Journal of the Scientific Agricultural Society of Finland Vol. 55: 183-301, 1983 Maataloustieteellinen Aikakauskirja POTATO VIRUSES IN FINLAND AND THEIR IDENTIFICATION Selostus: Suomessa esiintyvät perunavirukset ja niiden määrittäminen AARNE KURPPA Department of Plant Pathology University of Helsinki SF-00710 Helsinki 71, Finland Academic dissertation TO BE PRESENTED. WITH THE PERMISSION OF THE Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in AuditoriumViikki B 2 on November 4,1983 at 12 O'CLOCK. SUOMEN MAATALOUSTIETEELLINEN SEURA, HELSINKI https://www.c-info.fi/en/info/?token=yjQlYJQhElxde4nl.I5NTtg_tGFlZz3R9lnitCw.ocBgdmCHuLC26DMIYa12ipJt8KWHNlVyremMcaViKATk0PqXUWr08HKGt2e_FQnBwBAqDVI8an-SCmI8Gsy_koenhCRyi17_TwVllaCvhElNN0lVBL8HmMculUBI7XQ-Blg8PPtu8dKfYri8pagv9kQ7pv6CJsmJI8Q Preface This study was carried out at the Department of Plant Pathology of the University of Helsinki. I wish to express my sincere gratitude to my teacher, Professor Eeva Tapio, the Head of the Department, for the support she has given me in my work over a long period of time and for her constructive criticism of the manuscript. Special thanks are due to the personnel of the Department of Plant Pathology, especially to Mr, Pentti Heinänen, Mr. Tauno Koivunen and Mrs. Pirkko Korhonen and to my excellent student assistants Mrs. Marianne Heinonen M.Sc. and Mrs. Pirkko Martikainen M.Sc. for their invaluable technical help throughout this study. I also wish to thank Miss Heather MacKenzie B.Sc. for linguistic revision of the English text. Special thanks are also due to Mrs. Airi Visuri for typing the manuscript and Mrs. Kerttu Lehtinen for drawing the numerous figures. The Seed Potato Center, the State Seed Testing Station and the Finnish plant breeding stations and potato processing companies are thanked for providing research material. This study was supported by grants from the Finnish Cultural Foundation and the Society of Agronomists. I am grateful to the Scientific Agricultural Society of Finland for including this study in their series of publications. Finally, I wish to express my sincere thanks and gratitude to my wife for hergreat interest and patience during my work. Helsinki, May 1983 Aarne Kurppa CONTENTS ABSTRACT 189 INTRODUCTION 189 A. MATERIALS AND METHODS 191 1. Isolation of the viruses and theirbiological properties 191 1.1. The origin of the isolates 191 1.2. Isolation methods and the test plants used 191 1.3. Thermal inactivation point and dilution end point determination 194 1.4. Virus preservation and their propagation forpurification 194 2. Electron microscopy 195 3. Virus purification 195 4. Antiserum production and the properties of the antisera 196 5. Identification methods 197 5.1. Chloroplast agglutination, microprecipitin and cut leaf tests 197 5.2. Gel diffusion tests 197 5.3. ELISA test 198 6. Field experiments and statistical analysis of the results 199 B. THE VIRUSES AND THE VIRUS STRAINS FOUND IN FINLAND AND THEIR PROPERTIES 199 1. Potato virus X (PVX) 199 1,1. Symptoms in potatoes and test plants and virus strain classification 200 1.2. Sap properties and virus purification 202 1.2.1. Thermal inactivation point and dilution end point 202 1.2.2. Virus purification 203 1.2.3. Electron microscopy 203 1.3. Serological properties 204 1.3.1. Homologous titers of the antisera 204 1.3.2. Heterologous titers of the antisera 205 1.3.3. Immunogenesis of the degraded antigen 205 2. Potato virus S (PVS) 208 2.1. Occurrence, symptomology and transmission 209 2.2. Symptoms in test plants and isolate classification 210 2.3. Sap properties and virus purification 212 2.3.1. Thermal inactivation point and dilution end point 212 2.3.2. Electron microscopy and purification 213 2.4. Serolocical properties 213 2.4.1. Homologous titers of the antisera 213 2.4.2. Heterologous titers of the antisera 213 2.4.3. Immunogenesis of the degraded antigen 215 3. Potato virus M (PVM) 217 3.1. Occurrence and symptomology in potatoes 218 3.2. Symptoms in test plants and isolate classification 218 3.3. Sap properties and virus purification 219 3.4. Serological properties 220 3.4.1. Homologous titers of the antisera 220 3.4.2. Heterologous titers of the antisera 220 3.4.3. Immunogenesis of the degraded antigen 221 4. Potato virus Y(PVY) 224 4.1. Potato symptomology and virus transmission 225 4.2. Symptoms in test plants and strain classification 226 4.3. Sap properties 230 4.4. Virus purification 231 4.5. Serological properties 232 4.5.1. Homologous titers of the antisera 232 4.5.2. Heterologous titers of the antisera 232 5. Potato virus A (PVA) 237 5.1. Properties of the virus isolates that occur in Finland 237 5.1.1. Occurrence and symptomology in potatoes and test plants 237 5.1.2. Sap properties 238 5.1.3. Virus purification and serological identification 238 6. Potato leaf roll virus (PLRV) 240 6.1. Occurrence in Finland 240 6.2. Properties of the virus isolates 241 6.3. Virus identification 242 6.4. Virus purification and particle properties 243 7. Tobacco rattle virus (TRV) 246 7.1. Symptoms caused by the isolates occurring in Finland 248 7.2. Sap properties and virus purification 248 7.3. Serological properties 249 C. DIFFERENT METHODS OF IDENTIFYINGPOTATO VIRUSES 253 1. Methodological developments in virus identification 253 1.1. Traditional methods 253 1.2. Labelled antibody techniques 254 2. The suitability of these methods forpotato virus identification as tested in Finland 255 2.1. Chloroplast agglutination methods and the cut leaf method 255 2.2. Agar gel diffusion methods 256 2.2.1. Double diffusion test 256 2.2.2. Single diffusion test 257 2.2-3. Comparisons between the gel diffusion and the agglutination methods 258 2.3. ELISA method 260 2.3.1. Reaction specificity ofdifferent antibody preparates 260 2.3.2. Test sensitivity 264 2.3.3. Relationships between the viruses and the virus isolates as determined with the ELISA test 265 2.3.4. Virus identification in potato samples at different developmental stages 269 2.3.4.1. Identification of known isolates of PVX, PVM, PVS and PVY with indigenous test reagents 269 2.3.4.2. Identification of known isolates of PVY, PVA and PLRV with foreign test reagents 274 2.3.4.3. Identification of unknown viruses in naturally infected potatoes 277 2.3.5. Comparisons between the ELISA test, the chloroplast agglutination test and the A 6 cut leaf test 278 D. DISCUSSION 281 1. Properties of the viruses and the virus isolates 281 2. Identification methods 285 E. SUMMARY 288 REFERENCES 290 SELOSTUS 300 189 JOURNAL OF THE SCIENTIFIC AGRICULTURAL SOCIETY OF FINLAND Maataloustieteellinen Aikakauskirja Vol. 55:183—3Ol, 1983 Abstract. The occurrence of potato viruses in Finland and alternative methods for their identifica- tion are reported in this study. The following seven viruses were found to occur; potato viruses X, S, M, Y, A, potato leaf roll virus (PLRV) and tobacco rattle virus (TRV). The isolates of potato viruses X and Y included two clearly distinct strains. The other viruses had only minor variants. The most severe and important of the viruses was PVY, particularly its Y° strain. Other important viruses were PVM and PVA and potentially PLRV. PVY was found to be the most easily transmitted in the field. For serological identification of the viruses, indigenous antisera produced for selected isolates were mostly used. Significant serological variation was only found among the PVY isolates. The lowest concentration of PVX D-protein detected with the agar gel double diffusion test was 10 p.g/ml and 1 yu.g/ml with the single diffusion test. With the ELISA test 0.1 ng of virus /ml could be detected. For PVX the lowest relative value detected in potato leaf sap was a dilution of between 10 6 and 10'7 . The chloroplast agglutination test was too unreliable for detecting PVY in potato samples. Also the agar gel double diffusion test was found to be too insensitive for the identification of potato viruses but the single diffusion test could be used in certain cases. With the ELISA test potato viruses X, S, M and Y could be reliably detected in potato leaf sap and with almost the same accuracy in sap from sprouting tubers. The ELISA test was also found to give satisfactory results during routine testing for the identificationof PVA and PLRV in potato leaves or non-dormant tubers. Introduction Virus diseases in potatoes in Finland have been known to be important since 1924. At this time cultivars were imported from England and Germany for experimental purposes to the Department of Plant Pathology of the Central Agricultural Experiment Station in Tikkurila and it was noticed that they displayed severe viral symptoms (JAMALAINEN 1946). In particular, many of the varieties were greatly affected by leaf roll. When experiments were continued with the same potatoes in subsequent growing seasons, more and more potato plants became affected with leaf roll and the tuber yields remained poor. In the 1930’s and the early 1940’s virus diseases were commonly found in domestic potato breeding lines being grown at Tammisto near Helsinki (BRUMMER 1946). Even at this time certain diseases were known to be important. For example, severe secondary crinkle decreased the tuber yields of individual plants by 64 % on average and secondary streak decreased yields similarily by up to 93 %. Later studies, carried out in the 1950’s by POHJAKALLIO et ai. (1961), had similar findings. 