Figure 1.
Antagonistic activity of Bacillus species against X. axonopodis pv. citri XW19.
A 20 µl aliquot of X. axonopodis pv. citri XW19 suspension (OD620 = 0.3) was spread on an SYB agar plate. Following overnight incubation at 30°C, 20 µl of Bacillus suspension (OD620 = 0.3) was spotted (A) inside the stainless steel ring or (B) on a paper disc. The plates were incubated at 30°C, for 5 days. CK, 20 µl of sterile Milli-Q water was used as a control. The results represent the means and standard deviations (error bars) of a representative experiment. Different lowercase letters indicate significant differences (p<0.05) according to Tukey's HSD test.
Table 1.
Bacterial strains and plasmids used in this study.
Figure 2.
Phylogenetic tree of Bacillus species based on 16S rRNA gene sequences.
The tree was constructed using the neighbor-joining method and genetic distances were generated using the Kimura 2-parameter method. The numbers at the branches are bootstrap confidence percentages from 1000 bootstrapped trees. Alicyclobacillus acidocaldarius (GenBank accession no. AB089859) was used as the outgroup. The numbers in parentheses indicate the GenBank accession numbers.
Figure 3.
ITS-PCR fingerprint and UPGMA cluster analysis of Bacillus species.
(A) ITS-PCR fingerprint and (B) UPGMA cluster analysis. The UPGMA cluster analysis was based on ITS-PCR. M, GeneRuler™ 100 bp plus DNA ladder (Fermentas, Taipei, Taiwan).
Figure 4.
tDNA-PCR fingerprint and UPGMA cluster analysis of Bacillus species.
(A) tDNA-PCR fingerprint and (B) UPGMA cluster analysis. The UPGMA cluster analysis was based on tDNA-PCR. M, GeneRuler™ 100 bp plus DNA ladder (Fermentas, Taipei, Taiwan).
Figure 5.
BOXA1R-PCR fingerprint and UPGMA cluster analysis of Bacillus species.
(A) BOXA1R-PCR fingerprint and (B) UPGMA cluster analysis. The UPGMA cluster analysis was based on BOXA1R-PCR using the BOXA1R primer. M, GeneRuler™ 100 bp plus DNA ladder (Fermentas, Taipei, Taiwan).
Figure 6.
The effect of B. subtilis TKS1-1 and B. amyloliquefaciens WG6-14 on the disease severity of citrus bacterial canker on Mexican lime.
(A) Symptoms on upper (top panels) and lower (bottom panels) leaf surfaces of Mexican lime one month post -inoculation with X. axonopodis pv. citri TPH2 (OD620 = 0.3). Milli-Q water or B. subtilis TKS1-1 (TKS1-1) and B. amyloliquefaciens WG6-14 (WG6-14) culture suspensions were sprayed on the leaves of Mexican lime one day prior to inoculation with X. axonopodis pv. citri TPH2. (B) Number of cankers per cm2 on each leaf. All experiments were performed three times with similar results. The results are the means and standard deviations (error bars) of five replicates from one representative experiment. *, significantly different (p<0.05) from water control analyzed by one-way ANOVA and Tukey's HSD test. Scale bar, 1 cm.
Figure 7.
Colonization by X. axonopodis pv. citri strain TPH2 and B. subtilis TKS1-1 and B. amyloliquefaciens WG6-14 on leaf surfaces of Mexican lime observed by confocal laser scanning microscopy.
Leaf surfaces were spray inoculated with (A, E) X. axonopodis pv. citri strain TPH2 harboring pGTKan, and (B, C) B. subtilis strain TKS1-1, or (F, G) B. amyloliquefaciens strain WG6-14 1 day prior to inoculation with X. axonopodis pv. citri strain TPH2 harboring pGTKan. The photos were taken 1 day post-inoculation of the pathogen. Green, X. axonopodis pv. citri strain TPH2 expressing green fluorescent protein. Red, acridine orange stained cells. (D) and (H), merged images of (B, C) and (F, G), respectively. Arrow, stomata. Scale bar, 10 µm.
Figure 8.
The effect of application time and frequency of B. subtilis strain TKS1-1 on symptom development and disease incidence of citrus bacterial canker on navel orange grown in the greenhouse.
B. subtilis strain TKS1-1 endospore formulation (T, 109 CFU/ml) and X. axonopodis pv. citri XW19 (B, 108 CFU/ml) were used. Treatment T-B-T/1WK, strain TKS1-1 endospores were applied 24 h prior to inoculation with X. axonopodis pv. citri XW19, and then weekly post-pathogen inoculation for 4 weeks; treatment T-B-D/1WK, strain TKS1-1 endospores were applied 24 h prior to X. axonopodis pv. citri XW19 inoculation; treatment D-B-T/1WK, strain XW19 was inoculated, then strain TKS1-1 endospores were sprayed every week post-pathogen inoculation for 4 weeks; treatment D-B-D/1WK, only strain XW19 was inoculated. Without Bacillus treatment, Milli-Q water (D) was sprayed on the leaf surface. (A) Symptoms on upper (left) and lower (right) leaf surfaces after different treatments. Scale bars, 1 cm. (B) Disease incidence was rated 4 weeks post-inoculation. Bars indicate standard deviations. Columns that are top-labeled with different letters are significantly different (p<0.05) according to one-way ANOVA and Tukey's HSD test.