**2. Chromosomal polymorphism and genome organization in** *Botrytis cinerea* **and** *Colletotrichum* **spp.**

*Botrytis cinerea* and *Colletotrichum acutatum* are two species of phytopathogenic fungi that show a very high level of phenotypic diversity among isolates. These fungi show complex cycles of life and infection, including both sexual and asexual forms (Garrido et al., 2008; Vallejo et al., 2002). Also high levels of somatic variability appear when the fungi are grown "*in vitro*", depending on the medium, temperature, light and other factors, which even determine differences in cultural characteristics, production of reproductive structures and pathogenicty between others (Bailey & Jeger, 1992; Carbu, 2006; Garrido et al., 2009b; Rebordinos et al., 1997, 2000; Vallejo et al., 1996, 2001). These fungi do not show a high level of host specificity and they infect many different genera of hosts, adapting their infection strategy and metabolism to the environment conditions and kind of plant colonized. They are notoriously variable genera about which many fundamental questions relating to taxonomy, evolution, origin of variation, host specificity and mechanisms of pathogenesis remain to be answered (Bailey & Jeger, 1992; Elad et al., 2004).

Many research projects are aimed to study the genome organization and chromosomal polymorphism trying to find the origin of phenotypic variability showed by these fungi. In the past decades, several strategies have been tested on lower fungi such as *B. cinerea* and *Colletotrichum* spp., i.e. cytological karyotyping, analysis of progeny from crosses between strains, sexual hybridizations, etc. These assays looked for a relation between molecular and phenotypic variability (Carbu, 2006; Faretra & Antonacci, 1987; Faretra et al., 1988; Vallejo et al., 1996). Cytological studies showed a very high level of difficulty in this group of microorganisms due to small size and/or the difficulty to condense sufficiently the chromosomes to make them visible by microscope. These characteristics made difficult to obtain reliable information about the genome organization of these fungi, and the obtaining of conclusive results about their biological mechanisms of recombination and chromosomal polymorphisms.

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

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

disease of cultivated strawberry (Denoyes-Rothan et al., 2004; Garrido et al., 2009a).

of each method and showing the approaches carried out and the results obtained.

remain to be answered (Bailey & Jeger, 1992; Elad et al., 2004).

**and** *Colletotrichum* **spp.** 

polymorphisms.

**2. Chromosomal polymorphism and genome organization in** *Botrytis cinerea*

*Botrytis cinerea* and *Colletotrichum acutatum* are two species of phytopathogenic fungi that show a very high level of phenotypic diversity among isolates. These fungi show complex cycles of life and infection, including both sexual and asexual forms (Garrido et al., 2008; Vallejo et al., 2002). Also high levels of somatic variability appear when the fungi are grown "*in vitro*", depending on the medium, temperature, light and other factors, which even determine differences in cultural characteristics, production of reproductive structures and pathogenicty between others (Bailey & Jeger, 1992; Carbu, 2006; Garrido et al., 2009b; Rebordinos et al., 1997, 2000; Vallejo et al., 1996, 2001). These fungi do not show a high level of host specificity and they infect many different genera of hosts, adapting their infection strategy and metabolism to the environment conditions and kind of plant colonized. They are notoriously variable genera about which many fundamental questions relating to taxonomy, evolution, origin of variation, host specificity and mechanisms of pathogenesis

Many research projects are aimed to study the genome organization and chromosomal polymorphism trying to find the origin of phenotypic variability showed by these fungi. In the past decades, several strategies have been tested on lower fungi such as *B. cinerea* and *Colletotrichum* spp., i.e. cytological karyotyping, analysis of progeny from crosses between strains, sexual hybridizations, etc. These assays looked for a relation between molecular and phenotypic variability (Carbu, 2006; Faretra & Antonacci, 1987; Faretra et al., 1988; Vallejo et al., 1996). Cytological studies showed a very high level of difficulty in this group of microorganisms due to small size and/or the difficulty to condense sufficiently the chromosomes to make them visible by microscope. These characteristics made difficult to obtain reliable information about the genome organization of these fungi, and the obtaining of conclusive results about their biological mechanisms of recombination and chromosomal 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 variability (Vallejo et al., 2002).

