The effect of disinfectants on Clavibacter michiganensis subsp. sepedonicus and Erwinia carotovora subsp. atroseptica on different surface materials Hilkka Koponen, Merja Manninen, Pirkko Harju, Hanna Avikainen and Risto Tahvonen Kotonen, H., Manninen, M., Harju, P., Avikainen, H. & Tahvonen, R. 1992. The effect of disinfectants on Clavibacter michiganensis subsp. sepedonicus and Erwinia carotovora subsp. atroseptica on different surface materials. Agric. Sei. Finl. 1:597- 602. (Dept. PI. Biol,, P. O. Box 28, SF-00014 University of Helsinki, Finland, Techn. Res. Centre of Finland, Food Res. Lab., SF-02151 Espoo, Finland and Agric. Res. Centre of Finland, Inst. PI. Protect., SF-31600, Jokioinen, Finland.) The effect of seven disinfectants on Clavibacter michiganensis subsp. sepedonicus and Erwinia carotovora subsp. atroseptica was tested on metal, plastic and wood surfaces in laboratory trials. lobac P was the most effective disinfectant in the control of E. carotovora on clean and dirty surfaces. Ipasept and Menno-Ter-forte were effective on plastic surfaces, but dirt reduced their efficacy. The least effective preparations were Deskem-1, Virkon S and Korsolin. lobac P, Korsolin and Virkon S were the most effective disinfectants against C. michiganensis. The efficacy of Ipasept and Menno-Ter-forte was reduced by dirt. The least effective preparation was Deskem-1, Key words; disinfection, bacterial diseases of potato, organic material, potato black leg, potato ring rot Introduction Bacterial ring rot of potatoes, caused by the bac- terium Clavibacter michiganensis subsp. sepedoni- cus (Bradbury 1986), has caused sporadic but destructive outbreaks ofbacterial ring rot of potato- es. The black leg disease, caused by Erwinia caro- tovora subsp. atroseptica (Bradbury 1986), has been a major problem in seed potato production in Finland during the past few years. Bacterial ring rot and black leg disease outbreaks are the result from the use of infected seed potatoes, the bacteria being spread from diseased to healthy seed tubers during planting. Many chemicals have been recommended for the disinfection of storages, equipment and other sur- faces contamined by C. michiganensis subsp. sepe- donicus and E. carotovora subsp. atroseptica (Dykstra 1941, Knorr 1947, MacLachlan 1960, Letal 1977, Secor et al. 1988). Secondary infection of C. michiganensis subsp. sepedonicus on infested surfaces and equipment can be effect- ively eliminated by quaternary ammonium or iodi- ne based compounds (Secor et al. 1988). Quater- 597 Agric. Sei. Fint. 1 (1992) nary ammonium solutions have been used extens- ively in the potato industry for the disinfection of materials, equipment and storages that are used in the handling of potatoes. Since the control of E. carotovora subsp. atro- septica and C. michiganensis subsp. sepedonicus depends on preventing reinfection, it is essential that we have effective bacterial disinfectants at our disposal. The present experiment was designed to deter- mine the efficacy of some disinfectants against the ring rot and black leg organisms with and withouth organic material on wood, metal and plastic sur- faces of equipment and materials used in the handl- ing of seed potatoes. Material and methods Seven disinfectants (Table 1) were tested against bacterial diseases using the concentrations recom- mended by the manufacturer. The disinfectants were diluted in saline. One-to two-day old cultures of Clavibacter michiganensis subsp. sepedonicus (NCPPB 2137) and Erwinia carotovora subsp. atroseptica (NCPPB 549) obtained from the National Codec- Table 1. The disinfectants tested for control of bacterial diseases. tion of Plant Pathogenic Bacteria, England, were used. The surface materials, stainless steel (AISI 316), polyethene plastic and pine wood discs ofa diame- ter of approx. 