Inhibitory Influence of Organic and Inorganic Sodium Salts and ...

Gesunde Pflanzen (2015) 67:83–94 DOI 10.1007/s10343-015-0339-z

O r i g i n a l A rt i c l e

Inhibitory Influence of Organic and Inorganic Sodium Salts and Synthetic Fungicides Against Bean Root Rot Pathogens Muharrem Türkkan · İsmail Erper

Received: 11 December 2014 / Accepted: 11 March 2015 / Published online: 14 April 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract The efficacy of 20 organic and inorganic sodium salts, and two synthetic fungicides against eight bean root rot pathogens—Fusarium equiseti, F. proliferatum, F. semitectum, F. solani f. sp. phaseoli, F. verticillioides, Rhizoctonia solani AG4–HG I, Macrophomina phaseolina and Sclerotium rolfsii—were evaluated in this study. Accordingly to preliminary in vitro tests, only captan, benzoate and metabisulfite (2 %) were able to completely inhibit mycelial growth of all eight fungi. Moreover, no significant differences were observed among the inhibitory effect of these three compounds and EDTA (P ≤ 0.05). With few exceptions, the ED50 values indicated captan to have a greater effect against fungi than benzoate, EDTA and metabisulfite. However, captan, benzoate and EDTA all had MIC values that varied greatly from that of metabisulfite. Whereas captan, benzoate and EDTA showed fungitoxic activity against all fungi tested at concentrations greater than 0.1 %, metabisulfite showed fungitoxic activity against all fungi tested at concentrations of 0.025–0.25 %. Soil bioassays showed 0.25 % metabisulfite to completely inhibit mycelial growth of F. proliferatum, F. semitectum, R. solani AG–4 HG I, M. phaseolina and S. rolfsii, but not F. equiseti, F. solani f. sp. phaseoli and F. verticillioides. Higher concentrations of captan and benzoate were required to achieve total inhibition in soil bioassays when M. Türkkan () Faculty of Agriculture, Department of Plant Protection, Ordu University, 52200 Ordu, Turkey e-mail: [email protected] İ. Erper Faculty of Agriculture, Department of Plant Protection, Ondokuz Mayıs University, 55270 Samsun, Turkey

compared to metabisulfite, whereas EDTA was not able to completely inhibit growth of any of the fungi tested, even at the highest concentration. Moreover, the application of 1.0–2.0 % EDTA was found to be phytotoxic to bean seeds in terms of both seed germination and root elongation, whereas 0.1–0.75 % captan, 0.1–0.75 % benzoate and 0.1 % metabisulfite did not exhibit any phytotoxicity in terms of germination; 0.5 % captan, 0.1 % benzoate and 0.1 % metabisulfite did, however, have a negative effect on root elongation. The results of pH studies also demonstrated all eight fungi tested to be capable of growth in both acidic and basic environments, although the growth of some species was inhibited at the lowest value tested (pH 2), and the growth of all species was totally inhibited at the highest value tested (pH 12). Keywords Bean · Root rot pathogens · Alternative control · Toxicity · pH Inhibitorische Wirkung organischer und anorganischer Natriumsalze und synthetischer Fungizide gegen Wurzelfäuleerreger bei Bohnen Zusammenfassung Die Wirksamkeit von 20 organischen und anorganischen Natriumsalzen und zwei synthetischen Fungiziden gegen acht Wurzelfäuleerreger bei Bohnen – Fusarium equiseti, F. proliferatum, F. semitectum, F. solani f. sp. phaseoli, F. verticillioides, Rhizoctonia solani AG4–HG I, Macrophomina phaseolina und Sclerotium rolfsii – wurde in dieser Studie beurteilt. Vorläufigen Invitro-Tests zufolge konnten nur Captan, Benzoat und Metabisulfit (2 %) das Myzelwachstum aller acht Pilze vollständig hemmen. Außerdem wurden keine signifikan-

13

84

ten Unterschiede zwischen der hemmenden Wirkung dieser drei Verbindungen und von EDTA festgestellt (P ≤ 0,05). Mit wenigen Ausnahmen deuteten die ED50-Werte darauf hin, dass Captan eine stärkere Wirkung gegen Pilze hat als Benzoat, EDTA und Metabisulfit. Captan, Benzoat und EDTA wichen aber stark in ihren MHK-Werten von Metabisulfit ab. Während Captan, Benzoat und EDTA bei Konzentrationen über 0,1 % eine fungitoxische Wirkung gegen alle getesteten Pilze zeigten, zeigte Metabisulfit bei Konzentrationen zwischen 0,025 und 0,25 % eine fungitoxische Wirkung gegen alle getesteten Pilze. Boden-Bioassays ergaben, dass 0,25 % Metabisulfit das Myzelwachstum von F. proliferatum, F. semitectum, R. solani AG–4 HG I, M. phaseolina und S. rolfsii vollständig hemmt, nicht aber das von F. equiseti, F. solani f. sp. phaseoli und F. verticillioides. Höhere Konzentrationen als bei Metabisulfit waren von Captan und Benzoat erforderlich, um in Boden-Bioassays eine vollständige Hemmung zu erreichen, wohingegen EDTA das Wachstum keines der getesteten Pilze vollständig hemmen konnte, auch nicht in der höchsten Konzentration. Außerdem wurde der Einsatz von 0,1–2,0 % EDTA als phytotoxische für Bohnensaat erkannt, was die Saatkeimung und das Längenwachstum der Wurzeln betrifft, wohingegen 0,1 − 0,75 % Captan, 0,1 − 0,75 % Benzoat und 0,1 % Metabisulfit keine phytotoxische Wirkung auf die Keimung aufwiesen. 0,5 % Captan, 0,1 % Benzoat und 0,1 % Metabisulfit hatten allerdings negative Auswirkungen auf das Längenwachstum der Wurzeln. Die Ergebnisse von pH-Studien zeigten auch, dass alle acht getesteten Pilze sowohl in saurem als auch in basischem Milieu wachsen können, obwohl das Wachstum mancher Spezies beim geringsten getesteten pH-Wert (2) und das Wachstum aller Spezies beim höchsten getesteten pH-Wert (12) gehemmt wurde. Schlüsselwörter Bohne · Wurzelfäuleerreger · Alternative Bekämpfung · Toxizität · pH-Wert Introduction A number of soil-borne pathogenic fungi are widespread throughout common bean (Phaseolus vulgaris L.) production areas (Hall 1991). Fusarium solani f. sp. phaseoli, Macrophomina phaseolina, Rhizoctonia solani and Sclerotium rolfsii are among the most well-known pathogenic fungi associated with root and stem degradation and are reportedly responsible for severe growth reduction and yield losses in Africa, Asia, Europe, Latin America and the United States of America (Hagedorn and Inglis 1986; Abawi and Pastor Corrales 1990; Buruchara and Camacho 2000; Montiel Gonzalez et al. 2005; Andres Ares et al. 2006; Mwang’ombe et al. 2007). The pathogens Fusarium azukicola, Fusarium

