Chlorine Dioxide for Reduction of Postharvest Pathogen

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p Vol. 60, No /94/$ Chlorine Dioxide for Reduction of Postharvest Pathogen Inoculum during Handling of Tree Fruits RODNEY G. ROBERTS* AND STEPHEN T. REYMOND Tree Fruit Research Laboratory, Agricultural Research Service, U.S. Department ofagriculture, Wenatchee, Washington Received 18 February 1994/Accepted 1 June 1994 Alternatives to hypochlorous acid and fungicides are needed for treatment of fruit and fruithandling facilities. Chlorine dioxide was evaluated and found effective against common postharvest decay fungi and against filamentous fungi occurring on fruit packinghouse surfaces. In vitro tests with conidial or sporangiospore suspensions of Botrytis cinerea, Penicillium expansum, Mucor piriformis, and Cryptosporiopsis perennans demonstrated >99% spore mortality within 1 min when the fungi were exposed to aqueous chlorine dioxide at 3 or 5,ug * ml'. Longer exposure times were necessary to achieve similar spore mortalities with 1,ug * ml'. Of the fungi tested, B. cinerea and P. expansum were the least sensitive to Cl02. In comparison with the number recovered from untreated control areas, the number of filamentous fungi recovered was significantly lower in swipe tests from hard surfaces such as belts and pads in a commercial apple and pear packinghouse after treatment of surfaces with a 14.0 to 18.0,ig *mvl' Cl02 foam formulation. Chlorine dioxide has desirable properties as a sanitizing agent for postharvest decay management when residues of postharvest fungicides are not desired or allowed. Control of postharvest diseases of tree fruits, especially apples, pears, and cherries, is becoming increasingly difficult. The loss of the postharvest registration for benomyl (Benlate) in 1989 and the loss of the registration for dichloronitroaniline (Botran) in 1992 severely restricted the number of effective materials for control of postharvest decay in fruits. The loss of fungicides and reduced consumer acceptance of pesticide residues have fostered a growing interest in integrated management systems for postharvest diseases (1517, 27, 28). System components include but are not limited to mineral nutrition (7, 23, 24), harvest maturity and handling practices (4, 20), orchard sanitation (11), and development of biological control technology for postharvest diseases (5, 8, 10, 14, 19, 25). Alternative methods of disease control are needed, especially in the case of fruit exported to countries such as Japan which currently do not allow importation of fruit treated with postharvest fungicides. With the loss of several effective fungicides and decreasing residue tolerance for those that remain, sanitation of both fruit and environmental surfaces must take a more prominent position as a disease management tool. Chlorine, usually as hypochlorous acid (formed by the dissociation of sodium hypochlorite in water), has long been the standard material of the fruit and vegetable industries for sanitation of process waters and fruit. A level of 100 p,g of free chlorine ml' is currently recommended for control of postharvest pathogen spores in dump tanks and other recirculating water systems (28), although actual levels of free chlorine in process waters vary greatly from packinghouse to packinghouse. The disadvantages of chlorine as a primary disinfectant include extensive corrosion of metal equipment, reliance on manual monitoring of chlorine concentrations, sensitivity to organic load, effec * Corresponding author. Mailing address: U.S. Department of Agriculture, Agricultural Research Service, Tree Fruit Research Laboratory, 1104 N. Western Ave., Wenatchee, WA Phone: (509) Fax: (509) Electronic mail address: A03rro berts@attmail.com tiveness within a relatively narrow ph range, and the formation of chlorinated byproducts, including chloroform (9). Alternatives to hypochlorous acid such as ozone (21) and chlorine dioxide (22) have been