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. 2020 Jul 20;8(7):1080.
doi: 10.3390/microorganisms8071080.

Biological Control of Verticillium Wilt on Olive Trees by the Salt-Tolerant Strain Bacillus velezensis XT1

David Castro  1 , Marta Torres  2   3 , Inmaculada Sampedro  2   3 , Fernando Martínez-Checa  2   3 , Borja Torres  1 , Victoria Béjar  2   3
Affiliations

Biological Control of Verticillium Wilt on Olive Trees by the Salt-Tolerant Strain Bacillus velezensis XT1

David Castro et al. Microorganisms. .

Abstract

Verticillium wilt, caused by the pathogen Verticillium dahliae, is extremely devastating to olive trees (Olea europea). Currently, no successful control measure is available against it. The objective of this work was to evaluate the antifungal activity of Bacillus velezensis XT1, a well-characterized salt-tolerant biocontrol strain, against the highly virulent defoliating V. dahliae V024. In vitro, strain XT1 showed to reduce fungal mycelium from 34 to 100%, depending on if the assay was conducted with the supernatant, volatile compounds, lipopeptides or whole bacterial culture. In preventive treatments, when applied directly on young olive trees, it reduced Verticillium incidence rate and percentage of severity by 54 and ~80%, respectively. It increased polyphenol oxidase (PPO) activity by 395%, indicating an enhancement of disease resistance in plant tissues, and it decreased by 20.2% the number of fungal microsclerotia in soil. In adult infected trees, palliative inoculation of strain XT1 in the soil resulted in a reduction in Verticillium symptom severity by ~63%. Strain XT1 is biosafe, stable in soil and able to colonize olive roots endophytically. All the traits described above make B. velezensis XT1 a promising alternative to be used in agriculture for the management of Verticillium wilt.

Keywords: Bacillus velezensis; Olea europaea; Verticillium dahliae; antifungal activity; biocontrol; crop protection.

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Conflict of interest statement

DC was a full-time employee of Xtrem Biotech S.L., company that holds an exclusive license agreement on the patent that protects B. velezensis XT1 industrial explotation. All the authors declare that the research was conducted in the absence of any competing interests.

Figures

Figure 1
Figure 1
Biosafety evaluation of Bacillus velezensis XT1. (a) Escherichia coli MC4100 sensitivity assay. Pseudomonas putida KT2440, Burkholderia cepacia CC-Al74 and 0.2% (w/v) copper sulfate were used for comparison. Non-inoculated tryptic soy broth (TSB) was used as a control. (b) Caenorhabditis elegans N2 pathogenicity test. Pseudomonas aeruginosa PAO1 was used for comparison. E. coli OP50 was used as a control. (c) Comparison of LC50 values obtained in C. elegans N2 and average concentrations used in the field. Copper sulfate was used for comparison. Significant differences between the LC50 and the field concentration are indicated by asterisks (** p < 0.01, ***, p < 0.001).
Figure 2
Figure 2
In vitro antifungal activity of Bacillus velezensis XT1 against Verticillium dahliae V024. (a) Assay on solid media with the whole culture of strain XT1. Non-inoculated tryptic soy broth (TSB) was used as a control. (b) Assay on modified medium prepared with the half-diluted and undiluted supernatant of strain XT1. Control consisted of medium hydrated with distilled water. (c) Volatile compounds antifungal test. Control consisted of non-inoculated TSB. (d) Crude lipopeptide extract antifungal test. Control consisted of medium hydrated with distilled water. (e) Minimal inhibitory concentration (MIC) of the crude lipopeptide extract determined in a liquid assay.
Figure 3
Figure 3
Prevention of Verticillium wilt on young olive trees by Bacillus velezensis XT1. (a) PCR amplification of Verticillium dahliae in olive tree leaves. 1: Infected non-treated control, 2: infected B. velezensis XT1-treated plants 30 days after infection, 3: infected B. velezensis XT1-treated plants 90 days after infection, 4: molecular weight ladder. (b) Polyphenol oxidase (PPO) activity. Leaves of infected non-treated olive trees were used as control. (c) Incidence rate of infected non-treated control and infected XT1-treated olive trees. (d) Percentage of severity of infected non-treated control and infected XT1-treated olive trees. (e) Transversal cut of infected non-treated control and infected XT1-treated olive tree branches. (f) Whole plant aspect of infected non-treated control and infected XT1-treated trees. Significant differences with the control are indicated by asterisks (**, p < 0.01; ***, p < 0.001).
Figure 4
Figure 4
Palliative treatment of Verticillium wilt on diseased adult olive trees by Bacillus velezensis XT1. (a) Microscopic observation of V. dahliae isolated from affected trees. (b) Olive trees in the field experiment before and after the treatment with strain XT1. Control consisted of diseased non-treated trees.
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