Ex Vivo Application of Secreted Metabolites Produced by Soil-Inhabiting Bacillus spp. Efficiently Controls Foliar Diseases Caused by Alternaria spp

Abstract

Bacterial biological control agents (BCAs) are largely used as live products to control plant pathogens. However, due to variable environmental and ecological factors, live BCAs usually fail to produce desirable results against foliar pathogens. In this study, we investigated the potential of cell-free culture filtrates of 12 different bacterial BCAs isolated from flower beds for controlling foliar diseases caused by Alternaria spp. In vitro studies showed that culture filtrates from two isolates belonging to Bacillus subtilis and Bacillus amyloliquefaciens displayed strong efficacy and potencies against Alternaria spp. The antimicrobial activity of the culture filtrate of these two biological control agents was effective over a wider range of pH (3.0 to 9.0) and was not affected by autoclaving or proteolysis. Comparative liquid chromatography-mass spectrometry (LC-MS) analyses showed that a complex mixture of cyclic lipopeptides, primarily of the fengycin A and fengycin B families, was significantly higher in these two BCAs than inactive Bacillus spp. Interaction studies with mixtures of culture filtrates of these two species revealed additive activity, suggesting that they produce similar products, which was confirmed by LC-tandem MS analyses. In in planta pre- and postinoculation trials, foliar application of culture filtrates of B. subtilis reduced lesion sizes and lesion frequencies caused by Alternaria alternata by 68 to 81%. Taken together, our studies suggest that instead of live bacteria, culture filtrates of B. subtilis and B. amyloliquefaciens can be applied either individually or in combination for controlling foliar diseases caused by Alternaria species.

FIG 1

Screening of soil-inhabiting bacterial species for controlling Alternaria alternata in vitro. (A) Effects of different dilutions (10%, 1%, and 0.1%) of culture filtrates (CFs) of the bacterial isolates indicated on the x axis on the in vitro growth of A. alternata. Growth was normalized to growth in a no-CF control, considered 100%. Azoxystrobin (Heritage), a broad-spectrum fungicide, was used as a positive control, and its 10%, 1%, and 0.1% levels correspond to 5.0, 0.5, and 0.05 μg ml⁻¹ active ingredient in the assay solution. *** and **, statistically significant differences at P values of <0.001 and <0.01, respectively, compared to corresponding dilutions of B11-48. (B) Representative pictures showing the effects of CFs of B. amyloliquefaciens isolate B11-144 and B. subtilis isolates B11-128 (strong inhibition) and B11-48 (no inhibition) on growth of A. alternata on PDA plates. CF (25 μl) was applied at the four locations indicated by circles on the plates. Control plates contained only LB. Pictures were taken 10 days after the start of the experiment. (C) Fungicidal assays. Spores treated with 2% CFs for 24 h did not resume growth when washed of culture filtrates, showing that CFs of B11-128 and B11-144 have a fungicidal mode of action. (For B11-128, washed versus unwashed, P = 0.699 by t test [n = 4]; for B11-148, washed versus unwashed, P = 0.515 by t test [n = 4]). Error bars are standard errors of the means (SEM).

FIG 2

Dose-response analyses of culture filtrates of B. subtilis (B11-128) and B. amyloliquefaciens (B11-144). (A and B) Best-fit curves were generated from dose-response data consisting of 10, 5, 2.5, 1.25, 0.62, 0.31, 0.165, 0.078, 0.039, 0.019, 0.009, and 0.004% culture filtrates using the sigmoidal (four-parameter logistic) model. r² values for measuring goodness of fit of the curves are shown on the graphs. Sy.x, Sum of standard errors. (C) Representative pictures of A. alternata hyphae in a microtiter plate treated with the indicated dilutions of B11-128 and B11-144 culture filtrates. Pictures were taken 5 days after incubation. Bars = 100 μm.

