Effect of Sodium Bisulfite on Peroxidase Activity and Electrolyte Leakage in Maize in Relation to Sporulation of Bipolaris maydis

In this study, we sought to determine whether the increases in peroxidase activity and electrolyte leakage induced in maize (Zea mays L.) leaves by sodium bisulfite were causally related to the sodium bisulfite-induced increases in sporulation of the pathogen Bipolaris maydis race T on infected maize leaves. Pretreatment of detached leaves of maize inbred W64 A with sodium bisulfite (500 |ig/ml) for 24 h in the dark at 28°C increased peroxidase activity in the Tms cytoplasm (susceptible) isoline compared with the N cytoplasm (resistant) isoline. No such differences in peroxidase activity between the two isolines were observed when detached leaves were pretreated with double distilled water. The sodium bisulfite-induced increase in peroxidase activity persisted even when leaves pretreated with sodium bisulfite were inoculated with R maydis race T and subsequently incubated for 48 h in the dark at 28° C. Similarly, pretreatment with sodium bisulfite caused a greater increase in electrolyte leakage as well as in sporulation on the leaves of the susceptible than on those of the resistant isoline when compared with leaves not treated with sodium bisulfite. Sodium bisulfite showed no effect on sporulation in vitro. Leachates from the susceptible isoline pretreated with sodium bisulfite also caused greater increase in sporulation than those from the resistant isoline pretreated with sodium bisulfite. OHIO J. SCI. 90 (3): 71-76, 1990 INTRODUCTION Different kinds of stresses may induce a state of resistance or susceptibility of plants against pathogens depending upon the host-pathogen interactions (Byther and Steiner 1975, Chamberlain 1972, Daly et al. 1970, Stahmann et al. 1966). For example, chemicals such as ethylene have been shown to induce resistance of sweet potato to Ceratocystis fimbriata (Stahmann et al. 1966) and susceptibility of wheat to Puccinia graminis f. sp. tritici (Daly et al. 1970). In the above interactions, the possible involvement of peroxidase has been proposed, based on the role of peroxidase in several physiological processes (Gasper et al. 1982), including disease resistance (Hammerschmidt et al. 1982, Urs and Dunleavy 1974). Results, however, are not consistent. For example, ethylene-induced resistance of sweet potato to C. fimbriata as well as susceptibility of wheat to P. graminis f. sp. tritici were accompanied by an increase in peroxidase activity in either case. Thus, the role of increased peroxidase activity in either resistance or susceptibility is an open question. Chemicals and other stresses such as high temperature could alter the resistance or susceptibility of plants to infection through their effects on membrane permeability. It is known that ethylene affects membrane permeability (Abrams and Pratt 1967, Goodman et al. 1986). Similarly, high temperature stress could induce susceptibility in maize through its effect on membrane permeability as measured by increased electrolyte leakage (Garraway et al. 1989). This might result in the loss of host cells' constituents which might be used by an invading pathogen as a source of nutrients. Thus, chemicals such as 'Manuscript received 21 July 1989 and in revised form 1 December 1989 (#89-20). Present Address: Microbiologist, Institute for Microbial and Biochemical Technology, USDA Forest Service, Forest Products Laboratory, Madison, Wisconsin 53705. ethylene might function like other abiotic (Rist and Lorbeer 1984) or biotic (Stevens and Gudauskas 1982) stresses predisposing plants to disease. In addition to the above literature, we have conducted preliminary studies which indicate that exposure of maize {Zea mays L.) leaves to chemicals such as the reducing agent sodium bisulfite prior to inoculation with a fungal pathogen Bipolaris maydis race T appears to increase the severity of infection and disease (Akhtar and Garraway 1988, 1989). We, therefore, sought in the present study to define the relationship, if any, of peroxidase activity and electrolyte leakage to the above phenomenon. Specific objectives were to determine: 1) the effect of sodium bisulfite on peroxidase activity and electrolyte leakage in two isolines of maize which differ in their degree of susceptibility to B. maydis race T; and 2) the relationship of sodium bisulfite-induced peroxidase activity and electrolyte leakage with sporulation of B. maydis race T on infected leaves of these isolines. MATERIALS AND METHODS HOST: TWO isolines of the maize {Zea mays L.) inbred W64 A, i.e., normal (N) cytoplasm and Texas male sterile (Tms) cytoplasm were grown in the greenhouse as previously described (Birecka et al. 1975, Birecka and Garraway 1978). The fungal pathogen used in this study was Bipolaris maydis (Nisikado) Shoemaker (syn. Helminthosporium maydis Nisikado and Miyke, perfect stage Cochliobolus heterostrophus Drechlser) race T. Leaf samples of comparable age from 3-wk-old plants were detached from each isoline, washed with double distilled water, cut into pieces of about 5x2 cm in size, then placed on a sheet of Whatman No. 3 filter paper. Leaves thus prepared were floated either on 5 ml of double distilled water (control) or on 5 ml of an aqueous solution of sodium bisulfite (250, 500 and 1,000 ng/ml, J.A. Baker Chemical Company, Phillipsburg, NJ) for 24 h in the dark SODIUM BISULFITE AND MAIZE PEROXIDASE VOL. 90 at 28° C in a water-vapor saturated incubator. Sodium bisulfite-treated and control leaves were then inoculated with a B. maydis race T spore suspension (10,000-15,000 coniclia/ml) in double distilled water containing Tween20 (50 ni/100 ml) as a surfactant. They were then incubated for 48 h in the dark at 28° C. Previous studies had indicated that this incubation time (48 h) was optimum for fungal colonization of the tissues. As the sensitivity of the