**1. Introduction**

96 Soybean – Genetics and Novel Techniques for Yield Enhancement

Wildermuth, M.C., Dewdney, J., Wu, G. & Ausubel, F.M. (2001). Isochorismate synthase is required to synthesize salicylic acid for plant defence. *Nature* 414:562-565. Williamson, V.M. & Gleason, G.A. (2004). Plant-nematode interactions. *Current Opinion in* 

Wrather J.A. & S.R. Koenning. (2006). Estimates of disease effects on soybean yields in the

Young, L.D. (1992). Epiphytology and life cycle, *in* Riggs, R.D. & Wrather, A.W. (ed.), *Biology and management of the Soybean Cyst Nematode.* APS Press, St. Paul, pp 27-36.

United States 2003 to 2005. *Journal of Nematology* 38:173-180.

*Plant Biology* 6:327-333.

More than 100 fungal diseases affecting soybean (*Glycine max* (L.) Merr.) culture have been found worldwide, though only 35 of them are of great economic concern since they cause a 10 to 15% yield decrease (Ivancovich & Botta, 2003; Yeh & Sinclair, 1980).

In Argentina, "Late-cycle diseases" (LCD) constitute a complex of infections affecting the culture of this leguminous, particularly in the reproductive stages. The first symptoms appear from the onset of carpel formation, thus producing premature plant maturation, a reduction in yield (5 to 8%) and seed quality loss. Due to the climate conditions of the region, leaf blight and purple seed stain, is one of the LCD prevailing in the central-northern region of Santa Fe Province (Formento, 2005; Ivancovich & Botta, 2003).

The causal agent of the disease is the fungus *Cercospora kikuchii* (T. Matsumoto & Tomoyasu) M.W. Gardner, which produces irregular injuries on the leaves, forming reddish-purple necrotic areas, pale pink to dark purple stains on the seeds, together with cracks on the outer coat (Formento, 2005). *C. kikuchii* belongs to the *Cercospora* taxonomic complex*,* which is assumed to be host-specific (Crous & Braun, 2003), and therefore its species are normally identified from the phenotypic characteristics they show when grown on their natural substrate, being much more difficult to be characterized from artificial media (Almeida et al., 2005; Gams et al., 2007).

One of the most important factors determining the pathogenicity of this fungus is the production of cercosporin, a red exotoxin (Kuyama & Tamura, 1957; Upchurch et al.,1991). Another aspect to consider is the genetic variability found in some fungi, which has determined the description of different races; that is why the use of some methodology to detect inter- and intraspecific variations between isolates is recommended (Kuyama & Tamura, 1957). In this sense, the Random Amplified Polymorphic DNA (RAPD) technique allows the differentiation between strains since amplification focuses only on the whole genome (Tigano et al., 2003; Williams et al., 1990).

Although some Brazilian research groups have been studying aspects related with this phytopathogen (Almeida et al., 2003) only a few reports have been found in Argentina and, in particular in Santa Fe Province, about epidemiology and population structure of *C. kikuchii.* 

Phenotypic and Genotypic Variability in

stored and subsequently included in the strain collection.

(Fig. 2).

Gobernador Crespo.

incubation.

**2.2 Phenotypic characterization** 

*Cercospora kikuchii* Isolates from Santa Fe Province, Argentina 99

Tissue pieces were disinfected by immersion in 3% w/v sodium hypochlorite for 3 min, rinsed with sterile distilled water (Fig.1) and incubated in moist chambers at 26 ± 0.5 ºC, under alternate light cycles (16 h cold light and 8 h in the dark) (Salvador & Garrido, 1990).

Observation with stereoscopic magnifying lens (BOECO Germany, BTB 3-C) was performed from the 3rd day of incubation onwards. Once the conidial structure growth was verified, conidia were taken with a sterile needle and suspended in 1 mL sterile water so as to obtain a homogeneous suspension. To produce monosporic cultures, an aliquot (100 L) was spread over Potato Dextrose Agar (PDA) in a Petri dish and incubated as described above (Dunkle & Levy, 2000). Isolates were labeled with letter C and correlative numbers (Table 2),

> **Isolate Location Lot Cultivar/varietal** C14 Emilia A01 A5409 C15 Emilia A01 A5409 C16 Emilia A01 A5409 C17 Emilia A01 A5409 C18 Emilia A01 A5409 C19 Emilia A01 A5409 C20 Margarita A04 TJ2070 C21 Margarita A04 TJ2070 C22 Margarita A04 TJ2070 C23 San Justo A02 A4910 C24 Margarita A04 TJ2070 C25 G. Crespo A04 A8000 C26 Emilia B01 A5409 C27 G. Crespo A04 A8000 C28 Esperanza Lote Rural A7636 C29 Esperanza Lote Rural A7636 C30 Esperanza Lote Rural A7636 C31 Esperanza Lote Rural A7636 C32 Esperanza Lote Rural A7636

Table 2. Origin and designation of *Cercospora kikuchii* regional isolates. G. Crespo:

Each fungus was inoculated with a single touch in the center of a Petri dish containing PDA and incubated under the conditions previously described. Macro and microscopic observations were conducted after 15 days, taking into account color and diameter of the colony, red pigment production (cercosporin) and the presence or absence of typical elements of reproduction. For the latter case, plates were discarded only after 25 days of

Cercosporin production was confirmed following Jenns et al. methodology (Jenns et al, 1989) with slight modifications as described by González et al. (González et al., 2008).

The aim of the present work was to determine the occurrence of phenotypic and genotypic variability between isolates of *C. kikuchii* in various regions of Santa Fe Province.
