Introduction

The phytopathogenic fungi Cercospora sp. and Septoria sp. are responsible for major diseases in soybean production, namely Cercospora Leaf Blight and Septoria Brown Spot, respectively. Both diseases significantly reduce the photosynthetically active leaf area, leading to substantial yield losses (^{Hartman et al., 2015}). The widespread adoption of no-till farming, the extensive monoculture of soybean, and the effects of climate change have markedly increased the prevalence and severity of these diseases (^{Sautua et al., 2024}).

For Cercospora sp., estimated average annual yield reductions are approximately 10%, with potential losses ranging between 30% and 50%, depending on environmental conditions throughout the production cycle (^{Lavilla & Ivancovich, 2021}; ^{Hartman et al., 2015}). In Paraguay, the prevalence of this disease has been predominantly observed in commercial soybean crops from the R4 reproductive stage onward. Although no precise records exist regarding the specific yield losses caused by this pathogen in the country (^{Enciso-Maldonado, Fernández-Gamarra et al., 2021}; ^{Arrúa et al., 2021}), ^{Wrather et al. (2010)} estimated losses of 0.7 million tons in 2006, when total soybean output was recorded at 2,227,487 tons across a cultivated area of 3,641,186 hectares (^{Paraguayan Chamber of Cereal and Oilseed Exporters and Traders, 2025}).

With respect to Septoria sp., the etiological agent of Septoria Brown Spot in soybean, several studies have reported yield reductions between 8% and 34% (^{Hartman et al., 2015}). In regional production systems, this pathogen is commonly observed during reproductive stages, with symptom severity levels approaching 50% and recorded yield losses of approximately 9% (^{Enciso-Maldonado et al., 2021}).

Cercospora sp. infection results in symptoms affecting various plant structures, including hypocotyls, leaves, stems, petioles, pods, and seeds (^{Sautua et al., 2024}). In Paraguay, the primary soybean diseases associated with Cercospora sp. are Cercospora Leaf Blight and Cercospora Purple Seed Stain (^{Caballero-Mairesse et al., 2024}). Foliar symptoms initially manifest as irregular, reddish-purple or tan lesions due to cercosporin production. These lesions gradually expand and coalesce into necrotic areas, leading to early defoliation that predominantly affects upper leaves, while petioles often remain attached (^{Enciso-Maldonado, Fernández-Gamarra et al., 2021}).

Similarly, Septoria sp. infection initially presents as small, irregular brown lesions accompanied by a characteristic yellow halo. This disease may also lead to early defoliation (^{Hartman et al., 2015}).

The ability of these phytopathogens to grow and sporulate under controlled laboratory conditions has critical implications for understanding their epidemiology and designing effective management strategies. In Cercospora sp., the limited sporulation ability exhibited in artificial media poses a significant challenge for in-depth studies on the biology, taxonomy, and pathogenicity of the genus (^{Chand et al., 2013}; ^{Crous et al., 2019}). Additionally, sporulation is markedly influenced by environmental factors, particularly temperature and light intensity (^{Silva et al., 2016}; ^{Brunelli et al., 2006}). A frequent limitation is the rapid decline in the reproductive capacity of isolates, which frequently lose their ability to produce conidia when maintained in vitro, thereby hindering further studies (^{Yuliarni et al., 2013}).

To address these challenges, several culture media have been evaluated, including Oat Agar, Malt Agar, Potato Dextrose Agar, V8 Agar, and Soybean Leaf Infusion Agar (^{Alloati et al., 2015}; ^{Uppala et al., 2019}; ^{Kashiwa et al., 2021}). For Septoria species specifically, certain studies specifically highlight Malt Agar and Oat Agar as suitable media, emphasizing the importance of modifying environmental conditions, including light intensity and exposure to near-ultraviolet radiation, to induce sporulation (^{Quaedvlieg et al., 2013}; ^{Crous et al., 2019}).

Given the significance of these previous findings, this study aimed to assess the growth and sporulation of Cercospora sp. and Septoria sp. isolates in different culture media to establish the optimal in vitro conditions for their development.