190 POHJAKALLIO et ai. (1961) demonstrated that the same virus isolate could induce variable symptoms in different potato cultivars. POHJANHEIMO (1961) found that the cv. Tammisto Early remained latent after infection with PVY and was a dangerous carrier of the virus. The cultivar Jaakko was found by POHJANHEIMO (1962) to be an extremely sensitive indicator plant in determining PVY occurrence and transmission in the field. POHJANHEIMO (1962) also found that a higher percentage of infected potato plants occurred in the fields in the Southern parts of the country than in the other parts. Similar phenomena were found to occur in Sweden by ESBO (1946), who also demonstrated that high infection rates depend on a large aphid population being present in the crop. The first attempts at serological identification of potato viruses in Finland were carried out by AURA (1957). All domestic seed potato lots tested were found to contain high percentages of tubers infected with PVX and PVS. Only two lots, originating from imported English and Dutch seed potatoes, of 26 tested, were virus-free. No significant differences in the rate of tuber infection from potatoes grown in different parts of the country were found. Further studies on potato viruses were carried out in 1964—1966 by SEPPÄNEN (1972) who also studied the occurrence of PVY and PVA in Finland. The percent of PYX and PVS infected tubers in ’’merchant seed” was about 50 % and also the proportion on PVS in quality seed had reached 70 % on average. The percent occurrence of PVY was low in all tuber lots and PVA was not found at all. The importance of virus-free seed potatoes as a factor in attaining high yields was often emphasized in the 1960’s by HUOKUNA (1962), SEPPÄNEN (1963) and YLLÖ (1966), but it took several years before domestic virus-free seed potato production was initiated. The first indigenous cultivars, which were purified from viruses via heat treatment following tissue culture, were Pito and Tammisto Early and this was accomplished by TAPIO in 1972a. The growing seasons of 1973 and 1975, when there was a high rate of virus infection in potatoes, combined with this country’s dependence on foreign seed potatoes to make the need for wide scale domestic seed potato produc- tion commonly understood. A year later the Seed Potato Center was founded at Liminka near Oulu, and it was assigned the responsibility of producing healthy seed potatoes for Finland. For the production of healthy basic stocks of potatoes and the control of virus diseases during the course of large scale potato cultivation, reliable virus test methods are needed. The purpose of this study was to improve the test methods for potato virus identification. In order to apply new technological achievements in virus testing, basic research about the viruses and their isolates had to be carried out. Thus this study emphasizes the main properties of the important viruses that occur in potatoes in Finland as this knowledge is crucial for the application of modern serological methods such as the ELISA test. 191 A. Materials and methods 1, Isolation of the viruses and their biological properties 1.1. The origin of the isolates All viruses examined were isolated in Viikki at the Department of Plant Pathology of the University of Helsinki between 1975 1982. Some of the isolates were of foreign origin, these being obtained via imported seed potatoes. During the spring of 1975 hundreds of tons of seed potatoes were imported into Finland (SEPPÄNEN & HYTÖNEN 1977) and some of these were moderately infected with different viruses. Thus several isolates studied originated from this seed material. Also, many isolations made in the years after 1975 may be traced back to this imported material; particularly isolates from the cultivars Kaptah, Posmo, Prevalent, Prumex, Record and Saturna. Other virus isolates originated from material sent from field inspections to the State Seed Testing Station or came from seed potatoes sent for statutory winter testing to this institute between 1975 1982. Most of this seed material was produced by seed potato growers in the vicinity of the Seed Potato Center at Tyrnävä. Viruses were also isolated from potato cultivars and clones grown at the Hankkija Plant Breeding Institute at Tuusula and the Plant Breeding Insti- tute, Agricultural Research Centre (A.R.C.) at Jokioinen. As well, several isolations were made from potatoes grown at experimental stations in Central Finland at Laukaa and Maaninka and from potatoes produced for the starch industry by Hämeen Peruna Ltd in Hämeenlinna and Prestoperuna Ltd in Kotka. Moreover, viruses were also isolated every year from field experiments at the University Farm in Viikki. Random samples from virus-infected material were an important source of isolates. This material was collected from all over the country during the growing seasons and from tubers sent to the department during the winter- time. If isolates of certain viruses from the same source proved to have the same properties only one isolate was selected and maintained for further studies. The number of these isolates that were studied further is as follows: Potato virus X (PVX) 21, potato virus S (PVS) 18, potato virus M (PVM) 15, potato virus Y (PVY) 48, potato virus A (PVA) 4, potato leaf roll virus (PLRV) 11 and tobacco rattle virus (TRV) 3. 1.2. Isolation methods and the test plants used Most viruses were isolated from seedlings grown from virus infected tubers as part of a winter-testing program. Viruses were also isolated from infected leaf samples and tubers of different developmental stages that were collected in the field. Isolates of PYX, PVA and PVY were even taken from lesions initiated by the viruses in A 6 cut leaf tests. 192 For mechanical inoculations virus infected samples were ground with a mortar and pestle in 0.06 M or 0.1 M phosphate buffer at pH 7 (one part sample/5—lO parts buffer). Carborundum dusted leaves of test plants were inoculated using one’s forefinger and a cotton-tipped match or a muslin pad (for infectivity tests). Inoculated leaves were rinsed with tap water within I—2 minutes. Aphid transmission was used for the isolation of all viruses excluding PYX and TRY. This method was also used for further studies of the virus isolates. The aphid species normally used was Myzus persicae Sulz. For PLRV transmission experiments the species Aulacorthum solani Kltb. and Aphis nasturtii-frangulae Kltb. were used. For PYY transmission the use of Rhopalosiphum padi L. was studied. R. padi was raised on oats and the other aphid species were raised on potatoes. In aphid transmission experiments 3 X 10 test plants were used but for isolation purposes the number of test plants used was 10 of each species needed. Three aphids were moved with a brush onto each test plant. The acquisition feeding time for nonpersistent viruses was I—2 min and for the persistent (PLRV) virus 48—72 h. The inoculation feeding times were 18—24 h and 48 —72 h