**3. Phylogenetic relationships between strains of** *Colletotrichum* **spp. using telomeric fingerprinting**

*Colletotrichum acutatum* is a widely spread species that can be found throughout the world (Whitelaw-Weckert et al., 2007). *C. acutatum* causes anthracnose on a number of economically important crops, including woody and herbaceous crops, ornamentals, fruits, conifers and forage plants (Sreenivasaprasad & Talhinhas, 2005). It was classified as an organism of quarantine significance in Canada from 1991 to 1997, in the UK and the EU since 1993, and it can be found widely spread in the southwest region of USA (EPPO/CABI, 1997; Garrido et al., 2009a; Mertely and Legard, 2004). Investigations of *C. acutatum* were focused in two main aspects of the pathogen: i) cultural and morphological studies (Afanador-Kafuri et al., 2003; Denoyes-Rothan & Baudry, 1995; Garrido et al., 2008;) and ii) molecular approaches using molecular techniques including isoenzyme comparisons, Restriction Fragment Length Polymorphism (RFLP) analyses of mitochondrial DNA, Amplified Fragment Length Polymorphism (AFLP), AT rich analyses, Random Amplified Polymorphic DNA (RAPD), and ITS sequences analyses for specific PCR sequencing and identification (Buddie et al., 1999; Freeman et al., 1993; Garrido et al., 2009a, 2009b; Sreenivasaprasad et al., 1996; Talhinhas et al., 2005).

*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

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

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

**3. Phylogenetic relationships between strains of** *Colletotrichum* **spp. using** 

*Colletotrichum acutatum* is a widely spread species that can be found throughout the world (Whitelaw-Weckert et al., 2007). *C. acutatum* causes anthracnose on a number of economically important crops, including woody and herbaceous crops, ornamentals, fruits, conifers and forage plants (Sreenivasaprasad & Talhinhas, 2005). It was classified as an organism of quarantine significance in Canada from 1991 to 1997, in the UK and the EU since 1993, and it can be found widely spread in the southwest region of USA (EPPO/CABI, 1997; Garrido et al., 2009a; Mertely and Legard, 2004). Investigations of *C. acutatum* were focused in two main aspects of the pathogen: i) cultural and morphological studies (Afanador-Kafuri et al., 2003; Denoyes-Rothan & Baudry, 1995; Garrido et al., 2008;) and ii) molecular approaches using molecular techniques including isoenzyme comparisons, Restriction Fragment Length Polymorphism (RFLP) analyses of mitochondrial DNA, Amplified Fragment Length Polymorphism (AFLP), AT rich analyses, Random Amplified Polymorphic DNA (RAPD), and ITS sequences analyses for specific PCR sequencing and identification (Buddie et al., 1999; Freeman et al., 1993; Garrido et al., 2009a, 2009b;

environment (Garrido et al., 2009b).

information in the researches of these fungi (Fig. 1b).

encoding them (Fernández-Acero et al., 2011).

Sreenivasaprasad et al., 1996; Talhinhas et al., 2005).

**telomeric fingerprinting** 

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

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

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 directions (Garrido et al., 2009b).

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

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

Molecular Microbiology Applied to the Study of Phytopathogenic Fungi 147

2009b). In this study the number of strains studied was higher than those studied by FPGE, and although fingerprinting analyses did not make possible to study the chromosomal length polymorphisms among the isolates, the minimum numbers of estimated chromosomes are coincident with those obtained from FPGE analyses, showed in the last

The telomeric profiles obtained for each isolate of *C. acutatum* were analysed. UPGMA dendogram showed a representative grouping among the isolates, which was coincident with the grouping in the neighbuor-joining phylogenetic tree based on sequences of rDNA ITS regions (Garrido et al., 2009b). All the strains previously classified in the A2 molecular groups, also clustered in a large group with more than 70% of similarity based in this case in the telomeric fingerprinting profiles. These results proved the high level of similarity shows by these isolated, not only based in sequence similarity of one specific region but also in their genotypes and genome organization among *C. acutatum* strains, which suggests a common origin of the strains among the different molecular groups (Garrido et al., 2009b;

