Globodera rostochiensis (Woll.) Behrens ( Tylenchida , Heteroderidae), the only potato cyst nematode species found in Finland Jari Heikkilä and Kari Tiilikkala Heikkilä, J. & Tiilikkala, K. 1992. Globodera rostochiensis (Woll.) Behrens (Tylen- chida, Heteroderidae), the only potato cyst nematode species found in Finland. Agric. Sei. Finl. 1; 519- 525. (Univ. Helsinki, Dept. Zoology, SF-00100 Helsinki, Agric. Res. Centre ofFinland, Inst. PI. Protect., SF-31600 Jokioinen, Finland.) About 10 000 soil samples, 519 thereof infected with potato cyst nematode (PCN), were studied during 1984-1988. Cysts from infected samples were tested by isoelectric focusing to identify PCN species. All the infected samples were also tested with Hl-resistant (Satuma) and susceptible (Bintje) potato cultivars to separate resistance breaking populations. Cysts from the roots ofSatuma were tested by two-dimensional electrophoresis. The potato seed production area in Finland was found to be free ofPCN ofany kind. In other parts ofFinland all tested samples revealed G. rostochiensis banding pattern, but no G. pallida was found. Except for the most common pathotype Rol-Ro4, we only found Ro2. Key words: potato cyst nematode, PCN, Globodera rostochiensis, Globoderapallida, isoelectric focusing, two-dimensional electrophoresis Introduction In Finland, potatoes are grown commercially on about 41 000 hectares. The cultivated area extends from the southern coast (60° 00’N) up to the north (69° 00’N). The growing season for potatoes ex- tends from mid-May to the end of August in south- ern Finland. In the north the growing season is more than one month shorter (Mukula and Rantanen 1987). Potato cyst nematode (PCN), Globodera rosto- chiensis, was found in Finland for the first time in 1946 (Vappula 1954), and the first noticeable damage on commercial potato farms appeared in the early 1970 s (Sarakoski 1976a). Since the be- ginning of the 19705,PCN has been the most harm- ful pest ofpotatoes in Finland. Magnusson (1987) and Tiilikkala (1987, 1991) have studied the biological and physical fac- tors affecting the success of G. rostochiensis. In Finland, the whole life cycle of G. rostochiensis requires over 600 day degrees above 4.4°C. This means that G. rostochiensis is well adapted to the low soil temperatures and is able to develop in the whole potato growing area up to the polar circle. The northernmost observation in Finland is a few kilometers north of the Polar circle (Sarakoski 1976b). Considering that Globodera pallida needs 519 Agric. Sei. Finl. 1 (1992) https://www.c-info.fi/en/info/?token=Mm--yEk_m36DstK-.K8j6mnF2h8O1VxjC7jrPRw._c_hF4WoR6t0b99S34dpVr79oII3fJEgIjML9YcoiEyZy-OsvniRUkjQFuUN8kaMGUaz-fVcGphsuIUy9qNCU-tfDGs690JrhLXR4Il9KnJbDJDV6oTD5SmSvwrQL9kqLN6-2ZADUtiGXLEJFOFGHAotNZXWpfMU56ea6Y1PYktxcOUmsKTtkkPzI-SRTyARvua-TGNDEEEudiirIpLlwc8IFjrGdSXERXhxm7p6esYx46vBsTYIXjufsqOJ9yGoyVBCCNkiNLg8zTWU9CX-wH5u-wNh9B746eI7VJk fewer day degrees to reproduce than does G. rosto- chiensis (FOOT 1978, MUGNIERY 1978, FRANCO 1979), one would expect G. pallida to be an even more successful species in Finland. If G. pallida females develop faster and produce eggs sooner than G. rostochiensis at lower temperatures (We- bley and Jones 1981), this could lead to gradual replacement of G. rostochiensis. On the other hand, at 24°C G. rostochiensis produces more juveniles than does G. pallida (Webley and Jones 1981). Both species are common in Central Europe, and have been found in Sweden (Olsson 1985 a, 1985 b), Norway (ÖYDVIN 1973, 1978), Iceland (SIG- GEIRSSON and Van Riel 1975), and in Denmark (Jakobsen and Hansen 1983). In Finland, Mag- nusson (1979) pointed out that although some of her populations, tested with test plants in the green- house, did not fit into any pathotype group, no observations of G. pallida had been made till 1979. Reliable identification of species is a key factor whencontrol programs are evaluated. These testing procedures should also be applicable to routine plant protection practice. Testing methods for identification of species and pathotyping of potato cyst nematodes were studied to develop a system for the analysis ofpopulations with low numbers of larvae in the cysts. According to several authors (Fleming and Marx 