13

M. Türkkan, İ. Erper

spp. (F. culmorum, F. crookwellense, F. equiseti, F. lateritium, F. oxysporum, F. proliferatum, F. reticulatum, F. sporotrichoides and F. verticillioides), and Pythium ultimum and Sclerotinia sclerotiorum have also been recovered from bean plants in Japan, Mexica and Spain (Aoki et al. 2012; Montiel Gonzalez et al. 2005; Andres Ares et al. 2006). In Samsun, Turkey, a province where beans are intensively cultivated, Hatat and Özkoç (1997) found Fusarium spp., R. solani and Pythium spp. to be common, and Beldek (2013) reported the most prevalent pathogen isolated from among various Fusarium root-rot agents found on beans in Samsun to be Fusarium solani f. sp. phaseoli, followed by F. semitectum, F. verticillioides, F. equiseti and F. proliferatum. Root-rot disease on beans can be effectively controlled through a variety of agronomic practices including the use of resistant cultivars, crop rotation, solarisation, fungicidal seed treatment and soil fumigation (Abawi and Pastor Corrales 1990). While physical and biological treatments play a very important role in disease control, the application of fungicides, alone or in combination with other control methods, has been recommended at various stages of plant growth (Elad et al. 1980). For example, seed treatments with fungicides such as benomyl, captan, captafol, carboxin, metalaxyl, thiram, thiophanate-methyl, imazalil, tolclofosmethyl and propamocarb hydrochloride have been reported to reduce severity of the disease (Abawi and Pastor Corrales 1990). Whereas soil fumigation with fumigants such as methyl bromide, chloropicrin and vorlex has been shown to be the most effective method for controlling soil-borne fungi (Yuen et al. 1991; Abawi et al. 2006), the negative effects of these chemicals on both the environmental and public health have led to their banning in many countries (Fan et al. 2008), prompting a search for alternative methods of disease control. Natural compounds such as organic and inorganic salts may offer the greatest potential alternative to commercial fungicides in the control of bean root rot. Ammonium, sodium and potassium carbonate and bicarbonate were showed to be effective in inhibiting damage to fruits, field crops, vegetables and ornamentals caused by fungal pathogens (Depasquale and Montville 1990; Ziv and Zitter 1992; Punja and Gaye 1993; Palmer et al. 1997; Campanella et al. 2002; Arslan et al. 2006; Latifa et al. 2011). Sodium metabisulfite was demonstrated effectiveness in reducing potato silver scurf caused by Helminthosporium solani (Olivier et al. 1999; Hervieux et al. 2002), potato dry rot caused by Fusarium sambucinum (Mecteau et al. 2002) and F. solani var. coeruleum (Mecteau et al. 2008), and onion basal rot caused by Fusarium oxysporum f. sp. cepae (Türkkan and Erper 2014). The applications of potassium sorbate, sodium benzoate and sodium EDTA were reported to effectively control damage to oranges caused by Penicillium digitatum and P. italicum (Palou et al. 2002; ValenciaChamorro et al. 2008). Importantly, organic and inorganic

Inhibitory Influence of Organic and Inorganic Sodium Salts and Synthetic Fungicides

salts are generally recognized as safe (GRAS) by the United States Food and Drug Administration (USFDA) (Anonymous 2013), and their low level of toxicity to mammals has led to their widespread use by the food industry as preservatives, pH regulators and antimicrobial agents (Olivier et al. 1998). The aim of the present study was to evaluate the efficacy of 20 organic and inorganic sodium salts and two fungicides for the control of Fusarium spp. (F. equiseti, F. proliferatum, F. semitectum, F. solani f. sp. phaseoli, F. verticillioides), Macrophomina phaseolina, Rhizoctonia solani AG–4 HG I and Sclerotium rolfsii reported the causal agents of bean root rot in the provinces of Samsun and Ordu at the Black Sea Region of Turkey. The compounds demonstrating the highest toxicity in vitro were then evaluated in soil and seed germination/root elongation bioassays. The effects of pH on the mycelial growth of the fungi were also evaluated. Materials and Methods

85

isolate was obtained from the Biology Department at the Ondokuz Mayis University, Faculty of Arts and Sciences in Samsun, Turkey. Isolates were maintained on potato dextrose agar (PDA; BD Difco, Sparks, USA) slants stored at 4 °C for future as stock cultures. Compounds and Seeds The 22 compounds used in this study are listed in Table 1, along with their chemical composition and molecular weight. Sodium salts were purchased from Merck Chemicals (Darmstadt, Germany) and Sigma-Aldrich (Seelze, Germany), and fungicides were purchased from Turkish agrochemical manufacture Hektaş (Samsun, Turkey). With the exception of sodium molibdate, sodium nitrate and sodium sulfate, all the salts tested have been classified as food additives or GRAS compounds by the USFDA. Phaseolus vulgaris L. cv. Barbi seeds were purchased from Agrostar Tohumculuk (Antalya, Turkey).

Fungal Isolates

Antifungal Effects of Sodium Salts and Fungicides on Mycelial Growth of Fungi

The study was conducted using eight isolates of four fungal species. Fusarium spp. (F. equiseti, F. proliferatum, F. semitectum, F. solani f. sp. phaseoli and F. verticillioides), Macrophomina phaseolina and Sclerotium rolfsii were isolated from bean plants in Samsun and Ordu exhibiting symptoms of root rot, whereas the Rhizoctonia solani AG–4 HG I

A modification of Mecteau et al. (2002) was used to assay the effects of sodium salts and fungicides on fungal mycelial growth. Compounds (2 %, w/v) were added to autoclaved PDA medium cooled to 50 °C, and the pH of each treatment was measured using a pH meter (Hanna HI 2211, Hanna Instruments, Germany). For each compound,

Table 1 Compounds used in the study Compounds Sodium acetate Sodium benzoate Sodium bicarbonate Sodium carbonate Sodium chloride Sodium citrate dihydrate Sodium EDTA Sodium formate Sodium metabisulfite Sodium molibdate Sodium nitrate Sodium phosphate dibasic Sodium phosphate monobasic Sodium phosphate tribasic Sodium propionate Sodium succinate Sodium sulfate Sodium sulfite Sodium tartrate Sodium thiosulfate Captan Tolclophos methyl

Molecular weight (g/mol) 136.08 144.10 84.01 105.99 58.44 294.10 372.24 68.01 190.11 241.95 84.99 268.06 156.01 163.94 96.60 162.05 142.04 126.04 230.08 248.19 300.59 301.13

Chemical formule C2H3NaO2.3H2O C7H5NaO2 NaHCO3 Na2CO3 NaCl Na3C6H5O7.2H2O C10H14N2O8Na2.2H2O CHNaO2 Na2S2O5 Na2MoO4.2H2O NaNO3 Na2HPO4.7H2O NaH2PO4.2H2O Na3PO4 C3H5NaO2 C4H4Na2O4 Na2SO4 Na2SO3 C4H4Na2O6.2H2O Na2S2O3.5H2O C9H8Cl3NO2S C9H11Cl2O3PS

Company Merck (Darmstadt, Germany) Sigma-Aldrich (Seelze, Germany) Sigma-Aldrich (Seelze, Germany) Sigma-Aldrich (Seelze, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Sigma-Aldrich (Seelze, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Sigma-Aldrich (Seelze, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) Hektaş (Kocaeli, Turkey) Hektaş (Kocaeli, Turkey)