Fig. 2. Left.- Telomeric fingerprinting patterns obtained by telomeric hybridisation of Southern blots from HindIII-DNA digestions. Right.- Combined UPGMA dendograms with the *C. acutatum* isolates belonging to A2 group, based on Dice coefficients generated using a composite data set from individual experiments of each enzyme digestion (*Bam*HI, *Eco*RI,

*Hin*dIII and *Pst*I) hybridised with a telomeric probe.

section of this chapter.

Talhinhas et al., 2005).

the USA, and then it was observed in France and UK at the beginning of the 80s. It is not clear how the pathogen was introduced into production fields in Europe, but it is thought that the pathogen could have arrived since the American nurseries to the EU (Freeman & Katan, 1997). It should have arrived to France, UK and the Iberian Peninsula fields. The arrival of the pathogen was facilitated by the intense international trade between these countries related with strawberry crop. Therefore the fungus could be introduced by infected plants, contaminated soil associated with strawberry crowns at planting, and quiescent infections on strawberry leaves or fruits (Garrido et al., 2008, 2009b; Leandro et al., 2001, 2003).

In order to complete the phylogenetic classification of our *C. acutatum* strain collection, a different molecular approach was carried out. The results obtained were compared with those from the ITS sequences analyses. We used the profiles obtained after restriction enzymes digestions of genomic DNA, and then hybridized with a telomeric probe by Southern-blot hybrisization. Genomic DNA of *C. acutatum* strains were digested to completion with several restriction enzymes in independent experiments (*Bam*HI, *Eco*RI, *Hin*dIII and *Pst*I). Gel electrophoresis is an intermediate point of the complete protocol. It make possible to separate the DNA fragments obtained after the restriction enzymes digestions. In this case, we used a 1.5% agarose gel, and electrophoresis was carried out in a conventional horizontal tray for DNA electrophoresis (Bio-Rad). After separation of digested fragments, gels were blotting to Hybond-N membrans, being ready for subsequently hybridization (Garrido et al., 2009b).

For Southern-blot hybridization we used a telomeric probe to get hybridization in the telomeric regions. These regions are located at the end of the lineal chromosomes of most eukaryotic organisms, and they are named telomeres. Telomeres are regions of repetitive DNA sequences that protect the end of the chromosome from deterioration or from fusion with neighboring chromosomes. The repeated sequences is dependent of the species. For *C. acutatum* telomeres, we produced our telomeric probe, (TTAGGG)n, by PCR in the absence of a template using (TTAGGG)5 and (CCCTAA)5 primers as it was described by Ijdo et al. (Ijdo et al., 1991). The Hybond-N membranes were allowed to hybridize with the telomeric probe; films images were digitalized and telomeric profiles were analysed using Fingerprinting II software v3.0 (Bio-Rad).

The experimental setup described provided the possibility to obtain two different kinds of results/conclusions from the study: I) Selected restriction enzymes used for RFLP did not produce any cut in the telomeric regions of *C. acutatum* strains. Each band represents a physically distinct telomere extremity. Therefore, taking into consideration the higher number of telomeric extremities and then divided into two, we can determine the number of chromosomes among strains studied. II) The fingerprinting analyses of the telomeric profiles, carried out using Fingerprinting II software, make possible to produce phylogenetic trees based in the similarity of the profiles showed among the strains. Therefore, these results could be compared with those obtained from phylogenetic groups based on ITS sequences.