evaluated. The antimicrobial activity of chlorine dioxide in the presence of high levels of organic matter such as those found in immersion dump tanks and flume processing waters is not diminished as readily as is that of chlorine. Benarde et al. (3) demonstrated the increased effectiveness of chlorine dioxide over chlorine in killing Escherichia coli in sewage effluent and reported greater effectiveness of chlorine dioxide in situations in which high organic matter levels are present in the water. Tanner (26) demonstrated that chlorine dioxide had the highest biocidal activity on a microgrampermilliliter basis against several bacteria and a yeast in comparison with sodium hypochlorite, iodine, quaternary ammonium compounds, glutaraldehyde, and phenol. The reported efficacy of CdO2 against several postharvest pathogens in vitro and in vivo on pear fruit (22), against bacteria (3, 12, 13), and against protozoans (6) warranted further investigation of its disinfestation potential for the tree fruit industry and its suitability as a component of an integrated disease management system. (A preliminary report of some experiments has been presented elsewhere in a discussion of the potential role of chlorine dioxide in integrated management systems [15]). MATERIALS AND METHODS Preparation of aqueous chlorine dioxide. Aqueous solutions of chlorine dioxide were generated on demand at the pilotscale packingline facility at the Tree Fruit Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Wenatchee, Wash., with a small, waterdriven chlorine dioxide generator (Rio Linda Chemical Co., Sacramento, Calif.). The generator consisted of a solenoid valve connected to an oxidationreduction potential controller (Chemtrol 075; Rio Linda), which was in turn connected to a platinumplatinum redox electrode which measured the redox

2 VOL. 60, 1994 CHLORINE DIOXIDE REDUCES PATHOGEN INOCULUM 2865 potential in dump tank water. When the millivolt output from the redox probe fell below an empirically determined setpoint, the oxidationreduction potential controller actuated the solenoid valve, allowing water to flow through the generator. The water flow over two venturis drew 7.5% (wt/vol) aqueous sodium chlorite (Savolite Inc., Seattle, Wash.) and gaseous chlorine through separate rotometers into a reaction chamber filled with small Teflon cylinders, where C102 was formed by their reaction. The effluent from the generator was captured in opaque glass bottles which had been pretreated with concentrated C102 to satisfy any C102 binding sites as recommended by Aieta et al. (1); it was then removed to the laboratory for titration and dilution for use in in vitro studies. Determination of chlorine dioxide concentrations. Aqueous chlorine, chlorine dioxide, and chlorite concentrations in generator product streams were determined by potentiometric titration with phenylarsine oxide according to the method of Aieta et al. (1). Titrations were performed in triplicate for each sample, and the mean values were calculated for each oxychlorine species and residual chlorine. The product streams typically contained 26 to 82 (mean, 58),ug of C12 * ml 1, 384 to 425 (mean, 409),ug of C102 * ml', and 98 to 198 (mean, 141),ug of ClO2 ml1. The product streams were diluted to the desired concentrations with deionized water. Preparation of chlorine dioxide foam. Chlorine dioxide formulated as a foam was generated in much the same manner as aqueous C102, except that sodium chlorite was combined with an acidic detergent (Savolite) instead of gaseous chlorine, and the product stream was held in a 1,900liter holding tank connected to a series of drop stations in a commercial packing facility. Foam was generated at each drop station by combining the product stream from the reservoir with compressed air and was applied to hard surfaces by a handheld wand from which the foam was forcibly ejected. Chlorine dioxide concentrations in foam generator product streams were determined with a