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%\large\scshape\bfseries\sffamily 
Training Period Report\\
Magistre of Biotechnologies, University of Orsay/Paris-Sud\\
January 2007 -- June 2007\\
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\LARGE\scshape\bfseries\sffamily Downy Mildiew Adaptation to Fungicide Pressure:\\ Fitness Study

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\begin{flushright}
Student: \textbf{Pierre {\scshape Abadie}}\\
Laboratory: {\scshape Inra} Bordeaux, UMR Sant Vgtale\\
Under the responsability of: Marie-France {\scshape Corio-Costet}
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\scshape\bfseries\sffamily{Introduction}
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Downy mildew is a fungal-like disease that is specific to grapevine
and can attack all tissues with stomatas. Symptoms are yellow
"oilspots" or white down spots on leaves, brown areas on shoots, and
fruits rot. The causal agent is \emph{Plasmopara viticola}, a
biotrophic parasite in the family of Peronosporaceae (oomycota) that
can only develop on its host and doesn't survive without grapevine.

That disease was brought from United States and first occurred in
1878 in France. Today, it is still responsible for extensive damage
in all French vineyards. At present, chemical control remains the
most effective form of management, and this parasite is focusing
78\% of all fungicides applications in France. Many efficient
molecules have been developed, but resistant pathogen populations
emerged regularly and rather quickly, one example being general
resistance reported to QoI fungicides, that inhibitates mitochondrial
respiration by stopping the electron transport chain. In this study
we focuse on \emph{Plasmopara viticola}'s resistance to famoxadone,
which has been demonstrated to be due to a punctual mutation (G143A)
in the mitochondrial gene coding the cytochrom \textit{b}.

A major issue understanding the resistance process is to measure
pathogen fitness, i.e. the selective value of the pathogen. The
study's general goal is to acquire datas on dispersion and maintain
of \emph{P. viticola} resistant strains, in the optics of improving
fungicides applications management. My training period specific goal
focuses on the study of fitness and competitiveness of famoxadone
resistant and sensitive strains, in order to answer the following
question:

Is there a cost of resistance, i.e. are resistant strains less
competitive than sensitive ones in the absence of fungicide?

Detecting a cost of resistance in \emph{Plasmopara viticola} could
lead to interesting repercussions in the way fungicides are used in
wineyard.

Previous datas on resistant and sensitive strains fitness were
collected by measuring different parameters (growth rate,
sporulation rate, infectiousness, \dots), and didn't proved any clear global differences between strains. In this study, we set up a
competitiveness test between resistant and sensitive, in order to
detect or not a cost of resistance. The experiments can be separated
in three chronological points:
\begin{enumerate}
  \item From initial mixes of resistant and sensitive strains, we will mesure during eight weeks the evolution of each proportions.
  \item Molecular tools will be used to corroborate and improve the biological measures: Quantitative PCR will be performed in order to detect or not the G143A
  mutation.
  \item An evolutionnary model will be realized with fitness
  datas.
\end{enumerate}

\newpage

\section{Material and Methods}

\subsection{Competitiveness test}
Caracteristics and FIC\footnote{FIC = Fitness Index Complex = sporulation capacity $\times$ infection frequency $\times$ latency period $\times$ sporangia size $\times$ infection efficiency $\times$ growth rate} of \textit{Plasmopara viticola} isolates resistant and sensitive couples used are detailed in
table \ref{origines}.

\begin{table}[h]
\begin{center}
\begin{tabular}{|c|c|c|c|c|}
  \hline
  % after \\: \hline or \cline{col1-col2} \cline{col3-col4} ...
  \textbf{Strain} & \textbf{Origin} & \textbf{Year} & \textbf{FIC} & \textbf{QoI Resistance} \\
\hline
\hline
  COU15 & Petit Couhins (33) & 2003 & 0,648 & S \\
  MIC128 & Castillon (32) & 2005 & 0,636 & R \\
  \hline
  PIC59 & Couhins (33) & 2003 & 0,571 & R \\
FEM03 & Puligny-Montrachet (21) & 2003 & 0,731 & S \\
  \hline
  PAU32 & Pauillac (33) & 2003 & 0,389 & R \\
COU15 & Petit Couhins (33) & 2003 & 0,648 & S \\
\hline
PAU32 & Pauillac (33) & 2003 & 0,389 & R \\
MIC103 & Fleurie (69) & 2004 & 0,465 & S \\
\hline
\end{tabular}
\end{center}
\caption{Origin and FIC of resistant and sensitive strains used in
the competitiveness test}
\label{origines}
\end{table}

\begin{description}
\item[General protocol of downy mildiew pricking out:]

Fungus isolates are cultivated on detached leaves placed on
water-saturated filter paper in Petri dishes at 21C. All
experiments are performed with detached leaves of 4 weeks-old plants
of Cabernet-Sauvignon grown in greenhouses.