1 cm, were sterilized by autoclaving. The discs were transferred aseptically onto 24 hole Limbro plates. The plastic and wood discs were fixed onto the bottom of the plates using sterile adhesive material. The organic material used was a mull-peat mixture. One gram ofmull-peat was mixed in 50 ml of saline and autoclaved. The discs were contami- nated by applying 2 ml of the mull-peat mixture to the holes. The organic material was allowed to sediment on the surface of the discs. The extra fluid was removed from all holes aseptically with a Pas- teur’s pipette, and the plates with the discs were dried in an incubation chamber at +3O°C with the lid slightly open. E. carotovora subsp. atroseptica was grown in Erwinia LB broth (tryptone 10 g, yeast extract 5 g, NaCl 10 g, H2O 1000 ml) for one day at +25°C. The bacterial suspensions were diluted in fresh growth medium (1:10). Diluted bacterial suspensi- on (70 pi) was added onto the surface of the discs. The drops containing bacteria were allowed to stand at room temperature overnight (metal and plastic) or for 15 min (wood). The wood discs were tested on the same day the bacterial suspension had been applied. Any fluid remaining on the surface of the metal and plastic discs was removed by drying them in incubators (+3O °C). If a sufficient number of bacteria was not obtained by allowing the drops to stand on the surface, 2 ml of bacterial suspension (1:10 in fresh growth medium) was added into each hole and allowed to stand overnight at +25°C. The fluid was removed from the holes and bacteria were added once more and allowed to stand. Thereafter the discs were dried. The disinfection efficacy was tested at room tem- perature. Two ml of disinfection suspension or sa- line (control) was pipetted in the holes so that the disc surfaces containing bacteria remained under Disinfectant Active ingredient % Tested concentration % Deskem-1 Quaternary ammonium 0.2 compounds, 20.0 lobac P lodine, 1.8 3.0 Ipasept Quaternary ammonium 2.0 compounds, 2.8 Korsolin Glutaraldehyde, 10.0 3.0 Menno-Ter-Forte Quaternary ammonium 1.0 compounds, 32.5 Sodium hypochlo- Active chlorine, 10.0 10.0 rite (NaOCl) Virkon S Potassium 1.0 peroxysulphate, 60.0 598 Agric. Sd. Fint. 1 (1992) the fluid. The test was made with three replicates and it was repeated three times. The disinfectant was allowed to act for 20 min, whereafter the fluid was removed and the discs were placed into tubes containing 1.5 ml of inac- tivation solution (lecithin 1.5 g. Tween 80 18 ml, L-histidine 0.5 g, Na-thiosulphate 4 g, 0.25 N phos- phate buffer (pH 7.2) 5 ml and H2O 500 ml) for 5 min to inactivate the disinfectant. The bacteria and organic material possibly occurring on the surface of the discs were removed with cottonwool swabs. The bacterial count of the suspension was deter- mined by surface cultures on agar plates. E. caroto- vora subsp. atroseptica was cultured on nutrient agar plates (Difco); the plates were incubated for 3-4 days at +25°C. C. michiganensis subsp. sepedo- nicus was cultured on yeast glucose mineral oil agar (Lelliott and Stead 1987) and incubated for 6-7 days at +25°C. The trial consisted of three repli- cates and it was repeated three times. Virkon S was not tested at the same time as the other disinfect- ants, but the same test methods were applied. The efficacy percentage of disinfectant on bacte- ria was calculated using the formula: (Nc-Nd)* 100 Nc where Nc was the number of colony forming units developed in the control series in which the disin- fectant was replaced by saline, and Nd was the number of colony-forming units counted after ex- posure to the disinfectant. The values are means of three replications. Results lobac P was the most effective preparation in the control of Erwinia carotovora subsp. atroseptica on dirty and clean metal, plastic and wood surfaces. Menno-Ter-forte was effective on plastic surfaces, but organic material reduced its efficiency. Des- kem-l was the least effective on all surfaces. Also Virkon S (1%) and Korsolin were ineffective on all surface materials against E. carotovora subsp. atro- septica. Of the surface materials used, plastic was the easiest to disinfest (Table 2). Clavibacter michiganensis subsp. sepedonicus was more susceptible to the disinfectants than E. carotovora subsp. atroseptica. lobac P, Ipasept, Korsolin, Menno-Ter-forte and Virkon S were ef- fective in the control of C. michiganensis subsp. sepedonicus on metal and plastic surfaces. Organic material impaired the efficiency of Ipasept and Menno-Ter-forte. The wood surface was more dif- ficult to disinfest than metal and plastic surfaces. Korsolin, NaOCl and Virkon