Ergosterol: A Novel Indicator For Testing Fungal Infections in Grapes

Fungal infections in grapes, be it Botrytis cinerea, Penicillium spp., powdery mildew, or any other rot, is a frequent headache for vineyards, and are infections that threaten the quality of the grapes when picked at harvest, resulting in a lot of crop losses.  With the exception of when “botrytised” grapes, or noble rot, is desired in certain sweet wines, fungal rots are high on the “to kill” list of vineyard workers around the world.

Rot and other fungal infections in grapes are highly undesirable in the wine industry, both for negative organoleptic (sensory) changes caused by chemical interactions with the fungus, as well as potential negative health effects caused by some of the toxin created and released by the fungi during these

Photo By Rob & Lisa Meehan (Flickr: Someone Else's Grapes) [CC-BY-2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons

Photo By Rob & Lisa Meehan (Flickr: Someone Else’s Grapes) [CC-BY-2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons

infections of the grapes.  As a result of this, sorting the grapes at harvest is extremely important to ensure that you’re only using the highest quality grapes for making wine, and not introducing spoiled grapes that could dramatically lower the quality of the finished wine as well as harbor potential harmful toxins.

Currently, the most commonly used method for sorting spoiled grapes from healthy grapes, particularly for smaller outfits with fewer resources, is a simple visual observational method.  The workers observe the grapes coming along the sorter and they remove the grapes that appear to be infected.  This could be problematic, as sometimes grapes can be infected without showing any obvious outward signs, or they could simply be missed in the mass quantities of grapes rushing by.

The study presented to you today aimed to find a more reliable method for determining presence of fungal rot in grapes early on in the harvest and sorting process, that could potentially be used for quick sampling and testing right on the crush pad for more accurate sorting.

There have been a few studies examining different “markers” for fungal infections in grapes, though results have been mixed.  The study presented today turned their focus to ergosterol (ergosta-5,7,22-trien-3β-ol), which is a compound that is found in the majority of fungi and is found in their cell membranes and walls.  Ergosterol seems to be unique to fungal cell walls, as this compound is not found in any plant, animal, or bacterial cells.  As a result of this, it may be a very useful compound to test for in grapes, as if the grapes test positive for ergosterol, it could be a clear indication that a fungal rot is present, since ergosterol only originates from fungal cells.

Methods

10 strains of 5 mold species associated with various grape rots were tested for ergosterol content.

Riesling grapes harvested from two different sites in Germany were used for inoculation experiments.  After harvesting the grapes, they were split and sanitized with 70% ethanol, 0.35% sodium hypochlorite,

Photo By Calvero. (Selfmade with ChemDraw.) [Public domain], via Wikimedia Commons

Photo By Calvero. (Selfmade with ChemDraw.) [Public domain], via Wikimedia Commons

and again with 70% ethanol.  Grapes were allowed to dry, and then were injected with a solution containing the mold samples.  Control samples that were not injected with any mold were used for comparison.  All samples were placed in plastic boxes that held moist cellulose tissues inside and let to incubate in the dark for 7 days at 21oC.

Naturally contaminated grapes were also collected for analysis.  148 different grape samples (64 red and 84 white) were collected over three vintages in various location in Germany.  Grapes were separated into “completely sound” or 0% rot, or “rotten” categories.  The rotten category was not split up by degree of rot, but as long as the grape had any noticeable rot present, it was put into this category.  Blends of the two mashes were made for further analysis.

The following were measured in the grape mashes: ergosterol, fungal biomass, glucose, fructose, ethanol, glycerol, D-gluconic acid, malic acid, acetic acid, titratable acidity, tartaric acid, must density, and laccase activity.

Results

  • Fungal biomass was significantly different among the different species and strains found.
  • Nearly all of the fungal species studied (Ascomycetes) contained significant levels of ergosterol, with the exception of powdery mildew (Erysiphales), which instead contained ergosta-5,24(28)-dien-3β-ol (24 methylene cholesterol).
  • Grape rot metabolites: gluconic acid, glycerol, and ethanol; were present in significant levels in infected grapes, while control grapes (no rot) did not have any of these compounds present.
    • Control grapes were also lacking ergosterol, which was found only in those grapes that were infected by the different molds.
  • Laccase activity was not detected in grapes infected with Botrytis cinerea, even though it is thought that laccase activity is a good indicator of fungal infection.  This test appears unreliable for detecting rot

Conclusions

This study was a relatively simple one, in that the researchers were really only focused on one major question:  could ergosterol be used as an indicator for fungal infections in grapes?

According to the results of both laboratory and naturally infected grapes in this study, it appears as though ergosterol may be a very effective marker for infection, with the exception of powdery mildew, which should be further explored and developed into some sort of field-testing handheld device.

You may have noticed that grape rot metabolites such as gluconic acid, glycerol, and ethanol were also only found in infected grapes, so why not use though as indicators of fungal infection instead of or in addition to ergosterol?  According to the authors of this study, while these metabolites were, in fact, only found in infected grapes, testing for these compounds is often very time consuming, labor intensive, and expensive.  Testing for ergosterol, according to the authors, would be a more reliable approach that is

Copyright R. Yeamans 2013

Copyright R. Yeamans 2013

much more efficient and accurate, and could be developed to be incorporated into a quick and easy testing device for the vineyard.

Future studies are most certainly needed, as this is just the first in likely a series of studies, but from the initial results, it sounds like using ergosterol as universal indicator for grape rot (with the exception of powdery mildew) may be highly effective and a more reliable way to sort out infected grapes from the harvest.  Perhaps developing a simple-to-use handheld device that can easily test samples for ergosterol in the field could help improve the quality of wines by more efficiently removing infected grapes from healthy grapes.

What do you all think about this study?  If you work with grapes, do you have another test for rot (other than observational) that you’ve used and found reliable?  Please feel free to leave any of your comments!

Source: Porep, J.U., Walter, R., Kortekamp, A., and Carle, R. 2014. Ergosterol as an objective indicator for grape rot and fungal biomass in grapes. Food Control 37: 77-84.

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