Unlocking grapevines’ natural defences

Cultivar susceptibility compared

Raletsena started by developing an assay to compare the resistance of grape cultivars to the agents of grey mould (Botrytis cinerea), powdery mildew (Erysiphe necator), and downy mildew (Plasmopara viticola). The mildews are tricky to study because they only grow on living tissues, whereas grey mould can be cultured on artificial media.

For her assays, Raletsena cut Smartie-sized discs from young leaves of 10 grapevine cultivars: Baco Noir, Chelois, Deckrot, Douce Noire, G1-1227, Marechal Foch, Noiret, Red Globe, Regent, Sultana, and Ventura. Several of these were obtained from table-grape breeder Phyllis Burger of the ARC Infruitec-Nietvoorbij and were selected based on their field performance against powdery and downy mildew.

Leaf discs were placed in multi-well plates and inoculated with one of the three pathogens. Susceptibility was scored according to the time it took for the pathogens to cause leaf lesions.

Raletsena first ran the assays with leaves from field-grown vines and then repeated them with glasshouse-grown grapevine cuttings. Although there were minor differences in the results, cultivar performance was similar in both sets of assays.

Both field- and glasshouse-grown Sultana and Red Globe were highly susceptible to all three pathogens.
“We picked up infections as soon as we started our observations,” comments Raletsena.

The same degree of susceptibility was not seen for any other cultivars.

In contrast, both field- and glasshouse-grown Douce Noir showed a degree of resistance to all three pathogens, never exhibiting signs of infection within the first 48 hours after inoculation. Of the remaining cultivars, Regent and Ventura were the least susceptible to mildew pathogens and slightly resistant to grey mould.

“It’s interesting that Douce Noir, Regent, and Ventura are consistent in their resistance to Botrytis cinerea, Plasmopara viticola, and Erysiphe necator,” says Raletsena.

Fungal inhibition assessed

The initial leaf-disc assays demonstrated that cultivars differ in their susceptibility to grey mould, powdery mildew, and downy mildew. However, the results did not prove that differences in cuticular waxes are the cause. For the next phase of the project, Raletsena set out to test the antifungal effect of cuticular waxes using Botrytis cinerea spores.

“Most fungicides target the germination stage,” she says, “so it is important to see how the fungi behave during their germination stage when exposed to the different waxes.”

She extracted the wax from the same number of leaf discs from each cultivar. Botrytis cinerea spores were added to each wax extract, and their germination was monitored under the microscope. Images of the germinating spores were analysed with the help of specialised software.

Normal germination is rapid, with spores developing a germination tube at least twice the length of the spore.
“This happens within a couple of hours under ideal conditions,” remarks Young.

In a good environment, the spores will form infective structures called appressoria on their germination tubes, and a single spore may even have branched germination tubes. But in a stressful environment, germination is delayed, germination tubes are short, and appressoria form on abnormally short germination tubes or not at all.

Raletsena charted the percentage of spore germination over 10 hours for spores exposed to each wax extract versus control spores not exposed to wax extracts. The results support the hypothesis that the type or amount of cuticular wax compounds affects the degree of resistance relative to cultivar differences.

For example, wax extracted from Sultana leaf discs doesn’t significantly affect spore germination compared with the control. This aligns with the observation that Sultana leaf discs are highly susceptible to Botrytis cinerea infection.

Conversely, less than 20% of spores exposed to wax extracted from Douce Noire germinated after 10 hours, compared with 100% of the control spores. Douce Noire had the least susceptible leaf discs of the cultivars tested in the first part of the project.

Wax compounds analysed

Cuticular waxes are complex substances.
“These waxes are made of long-chain fatty acids and their derivatives, which are alkanes, aldehydes, primary and secondary alcohols, ketones, esters, and triterpenoids,” says Raletsena. Which of these many molecules are inhibiting plant pathogens?

To answer this question, Raletsena has been analysing wax extracts from the different cultivars using gas chromatography-mass spectrometry. So far, she has detected about 56 compounds in the waxes and found differences in the wax composition of the cultivars.

Once she has the profile of each cultivar’s wax, she can look for correlations between the presence or absence of wax compounds and resistance or susceptibility to pathogens. Other researchers have identified antimicrobial compounds in cuticular waxes, and Raletsena will also see whether any of these are present in her samples.

“If we know that a compound is present in the wax of a resistant cultivar, we can take the individual compounds and add them to a wax that doesn’t have antifungal activity to see if it slows germination,” says Young, adding that antifungal activity will probably require a combination of compounds.

Ultimately, he hopes to identify compounds from grapes that can be incorporated into edible wax, which could be applied to grapes to control postharvest decay. A better understanding of grapevine waxes could also inform breeding programmes, offering table-grape growers the prospect of someday producing fruit that naturally resists pathogens while still tasting great.