S were the most effective disinfectants on wood surface against C. michiganensis subsp. sepedonicus on clean and dirty surfaces. The organic material did not reduce the efficacy of Korsolin, NaOCl and Virkon S. The least effective disinfectant was Deskem-1 (Table 2). Discussion lobac P was effective in the control of both Clavi- hacter michiganensis subsp. sepedonicus and Emi- niä carotovora subsp. atroseptica on plastic, metal and wood surfaces. lobac P has been shown also in previous studies very effective in the control of C. michiganensis subsp. sepedonicus (Dinesen 1984, Bång 1987) and E. carotovora subsp. atroseptica (Bång 1987). Korsolin was effective against C. michiganensis subsp. sepedonicus, but the effect was not satisfac- tory enough against E. carotovora subsp. atrosepti- ca. According to Dinesen (1984), too, the efficacy of Korsolin (2 %) is good against C. michiganensis subsp. sepedonicus when the exposure time is at least 10 min. In the present study, Menno-Ter-forte was poor- ly effective against E. carotovora subsp. atrosepti- ca, but effective against C. michiganensis subsp. sepedonicus. These results are consistent with pre- vious data (Bång 1987). 599 Agric. Sei. Finl. 1 (1992) 600 Table 2. The effect of disinfectants against Erwinia carotovora subsp. atroseptica (E) and Clavibacter michiganensis subsp. sepedonicus (C) on clean and dirty metal, plastic and wood surfaces Surface Disinfectant Concentration Clean Dirty material % E C E C Efficiency % Metal Deskem-1 0.2 11 74 0 10 lobac P 3 82 100 88 100 Ipasept 2 63 100 0 35 Korsolin 3 4 100 28 100 Menno-Ter-forte 1 38 100 23 46 NaOCl 10 57 66 76 95 Virkon S 1 10 7 38 100 Plastic Deskem-1 0.2 0 74 0 2 lobac P 3 100 100 100 100 Ipasept 2 87 100 42 55 Korsolin 3 16 100 22 100 Menno-Ter-forte 1 % 100 76 65 NaOCl 10 33 65 46 100 Virkon S I 34 100 3 100 Wood Deskem-1 0.2 0 12 0 22 lobac P 3 100 20 100 1(K) Ipasept 2 59 73 45 10 Korsolin 3 3 100 14 100 Menno-Ter-forte I 44 5 38 19 NaOCl 10 23 100 28 90 Virkon S 1 10 100 5 100 Organic material reduced the efficacy of Menno- Ter-forte and Ipasept on all the materials investigat- ed, whereas the efficacy of lobac P was not affected by organic material. Also in other studies organic material has been shown to reduce the efficacy of disinfectants against bacteria (DINESEN and LOSCHENKOHL 1987), the efficacy of NaOCl and iodine based disinfectants in particular (Secor et al. 1988). In the present study this was not observ- ed. The differences may be due to the fact that the bacteria were tested in the presence of dry growth medium. On the basis of this and other studies (Letal 1977), quaternary ammonium solutions are not suitable for the control of E. carotovora subsp. atroseptica on wood and metal surfaces although these have been earlier recommended for disinfec- tion of black leg bacteria from these surfaces (Mac- Lachlan 1960). In this study, quaternary ammo- nium solutions (Ipasept and Menno-Ter-forte) gave good control of C. michiganensis subsp. sepedoni- cus on clean metal and plastic surfaces, but organic material reduced the efficacy of the compounds. A disinfectant applicable for the control of E. carotovora subsp. atroseptica and C. michiganen- sis subsp. sepedonicus should act also on dirty wood and metal surfaces, because organic material is always present in places where potato is being handled. Based on the data presented here, the use of lobac P may be applicable for the disinfection of wood, plastic and metal surfaces contamined by E. carotovora subsp. atroseptica and C. michiganen- sis subsp. sepedonicus. However, it must be taken into consideration that lobac P solution is corrosive to metals and an iodine based disinfectant may cause allergic reactions to the user in a confined environment. Korsolin and Virkon S are also effect- ive and NaOCl provided complete control against C. michiganensis subsp. sepedonicus. Hypochlorite solution is also corrosive to metals, and this must be taken into consideration when used. Before disinfection the surfaces should be cleaned from mull and plant debris, because organ- ic material reduces the disinfection capacity. These results are based on laboratory tests and before it is posible to recommend any disinfectant for use in practice it should be tested under conditions simu- lating practical conditions or in the field. Also the method of application should be tested, because it may affect the disinfection capacity. References BAno, U, 1987. Försök med desinfektionsmedel. Spor pota- tisodl. 