respectively. The aphids were killed by nicotine fumigation at the end of the inoculation feeding period. The test plants were then grown under normal greenhouse conditions for 3 8 weeks, depending on the virus and the host. The artificial illumination used in the greenhouses during the wintertime supplied 4000—5000 lux for 16 h per day. The temperature in the greenhouses was 20 C in the wintertime but varied between 20 C and 40 C in the summertime. All host range, symptomology and aphid transmission experiments and most of the virus isolations were carried out during the wintertime under good conditions. The test plants used in all experiments were young, healthy plants of equal size. The following test plant species were used: Chenopodium amaranticolor Coste & Reyn. Chenopodium quinoa Willd. Datura metel L. Datura stramonium L. Gomphrena globosa L. Lycopersicon chilense Dun. Lycopersicon esculentum Mill. cvs. Kotitomaatti and Nevskij Lycopersicon pimpinellifolium (Jusl.) Mill. Nicandra physaloides L. Nicotiana clevelandii Gray Nicotiana debneyi Domin Nicotiana glutinosa L. Nicotiana tabacum L. cv. Samsun Phaseolus vulgaris L. cvs. Red Kidney and Stella Physalis floridana Rydb. Pisum sativum L. cv. English sword Solarium chacoense Bitt. Solarium demissum A Lindl. (SdA) 193 Solanum demissum Y Cock. (SdY) Solanum demissum Y x Solanum tuberosum cv. Aquila (A6) Solanum tuberosum L. several cultivars Solanum rostratum Dun. Vida faba L. cvs. Hankkija’s Mikko and Pirhonen For virus isolation a wide host range was used because some test plants were chosen in order to show the symptoms caused by possible unwanted contamination. For isolation potato virus X was inoculated mechanically onto the following plant species: G. globosa, TV. glutinosa and TV. tabacum cv. Samsun. From local lesions that developed on G. globosa leaves the virus was transmitted to TV. glutinosa, in which it could be maintained for several months. Potato virus S was mechanically inoculated onto the following species: C. quinoa, TV. debneyi, TV. glutinosa, TV. tabacum cv. Samsun and S. rostratum. From local lesions on C. quinoa leaves the virus was transmitted again to TV. debneyi and L. esculentum cv. Nevskij, in which it could be maintained for I—4 months. The test plants used for aphid transmission were L. esculentum cv. Nevskij and TV. debneyi. Potato virus M was mechanically inoculated onto the following species: L. esculentum cv. Kotitomaatti, L. chilense, TV. debneyi and TV. tabacum cv. Samsun. The host used for aphid transmission was L. esculentum cv. Kotitomaatti in which the virus could be maintained for 2—4 months. Potato virus Y was mechanically inoculated onto the following species: S. demissum A, S. demissum Y, TV. glutinosa, TV. tabacum cv. Samsun, TV. physaloides and P. floridana. The virus was re-inoculated from the top leaves of S. demissum Y that exhibited systemic vein necrosis to TV. glutinosa, in which the virus could be maintained for 2—4 months. The hosts used for aphid transmission were TV. glutinosa and S. demissum Y. The test plant species used for potato virus A were TV. glutinosa, TV. tabacum cv. Samsun, TV. physaloides, P. floridana and S. demissum A. No aphid transmission experiments were carried out. The virus was maintained in TV. tabacum cv. Samsun for 1 3 months. Potato leaf roll virus was transmitted via aphids to D. stramonium, P. floridana, S. demissum Y and also to potato cv. Sieglinde. Virus isolates were maintained in P. floridana for 3—6 months or in potato tubers for up to 24 months. Tobacco rattle virus was isolated mechanically from potato tubers, roots and stocks. The test plant species used for isolation were C. amaranticolor, C. quinoa, TV. clevelandii, TV. debneyi, TV. glutinosa and TV. tahacum cv. Samsun. The virus was re-inoculated from local lesions in Chenopodium leaves to TV. clevelandii, in which it could be maintained for 2—4 months. 194 1.3. Thermal inactivation point and dilution end point determination The thermal inactivation point (TIP) and the dilution end point (DEP) were determined for some of the isolates of each virus; the isolates being selected according to their symptomology in test plants. TIP was determined by heating 1 ml of sap for 10 min in thin-walled tubes in a water bath followed by rapid cooling. Virus infected sap was diluted in cold distilled water for DEP determination. The virus sources and the indicator plants used were as follows: PVX N. glutinosa —» G. globosa PVS N. debneyi —* C. quinoa PVM L. esculentum —> L. esculentum PVY N. glutinosa —* S. demissum Y PVA N. tabacum —> S. demissum A TRV N. Clevelandit —> C. amaranticolor The lesions were counted and the other symptoms were observed within 6—21 days after inoculation. Potato virus M was tested serologically in the sap of test plants 3 weeks after inoculation. TIP and DEP values were not determined for potato leaf roll virus. 1.4. Virus preservation and their propagation for purification The virus isolates selected for further studies were maintained in test plants. Viruses were also preserved as dried material with calcium chloride or they were deep-frozen at —2O C. By using all these methods most of the virus isolates kept their infectivity throughout the course of the research program. Potato leaf roll virus isolates and isolates of PVA and PVY were also preserved in potato tubers. For virus purification, biologically pure virus isolates were propagated in the following hosts: PVX N. glutinosa and N. tabacum cv. Samsun PVS N. debneyi, L. esculentum cv. Nevskij and S. tuberosum cv. Pito PVM L. esculentum cv. Kotitomaatti and L. chilense PYY N. glutinosa and N. tabacum cv. Samsun PVA N. tabacum cv. Samsun PLRV P. floridana and S. tuberosum cv. Sieglinde For virus propagation 30—100 test plants were used simultaneously. Systemically infected leaves were collected every 10—20 days for Nicotiana spp. and L. esculentum plants or every 4—6 weeks for N. debneyi, P. floridana and S. tuberosum plants. The leaves were then deep-frozen at —2O C for later use or were used fresh (PYY, PVA, PLRV). The leaves infected with TRY were homogenised with a blender and the sap was deep-frozen to be further purified later (see KURPPA et ai. 1981). 195 2. Electron microscopy For electron microscopy virus particles were negatively stained in prepa- rations of plant sap or purified sap suspensions. The preparations were examined on carbon-coated grids and stained with either 1.5 % phos- photungstate at pH 6.5 or 2 % ammonium molybdate at pH 6.5. For PLRV studies thin sections of fixed plant material coated with epon were also examined. For these studies JEOL 100 S and JEOL 1008 transmission electron microscopes were used. The electron micrographs were taken at fixed magnifications of IOOOOx, 20000x, 30000 x or 50000 x and then enlarged to the final magnification. For electron microscope serology, antigen solutions of 10 pg/ml were used and the reaction end-point of an antiserum was taken as the greatest dilution of which antibody molecules could be seen attached to all virus particles (ROBERTS et al. 1979). This method was used to separate isolates of potato virus Y and isolates of tobacco rattle virus. 