Antifungal Effect of Various Salts Against Fusarium oxysporum f . sp . cepae , the Causal Agent of Fusarium Basal Rot of Onion

This study evaluated the efficacy of 26 different salts as possible alternatives to synthetic fungicides for the control of Fusarium oxysporum f.sp. cepae, the causal agent of Fusarium basal rot. Preliminary evaluation of salt tests was performed in vitro using 2% concentration (w/v). When compared to control, potassium acetate, potassium chloride, potassium nitrate, potassium phosphate dibasic, sodium chloride, sodium sulfate and trisodium phosphate significantly enhanced the mycelial growth of the fungus; diammonium phosphate had no significant effect; 15 other salts reduced mycelial growth to some extent (16.61-83.44%) (P ≤ 0.05); and ammonium bicarbonate, ammonium carbonate and sodium metabisulfite completely inhibited mycelial growth. No significant difference in the inhibitory effects of ammonium bicarbonate, ammonium carbonate and sodium metabisulfite was observed in vitro (P ≤ 0.05); however, the ED50, minimum inhibition concentration (MIC), and minimum fungicidal concentration (MFC) values indicated sodium metabisulfite to be more toxic to F. oxysporum f.sp. cepae than ammonium carbonate and ammonium bicarbonate. Soil tests showed that 0.4% sodium metabisulfite completely inhibited fungal growth, whereas only the highest concentrations of ammonium bicarbonate and ammonium carbonate tested (2%) were able to inhibit growth (P ≤ 0.05). The present study also showed that F. oxysporum f.sp. cepae capable of growth in both acidic and basic environments. While the fungus showed uninhibited growth at pH values between 6-9, growth decreased significantly at both higher and lower pH values (P ≤ 0.05) and was completely inhibited at pH 12.


Inroduction
Onion (Allium cepa L.) is one of the many economically important vegetable crops grown in Turkey.In 2010, the total area of onion cultivation in Turkey was 85.784 ha, production volume was 2.295.193tons, and the rate yield was 40.29 kg per hectare (FAO 2013).Fusarium basal rot caused by Fusarium oxysporum Schlechtend.: Fr. f.sp.cepae (H.N.Hans.)W.C. Snyder & H.N. Hans is one of the most devastating diseases of onion and causes important yield losses in all growing areas of the world (Brayford 1996).The pathogen delays seedling emergence and causes damping-off on onion seedlings as well as root and basal rot.Early symptoms of the disease include curving, wilting, yellowing and eventually dying-back of the leaves from the tips.In addition, diseased bulbs are discolored and the infected tissue appears brown and watery when the bulbs are cut open (Sumner 1995).
Various agronomic practices have been used to control Fusarium basal rot, including the use of resistant onion cultivars, long crop rotation, solarisation, treatments of seed with fungicides and soil fumigation (Özer & Köycü 1998;Cramer 2000).
The aim of the present study was to evaluate the efficacy of different salts in controlling F. oxysporum f.sp.cepae.In vitro testing was performed to determine the fungistatic effects of 26 salts, and those with the highest toxicity were evaluated in soil tests.Also, the effects of pH on mycelial growth of F. oxysporum f.sp.cepae were investigated in this study.

Fungal isolate
The F. oxysporum f.sp.cepae isolate used in this study was obtained from the culture collection of the Ankara University, Faculty of Agriculture, Department of Plant Protection.The isolate was maintained on potato dextrose agar (PDA; Merck, Darmstadt, Germany).PDA slants were stored at 4 °C and served as stock cultures for future use.

