Edited by: Sandra Torriani, University of Verona, Italy
Reviewed by: Antonio Santos, Complutense University of Madrid, Spain; Matthias Sipiczki, University of Debrecen, Hungary
*Correspondence: Ileana Vigentini
This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against
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Plants provide over 80% of the human diet. Just three cereal crops (i.e., rice, maize, and wheat) and two fruit crops (grape-berries and citrus fruits) provide 70% of energy intake and cope the production of 80% of the fermented beverages in the world (FAO,
Yeasts are unicellular fungi that have been isolated from different ecosystems and sources both natural and in connection with human activities. They can be found on/in fruits, including
The main aim of this work was to evaluate the antagonistic potential of yeasts isolated from grape berries collected from
Yeast strains were isolated between 2013 and 2016 from grape berries collected in Georgia, Italy, Romania, and Spain from
Origin and source of the yeast strains assayed in this work.
The mold strains used in this work were
The antagonistic activity of the 241 yeast isolates against
An estimation of the starting concentration of yeast cells capable to inhibit the mold growth was carried out by the following test. Fresh cultures of the yeasts that overcome the second step of selection were grown in YPD broth at 25°C for 3 days. PDA plates were prepared for each strain containing a different cell concentration, from 103 to 106 CFU/mL. When the plates solidified, 10 μL of conidial suspensions (3 × 105 spores/mL) of
The killer character assay was performed according to Stumm et al. (
In order to investigate the reason of the observed inhibitory effect, the previous selected strains were examined taking in consideration the production of cell wall lytic enzymes. Yeast fresh cultures were adjusted at a final concentration of 1 × 106 CFU/mL. To evaluate the proteolytic activity, 20 μL of the yeast suspension were spotted onto Skim Milk agar (Merck, Darmstadt, Germany); the formation of a clear halo around the colony after incubation at 25°C for 5 days indicated the enzymatic activity. Glucanase and chitinase activities were determined by replica plating technique. In this case, 20 μL of the yeast suspension were spotted onto YPD plates containing 0.2% β-glucan (Sigma, Town, Nation) and YPD plates containing 0.2% chitin (Sigma). Petri dishes were incubated at 30°C for 5 days. Colonies were rinsed off the plates with distilled water before staining the plates with 0.03% (w/v) Congo Red. A clear zone around the colony meant the presence of glucanase activity. Yeasts were screened for polygalacturonase production with the method described by Strauss et al. (
Selected yeast strains were also evaluated for their production of VOCs and hydrogen sulfide released against the molds
The capability to produce biofilm was evaluated following the protocol of Jin et al. (
In order to investigate the influence of iron concentration on the inhibitory activity of the selected yeasts the following test was carried out. PDA plates without added iron and plates with 5 and 20 μg/mL of FeCl3 were prepared spreading on plates a conidial suspension (3 × 105 spores/mL) of
In order to examine the effect of other potential metabolites derived from the primary or secondary metabolism of yeasts produced by antagonistic yeasts, the molds were grown in a medium containing the supernatant of a yeast culture. The yeast cultures were grown in 50 mL YPD broth at 25°C for 5–7 days in a shaker at 125 rpm. The cell growth was monitored by spectrophotometer measurements at 600 nm (Jenway 7315, Staffordshire, U.K.). When yeast cultures attained the stationary phase the supernatants were collected by centrifugation at 3,500 rpm for 5 min at 4°C (Rotina 380 R, Hettich Zentrifugen, Tuttlingen, Germany) and filtered by a 0.45 μm sterile membrane (Minisart, Goetting, Germany). Five, 0.5, and 0.05 mL of supernatants were mixed with warm (<45°C) and concentrated 5X PDA medium by adjusting the volume with sterile distilled water and poured in Petri dishes. When the plates solidified, 10 μL of conidial suspensions (3 × 105 spores/mL) of
The yeast strains showing an evident inhibitory activity by
The inhibiting activity of strains, that showed the best results in the previous tests, were compared to the commercial pesticide Switch®, Syngenta (37.5%