13

86

a 15 mL aliquot of ameliorated PDA medium was aseptically dispensed into a glass petri dish (8-cm-dia.), with an unamended PDA dish used as a control. A mycelial disk (5-mm-dia). cut from 7-day-old fungal cultures was placed in the center of each dish, and the dishes were sealed with Parafilm and incubated in the dark at 25 °C for 2–5 days. When the control fungal colonies had grown to the point of nearly covering the Petri dishes (5 days after inoculation with Fusarium spp., 3 days after inoculation with M. phaseolina and S. rolfsii, and 2 days after inoculation with R. solani), all colony diameters were measured at two perpendicular points. Mycelial growth values were recorded and converted into the inhibition percentages of mycelial growth inhibition (MGI) in relation to the controls using the formula MGI (%) = [(dc − dt)/dc]× 100, where dc represents mycelial growth diameter of the control and dt represents mycelial growth diameter of the amended Petri dish. Each of the experiments were repeated twice, with three replicates per treatment. The procedures described above were repeated using varying concentrations of each compound (0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 %) (w/v) in order to determine the concentrations required of each compound to achieve a 50 % reduction (ED50) in fungal mycelial growth and to completely inhibit fungal mycelial growth (i.e. MIC, the minimum inhibition concentration). ED50 values were determined by Probit analysis (IBM SPSS Statistic Program). Effects of pH on Mycelial Growth of Fungi The effect of pH on fungal mycelial growth was examined by using 1.0 N NaOH (Sigma-Aldrich, Seelze, Germany) or HCI (Merck, Darmstadt, Germany) to adjust PDA to pHs of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0, with an modified PDA Petri dish used as a control. A mycelial disk (5-mm-dia.) cut from 7-day-old fungal cultures was placed in the center of each dish (8-cm-dia.), and the dishes were sealed with Parafilm and incubated at 25 °C for 2–5 days. Fungal mycelial growth was determined by measuring colony diameter along two perpendicular points. The experiment was repeated twice with three replicates. Soil and Seed Germination/Root Elongation Bioassays Soil and seed germination/root elongation bioassays were conducted to further examine those compounds shown to strongly inhibit mycelial growth of all fungi in vitro at concentrations of 2 %, namely: captan, sodium benzoate, sodium EDTA and sodium metabisulfite. For each treatment, 45 g of 1:8 cornmeal-sand medium prepared according to Arslan et al. (2009) was placed in a glass Petri dish (8-cm-dia.) and sterilized by heating for 5 h in an oven (Ecocell LSIS-B2V/

13


EC111, MMM Group, Germany) at 130 °C. A mycelial disk (5-mm-dia.) cut from 7-day-old fungal cultures grown on PDA medium was placed in the center of the cornmeal-sand medium at a depth of 0.5 cm. Using sterile distilled water, compounds were prepared at concentrations of 0.1, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 % and 10 ml of solution was added to each Petri dish and incubated in the dark at 25 °C. After 5–7 days, a 5-cm scale was placed on the lid of each dish, which was photocopied using transparent paper to record images of fungal mycelial growth. The photocopies were digitally scanned (1200 UB Plus desktop scanner Mustek, Taiwan, Republic of China) and saved as 24-bit bmp files. Surface areas were digitally measured using the publicdomain software Digimizer (Version 4.0.0.0 for Windows 2005–2011 MedCalc Software bvba Broekstraat 52, 9030 Mariakerke, Belgium). Inhibition of mycelial growth was expressed as a percentage of mycelial growth of each sample in comparison to the control. For each treatment replicate, one dish was randomly selected for pH measurements, which was performed by adding 20 ml of deionized water to the 45 g cornmeal-sand medium and inserting the pH-meter electrode into the diluted sample. Seed germination/root elongation bioassays were performed according to Di Salvatore et al. (2008), with slight modification. Four layers of Whatman No. 1 filter paper were placed at the bottom of a glass Petri dish (10-cm-dia.) containing 10 ml of compound at varying concentrations [captan (0.1, 0.25, 0.5 and 0.75 %), sodium benzoate (0.1, 0.25, 0.5 and 0.75 %), sodium EDTA (1.0, 1.5 and 2.0 %) and sodium metabisulfite (0.1 and 0.25 %)], or 10 ml of sterile distilled water (control). Ten undamaged bean seeds nearly identical in size were placed in each dish, and the dishes were incubated in the dark at 25 °C. At the end of a 5-days incubation period, the germination rate was calculated as the ratio between the number of germinated seeds and the total number of seeds tested, and root elongation was defined as the length from the tip of the root to the radicle. The pH of each compound concentration used in the study was also determined. Statistical Analysis All statistical analysis was performed using the software package SPSS (Version 19, IBM, USA). Data were separately subjected to one-way analysis of variance (ANOVA), and the Tukey–HSD test was used to identify significant differences between means (P ≤ 0.05). Results The present study evaluated 20 organic and inorganic sodium salts and two fungicides for inhibitory activity against Fusarium equiseti, F. proliferatum, F. semitectum, F.


solani f. sp. phaseoli, F. verticillioides, Rhizoctonia solani, Macrophomina phaseolina and Sclerotium rolfsii at 2 % in vitro. Sodium benzoate, sodium metabisulfite and captan completely inhibited fungal mycelial growth, and sodium EDTA inhibited mycelial growth at rates between 91.53– 100 %; differences in the effects of these four compounds at 2 % were not statistically significant (P ≤ 0.05) (Table 2). Sodium carbonate and sodium phosphate tribasic inhibited mycelial growth of Fusarium spp. at rates 78.24–90.82 %, which were significantly lower than those of the abovementioned compounds (P ≤ 0.05), but significantly higher than those of sodium bicarbonate, sodium citrate dihydrate, sodium propionate and tolclophos methyl, which ranged from 20.30 to 78.63 %. Sodium bicarbonate, sodium propionate and tolclophos methyl had similar effects on the mycelial growth of F. proliferatum (59.08–63.70 %), F. semitectum (50.47–58.28 %) and F. solani f. sp. phaseoli (49.09–53.79 %) (P ≤ 0.05). The inhibitory effects of sodium carbonate and sodium phosphate tribasic against R. solani and M. phaseolina were also similar, as were the effects of sodium bicarbonate and tolclophos methyl (P ≤ 0.05), all of which were significantly higher than those of sodium citrate

87

dihydrate and sodium propionate. However, the differences between the inhibitory effects of these six compounds on the mycelial growth of S. rolfsii (90.36–100 %) were not statistically significant (P ≤ 0.05). Sodium molibdate exhibited great variation in terms of mycelial growth inhibition, showing the greatest effect against S. rolfsii (97.14 %), followed by M. phaseolina (81.23 %), F. solani f. sp. phaseoli (62.41 %), R. solani (22.66 %) and F. equiseti (15.52 %), and no inhibitory effect against F. proliferatum, F. semitectum and F. verticillioides. In general, the remaining compounds tested exhibited similar effects, with significant inhibitory activity against M. phaseolina, R. solani and S. rolfsii, but little or no inhibitory effect against any of the Fusarium species. Sodium acetate, sodium formate, sodium phosphate dibasic, sodium succinate, sodium sulfite and sodium thiosulfate had inhibitory effects ranging from 85.77 to 100.00 % against S. rolfsii. These were statistically higher than the effects of sodium chloride, sodium nitrate, sodium phosphate monobasic, sodium sulfate and sodium tartrate, which ranged from 7.86 to 27.16 %. Furthermore, sodium phosphate monobasic stimulated mycelial growth of all fungi except R. solani and S. rolfsii (P ≤ 0.05).

