Bikaverin as a molecular weapon: enhancing Fusarium oxysporum pathogenicity in bananas via rhizosphere microbiome manipulation

Abstract

Background: Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), poses a severe threat to global banana production. Secondary metabolites are critical tools employed by pathogens to interact with their environment and modulate host-pathogen dynamics. Bikaverin, a red-colored polyketide pigment produced by several Fusarium species, has been studied for its pharmacological properties, but its ecological roles and impact on pathogenicity remain unclear.

Results: This study investigated the role of bikaverin in Foc TR4, focusing on its contribution to pathogenicity and its interaction with the rhizosphere microbiome. Pathogenicity assays under sterile and autoclaved conditions demonstrated that bikaverin does not directly contribute to pathogenicity by affecting the infection process or damaging host tissues. Instead, bikaverin indirectly enhances Foc TR4's pathogenicity by reshaping the rhizosphere microbiome. It suppresses beneficial plant growth-promoting rhizobacteria, such as Bacillus, while promoting the dominance of fungal genera, thereby creating a microbial environment beneficial for pathogen colonization and infection. Notably, bikaverin biosynthesis was found to be tightly regulated by environmental cues, including acidic pH, nitrogen scarcity, and microbial competition. Co-culture with microbes such as Bacillus velezensis and Botrytis cinerea strongly induced bikaverin production and upregulated expression of the key bikaverin biosynthetic gene FocBik1. In addition, the identification of bikaverin-resistant Bacillus BR160, a strain with broad-spectrum antifungal activity, highlights its potential as a biocontrol agent for banana wilt management, although its stability and efficiency under field conditions require further validation.

Conclusions: Bikaverin plays an indirect yet important role in the pathogenicity of Foc TR4 by manipulating the rhizosphere microbiome. This ecological function underscores its potential as a target for sustainable disease management strategies. Future research should focus on elucidating the molecular mechanisms underlying bikaverin-mediated microbial interactions, using integrated approaches such as transcriptomics and metabolomics. Together, these findings provide a foundation for novel approaches to combat banana wilt disease and enhance crop resistance. Video Abstract.

Keywords: Bacillus; Banana wilt disease; Bikaverin; Foc TR4; Plant growth-promoting rhizobacteria; Rhizosphere microbiome; Secondary metabolites.

Fig. 1

Role of bikaverin in the vegetative growth and pathogenicity of Fusarium oxysporum f. sp. cubense TR4 (Foc TR4). a Schematic representation of the bikaverin biosynthetic gene cluster in the wild-type (WT) and the FocBik1 knockout mutant (ΔFocBik1). FocBik1, encoding a polyketide synthase, was replaced with a neomycin phosphotransferase II (NPTII) cassette in ΔFocBik1. b Quantification of bikaverin production in WT, ΔFocBik1, and uninoculated control cultures by LC–MS. Bars represent mean peak areas of bikaverin-specific ions. ΔFocBik1 exhibits no detectable bikaverin production. Data are presented as mean ± standard deviations. c Colony morphology of WT, ΔFocBik1, and complemented strain (Res-ΔFocBik1) on complete medium (CM), minimal medium (MM), and under various abiotic stress conditions. d Colony diameter of WT, ΔFocBik1, and Res-ΔFocBik1 after 7 days of growth on CM and MM. e Quantification of conidiation of WT, ΔFocBik1, and Res-ΔFocBik1 cultured in liquid complete medium. f Growth inhibition of WT, ΔFocBik1, and Res-ΔFocBik1 under abiotic stress conditions. Data are presented as mean ± standard deviations. Letters indicate statistical significance determined by one-way ANOVA with Duncan’s multiple range test (P < 0.05). g Disease symptom and index of banana plantlets inoculated with WT, ΔFocBik1, and Res-ΔFocBik1. Representative images of pseudo-stem browning are shown, with the corresponding disease index distribution across plantlets. h Infection and colonization of banana roots by GFP-tagged strains of WT and ΔFocBik1 visualized after 7 days of inoculation. Bright-field (BF), GFP fluorescence, propidium iodide (PI) staining, and merged images are presented. Arrows indicate vascular colonization by WT