1982,1983, Ohms and Heinicke 1983,Fox and Atkinson 1984, Marx and Fleming 1985, Fleming 1987), isoelectric focusing ofgeneral pro- teins is a useful method to identify potato cyst nematode species. Isoelectric focusing and staining of general proteins have been used successfully also to determine other plant parasitic species e.g. with Meloidogyne spp., (Dalmasso and Berge 1978, Lawson et al. 1984), and with animal parasites e.g. several Cestoda species (Dixon and Arai 1985, 1987). We concentrated on isoelectric focusing and on identification of species directly from mde soil samples. Some problems related to the identifica- tion of pathotype are also discussed. The aims of this study were 1) to investigate the probable disribution of Glohoderapallida in Fin- land, and 2) the establishment of Hl resistance breaking pathotypes. Material and methods Soil samples of about one litre per hectare (ca. 0.2 dl subsamples collected randomly from the area) were taken during routine farm inspection by the Plant Quarantine Office, or were sent by farmers in July-August during 1984-88. The total number of one litre samples was about 10000. The sampling area covered the whole country from southernmost Finland to north of the polar circle. The sampling intensity differed somewhat from area to area, being more intense in western and southernFinland (Fig-1)- Samples were washed either in a Fenwick can, 250 grams dry soil per wash, or in a Schuiling centrifuge, 1 dl dry soil per wash. Cysts were picked manually from the filter paper. Cysts were stored dry for several months in a refrigerator be- fore identification of species. The rest of the samples were stored at about 5°C, until used in plant tests. Isoelectric focusing (Fleming and Marx 1983), with slight modifications, was used in identification of species as follows. About 20 cysts per sample were soaked in 1% glycine for at least 24 hours in 1.5 ml microcentrifuge tubes. After soaking, cysts were crushed, homogenates fuged and 20 microliter samples were pipetted onto pieces of filter paper on the 1 mm thick polyacrylamide gel plates, pH range 3.5-9.5 (LKB, Bromma, Sweden). Separation of the proteins was performed for 70 minutes at 4°C. Gels were stained for general proteins with Goomassie Brilliant Blue. The rest of the soil was used for testing the samples with HI-resistant (Satuma) and suscept- ible (Bintje) potato cultivars. Pots of 2 dl, sub- merged in peat and filled with sample soil, were planted with both cultivars. Four replicates at least were used. The pots were kept in greenhouses at 18°C for eight weeks. Cysts formed on the roots of Satuma were picked and stored in a refrigerator for 520 Agric. Sei. Fin!. 1 (1992) pathotype testing. Larvae from field samples were counted to get an estimate of initial densities for plant tests. Approximately ten cysts per sample, when available, were crushed and larvae were counted under a microscope without diluting. Because isoelectric focusing separates species, but is not sensitive enough to identify pathotypes, 2-D electrophoresis (Ohms and Heinicke 1985) was used for pathotype identification of cysts de- veloped on the roots of the HI-resistant cultivar Satuma. Results All samples tested by isoelectric focusing revealed G. rostochiensis banding patterns. No G. pallida bands were seen. Plant tests directly confirmed the results in all (519) but twelve samples tested with Bintje and Satuma. No cysts had developed on the roots of Satuma, except for twelve samples (Table 1). But, because the isoelectric banding pattern clearly showed them to be G. rostochiensis , we must be dealing with some other pathotypes of G. rosto- chiensis than Rol or Ro4. These ”resistance break- ing” populations were found in different localities and on areas of several Agricultural Advisory Centers, which means that they probably are inde- pendent ofeach other. 2D-electrophoresis revealed Rol or Ro2 patho- type banding patterns. In the gels it is impossible to separate thesepathotypes from each other. The small number of larvae in cysts extracted from soil samples from the fields where ”resistance breaking” populations had been found (Table 1) Fig. I. Soil samples and sampling areas. Letters (a to t) indicate the sampling areas and white bars the total number ofsoil samples per area. Black bars present the numbers ofPCN infested samples. 