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. 2020 Apr 22;10(1):6857.
doi: 10.1038/s41598-020-63689-y.

Trichoderma virens Gl006 and Bacillus velezensis Bs006: a compatible interaction controlling Fusarium wilt of cape gooseberry

L F Izquierdo-García  1 , A González-Almario  2 , A M Cotes  1 , C A Moreno-Velandia  3
Affiliations

Trichoderma virens Gl006 and Bacillus velezensis Bs006: a compatible interaction controlling Fusarium wilt of cape gooseberry

L F Izquierdo-García et al. Sci Rep. .

Abstract

The combination of Trichoderma virens Gl006 and B. velezensis Bs006 as a consortium has high potential to control Fusarium wilt (FW) of cape gooseberry (Physalis peruviana) caused by Fusarium oxysporum f. sp. physali (Foph). However, the interactions between these two microorganisms that influence the biocontrol activity as a consortium have not been studied. Here, we studied the interactions between Gl006 and Bs006 that keep their compatibility under in vitro and greenhouse conditions. Antagonism tests between Gl006 and Bs006 inoculated both individually and in consortium against Foph strain Map5 was carried out on several solid media. The effect of supernatant of each selected microorganism on growth, conidia germination, biofilm formation and antagonistic activity on its partner was also studied. Biocontrol activity by different combinations of cells and supernatants from both microorganisms against Fusarium wilt was evaluated under greenhouse conditions. In vitro antagonism of the consortium against Foph showed a differential response among culture media and showed compatibility among BCA under nutritional conditions close to those of the rhizosphere. The supernatant of Bs006 did not affect the antagonistic activity of Gl006 and vice versa. However, the supernatant of Bs006 promoted the biocontrol activity of Gl006 in a synergistic way under greenhouse, reducing the disease severity by 71%. These results prove the compatibility between T. virens Gl006 and B. velezensis Bs006 as a potential tool to control Fusarium wilt of cape gooseberry.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Growth of T. virens Gl006 in PDB medium supplemented with different concentrations (1, 5, 10, 20 and 30%) of B. velezensis Bs006 cell-free supernatant. (b) Growth of B. velezensis Bs006 in LB medium supplemented with 1, 5, 10, 20 and 30% of T. virens Gl006 cell-free supernatant. Growth was expressed as optical density (OD630 nm) measured in a microplate reader at 24 and 48 hours. Columns with the same letter are not significantly different according to Tukey test (α = 0.05) at each time of evaluation. Bars on the columns represent standard deviation (n = 3).
Figure 2
Figure 2
Antagonistic activity of T. virens Gl006 after being exposed to Bs006-supernatant in dual confrontation test against F. oxysporum f. sp. physali strain Map5 (Foph). Gl006 (G) conidia were exposed to Bs006 cell-free supernatant (Sup) from 1 to 30%. Growth inhibition of Foph was measured according to diametral growth in the positive control - F in PDA medium, after seven days of incubation at 25 °C.
Figure 3
Figure 3
Antagonistic activity of Bs006 after being exposed to Gl006-supernatant in dual confrontation test against F. oxysporum f. sp. physali strain Map5 (Foph). Bs006 (B) cells exposed for six hours to Gl006 cell-free supernatant (Sup) from 1 to 30%. Growth inhibition of Foph was measured according to diametral growth in the positive control - F in PDA medium after seven days of incubation at 25 °C.
Figure 4
Figure 4
Biofilm formation of B. velezensis Bs006 in presence of T. virens Gl006 supernatant (a) and conidia (b). B. velezensis Bs006 was grown for 24 h in LB medium supplemented with Gl006-supernatant from 1 to 30% (SupGl1 to SupGl30) or viable conidia of T. virens Gl006 at 105 to 107 conidia.mL−1 (CoGlE5 – CoGlE7). Control: Growth of Bs006 in LB free of Gl006-supernatant and conidia. Columns with the same letter are not significantly different according to Tukey test (α = 0.05). Bars on the columns represent standard deviation (n = 9).
Figure 5
Figure 5
Growth inhibition of Foph colony in solid media by Gl006, Bs006 and the consortium. (ARE), artificial root exudates. soil solution + synthetic root exudates (SARE), soil solution (SS). Luria Bertani-Agar (LBA), Potato-Dextrose-Agar (PDA), PDA (50%) + LBA (50%) (LBA+PDA), Cape gooseberry root exudates (CRE), soil solution + Cape gooseberry root exudates (SCRE). Bars on the columns represent standard deviation between experimental units of three replicates in the time (n = 12). Columns with the same letter are not significantly different according to Tukey test (α = 0.05).
Figure 6
Figure 6
Microscopy observations of the interaction between T. virens Gl006 and B. velezensis Bs006 in jellified cape gooseberry root exudates under fluorescent microscope. (a) Germinated conidia of T. virens Gl006 in absence of B. velezensis. (b–d) Interaction between Gl006 conidia and Bs006 cells. Yellow arrows indicate Bs006 spores at the beginning of biofilm formation on Gl006 surface after 72 hours contact.
Figure 7
Figure 7
Environmental scanning microscopy images of non-germinated and germinated seeds of cape gooseberry inoculated with T. virens Gl006 and B. velezensis Bs006. (a) Surface of seed coat without inoculations, (b and c) co-inoculated seed with Gl006 and Bs006, (d) co-inoculated seedling with Bs006, Gl006 and Foph. Bs006: B. velezensis Bs006 cells, Gl006: T. virens Gl006 hypha, Foph: F. oxysporum f. sp. physali-Map5. Samples were observed at 24 hours after inoculation and incubation at 25 °C.
Figure 8
Figure 8
Effect of the combination of cells and supernatants of Gl006 and Bs006 on the progress of Fusarium wilt severity. Columns with the same letter are not significantly different according to Duncan's multiple range test (α = 0.05). Con = Conidia, Cel = cells, Bs = B. velezensis Bs006, Gl = T. virens Gl006. Sup = cell-free supernatant.
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