3. Virus purification Virus isolates were purified from leaves that were systemically infected. For the purification of viruses X, S and M the method described by SHEPARD (1972) was followed. Thus 0.5 M borate buffer at pH 8.2 was used for homogenise leaf material and polyethylen glycol was used as a solvent for virus concentration. For the purification of PVY a method described by STAGE-SMITH & TREMAINE (1970), based on ether clarification and differential centrifugation, was used initially. As well, modifications of this based on chloroform (1:1) and chloroform/buthanol 1/1:1 clarification were tried for the first steps in the purification procedures. A third procedure used for PVY purification was a slightly modified version of the method described by LEISER & RICHTER (1978). This method was also used for PVA purifications. For virus concent- ration high speed centrifugation was used. For PLRV purification two different methods were used. One was based on enzyme-assisted plant material dispersal as described by TAKANAMI & KUBO (1979) and the other was modified from the method described by CLARKE (1981). For better results with this modification the ELISA method was used to control all the steps in the purification procedure. TRY particles were purified as described by KURPPA et ai. (1981). Since the autumn of 1979 density gradient centrifugation has been used as the final step in separating impurities from virus particles. To prepare the gradient solutions of 20 % (for filamentous viruses) or 25 % (for other viruses) sucrose in buffer were first frozen in Beckman SW 27 tubes. The buffers used to make the gradients were the same ones which were used to resuspend the virus pellets during previous high speed centrifugation in all cases. The gradients were produced immediately before centrifugation by 196 bringing the tubes to room temperature a few hours before use, thus allowing the solutions to thaw slowly. The centrifugation times for each of the viruses were as follows: TRY 2.5 h, PYX, PVS, PVM, PYY and PVA about 3 h and PLRV 4 h at 24 000 rpm. After centrifugation in a Beckman L-50 ultracentrifuge with a Beckman SW- -27 rotor the gradients were fractionated by upward displacement, using an ISCO Model 640 density gradient fractionator. The highly purified virus particles in fractions were recovered from the sucrose solution by dilution and sedimentation for 70—100 minutes at 45 000 rpm in a Beckman 50-Ti rotor. The final sediments were resuspended in a low molar buffer with or without sodium azide depending on their purpose of use. 4. Antiserum production and the properties of the antisera A blood sample (normal serum) of about 5 ml was taken from each rabbit before the first injection. Antisera for potato viruses X, S, M and Y were produced by injecting 1 mg of antigen in 1 ml of buffer mixed 1:1 with Freunds adjuvant (complete adjuvant was used in the first injection and incomplete in the following injections) intramusculary. In each immuniza- tion program several injections were used, for details see tables 3, 10, 14 and 19. Antiserum collection was started 4—6 weeks after the first injection and after that the rabbits were bled over a period of several months at about two week intervals. Blood was taken from the marginal vein in the ear and 20—30 ml was taken at each bleeding. For antiserum separation blood samples were left for I—2 hours at room temperature to allow coagulation to occur and then were moved into a cold room overnight. The next day the clear serum solution was decanted out of the tubes and was centrifugated in a low speed centrifuge at 2000 g/15 min. The antiserum was stored in soft plastic tubes (NUNC) at —2O C or at 4 C in 1:1 glycerol. The antisera needed for the agar gel diffusion tests were produced by using degraded virus protein (D-protein) in the injections. To prepare the protein, purified virus suspensions of 1 - 2 mg/ml were mixed rapidly with equal volumes of 5 % pyrrolidine. The mixtures were dialysed immediately in 3 changes of buffer solution. The immunization and blood collection prog- rams are outlined in figs. 2, 11 and 18. To determine the titers of the antisera the microprecipitin test of van SLOGTEREN (1955) was used in cases where the antisera were produced using whole virus particles as the immunogens. For titration 0.02 ml of diluted antigen and antiserum solutions were mixed in a drop on a new plastic petri plate or on a Formwar coated petri plate made of glass. Evaporation from the drops was prevented by fixing a wet filter paper inside the cover of the petri plates. The antisera were diluted in 2 steps and the antigens in 4 steps starting from 0.5 mg/ml. A saline solution was used as the control in all tests. The test 197 results were read both after 4 hours and 20 hours of incubation. The titers of the antisera produced with degraded virus protein were determined via a double diffusion test (van SLOGTEREN 1955). The 1 % agar gels were 3 mm thick and holes were cut into them with a cork borer 4.5 mm in diameter. The distance between the central hole and the surrounding holes was 4 mm. For titer determination antigen dilutions of 0.5 mg/ml, 0.125 mg/ ml and 0.031 mg/ml were used. The gels were incubated at 4 C and were examined 1, 2 and 4 days later. To determine heterologous titers and the relationships between the viruses and virus isolates, the microprecipitin and gel diffusion tests were used with the antigen concentrations mentioned above. For comparison with foreign antisera, serum preparates of the following origin were tested: Danish antisera, Statens Forsogsvirksomhed i Plantekultur, Lyngby; Estonian antis- era, Jogeva Plant Breeding Station; Polish antisera, Institut Ziemniaka, Bonin; Hungarian antisera, Agrärtudomänyi Egyetem Burgonyanemesitö Osoport, Keszthely. To determine the serological relationships between Finnish virus isolates the EM-serological AVM (antibody-virus mixture) method (ROBERTS et al. 1979) and the ELISA test were used. 5. Identification methods 5.1. Chloroplast agglutination, microprecipitin and cut leaf tests Antisera produced for virus isolates in this study were compared with the antisera earlier produced at the Department of Plant Pathology and with the antisera of foreign origin in microprecipitin tests using purified virus and clarified virus-containing sap as antigens. Similar comparisons were also made with chloroplast agglutination tests using potato leaf sap (see van SLOGTEREN 1935, BALL 1961). The results obtained from the serological identification of PVY were compared with biological A- and TEr cut leaf tests (KÖHLER 1953, de BOKX 1974). An attempt was made to try to improve the reaction specificity and the reliability of the chloroplast agglutination test by adding 0.4 % sodium sulphite (STASZEWICZ 1977) or 1 % bentonite to the diluted antisera. Otherwise the tests were carried out as originally described. The results of the agglutination tests were read under the microscope after 2 hours of incubation at room temperature and the results of the cut leaf tests were read 7 days after incubation at 20—22 C with 2000 lux of continuous illumination. 5.2. Gel diffusion tests In order to identify potato viruses X, S and M by means of gel diffusion tests, leaf or tuber sap was mixed with an equal volume of 5 % pyrrolidine. 198 Otherwise the tests were made as described above. In routine tests the antiserum in a 1/8 dilution was placed in the central hole. Test plates were read after 2 days of incubation at 4 C. For single diffusion tests (MANCINI et al. 1964, SHEPARD & SECOR 1969) the gel plates were prepared by mixing undiluted antiserum with a 1 % agar gel solution at 50 C. Several antiserum dilutions were tested but in routine tests a final antiserum concentration of 1/50 in agar was used. Holes were cut in 2 mm thick agar with a cork borer 4.5 mm in diameter and the distance between the holes varied from 5 to 10 mm. The test samples were prepared as described for the double diffusion test. The results were read after 1,2, 4 and 24 hours of incubation at 4 C. 5.3. ELISA test The preparative steps, antibody purifications and conjugate preparations for the ELISA test were carried out as described by CLARK & ADAMS (1977). However, excluding the initial experiments, total immunoglobulin instead of the y-globulin fraction was used for coating the plates and for conjugate preparation, because in the course of further antibody purification heavy losses of antibodies were found to be caused by the DEAE-cellulose treat- ment. Microtitration plates made by several manufactures were tested but Lindbro microtitration plates 76301-05 or E.I.A. plates 76381-04 (Flow Laboratories, Hamden U.S.A.) and MicrostripR -plates (Eflab Ltd, Helsinki, Finland) were usually used. In all steps of the ELISA test (see CLARK & ADAMS 1977) 200 pi of reagents or samples were placed into the wells of the plates. To place the test samples into the wells a 1-channel pipette (Finn- pipette Ltd, Helsinki) was used. Test reagents were placed with a multichan- nel dispenser (Eflab Ltd, Helsinki). Reaction absorbances were measured with a Titertek Multiscan photome- ter (Eflab Ltd, Helsinki) at a wavelength of 405 nm. In routine tests absorbance measurements were carried out after 30—60 mins of substrate incubation at 22 C. In order to study the specificity and sensitivity of this method different incubation times were tested. Viruses were identified from dormant tubers, sprouted tubers and potato leaf sap. To determine the quantitative sensitivity of the test purified virus antigen was used. Relative sensitivity was determined by using serial dilutions of different virus infected plant material. The ELISA method was also used in the serological classifica- tion of virus isolates and to improve the purification methods for virus Y and potato leaf roll virus. The potato samples studied were selected so that comparisons could be made between the different virus identification methods. 