related genes in the TOR signaling pathway was significantly upregulated; simultaneously, the accumulation of acidic autophagy vacuoles in F. oxysporum cell indicated that the autophagy pathway was activated during apoptosis induced by C17 fengycin B. Therefore, this study provides new insights into the antifungal mechanism of fengycin.

Lipopeptides from Bacillus velezensis induced apoptosis-like cell death in the pathogenic fungus Fusarium concentricum.

AIMS: Stem rot caused by Fusarium concentricum is a new disease of Paris polyphylla reported by our research group. The present study investigates the growth inhibitory and apoptotic effects of Bacillus velezensis FJAT-54560 lipopeptide against F. concentricum. METHODS AND RESULTS: HPLC preparation and LC-MS analysis results show that the crude lipopeptides secreted by Bacillus velezensis FJAT-54560 isolated from Jasminum sambac consist of C14-17 iturin A, C14 fengycin B, C16 fengycin A/A2, C18 fengycin A, C20 fengycin B2, C21 fengycin A2, C22-23 fengycin A, C12-16 surfactin A, and C15 surfactin A derivatives. The mass ratios (g/g) of iturin, fengycin, and surfactin in lipopeptides are 2.40, 67.51, and 30.08%, respectively. Through inhibition zone and inhibition rate experiments, we found that crude lipopeptides and purified fengycin exhibit strong antifungal activity against F. concentricum, including accumulation of reactive oxygen species, loss of mitochondrial membrane potential, DNA fragmentation, Ca2+ accumulation, chromatin condensation, and phosphatidylserine externalization. Transcriptomic analysis indicates that crude lipopeptide-induced apoptosis in F. concentricum cells may be mediated by apoptosis-inducing factors and apoptosis mediators and can serve as a metacaspase-independent model. CONCLUSION: Lipopeptides from Bacillus velezensis FJAT-54560 can control the pathogenic fungus F. concentricum by inducing apoptosis.

First Report of Apiospora arundinis Causing Wet Root Rot of Pseudostellaria heterophylla in China.

Pseudostellaria heterophylla is one of the traditional medicines in China. From 2020 to 2022, postharvest wet root rot disease was observed with an incidence of 2~5% on the tuberous roots of the harvested P. heterophylla in Zherong county, Fujian province, China, which usually occurs under damp and unventilated conditions. The symptoms of the disease were as follows: white mycelia grew on the surface of tuberous root initially and gradually wrapped around the roots, the internal root tissue turned yellow and became wet decay finally. To identify the causal agent, a total of 20 samples with symptomatic tuberous roots were collected. Small pieces (3 mm2) were treated by surface disinfection with 75% ethanol and 1% NaOCl, then rinsed 3 times with sterile water. These treated pieces were transferred onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 7 d. Ten pure cultures were obtained using single-spore isolation method. The fungus colonies initially produced white aerial mycelium, subsequently exhibited yellow pigmentation. Mycelia were consisted of smooth, hyaline, branched, and septate hyphae. The conidia were solitary or clustered, brown or dark brown, smooth, ellipsoidal to spherical, 6.66 (5.50-7.81)×5.65 (4.17-7.22) µm (n=50) in size. The conidiophores were hyaline or pale brown and produced conidiogenous cells, which were pale brown, smooth, ampulliform, and 10.14 (8.82-15.30) um long (n=50). Based on these morphological characteristics, the fungus was identified as the genus Apiospora (Arthrinium). The rDNA-ITS region and partial ß-tubulin gene (BenA) were amplified using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences of isolates FJAT-32563 and FJAT-32564 were deposited in GenBank (ITS, OM920984 and OM920985; BenA, OM953823 and OM953824). All sequences had more than 99% similarity with those of A. arundinis strain CBS:106.12 (ITS, KF144883; BenA, KF144973). In the multilocus phylogenetic analysis (ITS + BenA), the two isolates clustered together with other strains of A. arundinis with 100% bootstrap support. The isolates were therefore identified as A. arundinis based on both morphological and molecular characteristics. To confirm the pathogenicity, fresh tuberous roots were selected and surface disinfected, then the roots were immersed with a quarter length in the conidial suspension (106/mL) for 30 min, whereas the control roots were immersed with sterile water (n=30). They were placed in petri dish with wet filter paper at 25±2℃, maintaining 80% relative humidity in the dark. The white aerial mycelium appeared at 5 days after inoculation, and wet root rot decaying occurred after inoculation for 21 days. The symptoms were similar to those described above, whereas the control roots were asymptomatic. The same fungus was re-isolated from the infected roots, showing similar morphological characteristics and molecular traits. Koch's postulates were completed and the pathogenicity test for the isolates has been repeated thrice. Previously, A. arundinis was reported to infect peach and sugarcane (Ji et al. 2020; Liao et al. 2022). To our knowledge, this is the first report of A. arundinis causing wet root rot of P. heterophylla in China. The disease would be a potentially new threat to this medicinal plant.