Table 2 Compound pH values and effects of 2 % compounds on the mycelial growth of bean root rot agents Compounds pH Inhibition of mycelial growth (%) F. equiseti F. F. F. solani F. M. R. solani proliferatum semitectum f. sp. phaseoli verticillioides phaseolina Sodium acetate 6.78 10.58 ghia −7.37 efghij −4.61 d 2.22 hij 25.23 f 53.67 g 34.28 f Sodium benzoate 6.26 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a Sodium bicarbonate 8.27 37.43 f 59.08 c 50.47 c 53.79 d 71.52 e 99.40 a 95.25 ab Sodium carbonate 10.74 78.24 bc 87.81 b 81.61 b 90.09 b 87.58 bc 100.00 a 100.00 a Sodium chloride 5.39 −10.90 jkl −10.63 fghij −25.80 ef 11.18 fg −6.95 ghi 16.12 i 41.49 e Sodium citrate 7.10 48.05 ef 55.46 c 60.32 c 35.56 e 20.30 f 92.41 b 79.99 c dihydrate Sodium EDTA 4.67 92.51 ab 100.00 a 100.00 a 100.00 a 91.53 ab 97.99 a 100.00 a Sodium formate 6.05 −0.53 hij −6.27 efghij −25.68 ef 14.22 fg −8.43 ghi 32.61 h 65.36 d Sodium metabisulfite 4.94 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a Sodium molibdate 7.23 15.52 g −4.16 efgh −2.56 d 62.41 c −7.02 ghi 81.23 cd 22.66 g Sodium nitrate 5.47 −15.31 kl −4.84 efghi −3.77 d 10.12 fgh −1.37 g 7.11 j 38.17 ef 8.03 12.17 gh −15.80 j −0.52 d 1.85 hij −11.92 i 83.66 c 13.60 i Sodium phosphate dibasic Sodium phosphate 4.90 −3.89 ijk −14.38 hij −25.98 f -3.23 j −11.11 hi −27.58 l 13.00 i monobasic Sodium phosphate 8.37 83.12 bc 85.29 b 83.58 b 90.82 b 84.37 bcd 100.00 a 100.00 a tribasic Sodium propionate 6.87 60.48 de 63.70 c 58.28 c 49.09 d 78.63 cde 91.98 b 93.42 b Sodium succinate 7.13 −1.63 hijk −12.33 ghij −14.36 def 17.77 f −13.59 i 73.29 ef 24.68 g Sodium sulfate 5.62 −22.43 l −2.15 efg −13.22 def 8.49 ghi 1.05 g 76.31 de 15.55 hi Sodium sulfite 8.47 −8.98 jkl −15.02 ij −4.75 d 15.71 fg −2.05 gh 82.15 c 80.83 c Sodium tartrate 6.02 −0.41 hij −1.07 ef −7.40 d 17.99 f −4.52 ghi 75.20 ef 12.67 i Sodium thiosulfate 5.49 17.60 g 25.07 d −11.09 de 7.90 ghi −0.85 g 69.96 f 21.04 gh Captan 5.12 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a Tolclophos methyl 6.05 73.89 cd 59.62 c 57.15 c 53.20 d 76.02 de 100.00 a 100.00 a Control 5.71 0.00 hij 0.00 e 0.00 d 0.00 ij 0.00 g 0.00 k 0.00 j a Means in each column followed by the same letter are not significant different according to the Tukey HSD (P ≤ 0.05)

S. rolfsii 93.54 100.00 100.00 100.00 19.62 90.36

a a a a bc a

98.81 100.00 100.00 97.14 21.60 87.29

a a a a bc a

7.86 cd 100.00 a 100.00 85.77 14.45 100.00 27.16 93.14 100.00 100.00 0.00

a a bcd a b a a a d

13

88

ED50 and MIC values of the compounds varied depending on the fungus species tested (Table 3). For example, tolclophos methyl had an ED50 value towards M. phaseolina, R. solani and S. rolfsii that was lower than the ED50 values of captan, sodium metabisulfite, sodium EDTA and sodium benzoate, even though these four compounds exhibited the greatest toxicity of all the compounds tested against all the fungi tested. Moreover, the MIC values of captan, sodium benzoate and sodium EDTA, which showed fungitoxic activity against all fungi tested at concentrations of 0.1 % or higher, were much higher than the MIC value of sodium metabisulfite, which showed fungitoxic activity against all fungi tested at concentrations of 0.025–0.25 %. ED50 values were not calculated for compounds that exhibited a small negative effect on mycelial growth of fungi or that stimulated mycelial growth of fungi. For the 22 compounds tested at 2 %, pH values ranged from 4.67 to 10.74 (Table 2). With the exception of S. rolfsii, mycelial growth of all fungi was reduced significantly at a low pH of 2–4 in comparison to controls (P ≤ 0.05) (Fig. 1). Unlike the other fungi tested, the mycelial growth of M. phaseolina was negatively affected in the pH range of 6–8 (P ≤ 0.05). While all five Fusarium species tended to show greater resistance than M. phaseolina, R. solani and S. rolfsii at high pH values, at the highest pH value (12), mycelial growth of all fungi was completely inhibited. Soil bioassays showed sodium metabisulfite, sodium benzoate and captan to have a much stronger inhibitory effect on fungal mycelial growth when compared to sodium EDTA, and in most cases, the difference was statistically significant (P ≤ 0.05) (Table 4). Sodium metabisulfite was able to completely eliminate mycelial growth of M. phaseolina, R. solani and S. rolfsii, even at a concentration as low as 0.1 %. Among the Fusarium species, sodium metabisulfite at 0.25 % was able to completely inhibit mycelial growth of F. proliferatum and F. semitectum, but not F. equiseti, F. solani f. sp. phaseoli and F. verticillioides. With the exception of F. verticillioides, however, the differences in the inhibitory effects of sodium metabisulfite at concentrations ranging between 0.25–2.0 % were not statistically significant among fungi (P ≤ 0.05). Sodium benzoate and captan were able to inhibit all fungi at the higher concentrations (1.0 and 1.0 %, respectively), whereas sodium EDTA was unable to completely inhibit mycelial growth, even at the highest concentration (2 %). Inhibition rates of sodium EDTA (in comparison to controls) were 78.86, 33.14, 37.82, 69.60, 51.55, 41.85, 74.13 and 81.51 % for F. equiseti, F. proliferatum, F. semitectum, F. solani f. sp. phaseoli, F. verticillioides, M. phaseolina, R. solani and S. rolfsii, respectively. In terms of soil pH, at the concentrations tested in the study, sodium EDTA, sodium metabisulfite and captan reduced soil pH, whereas sodium benzoate increased soil pH.