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 using a UV light system.

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

Molecular Microbiology Applied to the Study of Phytopathogenic Fungi 143

The *B. cinerea* strains showed a high level of CLPs, revealing the facility to support chromosomal rearrangements in this species, and could be the basis of the high degree of adaptability to the environmental conditions. Our group has also studied crosses between strains with different EK profiles. This study had as main aim to analyze the chromosomal rearrangements and chromosomal segregation in the crossed strains, in order to clarify the controversy appeared about the possibility that a high level of CLPs between strains, could inhibit meiosis (Zeigler, 1998), and therefore to be one possible reason to explain the low level of sexual reproduction that take place in *B. cinerea* under natural conditions (Carbu,

The crosses between strains produced fertile strains (more than 100 ascopores studied) and our results demonstrated that chromosomal rearrangements did not affect the capacity to reproduce sexually in *B. cinerea*. It was observed than only several isolates recovered the parental EKs. New chromosomes sizes were identified and some bands were lost from the parental to descendants EKs. All these results, along with a segregation analyses carried out in the decendants, represented strong evidence that some strains might not be haploid, and that aneuploidy and differences in ploidy levels are present in this species (Vallejo et al., 2002). Our group has also studied how during a long period of time, reproducing the fungus "*in vitro*", there were not detected changes in the EK of a given strain. All results together, proved that mitotic growth does not provide EK variability in this fungus, being the chromosomal rearrangements generated after meiotic recombination the causal agent of EK

In the case of the species *C. acutatum*, there were not data published about the EKs and CLPs among isolates until the last 2009 (Garrido et al., 2009b). PFGE had been used with other species of this genus, like *C. gloeosporioides* (Masel et al., 1993) and *C. lindemouthianum* (O´Sullivan et al., 1998). *Colletotrichum* spp. displayed an estimated genome sizes higher than *B. cinerea*. Protocols to separate the chromosomes molecules were carried out in two different experimental setups, including variations in the pulse of electric field, percentage of agarose gels and duration of the assays (Masel et al., 1993), i.e. for separation of larger chromosomal molecules in *C. gloeosporioides*, Masel et al. (1993) optimized an PFGE approach running a electrophoresis of seven days long. During this experiment, it was necessary to replace the running buffer each two days to obtain a better resolution in the final image. Similar protocols were used to resolve EK from *C. lindemouthianum*

The karyotype of *C. acutatum* was studied by our group in several strains isolated from different geographical origins. They had showed differences in the morphological characteristics in relation to the color and texture of mycelium, ratio of growth in different medium, pathogenicity and level of conidia production (Garrido et al., 2008, 2009b). Protocol to obtain *C. acutatum* protoplasts and the PFGE conditions to separate chromosomes were optimized based on our previous experience with *B. cinerea*. We optimized a PFGE running conditions to separate chromosomes between approximatele 0.1 and 9 Mb after only 72 h. of running. This protocol improved substantially those previously described for *Colletotrichum* spp., which took longer due to the two steps needed to resolve the complete karytopyes. Those longer protocols (Masel et al., 1993) were also tested, and we got the same number and sizes of chromosomal bands, proving the improvement of our

2006; Giraud et al., 1997).

variability in *B. cinerea* (Carbu, 2006).

strains(O´Sullivan et al., 1998).

optimized 72-hours protocol (Garrido et al., 2009b).

genome size for these fungi; the high level of CPL displayed by them, represented in the different EK profiles showed by the strains; and PFGE has made possible downstream applications such as Southern-blot analysis using different probes. All the results accumulated during the last years have provided a better understanding about the genome organization and the molecular bases of asexual and sexual reproduction of these fungi. They proved that polymorphism has been observed in both asexual and sexual fungi and most likely results from both mitotic and meiotic processes, especially in the case of *Botrytis cinerea* (Vallejo et al., 2002).

