2 Maataloustieteellinen A ikakauskirja Vol. 57: 223—230, 1985 Experiments on direct isolation of Pythium spp. from Finnish sugar beet soils MAURITZ VESTBERG Department of Plant Pathology, University of Helsinki* SF-00710 HELSINKI, Finland Abstract. Pythium spp. were successfully isolated directly from sugar beet soils by the method of Ricci et al. (1976). The isolates obtained were divided into four groups on the basis of morphological and growth habit characters. The groups showed large differences in in vitro pathogenicity. The importance of some factors affecting the estimation of Pythium propagule density is studied and discussed. Index words: Pythium, direct isolation, sugar beet soils Introduction Scientists have developed a diversity of techniques for the isolation of Pythium spp. from soil. Fundamentally these techniques are based on two principles (Bouhot 1979): 1. Introduction of baits (living or deadplant fragments) into the soil sample, allowing each propagule of theparasite to manifest its pres- ence by the colonization of the baits. 2. In- corporation of soil samples into an artificial selective medium, allowing each propagule of the parasite to develop into a colony. Baiting with plant materials has often been Present address: Agricultural Research Centre, Re- search Station of Central Finland, Juntula, SF-41340 LAUKAA, Finland used for studying specific soil fungi causing plant diseases. Materials used for detecting Pythium include apple (Hendrix& Campbell 1970), cucumber (Banihashemi 1970), pine- apple (Klemmer& Nakano 1962), citrus leaf (Grimm & Alexander 1970) and avocado (Zentmyer et al. 1960). Robertson (1975) developed a paper disc technique for the recovery of Pythium spp. from soil or water. For the isolation of Pythium spp. directly from soil Warcup’s (1950) soil-plate method, in which soil is dispersed in agar, has proved superior to the dilution-plate method. Ricci and coworkers (1976) developed the soil-plate method further by transferring soil-agar discs from a primary medium to a secondary isola- tion medium. Schmitthenner (1962) used a 223 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=O-qBRTfjCX-H04Wp.T9tNQXUg-gWqQeqafQoUYQ.V-3D95EQZM09PXpX3eE_bp9iYG4AfOL9ZSds-Ed0I0rNsuKqasXn-glXIxm5qDkuG1SgEQXA0aEGWFx4Lyf5RNr3vB8gmuIVrCykB91M6XkVhiDpVj8Ro3sUa7J5tnS9PABsK3gHWrN3Gzdkxfe1ZLclXR6CduDO3JmrVGND soil-particle technique for isolating Pythium ultimum Trow, and several other Pythium spp. from soil particles. Particles of 1, 3 and 7 mg mean weight gave amounts of Pythium comparable to the soil plate technique. Angell (1954) found that Pythium was pre- sent in the larger soil particles that sedi- mentated from a suspension and that bacteria were present in the colloidal part. Stanghel- lini and Hancock (1970) found that P. ultimum grew out from small drops dispersed on the surface of 3-day-old 2 % water agar and that this method could be quantified by making dilutions. Numerous selective media have been re- ported for the isolationof Pythium spp. from soil. Rose bengal (Sing & Mitchell 1961, Kerr 1963, Vaartaja& Bumbieris 1964, Ta- kahashi & Ozaki 1965, Vaartaja 1967, Flowers & Hendrix 1969, Martin 1970), gallic acid (Flowers & Hendrix 1969) and fungicides like PCNB (Vaartaja& Bumbieris 1964, Takahashi & Ozaki 1965, Ricci et al. 1976) and benomyl (Ricci et al. 1976) were used in these media before the introduction of the polyene antibiotics (Tsao 1970). The polyene antibiotic pimaricin has been widely used to suppress fungi other than Pythium spp. in selective media (Eckert& Tsao 1960, Sing & Mitchell 1961, Hine & Luna 1963, Mercetich&Fogle 1969). Several other anti- biotics, eg. penicillin, polymyxin, endomycin, streptomycin, nystatin and vancomycin are also mentioned for this purpose by Tsao (1970) in his review on selective media for isolating pathogenic fungi. This paper presents some preliminary ex- periments