Antifungal activities of Bacillus velezensis FJAT-52631 and its lipopeptides against anthracnose pathogen Colletotrichum acutatum.

This study was aim at investigating antifungal activities of Bacillus velezensis FJAT-52631 and its lipopeptides against Colletotrichum acutatum ex situ and in situ. The results showed that the strain FJAT-52631 and its crude lipopeptides (10 mg/ml) exhibited strong inhibitory effects on growth of C. acutatum FJAT-30256 with an inhibition rate of 75.3% and an inhibition zone diameter of 17.66 mm, respectively. Both the viable bacterial cultures and lipopeptides of FJAT-52631 could delay the onset of loquat anthracnose by 1 day and lower the incidence of loquat anthracnose in situ. The whole cultures of B. velezensis FJAT-52631 displayed a 50% biocontrol efficacy on loquat anthracnose at the fourth day after inoculation, but the crude lipopeptides not. The average lesion diameter of the whole-culture treated group was 5.62 mm, which was smaller than that of control group (6.81 mm). All the three types of lipopeptides including iturin A, fengycin, and surfactin A secreted from the strain FJAT-52631 exhibited antifungal activities. Among them, surfactin A displayed higher antifungal activity at a concentration of 1.25 mg/mL than other two lipopeptides even if at a concentration of 60 mg/mL. Thus, the results indicated that surfactin A produced by FJAT-52631 played a major role in the biocontrol of the loquat anthracnose. Scanning electron microscopy (SEM) observation revealed the structural deformities in the mycelia of C. acutatum. The above results suggested that the antifungal lipopeptides from B. velezensis FJAT-52631 would be potential in biocontrol against anthracnose disease of loquat caused by C. acutatum.

Effects of Polygonatum cyrtonema extracts on the antioxidant ability, physical and structure properties of carboxymethyl cellulose-xanthan gum-flaxseed gum active packaging films.

A novel film composed of Polygonatum cyrtonema extracts (PCE), xanthan gum (XG), flaxseed gum (FG) and carboxymethyl cellulose (CMC) was prepared (XFCP). Addition of PCE has decreased the light transmittance, while enhanced the UV blocking performance, antioxidant activity, tensile strength and elongation at break of XFCP due to polysaccharides, polyphenols, and flavonoid in PCE. Structural analyses by FTIR and XRD indicated the hydrogen-bonding interaction between PCE, XG, FG and CMC. It was found that compared with the control sample, XFCP2.5% with the lowest WVTR was able to prolong the shelf life of mango. The overall quality of mango was also improved in terms of lower decay rate, weight loss rate, total soluble solid, and polyphenol oxidase, higher titratable acidity, Vc, and superoxide dismutase than control mango upon 8 days of storage. This effectively expanded the application of PCE into food packaging in addition to merely as Chinese traditional medicine herbs.

First Report of Green Mold Disease Caused by Penicillium ochrochloron on Medicinal Plant Pseudostellaria heterophylla in China.

