The effect of disinfectants on Clavibacter michiganensis subsp.
sepedonicus and Erwinia carotovora subsp. atroseptica on different surface materials
HilkkaKoponen, MerjaManninen, PirkkoHarju,Hanna Avikainen and Risto Tahvonen
Kotonen, H., Manninen, M., Harju,P., Avikainen, H. &Tahvonen, R. 1992.The effect of disinfectantsonClavibactermichiganensissubsp. sepedonicusand Erwinia carotovorasubsp. atrosepticaondifferent surface materials.Agric. Sei.Finl. 1:597- 602. (Dept. PI. Biol,, P.O. Box28,SF-00014UniversityofHelsinki, Finland, Techn.
Res. Centre ofFinland, Food Res. Lab.,SF-02151 Espoo,Finland and Agric.Res.
CentreofFinland,Inst.PI. Protect., SF-31600,Jokioinen,Finland.)
The effect ofsevendisinfectantsonClavibacter michiganensissubsp. sepedonicusand Erwinia carotovora subsp. atrosepticawastestedonmetal,plasticand wood surfaces in laboratory trials. lobac P was the most effective disinfectantin the control ofE.
carotovoraonclean anddirtysurfaces.Ipaseptand Menno-Ter-forte wereeffectiveon plasticsurfaces, but dirt reduced theirefficacy. The least effective preparationswere Deskem-1,Virkon S and Korsolin.
lobacP, Korsolin and Virkon S were the most effective disinfectants againstC.
michiganensis.Theefficacyof Ipasept and Menno-Ter-fortewasreduced bydirt. The least effective preparationwas Deskem-1,
Key words;disinfection, bacterial diseases ofpotato,organic material,potatoblack leg, potatoring 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 ringrotofpotato- es.The black leg disease,caused by Erwinia caro- tovora subsp. atroseptica (Bradbury 1986), has beenamajor problem in seedpotatoproduction in Finland during thepastfew years. Bacterial ringrot and black leg disease outbreaksare the result from theuseof infected seedpotatoes, the bacteria being
spread from diseasedtohealthy seed tubers during planting.
Many chemicals have been recommended for the disinfection ofstorages, equipment and othersur- faces contamined by C. michiganensis subsp. sepe- donicus and E. carotovora subsp. atroseptica (Dykstra 1941, Knorr 1947, MacLachlan 1960, Letal 1977,Secoretal. 1988). Secondary infection of C. michiganensis subsp. sepedonicus oninfested surfaces and equipment can be effect- ively eliminated byquaternaryammoniumoriodi- nebased compounds (Secor etal. 1988).
Quater-
Agric.Sei. Fint. 1(1992)
nary ammonium solutions have been usedextens- ively in thepotato industry for the disinfection of materials,equipment and storages that areused in the handling ofpotatoes.
Since the control of E. carotovora subsp. atro- septica and C. michiganensis subsp. sepedonicus depends on preventing reinfection, it is essential thatwehave effective bacterial disinfectantsatour disposal.
Thepresent experimentwas designedto deter- mine the efficacy ofsomedisinfectants against the ring rotand 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 andmethods
Seven disinfectants (Table 1)were tested against bacterial diseases using the concentrationsrecom- mended by the manufacturer. The disinfectants werediluted 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 surfacematerials, stainless steel(AISI316), polyethene plastic and pine wood discs ofadiame- terof approx. 1cm, were sterilized by autoclaving.
The discs were transferred aseptically onto24 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-peatwasmixed in 50 ml of saline and autoclaved. The discs werecontami- nated by applying2 ml of the mull-peat mixtureto the holes. The organic material was allowed to sedimentonthe surface of the discs. Theextrafluid wasremoved from all holes aseptically withaPas- teur’s pipette, and the plates with the discs were dried in an incubation chamberat +3O°C with the lid slightly open.
E. carotovorasubsp. atrosepticawas grown in Erwinia LB broth (tryptone 10 g,yeastextract5 g, NaCl 10 g, H2O 1000ml) for one day at +25°C.
The bacterial suspensions were diluted in fresh growth medium (1:10). Diluted bacterial suspensi- on (70 pi) wasadded ontothe surface of the discs.
The drops containing bacteria were allowed to stand at room temperature overnight (metal and plastic)orfor 15 min(wood).The wood discswere testedonthesameday the bacterial suspension had been applied. Any fluid remaining onthe surface of the metal and plastic discs wasremoved 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 allowedtostand overnightat+25°C. The fluidwas removed from the holes and bacteria were added once more and allowed to stand. Thereafter the discs weredried.
The disinfection efficacywastestedatroomtem- perature. Two ml of disinfection suspensionor sa- line (control) waspipetted in the holesso that the disc surfaces containing bacteria remained under
Disinfectant Activeingredient % Tested concentration
%
Deskem-1 Quaternaryammonium 0.2
compounds, 20.0
lobacP lodine, 1.8 3.0
Ipasept Quaternaryammonium 2.0
compounds, 2.8
Korsolin Glutaraldehyde, 10.0 3.0
Menno-Ter-Forte Quaternaryammonium 1.0 compounds, 32.5
Sodiumhypochlo- Activechlorine, 10.0 10.0 rite (NaOCl)
Virkon S Potassium 1.0
peroxysulphate, 60.0 Agric. Sd.Fint. 1(1992)
the fluid. The testwas made with three replicates and itwasrepeated three times.
