Chitooligosaccharide enhanced the efficacy of Bacillus amyloliquefaciens CAS02 for the control of tobacco black shank

Abstract

Introduction: Tobacco black shank is a devastating soil-borne disease caused by the oomycete Phytophthora nicotianae, severely hamper tobacco production worldwide. However, the synergistic effect of biocontrol bacteria and marine polysaccharides/oligosaccharides on tobacco black shank control was few documented.

Methods: In this study, Bacillus amyloliquefaciens CAS02 (CAS02) and chitooligosaccharide (COS) were screened firstly, and their synergistic antagonistic effect against P. nicotianae and the underlying mechanism were investigated in vitro and in vivo.

Results: In vitro experiments showed that, compared with the application of CAS02 or COS alone, co-application of CAS02 and COS significantly increased the inhibition rate against P. nicotianae by 11.67% and 63.31%, respectively. Furthermore, co-application of CAS02 and COS disrupted the structure of mycelia to a greater extent. The co-application of CAS02 and COS showed synergistic effect, with the relative control effect maintained above 60% during the 60-day pot experiment, significantly higher than that of application CAS02 or COS alone. The combined application of CAS02 and COS reduced the relative abundance of P. nicotianae in the rhizosphere soil and increased the relative abundance of bacterial taxa potentially involved in disease suppression, such as Nocardioides, Devosia and Bradyrhizobium. Meanwhile, CAS02 and COS synergistically activated salicylic acid (SA), ethylene (ET), and hypersensitive response (HR) defense signaling pathways in tobacco plants.

Discussion: Our findings demonstrate that co-application of CAS02 and COS remarkably improve the relative control effect against tobacco black shank through multiple pathways and provide a promising strategy for the efficient green control of tobacco black shank.

Keywords: biocontrol bacteria; chitooligosaccharide; plant defense signal marker gene; rhizosphere soil microorganism; tobacco black shank.

Figure 1

Inhibitory effects of biocontrol bacteria (A) and marine polysaccharides/oligosaccharide (B) on Phytophthora nicotianae. CAS02, Bacillus amyloliquefaciens CAS02; 3 T33, Bacillus aureus 3 T33; BB22, Bacillus megaterium BB22; BBS23, Bacillus adenii BBS23, NATT0; Bacillus natto NATT0; 7, Bacillus alpine 7; SB1, Bacillus amyloliquefaciens SB1; SB2, Bacillus amyloliquefaciens SB2; COS, chitooligosaccharide; AOS, alginate oligosaccharides; CAOS, carrageenan oligosaccharide; EP, enteromorpha polysaccharide; FOS, fucoidan oligosaccharide.

Figure 2

Synergistic inhibitory activity of CAS02 and COS against P. nicotianae. (A) The relative inhibition rate of CAS02 and COS in the synergistic inhibition of P. nicotianae. Growth of P. nicotianae mycelium in control (B) and after application COS alone (C), CAS02 alone (D) and co-application of CAS02 and COS (E).

Figure 3

Effect of CAS02 and COS on the morphology (A) and ultrastructure (B) of the pathogen P. nicotianae. In both A and B (a,b) show healthy mycelia in control. (c,d) Present the effects of application CAS02 alone. (e,f) Present the effect of application COS alone. (g,h) Present the effect of synergistic application of CAS02 and COS.

Figure 4

The effect of combined application of biocontrol bacteria CAS02 and COS on the prevention and control of tobacco black shank and on tobacco plant growth. Tobacco black shank disease index and relative control effect at day 10 (A) and 25 (B). (C) Plant height. (D) Maximum leaf area. Control: No CAS02 suspension or COS added; CAS02: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) was added; COS: 36 mg COS was added; CAS02-COS: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) and 36 mg COS were added. Data are means ± SE (n = 3). Different letters on the bars indicate significant differences (p < 0.05).

Figure 5

Profiles of microbial community in the rhizosphere soil in different treatments. (A) The density of P. nicotianae in the rhizosphere soil in different treatments. (B) Principal component analysis (PCoA) of bacterial community composition in rhizosphere soils of different treatments at OTU level. (C) Venn diagram showing the number of specific and shared OTUs in rhizosphere soils among different treatments; (D) Relative abundance of bacteria at the phylum level (top 10) in rhizosphere soils of different treatments. Control: No CAS02 suspension or COS added; CAS02: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) was added; COS: 36 mg COS was added; CAS02-COS: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) and 36 mg COS were added. Data are means ± SE (n = 3). Different letters on the bars indicate significant differences (p < 0.05).

Figure 6

Two group comparisons showing the difference in the relative abundance of bacterial genera (top 15) in rhizosphere soils among different treatments. The distribution of the relative abundance of bacterial taxa between control and individual or co-application of CAS02 and COS (A–C) and between individual and co-application of CAS02 and COS (D–F). Control: No CAS02 suspension or COS added; CAS02: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) was added; COS: 36 mg COS was added; CAS02-COS: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) and 36 mg COS were added.

Figure 7

Relative expression levels of defense signaling marker genes. (A) PR1a/c. (B) PR2. (C) EFE26. (D) ACC oxidase. (E) SGT1. (F) H1N1. Control: No CAS02 suspension or COS added; CAS02: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) was added; COS: 36 mg COS was added; CAS02-COS: 25 mL CAS02 suspension (OD₆₀₀ value of 0.4) and 36 mg COS were added. Data are means ± SE (n = 3). Different letters on the bars indicate significant differences (p < 0.05).