Characterization of race 65 of Colletotrichum lindemuthianum by sequencing ITS regions

The present work aimed characterize isolates of C. lindemuthianum race 65 from different regions in Brazil by ITS sequencing. A total of 17 isolates of race 65, collected in the states of Mato Grosso, Minas Gerais, Paraná, Santa Catarina and São Paulo, were studied. Analysis of the sequences of isolates 8, 9, 12, 14 and 15 revealed the presence of two single nucleotide polymorphisms (SNPs) in the ITS1 region at the same positions. These isolates, when analyzed together with the sequence of isolate 17, revealed a SNP in the ITS2 region. The highest genetic dissimilarity, observed between isolates 11 and 3 and between isolates 11 and 10, was 0.772. In turn, isolates 7 and 2 were the most similar, with a value of 0.002 for genetic distance. The phylogenetic tree obtained based on the sequences of the ITS1 and ITS2 regions revealed the formation of two groups, one with a subgroup. The results reveal high molecular variability among isolates of race 65 of C. lindemuthianum.


Introduction
The common bean (Phaseolus vulgaris L.) is admittedly an important source of protein and energy (Vieira, Vieira, Euclydes, & Silva, 1983;Borém & Carneiro, 1998) and is a legume of great economic significance (Broughton et al., 2003) and agronomic interest across the world (Angioi et al., 2010).Moreover, it is the most important legume for human consumption and an important source of proteins, vitamins and minerals (Ca, Cu, Fe, Mg, Mn, and Zn) in the human diet, particularly in developing countries (Beebe, 2012).This crop is cultivated throughout the year under different temperature, light intensity, relative humidity and water availability conditions.These conditions favor the development of several pathogens, such as fungi (Paula Júnior & Zambolin, 2006).
The common bean anthracnose is one of the main fungal diseases affecting this crop, especially if the environmental conditions favor the pathogen's development, leading to yield losses of up to 100% when susceptible cultivars are used (Talamini et al., 2006; Damasceno e Silva, Souza, & Ishikawa, 2007).Colletotrichum lindemuthianum is a cosmopolitan pathogen and is therefore widely distributed in common bean-producing regions (Bianchini, Maringoni, & Carneiro, 2005).
Anthracnose occurs more severely in places where relative humidity conditions above 91% and temperatures ranging from 18° and 22°C predominate.The pathogen's development limits infection at temperatures higher than 24ºC and lower than 18ºC (Cháves, 1980;Kelly, Afanador, & Cameron, 1994).The control of anthracnose in common bean is greatly impaired due to the survival capacity of the fungus for several months in both the soil and infected crop residues (Sutton, 1992;Dillard & Cobb, 1993).Integrated measures are recommended for the efficient control of anthracnose, including the use of pathogen-free and fungicide-treated seeds, crop rotation with non-host plants for a period of 2 to 3 years, varietal resistance and chemical control (Rava, Purchio, & Sartorato, 1994).
C. lindemuthianum exhibits wide genetic variability, as indicated by the high number of races in the common bean-producing regions, which impairs the durability of cultivar resistance (Souza, Souza, & Mendes-Costa, 2007).The high number of existing physiological races and the complexity in the use of genetic resistance of the C. lindemuthianum fungus are evidence of wide virulence diversity (Pinto, Pereira, Mota, Ishikawa, & Souza, 2012).Brazil is notably the country with the highest pathogenic variability, with 73 pathogenic races distributed in 15 states; race 65 is present in 12 states (Nunes, Gonçalves-Vidigal, Lacanallo, & Coimbra, 2013).
Molecular tools have been widely used in the study of genetic diversity in many organisms, including fungi.One of those molecular tools is the sequencing of conserved regions, such as the internal transcribed spacer (ITS) in ribosomal DNA (rDNA), which has been used to establish molecular phylogenetic relationships within many groups of fungi (Stewart, Zaowei, Crous, & Szabo, 1999).The ITS region is divided into ITS1, located between genes 18S and 5.8S, and ITS2, which separates genes 5.8S and 28S; the ITS1 and ITS2 regions are transcribed and processed to yield ribosomal RNA (Hillis & Dixon, 1991;Schlotterer, Hauser, Huesler, & Tautz, 1994).Given the importance of C. lindemuthianum race 65, this work aimed to characterize isolates of C. lindemuthianum race 65 from several regions in Brazil by ITS sequencing.

