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IGEM Bordeaux 2015

The Problem

This year, iGEM Bordeaux’s project is focused on Downy Mildew

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 . For example, this year, in the Aquitaine region, over 80% of certain parcels are contaminated ref. Until the middle of the 20th century, research efforts were mainly concentrated on optimizing the application of cooper fungicides and developing new molecules and formulations for controlling the disease

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 midwestern 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. 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 (1900, 1905, 1906, 1910, 1912, 1913, 1916, 1917, 1930, and 1932), 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 (Cadoret, 1923,1931). 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)[1]

Common symptoms include necrosis of the stem or shoot, discoloration, brown spotting and yellowish-green tips of the leaves and mycelium invasion of the grapes. 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. Depending on the year, production of grapes in France has been estimated to be at a loss of 50% or more ref and the Aquitaine region is particularly affected due to the favorable climate and the economic importance of the wine industry. Thus, Downy mildew has been considered the most devastating disease caused by a filamentous pathogen to affect European vineyards and this has lead vineyards to search for effective measures to protect their vines. Unfortunately, most of these mesures have a bad environmental impact and pollute the surrounding soils.

Source: [1] Plasmopara viticola: a review of knoledge on downy mildew of grapevine and effective disease management. Phytopathol. Mediterr. (2011) 50, 3-44

Infection Mode of Downy Mildew

Downy mildew requires optimum conditions to reproduce and infect as warm, moist, and humid environment.

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's upper tissues through rainwater's splashes.

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's nutrients, which creates discolored and yellowish areas on the it's leaves called “oil stains”. After, on leaves bottom, conidiophores and conidia are formed. These symptoms cause damages to the leaves’ tissues and affect the plant’s 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.

Since repairing damaged tissues infected by downy mildew is impossible, the main solutions available to vinyards are preventive solutions, mainly through preventing primary infections. This is mainly 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, synthetic fungicides have replaced this chemical treatment and more and more research is being done on alternative eco-friendly preventive treatments.

Natural defenses of plants

Plant Immunization is based on the same principle as human immunization: activating it's natural defenses before contamination by an infectious agent. The concept is simple; it is to put 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.

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.

In 2015, vineyards are still threatened by the disease

Our iGEM team has been following this year's effect of mildew closely reading the official vineyard mildew bulletins available on the vinopole website. It is clearly shown that there is a significant increase of mildew infection on parcels that haven't been treated with copper sulfate compared to those that have been treated. Furthermore, the infection of mildew on treated parcels appears to be much more easily controled on parcels treated with copper sulfate. Evidently, without any alternative treatment, wine production in the region would be affected and this shows just how important our project is!

Different models (Caffi model, Potential systems model) take into account pluviometry, temperature, relative humidity and plant morphology to decide when are the best moments to apply the fungicides. However, even if these models have allowed vinyards to drastically reduce the quantities of fungicides used, they still cause environmental and sanitary problems in the surrounding regions.

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 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 loose 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. »

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