When a study of PFGE has made, it is usual to find chromosomal bands of different intensity and therefore it is important to consider several technical aspects that can have influence in the interpretation of the final results, and the conclusions obtained: i) a double band could be composed of two coumpounds of a couple of homologous chromosomes or of two heterologous chromosomes of similar size, and ii) two homologous chromosomes can differ in size and appear like two heterologous ones. Due to this fact, depending on the aims of the study, sometimes further hybridization studies are necessary in order to determine the linkage groups of each of the bands (Carbu, 2006; Vallejo et al., 1996).

*Botrytis cinerea* strains studied by our group were isolated from different hosts and geographical origins. We found different EK profiles between isolates, which did not follow any correlation with the host, year of isolation, or phenotypical characteristics. We have found that the number of chromosomal bands varied between 5 and 12, and they ranged between 1.80 and 3.8 Mb. These results made possible to estimate the minimal genome size of *B. cinerea* genome, found between 14.5 and 22.7 Mbp (Carbu, 2006; Vallejo et al., 1996, 2002) (Fig. 1a).

Fig. 1. A.- PFGE chromosomal separation of selected *B. cinerea* isolates. The molecular sizes were estimated using *Schizosaccharomyces pombe* (line 1 and 10), and *Hansenula wingeii* (line 6) chromosomes as reference molecular markers (Bio-Rad). B.- Southern-blot hybridization using a telomeric DNA probe to hybridise the PFGE separated chromosomal bands.

genome size for these fungi; the high level of CPL displayed by them, represented in the different EK profiles showed by the strains; and PFGE has made possible downstream applications such as Southern-blot analysis using different probes. All the results accumulated during the last years have provided a better understanding about the genome organization and the molecular bases of asexual and sexual reproduction of these fungi. They proved that polymorphism has been observed in both asexual and sexual fungi and most likely results from both mitotic and meiotic processes, especially in the case of *Botrytis* 

When a study of PFGE has made, it is usual to find chromosomal bands of different intensity and therefore it is important to consider several technical aspects that can have influence in the interpretation of the final results, and the conclusions obtained: i) a double band could be composed of two coumpounds of a couple of homologous chromosomes or of two heterologous chromosomes of similar size, and ii) two homologous chromosomes can differ in size and appear like two heterologous ones. Due to this fact, depending on the aims of the study, sometimes further hybridization studies are necessary in order to determine

*Botrytis cinerea* strains studied by our group were isolated from different hosts and geographical origins. We found different EK profiles between isolates, which did not follow any correlation with the host, year of isolation, or phenotypical characteristics. We have found that the number of chromosomal bands varied between 5 and 12, and they ranged between 1.80 and 3.8 Mb. These results made possible to estimate the minimal genome size of *B. cinerea* genome, found between 14.5 and 22.7 Mbp (Carbu, 2006; Vallejo et al., 1996, 2002) (Fig. 1a).

Fig. 1. A.- PFGE chromosomal separation of selected *B. cinerea* isolates. The molecular sizes were estimated using *Schizosaccharomyces pombe* (line 1 and 10), and *Hansenula wingeii* (line 6) chromosomes as reference molecular markers (Bio-Rad). B.- Southern-blot hybridization using a telomeric DNA probe to hybridise the PFGE separated chromosomal bands.

the linkage groups of each of the bands (Carbu, 2006; Vallejo et al., 1996).

*cinerea* (Vallejo et al., 2002).

The *B. cinerea* strains showed a high level of CLPs, revealing the facility to support chromosomal rearrangements in this species, and could be the basis of the high degree of adaptability to the environmental conditions. Our group has also studied crosses between strains with different EK profiles. This study had as main aim to analyze the chromosomal rearrangements and chromosomal segregation in the crossed strains, in order to clarify the controversy appeared about the possibility that a high level of CLPs between strains, could inhibit meiosis (Zeigler, 1998), and therefore to be one possible reason to explain the low level of sexual reproduction that take place in *B. cinerea* under natural conditions (Carbu, 2006; Giraud et al., 1997).