6. Field experiments and statistical analysis of the results Several field experiments were carried out to study virus transmission, viral symptomology in different potato cultivars and the yield losses caused by viruses. Suitable material for virus identification studies was simultane- ously obtained from these experiments. Only some of the results obtained from the field experiments are reported here. Those reported deal particularly with natural virus transmission and other important differences between the potato virus isolates. The main purpose of this work was to study virus properties in relation to their serological identification and to improve serological methods for reliable virus identification in routine work. Because of the aim of this study statistical analyses were seldom needed to verify the reliability of the results obtained. B. The viruses and the virus strains found in Finland and their properties 1. Potato virus X (PVX) Potato virus X, R/l :2.1/6:E/E:S/(Fu), is distributed worldwide in potato growing areas. It consists of elongated particles with a normal length of 515 nm. The virus was first described by Smith in 1931 (BERCKS 1970). The following synonyms have been used for the virus: ’’Healthy potato virus” (JOHNSON 1936), Potato latent virus, Potato mosaic virus and Solanum virus 1 (BERCKS 1970). The host range is mainly limited to the Solanaceae although some plants in other families are susceptible and may maintain the virus in the field (ALLEN & DAVIS 1981). Potato virus X causes mild mosaic of potato, mosaic and stunting of tomato, and mottle or necrotic ring spotting of tobacco. The virus is mainly transmitted by contact (BERCKS 1970) but some insects are probably able to transmit it mechanically on their mouthparts (WALTERS 1952). Transmission has also been reported by the fungus Synchytrium endobioticum (NIENHAUS & STILLE 1965). According to several field experiments the virus causes yield losses of 0-10 % (REESTMAN 1970, van der ZAAG 1977, MANZER et al. 1979) but also heavy yield losses of up to 74 % have been reported (KLINKOWSKI 1951). In good potato growing conditions the tuber yield losses have been 0.10—0.30 % for every one percent of virus infected plants found in the field (BONDE & MERRIAM 1951, BONDE 1953, BORCHARDT et al. 1964). 2 199 200 Potato virus X may have been detected serologically as early as the 1930’s (CHESTER 1937). 1.1. Symptoms in potatoes and test plants and virus strain classification All isolates caused mild symptoms in potatoes and some of them were completely latent. When virus X was isolated from plants with mixed infections visible symptoms were always found. Potato virus X was common in seed potato in Finland still in the 1970’s (Table 6, page 208). The typical symptoms caused by the isolates belonging to the common strain are mild mosaic and vein clearing. A strain type with different symptoms was found occurring naturally in the cvs. Ostara, Prumex and Saturna. Isolates of this strain causes leaf-narrowing, vein-clearing and faint chlorotic ring spots in potatoes (Fig. 3, page 206). These isolates were transmitted quickly in the field although only the mechanical means of transmission could be demonstrated (Fig. 1). The viruses isolated from potatoes were classified according to the symptoms they caused in test plants. No symptom variation was found in the following test plants: Chenopodium amaranticolor: Small chlorotic local lesions in 3 8 days; later the inoculated leaves became reddish and dropped. Fig. 1. Natural transmission of PVX in field experiments at Viikki during three successive growing seasons using seed material from the previous crop. 201 C. quinoa: Small chlorotic local lesions in 4—7 days. The inoculated leaves became chlorotic and dropped immaturely in 14—22 days. Gomphrena globosa: Necrotic local lesions in 2—4 days. The lesions grew larger and developed red margins in 6—B days (Fig. 4). The PVX isolates were classified into two strains according to the symptomology of the following test plants (see SALAMAN 1938, LADEBURG et al. 1950, VARMA et ai. 1970): I Common strain (’’mild mosaic” strain): Datura stramonium: Faint chlorotic local rings or no local symptoms; mild systemic mosaic and chlorosis in 6—9 days. Lycopersicon esculentum : Mild systemic mosaic and necrosis in 6—B days. A 6: No local symptoms; systemic chlorosis and necrosis in 10—14 days. Solanum chacoense: No local symptoms; systemic mottle and necrosis in 10—14 days. Nicotiana glutinosa : No local symptoms; systemic mosaic, mottle and stunt- ing in 7—lo days. N. tabacum cv. Samsum: No local symptoms; systemic vein clearing and faint mottle in 7—lo days (Fig. 5). The origin of the isolates of the ’’mild mosaic” strain was as follows XSFI, Tammisto Early, Tikkurila 1975 XSF2, Pito, Seed Testing Institute 1975 XSFS, Record, Hankkija Plant Breeding Institute 1976 XSFB, Veto, Plant Breeding Institute, A.R.C. 1976 XSF9, Kaptah, Danish imported seed 1976 XSFII, Hja’s Tuomas, Flankkija Plant Breeding Institute 1977 XSFIB, Bintje, Renko 1982 XSFI9, Record, Tyrnävä 1982 XSF2O, Bintje, Seinäjoki 1982 XSF2I, Record, Flämeenlinna 1982 II Ringspot strain: D. stramonium: Chlorotic local rings in 4—6 days; systemic mottle, mosaic and necrosis in 6—B days. L. esculentum: No local symptoms; systemic mosaic, yellowing and necrosis A6: Small necrotic local spots and lesions in 3—5 days; systemic chlorosis and deformation in B—l 4 days (Fig. 9). S. chacoense : Small necrotic local spots in 3—6 days; systemic chlorosis and necrosis in 8 14 days. N. glutinosa: Chlorotic or necrotic ringspots in 4—6 days; severe systemic mosaic and deformation in 7—lo days; occasionally systemic leaf necrosis (Fig. 6). N. tabacum cv. Samsun: Chlorotic and necrotic local rings in 4—6 days; systemic chlorotic and necrotic rings and vein clearing in 6—B days (Figs. 7 and 8). The isolates belonging to the ringspot strain of PVX originated as follows: XSF3, Saturna, Dutch imported seed XSF4, Ostara, 1975 1975 XSF6, Saturna, Prestoperuna Ltd XSF7, Prumex, 1976 1976 XSFIO, Saturna, XSFI2, Prumex, 1977 1977 XSFI3, Saturna, Loppi XSFI4, Saturna, Tyrnävä XSFIS, Ostara, Porvoo XSFI6, Ostara, Porvoo XSFI7, Saturna, Pyhtää 1979 1980 1982 1982 1982 Minor variants with similar host ranges and symptomology within these strains were grouped together and from these 4 type isolates were selected for further studies. 1.2. Sap properties and virus purification 1.2.1. Thermal inactivation point and dilution end point All isolates studied lost their infectivity at at least 72 C when virus- containing N. glutinosa sap was heated for 10 min. The isolates XSFI and XSF2 of the mild strain were inactivated at lower temperatures than the ringspot strain isolates XSF6 and XSFI3 (Table 1). Gradual inactivation was found to start at temperatures lower than 60 C. The mild strain isolates of PVX were inactivated when sap of systemically Table 1. The mean number of local lesions on Gomphrena globosa leaves inoculated with Nicotiana glutinosa sap containing PVX isolates heated for 10 min at different temperatures. Lesions/leaf (4 replicates) Temperature C Isolate XSFI XSF2 XSF6 XSFI3 rUnheated >3O >3O >3O >3O 54 >3O >3O >3O >3O 57 21.3 26.5 27.3 >3O 60 11.3 16.5 16.0 21.8 63 5.0 8.3 12.3 13.5 66 0.0 1.8 3.0 6.0 69 0.0 0.0 1.0 2.5 72 0.0 0.0 0.0 0.0 75 0.0 0.0 0.0 0.0 78 0.0 0.0 0.0 0.0 202 203 Table 2. The mean number of local lesions on Gomphrena glohosa leaves inoculated with diluted Nicotiana glutinosa sap containing PVX isolates. Lesions/leaf (4 replicates) Dilution Isolate XSFI XSF2 XSF6 XSFI3 10° >3O >3O >3O >3O infected N. glutinosa was diluted to 10 6 . The isolates of the ringspot strain lost their infectivity when diluted to 10”7 (Table 2). Longevity in vitro values were not carefully determined but all isolates were infective after 2 months of incubation at room temperature. 