The disinfectantwas allowedto actfor20min, 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 18ml, L-histidine0.5 g, Na-thiosulphate 4 g, 0.25 N phos- phate buffer (pH 7.2) 5 ml and H2O 500ml) for5 min toinactivate the disinfectant. The bacteria and organic material possibly occurring on the surface of the discswereremoved with cottonwool swabs.
The bacterial count of the suspension was deter- mined by surface cultureson agarplates. E. caroto- vora subsp. atroseptica was cultured on nutrient agarplates (Difco); the plates were incubated for 3-4 daysat+25°C. C. michiganensis subsp. sepedo- nicuswasculturedon yeastglucose mineral oil agar (Lelliott and Stead 1987)and incubated for6-7 days at+25°C. The trial consisted of three repli- catesand itwasrepeated three times. Virkon S was not tested atthe same timeas the other disinfect- ants,but the sametestmethodswere applied.
The efficacypercentage of disinfectantonbacte- ria was calculated using the formula:
(Nc-Nd)* 100 Nc
where Nc wasthe 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 afterex- posuretothe disinfectant. The valuesare meansof three replications.
Results
lobac P wasthe mosteffective preparation in the control of Erwinia carotovora subsp. atroseptica ondirty and cleanmetal,plastic and wood surfaces.
Menno-Ter-fortewas effectiveonplastic surfaces, but organic material reduced its efficiency. Des- kem-l was the least effectiveon all surfaces. Also
Virkon S (1%) and Korsolinwere ineffectiveon all surface materials against E.carotovorasubsp.atro- septica. Of the surface materialsused, plasticwas the easiest todisinfest(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 wereef- fective in the control of C. michiganensis subsp.
sepedonicus onmetal and plastic surfaces. Organic material impaired the efficiency of Ipasept and Menno-Ter-forte. The wood surfacewas moredif- ficult todisinfest than metal and plastic surfaces.
Korsolin, NaOCl and Virkon S were the most effective disinfectantson wood surface against C.
michiganensis subsp. sepedonicus on clean and dirty surfaces. The organic material did notreduce the efficacy ofKorsolin, NaOCl and Virkon S. The least effective disinfectant was Deskem-1 (Table 2).
Discussion
lobac P waseffective in the control of both Clavi- hacter michiganensis subsp. sepedonicus and Emi- niä carotovora subsp. atrosepticaonplastic, 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 waseffective against C. michiganensis subsp. sepedonicus, but the effectwasnot satisfac- toryenough against E.carotovorasubsp. atrosepti- ca. AccordingtoDinesen(1984),too, the efficacy ofKorsolin (2 %)is good against C. michiganensis subsp. sepedonicus when the exposure time is at least 10min.
In thepresentstudy, Menno-Ter-fortewaspoor- ly effective against E. carotovorasubsp. atrosepti- ca, but effective against C. michiganensis subsp.
sepedonicus. These results areconsistent with pre- vious data (Bång 1987).
Agric. Sei.Finl. 1(1992)
Table2.The effect of disinfectantsagainstErwiniacarotovorasubsp.atroseptica(E) and Clavibactermichiganensis subsp.
sepedonicus (C) onclean anddirty metal, plasticand wood surfaces
Surface Disinfectant Concentration Clean Dirty
material % E C E C
Efficiency %
Metal Deskem-1 0.2 11 74 0 10
lobacP 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
lobacP 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
lobacP 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 theefficacy of Menno- Ter-forte and Ipaseptonall the materials investigat- ed,whereas the efficacy of lobac P wasnotaffected by organic material. Also in other studies organic material has been showntoreduce 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 thepresent study thiswas notobserv- ed. The differences may be due tothe 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.
atrosepticaon 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 andMenno-Ter-forte)gave good control of C. michiganensis subsp.sepedoni- cus onclean metal and plastic surfaces,but organic material reduced the efficacy of the compounds.
A disinfectant applicable for the control of E.
carotovorasubsp. 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. Basedonthe data presented here,theuse of lobac P may be applicable for the disinfection of wood,plastic and metal surfaces contamined by E.
carotovorasubsp. atroseptica and C. michiganen- sis subsp. sepedonicus. However, it mustbe 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 Sarealso effect-
ive and NaOCl provided complete control against
C. michiganensis subsp. sepedonicus. Hypochlorite solution is also corrosivetometals,and thismustbe 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
resultsarebasedonlaboratorytestsand before it is posible torecommend any disinfectant for use in practice it should be tested under conditions simu- lating practical conditionsor in the field. Also the method of application should be tested,because it may affect the disinfectioncapacity.
References BAno,U, 1987.Försök med desinfektionsmedel. Sporpota-
tisodl.5,2; 32-34.