on direct isolation of Pythium spp. from Finnish sugar beet soils contaminated with the damping-off pathogen Pythium debaryanum auct. non Hesse. Materials and method Soil The soil used in the various experiments were from sugar beet fields where damping- off problems had occurred. There were two very finesandy soils (Mietoinen 60°40'N 21°52'E and Laitila 60°56'N 24°40'E), one clay soil from Salo (60°21 'N 23°4'E) and two soils were high in organic matter, a loamfrom Köyliö (61°6'N 22°20'E) and a peaty soil from Janakkala (60°47'N 24°40'E) Soil samples were stored in semi-moist con- ditions at room temperature until use. Agars and chemicals Agar media of the following types were used in the experiments: Difco corn meal agar » potato dextrose agar » bacto agar Martin’s medium (Martin 1950) of the following composition: agar 20 g, peptone 5 g, dextrose 10 g, KH 2P04 1 g, MgS04 x 7H 20 0.5 g, Rose bengal 30 mg/1 water SPB medium (Ricci et al. 1976): agar 25 g, malt 1 g, saccarose 5 g, Ca(NO 3 )2 Ig, KN0 3 250 mg, MgSQ4 X 7H 20 250 mg, KH 2P0 4 125 mg, citric acid 50 mg, micronutrient solution 1 mg/1 water. The micronutrient solution had the fol- lowing composition: FeS04 1 g/1, ZnS04 1 g/1, CuS04 1 g/1 and MnCl2 1 g/1 water. Commercial fungicides in the agar media were benomyl in the form of Benlate (Du Pont) as 50 % a.i. and PCNB as Avicol (Ke- mira) with 50 ®/o a.i. The antibiotic pimaricin was of Dutch origin, Brocades-Stheeman & Pharmacia. Suspensions of fungicides and pimaricin were used immediately after preparation or after short storage in a refrigerator at 4°C. Assessment of Pythium spp. The basis for the isolation of Pythium spp. is the Warcup (1950) soil-plate method as modified by Ricci et al. (1976). In this meth- od small amounts of soil are evenly dispersed in 2.5 % water agar at 40— 42°C. Citric acid, 50 mg/1, is added to the agar before auto- 224 claving. After solidification of the agar, round discs of 1 cm diameter are cut out and trans- ferred to a Pythium selective medium. The plates are incubated for 24 h at 15°C in dark- ness, after which they are exposed to normal day light and darkness. After 4 days the num- ber of plates with mycelia of Pythium are recorded and the number of propagules per gram of dry soil is calculated according to the MPN method. Statistical interpretation, MPN method In some experiments the number of propa- gules of Pythium was calculated using the most probable number method (MPN) (Maloy & Alexander 1958). This method is based on the appearance or non-appearance of Pythium and results are compared to the Poisson distribution. The method involves three successive dilutions for the calculation of the MPN. In the present experiments the dilution coefficient was 10and the number of replicates also 10. The MPN estimates of two soil samples dif- fer from one another at the 95 % level of probability when their ratio exceeds 3.3. The MPN estimate differs from a fixed norm at the same probability level when the mutual ratio is at least 2.3. Patogenicity of Pythium strains in vitro Strains of Pythium were transferred to CMA to which had been added 500 ppm of wheat germ oil for induction of oogonia. After 7 days 10 seeds of Monohill sugar beet were sown in each petri dish (0 90 mm) showing fungal growth. As control, plates with no transfers of Pythium were used. After 5 days at laboratory temperatures a disease index (D1,_,0) was calculated, given by the mean of the index for seed germination (10 — 1) and seedling death (1—10). Results Testing of methods Pythium spp. could not be isolated direct- ly from soil using the selective Martin’s medium with the dilution plate method, even though inhibitory substances like benomyl, streptomycin or pimaricin were used. The first experiments with the soil-plate method gave positive results in some cases and negative in others, this depending on several factors. Sieving the