colorimetric test kit (Chlorine Test Kit no. 2231; Hach Co., Loveland, Colo.). Effect of C102 on fungi. (i) Source and production of inoculum. Isolates of Mucorpiriformis E. Fisch. (isolate 8743, from d'anjou pear fruit), Botrytis cinerea Pers.:Fr. (isolate 8889, from Rainier cherry fruit), Penicillium expansum Link (isolate 874, from Red Delicious apple fruit), and Cryptosporiopsis perennans (Zeller et Childs) Wollenweb. (isolate 8781, from Golden Delicious apple fruit) were used singly in tests to determine the effect of contact time and concentration of chlorine dioxide on mortality of common postharvest pathogens. Sporangiospore suspensions of M. piriformis at 3 x 104 CFU/ml were prepared from 7 to 11dayold potato dextrose agar cultures grown at 15 C. B. cinerea and C. perennans cultures were grown on potato dextrose agar at 18 C under an alternating 16h/8h lightdark cycle with UV and fluorescent lighting. Conidial suspensions were prepared by scraping conidia from 7 to 11dayold cultures with an inoculating loop and suspending the harvested conidia in sterile deionized water. Conidial suspensions of P. expansum were prepared similarly, except that the cultures were grown at 18 C without an alternating lightdark cycle. Inoculum suspensions were adjusted to the desired concentrations with a hemacytometer. The term "fungi" in this paper refers exclusively to filamentous forms and excludes yeasts and yeastlike fungi. (ii) Contact time and concentration experiments. For each pathogen, conidial suspensions were pipetted into test tubes containing 1, 3, or 5 jig of chlorine dioxide * ml' and then vortexed. Onehundredmicroliter aliquots were removed from the test tubes at 0.5min intervals for 4.5 min, serially diluted, plated in triplicate onto malt extract agar (2), and incubated for 2 to 3 days. The number of resulting colonies was counted, and the number of viable CFU per milliliter was calculated and recorded for each sampling time at each C102 concentration. Each experiment was repeated once. (iii) Effect of chlorine dioxide foam on isolation of fungi from environmental surfaces. Environmental surfaces in a pear packinghouse in Wenatchee, Wash., were sampled before and after treatment with chlorine dioxide foam. Cardboard templates (100 cm2) were placed on the surfaces of cull belts, foam pads on cull chutes, flooring, and belts beneath waxing equipment. Areas within the templates were swabbed repeatedly with sterile cottontipped applicators moistened with sterile water, with as much of the liquid and solid material as possible picked up. Four replicate areas were sampled before and after application of 14 to 18,ug of chlorine dioxide foam * ml'. The interval of application of foam, rinsing, and subsequent sampling was ca. 45 min. After each sampling area was thoroughly swabbed, the cottontipped applicator was returned to a test tube containing 5 ml of sterile water and returned to the laboratory on ice. The tubes containing the swabs and the wash water were vigorously vortexed and then dilution plated onto malt extract agar amended with streptomycin and tetracycline. The plates were incubated at room temperature; fungal populations on the swabbed surfaces were then estimated from plate dilution counts of resulting colonies TABLE 1. Mean percent spore mortality of four fungal species after in vitro exposure to differing C102 concentrations and contact times Fungus C102 concn (,.g * ml1) % Spore mortality after indicated exposure time in min' C. perennans NS NS NS NS NS M. pinformis b 92.9 b 99.9 NS 99.9 NS NS a a a a P. expansum c 76.6 c 99.3 b 99.6 b 99.8 b b 99.9 b a a a a a a a a B. cinerea c 48.6 b 93.5 c 98.1 b 98.5 b b 99.2 a 99.7 b 99.9 a 99.9 a a 99.5 a a a a a Values are the means of two trials, each performed in triplicate. Values within a column for a particular fungus followed by the same letters do not differ significantly according to ANOVA of the arcsine square roottransformed data (P > F = ). NS, not significant.