521 Agnc. Sei. Finl. 1 (1992) Table 1.Average number ofcysts and larvae per cyst from Samma tests. Sample Initial population Satuma tests Cysts/ Larvae/ Cysts/ Larvae/ 250 g soil cyst plant cyst 53 170 98 34 11 112 286 38 35 40 121 70 97 18 37 135 19 50 3 5 146 245 66 263 61 204 29 54 3 33 246 128 68 25 63 258 280 87 51 82 284 145 42 23 35 322 26 19 348 145 18 1 26 460 75 75 48 23 was a remarkable and interesting point And so was the small number of cysts per plant, and especially the even smaller number oflarvae per cyst extracted from roots of Satuma in most ”resistance breaking” samples compared to the soil samples (Table I). The difference was statistically signific-ant (paired t-test, p=0.0221) between the mean number of lar- vae per cyst from soil samples and that from potato roots. Discussion The total number ofclearly visiblebands in isoelec- tric focusing of general proteins of G. rostochiensis and G. pallida is highly dependent on the quality of the sample and the sample preparation method used. However, there are a couple of species spe- cific and diagnostic major bands which are useful in identification of the species. Moreover, these diag- nostic bands are not particularly sensitive either to the quality of the sample or the way a sample has been prepared, although the position of thesebands may vary according to the preparation method. Our results are comparable with Fleming and Marx (1982, 1983), and Marx and Fleming (1985) be- cause of the same methodology. Our finding that G. rostochiensis seems to be the only PCN species in Finland is somewhat surpris- ing. The other species, G. pallida, exists in all neighbouring countries. Moreover, climatic and other environmental factors in Finland rather favor G. pallida at the expense of G. rostochiensis than prevent its existence. Our suggestion is simply that G. pallida has not yet invaded Finland. The potato seed production area along the west coast of the Baltic sea seems to be free of PCN of any kind. Commercial farmers often have contracts which provide the farmers to use seed potato of high quality. This may help to control the PCN problem on those areas. The census procedure is directed to commercial potato farms, and hence leaves small-scale house- hold farming out ofcontrol. Uncontrolled exchange of seed and machinery leaves at least theoretical possibilities that these small household gardens could function as a reservoir for PCN and G. pal- lida as well. Species can be identified by isoelectric focusing of general proteins. Our results indicate that G. rostochiensis is the only potato cyst nematode species in Finland, but thepathotype question needs more attention. Application of the two-dimensional electrophoresis in pathotype identificationhas been used successfully elsewhere, but in Finland there have been some difficulties. The sample quality must be good, which in our situation is difficult to achieve. The places where ”strange” pathotypes were found are few, indicating recent infection. Moreover, the small number of cysts, and espe- cially the small number of larvae per cyst limits the applicability of the system for routine use, and hence the informationneededand the purpose must be properly defined. When comparing for example the Dutch (BAKER 1987) and German (Heinicke pers. comm.) tradi- tions in the use of2D-electrophoresis in PCN ident- ification, there is one obvious difference in their level ofoperation. Bakker (1987) clearly emphas- izes the importance of exact determination of al- leles and hence the genetic structure of PCN popu- 522 Agric. Sei. Finl. 1 (1992) lations. This, of course, preconceives optimaliz- ation of the procedure which, in turn, makes the whole system less applicable to routine work. In Germany the approach is different, more practically oriented. When certain spots are found in the 20- gels, it automatically leads to the naming of certain pathotypes and to routine control procedures with resistant cultivars even though the exact pathotype and genetic background are unknown. In Finland the situation is easier because we have only one PCN species, and the number of populations where resistance breaking pathotypes have emerged is small. In this situation it is possible to handle all suspected cases individually by leaving them out of potato cultivation. This requires only the identifica- tion of abnormal cases which not necessarily re- quire any complicated methodology. The problem which remains is the possibility some cysts to de- velop on the roots of resistant cultivars in the ab- sence ofresistance breaking pathotypes. In that case no or only few larvae will develop. References Barker, J. 1987. Protein variation in cyst nematodes. PhD thesis. Wageningen, Netherlands. 