Two hundred and thirty-one yeast strains were isolated from grape berries samples of different vines: 85, 62, and 16 from a conventional, a biodynamic, and an organic vineyard, respectively. Sixty-seven yeasts were collected from
Yeast species occurrence and distribution of the isolated and identified from
FZ02 | 46 | 15 | 9 | 1 | ||||
CABMC2A | – | – | – | 1 | ||||
FZ03a | – | 2 | – | – | – | |||
HB09c | – | – | – | – | 1 | |||
CABMB1A | – | – | – | – | 1 | |||
HURM6B | – | – | – | – | 4 | |||
CAMB9A | – | 17 | 34 | 1 | 28 | |||
NUR3AM | – | – | – | – | 1 | |||
ROMA10 |
– | – | – | – | 5 | |||
CABM7C |
– | 9 | 8 | 1 | 1 | |||
CABM9C |
– | – | – | – | 5 | |||
ROMAM1A |
– | – | – | – | 2 | |||
– | – | – | – | 1 | ||||
– | – | – | – | 4 | ||||
HB01a | 4 | – | 1 | – | ||||
CABM8C | – | – | – | 1 | ||||
SEMA6B | – | – | – | – | 4 | |||
SEHM2A | – | – | – | 1 | ||||
EP02c | 3 | 4 | 1 | – | ||||
HURM4A | – | – | – | 1 | ||||
SEHUM7B | – | – | – | 1 | ||||
ARIM1B | – | – | – | 1 | ||||
CABMA3A | – | – | – | – | 1 | |||
SEHM1C | – | – | – | – | 1 | |||
PIEM5B | – | – | – | – | 1 | |||
HB02b | 4 | 1 | 3 | – | ||||
Total: | 85 | 62 | 16 | 67 |
Unfortunately, we encountered the problem that isolates ROMA1A, ROM10, CABM7C, and CABM9C (Table
All yeast isolates were subjected to a preliminary
Wildlife vines | 67 | 42 | 62.7 | 18 | 42.9 | 26.9 |
Biodynamic vineyard | 62 | 11 | 17.7 | 2 | 18.2 | 3.2 |
Organic vineyard | 16 | 1 | 6.2 | 0 | 0 | 0 |
Conventional vineyard | 85 | 6 | 7.1 | 0 | 0 | 0 |
Total isolates | 230 | 60 | 26.1 | 20 | 33.3 | 8.7 |
After the preliminary assay, a second
MICs were determined in triplicate for all yeast strains selected after dual assays against the different molds. The evaluation of the MIC revealed that the 20 yeasts significantly reduced the progress of hyphal growth of
Phenotypical assaying for yeast antagonistic activity against molds and their volatile organic compounds (VOCs) referred to mycelial growth reduction of
FZ02a | 28.0 | – | + | + | + | – | – | 0.3 | + | Positive with |
0.110 | ||
CABMC2A | 45.0 | – | – | – | – | – | – | 0 | + | Positive with |
0.030 | ||
SEHMA6A | 31.0 | – | + | – | – | – | – | 0 | – | Positive with |
0.042 | ||
CABM8A | 44.5 | – | + | – | – | – | – | 0.1 | + | Positive with |
0.010 | ||
CABCM1A | 35.8 | – | + | – | – | – | – | 0.2 | – | Positive with |
0.100 | ||
CAMM3A | 34.8 | + | + | – | – | – | – | 0.1 | – | Positive with |
0 | ||
CAMM6A | 40.5 | – | – | – | – | – | – | 0.3 | – | Negative | 0.010 | ||
SEHI3C | 25.8 | – | – | – | – | – | – | 0.1 | – | Positeive with |
0.030 | ||
SEHI1C | 21.0 | – | + | – | – | – | – | 0 | + | Positive with |
0.080 | ||
SEHM7C | 26.3 | – | – | – | – | – | – | 0.1 | – | Positive with |
0.150 | ||
CAMB9A | 27.7 | – | – | – | – | – | – | 0 | – | Negative | 0.034 | ||
CABMB1A | 18.7 | + | – | – | – | – | – | 0 | – | Positive with |
0.011 | ||
Control | 28.7 | + | – | – | – | – | – | 0.3 | + | Negative | 0.033 | ||
ROMA10 | 28.3 | + | – | – | – | – | – | 0 | – | Positive with |
0.070 | ||
CABM1A | 44.5 | – | + | – | – | – | – | 0.2 | + | Negative | 0.010 | ||
SEHIB8 | 37.0 | + | + | – | – | – | – | 0.2 | + | Positive with |
0.027 | ||
ROMMA1A | 46.5 | + | – | – | – | – | – | 0 | – | Positive with |
0.050 | ||
SEHMA6B | 26.7 | – | – | – | – | – | – | 0 | + | Positive with |
0.014 | ||
CABMC6C | 29.5 | + | – | – | – | – | – | 0 | – | Positive with |
0.360 | ||
CABMA3A | 40.0 | – | – | – | – | + | + | 0.1 | + | Positive with |
0.010 | ||
HB02b | 28.0 | – | – | – | – | – | – | 0 | – | Positive with |
0.110 |
Disease incidence by
FZ02a | – | – | – | – | + | – | – | – | + | + | + | + | |
CABMC2A | – | – | – | – | + | – | – | – | + | + | + | + | |
CABMB1A | + | – | – | – | + | + | + | + | + | + | + | + | |
SEHMA6A | + | – | – | – | + | + | + | + | + | – | – | – | |
CABM8A | + | – | – | – | + | + | + | + | + | + | – | – | |
CABCM1A | + | + | – | – | + | + | + | + | + | + | + | + | |
CAMM3A | + | + | – | – | + | + | + | + | + | + | + | + | |
CAMM6A | + | – | – | – | + | + | – | – | + | + | + | + | |
SEHI1C | + | – | – | – | + | – | – | – | + | + | + | + | |
SEHM7C | + | – | – | – | + | + | – | – | + | + | + | – | |
CAMB9A | + | + | + | + | + | + | + | + | + | + | + | + | |
SEHIC3 | – | – | – | – | + | + | + | + | + | + | + | + | |
Control | – | – | – | – | – | – | – | – | – | – | – | – | |
CABM1A | + | + | – | – | + | + | + | + | + | + | + | – | |
SEHIB8 | + | – | – | – | + | + | + | + | + | + | + | + | |
