Materials and methods

Phytopathogenic fungi. The fungal strains used in this study (Table 1) were obtained from the culture collection of the Laboratory of High Technology in Xalapa (LATEX) and came from different cultures of economic interest in the state of Veracruz, Mexico. Their identification has been supported through taxonomic keys (^{Romero-Cova, 1988}; ^{Mendoza-Zamora, 1996}; ^{Barnett and Hunter, 1998}) and for the Colletotrichum genus, pathogenicity assessments were done (^{Márquez-Fernández et al., 2013}). Likewise, a strain of I. lunata ATCC 12574 was used, due to the antibacterial activities reported in said species by ^{Espinoza et al., (2008)}. The cultivation of each one of the fungi was done in Petri dishes with potato dextrose agar (PDA, Difco), the incubation period varied for each species and oscillated between 7 and 12 days at 25 ± 2 °C until the mycelium covered 75 % of the surface of the culture medium.

Table 1 Phytopathogenic Fungi strains. 

The strains were isolated and morphologically identified through taxonomic keys (^{Romero-Cova, 1988}; ^{Mendoza-Zamora, 1996}; ^{Barnett y Hunter, 1998}) and through pathogenicity tests for the Colletotrichum genus (^{Márquez-Fernández et al., 2013}).

Bacterial strains. The bacterial strains used were: Staphylococcus aureus β-hemolytic (ATCC 25923), Streptococcus pneumoniae (ATCC 6303), Staphylococcus epidermis coagulase (+) (ATCC 14990), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 17933) (^{American Type Culture Collection, 2010}).

Fungal culture and extraction. For the liquid culture of the phytopathogenic fungi, circles of 1 cm in diameter were taken from each one of the fungal strains of a culture from 7 days of incubation, and were aseptically transferred to 250 mL Erlenmeyer flasks with 50 mL of potato broth with dextrose, where they incubated at 25 ± 2 °C for 15 days shaking them continuously at 150 rpm. Once the incubation period was over, the biomass produced by each fungal strain was filtered by vacuum filtration. Subsequently, it was dehydrated through lyophilization and was extracted with a mixture of methanol:chloroform (1:1) during 3 days, the solvent excess was eliminated through distillation with reduced pressure (^{Trigos et al., 2011}).

Antibacterial activity assessment of phytopathogenic fungi extracts against bacterial strains of clinical interest. The antibacterial activity was evaluated following the agar on a Kirby-Bauer disk diffusion method (^{Koneman et al., 2004}). The treatments were done in Petri dishes with Luria-Bertani agar, these were inoculated with each of the bacterial strains under review, with a cellular suspension with a turbidity equal to that of the 0.5 tube on the McFarland scale, equal to 1.5 x 10⁸ UFC/mL (^{Clinical and Laboratory Standards Institute, 2007}). Conversely, the dry fungal extracts were dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical Co.) to a concentration of 1 mg/mL, and from this solution serialized dilutions were done in order to obtain the different extract concentrations that were used in the test (20, 50 and 70 µg/mL). Subsequently, filter discs (6 mm in diameter) were placed on top of the Petri dishes, each of them permeated with 20 μL of each of the different extract concentrations of the phytopathogenic fungi. Permeated discs with DMSO served as a negative control and commercial discs with 25 μg of ampicillin were used as a positive control. Finally, the Petri dishes were incubated at 35 ± 2 ºC during 24 h. The inhibition zone diameters were measured in mm. The treatments were done with three repetitions for each concentration of each of the phytopathogenic fungi against each one of the bacterial strains assessed.

Statistical Analysis. The data obtained was analyzed with an ANOVA Dunnett, (P<0.0001) in order to detect differences between the inhibition zone diameters obtained by the extracts compared to the negative control.

Results and discussion

The statistical analysis of the obtained inhibition diameters showed that the Colletotrichum musae and C. gloeosporioides extracts had significant inhibition activity on the growth of Escherichia coli for the concentrations of 20, 50 and 70 µg/mL (P<0.0001), related to the negative control. Conversely, the significant differences in the inhibition diameters caused by the Idriella lunata extract on the growth of Staphylococcus aureus β-hemolytic, E. coli and Pseudomonas aeruginosa in concentrations of 50 and 70 µg/mL (P<0.0001) in relation to the negative control used (Table 2) were also registered. The results obtained show that the C. gloeosporioides and C. musae extracts are selective for E. coli. This could be useful in the treatment of domestic waste water primarily used in agriculture, especially in places where this resource is scarce (^{Pedrero et al., 2010}). Furthermore, the Idriella lunata extract showed activity against both Gram (-) and Gram (+) bacteria.

Table 2 Bacterial inhibition zone diameters taken from fungal extracts. 

¹Average of three repetitions ± the standard error of the measurement (SEM). *** indicate significant statistical differences regarding the negative control (Dunnett, p<0.0001). NA. Non-active.

When working with raw extracts and not with a pure chemical compound, the active concentrations are encouraging as they manifest a highly effective activity, that is also similar to previous results that have demonstrated the antimicrobial activity of other species of phytopathogenic fungi such as Menisporopsis theobromae and Idriella sp., against E. coli, S. aureus, P. aeruginosa, Erwinia carotovora, E. carotovora pv. atroseptica and Agrobacterium tumefaciens (^{Trigos et al., 2005}). Additionally, Curvularia lunata, Phytophthora drechsleri, Phytophthora capsici, Gliocladium spp, Neocosmospora vasinfecta resulted active against bacteria such as E. coli and P. aeruginosa (^{Trigos et al., 2006}). Similarly, ^{Espinoza et al. (2008)}, demonstrated that the 5-hydroxymethyl-2-furaldehyde and 1-n-Butyl- β-D-fructopyranoside obtained from the Idriella sp culture showed antibacterial activities against the phytopathogenic bacteria Xanthomonas axonopodis, Pectobacte-rium carotovorum, P. chrysanthemi and Erwinia amylovora with a Minimum Inhibitory Concentration (MIC) of 0.625 mg/mL.

Conclusions

Even though phytopathogenic fungi remain as the main cause of the partial or total yield decrease of crops and thus represent high financial losses, the results obtained place the Colletotrichum musae, C. gloeosporioides and Idriella lunata strains, for their antibacterial activity, as potential sources of bioactive compounds against bacterial strains of medical interest that represent the cause of common infections in our country. As a result, this work presents the possibility of continued research on the utilization of phytopathogenic fungi for medical applications, previous chemical, pharmacological, toxicological and clinical studies.