**1. Introduction**

138 Gel Electrophoresis – Advanced Techniques

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> Fungi is an extensive group of eukaryotic microorganisms, generally they are microscopic and usually filamentous. It is estimated that there are between 70,000 and 1.5 millions species of fungi, most of them are being discovering and describing (Agrios, 2005). Most of the known hundred thousand fungal species are strictly saprophytic, living on decomposing dead organic matter. About fifty species cause disease in human, and more than ten thousand species can cause disease in one (obligate parasite) or many kinds of plants (nonobligate parasites) (Fernández-Acero et al., 2007a).

> Phytopathogenic fungi are able to infect any tissue at any stage of plant growth. Plant pathogenic fungi show a complex life cycles, including both sexual and asexual reproduction stages (Agrios, 2005). Moreover, complex infection cycles and carbon assimilation is displayed (Garrido et al., 2010). These biological variability give them the possibility to develop its biological role from very climatologically different environments, since dry and desert zones until wet and hot regions in the tropic and equatorial area to the capacity to attack all plant tissues, from leaves to roots (Agrios, 2005).

> During the last decades, the development of molecular methods has lead the Scientifics community to accumulate a high quantity of information from different molecular approaches (Fernández-Acero et al., 2011; Garrido et al., 2009b). Advances into Genomics, Transcriptomics, Proteomics, and more recently, Metabolomics are transforming research into fungal plant pathology, providing better and more accurate knowledge about the molecular biology and infection mechanisms showed by these fungi (Garrido et al., 2010).

> Since 1992, our research group has been working with two of the most aggressive plant pathogens, which have been established such a model organisms for molecular and phytaphology studies: *Botrytis cinerea* and *Colletotrichum acutatum* (Fernández-Acero et al., 2006b, 2007a; Garrido et al., 2009b, 2010; Perfect et al., 1999). These genera include some of the most destructive plant pathogen species known. They induce worldwide diseases as, between others, the grey mould on grapes and the anthracnose on strawberries, respectively (Coley-Smith et al., 1980; Elad et al., 2004; Sutton, 1992). The losses caused by the phytopathogenic fungi *Botrytis cinerea* and *Colletotrichum acutatum* have been quantified

Molecular Microbiology Applied to the Study of Phytopathogenic Fungi 141

The development of Pulse- field gel electrophoresis (PFGE) resolved many problems found with cytogenetic studies in filamentous fungi. This technique has been widely used since the 90s for genomic characterization into fungal plant pathogens. PFGE allows the separation of large DNA molecules (DNAs from 100 bases to over 10 megabases (Mb) may be effectively resolved) which would all co-migrate in conventional agarose gels. This technique has proved to be a very useful tool to study aspects of genome organization in several yeast and fungi. It has led to the discovery that most species exhibit chromosome-length polymorphisms (CLPs), revealing a high level of intraspecific, and even population-level

Technically, PFGE resolves chromosome-sized DNAs by alternating the electric field between spatially distinct pairs of electrodes. The electrophoresis cell consists of an array with 24 horizontal electrodes arranged in a hexagon. Agarose gels are electrophoresed horizontally, submerged under recirculated buffer. The system (CHEF-"Clamped Homogeneous Electric Field" and PACE "Programmable Autonomously Controlled Electrodes", from BIO-RAD) provides highly uniform, or homogenous, electric fields within the gel, using an array of 24 electrodes, which are held to intermediate potentials to eliminate lane distortion. Thus, lanes are straight. The system maintains uniform field using patented Dynamic Regulation. The electrodes sense changes in local buffer conductivity due to buffer breakdown, change in buffer type, gel thickness, or temperature, and potentials. The preparation of samples for resolving chromosomal karyotypes by PFGE is not exempt of difficulty due to the biological characteristic of fungal cells. Fungus has to be growth in an optimal culture medium and mycelium harvest after determinate time which depends of the fungal species. This time is very important because is necessary to obtain the highest number of fungal cells in metaphase stage (Carbu, 2006; Garrido et al., 2009b). Chromosomes are condensed and highly coiled in metaphase, which makes them most suitable for visual analysis. After young mycelium is harvested, it is necessary to produce protoplasts using different mixes of lysing enzymes, which digest the fungal cell wall after incubation. Protoplast suspensions are mixed with low melting point agarose, adjusted to final concentration of 1 x 108 protoplast ml-1, and solidified plugs of agarose containing protoplast are digested with proteinase K. The digestion produces pores in the plasma membrane,

providing the possibility to extract the chromosomal by PFGE (Garrido et al., 2009b).

Gels are prepared with a special type of agarose. It depends of the DNA molecules sizes because there are different commercial preparations, some of them for DNA molecules higher than 10 Mb, i.e. PFGE TMMegabase agarose (Bio-Rad). Plugs are cast in the gel, and this is placed in the center of the hexagon formed by the 24 electrodes. Many parameters of the electrophoresis have to be optimized, since the type and concentration of running buffer, temperature of buffer, voltage and time of pulses, angles of electric fields. Depending of instrument setup, we can resolve the electrophoretic karyotype (EK) only with one experiment, like in the case of *Botrytis cinerea*; or even it could be necessary two different steps/running conditions, due to the high differences in sizes of the chromosomal DNA molecules. After electrophoresis, gels are stained using i.e. ethidium bromide and visualized

PFGE has been widely used by our group to study the genome organization and Chromosomal Polymorphisms (CPL) in *B. cinerea* and *C. acutatum*. We have determined the number and sizes of chromosomes in both species, and therefore we have estimated the

variability (Vallejo et al., 2002).

using a UV light system.

between 10 and 100 million of Euros per year in Europe (Fernández-Acero et al., 2007a). Losses caused by *B. cinerea* in French vineyards oscillate between 15% and 40%; in Holland, *B. cinerea* generates losses of about 20% of the flower crop; and, in Spain, the losses fluctuate between 20% and 25% of the strawberry crops (Fernández-Acero et al., 2007a). *Colletotrichum* spp. causes up to 80% plant death in nurseries and yield losses of >50%, being a major disease of cultivated strawberry (Denoyes-Rothan et al., 2004; Garrido et al., 2009a).

Our group has carried out an intense research activity of the molecular microbiology of these plant pathogens. These studies involve several molecular approaches in which the gel electrophoresis plays an important role. In this chapter, we will summarize the results obtained, and the molecular methods used for the study and characterization of the phytopathogen fungi *Botrytis cinerea* and *Colletotrichum* spp., all of them strongly related with different types of gel electrophoresis approaches and downstream protocols, including, between others, Pulse Field Gel Electrophoresis, agarose gel electrophoresis of DNA, Restriction Fragment Polymorphism Analyses, Southern-blot, Polyacrylamide Gel Electrophoresis and Two dimensional gel electrophoresis of proteins. These electrophoretic methods will be used to structure the development of chapter, describing the technical bases of each method and showing the approaches carried out and the results obtained.