Pseudostellaria heterophylla is one of the Chinese herbal medicines with high medicinal and economic values. From 2019 to 2021, postharvest green mold disease was observed with an incidence of 2~5% on the tuberous roots of the harvested P. heterophylla at eight locations in Zherong county, Fujian Province, China. The symptoms were as follows: white mycelial growth on the tuberous roots surface initially, then green mold layers forming, and the tuberous roots decaying finally. To identify the causal agent, a total of 20 symptomatic tuberous roots were collected. Small pieces (5 mm×5 mm) were treated by surface disinfestion with 75% ethanol and 1% NaOCl, then rinsed 3 times with sterile distilled water. These treated pieces were transferred onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 7 days. Twenty pure cultures were obtained by single-spore isolation method. Colonies on PDA medium initially appeared as white mycelium that developed grayish-green conidia with white margins. Mycelium was septate and colorless. Conidiophores were predominantly monoverticillate, occasionally biverticillate. Stipes was long and slender. Phialides were ampulliform to almost cylindrical with collula, 11.25 (7.80-23.50) µm long (n=50). Conidia were smooth walled and pale green, with globose to ellipsoidal shape, 2.75 (2.37-3.27)× 2.47 (2.18-3.13) µm (n=50). Based on these morphological characteristics, the isolates matched the description of the genus Penicillium. Genomic DNAs from two representative isolates (FJAT-32578 and FJAT-32579) were extracted with a fungal genomic DNA extraction kit. The rDNA ITS region and partial ß-tubulin gene (BenA) were amplified using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences of isolate FJAT-32578 and FJAT-32579 were deposited in GenBank (ITS, OM920986 and OM920987; BenA, OM953825 and OM953826). All sequences showed above 99% similarity to P. ochrochloron type strain CBS357.48 (ITS, NR111509; BenA, GU981672). In multilocus phylogenetic analysis (ITS + BenA), the two isolates from this study clustered together with other strains of P. ochrochloron with 100% bootstrap support. The two isolates were thus identified as P. ochrochloron based on both morphological and molecular characteristics. Pathogenicity tests were conducted in triplicate by inoculating the aseptic wounds with 10 µl of conidial suspension (1×106 conidia/ml) of the two isolates in the each healthy tuberous root (cv. Zheshen No.1). The experiment was conducted twice. All the inoculated tuberous roots were placed in sterilized Petri dishes with moistened filter paper, and incubated at 25 ± 2 °C. Fifteen days after inoculation, all inoculated tuberous roots demonstrated the same symptoms as those observed in the field conditions. The re-isolated fungi from the artificially infected tuberous roots were confirmed as P. ochrochloron using the method described above, while the control tuberous roots treated with sterile water did not develop symptoms, fulfilling Koch's postulates. To our knowledge, this is the first report of P. ochrochloron causing green mold disease on P. heterophylla in China, which would be a potentially new threat to the medicinal plant.

An avirulent Ralstonia solanacearum strain FJAT1458 outcompetes with virulent strain and induces tomato plant resistance against bacterial wilt.

BACKGROUND: Bacterial wilt (BW) caused by Ralstonia solanacearum (RS) is considered as one of the most destructive plant diseases. An avirulent strain of RS, FJAT1458, is a potential biocontrol agent of BW. In this study, the mechanism of FJAT1458 against BW was evaluated. RESULTS: FJAT1458 was tagged with the red fluorescent protein gene, and the resulting strain was named as FJAT1458-RFP. When FJAT1458-RFP and FJAT91-GFP (a virulent strain of RS labelled with the green fluorescent protein gene), were co-inoculated in potted tomato plants, the colonization of FJAT91-GFP reached an almost undetectable level at 7 days post-inoculation (dpi) in the roots and at 9 dpi in rhizosphere soil. When they were co-inoculated in a hydroponic tomato growing system, numbers of the two strains were similar at 3 dpi in the root tissues; however, FJAT91-GFP was not detected at 9 dpi while FJAT1458-RFP maintained 1.77 × 105 CFU g-1 . The inoculation of FJAT1458-RFP alone or combination with FJAT91-GFP significantly increased tomato root activity. Moreover, expression levels of the defense-related genes PR-1a, GLUA, and CHI3 in tomato roots were significantly up-regulated by FJAT1458-RFP and co-inoculation of FJAT1458-RFP and FJAT91-GFP at 5 dpi, compared to the control (water, CK) treatment. Noteworthy, expression levels of GLUA in the treatments of FJAT1458-RFP and FJAT1458-RFP + FJAT91-GFP were 12.22- and 12.05-fold higher than that in the CK at 5 dpi, respectively. CONCLUSIONS: The results suggested that the avirulent strain FJAT1458-RFP could suppress colonization of the virulent strain in tomato roots, and induce tomato plant resistance against BW. © 2022 Society of Chemical Industry.

Effect of metal ions on lipopeptide secretion from Bacillus subtilis strain FJAT-4: Negative regulation by Ca2.

