| Cucumber Fusarium wilt (CFW), a devastating soil-borne vascular fungal disease caused by Fusarium oxysporum f. sp. cucumerinum, survived in different types of soil, often threaten the quality and productivity of cucumber (Cucumis sativus L.) in the worldwide. So far, there are many reports about the pathogenic mechanism of F. oxysporum, such as theory of catheter obstruction, theory of toxin, cell wall degradation. However, efficient strategies for the management of Fusarium wilt have not been developed, which could be explained in part by our limited information regarding the biology of F. oxysporum f. sp. cucumerinum.Biofilms are dense, highly hydrated cell clusters that form on surfaces and are embedded in a self-produced gelatinous matrix comprising extracellular polymeric substances (EPSs). Moreover, the formation, maturation and dispersal of biofilms are key processes in the life cycle of many animal and plant bacterial pathogens. It has been reported that the pathogenic F. oxysporum involved human keratities could form biofilm. The aim of the present study was to investigate the interaction mechanisms between biofilm formation and virulence in F. oxysporum f. sp. cucumerinum through biofilm formation condition, biofilm characteristics, regulatory genes function analysis. The main results were as follows:1. Biofilm formation and conditions analysis in F. oxysporum f. sp. cucumerinum. Conidia of F. oxysporum f. sp. Cucumerinum (Foc-GD) were counted using a hemacytometer, and adjusted to 106 cfu/mL in RPMI 1640. Then 200 μL of the suspension was added to the wells of 96-well, flat-bottomed polystyrene microtiter plates and incubated at 28℃ without shaking to allow the conidia to settle and adhere to the bottom of the plate. Following the adhesion stage, the wells containing Foc-GD biofilms were washed gently two times with PBS to remove nonadherent cells using a microtiter plate washer. Fungal cells that remained attached to the plastic surface were considered true biofilms. The biofilms were mature after incubation at 28℃ for 48 h. At the end of the incubation, F. oxysporum f. sp. cucumerinum biofilms were quantified using a tetrazolium XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. The results showed that temperature, pH values, sugars influenced the biofilm formation. Foc-GD formed significantly strong biofilms under conditions of slightly acidic and neutral pH (pH5-7),28℃ and glucose as carbon source, respectively.2. Characteristics analysis of F. oxysporum f. sp. cucumerinum biofilms. The mature biofilms were stained by fluorescent nucleic acid stain and the architecture was examined using fluorescence microscope and confocal scanning laser microscopy (CSLM). The results showed that the biofilms formed by strain Foc-GD consisted of a highly organized architecture with red hyphal cells interwoven with green extracellular polysaccharide materials.Compared with planktonic cells, the mature biofilms were resistant to harsh environmental factors, such as high and low temperatures, UV light and fungicides. The results showed that the metabolic activity of biofilms was not affected by exposure to a relatively high temperature, whereas planktonic cells showed a significant reduction after being exposed to 45℃ for 30 min (62.25%) and 60 min (86.29%). The biofilms and planktonic cells did not show significant differences at 4℃ after incubation for 24h. At-10 and -20℃, biofilms were less susceptible to damage than planktonic cells. After 20,30 min of UV light irradiation, the percent metabolic activities of planktonic cells were significantly reduced by 52.29%,90.23%, but by no more than 29.18%,43.11% for biofilm cells respectively. After 24h incubation with 64 μg/mL carbendazim, prochloraz and propiconazole, respectively, the percent metabolic activity of biofilm cells was reduced by 33.8%,39.2% and 26.6%. In contrast, planktonic forms were reduced by 76.5%,73.2% and 50.7%.3. Cloning and expression of velvet protein FocVell in Foc-GD. The results showed that an open reading frame (ORF) of 1596 bp interrupted by a 94-bp intron. The gene encodes a predicted protein of 532 amino acids and was designated FocVell (GenBank Accession number:KJ716229). The predicted amino acid sequence of FocVell shared 97.56%,97.74%, and 78.29% identity with FfVell from F. fujikuroi (GenBank:FN548142), FvVel from F. verticillioides (GenBank:DQ274059), and FgVEA from F. graminearum (GenBank: JN635273), respectively. Real-time PCR results demonstrated that FocVell expression was low during early growth stages (24 and 36 h, p< 0.05), peaked at 48h, and then remained constant at 60 and 72 h (p< 0.05). Conidiophores typically proliferate after 48 h. These results were in complete agreement.4. Functional analysis of velvet protein FocVell in Foc-GD. To investigate the functions of FocVell gene in F. oxysporum f. sp. cucumerinum, gene deletion mutants were generated using a homology recombination strategy in the parental F. oxysporum f. sp. cucumerinum Foc-GD strain. The FocVell gene plays an important role in fungal growth, colony morphology, development, secondary metabolism, biofilm formation and virulence. In PDB medium, the deletion mutant △FocVell-3 produced greatly fewer conidia than the wild-type strain or the complemented strain △FocVell-3C (p< 0.05). Microscopic examination of hyphae revealed that △FocVell-3 displayed a characteristic flat colony phenotype with dramatically reduced aerial mycelium, grew markedly slower, exhibited a strong increase in hyphal branching than the wild-type strain Foc-GD and the complemented strain A Foc Vell-3 C on PDA medium. The sensitivity of the FocVell deletion mutant to various stresses was tested. Compared with the wild-type and complemented strain, △FocVell-3 exhibited significantly increased resistance to 1.0 M KC1,1.0 M sorbitol,0.05% Congo red and 5 mM caffeine (p< 0.05), but showed increased sensitivity to iprodione (16 μg/mL, p< 0.05) and prochloraz (0.2μg/mL, p< 0.05). These results suggested that FocVell may be associated with cell wall organization and cell wall integrity.The mutant △FocVell-3 showed significant defects in the thin biofilms that formed on the polystyrene surface, exhibiting heterogeneous hyphae and EPS production. Deletion of FocVell not only reduced the number of aggregative spores but also led to a delay in conidial germination. XTT reduction assays showed that quantification of the biofilms of wild-type and complemented strains were approximately 1.23-,1.52-,1.69-, and 2.56-fold that of △FocVell-3 after being incubated for 2,4,8, or 12 h, respectively (p< 0.05). Cucumber seedlings inoculated with conidia of the wild-type or complemented strain showed progressive wilt symptoms and usually died at 15dpi and the DSIs of Fusarium wilt were 93.7 and 92.3 respectively. In contrast, under the same conditions, plants inoculated with the △FocVell-3 mutant displayed a significantly lower DSI (37.3, p< 0.05), and most of the plants inoculated with this mutant survived the assay or developed only mild disease symptoms.5. F. oxysporum f. sp. cucumerinum was modified with the gfp gene using the sGFP expression vector. We observed that spore germinated and continued to adhere to the surface of cucumber seedling roots 24h after inoculation with a confocal scanning laser microscopy. The fungus penetrated the lateral root epidermis and into epidermal cells gradually. In the advanced stages of cucumber root colonization, the fungal pathogens infected taproot epidermis and vascular bundles. After 120h inoculation, at the late stages of cucumber root colonization, the vascular bundles were filled with fungal pathogens and hyphal layers were ovserved.These results demonstrated that the disruption of FocVell reduced the virulence and biofilm formation of F. oxysporum f. sp. cucumerinum. Our findings may provide a novel perspective on the pathogenic mechanism associated with biofilms of F. oxysporum f. sp. cucumerinum and theory support for comprehensive prevention and control of Cucumber Fusarium Wilt. |
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