The effect of disinfectants on fungi in pure culture and on different surface materials

The effect of eight disinfectants was tested on 11 fungi. The concentrations recommended by the manufacturers were mostly used in the present trials. Five and 15 min treatment times were too short for most preparations. The best efficacy was achieved at 60 min treatment lime. In pure cultures, all disinfectants tested were most effective against Pythium sp. Overall, the most effective preparation was Desinfektol EL which was effective against all fungi tested at 5 min treatment time. Wood surfaces were more difficult to disinfest than metal and plastic surfaces. Rhizoctonia solani and Verticillium dahliae were highly susceptible only to Desinfektol EL, and NaOCl controlled Pythium sp. and Botrytis cinerea. None of the preparations controlled completely the other test fungi. On metal surfaces the most effective disinfectants were Desinfektol EL and NaOCl; moderately effective were lobac P and Menno-Ter-forte. Korsolin was the least effective preparation. Verticillium dahliae and Phomopsis sderotioides were difficult to kill. The easiest fungi to disinfest were Pythium sp., Rhizoctonia solani, B. cinerea, Fusarium avenaceum, F. culmorum, F. oxysporum, Mycocentrospora acerina and Phoma foveata. Fungi grown on plastic surfaces were best controlled with Desinfektol EL. Also NaOCl, Menno-Ter-forte and lobac P were effective. Korsolin was the least effective preparation. The easiest fungi to eradicate were Pythium sp. and R. solani. The most difficult fungi were V. dahliae and P. sderotioides. Both peat and clay deteriorated the efficacy of the disinfectants except for Desinfektol EL.


Introduction
Farmers and agricultural advisors have been inquiring about the applicability of disinfectants for disin- fection of greenhouses and storages.There are sev- eral commercial disinfectants on the market.Many of the preparations available are intended for use in hospitals, food industry and domestic animal farms, less frequently for disinfection of greenhouses and storages.
There is little experimental data in the literature on the effect of disinfectants on plant pathogenic fungi and their use for control of plant pathogens (Brielmaier 1985, bång 1987, Böhmer 1983, LOSCHENKOHL et ai. 1990ai. , SUNDHEIM 1989)).
In greenhouses it is not necessary to disinfect the soil, because the growth substrate is changed after each growing period and plastic containers are used.However, it is still important to disinfect the benches and equipment, because many plant pathogens may survive for long periods of time in struc- tures and plant debris, even in soil, until the next growing season.Important plant diseases of cu- cumber and tomato seedlings in greenhouse include Pythium spp.and Rhizoctonia solani; diseases oc- curing later in the roots and stems of older plants include Phomopsis sclerotioides, Didymella bryoniae, Verticillium dahliae, Fusarium spp., Botrytis cinerea and Sclerotinia sclerotiorum (Fletcher 1984).These pathogens may also be carried over from one place to another by means of the equipment and pots.
The present study was undertaken in vitro to determine the effect of some commonly used disin- fectants on plant pathogenic fungi in pure culture and on wood, plastic and metal surfaces, as well as the effect of peat and clay on the efficacy of disin- fection.Some of the commonly used disinfectants, e.g.formaldehyde, are hazardous to the user's health.Therefore we tried to find altematives.The effect of disinfectants on germination of fungal sclerotia was also tested as surviving structures of fungi are more resistant than the mycelium and the conidia.An effective disinfectant to control plant pathogenic fungi in greenhouses and winter stor- ages ofpotatoes and vegetables was sought.

Disinfectants
A total of eight compounds (Table 1) were evaluat- ed for their effectiveness in killing fungi in vitro.The concentrations used in the trials were those recommended by the manufacturers.Any devi- ations from these concentrations are specified in the tables.The disinfectants were diluted in water.

Test fungi
Eleven fungi, Botrytis cinerea, Didymella bryoniae, Fusarium avenaceum (only in cloth and wood surface tests), F. culmorum, F. oxysporum, Mycocentrospora acerina, Phoma foveata, Phomopsis sclerotioides, Pythium sp., Rhizoctonia solani and Verticillium dahliae from the collection of the Department of Plant Biology, Section of Plant Patho- logy (HPP), University of Helsinki, were tested against disinfectants in the vegetative phase.The age of inoculum was about three-fourweeks.So the mycelia and the spores were well developed, but there were no resting bodies except chlamydospores or microsclerotia in F. oxysporum, M. aceri- na, P. sclerotioides and V. dahliae.The effect of the disinfectants on the germination of sclerotia was also tested on B. cinerea, R. solani, Sclerotium cepivorum, Sclerotinia sclerotiorum and Typhula sp.The names of the fungi are mainly according to Domsch et al. (1980).