AIMS: This study aimed to investigate the effect of metal ions on lipopeptide production by Bacillus subtilis strain FJAT-4 and the mechanism of negative regulation by Ca2+ . METHODS AND RESULTS: The quantitative measurement of lipopeptides in response to K+ , Na+ , Mg2+ and Ca2+ addition was carried out by LC-MS. The contents of fengycin and surfactin varied within the range of 116.24-129.80 mg/L and 34.03-63.11 mg/L in the culture media containing K+ , Na+ and Mg2+ , while the levels were 0.86 and 0.63 mg/L in the media containing Ca2+ . Ca2+ at a high concentration (45 mM) did not adversely affect the growth of strain FJAT-4, but caused significant downregulation of lipopeptide synthesis-related gene expression, corresponding to a decrease in lipopeptide production. This inhibition by Ca2+ was further investigated by proteomic analysis. In total, 112 proteins were upregulated and 524 proteins were downregulated in the presence of additional Ca2+ (45 mM). Among these differentially expressed proteins (DEPs), 28 were related to phosphotransferase activity, and 42 were related to kinase activity. The proteomics results suggested that altered levels of three two-component signal-transduction systems (ResD/ResE, PhoP/PhoR and DegU/DegS) might be involved in the control of expression of the fen and srfA operons of FJAT-4 under high calcium stress. CONCLUSIONS: The Ca2+ at the high concentration (45 mM) triggers a decrease in lipopeptide production, which might be attributed to the regulation of three two-component signal-transduction systems ResD/ResE, PhoP/PhoR and DegU/DegS. SIGNIFICANCE AND IMPACT OF THE STUDY: The regulatory effect of calcium on the expression of genes encoding lipopeptide synthetases can be applied to optimize the production of lipopeptides.

Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides.

BACKGROUND: There is an urgent need to discover biocontrol agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds. RESULTS: Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20 °C to 40 °C. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20 °C and 25 °C. CONCLUSIONS: The B. velezensis strain FJAT-46737 and its secreted lipopeptides could be used as new sources of potential biocontrol agents against several plant pathogens, and especially the bacterial wilt pathogen R. solanacearum.

Complete Genome Sequence of Ralstonia solanacearum FJAT-91, a High-Virulence Pathogen of Tomato Wilt.

Ralstonia solanacearum FJAT-91, which displays higher virulence toward plants belonging to the family Solanaceae, was isolated from a wilted tomato plant vessel in Fujian province, southeast China. Here, we report the complete genome sequence of R. solanacearum FJAT-91 using long-read single-molecule PacBio sequencing technology. The genome comprises a 3,873,214-bp circular chromosome and a 2,000,873-bp circular megaplasmid with an overall G+C content of 66.85%.

Draft Genome Sequence of Bacillus farraginis R-6540T (DSM 16013), a Spore-Forming Bacterium Isolated at Dairy Farms.

Bacillus farraginis R-6540(T) is a Gram-positive, aerobic, and spore-forming bacterium with very high intrinsic heat resistance. Here, we report the 5.32-Mb draft genome sequence of B. farraginis R-6540(T), which is the first genome sequence of this species and will promote its fundamental research.

Draft Genome Sequence of Bacillus shackletonii LMG 18435T, Isolated from Volcanic Mossy Soil.

Bacillus shackletonii LMG 18435(T) is a Gram-positive, aerobic, and spore-forming bacterium. Here, we report the 5.30-Mb draft genome sequence of B. shackletonii LMG 18435(T), which will promote its fundamental research and provide useful information for genomic taxonomy and phylogenomics of Bacillus-like bacteria.

Draft Genome Sequence of Bacillus plakortidis P203T (DSM 19153), an Alkali- and Salt-Tolerant Marine Bacterium.

Bacillus plakortidis P203(T) is a Gram-positive, spore-forming, and alkali- and salt-tolerant marine bacterium. Here, we report the 3.97-Mb draft genome sequence of B. plakortidis P203(T), which will promote its fundamental research and provide useful information for genomic taxonomy and phylogenomics of Bacillus-like bacteria.

Draft Genome Sequence of Bacillus humi LMG 22167T (DSM 16318), an Endospore-Forming Bacterium Isolated from Soil.

