The effect of disinfectants on Clavibacter michiganensis subsp. sepedonicus and Erwinia carotovora subsp. atroseptica on different surface materials

The effect of on and on different 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 andMenno-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, consistent


Introduction
Bacterial ring rot of potatoes, caused by the bacterium Clavibacter michiganensis subsp. sepedonicus (Bradbury 1986), has caused sporadic but destructive outbreaks ofbacterial ring rot of potatoes. The black leg disease, caused by Erwinia carotovora 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 surfaces contamined by C. michiganensis subsp. sepedonicus 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 effectively eliminated by quaternary ammonium or iodine based compounds (Secor et al. 1988). Quater-nary ammonium solutions have been used extensively 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. atroseptica 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 determine the efficacy of some disinfectants against the ring rot and black leg organisms with and withouth organic material on wood, metal and plastic surfaces of equipment and materials used in the handling of seed potatoes.

Material and methods
Seven disinfectants (Table 1) were tested against bacterial diseases using the concentrations recommended by the manufacturer. The disinfectants were diluted in saline.
The surface materials, stainless steel (AISI 316), polyethene plastic and pine wood discs ofa diameter 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 contaminated 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 Pasteur'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 suspension (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 temperature. Two ml of disinfection suspension or saline (control) was pipetted in the holes so that the disc surfaces containing bacteria remained under 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 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 inactivation solution (lecithin 1.5 g. Tween 80 18 ml, L-histidine 0.5 g, Na-thiosulphate 4 g, 0.25 N phosphate 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 determined by surface cultures on agar plates. E. carotovora subsp. atroseptica was cultured on nutrient agar plates (Difco); the plates were incubated for 3-4 days at +25°C. C. michiganensis subsp. sepedonicus 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 replicates and it was repeated three times. Virkon S was not tested at the same time as the other disinfectants, but the same test methods were applied.
The efficacy percentage of disinfectant on bacteria was calculated using the formula: 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. Deskem-l was the least effective on all surfaces. Also Virkon S (1%) and Korsolin were ineffective on all surface materials against E. carotovora subsp. atroseptica. 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 effective 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 difficult 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 ofKorsolin, NaOCl and Virkon S. The least effective disinfectant was Deskem-1 (Table   2). Discussion lobac P was effective in the control of both Clavihacter michiganensis subsp. sepedonicus and Eminiä 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 satisfactory enough against E. carotovora subsp. atroseptica. According to Dinesen (1984), too, the efficacy ofKorsolin (2 %) is good against C. michiganensis subsp. sepedonicus when the exposure time is at least 10min.
In the present study, Menno-Ter-forte was poorly effective against E. carotovora subsp. atroseptica, but effective against C. michiganensis subsp. sepedonicus. These results are consistent with previous data (Bång 1987). Organic material reduced the efficacy of Menno-Ter-forte and Ipasept on all the materials investigated, 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 observed. 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 disinfection 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. sepedonicus 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. michiganensis 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. michiganensis 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 effective 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 organic 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 simulating practical conditions or in the field. Also the method of application should be tested, because it may affect the disinfection capacity. Tehtyjen kokeiden perusteella vaalearengasmatä-ja tyvimätäbakteerien desinfiointiin perunavarastoissa voidaan suositella lobac P-valmistetta. Sitä käytettäessä tulee ottaa huomioon aineen metalleja (alumiini, kupari, messinki, galvanoitu pelti) syövyttävä vaikutus. Metallipinnat eivät saa olla valmisteen kanssa kosketuksissa yli 20 minuuttia. Korsolin, Virkon S ja NaOCl-valmisteet soveltuvat vaalearengasmätäbakteerin puhdistukseen. Myös natriumhypokloriitti syövyttää metallipintoja.