Among the fifty-two isolates analysed by telomeric fingerprinting, the number of band or telomeres oscillated between twelve and eighteen. Therefore, the minimum number of estimated chromosomes was from six to nine among *C. acutatum* isolates (Garrido et al.,

the USA, and then it was observed in France and UK at the beginning of the 80s. It is not clear how the pathogen was introduced into production fields in Europe, but it is thought that the pathogen could have arrived since the American nurseries to the EU (Freeman & Katan, 1997). It should have arrived to France, UK and the Iberian Peninsula fields. The arrival of the pathogen was facilitated by the intense international trade between these countries related with strawberry crop. Therefore the fungus could be introduced by infected plants, contaminated soil associated with strawberry crowns at planting, and quiescent infections on strawberry leaves or fruits (Garrido et al., 2008, 2009b; Leandro

In order to complete the phylogenetic classification of our *C. acutatum* strain collection, a different molecular approach was carried out. The results obtained were compared with those from the ITS sequences analyses. We used the profiles obtained after restriction enzymes digestions of genomic DNA, and then hybridized with a telomeric probe by Southern-blot hybrisization. Genomic DNA of *C. acutatum* strains were digested to completion with several restriction enzymes in independent experiments (*Bam*HI, *Eco*RI, *Hin*dIII and *Pst*I). Gel electrophoresis is an intermediate point of the complete protocol. It make possible to separate the DNA fragments obtained after the restriction enzymes digestions. In this case, we used a 1.5% agarose gel, and electrophoresis was carried out in a conventional horizontal tray for DNA electrophoresis (Bio-Rad). After separation of digested fragments, gels were blotting to Hybond-N membrans, being ready for

For Southern-blot hybridization we used a telomeric probe to get hybridization in the telomeric regions. These regions are located at the end of the lineal chromosomes of most eukaryotic organisms, and they are named telomeres. Telomeres are regions of repetitive DNA sequences that protect the end of the chromosome from deterioration or from fusion with neighboring chromosomes. The repeated sequences is dependent of the species. For *C. acutatum* telomeres, we produced our telomeric probe, (TTAGGG)n, by PCR in the absence of a template using (TTAGGG)5 and (CCCTAA)5 primers as it was described by Ijdo et al. (Ijdo et al., 1991). The Hybond-N membranes were allowed to hybridize with the telomeric probe; films images were digitalized and telomeric profiles were analysed using

The experimental setup described provided the possibility to obtain two different kinds of results/conclusions from the study: I) Selected restriction enzymes used for RFLP did not produce any cut in the telomeric regions of *C. acutatum* strains. Each band represents a physically distinct telomere extremity. Therefore, taking into consideration the higher number of telomeric extremities and then divided into two, we can determine the number of chromosomes among strains studied. II) The fingerprinting analyses of the telomeric profiles, carried out using Fingerprinting II software, make possible to produce phylogenetic trees based in the similarity of the profiles showed among the strains. Therefore, these results could be compared with those obtained from phylogenetic groups

Among the fifty-two isolates analysed by telomeric fingerprinting, the number of band or telomeres oscillated between twelve and eighteen. Therefore, the minimum number of estimated chromosomes was from six to nine among *C. acutatum* isolates (Garrido et al.,

et al., 2001, 2003).

subsequently hybridization (Garrido et al., 2009b).

Fingerprinting II software v3.0 (Bio-Rad).

based on ITS sequences.

2009b). In this study the number of strains studied was higher than those studied by FPGE, and although fingerprinting analyses did not make possible to study the chromosomal length polymorphisms among the isolates, the minimum numbers of estimated chromosomes are coincident with those obtained from FPGE analyses, showed in the last section of this chapter.

The telomeric profiles obtained for each isolate of *C. acutatum* were analysed. UPGMA dendogram showed a representative grouping among the isolates, which was coincident with the grouping in the neighbuor-joining phylogenetic tree based on sequences of rDNA ITS regions (Garrido et al., 2009b). All the strains previously classified in the A2 molecular groups, also clustered in a large group with more than 70% of similarity based in this case in the telomeric fingerprinting profiles. These results proved the high level of similarity shows by these isolated, not only based in sequence similarity of one specific region but also in their genotypes and genome organization among *C. acutatum* strains, which suggests a common origin of the strains among the different molecular groups (Garrido et al., 2009b; Talhinhas et al., 2005).