Bacillus humi LMG 22167(T) is a Gram-positive, aerobic, and spore-forming bacterium Here, we report the 4.80-Mb draft genome sequence of B. humi LMG 22167(T), which is the first genome sequence of this species and will promote its fundamental research.

High-Quality Draft Genome Sequence of Aneurinibacillus migulanus ATCC 9999T (DSM 2895), a Gramicidin S-Producing Bacterium Isolated from Garden Soil.

Aneurinibacillus migulanus ATCC 9999(T) (DSM 2895) is a Gram-positive, round-spore-forming, and gramicidin S-producing bacterium. Here, we report the 6.35-Mb high-quality draft genome sequence of A. migulanus ATCC 9999(T), which will provide useful information for the genomic taxonomy and phylogenomics of Bacillus-like bacteria.

Genome Sequence of Bacillus butanolivorans K9T (DSM 18926), an n-Butanol-Consuming Bacterium Isolated from Soil.

Bacillus butanolivorans K9(T) (DSM 18926) is a Gram-positive, spore-forming, strictly aerobic, and n-butanol-consuming bacterium. Here, we report the 5.68-Mb genome sequence of B. butanolivorans K9(T), which is the first genomic information of this species that will provide useful information for the genomic taxonomy and phylogenomics of Bacillus-like bacteria.

Draft Genome Sequence of Bacillus murimartini LMG 21005T, an Alkalitolerant Bacterium Isolated from a Church Wall Mural in Germany.

Bacillus murimartini LMG 21005(T) is a Gram-positive, spore-forming, and alkalitolerant bacterium isolated from a church wall mural. Here, we report the 4.17-Mb genome sequence of B. murimartini LMG 21005(T), which will accelerate the application of this alkalitolerant bacterium and provide useful information for genomic taxonomy and phylogenomics of Bacillus-like bacteria.

Draft Genome Sequence of Sporosarcina globispora W 25T (DSM 4), a Psychrophilic Bacterium Isolated from Soil and River Water.

Sporosarcina globispora W 25(T) (DSM 4) is a Gram-positive, round-spore-forming, and psychrophilic bacterium. Here, we report the 5.66-Mb genome sequence of S. globispora W 25(T), which will accelerate the application of this psychrophile and provide useful information for genomic taxonomy and phylogenomics of Bacillus-like bacteria.

Draft Genome Sequence of Bacillus pseudalcaliphilus PN-137T (DSM 8725), an Alkaliphilic Halotolerant Bacterium Isolated from Garden Soils.

Bacillus pseudalcaliphilus PN-137(T) (DSM 8725) is a Gram-positive, spore-forming, alkaliphilic, and halotolerant bacterium. Here, we report the 4.49-Mb genome sequence of B. pseudalcaliphilus PN-137(T), which will accelerate the application of this alkaliphile and provide useful information for genomic taxonomy and phylogenomics of Bacillus-like bacteria.

Bacillus taiwanensis sp. nov., isolated from a soil sample from Taiwan.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium (FJAT-14571(T)) was isolated from a soil sample in Taiwan. Strain FJAT-14571(T) grew at 20-40 °C (optimum 35 °C), pH 6-10 (optimum pH 8) and 0-2% (w/v) NaCl (optimum 0%). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain FJAT-14571(T) was a member of the genus Bacillus and was most closely related to Bacillus oceanisediminis DSM 24771(T) (96.2%). DNA-DNA relatedness between strain FJAT-14571(T) and B. oceanisediminis DSM 24771(T) was low (32.0% ± 0.88%). The diagnostic diamino acid of the peptidoglycan of strain FJAT-14571(T) was meso-diaminopimelic acid and the predominant menaquinone was MK-7 (96.6%). The major cellular fatty acids were iso-C15 : 0 (46.4%), anteiso-C15 : 0 (7.6%), iso-C17 : 0 (8.2%) and iso-C16 : 0 (10.0 %) and the DNA G+C content was 40.8 mol%. Phenotypic, chemotaxonomic and genotypic properties clearly indicated that strain FJAT-14571(T) represents a novel species within the genus Bacillus, for which the name Bacillus taiwanensis sp. nov. is proposed. The type strain is FJAT-14571(T) ( = DSM 27845(T) = CGMCC1.1 2698(T)).