Sporangias are taken from an infected leave with a brush to realize a suspension in permuted water, adjusted to 40000 sporangias.mL$^{-1}$ with the help of a Malassez cell. 24 drops of 15$µ$L are deposed per leaf. After one night at 22C to allow stomatas opening and leaf infection, drops are dryed with an air pomp and dishes are conserved during one week minimum at 21C.   

\begin{figure}[htb]
  \centering
  \includegraphics[height=4cm]{repiquage}\\
  \caption{Downy mildiew growth on a leaf (\textit{in vitro} experiment)}\label{repiquage}
\end{figure}

\item[Competitiveness test protocol:]
For each resistant/sensitive couple (see Table \ref{origines}), three initial proportions (20\% resistant, 50\% resistant and 80\% resistant) are realized at 40000 sporangias/mL and inoculated on four independant leaves for each proportion and each couple. After one week of growth, the mixes are pricked out on  four new leaves. The experiment is repeated during eight weeks. Every week, mixes samples are taken to perform a fungicide crible and a quantitative-PCR mesure to estimate the new resistant/sensitive proportions (see below).

The entire experiment is repeated independently twice a week.

\end{description}

\subsection{Fungicide crible}
In the same time leaves are inoculated with the resistant/sensitive inoculum suspension for the competitiveness test, this suspension is used to inoculate leaves disks containing fungicide. For each proportion and each couple, two Petri dishes containing eight disks are prepared. Disks are sprayed with famoxadone at 100mg/mL, and are dryed after one night of fungicide penetration. Three inoculum drops of 15$µ$L are deposed on each disk.

\begin{figure}[htb]
  \centering
  \includegraphics[height=4cm]{fongicide}\\
  \caption{The fungicide crible}\label{crible}
\end{figure}


After one week of growth, stains corresponding to resistant strain remaining are observed and evaluated using a visual ladder (see figure \ref{echelle}, leading to estimate the initial resistant proportion that was contained in the suspension deposed on the disks.
 

\begin{figure}[htb]
  \centering
  \includegraphics[height=3cm]{echelle}\\
  \caption{Visual ladder used in the fungicide crible}\label{echelle}
\end{figure}

\subsection{Quantitative Polymerase Chain Reaction (Q-PCR)}

\subsubsection{DNA extraction}
For each proportion of each couple, two tubes of 0,5mL of sporangias suspension and four little leaf disks containting three mildiew stains are prepared from the four leaves every week, and conserved at -20C. DNA extraction is performed from these samples:
\begin{itemize}
\item Samples are centrifugated and the pellet is iced at -80C and lyophilisyed overnight
\item After crushing the dryed pellet, 600$µ$L of CTAB\footnote{25mL of Tris 2M pH$_{8}$, 20mL of EDTA 0,5M pH$_{8}$, 41g of solid NaCl, 10g of CTAB, and 5g of PVP-40 completed to 1L with H$_{2}$O milliQ} (cetyl trimethylammonium bromide) at 65C are added
\item After one hour waiting, 400$µ$L of isoamylalcool-chloroform are added, and tubes are slowly shaken during ten minutes. Then, a centrifugation is performed at 13000rpm and 4C during ten minutes, and the aqueous phase is conserved.
\item 2/3 volume of cold isopropanol are added, and after shaking a two-hours wait is necessary to let the DNA precipitate.
\item Tubes are centrifugated ten minutes at 13000rpm and 4C, the pellet is conserved and rinsed with ethanol 70, and centrifugated again. Then the pellet is dryed, disolved in 50$µ$L of water and conserved at -20C.
\end{itemize}

\subsubsection{Q-PCR}
In order to measure the resistant/sensitive proportions, a strategy based on the detection of the G143A mutation (that confers resistance) has been set up. Two couples of primers are used:
\begin{itemize}
\item A first couple that is unspecific to resistant or sensitive isolates, and leads to the amplification of the mitochondrial \textit{b} cytochrom gene.
\item A second couple also leading to the amplification of the cytochrom \textit{b} gene, but in which one primer hybridates directly with the G143A mutation, and consequently is specific to resistant isolates. 
\end{itemize}

Quantitative PCR is performed with these two couples of primers, and the ratio between resistant-specific primers and unspecific primers gives us the resistant isolate proportion in the mix.

The following protocol is used: 5$µ$L of Master Mix SybrGreen\footnote{SybrGreen is a fluorophore that binds to double-strand DNA, it is used to quantify the PCR amplification during the cycles}, 0,1$µ$L of each primer and 2,5$µ$L of available DNA completed to 10$µ$L with H$_{2}$O are submitted to 30 cycles of quantitative PCR.