soil proved to be important and also even dispersal of the soil into the agar. The latter problem was solved by using a hand shaker. The soil was first poured into test tubes with melted agar at 42°C and thence after shaking into petri dishes. The use of suf- ficiently large soil samples proved important when soils of low propagule densities were tested. The direct isolation of Pythium using the modified soil-plate method of Ricci et al. (1976) proved very successful, irrespective of the kind of agar media used (Table 1). In sub- sequent experiments, therefore, this method was used as the basis for estimating Pythium propagule densities of soils. Some factors affecting the isolation method Fungal growth inhibitors The commonly used polyene antibiotic pimaricin inhibited the growth of six P. de- baryanum isolates out of eight (Table 2). No growth whatsoever was observed when pimar- icin was used at concentrations of 50 and 100 ppm. Reduced growth was noticed at 10 ppm. The fungicide PCNB together with benomyl inhibited completely the growth of one isolate and caused reduced growth in some others, es- pecially when PCNB was used at 100 ppm. Two isolates from the Janakkala site showed r>T (Seed germination, 10—1) 4- (Seedling death, 1—10)Ul.-.o - j 225 Table 1. Success in direct isolation of Pythium spp. from soil by different methods. Method Origin Isolation medium Success in iso- of soil lating Pythium Dilution-plate Janakkala Martin + 50 ppm benomyl + Laitila 20 ppm pimaricin CMA PDA Dilution-plate Laitila Martin + 10ppm benomyl + 100 ppm pimaricin + 100 ppm streptomycin sulphate Soil-plate Laitila Martin + 50 ppm PCNB + Soil-plate Laitila Water agar » » Salo Water agar +5O ppm citric acid + 15 ppm benomyl + 50 ppm PCNB » » Salo CMA +5O ppm citric acid +l5 ppm benomyl + 50 ppm PCNB » » Mietoinen Martin Soil-plate as Köyliö Martin + 15 ppm benomyl -I- 15 ppm + modif. by Ricci PCNB et al. (1976) » » Martin +l5 ppm PCNB + 5 ppm + pimaricin » » Martin + 15 ppmbenomyl + 15ppm + PCNB +lOO ppm streptomycin sulphate » » SPB + 100 ppm streptomycin + sulphate Table 2. Growth rate of 8 isolates of Pythium debaryanum on 7 agar media. Room temperature. Medium Daily growth rate (mm) of Pythium debaryanum Mietoinen Köyliö Janakkala Mean isolates isolates isolates 1 2 3 1 2 3 1 2 1 7.5 7.5 10.0 11.3 11.3 11.3 10.0 10.0 9.9 2 5.0 9.3 7.5 3.8 3.0 3.5 6.0 7.5 5.7 3 0 0 0 0 0 0 4.3 4.0 1.0 4 3.0 6.8 0 3.0 3.8 2.8 4.5 4.0 4.0 5 0 0 0 0 0 0 4.3 5.0 1.2 6 2.5 6.8 3.8 2.5 0 1.8 5.0 6.3 3.6 7 2.3 5.0 3.0 1.3 0 2.8 4.0 5.0 2.9 Media: 1 CMA + streptomycin sulphate 100 ppm 2 Martin’s medium 3 » » + pimaricin 50 ppm 4 » » + » 10 » + Benlate 10 ppm 5 » » + » 100 »+ » » » 6 » » + Brassicol 10 » + » » » 7 » » + » 100 » + » » » 226 Table 3. The influence of storage on the content ofPythium in soil from Köyliö with sugar beet and oats as pre- ceeding crops. Propagule densities estimated by the method of Ricci et al. (1976). Semi-moist storage of soil at room temperature. Amount of Number of positive petri-dishes/10 petridish Sugar beet field Oat field Soil tested Soil stored Soil tested Soil stored immediately 154 days immediately 154 days 14.0 10 10 9 10 2.5 10 10 13 0.8 8 9 1 1 0.2 8 6 0 2 no or only minor influence by the inhibitory agents used in this experiment. Soil properties Storage of the soil samples under semi- moist conditions at laboratory temperatures for 154 days did not affect their contents of Pythium propagules (Table 3). Drying the soil sample for 6 days before introduction into the agar medium gave fewer propagules as com- pared to drying overnight, especially when potato-dextrose agar was used as the isolation medium (Table 3). On the other hand, keeping the soil in the first isolation medium for 6 days gave a higher number of propagules as com- pared with keeping it in this medium over- night. Evaluation of the isolation method The method of Ricci et