Effect of the salts on mycelial growth
The effect of salts on F. oxysporum f.sp.cepae mycelial growth was assayed according to Mecteau et al (2002) with a slight modification.Salts (2%, w/v) were added to autoclaved and cooled PDA medium at 50 o C, and the pH of each treatment was measured using a pH meter (Hanna HI 2211, Hanna Instruments, Germany).The medium was dispensed aseptically into 7-cm-diameter Petri plates.Plates with unamended PDA were used as control.Mycelial disks of 5 mm in dia.from 7-day-old fungal cultures were placed in the center of the plates with 10 mL of PDA.The plates were then sealed with Parafilm and incubated at 24±1 °C.Mycelial growth was measured daily at two perpendicular colony diameters until the growth in the control plates reached the edge of the Petri plates.Mycelial growth values were converted into the inhibition percentage of mycelial growth inhibition (MGI) in relation to controls using the formula,

Effect of the salts on mycelial growth
The effect of salts on F. oxysporum f.sp.cepae mycelial growth was assayed according to Mecteau et al (2002) with a slight modification.Salts (2%, w/v) were added to autoclaved and cooled PDA medium at 50 o C, and the pH of each treatment was measured using a pH meter (Hanna HI 2211, Hanna Instruments, Germany).The medium was dispensed aseptically into 7-cm-diameter Petri plates.Plates with unamended PDA were used as control.Mycelial disks of 5 mm in dia.from 7-day-old fungal cultures were placed in the center of the plates with 10 mL of PDA.The plates were then sealed with Parafilm and incubated at 24±1 °C.Mycelial growth was measured daily at two perpendicular colony diameters until the growth in the control plates reached the edge of the Petri plates.Mycelial growth values were converted into the inhibition percentage of mycelial growth inhibition (MGI) in relation to controls using the formula, , where dc and dt represent mycelial growth diameters in controls and amended Petri plates, respectively.Each treatment was replicated 3 times and the experiment was repeated twice.

Effect of pH on mycelial growth
Mycelial growth of F. oxysporum f.sp.cepae was examined on adjusted PDA at pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 using 1.0 N NaOH (Riedel-de Haen AG, Buchs SG, Switzerland) or HCI (Merck, Darmstadt, Germany).The Petri plates containing unmodified PDA were used as controls.Mycelial disks of 5 mm in dia.from 7-day-old fungal cultures were placed in 9-cm-dia.plates that were then sealed with Parafilm and incubated at 24±1 °C for 7 days.Mycelial growth was determined by measuring colony diameter.Each experiment was replicated 3 times and repeated twice.

ED 50 , MIC and MFC values of the salts
Probit analysis was used to calculate concentrations of salts causing 50% reduction (ED 50 ) in mycelial growth of F. oxysporum f.sp.cepae (StatPlus, AnalystSoft Inc.).Mycelial growth was determined in PDA amended with where dc and dt represent mycelial growth diameters in controls and amended Petri plates, respectively.Each treatment was replicated 3 times and the experiment was repeated twice.

Effect of pH on mycelial growth
Mycelial growth of F. oxysporum f.sp.cepae was examined on adjusted PDA at pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 using 1.0 N NaOH (Riedel-de Haen AG, Buchs SG, Switzerland) or HCI (Merck, Darmstadt, Germany).The Petri plates containing unmodified PDA were used as controls.Mycelial disks of 5 mm in dia.from 7-dayold fungal cultures were placed in 9-cm-dia.plates that were then sealed with Parafilm and incubated at 24±1 °C for 7 days.Mycelial growth was determined by measuring colony diameter.Each experiment was replicated 3 times and repeated twice.

ED 50 , MIC and MFC values of the salts
Probit analysis was used to calculate concentrations of salts causing 50% reduction (ED 50 ) in mycelial growth of F. oxysporum f.sp.cepae (StatPlus, AnalystSoft Inc.).Mycelial growth was determined in PDA amended with salts at concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0% (w/v) as described above.Probit analysis was performed to determine the minimum inhibition concentration (MIC) value that completely inhibited mycelial growth.
Salt toxicity (fungistatic/fungicidal) was determined according to Thompson (1989) and Tripathi et al (2004).The inhibited fungal discs with no growth were taken from the salts amended Petri plates, and then re-inoculated separately into the fresh medium, and revival of their growth was observed for 9 days at 24±1 °C.The concentration that inhibited the fungi completely and irreversibly after transference to fresh medium was recorded as the minimum fungicidal concentration (MFC) value.

