**4. Discussion**

Soybean is one of the most important crops in Argentina, and it has been characterized by an incredible rate of adoption and growth. Twenty-one percent of the cultivated lands of Argentina are in Santa Fe Province, whose main crop, soybean, makes it the main national producer (Aizen et al., 2009; Penna & Lema, 2002). However, soybean crops are affected by several diseases which decrease the total production.

This study aims at contributing to the knowledge of one of the most frequent soybean phytopathogens. Phenotypic and genotypic variations among isolates of *C. kikuchii* from infected soybean corresponding to the centre-northern region in Santa Fe Province, geographically separated but with similar climatic conditions, were studied.

When grown on PDA, and considering micromorphology, the isolated fungi showed colonies with different macroscopic aspects. These results would be in agreement with those reported by Almeida et al. (2005) on Brazilian isolates of *C. kikuchii.* The red pigment observed around some of the colonies was caused by the presence of cercosporin, a pathogenicity factor of *C. kikuchii,* as described by many authors (Assante et al., 1997; Fajola, 1978; Upchurch et al., 1991).

In general, fungi belonging to genus *Cercospora*, as well as other similar genera, exhibit great difficulty for sporulating on artificial culture media (Avila de la Calle et al., 2004; Cadwell, 1994; Yeh & Sinclair, 1980). This limitation was also proved in this study.

Information about which is the optimum medium for studying *Cercospora* sporulation is diverse and confuse, there being no uniform criteria between the different authors. This particular fact evidences the great "inter" and "intra" variability among species belonging to this genus (Brunelli, 2004; Cai, 2004; Chen et al., 1979; Jenns et al., 1989; Salvador & Garrido, 1990).

Given that genus *Cercospora* belongs to the group of Dematiaceous (dark-colored) fungi, it shows septated and olive-brown pigmented hyphae. It is worth mentioning that the number of conidia, its size and number of septa are affected both by the environment and the culture media used (Cai, 2004). Yeh & Sinclair (1980) reported that conidiophore and conidia size differed between isolates even when incubated under the same conditions.

Since typing is a necessary first step in knowing pathogens (Redondo et al., 2009), techniques based on DNA polymorphisms are especially valuable to enhance epidemiological studies.

The AGNES cluster analysis resulted in a dendrogram that showed great genetic distances between different fungi (Figure 8). Cluster A included six isolates, C14, C15 and C16 in cluster A1 and C17, C18 and C19 in A2, all of them from the same origin (Emilia, Table 2). Cluster B included three fungi, which were grouped in two subclusters. B1 included C26 which, although isolated from Emilia, belonged to another lot, and B2 included C20 and C24,

Cluster C included both strains NBRC (C6711 and C6715) (subcluster C1) and C32 isolated from Esperanza (subcluster C2). Cluster D included 5 fungi, C23 and C25 from different origin and cultivar (subcluster D1) and C27 (same origin than C25), C28 and C29 isolated from Esperanza (subcluster D2) (Table 2). Finally, clusters E and F included C30 and C31,

Soybean is one of the most important crops in Argentina, and it has been characterized by an incredible rate of adoption and growth. Twenty-one percent of the cultivated lands of Argentina are in Santa Fe Province, whose main crop, soybean, makes it the main national producer (Aizen et al., 2009; Penna & Lema, 2002). However, soybean crops are affected by

This study aims at contributing to the knowledge of one of the most frequent soybean phytopathogens. Phenotypic and genotypic variations among isolates of *C. kikuchii* from infected soybean corresponding to the centre-northern region in Santa Fe Province,

When grown on PDA, and considering micromorphology, the isolated fungi showed colonies with different macroscopic aspects. These results would be in agreement with those reported by Almeida et al. (2005) on Brazilian isolates of *C. kikuchii.* The red pigment observed around some of the colonies was caused by the presence of cercosporin, a pathogenicity factor of *C. kikuchii,* as described by many authors (Assante et al., 1997; Fajola,

In general, fungi belonging to genus *Cercospora*, as well as other similar genera, exhibit great difficulty for sporulating on artificial culture media (Avila de la Calle et al., 2004; Cadwell,

Information about which is the optimum medium for studying *Cercospora* sporulation is diverse and confuse, there being no uniform criteria between the different authors. This particular fact evidences the great "inter" and "intra" variability among species belonging to this genus (Brunelli, 2004; Cai, 2004; Chen et al., 1979; Jenns et al., 1989; Salvador & Garrido,

Given that genus *Cercospora* belongs to the group of Dematiaceous (dark-colored) fungi, it shows septated and olive-brown pigmented hyphae. It is worth mentioning that the number of conidia, its size and number of septa are affected both by the environment and the culture media used (Cai, 2004). Yeh & Sinclair (1980) reported that conidiophore and conidia size

Since typing is a necessary first step in knowing pathogens (Redondo et al., 2009), techniques based on DNA polymorphisms are especially valuable to enhance

both isolated from the same region (Margarita) and cultivar (Table 2).

(isolated from Esperanza) and C21 and C22 (from Margarita), respectively.

geographically separated but with similar climatic conditions, were studied.

