**3.2 Genotypic characterization**

102 Soybean – Genetics and Novel Techniques for Yield Enhancement

When considering PDA cultures, the colonies, with sizes between 26-50 mm diameter, appeared as white, green-greyish and pink colored, depending on the isolate. The reverse side showed green, brown or red-brown color. All of them presented a reddish touch on their edge and abundant colorless exudate which became amber to ochre with the passing of time. The pigment diffused to the medium in some of them. Fructification was observed after 15 days of incubation, though sporulation was scarce and unevenly distributed over

Fig. 3. Growth of *Cercospora kikuchii* on natural substratum (400x)

Fig. 4. *Cercospora kikuchii.* Single conidium (400x)

**3. Results** 

the colony surface.

**3.1 Phenotypic characterization** 

A total of 90 bands were obtained, 100% of them were polymorphic. Amplification size ranged between 107 bp and 2750 bp, an average of 51 amplified fragments/oligonucleotide being produced. OPA-01 was the oligonucleotide which produced the least amount of bands (1) and OPA 12 the most (23) amplified fragments (Table 5). Amplifications with OPA-02 and OPA-06 were not satisfactory.


Figure 5 shows band profiles obtained with oligonucleotide OPA-14.

Table 5. Total number of bands and polymorphic bands obtained with the oligonucleotides selected for RAPD

A FANNY cluster analysis with k= 6 and r =1.2 (optimum combination) was carried out. Few distances near 0.2 were observed (Fig 6). C21 and C22 (isolated from Margarita and

Phenotypic and Genotypic Variability in

6715.

*Cercospora kikuchii* Isolates from Santa Fe Province, Argentina 105

Fig. 7. Silhoutte plot of FANNY (x= average distance; k=6; membership exponent (r) = 1.2). C17, C15, C14, C18, C19, C16, C20, C24, C26, C23, C21, C22, C32, C28, C27, C25, C29, C31, C30: regional isolates; C6711: *Cercospora kikuchii* NBRC 6711; C6715: *Cercospora sojina* NBRC

Fig. 8. Dendrogram of AGNES (x= average distance, method = Ward). C14, C15, C16, C17, C18, C19, C26, C20, C24, C32, C23, C25, C27, C28, C29, C30, C31, C21, C22: regional isolates;

C6711: *Cercospora kikuchii* NBRC 6711; C6715: *Cercospora sojina* NBRC 6715.

same cultivar) were strongly related, as well as C30 and C31, both of them isolated from Esperanza (same cultivar). Great genetic distances for the remaining fungi were detected (Fig.7).

Given the optimum combination obtained with Fuzzy analysis (FANNY), the AGNES algorithm was applied considering the number of clusters found (k=6).

Fig. 5. Sample gel with patterns produced by *Cercospora kikuchii* isolates and NBRC strains using OPA-14. MM: molecular marker 100 bp; C14, C15, C16, C17, C18, C19, C26, C20, C21, C22, C24, C23, C25, C27, C28, C29, C30, C31, C32: regional isolates; C6711: *Cercospora kikuchii* NBRC 6711; C6715: *Cercospora sojina* NBRC 6715.

Fig. 6. Average Distance Matrix. C32, C31, C30, C29, C28, C27, C25, C23, C24, C22, C21, C20, C26, C19, C18, C17, C16, C15, C14: regional isolates; C6711: *Cercospora kikuchii* NBRC 6711; C6715: *Cercospora sojina* NBRC 6715.

same cultivar) were strongly related, as well as C30 and C31, both of them isolated from Esperanza (same cultivar). Great genetic distances for the remaining fungi were detected

Given the optimum combination obtained with Fuzzy analysis (FANNY), the AGNES

Fig. 5. Sample gel with patterns produced by *Cercospora kikuchii* isolates and NBRC strains using OPA-14. MM: molecular marker 100 bp; C14, C15, C16, C17, C18, C19, C26, C20, C21, C22, C24, C23, C25, C27, C28, C29, C30, C31, C32: regional isolates; C6711: *Cercospora kikuchii*

Fig. 6. Average Distance Matrix. C32, C31, C30, C29, C28, C27, C25, C23, C24, C22, C21, C20, C26, C19, C18, C17, C16, C15, C14: regional isolates; C6711: *Cercospora kikuchii* NBRC 6711;

NBRC 6711; C6715: *Cercospora sojina* NBRC 6715.

C6715: *Cercospora sojina* NBRC 6715.

algorithm was applied considering the number of clusters found (k=6).

(Fig.7).

Fig. 7. Silhoutte plot of FANNY (x= average distance; k=6; membership exponent (r) = 1.2). C17, C15, C14, C18, C19, C16, C20, C24, C26, C23, C21, C22, C32, C28, C27, C25, C29, C31, C30: regional isolates; C6711: *Cercospora kikuchii* NBRC 6711; C6715: *Cercospora sojina* NBRC 6715.

Fig. 8. Dendrogram of AGNES (x= average distance, method = Ward). C14, C15, C16, C17, C18, C19, C26, C20, C24, C32, C23, C25, C27, C28, C29, C30, C31, C21, C22: regional isolates; C6711: *Cercospora kikuchii* NBRC 6711; C6715: *Cercospora sojina* NBRC 6715.

Phenotypic and Genotypic Variability in

for the re-infection in the next campaign (Sillón, 2007).

environmental adaptation and selective pressure and migration.

environment and the interaction with another organisms.

*Cercospora kikuchii* Isolates from Santa Fe Province, Argentina 107

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

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,

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

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

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

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

fact that they come from regions naturally different from those selected for this work.

numerous bands, its reading and interpretation becoming thus very difficult.

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

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