Biological material
The present work was conducted under greenhouse conditions and at Laboratório de Melhoramento de Feijão Comum e de Biologia Molecular of the Núcleo de Pesquisa Aplicada à Agricultura (Nupagri), Universidade Estadual de Maringá, Paraná State, Brazil.A total of 17 isolates of race 65, collected in the states of Mato Grosso, Minas Gerais, Paraná, Santa Catarina and São Paulo, were studied.
Twelve isolates were obtained from the mycology collection of the Núcleo de Pesquisa Aplicada à Agricultura at the Universidade Estadual de Maringá and five were kindly provided by Ms. Tamires Ribeiro of the Instituto Agronômico de Campinas, Campinas, São Paulo State, Brazil.

Growth of mycelial mass
The monosporic cultures of C. lindemuthianum were first replicated to Petri dishes with potato dextrose agar (PDA) medium (Menezes & Silva-Hanlin, 1997), and spore discs (ascospores) were then replicated and transferred to beakers containing liquid potato dextrose (PD) medium, according to the methodology proposed by Kado and Hesket (1970).
Subsequently, the mycelial mass was filtered, washed with distilled water and dried using autoclaved filter paper.After drying, the mycelium was wrapped in aluminum foil, properly labeled, and stored at a temperature of -20ºC.Carbonell et al. (1999) conducted a study focusing on the reaction of cultivars recommended for the State of São Paulo and on the identification of pathogen races.Nine races were identified, and races 65, 81 and 89 were predominant.However, when two isolates of race 31, two of race 65 and three of race 81 were inoculated into cultivars recommended for cultivation in the state, differences among races were observed.Such differences suggested that the set of differential cultivars of C. lindemuthianum was not sufficient to differentiate the pathogenicity diversity of the isolates evaluated due to possible interactions and existing gene interactions between the genes for pathogen resistance.
Isolate 16 from São Paulo state had only one SNP in the ITS2 region, which showed it to be divergent from all race 65 isolates and the sequences obtained in BLAST.On the other hand, the isolate 4 from the State of Minas Gerais revealed the presence of one SNP in the ITS1 region and one in the ITS2 region, both are different from the SNPs observed in the other isolates.Studies conducted by Talamini et al. (2006) noted the presence of molecular variability between and within isolates belonging to race 65 from the State of Minas Gerais.
Davide and Souza ( 2009) conducted a study to evaluate pathogenic variation within race 65.Six isolates collected in the State of Minas Gerais were inoculated into the twelve differential cultivars and into seven commercial cultivars; these cultivars were either resistant or susceptible to race 65 depending on the isolate inoculated, showing the presence of pathogenic variation within race 65.Pinto et al. (2012) analyzed 74 isolates of C. lindemuthianum in two regions of the State of Minas Gerais and observed the occurrence of six pathogenic races, with predominance of race 65 in the two regions.
The race 65 isolates 13, 14, 15 and 16 from the State of São Paulo used in this study showed differences between them and from all the other race 65 isolates, isolate 16 was the most dissimilar because it had SNPs in the region of the 5.8S gene and the ITS2 region that differed from all the other isolates of this race, a finding that suggests high genetic variability between and within race 65.