Soil tests
Soil tests were performed using a cornmealsand medium to further evaluate those salts that had completely inhibited mycelial growth at 2% concentrations in vitro.The medium was prepared as described by Arslan et al (2009) using a 1:8 ratio of sand to corn.Medium (45 g) was placed in glass 7-cm-dia.Petri plates and sterilized in an oven (Ecocell LSIS-B2V/EC111, MMM Group, Germany) at 130 °C for 5 h.Mycelial disks (5-mm dia.) were taken from 7-day-old fungal cultures grown on PDA medium and placed in the center of cornmeal-sand medium at a depth of 0.5 cm.Salt concentrations (described above) were prepared using sterile distilled water, 10 mL of solution was added to each Petri plate, and plates were incubated in the dark at 24±1 °C for 7 days.Images of fungal mycelial growth were photocopied on transparent paper, with a 5 cm scale placed on the lid of each plates.Images were then digitally scanned using a Mustek 1200 UB Plus desktop scanner (Mustek, China) and saved as bmp 24 bit files.Surface areas were digitally measured by using Digimizer (Version 4.0, Belgium).Inhibition of mycelial growth was expressed as a percentage of mycelia growth of each sample in comparison to the control.
In addition, pH values of the cornmeal-sand medium were determined by randomly sampling 45 g of medium per treatment replicate.Measurements were performed by inserting the electrode of the pH meter into the sample after adding 20 mL of deionized water.

Statistical analysis
Results were separately subjected to one-way analysis of variance (ANOVA) using the IBM SPSS Statistics Program (Version 19, Property of SPSS, Inc., IBM Company, USA).Significant differences between means were determined using the Tukey-HSD test (P ≤ 0.05).

Results and Discussion
This study evaluated the inhibitory activity of 26 inorganic and organic salts against Fusarium oxysporum f.sp.cepae in vitro at 2% concentration.Ammonium bicarbonate, ammonium carbonate and sodium metabisulfite completely inhibited mycelial growth of the fungus whereas potassium sulfate and sodium carbonate strongly reduced mycelial growth.Sodium citrate dihydrate, sodium bicarbonate, ammonium molybdate, potassium carbonate inhibited mycelial growth to a lesser extent (43.65-68.10%)(Figure 1).
The differences in the inhibitory effects of the salts were in general statistically significant (P ≤ 0.05).Although sodium carbonate had a grater inhibitory effect than potassium sulfate, the difference between the two was not statistically significant (P ≤ 0.05).These results are in line with those reported by previous studies.Arslan et al (2009) reported that mycelial growth of F. oxysporum f.sp.melonis was totally inhibited by ammonium bicarbonate, potassium benzoate, potassium sorbate and sodium benzoate, but not by sodium carbonate, sodium bicarbonate, or sodium citrate dihydrate.Mills et al (2004) determined that mycelial growth and spore germination of Alternaria alternata, Botrytis cinerea, Fusarium solani var.coeruleum, Phytophthora erythroseptica, P. infestans, Verticillium albo-atrum, and V. dahliae were strongly limited by sodium metabisulfite and propyl-paraben.The present study revealed that 10 salts (ammonium acetate, ammonium chloride, ammonium nitrate, ammonium sulfate, calcium acetate, calcium chloride, diammonium phosphate, potassium bicarbonate, sodium acetate trihydrate and sodium phosphate dibasic) inhibited the mycelial growth of F. oxysporum f.sp.cepae to a certain extent (6.22 and 31.62%).Diammonium phosphate, had the lowest inhibitory effect, which did not differ significantly from the control (P ≤ 0.05).No statistically significant differences were observed among ammonium acetate, ammonium nitrate, calcium acetate, calcium chloride and potassium bicarbonate or between ammonium chloride and sodium phosphate dibasic (P ≤ 0.05), all of which had significantly higher inhibitory effects than diammonium phosphate and the control.These results are in line with those of previous studies.Abdel-Kader et al (2012) found that calcium chloride concentrations of 1% and 2% gradually reduced mycelial growth of F. solani and F. oxysporum by 22.2% and 33.3%, respectively, whereas no further reductions were observed at a higher concentration (4%).Bigss et al (1997) reported that calcium acetate and calcium chloride did not inhibit growth of Monilinia fructicola on PDA as strongly as 4 other calcium salts (calcium oxide, calcium propionate, calcium pyrophosphate, calcium cilicate).However, Reid et al (2001) reported that sodium chloride was more effective than other chloride salts (calcium chloride, ammonium chloride and manganese chloride) in controlling Fusarium crown and root rot caused by F. oxysporum f.sp.asparagi and F. proliferatum.This contrasts with the finding of the present study showing calcium and ammonium chloride to be more effective than sodium chloride in inhibiting mycelial growth of F. oxysporum f.sp.cepae.Moreover, the present study found that sodium chloride, sodium sulfate, trisodium phosphate, potassium acetate, potassium chloride, potassium nitrate and potassium phosphate dibasic did not reduced the mycelial growth of F. oxysporum f.sp.cepae, but in fact had a stimulatory effect on mycelial growth when compared to the control (P ≤ 0.05).Potassium phosphate dibasic had the greatest stimulatory effect, followed by potassium acetate, potassium chloride and trisodium phosphate.No differences were found in the effects of potassium nitrate, sodium chloride and sodium sulfate (P ≤ 0.05).This is in line with previous studies such as Elmer (2002), which found no reductions in the severity of Fusarium wilt caused by F. oxysporum f.sp.cyclaminis when 0.25 g and 0.50 gL -1 concentrations of sodium chloride were