Materials and methods

Fungal Strains

The Cercospora sp. isolate was obtained from CEMIT-UNA and the Phytopathology Laboratory of Universidad Católica (Hohenau Pedagogical Unit) within the framework of the PINV01-152 Project, funded by CONACYT. The Septoria sp. isolate, in turn, was supplied by the Paraguayan Institute of Agricultural Technology (IPTA), Capitán Miranda Research Center, Itapúa, Paraguay. Both isolates are currently being subjected to molecular identification.

Preparation of culture media

For the preparation of Soybean Leaf Decoction Agar (SLDA), 200 g of fresh soybean leaves, 10 g of sucrose, 15 g of agar, and 1000 mL of distilled water were used. The soybean leaves were placed in water in a pot and heated to boiling, maintaining a gentle simmer for 15-20 minutes. The mixture was then filtered to remove solid residues, retaining only the resulting liquid. Subsequently, in a sterile medium preparation container, 10 g of sucrose and 15 g of agar were dissolved in the decoction liquid, ensuring a homogeneous mixture.

For the preparation of Soybean Grain Decoction Agar (SGDA), 200 g of soybean seeds, 10 g of sucrose, 15 g of agar, and 1000 mL of distilled water were used. The soybean seeds were placed in water in a pot and heated to a boil, maintaining a gentle simmer for 15-20 minutes. The mixture was then filtered to remove solid residues, retaining only the liquid fraction. In a sterile medium preparation vessel, 10 g of sucrose and 15 g of agar were dissolved in the decoction liquid, ensuring complete dissolution and a homogeneous mixture (^{SCABUSA, 2021}).

Finally, for the preparation of Potato Dextrose Agar (PDA) medium, 200 g of potato, 10 g of dextrose, 20 g of agar, and 1000 mL of distilled water were used. The potatoes were placed in water in a pot and heated to a boil. The mixture was then filtered to remove solid particles, retaining only the liquid fraction. In a sterile medium preparation vessel, 10 g of dextrose and 20 g of agar were dissolved in the decoction liquid, ensuring complete dissolution and uniform mixing.

Each culture medium was transferred to autoclavable glass containers and sterilized at 121°C for 15 minutes in an autoclave. For the transfer of the medium into Petri dishes, the sterilized mixture was cooled to approximately 50°C and aseptically transferred into the Petri dishes inside a laminar flow hood. Finally, the medium was allowed to solidify at room temperature and then stored under appropriate conditions until required for use.

Growth and Sporulation Assay

Each medium (ADH, ADG, and PDA) was prepared in five replicates. A 5 mm diameter plug of actively growing mycelium, obtained from pure pathogen cultures, was placed at the center of each plate. The inoculated plates were incubated at 24 ± 1°C under controlled conditions for the entire experimental period.

Evaluated Variables

Mycelial growth and sporulation were evaluated. Colony diameter was measured every three days using a sterile ruler or a digital caliper, up to day

15. Sporulation was quantified at the end of the experiment using a Neubauer chamber, and results were expressed as the mean number of spores per cm² of colony.

Statistical Analysis

An analysis of variance (ANOVA) was conducted using Infostat version 2020 (^{Di Rienzo et al., 2020}) to compare the mean mycelial growth among culture media. For sporulation, the mean spore production per cm² was determined for each medium to identify the optimal medium for spore production in each pathogen.

Figure 1 In vitro growth of Cercospora sp. in different culture media. 

Results

Under the evaluated experimental conditions, none of the studied phytopathogenic fungi exhibited sporulation, as only sterile mycelial growth was observed.

Significant differences in mycelial growth were detected among the tested culture media. On day 3, Cercospora sp. (Fig. 1) showed variations in its development, with the ADG medium supporting the highest average growth (3.63 ± 0.11 mm), while ADH exhibited the lowest (2.50 ± 0.11 mm). From day 6 to day 15, PDA significantly promoted the most extensive mycelial expansion, reaching a final mean of 21.44 mm on day 15, surpassing the growth observed in ADG (19.39 mm) and ADH (18.19 mm).