1994; Yeh & Sinclair, 1980). This limitation was also proved in this study.

differed between isolates even when incubated under the same conditions.

several diseases which decrease the total production.

**4. Discussion** 

1978; Upchurch et al., 1991).

epidemiological studies.

1990).

Genetic variability could be found both between isolates from the same region and between those from different regions, thus confirming previous results (González et al., 2008; Lurá et al., 2007), as reported by Almeida et al. and Cai for *C. kikuchii* and other species (Almeida et al., 2003, 2005; Cai, 2004). No isolates turned out to be 100 % similar. These findings are not easy to be explained. However, it is essential to consider that this fungus is transmitted through the soybean seeds and, being necrotrophic, it can survive in the stubble. As a great increase in the soybean producing area has occurred in Argentina in the last decade, the turnover of seeds from traditional to new production areas has increased accordingly (Secretaría de Agricultura, Ganadería, Pesca & Alimentos [SAGPyA], 2003). The survival of the fungus in the stubble, on the other hand, makes it the main source of primary inoculum for the re-infection in the next campaign (Sillón, 2007).

According to Pujol Vieira dos Santos et al. (2002) and Stenglein & Ballati (2006), many factors could have been affecting polymorphism analysis, e.g. the intraspecific variants of a pathogen, the number of samples selected for analysis, genetic flow between populations, environmental adaptation and selective pressure and migration.

Other factors to be considered, which could explain the genetic variability detected in the study, would be the changes in DNA within the populations of filamentous fungi. As reported by MacDonald (1997), these changes are the consequence of mutations, deletions, pairing systems or gene migration or flow, and population selection, since sexual reproduction is not known in *C. kikuchii* (Almeida et al., 2005). Daboussi Capy (2003) and Kempken & Kück (1998), on the other hand, state that changes mediated by transposable elements, together with transposition and recombination, provide a wide range of genetic variation, which is useful for the natural self-adaptation of the population to the changing environment and the interaction with another organisms.

With reference to the strains *C. kikuchii* NBRC 6711 and *C. sojina* NBRC 6715, from the Culture Collection of the National Institute of Technology and Evaluation (NITE), Japan, no data were found concerning either their isolation source, location or country of origin. The low similarity they showed, as compared to the regional isolates, could be attributed to the fact that they come from regions naturally different from those selected for this work.

As regards the two oligonucleotides that gave unsatisfactory results to amplification, very few bands were detected with OPA-06, which could be accounted for considering that efficiently amplified DNA regions must be located between two sites complementary to the primers and separated by a distance of a few kb (Williams et al., 1990). In the *C. kikuchii* under study, the sites for this primer could be less frequent, which made the amplification of fragments technically impossible. OPA-02, on the other hand, showed a pattern with numerous bands, its reading and interpretation becoming thus very difficult.

The 6 groups identified by the clustering techniques allowed to distinguish both genetic variability among them and similarity among the fungi belonging to the same group. After comparing the results of both clustering methods, a high genetic homogeneity could be observed in two out of the six ones (third and sixth FANNY groups with the corresponding F and E AGNES groups) since Silhouette coefficient associated to them turned out to be higher than 0.70 with FANNY algorithm and agglomerative coefficient in AGNES technique was close to zero for the same two clusters. Besides, clusters A and C (AGNES) corresponded with the 1st and 4th FANNY clusters, respectively. As regards the rest of the

Phenotypic and Genotypic Variability in

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Cai, G. (2004). *Cercospora Leaf Blight of soybean: Pathogen vegetative compatibility groups,* 

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fungi, little homogeneity was confirmed between the isolates comprising clusters B and D generated by AGNES and clusters 2 and 5 obtained with FANNY.

It was shown that *C. kikuchii* isolates from the same geographic region appeared in different groups. Molecular analyses showed intraspecific variability within *C. kikuchii* isolates from soybean collected in different regions, so it was difficult to establish a relationship between this variability and that of the soybean cultivars from which *C. kikuchii* isolates were obtained. Similar results had been previously reported by González et al. (2008) and Almeida et al. (2005). According to the results here obtained, Argentinian populations of *C. kikuchii* are phenotypically, genotypically and geographically variable. In agreement with Almeida et al. (2005), who consider that this pathogen is easily transmitted by seeds, it is not surprising to find the same haplotypes in different regions.

In Argentina, and in this region in particular, there has been a rapid increase in the soybean producing area since 1970 (Aizen et al., 2009); therefore, the traffic of seeds from traditional to new areas could be responsible for the geographical variability since *C. kikuchii* is a seed borne pathogen. Unfortunately, an insufficient number of isolates was obtained from each area to permit the evaluation of gene flow among populations more accurately.

For countries like Argentina, with large soybean areas it is very important to know the variability of the pathogen in advance in order to prevent resistant cultivars when sown in different areas.

Few studies have been reported concerning the molecular characterization of genus *Cercospora*. Therefore, widening the scope of knowledge about this pathogen biology, and developing strategies to control the cultures intended for human and/or animal use in this region, therefore, would be a great contribution of this work.
