Fig 1.
Antifungal activity of B. amyloliquefaciens S76–3 against FG on PDA plates 5 dpi (28°C).
Fungal plug was placed in the center of PDA plate and S76–3 was inoculated 3 cm from the fungal plug (S76–3). Fungal plug inoculated on PDA plate center was used as control (CK).
Fig 2.
RP-HPLC analysis of lipopeptides from strain S76–3, and detection of their antagonistic activity against FG.
Iturin, plipastatin, and surfactin were analyzed and collected by RP-HPLC, and their antifungal activities were detected on PDA plates 4 dpi (28°C). Aliquots of 20 µl of each (10 mg/ml) were added to filter disks and placed 3 cm away from the fungal plug. 20 µl of methanol on a filter disk was used as control.
Fig 3.
ESI-CID-MS analysis of iturin A (m/z 1043.35) and plipastatin A (m/z 1463.90).
CID spectrum of precursor ion of iturin at m/z 1043.35 (A), and the theoretical b-type and y-type fragment ions dissociated from ring-opening reactions at Gln-Pro (B, top panel) and Asn-Tyr (B, bottom panel). CID spectrum of precursor ion of plipastatin A at m/z 1463.90 (C), and common breakage and two typical fingerprint ions for identification of plipastatin A (D, top panel) and plipastatin B (D, bottom panel) molecules. Carbon number in acyl acid chains for iturin A (m/z 1043.35) is 14 (βAA), and for plipastatin A (m/z 1463.90) is 16 (n = 13).
Fig 4.
Primary structures of representative members within the three lipopeptides families produced by Bacillus spp.
Plipastatin A/B with L-Tyr4 and D-Tyr10 was K+salt, with D-Tyr4 and L-Tyr10 was free form or TFA salt [16,26,27].
Table 1.
Identification of lipopeptides extracted from strain S76–3 through RP-HPLC and ESI-CID-MS.
Table 2.
Conidia germination inhibition tests of iturin A and plipastatin A against FG in half-strength YPG medium 12 hpi.
Fig 5.
Optical and fluorescence microscopy analyses of FG treated with and without iturin A and plipastatin A.
In control treatment (CK), conidia showed typical canoe shape and widespread fluorescence; hyphae had equal widths, even surfaces, and symmetrically distributed branching, with strong fluorescence along hyphae. In iturin A or plipastatin A treatments (iturin A, plipastatin A), conidia showed substantially deformed and damaged morphology, lateral expansion, and very weak or absent distribution of fluorescence. Iturin A caused substantial condensation and massive conglobation along hyphae with uneven surfaces, expanded widths, restricted branching, and very faint fluorescence in most regions. Plipastatin A caused severely distorted and condensed hyphae with increased vacuole sizes and deformed and conglobated apical tips, with interrupted fluorescence along hyphae. Bar = 25 µm.
Fig 6.
Dynamic processes of hyphae of FG as affected by iturin A or plipastatin A.
Hyphae in control treatment (CK) had active apical growth with equal widths and many quickly elongating branches (circle); iturin A at MIC caused distortion and conglobation along hyphae, with lateral expansion and very restricted apical growth (iturin A, arrow); plipastatin A at MIC caused formation of conglobated structures, especially in young hypha and branch tips, resulting in either branch conglobation or restriction of apical growth (plipastatin A, arrow). The processes last 200 min. Bar = 25 µm.
Fig 7.
TEM analysis of FG conidia and hyphae treated with iturin A or plipastatin A.
Conidia and hyphae lacking lipopeptide treatment (CK) showed dense cytoplasm with typical vacuoles, regular septa, and intact plasma membranes attached to electron-dense cell walls; Iturin A and plipastatin A caused complete structural destruction of conidia and hyphae, disorganized and sparse cytoplasm, uneven widths and gapped cell walls (cw with arrows) with indiscernible layers, dissociated cell organelles, and discontinuous plasma membranes. cy cytoplasm; va, vacuole; cw, cell wall; pm, plasma membrane; s, septum.