Similarity and dissimilarity measures
The results of the sequence analysis based on the methodology proposed by Tamura et al. (2011) using the Mega software indicated that the highest genetic distance occurred between isolates 10 and 11 of race 65 from Santa Catarina, with a value of 0.772 (Table 1).High dissimilarity was also observed between isolates 3 and 11 from Mato Grosso and Santa Catarina, respectively, with the same value of 0.772.In turn, the most similar isolates were 2 and 7, collected in Mato Grosso and in Santa Catarina, which had a genetic distance of 0.002 (Table 1).
Isolates 15 from the State of São Paulo and 9 from the State of Santa Catarina showed a genetic distance of 0.004.The isolates of race 65 from the State of Santa Catarina used in this study showed high dissimilarity, as observed in Table 1.According to Alzate-Marin et al. (2001), wide of polymorphism were identified between isolates in studies of genetic variability in pathotypes of C. lindemuthianum and can be used in studies of genetic diversity between pathotypes.Thomazella et al. (2002) used random amplified polymorphic DNA (RAPD) markers to identify genetic variability in different isolates of race 65 collected in the State of Paraná, showing molecular variability within that race.
Isolates classified as belonging to race 65 differ in their reactions to several common bean genotypes.Isolates 15 and 8, collected in São Paulo and in Santa Catarina respectively, and isolates 15 and 14, collected in São Paulo, had genetic distances of 0.006.Three isolates of race 65 collected in the State of Santa Catarina (8, 9 and 12) and two in São Paulo (14 and 15) had genetic distances of 0.009 between them.
According to the results presented here, the isolates from the states of Santa Catarina and São Paulo showed wide genetic variability among them.In a survey of races, Alzate-Marin & Sartorato (2004) identified races 65, 73 and 81 as the most frequent and widely distributed in Brazil, and these races are commonly found in the States of Paraná, Santa Catarina, Goiás and Distrito Federal.
The phylogenetic tree based on the sequences of the ITS1 and ITS2 regions revealed the separation of the isolates in two groups, but within the group where 5 isolated from the State of Santa Catarina belonging to race 65, there was a formation of a subgroup with the races 23, 31, 73, and 1096 obtained from the database together with the isolated 11 from Santa Catarina.
Figure 3 illustrates that isolates 3 (Mato Grosso), 10 and 11 (Santa Catarina) belong to the same group, but isolates 3 and 10 are very close to, but distant from the isolated 11 belonging to another subgroup.Isolated 2 (Mato Grosso) and 7 (Santa Catarina) are very close within the same group.Group I consists of five isolates of race 65, the isolates of races R23, R2047, race 89 and C. orbiculare and C. trifolii.In turn, Group II consists of twelve isolates of race 65, the isolates of race 17, race 2, MAFF 305390, R23.19, Rec15, the subgroup with isolate 11 from the State of Santa Catarina belonging to race 65, and races 23, 31, 73 and 1096.Gonçalves-Vidigal and Kelly (2006) used an isolate of race 65 that overcame resistance in cultivar BAT93 that differed from the one used by Alzate-Marin et al. (2007) for which BAT93 showed resistance.In the present study, two groups within race 65 were observed in the phylogenetic tree.
Likewise, Ishikawa, Souza, and Davide (2008) worked with 13 isolates of race 65 from the State of Minas Gerais from different regions and years and used RAPD analysis to identify 11 groups with the phylogenetic tree, which demonstrates the presence of variability within race 65.Santos, Antunes, Rey, and Rosseto (2008) reported pathogenic variability within isolates of race 65, 73 and 81, and two isolates of the same race were inoculated into 37 cultivars used in this study.
These results demonstrate the high variability of the pathogen, suggesting the existence of new races or sub-races that cannot be detected through the current series of differential cultivars.

Conclusion
The highest variability for the 17 isolates of C. lindemuthianum race 65 was observed in the ITS1 region.From a total of 11 SNPs detected, six SNPs occurred in and a change from G to A at position 199.Isolate 4 from Paraná showed a SNP at position 120, where G was replaced by C, and isolate 14 from São Paulo had SNPs, a change from G to T at position 119, a change from A to G at position 156, and a change from G to A at position 199.Carneiro (1999) identified 13 races of C. lindemuthianum, among which races 81, 65 and 73 were the most frequent in a study of pathogen variability conducted with 70 isolates collected in the State of Paraná.

Figure 3
Figure3shows the formation of two distinct groups.Five isolates of race 65 are restricted to group I and twelve isolates to group II, with one (1) elonging to the subgroup, which demonstrates the high genetic variability of this race.

Figure 3 .
Figure 3. Phylogenetic tree of the 32 isolates of Colletotrichum based on the DNA sequences in the IST1 region, gene 5.8S and the ITS2 region.