Fig. 2

Environmental and biological factors induce bikaverin production in Fusarium oxysporum f. sp. cubense TR4 (Foc TR4). a Bikaverin production by wild-type (WT) and FocBik1 knockout mutant (ΔFocBik1) strains cultured in minimal medium with yeast extract (YE) or sodium nitrate (NaNO₃) as the nitrogen source under different pH conditions (pH 4, pH 7, and pH 9). b Relative expression levels of FocBik1 in WT strains grown in YE or NaNO₃ at pH 4, 7, and 9. The mycelium cultured at pH 7 was used as the reference samples. Data are presented as mean ± standard deviations. Letters indicate statistical significance determined by one-way ANOVA with Duncan’s multiple range test (P < 0.05). c Dual culture assays of WT and ΔFocBik1 strains with Botrytis cinerea, Bacillus velezensis, and Actinomycetes strain DMS16689. d Relative expression levels of FocBik1 in WT strains during dual culture with B. cinerea, B. velezensis, and Actinomycetes. Data are presented as mean ± standard deviations. Letters indicate statistical significance determined by one-way ANOVA with Duncan’s multiple range test (P < 0.05)

Fig. 3

Antimicrobial activity of bikaverin extracted from Fusarium oxysporum f. sp. cubense TR4 (Foc TR4). a Schematic representation of bikaverin extraction from wild-type (WT) and FocBik1 knockout mutant (ΔFocBik1) cultures. b Inhibitory effects of WT and ΔFocBik1 extracts on Bacillus velezensis and Actinomycetes strain DMS16689. Bacterial growth was monitored in the presence of 1‰ and 2‰ WT or ΔFocBik1 extracts. The 1‰ and 2‰ WT extracts corresponded to 1.25 and 2.5 μg mL.⁻¹ bikaverin, respectively. c Growth curves of B. velezensis and Actinomycetes strain DMS16689 in the presence of WT and ΔFocBik1 extracts. Data are presented as mean ± standard deviations. d Colony growth of Foc TR4, Colletotrichum gloeosporioides, and Botrytis cinerea on agar plates supplemented with WT and ΔFocBik1 extracts. e Quantification of colony diameters for fungal growth under different treatments. Data are presented as mean ± standard deviations. Letters indicate statistical significance determined by one-way ANOVA with Duncan’s multiple range test (P < 0.05)

Fig. 4

Bikaverin does not directly regulate the pathogenicity of Foc TR4 under sterile conditions. a Experimental design for evaluating the pathogenicity of wild-type (WT) and bikaverin-deficient mutant (ΔFocBik1) strains in tissue-cultured banana seedlings. Seedlings were inoculated with conidial suspensions of WT or ΔFocBik1 and grown either in sterile Murashige and Skoog (MS) medium or autoclaved soil. b Disease symptoms of banana seedlings grown in sterile MS medium. c Disease symptoms of banana seedlings grown in autoclaved soil

Fig. 5

Bikaverin reshapes the bacterial and fungal communities in the rhizosphere of banana plants. a Principal coordinate analysis (PCoA) of bacterial community composition in the rhizosphere of uninoculated control (CK), wild-type (WT), and bikaverin-deficient mutant (ΔFocBik1) treatments based on 16S rRNA sequencing. b Alpha diversity indices (ACE and Shannon) of bacterial communities. Data are presented as mean ± standard deviations (n = 6 biologically independent replicates). Letters indicate statistical significance (P < 0.05). c Taxonomic profiles of bacterial communities at the phylum and genus levels, showing the top 12 most abundant taxa. d PCoA of fungal community composition in the rhizosphere based on ITS sequencing. e Alpha diversity indices (ACE and Shannon) of fungal communities. Data are presented as mean ± standard deviations (n = 6 biologically independent replicates). Letters indicate statistical significance (P < 0.05). f Taxonomic profiles of fungal communities at the family and genus levels, showing the top 12 most abundant taxa

Fig. 6

Isolation and characterization of bikaverin-resistant bacteria with biological control potential. a Growth curves of bacterial isolates, S64, S76, S78, R91, R112, R128, R131, R151, R158, and R160, in liquid medium supplemented with 5‰ and 10‰ extracts from WT or ΔFocBik1 cultures. The 5‰ and 10‰ WT extracts corresponded to 6.25 and 12.5 μg mL.⁻¹ bikaverin, respectively. b In vitro inhibition of Foc TR4 by bacterial isolates. Foc TR4 was co-cultured with bacterial strains on agar plates. c Quantification of inhibition percentages for Foc TR4 colony growth in co-culture experiments. Data are presented as mean ± standard deviations. d Scatter plots of relative growth under bikaverin treatment versus inhibition percentages of Foc TR4 by bacterial isolates at 9, 12, 15, and 18 h. The red spot indicates strain R160, and the green one indicates strain R131

Fig. 7

The schematic illustrates the functional role of bikaverin in Fusarium oxysporum f. sp. cubense TR4 (Foc TR4) during banana wilt disease progression. During competition with other rhizosphere microbes, bikaverin biosynthesis is upregulated, suppressing key PGPRs such as Bacillus spp. The resulting disruption of the microbial community gives Foc TR4 a competitive advantage, facilitating root colonization and infection, ultimately leading to wilt symptoms in banana plants