Abstract
One of the major and yet unsolved threats for viticulture is the group of vascular fungal infections, the so-called grapevine trunk diseases. Besides their latent nature and the enormous number of associated pathogens, their control is also hampered by the lack of effective fungicides, directing growing attention toward the use of biocontrol agents. In the present study the isolation, identification, and characterization of a bacterial strain are presented, showing biocontrol potential against some main causal agents of grapevine trunk diseases. The strain was isolated from the wood of an asymptomatic grapevine and selected for the fungicidal activity against the pathogen Phaeomoniella chlamydospora. According to 16S rDNA, gyrA, and gyrB sequences, the isolate belongs to Bacillus velezensis species. Confrontation tests with the bacterium or with its fermentation broth further revealed growth inhibition and fungicide activity against Botryosphaeria dothidea, Eutypa lata and Diaporthe ampelina pathogens. Fractionation of the bacterial culture filtrate suggests that the antifungal agents secreted by the B. velezenzis isolate are mainly lipoproteins. Phytotoxicity tests were also carried out with the isolate, showing no harmful effects on grapevine foliar disks.
Introduction
Grapevine (Vitis vinifera) is an especially spray-demanding crop, mostly because of some well-known fungal diseases like powdery-, or downy-mildew, black rot, and bunch rot. Contrary to the above diseases, there is no efficient chemical control method for grapevine trunk diseases (GTDs). This group of fungal infections consists of five different syndromes as Botryosphaeria dieback (main causal agents: Diplodia spp., Neofusicoccum parvum), black foot disease (Ilyonectria spp., Campylocarpon spp., Cylindrocarpon spp.) Eutypa dieback (Eutypa lata, Eutypella spp.), Esca complex (Phaeomoniella chlamydospora, Phaeoacremonium minimum, Fomitiporia mediterranea), and Phomopsis disease (Diaporthe spp.). The necrotrophic pathogens of these syndromes infect and colonize the woody tissues of the trunk, therefore they are protected from sprayed fungicides (Bertsch et al., 2013). The only known practical method for the prevention of the development of GTDs is the treatment of plants with sodium arsenite (Fussler et al., 2008). However, its use is prohibited in the European Union since the early 2000s, because of its harmful effects on the environment and human health. Therefore, GTDs have a great negative impact on the grapevine and wine industry with an estimated annual economical loss of more than a billion dollars worldwide (Fussler et al., 2008; De la Fuente et al., 2016). The lack of effective control methods against GTDs, led to increasing attention toward the use of biocontrol agents (Mondello et al., 2018), with a special emphasis on endophytic microorganisms isolated from grapevine tissues (Silva-Valderrama et al., 2021). Several studies examined the potential use of well-known biocontrol species like the bacterium Bacillus subtilis (Trotel-Aziz et al., 2019; Leal et al., 2023), or fungal species like Clonostachys rosea (Billar de Almeida et al., 2020; Silva-Valderrama et al., 2021; Geiger et al., 2022), and members of the genus Trichoderma (Fourie et al., 2001; Berbegal et al., 2020; Pollard-Flamand et al., 2022) against GTDs. Another promising biocontrol organism is Bacillus velezensis, which is extensively investigated in this context during the latest years (Blundell et al., 2021; Bustamante et al., 2022; Langa-Lomba et al., 2023). In the present study, the characterization of the biocontrol potential of a B. velenzensis isolate is presented.
Material and methods
Isolation of endophytic bacteria from grapevine wood
Xylem samples were obtained as described previously (Adejoro et al., 2023) with minor modifications. Fifteen asymptomatic grapevines (Cabernet sauvignon) were sampled in the vineyard of the Eszterházy Károly Catholic University in August of 2022 by drilling. The bark was removed from the trunks at an approximately 2 cm2 surface and disinfected with 70 %v/v ethanol. The drill was also disinfected in the same way between samplings to avoid cross-contamination. Obtained wood flakes were collected in sterile test tubes. Small portions of the samples were inoculated into 5 mL of Luria Bertani liquid medium (LB; 1 %m/v tryptone; 1 %m/v sodium chloride; 0.5 %m/v yeast extract) and incubated on a rotary shaker for 48 h (25 °C, 120 rpm). Dilutions of the cultures at rates of × 10−4, × 10−5, × 10−6, and × 10−7 were prepared in sterile distilled water and 50 µL portions were streaked on the surface of solid LB media and incubated at 25 °C for 3 days. Colonies with different morphology were picked from the plates and subjected to two rounds of streaking to prepare pure cultures. A total of 57 isolates were obtained and preserved in 50 %v/v glycerol at −80 °C.