Cloth test
In the cloth test the fungi were grown on synthetic (nylon voile) gauze pieces (size approx. 1 x 1 cm) on PDA plates at room temperature in normal daylight.When visible fungal mycelium was spread from the inoculum plug to the cloth pieces on PDA surface, they were immersed in the test solutions for 5, 15, 30 and 60 min.After treatment the upper side of the fungi growing test material was placed upwards on PDA, each on a separate plate.The treatments were replicated five times with four plate per replicate.Sterile water was used as a control treatment.The plates were tabulated when the culture in the control treatment covered the entire plate.The results were evaluated on a rating scale of 0-2: o=no growth, l=growth inhibit- ed, 2=growth like in the control treatment.The figures were converted to efficiency percentages.

Sclerotia! test
With the exception of Typhula sp. which was grown at +5°C, the fungi were grown at room tem- perature on PDA plates to produce sclerotia.The sclerotia ofRhizoctonia solani were grown on cabbage pieces.The sclerotia (20 pcs) in a gauze bag were immersed in the disinfection solutions for 60 min.After treatment each sclerotium was placed on a plate of its own for evaluation of viability of the sclerotia.The results were evaluated when the control culture covered the entire plate.

Surface material
The tested surface materials were untreated pine, stainless steel and polyethene plastic discs.They were approx. 3mm in thickness and approx. 1 cm in diameter.The discs were sterilized by autoclav- ing (wood and metal) or with 90% ethanol (plastic).The test fungi were grown on these surfaces as described above in the cloth test and immersed in the test solution for 30 and 60 min.The results were evaluated as + (fungal growth) or as -(no fungal growth).The figures in the tables are efficiency percentages.
The effect of organic material (peat and clay) on the disinfection efficiency was evaluated on polyethene plastic and steel surfaces.Plastic and steel discs approx. 1 cm in diameter and approx.3 mm in thickness were used.The discs were contaminated with sterilized peat (15 g peat/5 dl water) and clay (80 g clay soil/8 dl water) suspension which was allowed to dry on the surface of the discs.The tests were carried out as described above in the surface material test except that the exposure time was 60 min.

Results
In the cloth test the most effective disinfectant was Desinfektol EL which completely inhibited the growth of all test fungi.Moderately effective disin- fectants were also lobac P, Menno-Ter-forte and NaOCl, although the growth inhibiting effect was not consistent in all fungi.Ipasept, Korsolin, Talo- set and Virkon S (1%) were weak disinfectants (Table 2).
In the cloth test the disinfectants were very effective against Pythium sp. and moderately effective against mycelia of Rhizoctonia solani.Most fungi were, however, difficult to eradicate.These include e.g.Verticillium dahliae and Mycocentrospora acerina.A treatment time of 5 or 15 min was too short.The most effective treatment time was 60 min (Table 2).
The metal surface was effectively disinfested from fungi with Desinfektol EL and NaOCl.Menno-Ter-forte and lobac P were effective against all test fungi except V. dahliae and Pho- mopsis sclerotioides (Table 5).
Organic material (peat and clay) did not reduce the efficacy of Desinfektol EL and NaOCl on metal surfaces.lobac P and Menno-Ter-forte were also moderately effective on surfaces contaminated with peat.Peat did not reduce the efficacy of the disin- fectants against Pythium sp, R. solani.Ftisarium oxysporum, F. culmorum and Phoma foveata (Table 5).
Clay reduced most the efficacy of Ipasept, Kor- solin, Menno-Ter-forte and Taloset on metal sur- faces.V. dahliae and B. cinerea were difficult to eradicate.Inversely, metal surfaces contaminated with clay were easy to disinfest from Pythium sp. and rather easy from D. bryoniae, F. culmorum and R. solani (Table 5).Metal surfaces contaminated with clay were more difficult to disinfest than metal surfaces contaminated with peat.
The plastic discs were effectively disinfested from fungi with Desinfektol EL and NaOCl; slightly less effective disinfectants were lobac P and Menno-Ter-forte.Pythium sp., Mycocentrospora acerina and R. solani were highly susceptible to disinfectants (Table 6).
The plastic discs contaminated with peat were most effectively disinfested with Desinfektol EL and Menno-Ter-forte; effective disinfectants were also lobac P and NaOCl.Weak, disinfectants were Ipasept, Taloset and Korsolin (Table 6).Pythium sp. and P. foveata were easy to eradicate, whereas V. dahliae, P. sclerotioides and F. oxysporum were not susceptible to disinfectants (Table 6).