3 2866 ROBERTS AND REYMOND and expressed as CFU per 100 cm2. The experiment was repeated at the same location 3 years later. (iv) Efficacy of chlorine dioxide treatment of immersion dump tank water. To evaluate the performance of a chlorine dioxide generator system in a commercial environment, a chlorine dioxide generator system, including process control and reagents as described above for in vitro tests, was installed and operated on an apple presize line (into which fruit from storage is placed for sorting and sizing) at an apple packinghouse in Wenatchee, Wash. The recirculating water system held ca. 38,000 liters of water, and the water was changed weekly. The performance of the system was evaluated during an 8h period; the evaluation was done four times on successive days by taking 50ml grab samples at intervals not exceeding 2 h. To estimate fungal contamination, samples were dilution plated onto malt extract agar amended with streptomycin and tetracycline, and then the number of fungal CFU per milliliter was estimated from colony counts. No attempt to distinguish between pathogenic and saprophytic fungi during plate counts was made. Because these experiments were conducted in commercial packing facility, untreated controls (no chlorine dioxide) were not included. Determination of chlorine dioxide residuals in these samples was done with a colorimetric test kit. Statistical analysis. Percent spore mortality data of individual fungi were analyzed by analysis of variance of the arcsine square roottransformed data, with mean separation by the WallerDuncan kratio t test at an a. of CFU isolated from packinghouse surfaces before and after chlorine dioxide foam treatment were analyzed by oneway analysis of variance (18) at an a of 0.05, with mean separation by least significant difference. RESULTS AND DISCUSSION In vitro tests. Percent spore mortality and viablecfupermilliliter data from in vitro dosing experiments are presented in Table 1. The four pathogens varied in sensitivity to C102 at 1.0,ug * ml'. C. perennans was the most sensitive, followed by M. pirifornis, P. expansum, and B. cinerea. All conidia of C. perennans were killed by all combinations of C102 and contact times. The minimum doseexposure combinations for 100% spore mortality in aqueous suspensions were 1.0 ug * ml 1 for 0.5 min for C. perennans, 1.0 ug ml' for 4.0 min or 3.0 ug* ml' for 0.5 min form. piriformis, 3.0,g* ml1 for 2.0 min or 5.0 ug * ml' for 0.5 min for P. expansum, and 5 ug * ml' for 2.0 min for B. cinerea. Dump tank The relationship between C102 concentration and the number of viable fungal propagules determined in in vitro tests was generally confirmed in tests conducted in a commercial packinghouse dump tank. On day 1 (Fig. 1), CO2 levels ranged from nearly 5 to just below 3 ug * ml', and no viable fungal spores were detected in plating assays. On the morning of day 2, the initial C102 concentration was 2.0,ug* ml', and approximately 20 CFU of filamentous fungi ml' was detected (Fig. 1). A manual adjustment at the controller to increase the C102 concentration caused the tank to become overcharged (6,ug ml' at 1000 h), and plate counts were 0 CFU/ml for this sampling time and thereafter on day 2. The overcharged condition of the dump tank during day 2 of the commercial experiment led to offgassing of chlorine dioxide and caused respiratory discomfort for some workers, and even at 3.0 ug * ml' chlorine dioxide can cause worker discomfort in enclosed environments unless special ventilation equipment is used. Such ventilation equipment would therefore be necessary in indoor situations to prevent worker exposure to excessive chlorine dioxide vapors. On day 3 (Fig. x '.Ṛ 200 I E Day Day3 1600$ > Day _ ~~~ HOURS FIG. 1. Effect of residual chlorine dioxide concentration on the number of filamentous fungal CFU in a commercially operated presize immersion dump tank during a 4day period. 1), the initial C102 concentration was again 2.0,ug * ml', and the concentration was manually adjusted to maintain a residual of approximately 1.0,ug * ml'. As the C102 concentration fell, the number of filamentous fungi increased to approximately 70 CFU/ml and then fell to approx. 20 CFU/ml at the final sampling time. On day 4 (Fig. 1), C102 concentrations were maintained between 0.8 and 1.2 jig ml', and the number of CFU per milliliter varied between 0 and approximately 1,500. During the 4 days of testing, levels of viable fungal propagules varied when C102 residuals were maintained at 2.0 Lg * ml' or lower, but no viable fungi were detected when C102 residuals were 3.0,ig * ml' or higher. The variation in numbers of viable fungal propagules when C102 concentrations are 2.0,ug mll or lower can be explained by (i) the increased time needed to kill spores at lower C102 concentrations and (ii) variable rates of ingress of fungal propagules into the water system. The dump tank used was part of a recirculating presize flume system. Random variation in the amount of decay and sporulation in each lot (bin) of fruit introduced into the system could cause the fluctuation in the number of CFU per milliliter observed in dilutionplated samples. Because spore mortality occurs more rapidly at 3.0,ug ml', this residual concentration appeared to maintain the system with no detectable fungal CFU. Spotts and Peters (22) evaluated chlorine dioxide at 1 and 10 APPL. ENVIRON. MICROBIOL. 200