1.2.2. Virus purification The following maximum yields of purified virus preparates were obtained with the method described by SHEPARD (1972): XSFI = 360 mg, XSF2 = 480 mg, XSF6 = 540 mg and XSFI3 = 650 mg/1 kg systemically infected N. glutinosa leaves. The purity of the preparates before density gradient cen- trifugation was fairly good. In 5—35 w/v and 10—40 w/v linear sucrose gradients the virus moved as a single narrow band. All purified virus preparates could be stored in 0.05 M borate buffer of pH 8.2 containing 0.02 % sodium azide at 4 C for at least 2 years without loss of infectivity. The A260/A2BO ratio was 1.20 for all isolates. 1.2.3. Electron microscopy Numerous filamentous virus particles were found in all negatively stained preparates made from infected potato or test plant leaf sap. The typical particle length in PTA (1.5 %, pH 6.5) stained preparates was 510—520 nm and the typical diameter was 12 nm. The virus particles, as seen with the electron microscope, were slightly bent and often occurred in groups of several particles (Fig. 59, page 251). The virus could usually be reliably separated from other filamentous potato viruses because of its very high particle consentration, typical particle length and habit of aggregation. In purified preparations the virus particles kept their typical properties. However, broken particles of different length were found more frequently than in sap preparates. 1.3. Serological properties 1.3.1. Homologous titers of the antisera All of the 4 PVX isolates studied proved to be good immunogens when injected intramusculary into rabbits. Titers high enough for serological tests (1/2048 1/8192 in microprecipitin tests) were reached 3—5 weeks after the first injection (Table 3). Repeated injections did not significantly raise the titers of the antisera, and the titers remained high for several weeks after the final injection. The isolates of the ringspot strain had higher titers than those of the mild mosaic strain and they also reached a higher level more quickly. The non-specific titers of all antisera were 1:4 or more lower when they were determined with the microprecipitin or the chloroplast agglutination test. Table 3. Injection and sampling schedules, and homologous titers of the antisera as determined with the microprecipitin test. —* = injection. Time in Virus isolate and titer of the antiserum weeks XSFI XSF2 XSF6 XSFI3 0 -> -> -> 1 _,_„_,_,. 3 -»1/512 -»1/312 -»1/2048 ->l/4096 4 5 1/2048 1/2048 1/40% 1/4096 6 -» 7 1/2048 -»1/2048 1/4096 8 1/8192 9 -.1/1024 1/4096 -» 10 1/4096 11 1/2048 1/8192 1/8192 -» 12 13 1/2048 1/4096 1/8192 1/8192 14 15 1/1024 1/2048 1/4096 1/8192 16 -» -» 17 1/4096 1/4096 18 1/4096 1/4096 19 20 1/2048 1/4096 21 22 1/2048 1/2048 23 24 1/1024 25 -» 26 27 1/4096 28 29 1/4096 30 31 32 1/2048 204 205 1.3.2. Heterologous titers of the antisera Slight serological strain specificity was found when antisera taken in the early stage of sampling (5 weeks from the Ist injection) were tested with heterologous antigens. The heterologous reactions in the microprecipitin test were always rather strong and the lowest heterologous titer determined was 1/1024 (Table 4). The antisera for isolates of the same strain were almost identical. Table 4, Homologous and heterologous titers of the antisera of PVX isolates as determined with the microprecipitin test; antigen concentrations; 250—4 pg/ml. Antigen The antiserum and its titer XSFI XSF2 XSF6 XSFI3 XSFI 1/2048 1/2048 1/1024 1/1024 XSF2 1/2048 1/2048 1/1024 1/2048 XSF6 1/1024 1/1024 1/4096 1/4096 XSFI3 1/1024 1/1024 1/2048 1/4096 The antisera of foreign origin reacted moderately with the antigens studied. The Polish antiserum had a significantly weaker reaction than the other antisera (Table 5). All the foreign antisera could be used, however, for potato virus X identification in our country. Table 5. The highest titers determined for 2 indigenous and 4 foreign antisera against PVX antigens XSF2 and XSF6 with the microprecipitin test; antigen concentration: 250—4pg/ml. Antigen The antiserum and its titer XSF2 XSF6 X Estonian X Polish X Danish X Hungarian XSF2 1/8192 1/2048 1/2048 1/512 1/4096 1/2048 XSF6 1/2048 1/8192 1/1024 1/512 1/1024 1/1024 1.3.3. Immunogenesis of the degraded antigen The purified PYX preparate (XSFI), degraded with pyrrolidine, caused an increase in antibody formation when injected into rabbit. The titer rose slowly and then only remained at the same level for a few weeks. The highest titer determined with the double diffusion test was 1/128 and it was reached 4 weeks from the beginning of the injection schedule. The same titer was reached again later when the injections were repeated (Fig. 2). The heterolog- ous titers of the antiserum against the other PYX isolates were as follows; XSF2 = 1/128, XSF6 = 1/64 and XSFI3 = 1/64. The most suitable concentration of degraded virus protein for the double diffusion test was 250—500 pg/ml. A higher concentration of antigen was responsible for antigen/antibody disproportion, which could be detected as a double precipitin line or a broad diffusive precipitin line. A significantly 206 lower antigen concentration was responsible for a sharp but weakly visible precipitin line. Fig. 2, PVX-D-antiserum production procedure and the titer of the antiserum as determined by the double diffusion test during the course of programme. Fig. 3. Chlorotic ringspots induced by the ringspot strain of PVX in potato cv. Saturna leaves. Fig. 4. Necrotic local lesions in Gomphrena globosa leaves as induced by each PVX isolate. 207 Fig. 5 Vein clearing and vein mosaic in Nicotiana tabacum cv. Samsun induced by the most mild strain Fig. 6. Severe yellow mosaic symptoms in Nicotiana glutinosa infected with the ringspot strain of PVX, isolate XSFI4. Fig. 9. Local necrotic spots and lesions of different sizes in Solarium demissum leaves induced by the ringspot strain isolates of PVX. Figs. 7 and 8. Systemic chlorotic and necrotic rings and concentric rings in Nicotiana tabacum cv. Samsun leaves induced by the ringspot strain of PVX, isolate XSF 10 (Fig. 7) and isolate XSFI4 (Fig. 8). 2. Potato virus S (PVS) Potato virus S, */* i*/*:E/E:S/Ap, is a virus with straight to slightly curved filamentous particles c. 650 X 12 nm (WETTER & BRANDES 1956). PVS is a member of the Carla-virus group (HARRISON et al. 1971). The virus was first described by de BRUYN OUBOTER (1952) and ROZENDAAL (1952). The virus is sap and aphid transmitted (BODE & WEIDEMANN 1971). It is found all over the world in cultivated potatoes and is nowadays probably the most common of the potato viruses (KOWALSKA 1978). Potato virus S is widely reported as an inducer of mild diseases (van der ZAAG 1977, MANZER et al. 1978, 1979) but it can also cause significant yield losses in potato tubers of up to 20 % (WETTER 1971). The virus causes few or no symptoms alone but in mixed infections with potato virus M the symptoms are easily visible (HUNNIUS 1976). The host range of PVS is narrow and its importance is restricted to potatoes. However other susceptible species exist and they belong mainly to the genera Chenopodium, Lycopersicon, Nicotiana and Solanum (BAGNALL et al. 1956, HORVATH 1964, VULIC & HUNNIUS 1967, ROSS 1968, de BOKX 1970b). Table 6. The occurrence of the most common potato viruses in commercial seed potato lots tested at the Department of Plant Pathology in 1975 and 1976. Cuhivar % of tested tubers infected with a virus PVX PVS PVM PVY 8 26 59 60 8 9 3 39 9 37 10 8 33 14 11 Record 1 1 42 67 31 2 0 0 2 0 3 0 0 0 0 4 10 0 0 5 0 4 12 2 6 0 0 0 0 7 0 2 3 0 Jaakko 1 14 41 9 17 2 27 59 13 12 208 2.1. Occurrence, symptomology and transmission Potato virus S was very common in potatoes grown in Finland until the end of the 1970’5, when the use of