pathotoxin produced by this fungus appeared to be higher in the dark than in the light, all incubations were carried out in the dark. The spore suspension used as inoculum was prepared from cultures grown on glucose-L-asparagine agar medium for 7 days at 28° C (Garraway 1973b). PATHOGEN: A single spore isolate of B. maydis race T collected from a maize seed grown in Franklin County, OH, in 1970 (ATCC # 36180) was cultured on a glucoseL-asparagine agar medium in the dark at 28° C for 7 days as previously described (Garraway 1973b). Results obtained with this isolate in our previous preliminary studies were similar to those with other isolates of race T. DETERMINATION OF PEROXIDASE ACTIVITY: Procedures for extraction of peroxidase (donor: oxidoreductase: E.C. 1.11.1.7) were similar to those used previously (Birecka et al. 1975, Birecka and Garraway 1978, Garrawjay 1973a). The tissue was homogenized in 5 ml of 10 mM sodium phosphate buffer (pH 6) using a Brinkmanri polytron homogenizer. The resulting homogenate was centrifuged at 20,000 g for 5 min at 4° C. The supernatant was assayed for the buffer-extractable, or soluble peroxidase activity. To recover salt-extractable, or ionically bound peroxidase activity, the washed pellet was resuspended in 5 ml of NaCl in 10 mM phosphate buffer (pH 6), stirred at 4° C for 1 h, and centrifuged at 20,000 g for 5 min at 4° C. The supernatant was then assayed. Our data are based solely on the activity of the soluble and ionically bound peroxidase fractions, as they constitute about 90% of the total peroxidase activity in maize and are recovered with ease and speed. Cadena-Gomez and Nicholson (1987), using the same method for peroxidase extraction in maize, have also recovered about 93% of the total peroxidase activity. The reaction mixture for the peroxidase assay included 200 |il of enzyme solution, 1300 LLI of sodium phosphate buffer (10 mM pH 6), 500 JLLI of 50 mM guaiacol and 100 LLI of 100 mM H2O2. One unit of the peroxidase activity has been defined as the change of 1.0 absorbance unit at 470 nm per minute per gram fresh weight of leaf tissues. DETERMINATION OF ELECTROLYTE LEAKAGE: Detached leaves were floated either on double distilled water or on an aqueous solution of sodium bisulfite (500 Lig/ml) for 24 h in the dark at 28° C, inoculated with B. maydis race T as described above, then incubated in the dark for 24 h at 28° C. These leaves were cut into 3 cm sections, then washed with double distilled water. Each leaf section was immersed in 25 ml of sterile double distilled water in screw cap vials (40 ml-capacity). They were further incubated in the dark for 24 h at 28° C. To determine the quantity of electrolyte leakage, the change in conductance of the double distilled water with immersed leaf sections was measured at 0 and 24 h using a conductivity bridge (model 4959) and a conductivity cell (k=0.01, Leeds and Northrup Co., Philadelphia, PA). The amount of electrolyte leakage is expressed as micromhos (M, mhos) per milligram dry weight of leaf tissue. DETERMINATION OF SPORULATION ON INFECTED MAIZE LEAVES: Detached leaves of both isolines were floated either on double distilled water or on an aqueous solution of sodium bisulfite (500 Lig/ml) for 24 h in the dark at 28° C and inoculated with B. maydis race T as described above. They were then incubated in the dark for 48 h at 28° C in a water-vapor saturated incubator as reported previously (Birecka et al. 1975). At the end of the incubation period, infected leaf sections were placed in screw cap vials (15 ml-capacity) containing 3 ml of preservative solution (5% colorox, 20% ethanol and 2% NaOH) to inactivate the conidia. These vials were then agitated to dislodge conidia from the leaf surface. Conidium concentrations were determined with a hemacytometer as described previously (Garraway 1973b). Results were expressed as the number of B. maydis race T conidia produced per milligram dry weight of leaf tissues. DETERMINATION OF SPORULATION IN VITRO: TO determine the effect of an aqueous solution of sodium bisulfite (500 Lig/ml) on sporulation and mycelial dry weight of B. maydisra.ce T in vitro, standard glucose-L-asparagine agar medium was prepared. Then this medium was either nonamended (double distilled water control) or amended with an aqueous solution of sodium bisulfite (500 jig/ml). Sporulation and mycelial dry weight were measured after 7 days of incubation in the dark at 28° C. To study the effect of leachates on B. maydis race T sporulation, detached leaves of both isolines were floated either on double distilled water or on an aqueous solution of sodium bisulfite (500 (J.g/ml) for 24 h in the dark at 28° C, inoculated with B. maydisra.ee T as described above and then incubated in the dark for 24 h at 28° C. The leaves were cut into 3 cm sections, rinsed and immersed in 50 ml of double distilled water in a beaker. After 12 h immersion, double distilled water containing leachates was autoclaved at 121° C for 20 min at 15 PSI and then used to constitute 20 ml of 2% agar media. This experimental media either non-amended (water agar) or amended with double distilled water containing leachates (leachates agar) was seeded with 0.5 ml of a sterile B. maydisra.ee T spore suspension (30,000-40,000 conidia/ml). The conidia were uniformly distributed on the surface of each plate with a sterile spreader. Seeded plates were incubated in the dark at 28° C for 7 days. Conidia were collected from the culture by scraping and washing with 1 ml preservative solution and the number per ml was determined. Sporulation was expressed as conidia per milligram dry weight of fungus. Inoculation procedures, media preparation and sporulation, conidia and mycelial measurements have been described previously (Garraway 1973b). All experiments involving peroxidase, electrolyte leakage, sporulation in vivo and in vitro were repeated three times with five replicates in each.