al. (1976) for the direct isolation of Pythium species from soil gave somewhat differing amounts of Pythium propagules depending on the type of isolation agar used (Table 4). Consistent trends were also noticed. The amount of propagules was the highest in the Köyliö soil on each of the four isolation media. On average, the num- ber of propagules was highest on CMA and lowest on PDA. The direct isolation of Pythium from soil yielded several species and strains. From the sugar beet soils of Köyliö, Salo, Laitila and Janakkala four different types of Pythium could be recovered on corn meal agar: 1. Oogonia with cell wall projections 2. » » smooth cell walls of the type P. debaryanum P. ultimum. Mycelial growth irregular growth on CMA 3. Like type 2, but with far more abundant mycelial growth on CMA 4. No oogonia observed. Mycelial growth radial on CMA. Type 1 had the lowest DI in the pathogeni- city test. Most of the transfers of this group showed a DI of 1, which is the same as in the control (Fig. 1). Transfers of Pythium type 2 showed a wide diversity of pathogenicity, while the third group consisted of highly pathogenic transfers with a DI peak of B—9.8 —9. The fourth group of Pythium showed low to moderate pathogenicity. When Pythium spp. were isolated directly from soil, sevaral slow-growing fungi oc- Table 4. Pythium densities in four soils on four media. Estimation according to the MPN method. Origin No of Pythium propagules/g dry soil as of soil estimated by the MPN methodestimated by the MPN method Martin’s SPB CMA PDA medium medium Köyliö 205 a 75a 205 a 40b Salo 7a 2b 60c 13ad Laitila 23a 14a 105 b 12a Janakkala 20a 18a 50a 16a Mean 64ab 27a 105 b 20a Row values marked with different letters differ signifi- cantly (P<0.05) 227 curred on the isolation media. In Table 5 are summarized the fungi of four sugar beet soils and their occurrence on four agar media. There were differences between the occurrence of fungi on different media. On the whole, CMA showed only a few species, this being due to the abundant growth of bacteria on this medium. Chrysosporium pannorum (Link) Hughes and Penicillium spp. were the most common fungi on Martin’s medium, PDA and SPB, while for instance Acremonium spp. could be found especially on Martin’s medium and SPB and Zygorhynchus spp. on Martin’s medium and PDA (Table 5). Discussion Pythium spp. were readily isolated directly from sugar beet soils by the soil-plate method (Warcup 1950) as modified by Ricci et al. (1976). Even common media like CMA and PDA could be used as isolation media in this method. However, cultures of Pythium ob- tained on Martin’s medium (1950) and the SPB medium were more easily recognizable than cultures on CMA or PDA. In the present investigation Pythium species were not identified, but they were grouped on the basis of morphological and growth habit characters. There were four groups of dif- fering pathogenicity in vitro. These four groups certainly do not represent the whole Pythium flora of the soil samples studied, be- Table 5. Occurrence of fungi other than Pythium spp. on agar media used for direct quantitativeisolation ofPythium in soil. 114 petri-dishes with Martin’s agar, 118 dishes with PDA, 82 dishes with CMA and 125 petri- dishes with SPB. Fungus Number of petri-dishes showing fungal growth on different media Martin’s PDA CMA SPB agar Absidia sp. 0 2 0 0 Acremonium spp. 12 0 1 13 Alternaria alternata (Fr.) Keissl. 