FIG 3

Effects of heat and proteinase K treatment and pH on the anti-Alternaria activity of culture filtrates of B. subtilis (B11-128) and B. amyloliquefaciens (B11-144). (A to D) Best-fit curves were generated from dose-response data using the sigmoidal (four-parameter logistic) model as for Fig. 2. Curve fits of control and treatment (autoclaved or proteinase treated) were compared using the null hypothesis that a single curve fits both data sets. P values for significance of tests are 0.485 (A), 0.285 (B), 0.678 (C), and 0.893 (D), which are all nonsignificant (P > 0.05). r² values for measuring goodness of fit of the curves are shown on the graphs. (E) For assaying the effect of pH on anti-Alternaria activity, growth inhibition was normalized to a no-CF control at pH 7.0. Statistical comparison showed no significant effect of pH on the activities of B11-128 and B11-144 (P > 0.1). Data shown are means ± SEM (n = 4).

FIG 4

Interaction of B. subtilis (B11-128) and B. amyloliquefaciens (B11-144) culture filtrates in controlling A. alternata. (A) Bar graph comparing effects of combining culture filtrates of B11-128 and B11-144 on A. alternata growth. CFs of B11-128 alone, B11-144 alone, and B11-128 and B11-144 combined were used, respectively, at the following percent dilutions: 2, 2, and 1 + 1 (bars A), 1, 1, and 0.5 + 0.5 (bars B), 0.3, 0.3, and 0.15 + 0.15 (bars C), 0.2, 0.2, and 0.1 + 0.1 (bars D), 0.14, 0.14, and 0.07 + 0.07 (bars E), 0.1, 0.1, and 0.05 + 0.05 (bars F), and 0.02, 0.02, and 0.01 + 0.01 (bars G). Growth data were normalized to the growth in an untreated control. Error bars are SEM. (B) Micrographs showing effects of B11-128 and B11-144 used individually or in mixtures at the indicated dilutions. Bars = 100 μm.

FIG 5

In vivo control of Alternaria spp. by culture filtrates from B. subtilis (B11-128) and B. amyloliquefaciens (B11-144). (A to D) Results of detached-leaf assays; (E) results of whole-plant assays. (A, C, and D) Bar graphs showing average lesion sizes caused by Alternaria spp. on poinsettia (A), dieffenbachia (C), and tomato (D). (B) Photographs showing control of A. alternata on poinsettia leaves. Pictures were taken 7 days postinoculation. Culture filtrates of B11-128 and B11-144 significantly reduced lesion sizes. (E) Bar graph showing number of lesions per plant in response to CFs from B11-128 and B11-144. H₂O→Aa, water control application followed by A. alternata inoculation; LB→Aa, 2% LB control application followed by A. alternata inoculation; CF→Aa, 2% culture filtrate application followed by A. alternata inoculation; Aa→CF, A. alternata inoculation followed by 2% culture filtrate application; CF→Aa→CF, 2% culture filtrate was applied both pre- and postinoculation with A. alternata; Azox→Aa, Azoxystrobin (Heritage) spray followed by A. alternata inoculation. Error bars indicate standard errors of the means.

FIG 6

(A to F) Total ion current chromatograms of water-soluble (A, B, and C) and methanol-soluble (D, E, and F) fractions of acid-precipitated lytic peptides of B. subtilis (B11-128), B. amyloliquefaciens (B11-144), and B. thuringiensis (B11-48). Two distinct clusters of peaks were observed at retention times of 12 to 20 min (cluster 1) and 23 to 26 min (cluster 2). Cluster 1, which was observed only in water-soluble fractions, displayed similar peak patterns in the bioactive (B11-128 and B11-144) and inactive (B11-48) strains. In contrast, cluster 2 peaks were present in the bioactive culture filtrate but not in the inactive culture filtrate. (G and H) Mass spectra (m/z) displaying the overall distribution of water-soluble cyclic lipopeptides in CFs of B. subtilis (B11-128) and B. amyloliquefaciens (B11-144). The mass spectra correspond to an average of cluster 2 (retention time, 23 to 26 min). In panel G, prominent peaks are labeled a to e.