In vitro evaluation of antifungal activity of some agricultural fungicides against two saprolegnoid fungi infecting cultured fish

2.1 Isolation of saprolegnoid strains

Two strains of Saprolegnia spp. (S. parasitica and S. diclina) were isolated from infected tilapia fish (Oreochromis niloticus) using sesame baiting technique. The fungal strains were purified, identified according to the methods described by Willoughby (1985) and Johnson et al. (2002) and preserved in PDA slant till used.

2.2 Preparation of zoospores suspensions

Zoospores suspensions of S. parasitica and S. diclina were prepared by the method described by Diéguez-Uribeondo et al. (1994). Colony discs (7 mm) of actively growing S. parasitica and S. diclina were immersed in flasks containing (50 ml) of sterilized aquarium water supplemented with antibiotic mixture and (0.1%) tween-20 to eliminate bacterial growth and facilitate zoospores separation from saprolgenoid mycelium. The flasks were incubated at 20 ± 2 °C for 18 hrs and emerging zoospores were harvested and counted using haemocytometer slide. The concentration of the isolated zoospores was diluted and adjusted to 1.2 × 10⁵ (zoospores /ml) through measuring their absorbance at wave length (580 µm) using spectrophotometer.

2.3 Fungicides

Seven fungicides including azoxytrobin (Tazer, 25.0%), cyazofamid (Segway_O, 34.5%), etridiazole (Terramster_4EC, 44.3%), fluopicolide (Adorn, 39.5%), mefenoxam (Subdue_Maxx, 22.0%), Metalaxyl (Sebring_480FS, 44.08%) and Propamocarb (Banol, 66.5%) were screened for their antifungal activities against two saprolegnoid fungi using food poisoned technique in potato dextrose broth medium.

2.4 Antifungal assay of fungicides

Antifungal activities of these fungicides were evaluated on two saprolegnoid fungi (S. parasitica and S. diclina) using potato dextrose broth medium by food poisoning technique according to the method described by Bhagwat and Datar (2014). The fungicides were dissolved in 5 ml of methanol then mixed with 45 ml of sterile potato dextrose broth medium (PDB) to attain final concentration of 0.25 mg/ml of each fungicide. For control, 5 ml of methanol was added to PDB medium and 1.0 ml of each zoospores suspensions was inoculated to the treatment flasks and control sets. After the incubation of the flasks at 20 ± 2 °C for 7 days, their contents were filtered (Whatman No.1) and biomass of the gained mycelium was weighed after drying at 70 °C for three successive days till their weights remain constant. The inhibition of fungal growth correlated with fungicides treatment was calculated as percentage from the fungal mycelial growth in control

2.5 Fungicidal analysis of effective fungicides compared with reference fungicide (Malachite green).

Four of seven fungicides belonging to 4 different chemical categories were found to be effective against the two saprolegnoid fungi (Table 1). The chemo-physical properties of these effective fungicides were illustrated in Table 2 and their antifungal activities were conducted to evaluate their efficiency in controlling saprolegnoid fungi. To determine minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of the effective fungicides, different concentrations of the concerned fungicides based on their active ingredients (0.0, 25, 50, 100, 150, 200, and 250 ppm) were prepared by dissolving their requisite amount in 5 ml of methanol, sterilized through Millipore filter (0.22 µm) then mixed with 45 ml of sterile potato dextrose broth medium to attain the final concentrations. To compare efficacy of agricultural fungicides with that of malachite green (reference fungicide) in controlling saprolegnoid fungi, different concentrations (0.0, 0.125, 0.250, 0.50, 1.0, 2.0 ppm) of malachite green were prepared by dissolving 1 mg in100 ml of methanol as a stock solution and their requisite amount were mixed with flasks containing 45 ml of potato dextrose broth. The flasks were inoculated with 1 ml of saprolegnoid zoospores suspensions and were incubated at 20 ± 2 °C for 7 days. After incubation, they were filtered and biomass of the saprolegnoid fungi were weighed after drying at 70 °C for three successive days. The fungicidal efficiency of fungicides were evaluated and their MIC & MFC were determined then compared with those of malachite green as reference fungicide with three replicates.

2.6 Acute fish toxicity test of the effective fungicides

Acute fungicides toxicity test was performed with fingerlings of tilapia fish (Oreochromis niloticus) using six concentrations of the both effective fungicides (azoxystrobin and metalaxyl). Stock solutions of azoxystrobin and metalaxyl were prepared with the concentrations of (0.0, 50, 100, 200, 300, 400 and 500 ppm). A total of 70 clinically normal tilapia fingerlings with average body weight (10 ± 0.5 gm) were used. These fish were held in seven glass aquaria of dimensions (30 × 50 × 80 cm) containing 60 L of water and equipped with an air supply and dechlorinated tap water in a rate of 10 fish/aquarium. Water temperature was maintained at 23 ± 2 °C and pH was adjusted to 7.0 ± 0.5 through the experiment period. Fish were acclimatized to lab. conditions one week before the experiment during which they were fed on commercial diet. Seven experimental fish groups were exposed to different concentrations of azoxystrobin and metalaxyl and kept for 96 hrs. in aerated water without fish feeding. Dead fish were discarded immediately from the glass aquarium to avoid water deterioration. The number of dead fish was estimated and their mortality percentages were calculated after 24, 48, 72 and 96 hrs. of exposure for each fungicide concentration respectively.