healthy seed potato became established (AURA 1957, SEPPÄNEN 1972, 1974, YLLÖ 1975). Some of the most widely grown cultivars were totally infected with virus S (Table 6). The virus occurred in most cultivars showing only very mild symptoms and it was impossible to decide with the naked eye whether a potato plant was infected or not. In some cultivars, e.g. Pito and Sanna, symptoms of interveinal mosaic and chlorotic spots between the veins, were easily visible (Fig. 12, page 216). When symptoms existed they were clearest at the time of blooming or immediately after it. The symptoms were more typical of the cultivars than of the virus isolates. Natural transmission in the field was very rapid if the cultivar was susceptible. The cultivars susceptible to virus S were simultaneously suscep- tible to virus M (Fig. 10). Most PVS isolates were readily aphid transmitted. However, significant variation in aphid transmission between the virus isolates were found (Table 7). Similar symptoms appeared 24—30 days after inoculation in test plants that were mechanically inoculated. Fig. 10. Natural transmission of PVS and PVM in field experiments at Viikki during three successive growing seasons using seed material from the previous crop. 209 210 Table 7. Aphid transmission of PVS isolates. Three wingless Myzus persicae aphids were transferred onto each Nicotiana debneyi plant. The acquisition timewas 2 min and the inoculation time was 18-20 h. Three replicates of 10 test plants were used. For the origin of the isolates see text. % of plants infected with PVS Virus isolate SSFI SSF4 SSF6 SSF7 SSFB SSFIO SSFI4 SSFIS 13.3 0.0 6.7 6.7 10.0 26.7 30.0 36.7 2.2. Symptoms in test plants and isolate classification Most isolates of PVS induced similar symptoms in certain test plants, however some differences were found. Similar symptoms were found in the following host species: Chenopodium amaranticolor: Chlorotic local lesions I—2 mm in diameter in 15—20 days. C. quinoa: Chlorotic local lesions I—2 mm in diameter in 12—20 days (Fig. 14). Solanum rostratum: Numerous small necrotic local lesions in 20—30 days. Systemically infected leaves showed similar symptoms. Leaf drop of older leaves occurred (Fig. 13). Datura metel Lycopersicon esculentum cv. Kotitomaatti Nicandra physaloides Latent systemic Physalis floridana infection was found. Solanum demissum Y Gomphrena globosa Nicotiana clevelandii N. glutinosa These plants did not become infected.N. tahacum cv. Samsun Phaseolus vulgaris cv. Stella Uniform variation in the severity of the symptoms was found in L. chilense and L. esculentum cv. Nevskij. According to these symptoms the virus isolates were classified into the following 3 groups: Group I L. chilense: Systemic epinasty, mild vein-clearing and stunting in 10—14 days. Later also leaf-curling and leaf-drop occurred. No necrosis was found. L. esculentum cv. Nevskij: Latent systemic infection initially; later leaf- curling of the oldest leaves; no necrosis. The following isolates were included in this group: SSF4, Kaptah (Danish), Prestoperuna Ltd 1976 SSF6, Hja’s Tuomas, Hankkija Plant Breeding Institute 1976 211 SSFB, Veto, Plant Breeding Institute, A.R.C. 1976 SSFII, Hja’s Timo, Hankkija Plant Breeding Institute 1978 Group II L. chilense: Systemic epinasty and vein-clearing in 10—14 days; stunting and leaf necrosis starting from the basal leaves in 18—22 days. L. esculentum cv. Nevskij: Stunting and faint epinasty in 10—15 days; necrosis in the basal leaves in 15—20 days. The following isolates were included in this group: SSFI, Jaakko, Tikkurila 1975 SSF3, Pito, Seed Testing Institute 1975 SSFS, Prestoperuna Ltd 1976 SSF9, Record, Hankkija Plant Breeding Institute 1976 SSFI3, Veto, Plant Breeding Institute, A.R.C. 1978 SSFI6, Saturna, Liminka 1980 SSFI7, Posmo, Viikki 1980 SSFIB, Eigenheimer, Porvoo 1982 Group 111 L. chilense: Severe systemic epinasty and leaf-curling in 10—14 days; severe foliar necrosis and leaf-drop in 18—22 days. L. esculentum cv. Nevskij: Leaf-curling and stunting in 10—15 days; necrosis and leaf-drop starting from the basal leaves in 15—20 days (Fig. 15). The following isolates were included in this group: SSF2, Saturna, Dutch imported seed 1975 SSF7, Sanna, Plant Breeding Institute, A.R.C. 1976 SSFIO, Prevalent, Prestoperuna Ltd 1977 SSFI2, Sanna, Plant Breeding Institute, A.R.C. 1978 SSFI4, Ostara, Viikki 1979 SSFIS, Pito, Ahvenanmaa 1979 N. debneyi showed the following variation in symptoms between PVS isolates: All isolates induced systemic vein chlorosis and chlorotic areas between the veins in 20—30 days followed by marginal leaf necrosis in 40—50 days. Large chlorotic rings were caused by the isolates SSF7 and 12 (Fig. 16) and large chlorotic spots by the isolates SSFI,IS and 18. Chlorotic lesions ofequal size were induced by the other isolates (Fig. 17). Symptom variation between the isolates was minimal. The isolates selec- ted for further studies had the following properties: SSFI: Severe chlorosis in potatoes; not easily transmitted by aphids. SSF4: Some ’’rough” symptoms in potatoes; no aphid transmission was detected. SSF6: No visible symptoms in potatoes and mild symptoms in the test plants; not easily transmitted by aphids. 212 SSF7: Chlorosis in potatoes and chlorotic rings in N. dehneyi; severe symptoms in L. chilense and L. esculentum cv. Nevskij; not easily transmitted by aphids. SSFI4; Chlorosis in potatoes and severe symptoms in L. chilense and L. esculentum cv. Nevskij; readily transmitted by the aphids. 2.3. Sap properties and virus purification 2.3.1. Thermal inactivation point and dilution end point The values obtained for the thermal inactivation points of the virus isolates were almost equal and so it was not possible to use this test as a criterium for classification (Table 8). Table 8. The mean number of lesions on Chenopodium quinoa leaves after inoculation with heated sap of Nicotiana dehneyi infected with PVS isolates. Three replications were done. Lesion number Temperature in C Isolate SSFI SSF4 SSF6 SSF7 SSFI4 Unhealed 51.3 76.0 57.7 81.7 49.7 45 24.3 41.0 32.0 27.7 31.3 48 14.0 21.3 16.3 16.0 19.0 51 6.0 7.7 7.7 9.0 3.7 54 3.3 5.3 2.0 4.7 1.3 57 0.0 2.3 1.5 2.0 0.0 60 0.0 0.0 0.0 0.0 0.0 63 0.0 0.0 0.0 0.0 0.0 The dilution end point values were between 10‘2 and 10'3 . No significant differences in dilution end point values were found but a relationship between them and the thermal inactivation point values was obvious (Table 9). Table 9. The mean number of lesions on Chenopodium quinoa leaves after inoculation with diluted sap of Nicotiana dehneyi. Three replications were done. Lesion number Dilution Isolate SSFI SSF4 SSF6 SSF7 SSFI4 Undiluted 44.3 69.0 47.3 74.7 36.0 10' 11.0 19.7 13.7 17.0 8.3 10"2 1.3 3.7 1.3 2.0 0.7 10 3 0.0 0.0 0.0 0.0 0.0 10'" 0.0 0.0 0.0 0.0 0.0 213 2.3.2. Electron microscopy and purification Numerous virus particles were seen in negatively stained preparates of PVS infected plant sap. A length of c. 650 nm and a diameter of 12 nm were typical of the virus particles. The particles were straight or slightly flexuous and usually occurred separately (Fig. 60, page 251). Broken particles or particles joined from end to end were seldom seen. In virus purification L. esculentum cv. Nevskij proved to be the host in which the highest virus concentration occurred. The purity of the virus preparate was, however, best when it was prepared from potato leaves. A partially purified preparate from L. esculentum cv. Nevskij contained the highest amount of impurities. In density gradient centrifugation the virus moved to one narrow band. In electron microscope preparates it was found to contain homogeneous and mainly intact particles. The following maximum yields of purified virus were obtained for the PVS isolates: SSFI from 5. tuberosum cv. Pito SSF4 from N. debneyi 18 mg/kg 21 ” SSF6 from N. debneyi 19 SSF7 from N. debneyi 27 SSF7 from L. esculentum cv. Nevskij 31 SSFI4 from N. debneyi 22 SSFI4 from L. esculentum cv. Nevskij 28 All purified virus preparates were infectious after being stored for 2 years in 0.05 M borate buffer which contained 0.02 % sodium azide at pH 8.2. 