IGEM Bordeaux 2015

The Problem: Downy mildew

In 2015, vineyards are still threatened by the disease

Downy mildew is a disease caused by an oomycete (fungus-like eukaryotic microorganism) called Plasmopara viticola . It is unfortunately famous in the Aquitaine region because it affects tens of hectares of Bordeaux vineyards every year and threatens wine production . Until the middle of the 20th century, research efforts were mainly concentrated on optimizing the application of cooper fungicides such as "Bouillie Bordelaise" and developing new molecules for controlling the disease. Now, more and more alternative treatments are being tested and effective models which take into account meteorologic conditions are being developed. This could drastically change the environmental impact of winemaking.

Our iGEM team has been following this year's effect of mildew closely reading the official vineyard mildew bulletins available on the vinopole website. In the past few months ( June 2015 ) there has been another violent attack of mildew on the grapevines in the Aquitaine region. Up to 60% of wine grapes have been infected on certain parcels [4] and the vice president of the agriculture chamber, Patrick Vasseur, hasn't been underestimating the economic significance this could have since the wine production will evidently be affected. He calls the situation "exceptional" since "even the main branches are affected"

Serge Audubert, head of 3 castles in the region and owning a total of 24 ha, has been watching the effects on his land. On his 17 ha of château-laborde grapevines, in Saint-Médard-de-Guizières, 2ha are severly touched. « the leaves, the branches, the grapes, everything is affected. We are going to lose at least 50% of the grapes on these 2 ha. » On the first of May, this vineyard observed a spot on a branch, nothing severe especially since the « Bulletin de santé du végétal » (plant health review) which came out a few days before clearly states that the conditions aren't favorable for contaminations. As a precaution, Serge Audubert starts his preventive treatments on the 7th of May. On the 15th of May, the outburst starts, shocking the entire region: « I have been living here since 1987. I have never seen something like this. Informatics models were supposed to alert us when mildew evolution becomes dangerous. » Without better models and solutions to prevent mildew infections, winemakers in the region remain threatened every year!

(a) sporulation on abaxial leaf surface.

(b) Lesions on adaxial leaf surface caused by secondary infections in July.

(c) sporulation on a grape cluster before bloom.

(d) Symptoms on a developed cluster in summer.

Figure 1: Pictures of various stages of mildew infection [1]

A bit of History

Downy Mildew was originally observed in the United States of America in 1834 and has been most abundantly found in the Northern and mid-Western areas of the United States. Shortly after, the pathogen was introduced in European countries where it played a devastating role in the yield and production of their wine. In 1878, the first cases of Downy mildew were observed in France (in the region of Lyon) and also in Swizerland and Italy. While some North American species have become resistant to this parasite, European species such as Vitis vinifera (the grapevine used for wine) are extremely sensitive. From the beginning of the 20th century, the disease was clearly a huge problem for European viticulture. In the years in which weather conditions were favorable and sufficient control measures were not yet availabe or were not applied, serious damage was caused to viticulture in Germany, France and Switzerland. For example in 1915, 70% of the French grape crop was lost to P. viticola ([2],[3]). In 1930, 20 million hl of wine were lost in France. From 1907 to 1916, downy mildew was responsible for a 33% reduction in the total vine-growing area in the Baden province of Germany (Müller, 1938). During the Second World War, this disease also caused considerable damage. However, the lack of copper for pesticides contributed to this situation more than unfavourable weather conditions (Hadorn, 1942). As an example, it was reported that the normal Swiss copper requirements for agriculture in 1942 would have amounted to 1550 tons but due to the war situation, the government allocated only 690 tons, of which 300 tons were allocated for the potato crop and 320 tons were allocated for viticulture. The remaining copper was allocated for orchards, beans, tomatoes, celery and onions. [1]

Favorable conditions in Aquitaine

Downy mildew requires a warm, moist, and humid environment to reproduce and infect the plant, which is the case in the Aquitaine region . Generally, a correlation exists between low rainfall during the winter-spring period and slight epidemics. Mature oospores germinate best if their outer walls are ruptured, possibly as a result of a light freeze and sufficient humidity. The germination of oospores requires soil temperatures of 12 to 13°C and moisture. Common infection symptoms include necrosis of the stem or shoot, discoloration, brown spotting and yellowish-green tips of the leaves and mycelium invasion of the grapes (Figure 1). [1] These symptoms gravely affect the plant's photosynthetic ability and it's grape production. Thus, Downy mildew has been considered the most devastating disease caused by a filamentous pathogen to affect European vineyards and this has lead them to search for effective measures to protect their vines. Unfortunately, most of these measures use copper sulfate which pollutes the surrounding soils.

Literature Cited:

[1] Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathol. Mediterr. (2011) 50

[2] Bouillies bleues et vignes bleues. 7e année d'expériences. Progres Agricole et Viticole. (1923) 40, 380-382 (2011) 50

[3] Les leçons du mildiou en 1930 Progres Agricole et Viticole (1931) 95, 187-188

[4] Enquête sur une attaque imprévue de mildiou La Vigne, la revue du monde viticole 11.06.2015

Infection Mode of Downy Mildew

In winter, Plasmopara viticola is present on dead leaves on the ground as oospores. They are inactive and do not produce any symptoms. When rain falls during spring, these eggs grow and release zoospores when the temperature exceeds 11 degrees. The zoospores will be able to spread and infect the plant upper tissues through rainwater splashes. [5]

The primary contamination begins by the emission of a filament through the stomatal area where the parasite begins to develop sinkers from which is formed the mycelial network. These sinkers help to feed Plasmopara viticola by stealing the plant nutrients, which creates discolored and yellowish areas on the leaves called “oil stains”. After, on leaves bottom, conidiophores and conidia are formed. These symptoms cause damages to the leave tissues and affect the plant photosynthetic ability, which slows down the maturity of the plant.