4 VOL. 60, 1994 CHLORINE DIOXIDE REDUCES PATHOGEN INOCULUM 2867 TABLE 2. Mean numbers of CFU of fungi from hard surfaces in a commercial pear packinghouse before and after treatment with 14 to 18 jig of chlorine dioxide foam ml' Trial CFU/100 cm2' Cull belt Floor Foam pad Cull chute Waxer belt 1 Before treatment 2.57 x 104 a 4.85 x 103 a 1.75 x 104 a 7.90 x 102 a After treatment 2.95 x 103 b 2.65 x 102 b 5.38 x 102 b 2.62 x 102 a 2 Before treatment 1.07 x 104 a 5.30 x 103 a 3.42 x 105 a 6.50 x 102 a After treatment 1.60 x 103 b 4.97 x 10' b 8.82 x 102 b 2.27 x 101 b a Values are the means for four replicate areas. Values within a column for a particular trial followed by the same letter do not differ significantly at an a of Mean separation was done by leastsignificantdifference values from oneway analysis of variance.,ug * ml' for control of pear decay; they reported that 1 ug * ml' was not effective in reducing the pathogen inoculum but that 10,ug * ml' was effective but probably not economical. Our data support their conclusion that 1 pug of C102 * ml ' is inadequate to control spore loads in dump tank water, but we conclude that 3 to 5,ug of C102 ml' can provide an effective residual concentration to control spore loads. Considering the efficacy of 3 and 5,ug of C102 * ml', we agree that maintaining a 10,ug *mfll C102 residual would be expensive and unnecessary. C102 foam. Although the organic debris was not cleaned from environmental surfaces before the sanitizing treatment, isolation of fungi from hard surfaces in a commercial packinghouse was significantly reduced (ot = 0.05) after treatment with chlorine dioxide foam, with the exception of the waxer belt in trial 1 (Table 2). The greatest reductions were observed on the most contaminated surfaces, such as cull belts and chutes. The reduction in the number of fungal propagules may be attributable to the relatively high concentration of C102 in the foam formulation in comparison with aqueous formulations and to the tendency for the foam to persist on surfaces, extending contact time. Because the probability of decay development is strongly influenced by the inoculum concentration, it seems prudent to implement a regular program of prophylactic treatment of packinghouse surfaces to minimize or prevent the growth and sporulation of fruit decay fungi, especially P. expansum. With the emergence of new postharvest disease control technologies and practices, including implementation of biological control and integrated management, the management of pathogen populations will become increasingly important (8, 14, 15). A characteristic common to several biological control systems is the decreased efficacy of biological treatments as the pathogen concentration increases (8, 14), and it has been suggested (15) that inoculum reduction methods will be critical to the successful implementation of biological control systems. Our data show that chlorine dioxide can be used to reduce the inoculum in both aqueous and hardsurface environments and that it may ultimately prove a valuable tool in integrated management of postharvest diseases of fruits and vegetables. ACKNOWLEDGMENTS We appreciate the cooperation of Rio Linda Chemical Company; Scott Ager and Camilo Cheng of Savolite Inc.; Gary Apel of Michelsen Packaging, Inc., Yakima, Wash.; Nate Reed of Stemilt Growers, Inc., Wenatchee, Wash.; and Bert Navone of the Auvil Fruit Company, Orondo, Wash., throughout the project. We also thank David Sugar, Gary Apel, and Eugene Kupferman for their constructive reviews of the manuscript. REFERENCES 1. Aieta, E. M., P. V. Roberts, and M. Hernandez Determination of chlorine dioxide, chlorine, chlorite, and chlorate in water. J. Am. Water Works Assoc. 76: Atlas, R. M., and L. C. Parks Handbook of microbiological media. CRC Press, Inc., Boca Raton, Fla. 3. Benarde, M. A., B. M. Israel, V. P. Olivieri, and M. L. Granstrom Efficiency of chlorine dioxide as a bactericide. Appl. Microbiol. 13: Boonyakiat, D., P. M. Chen, R. A. Spotts, and D. G. Richardson Effect of harvest maturity on decay and postharvest life of 'd'anjou' pear. Sci. Hortic. 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