Discussion
In the laboratory tests of BÖHMER (1983), Menno- Ter-forte (0.5%) has been shown to inhibit the growth of Fusahum oxysporum conidia at 5 min treatment, but NaOCl (1%) did not inhibit the growth of the fungus.In the present laboratory tests, 70 % control was achieved over/ 7 , oxysporum Table 5.The effect of disinfectants and organic material on fungi on metal surfaces.Treatment time 60 min.Disinfectants: 1 = Desinfektol EL, 2 = lobac P, 3 = Ipasept, 4 = Korsolin, 5 = Menno-Ter-forte, 6 = NaOCI, 7 = Taloset 3 %, 8 = Virkon S. The recommended concentrations of the disinfectant (e.g.Menno-Ter-forte) were shown to con- trol the conidia of F. avenaceum, F. culmorum, F. oxysporum and Verticillium dahliae after 10 min treatment (Brielmaier 1985).In the present labor- atory tests, Menno-Ter-forte did not kill F. äveriä- ceum and V. dahliae after 60 min treatment.This is probably due to the fact the fungal cultures used in the trials contained in addition to conidia fungal mycelia and chlamydospores which are more re- sistant to disinfectants than the conidia.
In the present tests, the disinfectants were most effective against Pythium sp.In Oomycetes the chemical composition of the cell wall is different from that of Deuteromycetes or Ascomycetes, which is probably the reason for the differences in efficacy between the fungal species.Also in other studies Oomycetes have been shown more suscept- ible to disinfectants than Deuteromycetes (BAANDRUP 1983).In an agar test carried out in Sweden, Korsolin (1%) and Menno-Ter-forte (0.5%) did not inhibit the growth of Botrytis cine- rea, Didymella bryoniae and Phomopsis scierotioi- des at I min treatment time (JOHANSSON 1985).In the present study, 60 min treatment time was too short for Korsolin to kill the fungi, whereas for Menno-Ter-forte (1%) 60 min exposure time was quite sufficient to kill D. hryoniae and P. scleroti- oides.The results show that Menno-Ter-forte requires at least 60 min exposure time and the con- centration ofcompound should not to be lower than 1 %.
In the present study, the sclerotia of the fungi proved very resistant to disinfectants.None of the disinfectants provided complete control over the sclerotia ofSclerotium cepivorum, Sclerotinia scle- rotiorum and Rhizoctonia solani.Only Desinfektol EL inhibited the germination of sclerotia ofB.cine- rea and S. sclerotiorum and NaOCl that of B. cine- rea and Typhula sp. at 60 min treatment time.A treatment time of 60 min with Menno-Ter-forte (2%) did not prevent the growth of S. sclerotiorum although, according to BÖHMER (1985), soaking of sclerotia for 2 h in Menno-Ter-forte (1%) inhibited their growth.It is probably more important to use an exposure time ofat least 2 h for sclerotial control than higher concentration.During the longer exposure time the disinfectant will penetrate deeper in the tissues of sclerotia than during a short exposure time.Desinfektol EL may be recommended to control sclerotia of B. cinerea and S. sclerotiorum, and NaOCl to control B. cinerea and Typhula sp. at one hour exposure time.
Fungi growing on wood surface were difficult to disinfest.Only Desinfektol EL controlled com- pletely V. dahliae and R. solani, and NaOCl Pythium sp. and B. cinerea.Other disinfectants did not give complete control over the test fungi.Studies carried out in Sweden have shown that lobac P (3 and 5%) and Menno-Ter-forte (1 and 5%) do not disinfest wood pieces infected with Phoma and Fusarium (BÅNG 1987).Also in this study lobac P and Menno-Ter-forte were ineffective against P. foveata, F. avenaceum, F. culmorum and F. oxy sporum growing on wood surface.
In the present study, Korsolin proved to be a weak disinfectant.It did not kill fungi growing on wood surface.Also in other studies (SUNDHEIM 1989) Korsolin has been shown ineffective against fungi growing on wood surfaces.
In studies carried out in Norway, Menno-ter- forte (5 %) has been effective on wood surface against B. cinerea, but ineffective against P. sclero- tioides and D. hryoniae (SUNDHEIM 1989).In the present study, the recommended concentration (1 %) of Menno-Ter-forte was ineffective on wood surface against all test fungi.How long the fungus has grown on the wood surface will affect besides disinfectant concentration the efficacy of disinfec- tion as the mycelium of fungi will partly grow in tree tissues, causing differences in test results.
The present study showed that organic material reduces the efficacy of most disinfectants.It is therefore important to clean thoroughly the sur- faces before disinfection.In the laboratory tests, Desinfektol EL proved to be the most effective disinfectant on wood, steel andplastic surfaces contaminated with peat and clay.Not even organic material reduced the efficiency of the preparation.
In the present study, the test fungi cultures were under one month old, and the viability of the co- nidia and the mycelia was very high.In practice the situation is very different, the fungal spores, mycelia and resting bodies in cold storages and green- houses are not freshly grown, and the viability of pathogens has probably decreased.Thus, disinfect- ants with a wide effect on fungi e.g.NaOCl, lobac P and Menno-Ter-forte can be used on steel and plastic surfaces although the efficacy is not perfect.For disinfection of wood surfaces, only NaOCl and Desinfektol EL, which were effective against some test fungi may be recommend.Also other limita- tions, such as safety and corrosivenes, may affect the choice of disinfectant to be used.

Table 1 .
The disinfectants tested for control of fungi.

Table 3 .
The effect of disinfectants on the germination of sclerotia after 60 min treatment.