2 10 0 Chrysosporium pannorum (Link) Hughes 5 23 0 32 Doratomyces stemonitis (Pers. ex Steud.) Morton & G. Sm. 0 10 4 Fusarium spp. 0 0 0 2 Geotrichum candidum Link ex Leman 3 0 0 0 Graphium sp. 0 0 10 Moniliella sp. 17 2 0 Monodictys levis (Wiltsh.) Hughes 0 0 0 1 Morlierella sp. 10 0 0 Mucor spp. 12 0 0 Paecilomyces sp. 2 0 0 0 Penicitlium spp. 20 10 0 7 Ulocladium botrytis Preuss 7 3 0 1 Zygorhynchus sp. 15 15 0 1 Unidentified fungi 2 3 0 4 228 Fig. I. Percentage distribution of four types of Pythium transfers according to their disease indices as es- timated in vitro. cause only one isolation method was used. In fact, it is well known that the application of different isolation methods well yield a range of Pythium species and that no single method yields the whole Pythium flora of the soil (Hendrix & Campbell 1973). For example, the Flowers & Hendrix gallic acid medium is good for isolating P. acanthicum Drechs. P. oligandrum Drechs. group, P. ultimum Trow and P. vexans de Bary, while the Kerr medium is best for P. afertile Kanouse & Humphrey, P. aphanidermatum (Edson) Fizp. and the P. dissotocum Drechs. P. perniciosum Serbinow group (Hendrix & Campbell 1970). To approach a total quali- tative estimate of the Pythium flora, several different techniques should thereforebe used on each soil sample. Most of the numerous Pythium- media available contain one or several antimicrobial agents. The polyene antibiotic pimaricin in- hibits the growth of almost all fungi, except a small group including the pythiaceous fungi (Tsao 1970). However, reports of inhibition of some Pythiaceae fungi by pimaricin also exists (Hine 1962). In the present study, the growths of several isolates of the pathogenic P. debaryanum isolated from sugar beet seed- lings were strongly inhibited by pimaricin. Therefore, pimaricin will not be used in fur- ther investigations. PCNB and benomyl, on the other hand, did not inhibit, or only slightly inhibited the growth of P. debaryanum. The estimation of Pythium was quantified by making dilutions of test soil into water agar according to the method of Ricci et al. (1976). Although the method proved success- ful, it has some practical shortcomings. It is laborious and in vitro and in vivo tests are needed to separate pathogenic and nonpatho- genic species or strains from each other. The method proved reliable with Pythium in- oculum densities of 10or more propagules per gram of soil. At very low densities, ie. 1 pro- pagule or less per gram of soil, the method is not feasible because the amount of soil to be incorporated into the water agar is too high. The number of Pythium propagules per unit of soil is only one of several factors express- ing the likelyhood of plant disease. However, this knowledge might be valuable, for exam- ple, when establishing a certain minimum in- oculum level above which possibilities for dis- ease development exists. Acknowledgements: I wish to thank Professor Eeva Tapio for critical reading of the manuscript and for her support as head of the department. I am also grateful to Dr. Kyösti Rainioko (Agr. For.) and Mr. Nils Nuormala (Agronomist), who representing the Sugar Beet Research Centre, have encouraged mein my work. The English text was kindly revised by Mr. Peter Joy (M. Agr. For.). References Anoell, H.R. 1954. Partial segregation of bacteria and isolation of Pythium from the coarser soil fractions. Austr. J. Agric. Res. 5: 702—705. Banihashemi, Z. 1970. A new technique for isolation of Phytophlhora and Pythium species from soil. PI. Dis. Rep, 54: 261—262. Bouhot, D. 1979. Estimation of inoculum density and inoculum potential: Techniques and their value for dis- ease prediction, p. 21—34. In »Soil-borne plant patho- gens». Eds. Schippers, B. & Gams, W. Academic Press. New York. 686 pp. Eckert, J.W. & Tsao, P.H. 1960. A preliminary report on the use of pimaricin in the isolation of Phytophlhora spp. from root tissues. PI. Dis. Rep. 44: 660—661. Flowers, R.A. & Hendrix, J.W. 1969. Gallic acid in a procedure for isolation of Phytophlhora parasitica var. nicotianae and Pythium spp. from soil. Phytopath. 59: 725—731. Grimm, G.R. & Alexander, A.F. 1970. Citrus leaf pieces as trap for soil-borne Phytophlhora spp. Phytopath. 