PMC

Identification of herbicolins A and B in Pantoea agglomerans 9Rz4 supernatants by liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS). (A) Base peak chromatogram of the LC‐HRMS analysis of the supernatants of wild‐type 9Rz4 and mutants in the herbicolin A biosynthetic cluster (e.g. NB1, NB7 and NB9). The insert shows a zoomed region of the extracted ion chromatogram corresponding to an m/z of 569.8452 ± 0.0050 (theoretical value for [M + 2H]²⁺ in herbicolin B), confirming the presence of herbicolin B at 4.46 min in the supernatant of wild‐type 9Rz4 and its absence in those supernatants of mutants in the herbicolin A biosynthetic cluster. The peak at 4.12 min is originated by fragmentation of herbicolin A, with loss of the glucose unit. (B) Expansion of the HRMS spectrum of herbicolin A. (C) Expansion of the HRMS spectrum of herbicolin B. In (A–C), 5 μl supernatants of bacteria grown in minimal medium with glucose as sole carbon source was used for the LC‐HRMS analyses. (D) Chemical structures of herbicolins A and B.

Reduction of incidence of disease symptoms (necrosis and/or bacterial ooze) in immature pear fruit by P. vagans C9-1 and antibiotic-deficient derivatives CIR591 (herbicolin I⁻/pantocin A⁺), CIR638 (herbicolin I⁺/pantocin A⁻), and CIR600 (herbicolin I⁻/pantocin A⁻). Immature pear fruits were treated with buffer or 5 × 10⁶ CFU ml⁻¹ wild-type C9-1 or derivatives (A) or buffer or 5 × 10⁵ CFU ml⁻¹ of wild-type C9-1 or antibiotic-deficient mutants (B) and subsequently challenged with 5 × 10⁵ CFU ml⁻¹ E. amylovora Ea110. Vertical lines indicate standard errors of the means. Similar letters above bars for a time point indicate that the transformed incidence of symptoms between treatments is not significantly different according to Fisher's protected least significant difference at P = 0.05.

Expression of the herbicolin A biosynthetic cluster throughout the growth phase in Pantoea agglomerans 9Rz4. (A) β‐galactosidase activity (filled symbols) throughout growth (open symbols) measured from a chromosomal fusion hbcB::lacZ (strain NB1) in P. agglomerans 9Rz4 in LB medium at 25°C. Data are the mean and standard deviation of three biological replicates. β‐galactosidase activity was measured as described previously (Miller, ) using 2‐Nitrophenyl‐β‐d‐galactopyranoside (Sigma‐Aldrich; Cat No. N1127) as substrate. (B) Herbicolin A production by P. agglomerans 9Rz4 throughout growth in LB medium at 25°C. For the bioassays, a Schizosaccharomyces pombe top agar lawn was prepared and 300 μl of filter‐sterilized supernatants of 9Rz4 was added to holes punched in the S. pombe bioassay plates. The bioassays were repeated three times and representative pictures are shown. Pictures were taken after 48 h of incubation at 30°C. The size of the inhibition halos is indicative of the susceptibility of S. pombe to herbicolin A. Bar, 5 mm.

Antifungal activities of herbicolins A and B. Bioactivities of Pantoea agglomerans 9Rz4 and derivative mutant strains defective in the herbicolin A gene cluster (e.g. strains NB1, NB7 and NB9) are shown. The bioassays were conducted as previously described (Matilla et al., ). Fungi are present in the top agar lawns and the size of the inhibition halo determines their susceptibility to herbicolin A produced by 9Rz4. All the fungi strains tested in this study are listed in Table . Gmt, Gaeumannomyces graminis var. tritici. Numerical values at the bottom right of each bioassay represent the mean and standard deviation of the radius of the inhibition halo of three biological replicates. Bars, 5 mm.