DNA was isolated from 2 days old liquid cultures (LB, 25 °C, 120 rpm) according to a method described previously (Dashti et al., 2009). For the identification of bacterial isolates partial 16S rDNA, gyrA, and gyrB genes were amplified in polymerase chain reactions according to Wang et al. (2022).
Screening of bacterial strains for antifungal activity
Bacterial isolates were screened for antifungal activity by a plate method previously developed for the study of yeasts killer activity (Woods and Bevan, 1968). Plates containing solid yeast extract-glucose medium (YG, 1 %m/v yeast extract, 2 %m/v glucose, 2 %m/v agar) amended with 0.003 %m/v methylene blue were massively inoculated with a 106 cell/mL conidial suspension of P. chlamydospora strain P46 (Table 1) and dried. Subsequently, bacterial isolates were inoculated on the surface of the plates as ∼1 cm long streaks and cultures were incubated at 25 °C for 5 days. Dead cells take up the methylene blue in a higher amount and oppositely to the living cells they are unable to convert it to colorless leucomethylene blue. As a result, dead fungal cells stained dark blue around bacterial colonies with fungicidal activity.
Fungal isolates used in the present study
| Strain ID | Species | Sequenced loci | Grapevine trunk disease | Reference |
| 15/5 | Botryosphaeria dothidea | ITS, EF | Botryosphaeria dieback | Geiger et al. (2022) |
| 63C/2 | Diaporthe ampelina | ITS, EF, ACT | Phomopsis disease | Geiger et al. (2022) |
| T15/2 | Eutypa lata | ITS | Eutypa dieback | Geiger et al. (2022) |
| P46 | Phaeomoniella chlamydospora | ITS | Esca | Karácsony et al. (2023) |
ITS: internal transcribed spacer; EF: partial transcription elongation factor 1-α gene; ACT: γ-actin gene.
In vitro confrontation tests
All experiments were done in duplicate.
Examination of bacterial culture filtrates
Preparation of culture filtrates
To test the effects of secreted bacterial metabolites on GTD pathogens, liquid cultures were prepared. YG media were inoculated with bacterial isolates and subjected to a three days incubation at 25 °C, with 120 rpm shaking. Fermentation broths were centrifuged (4,000 g, 15 min) and supernatants were filtered through a 0.22 µm pore size membrane to obtain sterile culture filtrates.
Fungal growth inhibition
Solid, two-fold concentrated YG medium was mixed with an equal volume of bacterial culture filtrate or liquid YG medium as control and poured into Petri dishes. Actively growing mycelia of tested GTD fungal pathogens (Table 1) were inoculated on the media and incubated at 25 °C. Mycelial growth was measured at 2, 6, 8, and 9 dpi and RGI% was calculated as in the case of in vitro confrontation tests. All measurements were done in triplicates.
Fungicidal activity
Viability of fungal cells were measured by the use of fluorescein diacetate. Fluorescein diacetate is a widely used dye for the detection and quantification of viability in wide range of organisms including fungi (Gaspar et al., 2001). The substance is converted to the fluorescent molecule fluorescein as the action of intracellular esterases indicating living cells. The formed fluorescein can be either detected by microscopy (Calich et al., 1979) or quantified by a fluorometer after extraction of the biomass with acetone (Gaspar et al., 2001). Mycelial disks (3 mm in diameter) were cut from the actively growing margins of fungal colonies (Table 1) on PDA media, and placed in 500 µL culture filtrate or distilled water as a control. Thereafter, mycelia were incubated at 25 °C for 6 h 25 µL volumes of fluorescein diacetate solution (2 mg mL−1 in acetone) were added to the samples, and incubation continued for an additional 1 h. Reactions were stopped by adding 500 µL acetone. After a 30 min incubation at room temperature and a brief centrifugation (5,000 rpm, 5 min) supernatants were removed and the fluorescence of released fluorescein was measured by QFX Fluorometer (DeNovix Inc., United States) using the FITC (fluorescein isothiocyanate) program. All experiments were done in triplicates.
Fractionation of the bacterial culture filtrate
Different fractions of the 5 mL portions of bacterial culture filtrates were also tested for fungicidal activity. Proteins were precipitated by the addition of 0.7 g mL−1 ammonium sulfate and incubation at 4 °C for 16 h (Wingfield, 1998). After centrifugation (4,000 g, 20 min) supernatants were removed and the pellets were dried. Extraction of 5 mL culture filtrates with organic solvents was carried out two times by an equal volume of chloroform, or ethyl acetate (Maung et al., 2021). The organic extracts were pooled and dried at 60 °C for 16 h in a fume hood. The ammonium sulfate precipitates and the dried organic extracts redissolved in 5 mL distilled water and subjected to fungicidal activity measurement as described above, using Bdo as the test organism.