cepae (See Table 1 for salt details, 27=control) Şekil 1-% 2 konsantrasyonda Fusarium oxysporum f.sp. cepae'nın miselyal gelişimi üzerine tuzların etkileri (Tuzlar için Çizelge 1'e bakınız, 27=kontrol)
The differences in the inhibitory effects of the salts were in general statistically significant (P ≤ 0.05).Although sodium carbonate had a grater inhibitory effect than potassium sulfate, the difference between the two was not statistically significant (P ≤ 0.05).These results are in line with those reported by previous studies.Arslan et al (2009) reported that mycelial growth of F. oxysporum f.sp.melonis was totally inhibited by ammonium bicarbonate, potassium benzoate, potassium sorbate and sodium benzoate, but not by sodium carbonate, sodium bicarbonate, or sodium citrate dihydrate.Mills et al (2004) determined that mycelial growth and spore germination of Alternaria alternata, Botrytis cinerea, Fusarium solani var.coeruleum, Phytophthora erythroseptica, P. infestans, Verticillium albo-atrum, and V. dahliae were strongly limited by sodium metabisulfite and propyl-paraben.The present study revealed that 10 salts (ammonium acetate, ammonium chloride, ammonium nitrate, ammonium sulfate, calcium acetate, calcium chloride, diammonium phosphate, potassium bicarbonate, sodium acetate trihydrate and sodium phosphate dibasic) inhibited the mycelial growth of F. oxysporum f.sp.cepae to a certain extent (6.22 and 31.62%).Diammonium phosphate, had the lowest inhibitory effect, which did not differ significantly from the control (P ≤ 0.05).No statistically significant differences were observed among ammonium acetate, ammonium nitrate, calcium acetate, calcium chloride and potassium bicarbonate or between ammonium chloride and sodium phosphate dibasic (P ≤ 0.05), all of which had significantly higher inhibitory effects than diammonium phosphate and the control.These results are in line with those of previous studies.Abdel-Kader et al (2012) found that calcium chloride concentrations of 1% and 2% gradually reduced mycelial growth of F. solani and F. oxysporum by 22.2% and 33.3%, respectively, whereas no further reductions were observed at a higher concentration (4%).Bigss et al (1997) reported that calcium acetate and calcium chloride did not inhibit growth of Monilinia fructicola on PDA as strongly as 4 other calcium salts (calcium oxide, calcium propionate, calcium pyrophosphate, calcium cilicate).However, Reid et al (2001) reported that sodium chloride was more effective than other chloride salts (calcium chloride, Figure 1-Effects of salts at 2% concentration on mycelial growth of Fusarium oxysporum f.sp.cepae (See Table 1 for salt details, 27=control) Şekil 1-% 2 konsantrasyonda Fusarium oxysporum f.sp.cepae'nın miselyal gelişimi üzerine tuzların etkileri (Tuzlar için Çizelge 1'e bakınız, 27=kontrol) applied to potting mix.Similarly, Palmer et al (1997) reported that out of 26 salts tested, sodium chloride, sodium phosphate monobasic, sodium sulfate and sodium thiosulfate had no effect on the mycelial growth of Botrytis cinerea.Moreover, sodium phosphate monobasic was found to provide additional nutrients for development of pathogen.In contrast to the findings of the present study, Mecteau et al (2002Mecteau et al ( , 2008) ) reported that trisodium phosphate completely inhibited mycelial growth of F. sambicinum and F. solani var.coereuleum at 0.2 M (7.6%) on PDA.This implies that the sensitivity of Fusarium spp. to trisodium phosphate may vary and that higher concentrations of the salt may be required to completely inhibit mycelial growth of F. oxysporum f.sp.cepae.