2.7 Statistical analysis.

The experiments were set up in three replicates for each treatment and the data were reported as mean ± SE (standard error).The data were also analyzed statistically with GraphPad Prism 5.0 (GraphPad Software,Inc., La Jolla, CA, USA) using One-way analysis of variance (ANOVA) and differences among the means were determined for significance at P ≤ 0.05.

3.1 Antifungal assay of fungicides

Seven fungicides conventionally used in agricultural field were screened for their antifungal activities at concentration of (0.25 mg/ml) against two saprolegnoid fungi (Table 1). Among these, four fungicides (azoxystrobin, cyazofamid, etridiazole and metalaxyl) were found to be effective against (S. diclina and S. parasitica) while the other three fungicides (fluopicolide, mefenoxam and propamocarb) showed a slight inhibitory effect at the same concentration. Both of azoxystrobin and metalaxyl were the most effective fungicides in suppressing mycelial growth of the saprolegnoid fungi followed by cyazofamid and etridiazole. Azoxystrobin and metalaxyl showed a highest antifungal activities as mycelial growth of the concerned fungi were absolutely inhibited at 0.25 mg/ml while cyazofamid and etridiazole showed a moderate activities in suppressing their mycelial growth at the same concentration. Cyazofamid reduced mycelial growth of S. diclina and S. parasitica to 44.58 and 34.01% while etridiazole retarded their mycelial growth to 39.24 and 27.21% respectively.

Hence, the chemo-physical properties of the four effective fungicides were illustrated in Table 2 and were also chosen for further antifungal investigations. The MIC and MFC of these effective fungicides were employed by food poisoning technique to assess their fungistatic and fungicidal properties in comparison with malachite green as a reference fungicide.

3.2 Fungicidal analysis of effective fungicides compared with reference fungicide (Malachite green)

Saprolegnia diclina was more sensitive to malachite green as its mycelial growth was inhibited to 55.77% at concentration of 0.5 ppm while mycelial growth of S. parasitica was inhibited to 50.29% at the same concentration respectively. The fungicidal effect of malachite green was obvious at concentration of 0.25 ppm and increased in proportion to malachite green concentrations reached to maximum at concentration of 1 ppm inhibiting completely mycelial growth of the two saprolegnoid fungi (Table 3). Malachite green was strongly effective against S. diclina and S. parasitica with MIC of 0.125 & 0.25 ppm respectively while minimal fungicidal concentrations (MFC) of both fungal strains was 1 ppm. The concentration effect of the effective fungicides on the mycelial growth of the saprolegnoid fungi is shown by plotting their concentrations against mycelial growth of the saprolegnoid fungi (Fig. 1). Growth inhibitions of saprolegnoid fungi were increased with fungicides concentrations reached to maximum at 200 ppm and 250 ppm for azoxystrobin and metalaxyl while a higher concentration was required for cyazofamid and etridiazole respectively to attain the same effect. Moreover, results reported in Table 4 revealed that azoxystrobin was strongly effective fungicide and showed fungicidal and fungistatic activities against S. diclina and S. parasitica reducing their mycelial growth to 70.88 and 55.64% with MIC of 150 ppm while their growth was completely inhibited with MFC of 200 ppm respectively. Moreover, metalaxyl also showed a strong antifungal activity against the saprolegnoid fungi inhibiting mycelial growth of S. diclina and S. parasitica to 83.08 and 60.23% with MIC of 200 ppm and completely suppressed their mycelial growth with MFC of 250 ppm respectively. In contrast, antifungal activities of cyazofamid and etridiazole were confirmed to be moderately effective against the saprolgnoid fungi with MIC of 250 ppm and higher concentrations were required to suppress completely their mycelial growth. All mycelial growth inhibition of the effective fungicides were reported to be significant for all treatments at the level of (P < 0.05).

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Fig. 1

Effect of different concentrations of fungicides on mycelial growth of Saprolegnia parasitica and S. diclina.

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3.3 Acute fish toxicity test of the effective fungicides:

On the other hand, the acute toxicity of the two effective fungicides in tilapia fish was performed by exposing fish in aquaria containing different concentrations of azoxystrobin and metalaxyl for 96 hrs. Results indicated the variable susceptibility of the exposed tilapia fish to the tested fungicides. All fish were died at 400 ppm of azoxystrobin after 48hrs of exposure while 60% of fish moralities were recorded at the same concentration for metalaxyl fungicide. Moreover, 20, 40, and 70% of accumulative mortalities were observed at concentrations of 100, 200 and 300 ppm of azoxystrobin exposure for 96hrs respectively while no fish mortality was recorded at 50 ppm. On the other hand, no fish mortalities was attained at 50 and 100 ppm and only 10 and 40% of mortalities were detected at 200 and 300 ppm of metalaxyl exposure for 96 hrs respectively. Median lethal concentration of acute toxicity assay for the effective fungicides were shown in Fig. 2. According to plotted graph of fungicides toxicity assay, the lethal concentration causing 50% of fish mortalities after 96 hrs of exposure (LC₅₀) was recorded at 225 ppm for azoxystrobin while metalaxyl was recorded at 360 ppm respectively.

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Fig. 2

Acute toxicity of the effective fungicides on tilapia fish during their exposure for 96 hrs.

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