\section{Results}
\subsection{Biological competitiveness test}
\subsubsection{Measures standardization}
In order to derminate the resistant/sensitive strains proportions in the course of time, we have to assess wether the visual notation of the fongicide crible is correct. A standard scale is realized by inoculating known proportions on leaf disks (0 - 10 - \dots - 100\% of resistant) under fungicide application, and after one week of growth the stains are notated between 0 and 5 using the visual ladder. Results are shown in figure \ref{gamme}.

\begin{figure}[htb]
  \centering
  \includegraphics[height=9cm]{gamme}\\
  \caption{Standart scales for the four couples (error: Standart Error; dotted line: linear regression)} \label{gamme}
\end{figure}

As we can see on those graphs (fig \ref{gamme}), there is a good correlation between the percentage of resistants and the visual notation, under the fungicide crible. Indeed, inoculating at 40000 sporangias/mL extends the visual notation from 0 to 5 in a linear way. That concentration gives us the better precision for a visual notation, and is conserved for the following experiments.  

\subsubsection{Competitiveness test (biological results)}
Evolution of resistant/sensitive proportions is determined during eight weeks, using the fongicide crible. Two independant experiments are realized. Results of the first experiment are shown in figure \ref{competlundi}.

\begin{figure}[htb]
  \centering
  \includegraphics[height=9cm]{competlundi}\\
  \caption{Evolution of resistant/sensitive proportions during eight weeks, starting from three known proportions (20\%, 50\%, 80\%) [First Test].} \label{competlundi}
\end{figure}

At the time, a statistical study of those graphs hasn't been realized yet. For instance, a statistical work on the slopes point by point is planned. So the analyse of those graphs cannot be deeply done yet, we can only observe general tendencies. In figure \ref{competlundi}, the evolution of proportions is comparable between the different couples and the different initial proportions: we can't detect any significant variation during eight weeks. The little variations observed may be due to aleatory variation of mildiew growth, and can't be interpreted as a genuine variation of the resistant/sensitive proportion. 

Results of the second experiment are shown in figure \ref{competmercredi}.

\begin{figure}[htb]
  \centering
  \includegraphics[height=9cm]{competmercredi}\\
  \caption{Evolution of resistant/sensitive proportions during eight weeks, starting from three known proportions (20\%, 50\%, 80\%) [Second Test].} \label{competmercredi}
\end{figure}

In this second test, we are able to see significant variations of the evolution of the proportions. For the couple COU15 MIC128, the evolution is similar to the one in the first experiment. But for the remaining couples PAU32 COU15, PAU32 MIC103 and PIC59 FEM03, we observe an extinction of the resistant isolates PAU32 and PIC59 after eight weeks.

These results are consistent with the available datas on fitness (see table \ref{origines}): PAU32 and PIC59 have a low fitness compared to COU15, MIC103 and FEM03. According to these results, it appears that they may also be less competitive than the sensitive isolates they are associated with.

\newpage
\subsection{Molecular analysis of the competitiveness test}
At the time, the quantification of the evolution of the proportions using the quantitative PCR method is working, but hasn't been performed on all the samples yet. Meanwhile, The first available results (datas not shown) seem to corroborate the biological mesures using the fungicide crible.

\subsection{Modelisation}
A model of evolution\footnote{realised with Stella$^{\begin{scriptsize}\textregistered\end{scriptsize}}$ software} of a resistant/sensitive isolates mix with or without fungicide application is beeing realized using the fitness datas previously measured in the laboratory, to see if the experiment results are consistent or not with a theorical modelisation.  


\section{Discussion and perspectives}
This study is inserted in a larger project focusing on understanding the fitness of pathogenic fungi, and previous datas on downy mildiew's fitness didn't show any significant global differences between resistant and sensitive isolates, despite the existence of local variations. Here, by focusing on seven isolates with known FIC, we are able to demonstrate a possible correlation between competitiveness and fitness of fungi. Thus, these results may confirm the FIC previously collected, and so could represent the approval of the measure of fungi fitness, which is still an open challenge, due to the complexity of the implied factors.

By studying these isolates couples, we are able to detect a cost of resistance in some famoxadone-resistant strains. However, the results of quantitative PCR measures and an statistical work are expected to corroborate or reject this hypothesis. Even if costs of resistance have already been detected \textit{in vitro} by studying artificial mutants and isogenic strains, our experiments on strains coming directly from wineyards is a more complex problem, because of the diversity and the strenght of the selective forces that operate \textit{in natura}.





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