Fig. 2. Left.- Telomeric fingerprinting patterns obtained by telomeric hybridisation of Southern blots from HindIII-DNA digestions. Right.- Combined UPGMA dendograms with the *C. acutatum* isolates belonging to A2 group, based on Dice coefficients generated using a composite data set from individual experiments of each enzyme digestion (*Bam*HI, *Eco*RI, *Hin*dIII and *Pst*I) hybridised with a telomeric probe.

Molecular Microbiology Applied to the Study of Phytopathogenic Fungi 149

commercial kits are available for extraction of fungal DNA, they can represent a high cost per sample analysed, and they are not always totally reliable in not co-extracting PCR inhibitors, needed a dilution of samples prior to PCR reactions. The optimised protocol did not co-extracted PCR-inhibitors from any samples, and therefore, the sensitivity of the detection protocol is improved using this DNA extraction protocols (Garrido et al., 2011).

To date, conventional PCR has been a fundamental part of fungal molecular diagnosis, but it shows several limitations: i.e. gel-based methods, possibility of quantification, sensitivity, etc. The development of real-time PCR has been a valuable response to these limitations (Garrido et al., 2011). This technology improve the sensitivity, accuracy and it is less timeconsuming that conventional end-point PCR. For development and optimization of *Colletotrichum* diagnosis protocols, the commonly-used ribosomal RNA genes were used, because of the highly variable sequences of the internal transcribed spacers ITS1 and ITS2, which separate the 18S/5,8S and 5,8S/28S ribosomal RNA genes, respectively (Garrido et al., 2009a). Specific genus and species sets of primers and probes were designed for real-time PCR amplifications using TaqMan® chemistry technology. This system consists of a fluorogenic probe specific to the DNA target, which anneals to the target between the PCR primers; TaqMan® tends to be the most sensitive and simply methods for real-time PCR

The specificity of all assays was tested using DNA from isolates of six species of *Colletotrichum* and from DNA of another nine fungal species commonly found associated with strawberry material. All the new assays were highly specific for *Colletotrichum* spp., *C. acutatum* and *C. gloeosporioides*, no cross-reactions were observed with either related plant pathogens or healthy strawberry plant material. The sensitivity of the new real-time PCR assays was compared with that of previously published conventional PCR assays; they were confirmed to be 100 times more sensitive than the latter. The *C. acutatum*-specific real-time PCR assay was also compared with an existing ELISA assay for the diagnosis of this pathogen. Real-time PCR permitted the detection of the pathogen in samples that gave negative results for *C. acutatum* using ELISA. The real-time PCR assay detected the equivalent of 7.2 conidia per plant inoculated with a serial dilution of *C. acutatum* spores,

demonstrating the high degree of sensitivity of the method (Garrido et al., 2009a).

The new protocols were tested for monitoring the development of anthracnose disease in strawberry in the field in the south of Spain. The real-time PCR results showed a progressive increase of target DNA between January and June. The results showed that an increase in lesion development was accompanied by an increase in the amount and incidence of the pathogen as the season progressed. These results showed that new methods are suitable for diagnosis, identification and monitoring of the disease using field samples of strawberry and also, they permitted the detection of the pathogens from artificially infected symptomless plant material. Therefore, the methods described, based on real-time PCR, proved useful for studying the epidemiological routes of these strawberry pathogens in

In spite of the advances done by the described techniques above, nowadays proteomics is the most realistic and effective set of tools to unravel complex mixtures of proteins,

detection (Garrido et al., 2009a, 2011).

fields and nurseries (Garrido et al., 2009a, 2011).

**5. Proteomics approaches of phytopathogenic fungi** 