Bradbury,J.F. 1986.Guide toplant pathogenic bacteria.
CAB InternationalMycologicalInstitute. 332p.
Dinesen, I.G. 1984.Desinfektionsmidlers effektpå Coryne- hacterium sepedonicum (kartoflens ringbakteriose).
Tidsskr. Planteavl 88: 413-415.
& Loschenkohl, B. 1987.Desinfektionsmidlers effekt
undersmudsbelastning. 4.Danske Plantevasrnskonferen- ce.Sygdommeogskadedyr 1987: 93-100.
Dykstra, T,P. 1941.Results ofexperiment in control of bacterialringrotofpotatoes in 1940. Am.Potato J. 18;
27-55.
Knorr, L. C. 1947.Field testing of disinfectants for the control ofpotatoringrotbacteriaonwooden and metallic surfaces.Am.Potato J.24: 141-150.
Lelliott, R. A. & Stead, D. E. 1987. Methods for the diagnosisofbacterial diseases ofplants. MethodsinPlant Pathology. Blackwell Scientific Publications. Oxford.
Vol.2. 216p.
Letal, J.R. 1977. Efficacy of disinfectants againstpotato ringrotandblacklegbacteria.Am.Potato J.54:405-409.
LOSCHENKOHL, 8.,DINESEN, I. G. & Thinogaard,K. 1990.
Atpwvingafdesinfektionsmidler. Gartn. Tid. I()6:1324- 1325.
MacLachlan, D. S. 1960.Disinfectants and potatoring rot control.Am,Potato J.37: 325-337.
Secor, G. A., De Buhr, L. & Gudmestad, N. C. 1988.
SusceptibilityofCorynebacterium sepedonicum todes- infectantsinvitro. Plant Dis.72: 585-588.
Manuscriptreceived June 1992 Hilkka Koponen
Departmentof PlantBiology P.O.Box28
SF-00014UniversityofHelsinki,Finland MerjaManninen
PirkkoHarju
Technical Research Centre of Finland Food Research Laboratory
SF-02151Espoo,Finland Hanna Avikainen Risto Tahvonen
AgriculturalResearch Centre of Finland Institute of Plant Protection
SF-31600Jokioinen,Finland
Agric. Sei.Fint. 1 (1992)
SELOSTUS
Desinfiointiaineiden vaikutus Clavihacter michiganensis subsp. sepedonicus jaErwinia carotovorasubsp. atrosepticu bakteereihin eri pintamateriaaleilla
HilkkaKoponen, MerjaManninen, PirkkoHarju,Hanna Avikainen jaRisto Tahvonen
Helsingin yliopisto, Valtion teknillinen tutkimuskeskusjaMaatalouden tutkimuskeskus
Seitsemän desinfiointiaineen tehoa testattiin perunan vaa- learengasmätä-(Clavihactermichiganensis subsp. sepedoni- cus) japerunan tyvimätä-(Erwinia carotovorasubsp.alro- septica)bakteereihin liatuillaja puhtailla metalli-,muovi-ja puupinnoilla laboratoriokokeissa. Testattavat valmisteet oli- vatDeskem-1,Ipasept jaMenno-Ter-forte (kvartaarisia am- moniumyhdisteitä),lobacP(jodi),Korsolin(glutaraldehydi), natriumhypokloriitti (aktiivinenkloori)jaVirkon S (kalium- peroksisulfaatti).
lobacPoli tehokkain valmisteE.carotovorasubsp.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 kaikillapinnoilla. Myös 1% Virkon S ja Korsolin tehosivat bakteeriin huonosti. Pintamateriaaleista muovi olihelpoin puhdistaa.
C. michiganensis subsp.sepedonicusoli desinfiointiaineil-
le herkempi kuinE. carotovorasubsp. atroseptica.lobacP, Ipasept, Korsolin,Menno-Ter-fortejaVirkon S olivat tehok- kaita C. michiganensissubsp. sepedonicus-bakteeria vastaan metalli-ja muovipinnoilla. Lika heikensiIpaseptin jaMenno- Ter-forten tehoa.Puupinta olivaikeampi puhdistaakuinme-
talli-ja muovipinnat. Parhaitenpuupinnalla toimivat Korso- lin,NaOCljaVirkon S. Teholtaan heikoin valmiste oli Des- kem-1.
Tehtyjen kokeiden perusteella vaalearengasmatä- ja tyvi- mätäbakteerien desinfiointiinperunavarastoissavoidaan suo- sitella lobac P-valmistetta. Sitäkäytettäessätulee ottaa huo- mioon aineenmetalleja(alumiini,kupari, messinki, galvanoi- tupelti)syövyttävä vaikutus. Metallipinnat eivätsaaolla val- misteen kanssa kosketuksissayli 20minuuttia.Korsolin, Vir- kon S ja NaOCl-valmisteet soveltuvat vaalearengasmätäbak- teerin puhdistukseen. Myös natriumhypokloriitti syövyttää metallipintoja.
Agric. Sei.Fin!. 1(1992)