During the secondary contamination , the conidia are transformed into zoospores that contaminate the surrounding tissues, weakening the plant even more and creating unreparable lesions .

Literature Cited:

[5] Biologie du mildiou de la vigne. Cycle du Mildiou de la vigne. BASF

Figure 2: Schematic representation of mildew infection stages over time

Common Solutions

Since repairing damaged tissues infected by downy mildew is impossible, the majority of the solutions available to vineyards are preventive solutions, mainly through preventing primary infections. This is done by spraying fungicides on the organs that are most infected: leaves and stems. The most efficient preventive treatment was discovered at the end of the 19th century: a solution made of copper sulfate also known as "Bouillie Bordelaise", the only treatment used until the end of the 20th century.

Recently, more models and devices designed to be used by individual growers measure temperature, humidity and leaf wetness and provide treatment recommendations based on algorithms similar to the Goidànich model. This general algorithm defined as the 3–10 spray strategy, is prescribed when the average temperature is above 10°C, more than 10 mm of rain have fallen within 24 h and shoot length in the vineyard is at least 10 cm. Despite evident imprecision due to the strict parameters, this general model can reliably predict the first risk period and recommend thereafter a treatment schedule that will allow growers to prevent development of severe downy mildew in vineyards.

The weakness of this type of model is that the number of recommended pesticide sprays is usually greater than what is needed to avoid an epidemic, particularly at the beginning of the season. In 2006, Swiss researchers applied a concept based on a tolerance threshold for Downy mildew under the particular climatic conditions of southern Switzerland and were able to eliminate half of the recommended treatments. (Jermini et al., 2006). This paradigm change, from a focus on the pathogen and the disease toward a threshold concept, requires detailed knowledge of the host and its relationship with the environment and human activities but opens the path for a new era of pesticide applications. However, these are highly complex interactions and there is little available data describing them. The information that is available is heavily biased by site, year and cultivar factors, and so cannot be readily used for simulation and modeling activities. [1] More similar studies could help reduce the use of pesticides in the region.

Plant natural defenses

Plant immunization is based on the same principle as human immunization: activating its natural defenses before contamination by an infectious agent. The concept is simple; putting the plant in contact with a molecule able to activate it's natural defenses: an elicitor . In nature, there are many elicitors produced by micro-organisms (exogenous elicitors) or by the plant itself when it is attacked (endogenous elicitors). The presence of an elicitor in the plant triggers a series of cellular reactions including the production of molecules to strengthen the resistance of cell walls , but also the production of plant antibiotics such as phytoalexins or defense proteins. These compounds have antifungal and antibacterial properties. The external application of a natural elicitor or a similar synthetic molecule thus results in the production of phytoalexins or defense proteins in the absence of any pathogen. The "immunized" plant is ready to fight back if attacked.

First, the cell wall forms a physical barrier which prevents the penetration of most microbes. Then, if some pathogens are able to cross this wall, the infection depends on the ability of the plant to perceive and to trigger defense reactions that would prevent the development of the disease. This recognition is done with certain compounds, known as elicitors from the pathogen or plant. The fixing of an elicitor to a plant cell receptor initiates a cascade of events that leads to the synthesis of defense compounds . The best known are antimicrobial compounds such as phytoalexins and Pathogenesis Related proteins .

There are two types of defenses: "passive" and "active". Natural defense reactions of plants are passive when these components were preformed prior to infection. They remain active until the pathogen penetration. Active defenses are induced upon recognition of the pathogen and remain active during infection.

Literature Cited:

[6] Stimulation of plant natural defenses Sciences de la vie.2001 Oct;324(10):953-63

Figure 3: Schematic representation of the plant's imune system

Consequences of the infection

Delta-viniferin is a grapevine phytoalexin produced during infection by Plasmopara viticola . Phytoalexins are antimicrobial and often antioxidative substances synthesized by plants that accumulate rapidly at areas of pathogen infection. Phytoalexins produced in plants act as toxins to the organism. They damage the cell wall, delay maturation, disrupt metabolism or prevent reproduction of the pathogen. When phytoalexin biosynthesis is inhibited, the susceptibility of infection in plant tissue increases showing its importance in defense mechanism . While, when a plant cell recognizes particles from damaged cells or from the pathogen, the plant launches a two-pronged resistance: a general short-term response and a delayed long-term specific response.

During the short-term response , the plant deploys reactive oxygen species such as superoxide and hydrogen peroxide to neutralize invading cells. The short-term response corresponds to the hypersensitive response, in which cells surrounding the site of infection are signaled to undergo apoptosis (programmed cell death), in order to prevent the spread of the pathogen to the rest of the plant.

The long-term response , also called systemic acquired resistance (SAR), permits a communication of damaged tissues with the rest of the plant using plant hormones such as jasmonic acid, ethylene, abscisic acid or salicylic acid. The reception of the signal leads to changes within the plant inducing genes that protect from more pathogen intrusion like genes coding enzymes involved in the production of phytoalexins. Also, if jasmonates or ethylene is released from the wounded tissue, neighboring plants also synthesize phytoalexins in response.

Literature Cited:

[7] Les Stimulateurs naturels de défense (SDN) de la Vigne Chambre d'agriculture Indre et Loire

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