13


In terms of seed germination/root elongation, captan and sodium benzoate did not have a significant inhibitory effect on seed germination in comparison to controls at any of the concentrations tested (P ≤ 0.05), whereas 0.25 % sodium metabisulfite inhibited seed germination at a rate of 26.67 %, and sodium EDTA inhibited seed germination at rates between 40 and 100 %, depending on concentration (Table 5). Moreover, sodium EDTA at all concentrations caused in necrosis of root tips of germinated seeds. With the exception of captan at 0.1–0.25 %, captan, sodium benzoate and sodium metabisulfite at all concentrations also had a negative affect on root elongation in comparison to controls (P ≤ 0.05). In addition, the pH values of the seed germination/root elongation bioassays were similar to those of the soil bioassays. Discussion The effectiveness of organic and inorganic sodium compounds in controlling various plant diseases has been examined in a number of studies (Hervieux et al. 2002; Mecteau et al. 2002; Mills et al. 2004; Latifa et al. 2011; Türkkan and Erper 2014). Gould and Russell (1991) attributed the ability of sulfiting agents to inhibit the growth of bacteria, yeast and molds to sulfurous acid (SO2.H2O) and bisulfite ion (HSO3−) and to carbonyl group reactions that inhibit the formation of FAD + , RNA and DNA. Furthermore, a more recent study by Russell (2005) demonstrated that bisulfite ions are able to survive under acidic conditions. Sodium metabisulfite has been shown to inhibit mycelial growth and conidiation of the potato pathogens F. sambucinum (Mecteau et al. 2002), F. solani var. coereuleum (Mecteau et al. 2008) and H. solani (Hervieux et al. 2002) and a wide range of postharvest potato pathogens (Mills et al. 2004) in vitro at a concentration of 0.2 M (3.8 %). In addition, Türkkan and Erper (2014) reported sodium metabisulfite at 2 % to effectively inhibit mycelial growth of the onion pathogen Fusarium oxysporum f. sp. cepae; ED50 and MIC values were reported to be 2 Sodium benzoate 0.180 Sodium bicarbonate >2 Sodium carbonate 1.209 Sodium chloride ND Sodium citrate dihydrate >2 Sodium EDTA 0.080 Sodium formate ND Sodium metabisulfite 0.066 Sodium molibdate >2 Sodium nitrate ND Sodium phosphate dibasic >2 Sodium phosphate monobasic ND Sodium phosphate tribasic 0.417 Sodium propionate 1.073 Sodium succinate >2 Sodium sulfate ND Sodium sulfite >2 Sodium tartrate >2 Sodium thiosulfate >2 Captan 0.015 Tolclophos methyl 0.214 a The concentration that caused 50% reduction

>2 0.5 >2 >2 >2 >2 >2 >2 0.25 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 1 >2

NDc 0.095 >2 0.947 ND >2 0.165 ND 0.046 ND ND ND ND 0.760 0.852 ND ND ND ND >2 0.032 >2 >2 0.5 >2 >2 >2 >2 1.5 >2 0.25 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 1 >2

>2 0.221 1.617 0.815 ND >2 0.090 ND 0.083 >2 ND >2 ND 0.669 1.189 ND ND >2 ND ND 0.013 >2

>2 1 >2 >2 >2 >2 2 >2 0.25 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 0.75 >2

ND 0.155 1.915 0.379 >2 >2 0.089 >2 0.066 1.453 >2 >2 ND 0.721 >2 >2 >2 >2 >2 ND 0.020 >2

>2 1 >2 >2 >2 >2 1.5 >2 0.25 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 1 >2

Table 3 ED50 and MIC values (%, w/v) of compounds inhibiting mycelial growth of bean root rot agents Compounds F. equiseti F. proliferatum F. semitectum F. solanif. sp. phaseoli ED50a MICb ED50 MIC ED50 MIC ED50 MIC >2 0.111 0.201 0.098 >2 0.248 0.084 >2 0.022 0.982 >2 0.631 ND 0.129 0.218 0.604 1.127 0.294 0.923 1.040 0.004 2 0.75 >2 1.5 >2 >2 >2 >2 0.1 >2 >2 >2 >2 1 >2 >2 >2 >2 >2 >2 0.5 0.75

MIC >2 0.189 0.228 0.108 >2 1.192 0.056 0.709 0.024 >2 >2 >2 >2 0.295 0.081 >2 >2 0.451 >2 >2 0.005 2 0.5 >2 >2 >2 >2 >2 >2 0.1 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 >2 0.75 >2

MIC

ED50 >2 0.113 1.398 0.733 >2 >2 0.150 ND 0.048 >2 ND >2 ND 0.973 0.279 ND >2 >2 >2 ND 0.023 0.624

R. solani

F. verticillioides M. phaseolina

>2 1 >2 1 >2 >2 1.5 >2 0.1 >2 >2 >2 >2 1 >2 >2 >2 >2 >2 >2 0.75 0.25

MIC 0.096 0.027 0.078 0.055 >2 0.894 0.322 0.090 0.005 0.499 ND 1.209 >2 0.652 0.014 0.760 >2 0.010 >2 0.466 0.009 2 0.1 0.25 0.1 >2 >2 >2 1 0.025 >2 >2 >2 >2 2 0.250 >2 >2 0.5 >2 >2 0.5 0.1

MIC

Inhibitory Influence of Organic and Inorganic Sodium Salts and Synthetic Fungicides 89

13

90


Fig. 1 Effects of pH on the mycelial growth of bean root rot agents (bars represent standard errors of the means)

The present study found that few organic compounds strongly inhibited mycelial growth of all eight fungi at 2 %. Sodium benzoate completely inhibited mycelial growth of the fungi at concentrations of 0.1–1.0 %, as did captan at 0.5–1.0 %. In contrast, sodium EDTA, even at the highest concentration tested, was unable to completely inhibit mycelial growth of F. equiseti, F. verticillioides, M. phaseolina and S. rolfsii. These findings are in line with those of several previous studies. Mecteau et al. (2002) found that sodium benzoate at 0.2 M (3.8 %) completely inhibited F. sambucinum mycelial growth and it had a lower ED50 value than both sodium propionate and sodium citrate for F. sambucinum spore germination. Nisa et al. (2011) demonstrated that captan at 0.2 % was unable to completely eliminate mycelial growth of F. oxysporum, and Latifa et al. (2011) reported disodium EDTA to show no fungicidal effect against P. italicum at concentrations below 150 mM (5.6 %). In the present study, sodium propionate, sodium citrate and tolclophos methyl at 2 % only partially succeeded in inhibiting mycelial growth of all five Fusarium species tested, and sodium acetate, sodium formate, sodium succinate and sodium tartrate at 2 % were almost completely ineffective. This is in line with Mecteau et al. (2008), who reported that sodium citrate and sodium propionate had greater inhibitory effects on the mycelial growth of F. solani var. coeruleum than other sodium salts (acetate, formate, lactate, succinate and tartrate) at 0.2 M; however, unlike the present study, no statistically significant differences were found between the inhibitory effects of sodium benzoate, sodium citrate and sodium propionate. This finding is also in line with Arslan

