DOI: 10.20350/digitalcsic/16335
title: Supplementary Material to: Herbicolin A production and its modulation by quorum sensing in a Pantoea agglomerans rhizobacterium bioactive against a broad spectrum of plant-pathogenic fungi

description: Supplementary material table and figures
description: Table S1. Bacteria, oomycete, fungi, phages, plasmids and oligonucleotides used in this study. Figure S1. Halos of antibiosis of filter-sterilized supernatants of Pantoea agglomerans 9Rz4 grown in different culture media. Growth inhibition of the ascomycete yeast Schizosaccharomyces pombe (herbicolin A sensitive) with culture supernatants of P. agglomerans 9Rz4 and the herbicolin A deficient mutant NB1 grown overnight in LB, minimal medium (MM), YE and Strobel medium (Strobel et al., 1999) at 25 ºC. For the bioassays, a S. pombe top agar lawn was prepared in YE-agar and 300 µl of filter-sterilized supernatants were added to holes punched in the S. pombe bioassay plates. The size of the inhibition halos is indicative of the susceptibility of S. pombe to herbicolin A. The bioassays were repeated three times and representative pictures are shown. Picture were taken after 48 h of incubation at 30 ºC. The radius of the halos from three biological replicas is 7.0 ± 0.2 mm (LB), 4.1 ± 0.1 (MM), 6 ± 0.1 mm (YE) and 1.8 ± 0.05 mm (Strobel medium). Bars, 5 mm. Figure S2. Pantoea agglomerans 9Rz4 maize root colonization and its effect on plant growth. A, Maize plants 10 days after inoculation with 9RZ4. Non-inoculated plants were included as control. B, Root weight of maize plants shown in Fig. S2A. Shown are mean and standard deviation of six different plants. No statistically significant differences in root weight were observed between inoculated and non-inoculated plants. C, Maize root colonization assays of P. agglomerans 9Rz4 and its mutant strain NB1. Shown are mean and standard deviation of six different plants. In A-C, sterilization, germination and inoculation of maize seeds was carried out as described previously (Matilla et al., 2007), with minor modifications. Briefly, sterile maize seeds were incubated for 45 min at 30 ºC with a 107 CFU/mL of Pantoea agglomerans 9Rz4 strains. Thereafter, seeds were rinsed with sterile deionized water and planted in 50 mL tubes containing 40 g of sterile washed silica sand and 10% (v/w) plant nutrient solution supplemented with Fe-EDTA and micronutrients, as described previously (Matilla et al., 2007). Plants were maintained at 24 ºC with a daily light period of 16 h for 10 days. Figure S3. Effect of plasmid carriage on the antifungal properties of P. agglomerans 9Rz4. Bioactivity against Verticillium dahliae of 9Rz4 and a 9Rz4 variant (9Rz4-W) lacking plasmid p9Rz4_1. Pictures were taken after 96 h of incubation at 25 °C. Figure S4. Herbicolins A and B production is reduced in the quorum sensing mutant defective in the acyl-homoserine lactone synthase PagI. Abundance of herbicolins A and B relative to the wild type 9Rz4 in the supernatants of the P. agglomerans 9Rz4 strains. Data are the mean and standard deviations from three biological replicates and correspond to the intensity (area under the peak) of “extracted ion chromatograms” (EIC) shown in Fig. 6B. Note that the areas derived from the EICs are not comparable between compounds (e.g. herbicolin A vs herbicolin B) as these areas depend on the ionization efficiency of each compound. As shown in Fig. 1A, herbicolin B is found at trace levels in the 9Rz4 supernatants based on LC-HRMS analyses. Figure S5: Complementation of herbicolins A and B production in the quorum sensing mutant defective in the acyl-homoserine lactone synthase PagI. A, Growth inhibition of Schizosaccharomyces pombe with culture supernatants of Pantoea agglomerans strains. For the assays, P. agglomerans strains were inoculated at an initial OD660 of 0.05 in 10 mL of LB medium containing 1 mL supernatants of overnight cultures of P. agglomerans NB1 (herbicolin A defective; wild type in acyl-homoserine lactone production) or P. agglomerans PagI (defective in the synthesis of acyl-homoserine lactones) grown in LB medium. Then, bacterial cultures were grown overnight at 25 ºC, at which time the supernatants were collected, filter-sterilized and characterized chemically and biologically. For the bioassays, a Schizosaccharomyces pombe top agar lawn was prepared and 300 µL of filter-sterilized supernatants were added to holes punched in the S. pombe bioassay plates. Pictures were taken after 48 h of incubation at 30 ºC. Numerical values indicate the mean and standard deviation of the radius of the inhibition halo of three biological replicates. Bars, 5 mm. B, Extracted ion chromatograms (EIC) corresponding to an m/z of 659.385 ± 0.005 (theoretical value for [M+2H]2+ in herbicolin A) and to an m/z of 569.845 ± 0.005 (theoretical value for [M+2H]2+ in herbicolin B). C, Abundance of herbicolins A and B relative to the wild type strain 9Rz4 in the supernatants of the P. agglomerans 9Rz4 strains through the measurement of peak areas in the EICs shown in Fig. S5B. In B and C, note that the areas derived from the EICs are not comparable between compounds (e.g. herbicolin A vs herbicolin B) as these areas depend on the ionization efficiency of each compound. As shown in Fig. 1A, herbicolin B is found at trace levels in the 9Rz4 supernatants based on LC-HRMS analyses.