159p. Dalmasso, A & Berge, J.B. 1978.Molecular Polymorphism and Phylogenetic Relationship in some Meloidogyne spp. : Application to the taxonomy of Meloidogyne. J. Nematol. 10: 323-332. Dixon, B.R. & Arai, H.P. 1985. Isoelectric focusing of sol- uble proteins in the characterization of three species of Hymenolepis (Cestoda). Can J. Zool. 63: 1720-1723. & ARAL H.P. 1987. An investigation ofhost influence on soluble protein banding profiles of Hymenolepis spp. (Cestoidea), using isoelectric focusing. Can. J. Zool. 65: 2471-2474. Fleming, C.C. 1987.The use of isoelectric focusing as a tool in the management of the potato cyst nematodes Glo- hodera rostochiensis and Globodera pallida. 11th In- tern. Congress ofPlant Protection. Manilla, Philippines. & Marks, R.J. 1982. A method for the quantitative estimation of Globodera rostochiensis and Globodera pallida inmixed-species samples. Record ofAgricultural Research 30: 67-70. & Marks, R.J. 1983. The identification ofthe potato cyst nematodes Globodera rostochiensis and G. pallida by isoelectric focusing ofproteins on polyacrylamide gels. Ann, Appi. Biol. 103: 277-281. Foot, M.A. 1978. Temperature responses of three potato- cyst nematode populations from New Zealand. Nemato- logica 24:421-427. Fox, P.C. & Atkinson, H.J. 1984. Isoelectric focusing of general proteins and specific enzymes from pathotypes of Globodera rostochiensis and G.pallida. Parasitology 88: 131-139. Franco, J. 1979. Effect of temperature on hatching and multiplication of potato-cyst nematodes. Nematologica 25: 237-244. Jakobsen, J. & Hansen, L.M. 1983. Identification ofpatho- types of potato cyst nematodes (PCN) collected from infected private gardens. Plant diseases and pests in Den- mark 1982. Lyngby. Lawson, E.C., Carter, Jr, G.E. & Lewis, S.A. 1984. Appli- cation of isoelectric focusing to the taxonomic identifica- tion ofMeloidogyne spp.. J. Nematol. 16: 91-96. Magnusson, M.L. 1979. The occurrence of different patho- types of the potato cyst nematode, Globodera rosto- chiensis, in Finland. Ann. Agric. Fenn. 18: 154-159. 1987. Cultivated crops and climatic factors affecting the yellow potato cyst nematode, Globodera rostochiensis Behrens. Plant protection reports. SLU Uppsala. 36 p. Marks, R.J. & Fleming, C.C. 1985. The use of isoelectric focusing as a tool in the identification and management of potato cyst nematode populations. Eppo Bull. 15: 289-297. Mugniery, D. 1978. Vitesse de developpement, en fonction de la temperature, de Globoderarostochiensis et G. pal- lida (Nematoda: Heteroderidae). Rev. Nematol. 1: 3-12. Mukula, J. & Rantanen, O. 1987. Climatic risks to the yield and quality of field crops in Finland. Ann. Agric. Fenn. 26: 1-18. Ohms, J.P. & Heinicke, D.H.K. 1983.Pathotypen desKartof- felnematoden I. Schnellbestimmung der Arzugehörigkeit mittels Isoelektrofocussierung. Zeitschrift fur Pflanzen- krankheiten und Pflanzenschutz 90: 258-264. Olsson, E. 1985a. Die Kartoffelnematoden Globodera ros- tochiensis (Wolf) Behrens und G. pallida (Stone) Beh- rens und ihre Pathotypen in Schweden. Potato Research 28: 497-506. 523 Agric. Sei. Finl. 1 (1992) 1985b. Morphological-taxonomical studies andpathotype classification in potato cyst nematodes. Eppo Bull. 15: 281-283. Sarakoski, M L. 1976a. The distribution of the potato cyst nematode, Heterodera rostochiensis Wollenweber, in Finland. Ann. Agric. Fenn. 15: 111-115. 1976b. Potato cyst nematode, Heterodera rostochiensis , discovered in Finnish Lapland. Nematologica 22: 223- 225. Siooeirsson, E.I. & Van Riel, H.R. 1975. Snikjupradormar i plöntum a Islandi. Rannsoknastofhunin Nedri As, Hver- agerdi. Island. Skyrdsla 20. 