SEHMA6B | + | – | – | – | + | + | + | – | + | + | + | + | |
CABMC6C | + | + | – | – | + | + | + | + | + | + | + | + | |
CABMA3A | + | + | + | + | + | + | + | + | + | + | + | + | |
HB02b | – | – | – | – | + | – | – | – | + | + | + | + |
From over the 20 yeast strains assayed for the killer character, only
All yeasts that passed the dual test were evaluated for extracellular enzymatic activities (β-1, 3-glucanase, proteolytic, and pectinolytic activities). Twelve out of the 20 yeast strains were able to hydrolyze at least one of the assayed compound (milk proteins, pectin, glucan, and chitin). Only five yeast strains (4
Percentage data concerning production of VOCs and hydrogen sulfide release among the 20 yeast strains selected showed that 10 yeast strains (3
Only yeast strains of
Antagonistic activity of most of the selected strains were not significantly influenced by tested FeCl3 concentrations showing that inhibition activity of these yeasts against
Yeast primary or secondary metabolism generates numerous compounds as products of the transformation of the carbon, nitrogen, or sulfur sources. Two of the most common substances released are acetic acid and hydrogen sulfide that have antimicrobial effect. Table
The results of the efficacy of the 20 selected strains in reducing molds berry rots are reported in Table
The three yeast strains which showed a better antagonistic effectiveness against the studied molds taking into account the above described experiments, were subjected to a comparative
Comparative
SEHMA6A | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3.00 | |
CABMB9A | 2 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 2.89 | |
SEHMA6B | 2 | 2 | 2 | 2 | 1 | 1 | 1 | 3 | 2 | 1.78 | |
Commercial fungicide | 1 | 2 | 2 | 3 | 3 | 3 | 2 | 2 | 3 | 2.33 | |
Control | 4 | 4 | 4 | 4 | 4 | 4 | 3 | 3 | 3 | 3.67 |
The control of fungal diseases and mycotoxins contamination during grape maturation and post-harvesting is currently based on treatments with chemical fungicides. However, the environmental dispersion, the progressive loss of effectiveness, the emergence of resistant strains, and the increasing level of residues in table grape and wine (Marssat et al.,
Our results pointed out that there is a greater number of species found on wildlife vines (23), compared to cultivated ones, with only seven species. This is in line with other studies, which demonstrated that the biodiversity level of yeasts community is influenced by human activities (Cordero-Bueso et al.,
The minimum inhibitory concentrations (MICs) assays, defined as the lowest concentrations of yeasts resulting in complete growth inhibition of the molds, have shown that a concentration of 105 cells/mL is enough to reduce the progress of
Since several mechanisms of action are involved in the biocontrol activity of the antagonistic yeasts, we have examined the main modes of actions, such as iron depletion, cell wall degrading enzymes, diffusible, and volatile antimicrobial compounds, and biofilm formation on the 20 selected yeast strains. Within this group
The yeast metabolism leads to the formation of acetate and ethyl acetate, which are by-products with inhibitory action against molds in storing cereals (Fredlund et al.,
Little is known about the role of biofilms in the biocontrol activity of yeast used to control fungal diseases and the mechanisms involved in their formation. In this work,
Iron is essential for fungal growth and pathogenesis, thus, competition for this metal is functional for counteracting of pathogenic molds. Sipiczki (
Comparison of the three selected antagonistic yeast strains against
Actually, several yeast strains tested in the
Though variable performances in field can be a significant constraint for its practical implementation (Stewart,
In conclusion, this investigation on antagonism patterns in new yeast isolates, over all from
GC contributed to the design of the work, to the yeast isolation, and identification, to the
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
PRiSM: Project approved by the Andalucía Talent Hub Program launched by the Andalusian Knowledge Agency, co-funded by the European Union's Seventh Framework Program, Marie Skłodowska-Curie actions (COFUND—Grant Agreement n° 291780) and the Ministry of Economy, Innovation, Science, and Employment of the Junta de Andalucía, Spain.
YeSVitE: Yeasts for the Sustainability in Viticulture and Oenology (
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