The crosses between strains produced fertile strains (more than 100 ascopores studied) and our results demonstrated that chromosomal rearrangements did not affect the capacity to reproduce sexually in *B. cinerea*. It was observed than only several isolates recovered the parental EKs. New chromosomes sizes were identified and some bands were lost from the parental to descendants EKs. All these results, along with a segregation analyses carried out in the decendants, represented strong evidence that some strains might not be haploid, and that aneuploidy and differences in ploidy levels are present in this species (Vallejo et al., 2002). Our group has also studied how during a long period of time, reproducing the fungus "*in vitro*", there were not detected changes in the EK of a given strain. All results together, proved that mitotic growth does not provide EK variability in this fungus, being the chromosomal rearrangements generated after meiotic recombination the causal agent of EK variability in *B. cinerea* (Carbu, 2006).

In the case of the species *C. acutatum*, there were not data published about the EKs and CLPs among isolates until the last 2009 (Garrido et al., 2009b). PFGE had been used with other species of this genus, like *C. gloeosporioides* (Masel et al., 1993) and *C. lindemouthianum* (O´Sullivan et al., 1998). *Colletotrichum* spp. displayed an estimated genome sizes higher than *B. cinerea*. Protocols to separate the chromosomes molecules were carried out in two different experimental setups, including variations in the pulse of electric field, percentage of agarose gels and duration of the assays (Masel et al., 1993), i.e. for separation of larger chromosomal molecules in *C. gloeosporioides*, Masel et al. (1993) optimized an PFGE approach running a electrophoresis of seven days long. During this experiment, it was necessary to replace the running buffer each two days to obtain a better resolution in the final image. Similar protocols were used to resolve EK from *C. lindemouthianum* strains(O´Sullivan et al., 1998).

The karyotype of *C. acutatum* was studied by our group in several strains isolated from different geographical origins. They had showed differences in the morphological characteristics in relation to the color and texture of mycelium, ratio of growth in different medium, pathogenicity and level of conidia production (Garrido et al., 2008, 2009b). Protocol to obtain *C. acutatum* protoplasts and the PFGE conditions to separate chromosomes were optimized based on our previous experience with *B. cinerea*. We optimized a PFGE running conditions to separate chromosomes between approximatele 0.1 and 9 Mb after only 72 h. of running. This protocol improved substantially those previously described for *Colletotrichum* spp., which took longer due to the two steps needed to resolve the complete karytopyes. Those longer protocols (Masel et al., 1993) were also tested, and we got the same number and sizes of chromosomal bands, proving the improvement of our optimized 72-hours protocol (Garrido et al., 2009b).

Molecular Microbiology Applied to the Study of Phytopathogenic Fungi 145

Sreenivasaprasad & Talhinhas (2005) studied *C. acutatum* populations from several hosts and different geographical origins. They established molecular groups based on sequences analyses of the internal transcribed spacers (ITS) of ribosomal DNA polymorphic regions (Sreenivasaprasad & Talhinhas, 2005). ITS regions have been widely used on molecular approaches for studying relationship between microorganisms, and it is also very useful regions for designing molecular approaches to identification and diagnostic protocols, due to the high variability showed by the sequences among species and even strains (Garrido et al., 2009a). The classification carried out by Sreenivasaprasad & Talhinhas (2005), established eight molecular groups for *C. acutatum* species. These molecular groups have been widely used to study the genotypic and phenotypic diversity of this fungus, and to

During the last years, we carried out a study to classify a worldwide collection of *C. acutatum* strains isolated from thirteen countries (Australia, Canada, France, Germany, Japan, The Netherlands, New Zealand, Norway, Portugal, Spain, Switzerland, USA and UK). For this purpose we used two different molecular approaches in order to study the phylogenetic relationship between strains: i) a sequencing analysis of the internal transcribed spacers (ITS) of the 5.8S ribosomal DNA polymorphic regions; ii) a telomeric fingerprinting study by Southern-blot hybridization, using a telomeric probe after RFLP

