Fig 1.
Neighbour-joining phylogenetic tree of strain LM2303.
The tree was constructed using the Neighbour-joining method by MEGA 7.0based on 2351 single-copy core genes from 15 related strains with 1500 replications in bootstrap test. Bar, 0.05 nucleotide substitutions per site.
Table 1.
Comparison of secondary metabolite biosynthetic gene clusters in the B. velezensis strain.
Fig 2.
Antimicrobial activity of strain LM2303 against various pathogenic microbes.
a1: a 6-mm agar plug of F. graminearum is inoculated on the center of PDA plate for 6d at 28°C. a2-3: strain LM2303 is simultaneous inoculated with 3cm apart from the plug of F. graminearum. b1: F. culmorum, b2: A. flavus, b3: F. moniliforme, b4: Coniothyrium olivaceum, b5: Rhizomorpha Roth. ex Fr, b6: Alternaria tenuissima, c1: X. campestris, c2: Staphylococcus aureus, c3: Sarcine luted.
Fig 3.
Fuorescence microscopy analyses of F. graminearumon hyphae treated with LM2303 CLPs.
(A) untreated hyphae under white light. (B) untreated hyphae under green fluorescence. (C) treated hyphae under white light. (D) treated hyphae under green fluorescence. Hyphae were treated with 200 mg/mL LM2303 lipopeptides for 4h min at 28°C. All observations were performed by using confocal laser scanning microscopy (Olympus FV1200). Bar = 50μm. Propidium iodide is a popular red-fluorescent nuclear and chromosome counterstain, which is not permeant to normal cell membranes. So PI was used at 1mg/mL as a vital stain based on visualizing membrane permeability.
Fig 4.
The biosynthetic gene cluster and MS analysis of Fengycin A in strain LM2303.
Ions of m/z values 1449.7838, 1463.8002, 1477.8156, 1491.8315 and 1505.8478 were assigned to C13-17 Fenygcin B [M+H]+, repectively.
Fig 5.
The biosynthetic gene cluster and MS analysis of Iturin A in strain LM2303.
Ions of m/z values 1043.5482, 1057.5630, 1071.5787 and 1085.5953 were assigned to C14-17 Iturin A [M+H]+, and ions of m/z values 1065.5297, 1079.5441, 1093.5592 and 1107.5570 were assigned to C14-17 Iturin A [M+Na]+, repectively.
Fig 6.
The biosynthetic gene cluster and MS analysis of Surfactin A in strain LM2303.
Ions of m/z values 994.6405, 1008.6565, 1022.6711, 1036.6881 and 1050.7035 were assigned to C12-17 Surfactin A [M+H]+, and ions of m/z values 1002.6056, 1016.6213, 1030.6367, 1044.6508, 1058.6680 and 1072.6837 were assigned to C12-17 Surfactin A [M+Na]+, repectively.
Fig 7.
Secondary metabolite gene clusters with antibacterial metabolites in strain LM2303.
The above gene clusters were identified by antiSMASH 4.0.0rc1 and further Blastp alignment. The biosynthetic gene cluster with single letter codes above each ORF.
Fig 8.
MS analysis of antibacterial secondary metabolites from strain LM2303.
(A) m/z 1336.4773: plantazolicin [M+H]+, m/z 1354.4850: hydrolyzed plantazolicin [M+H]+; (B) m/z 271.1288: Bacilysin [M+H]+; (C1) m/z 581.4113: Bacillaene A [M+H]+, m/z 603.4241: Bacillaene A [M+Na]+; (C2) m/z 583.4271: Bacillaene B [M+H]+, m/z 605.4401: Bacillaene B [M+Na]+; (D1) m/z 489.3568: 7-o-malonyl macrolactin A [M+H]+, m/z 511.3701: 7-o-malonyl macrolactin A [M+Na]+; (D2) m/z 525.3752: 7-o-succinyl macrolactin A [M+Na]+.
Fig 9.
Colonial morphology and biofilm of strain LM2303.
(A) Colonial morphology of strain LM2303 in NA, (B) biofilm of strain LM2303 under different culture conditions.
Table 2.
Genes and gene clusters involved in bacterium-plant interactions in the LM2303 genome.
Fig 10.
Hypothetical model on the mode of actions of B. velezensis LM2303 in control of FHB in wheat.
(A) an untreated wheat, (B) wheat treated with strain LM2303. The illustration shows our present knowledge about the complex interactions in the tripartite system consisting of biocontrol bacterium (B. velezensis LM2303, light green rod), pathogen (F. graminearum, red filled circle) and plant (wheat). Strain LM2303 colonizes the surfaces of wheat tissues relying on its good motility and efficient biofilm formation, and subsequently produces a variety of secondary metabolites so as to form a protective zone (light blue). Cyclic lipopeptides (green circle) act the directly antagonism against F. graminearum and other pathogenic fungi by inhibit the conidia germination and mycelia growth, and the antibacterial metabolites (yellow circle) act the antibacterial activity against wheat bacterial diseases such as black embryo and bacterial leaf streak or alter the bacterial community, which may enhance the survival of strain LM2303. Meanwhile, surfactin and volatiles activate the defense response in wheat, providing an inner ISR-mediated protection against pathogens (green lines). And the promotion of wheat growth (yellow lines) also work effectively by means of nutrient competition and the action of growth-promoting hormones.