13

et al. (2009), who found that sodium citrate at 2.0 % had no fungicidal effect against F. oxysporum f. sp. melonis, M. phaseolina, R. solani AG–4 and Sclerotinia sclerotiorum. Organic acid salts are presumed to inhibit fungal growth by disrupting cell-membrane permeability, interrupting the Krebs cycle and disturbing the intercellular acid-base equilibrium, with or without accumulation of toxic ions (Brul and Coote 1999). According to Davidson et al. (2005), since the antimicrobial activities of organic acids stem primarily from their undissociated forms, the fungicidal effects of these acids are pH-dependent and thus heavily influenced by both the pKa of the acid and the pH of the environment in which it resides. However, the findings of the present study suggested that the fungicidal effects of organic acid salts were not solely a function of pH—considering that all the salts tested at 2 % (except of sodium EDTA) had pH values ranging between 6.02 and 7.13, and considering that no inhibitory effects on mycelial growth of all the fungi except M. phaseolina was observed between pH values of approximately 6–8. In fact, according to El-Shenawy and Marth (1988), the inhibitory effects of organic acid salts vary depending on temperature at the time of application. Inorganic carbonate and bicarbonate salts including ammonium, sodium and potassium were shown to inhibit fungal mycelial growth in numerous previous studies (Palmer et al. 1997; Olivier et al. 1998; Palou et al. 2001; Arslan et al. 2009; Latifa et al. 2011). Arslan et al. (2009) reported the concentrations of sodium carbonate required to completely inhibit mycelial growth varied among different fungal pathogens (M. phaseolina and R. solani AG–4, 0.4 %;

0.000 5.62 0.00 ia 0.00 h 0.00 h 0.00 i 0.10 5.46 49.17 fg 57.55 bc 38.66 d 34.21 ef 0.25 5.65 68.07 de 68.91 b 49.80 c 67.81 d 0.50 5.80 95.39 a 100.00 a 68.35 b 88.75 ab 0.75 5.85 100.00 a 100.00 a 96.44 a 100.00 a 1.00 5.94 100.00 a 100.00 a 100.00 a 100.00 a 1.50 6.05 100.00 a 100.00 a 100.00 a 100.00 a 2.00 6.10 100.00 a 100.00 a 100.00 a 100.00 a Sodium EDTA 0.10 5.13 24.72 h 7.78 gh −0.16 h 7.87 hi 0.25 5.05 38.09 gh 14.31 fgh 2.63 gh 15.84 gh 0.50 4.98 42.94 g 18.92 efg 10.57 g 18.65 gh 0.75 4.91 59.12 ef 24.56 defg 20.69 f 23.65 fg 1.00 4.87 60.72 ef 27.40 def 24.41 ef 33.63 ef 1.50 4.70 68.59 de 29.47 def 32.57 de 46.71 e 2.00 4.60 78.86 cd 33.14 de 37.82 d 69.60 cd Sodium metabisulfite 0.10 5.43 79.91 cd 67.80 b 72.29 b 81.91 bc 0.25 5.25 92.87 abc 100.00 a 100.00 a 92.47 ab 0.50 5.12 100.00 a 100.00 a 100.00 a 100.00 a 0.75 5.06 100.00 a 100.00 a 100.00 a 100.00 a 1.00 5.02 100.00 a 100.00 a 100.00 a 100.00 a 1.50 4.91 100.00 a 100.00 a 100.00 a 100.00 a 2.00 4.90 100.00 a 100.00 a 100.00 a 100.00 a Captan 0.10 5.51 66.82 de 43.31 cd 55.30 c 88.06 ab 0.25 5.36 80.27 bcd 72.52 b 90.63 a 90.02 ab 0.50 5.31 92.62 abc 93.25 a 98.57 a 97.16 a 0.75 5.29 95.14 ab 99.10 a 99.57 a 98.67 a 1.00 5.21 100.00 a 100.00 a 100.00 a 100.00 a 1.50 5.16 100.00 a 100.00 a 100.00 a 100.00 a 2.00 5.13 100.00 a 100.00 a 100.00 a 100.00 a a Means in each column followed by the same letter are not significant different according to the Tukey HSD (P ≤ 0.05)

Control Sodium benzoate 0.00 36.06 51.07 75.90 100.00 100.00 100.00 100.00 18.27 24.37 28.44 35.73 38.78 40.22 51.55 62.36 89.67 100.00 100.00 100.00 100.00 100.00 72.77 82.47 95.62 97.99 100.00 100.00 100.00

j gh f d a a a a i i hi gh g g f e bc a a a a a d cd ab ab a a a

0.00 34.36 53.07 100.00 100.00 100.00 100.00 100.00 16.92 19.62 21.59 22.84 25.39 37.19 41.85 100.00 100.00 100.00 100.00 100.00 100.00 100.00 85.15 100.00 100.00 100.00 100.00 100.00 100.00

h e c a a a a a g fg fg fg f de d a a a a a a a b a a a a a a

Table 4 Soil bioassay results showing the effects of captan, sodium EDTA, sodium benzoate and sodium metabisulfite on bean root rot agents Compounds Concentration pH Inhibition (%) (% w/v) F. equiseti F. proliferatum F. semitectum F. solani F. verticilloides M. phaseolina f. sp. phaseoli 0.00 26.22 65.25 100.00 100.00 100.00 100.00 100.00 −3.09 0.84 19.11 34.23 43.08 46.61 74.13 100.00 100.00 100.00 100.00 100.00 100.00 100.00 85.07 98.32 100.00 100.00 100.00 100.00 100.00

R. solani h g d a a a a a h h g f e e c a a a a a a a b a a a a a a

0.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 14.14 18.02 24.93 31.06 71.60 77.52 81.51 100.00 100.00 100.00 100.00 100.00 100.00 100.00 90.45 100.00 100.00 100.00 100.00 100.00 100.00

S. rolfsii h a a a a a a a g fg ef e d cd c a a a a a a a b a a a a a a

Inhibitory Influence of Organic and Inorganic Sodium Salts and Synthetic Fungicides 91

13

92 Table 5 Effect of sodium benzoate, sodium EDTA, sodium metabisulfite and captan on germination and root elongation of bean seeds 5 days after application Compounds Concentra- pH GerminaRoot tion (% w/v) tion (%) lenght (cm) Control 0.000 7.96 100.00 aa 39.10 b Sodium benzoate 0.10 7.30 96.67 a 28.31 c 0.25 7.38 93.33 ab 24.98 cd 0.50 7.40 90.00 ab 18.59 de 0.75 7.47 90.00 ab 14.13 ef Sodium EDTA 1.00 4.65 60.00 c 4.81 fg 1.50 4.59 10.00 d 0.41 g 2.00 4.57 0.00 d 0.00 g Sodium 0.10 4.29 100.00 a 28.04 cd metabisulfite 0.25 4.25 73.33 bc 6.72 fg Captan 0.10 6.49 96.67 a 57.82 a 0.25 6.19 93.33 ab 47.82 b 0.50 5.46 93.33 ab 29.41 c 0.75 5.12 90.00 ab 27.11 cd a Means in each column followed by the same letter are not significant different according to the Tukey HSD (P ≤ 0.05)