Phytotoxicity test
Phytotoxicity tests were done as described previously (Karácsony et al., 2023) with minor modifications. Young leaves from one-year-old V. vinifera Cabernet sauvignon potted cuttings were removed, extensively washed with sterile distilled water, and foliar disks with 5 mm diameter were cut. Five foliar disks were placed in a petri dish (d = 55 mm) containing 10 mL of ten-fold diluted bacterial culture filtrate or distilled water as a control. The foliar disks were incubated at room temperature with ambient light conditions for 6 days and photographed.
Statistical analysis
Significances of differences were calculated by One-way ANOVA test using GraphPad Prism 5 software (GraphPad Software, San Diego California USA, www.graphpad.com) demo version.
Results and discussion
A total of 57 bacterial strains were isolated from asymptomatic grapevines in 2022, in Eger wine region. Preliminary screening of the isolates for fungicidal activity against Pch led to the selection of 10 positive strains according to the blue discoloration of fungal cells around the bacterial colonies (Fig. 1). The species Pch was selected for preliminary screening because of its slow growth rate and its high significance as the pioneer pathogen of the possibly most important GTD, the Esca disease complex (Bertsch et al., 2013).
Representative photograph of a plate used for the screening of bacterial isolates for antifungal activity against Pch (YG medium + methylene blue, 25 °C, 5 dpi). Arrows indicate bacterial colonies with a dark blue halo on the mycelial mat, suggesting fungicidal activity
Citation: Acta Phytopathologica et Entomologica Hungarica 58, 2; 10.1556/038.2023.00192
The 10 selected isolates were subjected to in vitro confrontation tests against representative causal agents of the most important GTDs (Fig. 2). One bacterial strain (ID TKP3/1) showed significant growth inhibition against all tested fungal species. The RGI% values were somewhat lower in the case of Bdo (28%) and Dam (23%), while Ela and Pch suffered high growth inhibition (65% and 100% respectively) when co-inoculated with TKP3/1. The following experiments were focused on isolate TKP3/1 to further reveal its biocontrol potential. The sequencing results suggested, that the isolate belongs to the B. velezensis species. All three examined loci showed high similarity to strains of this taxon: partial 16S RNA (GenBank Accession: OR514235) showed 100% similarity with B. velezensis strain YA215; gyrA sequence (GenBank Accession: OR513919) was 99.89% similar to B. velezensis strain Bac57, and gyrB (GenBank Accession: OR513920) was 97.79% identical with B. velezensis strain SRCM100072. While this species attracts attention as a biocontrol agent only in recent years, it is already commercialized (previously identified as B. amyloliquifaciens) and efficiently used against several plant pathogenic fungi (Rabbee et al., 2023). B. velezensis is also recognized as a potential antagonist of GTDs pathogens suggested by in vitro tests (Blundell et al., 2021; Bustamante et al., 2022; Boiu-Sicuia et al., 2023) as well as in planta experiments (Langa-Lomba et al., 2023). While the efficient growth inhibition of Bdo, Ela, and Dam (besides some other GTD-associated fungi) by B. velezensis is reported previously in the above studies, to the best of our knowledge this study is the first observation of the antagonism of this species against Pch. The antifungal mode of action of B. velenzensis against GTD pathogens is mostly based on its secreted metabolites (Bustamante et al., 2022). Our results on the effect of TKP3/1 culture filtrate on GTD fungi are in accordance with these previous results. Fungal growth tests in the presence of 50 %v/v cultural filtrate of TKP3/1 isolate (Fig. 3) showed significant growth inhibition for Bdo (81 RGI%), Ela (25 RGI%), Dam (56 RGI%), as well as Pch (100 RGI%).
Percental mean growth inhibition values and standard deviances for the tested bacterial isolates against GTD pathogenic fungi in dual culture assays. Letters mark significantly differing (P < 0.05) groups of datasets
Citation: Acta Phytopathologica et Entomologica Hungarica 58, 2; 10.1556/038.2023.00192
Percental growth inhibition values of GTD pathogenic fungi in the presence of 50 %v/v culture filtrate (YG medium, 25 °C, 120 rpm, 3 days) of strain TKP3/1. Letters mark significantly differing (P < 0.05) groups of datasets
Citation: Acta Phytopathologica et Entomologica Hungarica 58, 2; 10.1556/038.2023.00192
Besides its growth inhibition ability, the culture filtrate of strain TKP3/1 is also tested for fungicidal activity. Results showed a significant decrease in the viability of cells in the presence of TKP3/1 culture filtrate in the case of all fungi, compared to control mycelia treated with distilled water (Fig. 4). This result suggests, that B. velezensis strain TKP3/1 may be suitable for the treatment of grapevines previously infected by GTD pathogens, its applicability is not limited to a preventive effect.