In terms of pH, the present study found pH values to range from 2.94 (potassium sulfate) to 10.75 (sodium carbonate) at 2% salt concentrations (Figure 1).Whereas 23 salts had pH values that differed significantly from the control (5.65), the pH values of 3 salts (calcium chloride, potassium chloride and sodium sulfate) did not differ significantly from the control (P ≤ 0.05).However, calcium chloride inhibited mycelial growth (28.18%),both potassium chloride and sodium sulfate stimulated mycelial growth (-9.19% and -7.19%, respectively).Ammonium bicarbonate, ammonium carbonate and sodium metabisulfite had pH values of 7.80, 7.76 and 4.83, respectively, and all 3 salts completely inhibited mycelial growth (Figure 1).Overall, this study showed that F. oxysporum f.sp.cepae was able to grow both in acidic and basic environments (Figure 2).While no negative effects were observed with pH values ranging from 6 to 9, mycelial growth was significantly inhibited at both higher and lower pH values (P ≤ 0.05) and halted completely at a pH of 12.Moreover, no significant differences were found between mycelial growth of the fungus at pH 5, 10 and the unmodified pH (5.65) (P ≤ 0.05).In other words, toxic effects of salts appear to be pHindependent.In line with this finding, a previous study also found that the pH values of organic and inorganic salts have a minor role in their toxic effects (Mecteau et al 2002).
Of the various salts examined in the present study, sodium metabisulfite had the most toxic effect on the mycelial growth of F. oxysporum ammonium chloride and manganese chloride) in controlling Fusarium crown and root rot caused by F. oxysporum f.sp.asparagi and F. proliferatum.This contrasts with the finding of the present study showing calcium and ammonium chloride to be more effective than sodium chloride in inhibiting mycelial growth of F. oxysporum f.sp.cepae.Moreover, the present study found that sodium chloride, sodium sulfate, trisodium phosphate, potassium acetate, potassium chloride, potassium nitrate and potassium phosphate dibasic did not reduced the mycelial growth of F. oxysporum f.sp.cepae, but in fact had a stimulatory effect on mycelial growth when compared to the control (P ≤ 0.05).Potassium phosphate dibasic had the greatest stimulatory effect, followed by potassium acetate, potassium chloride and trisodium phosphate.No differences were found in the effects of potassium nitrate, sodium chloride and sodium sulfate (P ≤ 0.05).This is in line with previous studies such as Elmer (2002), which found no reductions in the severity of Fusarium wilt caused by F. oxysporum f.sp.cyclaminis when 0.25 g and 0.50 gL -1 concentrations of sodium chloride were applied to potting mix.Similarly, Palmer et al (1997) reported that out of 26 salts tested, sodium chloride, sodium phosphate monobasic, sodium sulfate and sodium thiosulfate had no effect on the mycelial growth of Botrytis cinerea.Moreover, sodium phosphate monobasic was found to provide additional nutrients for development of pathogen.In contrast to the findings of the present study, Mecteau et al (2002Mecteau et al ( , 2008) ) reported that trisodium phosphate completely inhibited mycelial growth of F. sambicinum and F. solani var.coereuleum at 0.2 M (7.6%) on PDA.This implies that the sensitivity of Fusarium spp. to trisodium phosphate may vary and that higher concentrations of the salt may be required to completely inhibit mycelial growth of F. oxysporum f.sp.cepae.