Significant differences in colony development of Septoria sp. (Fig. 2) were observed from day 3 to day 15, with the ADG medium supporting the highest average mycelial expansion on day 15 (8.84 mm). In contrast, the PDA medium exhibited the lowest growth rate for this pathogen (3.65 mm). This study constitutes the first documented use of the ADG medium for assessing the in vitro growth dynamics of Septoria sp., highlighting its potential application in mycological research.

Figure 2 In vitro growth of Septoria sp. in different culture media. 

Discussion

No sporulation was detected in any of the evaluated culture media, as only sterile mycelium was observed in both analyzed phytopathogens. This difficulty in conidia production by Cercospora species grown in artificial media aligns with previous literature reports (^{Crous et al., 2019}).

^{Yeh and Sinclair (1980}) reported that various culture media, including potato dextrose agar, carrot leaf decoction agar, V8 agar, and mature soybean residue decoction agar, did not significantly affect the sporulation of Cercospora kikuchii isolates. These authors recommended specific media (V8A, DSPT, and CLDA) under controlled environmental conditions (25°C with an alternating 12-hour photoperiod) to optimize conidia production. In contrast to their findings, none of the tested media successfully induced sporulation in our experiment.

Similar findings were reported by ^{Beckman and Payne (1983}) for Cercospora zeae-maydis, who indicated that serial subculturing of the fungus generally led to limited conidiation and the development of sterile mycelium. These researchers observed a significant influence of the culture medium on mycelial growth and colony morphology, noting that V8 medium favored sporulation, whereas PDA promoted mycelial growth. This latter aspect aligns with our findings, as PDA significantly enhanced the growth of C. kikuchii.

^{Vathakos and Walters (1979}) reported that C. kikuchii exhibited vegetative growth only in media prepared with carrot leaf decoction and immature or senescent plant tissues from various crops. Additionally, these authors documented abundant sporulation under specific lighting conditions (Gro-Lux fluorescent lamps), noting that subculturing from conidia yielded greater spore production than from mycelium. Furthermore, ^{El-Gholl et al. (1982}) and ^{Vathakos and Walters (1979)} identified V8 agar as a suitable medium for inducing sporulation in Cercospora sp.. However, since this medium is not commercially available in Paraguay, there is a pressing need to identify viable local alternatives.

Regarding Septoria glycines, ^{Bertagnolli et al. (1986}) evaluated various culture media and identified Fries medium as the most effective for inducing sporulation. ^{Zalewska (2012}), while studying related species such as S. carvi, suggested that both temperature and the composition of the culture medium (malt medium and malt with leaf extract decoction) are key factors for optimizing fungal growth and sporulation. Although no sporulation of Septoria sp. was observed in our study, ADG medium significantly promoted its vegetative growth, marking the first documented use of this medium for this species.

^{Saidi et al. (2012}) also emphasized the importance of culture medium composition, temperature, and light conditions as critical factors for efficient sporulation induction in Septoria species. This highlights the multifactorial nature of sporulation and supports our findings, suggesting that the absence of sporulation could be attributed not only to the culture medium used but also to additional environmental factors such as temperature and photoperiod.

Whereas the growth and sporulation of Cercospora sp. and Septoria sp. are multifactorial processes influenced by a range of variables. Future research should aim at the integrated optimization of culture medium, light conditions, and temperature to enhance spore production under in vitro conditions.

Conclusions

Under the evaluated experimental conditions, the culture media allowed mycelial growth of Cercospora sp. and Septoria sp. but did not induce sporulation. PDA medium was the most effective in promoting mycelial growth in Cercospora sp., whereas ADG medium favored the development of Septoria sp. . Given the absence of sporulation, further research should investigate variations in environmental factors such as temperature and photoperiod, as well as alternative culture media formulations, to enhance in vitro sporulation in both pathogens. This study provides fundamental insights for selecting appropriate media in future research on the growth and management of these important soybean phytopathogens.