60; 1294. Hendrix, F.F. Jr. & Campbell, W.A. 1970. Distribution of Phytophlhora and Pythium species in soils in the Continental United States. Can. J. Bot. 48: 377—384. Hine, R. 1962. Effect of streptomycin and pimaricin on growth and respiration of Pythium species. Mycologia 54: 640—646. Hine, R.B. & Luna, L.V. 1963. A technique for isolating 229 Pythium aphanidermatum from soil. Phytopath. 53: 727—728. Kerr, A. 1963. The root xoi-Fusarium wilt complex of peas. Austr. J. Biol. Sci. 16: 55—69. Klemmer, H.W. & Nakano, R.Y. 1962. Techniques in isolation of Phythiaceous fungi from soil and diseased pineapple tissue. Phytopath. 52: 955—956. Martin, J.P. 1950. Use of acid, rose bengal and strep- tomycin in the plate method for estimating soil fungi. Soil Sci. 69: 215—232. Mercetich, S.M. & Fogle, H.W. 1969. Role of Pythium in damping-off of peach. Phytopath. 59: 356—358. Ricci, P.; Toribio, J.A. & Messiaen, C.M. 1976. I La dynamique des populations de Pythium dans les sols Maraichers de Guadeloupe. Methodes d’etude. Ann. Phytopath. 8: 51—63. Robertson, G.J. 1975. A paper disc technique for the recovery of Pythium spp. from soil or water. N.Z. J. Agric. Res. 18: 409—410. Scmitthenner, A.F. 1962. Isolation of Pythium from soil particles. Phytopath. 52: 1133—1138. Sing, R.S. & Mitchell, J.E. 1961. A selective method for isolation and measuring the population ofPythium in soil. Phytopath. 51: 440—444. Stanohellini, M.E. & Hancock, J.G. 1970. A quantita- tive method for the isolation of Pythium ultimum from soil. Phytopath. 60: 551 —552. Takahashi, N. & Ozaki, T. 1965. Ecological and taxo- nomic studies on Phythium as pathogenic soil fungi. IV The isolation methods of Pythium. Bull. Univ. Osaka Prefect., Ser. B 17: I—lo. Tsao, P.H. 1970. Selective media for isolation of patho- genic fungi. Ann. Rev. Phytopath. 8; 157—186. Vaartaja, O. 1967. Reinfestation of streilized nursery seedbeds by fungi. Can. J. Microbiol. 13: 771—776. Warcup, J.H. 1950. The soil-plate method for isolation of fungi from soil. Nature 166: 117—118. Zentmyer, G.A., Gilpatrick, J.D. & Thorn, W.A. 1960. Methods of isolating Phytophthora cinnamomi from soil and from host tissue. Phytopath. 50: 87. Ms received September 9, 1985 SELOSTUS Pythium-sienen suora eristäminen suomalaisista sokerijuurikasmaista Mauritz Vestberg Helsingin yliopiston kasvipatologian laitos Vuodesta 1978 alkaen on Helsingin yliopiston kasvi- patologian laitoksen ja Sokerijuurikkaan Tutkimuskes- kuksen välisenä yhteistyönä tutkittu sokerijuurikkaan tai- mipoltetta. Tämä kirjoitus käsittelee taimipoltteen tär- keimmän aiheuttajan, Pythium- sienen eristämistä maasta. Pylhium-sientä pystyttiin eristämään maasta ranskalai- sella menetelmällä, jossapieniä määriä kuivaa ja seulot- tua maata (3, 30, 300 ja 3000 mg) sekoitettiin 42°C:een vesiagariin. Jähmettyneestä agarista siirrettiin n. 1 cm kokoisia agarkiekkoja muhineen petrimaljoihin Martinin alustalle, johon oli lisätty benomyyliä ja PCNB:tä mui- den paitsi Pythium-sienen kasvun hidastamiseksi. Petri- maljat pidettiin ensin 1 vrk pimeässä + 15°C;eenlämpö- tilassa ja tämän jälkeen 5 vrk normaaleissa laboratorio- olosuhteissa. Tämän jälkeen tehtiin havainnot mahdol- lisista Pythium-kasvustoista. Saatujen lukujen perusteella laskettiin näytteille todennäköisin itiötiheys/g ilmakuivaa maata. Käytetyllä menetelmällä saatiin tutkituista juurikas- maista esiin sekä tautia aiheuttavia että saprofyyttisiä la- jeja jakantoja. Nämä jaettiin morfologisten jakasvuta- paominaisuuksiensa perusteella neljään ryhmään. Ryh- mien välillä todettiin patogeenisuuseroja. Yllä selostetulla menetelmällä saatu tieto on käyttökel- poista esim. verrattaessa käsittelyjä keskenään tai mää- ritettäessä sitä alinta Pythium- sienen määrää, jonkaylä- puolella taimipoltteenpuhkeamisvaara kasvaa. Tällä me- netelmällä on myös haittapuolensa. Hyvin alhaisia Pythium- sienen itiötiheyksiä ei voida määrittää luotetta- vasti. Patogeenisten lajien ja kantojen erottaminen sapro- fyyttisistä vaatii laajoja laboratorio- jakasvihuonekokeita. 230