32 p. Tiilikkala, K, 1987.Life cycle of the potato cyst nematode in Finland. Ann. Agric. Fenn. 26: 171-179. 1991. Effect of crop rotation on potato cyst nematode, Globodera rostochiensis, and potato yield. Eppo Bull. 21:41-47. Vappula, N.A. 1954. Nematod problem i Finland. Nord. Jordbr. forskn. 36: 323-325. Webley, D.P. & Jones, F.G.W. 1981. Observations on Glo- bodera pallida and Globodera rostochiensis on early potatoes. PI. Path. 30: 217-224. Öydvin, J. 1973. The usefulness of some larval dimensions to distinquish Heterodera rostochiensis and II pallida. Nematologica 19: 435-442. 1978. Studies on potato cyst-nematodes, Glohodera spp. (Skarbilovich), and the use of plant resistance against G. rostochiensis (Wolf) in Norway. Växtskyddsrapporter. SLU Uppsala. 37 p. Manuscript received May 1992 Jari Heikkilä University of Helsinki Department ofZoology SF - 00100 Helsinki, Finland Kari Tiilikkala Agricultural Research Centre of Finland Institute of Plant Protection SF - 31600 Jokioinen,Finland 524 Agric. Sei. Finl. 1 (1992) SELOSTUS Globodera rostochiensis Woll. (Behrens) on toistaiseksi ainoa Suomesta löydetty peruna-ankeroislaji Jari Heikkilä jaKari Tiilikkala Helsingin yliopisto ja Maatalouden tutkimuskeskus Peruna-ankeroislajeja on kaksi: Globodera rostochiensis ja G. pallida, joilla molemmilla on useita patotyyppejä määri- teltynä sen mukaan millä testiperunoilla ne lisääntyvät. G. rostochiensis on yleisempi laji Euroopassa, joskin G. pallida on lisääntynyt alueilla, joilla ankeroisenkestäviä perunalajik- keita on viljelty intensiivisesti. G. pallida -lajia esiintyy myös Tanskassa sekä Ruotsin ja Noijan eteläosissa. Ulko- maisten tutkimusten mukaan G. pallida on hyvin sopeutunut alhaisiin lämpötiloihin, joten se todennäköisesti myös me- nestyisi Suomessa paremmin ja olisi vahingollisempi kuin täällä jo oleva G. rostochiensis. Tämän tutkimuksen tavoitteena oli selvittää: a) esiintyykö G. pallida Suomessa, b) onko Suomessa ankeroisenkestä- villä perunalajikkeilla lisääntyviä G. rostochiensis -lajin pa- totyyppejä sekä c) miten bioteknisiä menetelmiä voidaan soveltaa peruna-ankeroisen lajimääritykseen. Tutkimus tehtiin pääosin Maa- ja metsätalousministeriön varoin MTTK:n jakasvinsuojeluviranomaisten yhteistyönä. Tutki- tut maanäytteet saatiin Maatilahallituksen ankeroiskartoituk- sen yhteydessä sekä MTTK:n neuvontaan tulleista lähe- tyksistä. Näytteitä oli yhteensä noin 10 000 ja niistä laji- ja patotyyppianalyyseihin sopivia elinvoimaisia ankeroisia oli 519 näytteessä. Näytteitä saatiin kaikkien maatalouskes- kusten alueilta, joskin pääosa oli otettu Etelä-, Keski- ja Pohjois-Pohjanmaalta. Puhtaiden näytteiden suuri määrä osoitti, että peruna-an- keroinen ei ole vielä yleistynyt Pohjanmaalla eikä se uhkaa välittömästi siemenperunatuotannon jatkumista Siemenpe- runakeskuksen toimialueella. Kaikki löydetyt ankeroiset kuuluivat G. rostochiensis -lajiin, joten G. pallida ei ole toistaiseksi levinnyt lainkaan maahamme tai se on erittäin harvinainen. Valtaosa löydetyistä ankeroisita oli tyyppiä ROl/RO4, joka ei lisäänny ankeroisenkestävillä (andigena-re- sistenteillä) lajikkeilla. Kahdentoista näytteen ankeroiset li- sääntyivät testikasvina käytetyllä Saturnalia jane olivat alus- tavien tutkumusten mukaan tyyppiä RO2. Tämän uuden anke- roistyypin, elins. “resistenssin murtajan” todettiin esiintyvän piilevänä jo useiden maatalouskeskusten alueella, joten an- keroisenkestävien perunalajikkeiden (Satuma, Stina, Hertha, Aminca, Prevalent, Provita) jatkuva viljely samalla paikalla voi johtaa resistenssin murtajien valikoitumiseen vallitse- vaksi missä tahansa Suomessa. Tämän tutkimuksen mukaan lajin määritys voidaan tehdä luotettavasti ja nopeasti perunoiden juurista poimituista kys- toista isoelektrisellä fokusoinnilla mikäli kystoissa on run- saasti toukkia. Lajin sisäisten erojen eli patotyyppien määri- tykseen tarvitaan testikasveja tai bioteknistä analytiikkaa, jonka soveltamisesta ja yhdenmukaisesta käytöstä ei ole päästy sopimukseen Euroopan ja Välimeren maiden kasvin- suojelujäijestössä (EPPO). Vuosittain löydettävien uusien resistenssinmurtajien määrä jäänee toistaiseksi niin vähäi- seksi, että patotyyppien määrittäminen on helpointa tehdä kansainvälisenäyhteistyönä jonkun Keski-Eurooppalaisen laboratorion kanssa. 525 Agric. Sei. Fint. 1 (1992)