In terms of pH, the present study found pH values to range from 2.94 (potassium sulfate) to 10.75 (sodium carbonate) at 2% salt concentrations (Figure 1).Whereas 23 salts had pH values that differed significantly from the control (5.65), the pH values of 3 salts (calcium chloride, potassium chloride and sodium sulfate) did not differ significantly from the control (P ≤ 0.05).However, calcium chloride inhibited mycelial growth (28.18%),both potassium chloride and sodium sulfate stimulated mycelial growth (-9.19% and -7.19%, respectively).Ammonium bicarbonate, ammonium carbonate and sodium metabisulfite had pH values of 7.80, 7.76 and 4.83, respectively, and all 3 salts completely inhibited mycelial growth (Figure 1).Overall, this study showed that F. oxysporum f.sp.cepae was able to grow both in acidic and basic environments (Figure 2).While no negative effects were observed with pH values ranging from 6 to 9, mycelial growth was significantly inhibited at both higher and lower pH values (P ≤ 0.05) and halted completely at a pH of 12.Moreover, no significant differences were found between mycelial growth of the fungus at pH 5, 10 and the unmodified pH (5.65) (P ≤ 0.05).In other words, toxic effects of salts appear to be pH-independent.In line with this finding, a previous study also found that the pH values of organic and inorganic salts have a minor role in their toxic effects (Mecteau et al 2002).MIC values were consistent with ED 50 values in all the salts tested, with the exception of ammonium bicarbonate.Moreover, ammonium bicarbonate had a lower ED 50 value than ammonium carbonate, whereas ammonium carbonate had lower MIC value (0.55%).However, sodium metabisulfite had the lowest MIC value with 0.17% (Table 2).These results are supported by previous studies.Mecteau et al (2002) found that aluminium acetate, aluminium lactate, aluminium chloride, sodium benzoate, sodium metabisulfite and potassium sorbate had a higher toxic effect, and sodium carbonate and sodium citrate dihydrate had a lower toxic effect on Fusarium sambucinum spores.Ammonium bicarbonate showed greater fungucidal activity against F. oxysporum f.sp.melonis than potassium carbonate, potassium bicarbonate, sodium bicarbonate, sodium carbonate and sodium citrate dihydrate (Arslan et al 2009).Similarly, Montville & Shih (1991) observed that ammonium bicarbonate at 1 and 2% completely inhibited monocultures of F. graminearium, Aspergillus ochraceus and Penicillium griseofulvum on cracked corn.The present study showed sodium metabisulfite to have a higher fungistatic effect than ammonium bicarbonate and ammonium carbonate (Table 2).Whereas fungal discs taken from medium amended with 0.2-2.0% of sodium metabisulfite did not grow in the fresh medium for 9 days, discs taken from medium amended with 0.6% ammonium carbonate grew in fresh medium after 4 days, discs taken from medium amended with ammonium bicarbonate did not grow for 9 days.In contrast, Arslan et al (2009) reported ammonium bicarbonate to have an MFC value of 1% for F. oxysporum f.sp.melonis and an ED 50 value of 0.35%, which was nearly twice as high as the value reported in the present study (ED 50 =0.19).These results indicate that F. oxysporum f.sp.cepae is more sensitive to ammonium bicarbonate than F. oxysporum f.sp.melonis.In soil tests, sodium metabisulfite had a higher toxic effect on F. oxysporum f.sp.cepae than ammonium bicarbonate and ammonium carbonate (Table 3).Sodium metabisulfite at a 0.4% concentration completely inhibited the fungus, whereas ammonium bicarbonate and ammonium

Table 2 -ED 50 , MIC, and MFC values (%, w/v) of the salts inhibiting the mycelial growth of Fusarium oxysporum f.sp. cepae
ND: Not determined