Anti-Aflatoxigenic Burkholderia contaminans BC11-1 Exhibits Mycotoxin Detoxification, Phosphate Solubilization, and Cytokinin Production

Abstract

The productivity and quality of agricultural crops worldwide are adversely affected by disease outbreaks and inadequate nutrient availability. Of particular concern is the potential increase in mycotoxin prevalence due to crop diseases, which poses a threat to food security. Microorganisms with multiple functions have been favored in sustainable agriculture to address such challenges. Aspergillus flavus is a prevalent aflatoxin B₁ (AFB₁)-producing fungus in China. Therefore, we wanted to obtain an anti-aflatoxigenic bacterium with potent mycotoxin detoxification ability and other beneficial properties. In the present study, we have isolated an anti-aflatoxigenic strain, BC11-1, of Burkholderia contaminans, from a forest rhizosphere soil sample obtained in Luzhou, Sichuan Province, China. We found that it possesses several beneficial properties, as follows: (1) a broad spectrum of antifungal activity but compatibility with Trichoderma species, which are themselves used as biocontrol agents, making it possible to use in a biocontrol mixture or individually with other biocontrol agents in an integrated management approach; (2) an exhibited mycotoxin detoxification capacity with a degradation ratio of 90% for aflatoxin B₁ and 78% for zearalenone, suggesting its potential for remedial application; and (3) a high ability to solubilize phosphorus and produce cytokinin production, highlighting its potential as a biofertilizer. Overall, the diverse properties of BC11-1 render it a beneficial bacterium with excellent potential for use in plant disease protection and mycotoxin prevention and as a biofertilizer. Lastly, a pan-genomic analysis suggests that BC11-1 may possess other undiscovered biological properties, prompting further exploration of the properties of this unique strain of B. contaminans. These findings highlight the potential of using the anti-aflatoxigenic strain BC11-1 to enhance disease protection and improve soil fertility, thus contributing to food security. Given its multiple beneficial properties, BC11-1 represents a valuable microbial resource as a biocontrol agent and biofertilizer.

Keywords: biocontrol agent; biofertilizer; mycotoxin detoxification; phosphate-solubilizing microorganisms; phytohormone.

Figure 1

Inhibition of A. flavus. (A) Bacterial suspension of BC11-1 exhibits inhibition of the growth of A. flavus. (B) Inhibition of the growth of A. flavus on PDA plate containing cell-free supernatant (CFS) of BC11-1. (C) Alterations in the ultrastructure of A. flavus after treatment with the CFS of BC11-1 extract. CK1: normal growth of A. flavus on PDA plate; CK2: growth situation of A. flavus on PDA plate containing 25 mL sterile water as a negative control; CK3: normal ultrastructure of A. flavus; CW: cell wall; FL: fibrillar layer.

Figure 2

Antifungal activity of BC11-1 against several phytopathogens and biocontrol agents. (A) BC11-1 antifungal analysis. BC11-1 (top) demonstrated antifungal activity, relative to the negative control (bottom), against B. cinerea, F. solani, P. asparagi, F. graminearum, R. solani, A. tenuissima, and C. oryzae; (B) BC11-1 had a negligible inhibitory effect on Trichoderma species. (C) Quantitative assessment of the antifungal activity of BC11-1 against different fungi. Data represent the mean inhibition ratio (n = 4). Different lowercase letters above the bars indicate significant differences in the level of inhibition of the different fungi by BC11-1, relative to the control (p < 0.05; Tukey’s test).

Figure 3

Mycotoxin detoxification activity of BC11-1. (A) AFB₁ and ZEN detoxification ratio by extracellular metabolites of BC11-1. (B) CFS derived from BC11-1 inhibited the hyphal growth and spore formation in A. flavus (B1) and reduced AFB₁ content, compared to the control (sterile water) (B2).

Figure 4

Biochemical assessment of BC11-1. A single BC11-1 colony was inoculated in 10 mL of LB broth and cultured overnight at 28 °C on a rotary shaker. Then, 50 µL of suspension was placed into holes in the center of the various assay plates. The formation of a halo surrounding the colony revealed the ability of the BC11-1 to produce extracellular protease (A) and a siderophore (B). The assays also demonstrated phosphate solubilization activity (C). No evidence of extracellular amylase (D) or β-1 and 3-glucanase (E) activity was observed.

Figure 5

Quantitative assessment of phosphate solubilization and plant growth-promoting activity of BC11-1. (A) After being exposed to BC11-1 for 0, 1, 2, 3, 4, 7, and 10 d, dynamic change in soluble phosphate content in NBRP broth media and the pH value. Data represent the mean ± SD (n = 3). Data represent the mean ± SD (n = 3). (B) Three groups of rice seedlings were subjected to different treatments (from left to right), as follows: Group I received tap water only; Group II received tap water containing 5 g/L Ca₃(PO₄)₂; and Group III received tap water containing either 60 mL or 180 mL phosphate solution produced by BC11-1. (C) Phosphate content in plants in the three different groups. Data represent the mean ± se (n = 3). Different lowercase letters above the bars indicate significant differences in the level of phosphorus in plants in the three different groups of fungi (p < 0.05) as indicated by a Tukey’s test.

Figure 6

Analysis of cytokinin production by BC11-1 in cells and CFS. (A) Promotion of tillering promotion by CFS of BC11-1. CK: rice seedlings treated with water as negative control. (B) Isopentenyl adenosine (IPA) in BC11-1 cells and cell-free supernatant (CFS) over culture time. Data represent the mean ± se (n = 3).

Figure 7

Pan-genome analysis. (A) Venn diagram indicating the core gene number (common to all strains) and strain-specific gene number in eight B. contaminans strains, including four biocontrol strains, MS14 (GenBank: ASM102914v1), CH1 (GenBank: ASM472362v1), NZ (GenBank: ASM336316v1), and XL-73 (GenBank: ASM975562v1), and four pathogenic strains, toggle 1 (GenBank: ASM1822378v1), SCAID TST1-2021 (GenBank: ASM1991536v1), LMG 23361 (GenBank: ASM175838v2), and SBC01 (GenBank: ASM1688794v1). (B) Dispensable gene heat map (left, dispensable gene cluster; top, strain cluster; Colors represent the identity of dispensable genes in the eight B. contaminans strains).