5,2; 32-34. Bradbury, J. F. 1986. Guide to plant pathogenic bacteria. CAB International Mycological Institute. 332 p. Dinesen, I. G. 1984. Desinfektionsmidlers effekt på Coryne- hacterium sepedonicum (kartoflens ringbakteriose). Tidsskr. Planteavl 88: 413-415. & Loschenkohl, B. 1987. Desinfektionsmidlers effekt under smudsbelastning. 4. Danske Plantevasrnskonferen- ce. Sygdomme og skadedyr 1987: 93-100. Dykstra, T, P. 1941. Results of experiment in control of bacterial ring rot of potatoes in 1940. Am. Potato J. 18; 27-55. Knorr, L. C. 1947. Field testing of disinfectants for the control ofpotato ring rot bacteria on wooden and metallic surfaces. Am. Potato J. 24: 141-150. Lelliott, R. A. & Stead, D. E. 1987. Methods for the diagnosis ofbacterial diseases ofplants. Methods in Plant Pathology. Blackwell Scientific Publications. Oxford. Vol. 2. 216 p. Let al, J. R. 1977. Efficacy of disinfectants against potato ring rot and blackleg bacteria. Am. Potato J. 54:405-409. LOSCHENKOHL, 8., DINESEN, I. G. & Thinogaard, K. 1990. Atpwvingaf desinfektionsmidler. Gartn. Tid. I()6: 1324- 1325. MacLachlan, D. S. 1960. Disinfectants and potato ring rot control. Am, Potato J. 37: 325-337. Secor, G. A., De Buhr, L. & Gudmestad, N. C. 1988. Susceptibility of Corynebacterium sepedonicum to des- infectants in vitro. Plant Dis. 72: 585-588. Manuscript received June 1992 Hilkka Koponen Department of Plant Biology P. O. Box 28 SF-00014 University of Helsinki, Finland Merja Manninen Pirkko Harju Technical Research Centre of Finland Food Research Laboratory SF- 02151 Espoo, Finland Hanna Avikainen Risto Tahvonen Agricultural Research Centre of Finland Institute of Plant Protection SF-31600 Jokioinen, Finland 601 Agric. Sei. Fint. 1 (1992) SELOSTUS Desinfiointiaineiden vaikutus Clavihacter michiganensis subsp. sepedonicus ja Erwinia carotovora subsp. atrosepticu bakteereihin eri pintamateriaaleilla Hilkka Koponen, Merja Manninen, Pirkko Harju, Hanna Avikainen jaRisto Tahvonen Helsingin yliopisto, Valtion teknillinen tutkimuskeskus ja Maatalouden tutkimuskeskus Seitsemän desinfiointiaineen tehoa testattiin perunan vaa- learengasmätä- ( Clavihacter michiganensis subsp. sepedoni- cus) ja perunan tyvimätä- ( Erwinia carotovora subsp. alro- septica) bakteereihin liatuilla ja puhtailla metalli-, muovi- ja puupinnoilla laboratoriokokeissa. Testattavat valmisteet oli- vat Deskem-1, Ipasept ja Menno-Ter-forte (kvartaarisia am- moniumyhdisteitä), lobac P (jodi), Korsolin (glutaraldehydi), natriumhypokloriitti (aktiivinen kloori) ja Virkon S (kalium- peroksisulfaatti). lobac P oli tehokkain valmiste E. carotovora subsp. atro- septica- bakteeria vastaan likaisilla ja puhtailla metalli-, muovi- ja puupinnoilla. Menno-Ter-forte oli melko tehokas muovipinnoilla, mutta lika heikensi sen tehoa. Deskem-1 :n teho oli huonoin kaikilla pinnoilla. Myös 1 % Virkon S ja Korsolin tehosivat bakteeriin huonosti. Pintamateriaaleista muovi oli helpoin puhdistaa. C. michiganensis subsp. sepedonicus oli desinfiointiaineil- le herkempi kuin E. carotovora subsp. atroseptica. lobac P, Ipasept, Korsolin, Menno-Ter-forte ja Virkon S olivat tehok- kaita C. michiganensis subsp. sepedonicus- bakteeria vastaan metalli- ja muovipinnoilla. Lika heikensi Ipaseptin ja Menno- Ter-forten tehoa. Puupinta oli vaikeampi puhdistaa kuin me- talli- ja muovipinnat. Parhaiten puupinnalla toimivat Korso- lin, NaOCl ja Virkon S. Teholtaan heikoin valmiste oli Des- kem-1. Tehtyjen kokeiden perusteella vaalearengasmatä- ja tyvi- mätäbakteerien desinfiointiin perunavarastoissa voidaan suo- sitella lobac P-valmistetta. Sitä käytettäessä tulee ottaa huo- mioon aineen metalleja (alumiini, kupari, messinki, galvanoi- tu pelti) syövyttävä vaikutus. Metallipinnat eivät saa olla val- misteen kanssa kosketuksissa yli 20 minuuttia. Korsolin, Vir- kon S ja NaOCl-valmisteet soveltuvat vaalearengasmätäbak- teerin puhdistukseen. Myös natriumhypokloriitti syövyttää metallipintoja. 602 Agric. Sei. Fin!. 1 (1992)