Effect of 6h treatment with culture filtrate of strain TKP3/1 (CF: YG medium, 25 °C, 120 rpm, 3 days) or distilled water (DW) on the ability of GTD pathogens mycelia to hydrolyze fluorescein diacetate. Asterisks mark the significance of differences (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001)
Citation: Acta Phytopathologica et Entomologica Hungarica 58, 2; 10.1556/038.2023.00192
To get further information on the active antifungal agents secreted by strain TKP3/1, its culture filtrate was subjected to fractionation and subsequent fungicide assays using Bdo as test organism (Fig. 5). The fungicidal activity of the culture filtrate is retained after ammonium sulfate precipitation or in the extracts of the organic solvents chloroform and ethyl acetate. The precipitation of the fungicidal agents by ammonium sulfate suggests their macromolecular nature, while their solubility in organic solvents suggests a hydrophobic character. These results are in accordance with the high number of previously identified antimicrobial lipopeptides in B. velezensis, and with several reports suggesting their crucial role in the antifungal activity of this bacterial species (Fazle Rabbee and Baek, 2020; Liu et al., 2020; Platel et al., 2021; Yu et al., 2022; Xiong et al., 2022).
Effect of 6h treatment with fractions of culture filtrate of strain TKP3/1 (YG medium, 25 °C, 120 rpm, 3 days) or distilled water (DW) on the ability of Bdo mycelia to hydrolyze fluorescein diacetate. Chl: chloroform extract; EtAc: ethyl acetate extract; AS: ammonium sulfate precipitate. Asterisks mark the significance of differences (**P < 0.005)
Citation: Acta Phytopathologica et Entomologica Hungarica 58, 2; 10.1556/038.2023.00192
While B. velezensis is in the focus of researches related to plant disease management, relatively few studies are available on its effectiveness against GTDs. Dual culture assays previously pointed out, that B. velezensis is an antagonist of Botryosphaeria dieback causal agents N. parvum and Diplodia seriata (Blundell et al., 2021; Bustamante et al., 2022; Boiu-Sicuia et al., 2023). The RGI% of the bacterium against these pathogens was between 30 and 75 depending on the pathogen and the bacterial isolate, while TKP3/1 showed 28 RGI% against another Botryiosphaeriaceae pathogen, Bdo in this study. Previously reported RGI% of B. velezensis against Ela was between 75 and 80 (Bustamante et al., 2022; Boiu-Sicuia et al., 2023) contrary to the 25 RGI% for TKP3/1. Strain TKP3/1 was exceptionally effective against Dam (56 RGI%) compared to a previously studied strain with 20 RGI% (Blundell et al., 2021). There are no literature data on the efficacy of B. velezensis as an antagonist of Pch, while the strain tested in this study showed 100% inhibition.
In the case of biocontrol agents, it's a straightforward prerequisite, that it does not damage the treated plant. While B. velezensis is broadly acknowledged as a potent biocontrol agent, its phytopathogenic behavior was also reported in some hosts like onion (Hwang et al., 2012), potato (Wang et al., 2017), or peach (Zeng et al., 2022). To examine the potential toxicity of B. velezensis TKP3/1 on V. vinifera, its cultural filtrate was subjected to phytotoxicity tests, using grapevine foliar disks (Fig. 6). After 6 days of treatment no harmful effects observed, suggesting that B. velezensis TKP3/1 strain can be applied on grapevines safely.
Effect of 6 days treatment with distilled water (a), or ten-fold dilution of culture filtrate of strain TKP3/1 (b: YG medium, 25 °C, 120 rpm, 3 days) on grapevine foliar disks
Citation: Acta Phytopathologica et Entomologica Hungarica 58, 2; 10.1556/038.2023.00192
In conclusion, the characteristics of the B. velezensis TKP3/1 strain presented in this study suggest its practical usability as a biocontrol agent against GTDs. Since the strain was isolated from the same niche as the pathogens, it can access these fungi in field conditions. The in vitro experiments with dual cultures and culture filtrates suggest, that the antagonistic activity of TKP3/1 against GDT pathogens is based on small secreted lipopeptides, which possibly can be transported by the vascular system of grapevine. This eliminates the need for direct contact between the pathogens and the biocontrol agent to achieve efficient antagonism. These secreted molecules also seem to be harmless for the grapevine, according to the phytotoxicity tests. While these preliminary results on the ability of the strain TKP3/1 to control GTD pathogens are promising, several further (e.g. in planta, in field) investigations should be carried out to prove this hypothesis.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
This work was financed by the NRDI Fund (projectID: TKP2021-NKTA-16). Kálmán Zoltán Váczy was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and the Bolyai + New National Excellence Program of the Ministry for Innovation and Technology.
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