In total, eighty-one 5.8S-ITS sequences were studied, several strains were sequenced by our group, and other ones used from databases such as reference sequences for allocating our strains in the previously established molecular groups for *C. acutatum*. ITS regions, including 5.8S rDNA, were amplified by conventional PCR using universal primers ITS1 and ITS4 (White et al., 1990). After PCR amplification, products were loaded in a conventional 1% agarose gel for conventional DNA electrophoresis. Products were cut from the gels using a purification kit, DNA was quantified, and subsequently sequenced in both

The phylogenetic study carried out with the sequences allowed us to allocate the strains into *C. acutatum* molecular groups described by Sreenivasaprasad & Talhinhas (2005), but the analysis of bootstrap in the neighbout-joining phylogenetic tree, published by Garrido et al. (2009), showed interesting data about the molecular groups. In base of that analyses, the nine molecular groups previously described (Whitelaw-Weckert et al., 2007), could be grouped in only four groups. Our results proved that A1, A2, A5 A8 and A9 subgroups showed a bootstrap support of 90%, and therefore could be considered such as large group in base to the analyses of the sequences of ITS regions (Garrido et al., 2009b). The same result was observed for subgroups A6 and A4, since these subgroups clustered together with a strong bootstrap support of 91% (Garrido et al. 2009). Our results supported a new classification into four molecular groups instead the nine previously described for this

The phylogenetic analyses showed that the majority of the strains studied grouped in the group A2. This happened because many strains from Spain were included in the analyses. The results proved the high level of similarity between *C. acutatum* strains isolated from Spain. It is also interesting that the A2 group included, principally, isolates from Spain, Portugal, France, UK and USA. *C. acutatum* was first described in the southwest region of

classify isolates from different origin (Whitelaw-Weckert et al., 2007).

digestions of genomic DNA (Garrido et al., 2009b).

species in base to the ITS sequences (Garrido et al., 2009b).

directions (Garrido et al., 2009b).

*C. acutatum* strains showed EK profiles containing between six and nine chromosomal bands with different sizes ranging from 0.1 and 8 Mb. The total minimal genome size estimated for *C. acutatum* ranged between 29 and 36 Mb, which is similar to that previously described for other species of *Colletotrichum* (Masel et al., 1993; O´Sullivan et al., 1998). We observed CLPs between strains studies but further analyses with a high number of isolates could be necessary in order to obtain strong conclusions about the CLPs showed by the species and how this variability could affect the sexual and asexual reproduction of this species in the environment (Garrido et al., 2009b).

PFGE gels from *B. cinerea* and *C. acutatum* were used in downstream applications, like Southern-blot analyses. Gels were transferred to Hybond-N membranes and they hybridised with a telomeric probe confirming that all the bands represented chromosomes. The description of Southern-blot analyses will be described in the next section, but it proved how PFGE, not only provides the possibility to obtain interesting conclusions about the biology and genome organization of these fungi, but also gel electrophoresis techniques are often the starting point for interesting downstream applications that provide more information in the researches of these fungi (Fig. 1b).

In our PFGE studies in *B. cinerea* and *C. acutatum*, it has not been observed a higher EKs variability that showed by phenotypic characteristics among strains (Carbu, 2006; Garrido et al., 2008, 2009a, 2009b; Rebordinos et al., 2000; Vallejo et al., 1996). Phenotypic features were very highly variable between strains with the same EKs. Therefore, we cannot conclude that there is a direct relation between morphological, physiological and pathogenic variability directly related with heterokaryosis, aneuploidy and a variable level of ploidy among strains. New proteomics approaches to *B. cinerea* and *Colletotrichum* spp., which will be described during next pages, is contributing with very interesting data, that in conjunction with genomic information, disclose that phenotypic variation is more related with the synthesis of proteins and their post-transductional modifications, and not only by genotypes encoding them (Fernández-Acero et al., 2011).