S. sclerotiorum, 0.1 %), and in this study, sodium carbonate was able to completely inhibit mycelial growth of M. phaseolina, R. solani AG–4 HG I and S. rolfsii at 1.5, 1.0 and 0.1 %, respectively, but was unable to completely inhibit mycelial growth in any of the Fusarium species tested, even at the highest concentrations. These findings indicate that the sensitivity of fungus species to sodium carbonate may be variable. Olivier et al. (1998) reported sodium carbonate to be more effective than sodium bicarbonate against H. solani. Palou et al. (2001) found sodium carbonate as well as sodium bicarbonate at concentrations of 4.0 % to demonstrate fungistatic rather than fungicidal activity against P. italicum, which is consistent with the present study. With few exceptions, the remaining eight inorganic salts tested in the present study (sodium chloride, sodium molibdate, sodium nitrate, sodium phosphate dibasic, sodium phosphate monobasic, sodium phosphate tribasic, sodium sulfate and sodium thiosulfate) were completely ineffective in inhibiting mycelial growth of any of the fungi tested at 2 %. In a study by Punja and Grogan (1982), sodium phosphate dibasic was found to be more effective than sodium chloride, sodium nitrate and sodium sulfate in preventing germination of S. rolfsii sclerotia, and Mecteau et al. (2002) and Hervieux et al. (2002) found sodium phosphate tribasic dodecahydrate at 200 mM (7.6 %) to completely inhibit mycelial growth of F. sambucinum at pH 12.7 and H. solani at pH 12.3, respectively. In contrast, Palmer et al. (1997) demonstrated potassium and sodium monobasic phosphates to provide additional nutrients for the development of B. cinerea in acidic culture media. Based on the results of in vitro testing in the present study, soil and seed germination/root elongation bioassays

13


were conducted with captan, sodium benzoate and sodium metabisulfite, and sodium EDTA. According to the findings of soil bioassay, captan, sodium benzoate, sodium metabisulfite at concentrations of 0.1 % and higher completely inhibited mycelial growth of all eight fungi tested, whereas sodium EDTA was shown to be ineffective in inhibiting mycelial growth. Previous studies have reported applications of sodium metabisulfite, captan and sodium benzoate to offer protection against various plant diseases. Sodium metabisulfite application has been reported to control pear fruit rot caused by Alternaria alternata and Mucor piriformis; potato dry rot caused by Fusarium sambucinum; alfalfa seed rot caused by Colletotrichum trifolii, R. solani, F. equiseti and F. incarnatum; and onion basal rot caused by F. oxysporum f. sp. cepae (El-Sheikh-Aly et al. 1998; Al-Askar et al. 2013; Türkkan and Erper 2014). Both captan and carboxin have been shown to significantly reduce the severity of blight on bentgrass caused by S. rolfsii, with small amounts of carboxin applied in combination with small amounts of either captan or ammonium bicarbonate offering better disease control than larger amounts of carboxin or captan alone (Punja et al. 1982). Moreover, potassium sorbate dips (pH 6) applied to fresh market carrots reduced black root rot caused by Chalara elegans (Punja and Gaye 1993), and sodium benzoate (pH 7.7), potassium sorbate (pH 7.9) and potassium benzoate (pH 7.6) at higher pH levels were able to decrease Penicillium decay in oranges by 70.9, 71.4 and 54.3 %, respectively (Palou et al. 2002). In the present study, the findings of soil bioassay showed sodium benzoate at pH values of 5.46 (0.1 %)—5.94 (1.0 %) and captan at pH values of 5.21 (1.0 %)—5.36 (0.25 %) completely inhibited mycelial growth of all eight fungi, whereas sodium EDTA at 4.60 (2.0 %) pH value had inhibition rates of only 33.14–81.51 %. Moreover, depending on concentration, sodium EDTA showed medium to severe phytotoxicity in terms of both seed germination and root elongation bioassays, whereas captan, sodium benzoate and sodium metabisulfite demonstrated no phtotoxicity in terms of seed germination at any concentration, although phytotoxicity was observed in terms of root elongation especially at higher concentrations. These results are in line with Araùjo and Monteiro (2005), who reported root growth to be more sensitive to metal toxicity than germination. In another phytotoxicity study, Ziv and Zitter (1992) found the application of ammonium, potassium and sodium bicarbonates at rates of 1–5 % to healthy cucurbit plants and those infected with powdery mildew (Sphaerotheca fuliginea) resulted in beige necrotic spots on plant leaves. In conclusion, the present study found sodium metabisulfite and sodium benzoate, alone or in combination with other safe treatments, to be effective in controlling bean root rot caused by F. equiseti, F. proliferatum, F. semitectum, F. solani f. sp. phaseoli, F. verticillioides, R. solani AG–4 HG


I, M. phaseolina and S. rolfsii. However, before any definitive recommendations can be made in this regard, further studies are needed to examine the possible negative effects of these salts on soil pH. This is especially true for sodium benzoate, which was unable to effectively control fungal mycelial growth, except at high concentration. Possible phytotoxicity may also be avoided through post-emergence application of these salts into soil. Acknowledgements The authors thank Prof. Dr. Gürsel Karaca, Plant Protection Department, Agriculture Faculty, Süleyman Demirel University, for her careful proofreading, and Dr. Melike Çebi Kılıçoğlu for Rhizoctonia solani AG–4 HG I isolate.

References Abawi GS, Pastor Corrales MA (1990) Root rots of beans in Latin America and Africa: diagnosis, research methodologies, and management strategies. Centro Internacional de Agricultura Tropical (CIAT), Cali, p 114 Abawi GS, Ludwig JW, Gugino BK (2006) Bean root rot evaluation protocols currently used in New York. Annu Rep Bean Improv Coop 49:83–84 Al-Askar AA, Ghoneem KM, Rashad YM (2013) Management of some seed-borne pathogens attacking alfalfa plants in Saudi Arabia. African J Microbiol Res 7(14):1197–1206 Andres Ares JL, Rivera Martinez A, Pomar Barbeito F (2006) Short communication. Telluric pathogens isolated from bean plants with collar and root rots in northwest Spain. Spanish J Agricult Res 4(1):80–85 Anonymous (2013) U.S. Food and Drug Administration (FDA). http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/ SCOGS/ucm084104.htm. Accessed 2 July 2014 Aoki T, Tanaka F, Suga H, Hyakumachi M, Scandiani MM, O’Donnell K (2012) Fusarium azukicola sp. nov., an exotic azuki bean rootrot pathogen in Hokkaido, Japan. Mycologia 104(5):1068–1084 Araùjo ASF, Monteiro RTR (2005) Plant bioassays to assess toxicity of textile sludge compost. Sci Agric Piracicaba Brazil 62:286–290 Arslan U, Ilhan K, Karabulut OA (2006) Evaluation of food additives and low-toxicity compounds for the control of bean rust and wheat leaf rust. J Phytopath 154:534–541 Arslan U, Kadir I, Vardar C, Karabulut OA (2009) Evaluation of antifungal activity of food additives against soilborne phytopathogenic fungi. World J Microbiol Biotech 25:537–543 Beldek Ö (2013) Determination of pathogenicities and Fusarium species agent of root rot and wilt in bean growing areas in Samsun province. Master Thesis. University of Ondokuz Mayıs, Samsun. 69 pp Brul S, Coote P (1999) Preservative agents in foods—mode of action and microbial resistance mechanisms, a review. Int J Food Microbiol 50:1–17 Buruchara RA, Camacho L (2000) Common bean reaction to Fusarium oxysporum f. sp. phaseoli, the cause of severe vascular wilt in Central Africa. J Phytopath 148:39–45 Campanella V, Ippolito A, Nigro F (2002) Activity of calcium salts in controlling Phytophthora root rot of citrus. Crop Protect 21:751–756 Davidson PM, Sofos JN, Branen AL (2005) Antimicrobials in food, 3rd edn. CRC Press, USA, p 721 DePasquale DA, Montville TJ (1990) Mechanism by which ammonium bicarbonate and ammonium sulfate inhibit mycotoxigenic fungi. Appl Environment Microbiol 56:3711–3717

93

Di Salvatore M, Carafa AM, Carratù G (2008) Assessment of heavy metals phytotoxicity using seed germination and root elongation tests: a comparison of two growth substrates. Chemosph 73(9):1461–1464 Elad Y, Katan J, Chet I (1980) Physical, biological, and chemical control integrated for soilborne diseases in potatoes. J Phytopath 70:418–422 El-Sheikh-Aly MM, Felaifel MSA, Fouad NA, Badawy HMA (1998) Comparative effectiveness of some fungicides and salts applied preharvest or postharvest for controlling pear fruit rots. Annals Agricul Sci Cairo 1:135–149. (Special Issue) El-Shenawy MA, Marth EH (1988) Sodium benzoate inhibits growth of or inactivates Listeria monocytogenes. J Food Prot 51:525–530 Fan CM, Xiong GR, Qi P, Ji GH, He YQ (2008) Potential biofumigation effects of Brassica oleracea var. caulorapa on growth of fungi. J Phytopath 156:321–325 Gould GW, Russell NJ (1991) Sulphite. In: Russell NJ, Gould GW (eds) Food preservatives. Blackie, Glasgow, p 72 Hagedorn DJ, Inglis DA (1986) Handbook of bean diseases. Cooperative Extension Publications, Wisconsin, p. 28 Hall R (1991) Compendium of bean diseases, APS Press, USA, p. 102 Hatat G, Özkoç İ (1997) Bean root rot disease incidence and severity in Samsun and fungi associated with bean roots and soils. Turk J Agricult Forest 21:593–597 Hervieux V, Yaganza ES, Arul J, Tweddell RJ (2002) Effect of organic and inorganic salts on the development of Helminthosporium solani, the causal agent of potato silver scurf. Plant Dis 86:1014–1018 Latifa A, Idriss T, Hassan B, Amine SM, El Hassane B, Abdellah ABA (2011) Effects of organic acids and salts on the development of Penicillium italicum: the causal agent of citrus blue mold. Plant Path J 10:99–107 Mecteau MR, Arul J, Tweddell RJ (2002) Effect of organic and inorganic salts on the growth and development of Fusarium sambucinum, a causal agent of potato dry rot. Mycol Res 106:688–696 Mecteau MR, Arul J, Tweddell RJ (2008) Effect of different salts on the development of Fusarium solani var. coeruleum, a causal agent of potato dry rot. Phytoprot 89:1–6 Mills AAS, Platt HW, Hurta RAR (2004) Effect of salt compounds on mycelial growth, sporulation and spore germination of various potato pathogens. Posthar Biol Technol 34:341–350 Montiel Gonzalez L, Gonzalez Flores F, Sanchez Garcia BM, Guzman Rivera S, Gamez Vazquez FP, Acosta Gallegos JA, Rodriquez Guerra R, Simpson Williamson J, Cabral Enciso M, Mendoza Elos M (2005) Fusarium species on bean (Phaseolus vulgaris L.) roots causing rots, in fives states of Central Mexico. Rev Mex Fitopatol 23(1):1–10 Mwang’ombe AW, Thiong’o G, Olubayo FM, Kiprop EK (2007) Occurrence of root rot disease of common bean (Phaseolus vulgaris L.) in association with bean stem maggot (Ophyiomia sp.) in Embu District, Kenya. Plant Path J 6(2):141–146 Nisa T, Wani AH, Bhat MY, Pala SA, Mir RA (2011) In vitro inhibitory effect of fungicides and botanicals on mycelial growth and spore germination of Fusarium oxysporum. J Biopest 4(1):53–56 Olivier C, Halseth DE, Mizubuti ESG, Loria R (1998) Postharvest application of organic and inorganic salts for suppression of silver scurf on potato tubers. Plant Dis 82:213–217 Olivier C, MacNeil CR, Loria R (1999) Application of organic and inorganic salts to field-grown potato tubers can suppress silver scurf during potato storage. Plant Dis 83:814–818 Palmer CL, Horst RK, Langhans RW (1997) Use of bicarbonates to inhibit in vitro colony growth of Botrytis cinerea. Plant Dis 81:1432–1438 Palou L, Smilanick JL, Usall J, Vinas I (2001) Control of postharvest blue and green molds of oranges by hot water, sodium carbonate and sodium bicarbonate. Plant Dis 85:371–376

13

94 Palou L, Usall J, Smilanick JL, Aguilar MJ, Vinas I (2002) Evaluation of food additives and low-toxicity compounds as alternative chemicals for the control of Penicillium digitatum and Penicillium italicum on citrus fruit. Pest Manag Sci 58:459–466 Punja ZK, Grogan RG (1982) Effects of inorganic salts, carbonate-bicarbonate anions, ammonia, and the modifying influence of pH on sclerotia germination of Sclerotium rolfsii. Phytopath 72:635–639 Punja ZK, Gaye MM (1993) Influence of postharvest handling practices and dip treatments on development of black root rot on fresh market carrots. Plant Dis 77:989–995 Punja ZK, Grogan RG, Unruh T (1982) Comparative control of Sclerotium rolfsii on golf greens in northern California with fungicides, inorganic salts, and Trichoderma spp. Plant Dis 66:1125–1128 Russell AD (2005) Mechanisms of action, resistance, and stress adaptation. In: Michael Davidson P, Sofos JN, Branen AL (eds). Antimicrobials in food, 3rd edn.Boca Raton, CRC Press, pp 633–657 Türkkan M, Erper I (2014) Evaluation of antifungal activity of sodium salts against onion basal rot caused by Fusarium oxysporum f. sp. cepae. Plant Prot Sci 50:19–25 Valencia-Chamorro SA, Palou L, del Rio MA, Perez-Gago MB (2008) Inhibition of Penicillium digitatum and Penicillium italicum by hydroxypropyl methylcellulose-lipid edible composite films containing food additives with antifungal properties. J Agricult Food Chem 56:11270–11278 Yuen GY, Schroth MN, Weinhold AR, Hancock JG (1991) Effects of soil fumigation with methyl bromide and chloropicrin on root health and yield of strawberry. Plant Dis 75:416–420

13

M. Türkkan, İ. Erper Ziv O, Zitter TA (1992) Effects of bicarbonate and film-forming polymers on cucurbit foliar diseases. Plant Dis 76:513–517

Muharrem Türkkan, He was born on the 30th of August 1975 in Karabük, Turkey. He completed his doctorate in 2008 at Department of Plant Protection, Faculty of Agriculture, Ankara University, in Ankara. He is now holding permanent position at Department of Plant Protection, Faculty of Agriculture, Ordu University as an Assistant Professor of Phytopathology. Türkkan worked as Post doc fellow at Universita’ degli Studi di Napoli Federico II (Italy), funded by the Scientific and Technological Research Council of Turkey, from January to July 2010. His fields of interest are mycology, soilborne fungal pathogens and using of natural alternatives to synthetic fungicides for control of fungi.