2.4. Serological properties 2.4.1. Homologous titers of the antisera All of the purified PVS isolates proved to be good or moderately good immunogens (Table 10). Reasonably high titers for serological tests were reached in 3—5 weeks. The titers of certain isolates remained somewhat lower than those of some others in spite of repeated injections. The titers were stable for several weeks. Non-specific titers were 1/8 or lower in the chloroplast agglutination and microprecipitin tests. 2.4.2. Heterologous titers of the antisera All Finnish PVS isolates reacted strongly against any antisera produced in this study. The lowest heterologous titer as determined by the microprecipi- tin test was 1/128. The maximum heterologous reaction was normally found 214 Table 10. Injection and sampling schedules of antiserum production by PVS isolates and titers as determined by the microprecipitin test. —> = injection. Time Virus isolates and their titers in weeks SSFI SSF4 SSF6 SSF7 SSFI4r 1/2048 1/1024 1/512 1/2048 1/1024 1/1024 1/512 1/2048 1/2048 -.1/1024 -.1/512 -.1/256 -»1/1024 1/2048 1/2048 1/1024 1/512 1/2048 -.1/1024 1/1024 1/1024 1/256 1/2048 iy 20 -.1/1024 1/512 21 1/1024 24 -.1/1024 1/1024 28 1/1024 when the antigen concentration was 30 fig/ml as determined by the micropre- cipitin test. The isolates SSFI, SSF4 and SSF6 were serologically closely related but the isolates SSF7 and SSFI4 differed significantly from the main group and from each other (Table 11). Remarkable variation in the heterologous titers of the foreign antisera was Table 11. Assessment of serological relationships between the PVS isolates by the microprecipitin test. Antigen Antisera and their titers SSFI SSF4 SSF6 SSF7 SSFI4 SSFI 1/2048 1/1024 1/512 1/256 1/256 SSF4 1024 2048 512 512 512 SSF6 1024 1024 1024 256 512 SSF7 512 512 512 512 256 SSFI4 256 256 256 128 2048 Table 12. The highest titers of 3 indigenous and 4 foreign antisera used in determining the Finnish type isolates of PVS. Antigen Antisera and their titers SSI-1 SSF7 SSFI4 Danish Estonian Polish Hungarian SSI-'l 1/2048 1/256 1/256 1/2048 1/1024 1/128 1/256 SSF7 512 512 256 512 256 1/32 1/64 SSFI4 256 128 2048 256 256 1/64 1/64 found. All the antisera reacted with the Finnish PVS isolates but only the Danish and the Estonian antisera were suitable for routine work. The foreign antisera were more closely related to the isolates SSFI, 4 and 6 than to the others (Tables 11 and 12). 2.4.3. Immunogenesis of the degraded antigen Potato virus S, isolate SSFI, degraded with pyrrolidine and then injected into rabbits, gave rise to antibody formation. The titer of the antiserum rose slowly and repeated injections were needed to keep it at a standard level (Fig. 11). The highest titer of the antiserum was 1/32 as determined with the agar gel double diffusion test and it was reached 4 weeks after the first in- jection. The same level was obtained again later but some of the reaction spe- cificity was lost. In comparison, the titers of the other degraded PVS- proteins were as follows: SSF4 = 1/32, SSF7 = 1/16, SSFI4 =l/8. The ideal protein concentration in the titers was 125 jig or 250 jig/ml. A higher concentration was responsible for a broader, diffuse precipitin line or a double one and with a lower concentration only a sharp, weakly visible 3 Fig. 11. PVS-D-antiserum production procedure and the titer of the antiserum as determined by the double diffusion test during the course of the programme. 215 216 Fig. 14. Local diffuse chlorotic lesions in Chenopodium quinoa leaves induced by all PVS isolates. Fig. 12. Chlorotic spots between the veins of the leaves of potato cv. Pito infected with the PVS isolate SSF3. Fig. 13. Systemic necrotic spots in Solarium rostratum leaves induced by all PVS isolates Fig. 15. Systemic leaf curling and necrosis starting from the basal leaves of Lysopersicon esculentum cv Nevskij as caused by the PVS isolate, SSFI2. 217 3. Potato virus M (PVM) Potato virus M, R/1:2.4/5.4:E/E:S/Ap (PROLL et al. 1981) is distributed worldwide in cultivated potatoes and diseases caused by it have been reported since the 1920’s (SCHULZ & FOLSOM 1923). The virus itself, however, was not studied until the 1950’s (BAGNALL et al. 1956, ROZEN- DAAL & van SLOGTEREN 1958, BRANDES et al. 1959, WETTER & VOLK 1960, ROSS 1968, KOWALSKA 1978, PROLL et al. 1978, PROLL & RICHTER 1979, PROLL et al. 1981). The virus has several synonyms, the most well-known of which are Kartoffel-K-Virus, Kartoffel-Rollmosaik-Virus, Potato interveinal mosaic virus, Potato leaf rolling mosaic virus, Potato paracrinkle virus, Potato virus E, Solanum virus 7 and Solanum virus 11 (WETTER 1972). Potato virus M has straight to slightly curved filamentous particles c. 650 X 12 nm (BRANDES et al. 1959). It is sap and aphid transmitted (WETTER 1972). Its host range is narrow with most species belonging to the Solanaceae (BAGNALL et al. 1956, BRANDES et al. 1959, ROSS 1968) but susceptible species in a few other families have also been found. Suitable hosts among them are: Datura metel (KAHN & MONROE 1970); Gomphrena globosa (BAGNALL et al. 1959); Phaseolus vulgaris (HIRUKI 1970, HORVATH 1972) and some Chenopodium species (HORVATH & de BOKX 1972). The economical importance of the virus is restricted to the damage it causes to potatoes. Remarkable variation in disease severity occurs among different potato cultivars and different virus strains or isolates (BEEMSTER & ROZENDAAL 1972). Examples of very susceptible cultivars are Arran Victory Figs. 16 and 17. Large systemic chlorotic rings or chlorotic + necrotic spots and vein mosaic in Nicotiana debneyi infected with PVS isolate SSF7 (Fig. 16) and isolate SSFI4 (Fig. 17). 218 (BRANDES & WETTER 1963) and Uran (CHRZANOWSKA 1976). In these cultivars the virus causes severe stunting and leaf-rolling symptoms. Losses in tuber yields of up to 60 % have been reported (CHRZANOWSKA 1976). In most potato cultivars the virus causes mild disease with yield losses of between 5 and 20 % (O’BRIEN & RICH 1976). In diseases caused by a mixture of several viruses PVM greatly increases the level of disease in the potato (HUNNIUS 1976). 3.1. Occurrence and symptomology in potatoes Potato virus M was rather common in commercial seed potatoes in Finland tested in 1975 and 1976. Also the potatoes produced at our plant breeding stations were severely infected with the virus. In imported seed potato the virus was seldom detected at that time (Table 6). All of the potato cultivars that were tested-in field experiments were susceptible to the virus but large differences in disease severity between the cultivars were found (Fig. 10). The natural rate of infection among the cultivars in the field reached the following levels during the third experimen- tal year when the seed potatoes used were obtained from the previous crop: Sanna 79 % PVM-infected tubers, Veto 49 %, Bintje, Ostara and Pito 30 %, Hankkija’s Tuomas 29 %. The most tolerant cultivars were Posmo and Saturna with 1 % infection and Hankkija’s Timo with 4 % infection. In crops of other cultivars between 7 and 10 % of the tubers were detected as being infected with PVM. Potato virus M induced severe stunting and leaf-rolling symptoms in the cultivars Bintje, Hankkija’s Tuomas and Puikelo (Figs. 19 and 22, page 223). On average the tuber yield of infected plants was about 50 % lower than that of healthy plants. The main reason for the yield losses was smaller tuber size. The symptoms typical of PVM were detectable in these cultivars even in cases of severe mixed infections with potato virus Y (Fig. 23). Visible leaf-rolling symptoms were also detected in the following cultivars: Prevalent, Record, Sanna, Hankkija’s Tanu and Hankkija’s Timo. In the other cultivars the virus was latent or only mild interveinal mosaic or some leaf-rolling was observed (Fig. 20). 3.2. Symptoms in test plants and isolate classification All 15 isolates studied could be transmitted both mechanically and by aphids to L. esculentum cv. Kotitomaatti. The infection remained latent but could be detected serologically within 8— 12 days after transmission. The virus induced symptoms in the following test plants: Chenopodium quinoa: Variable number of chlorotic local lesions in 10—14 days. 219 Datura metel: A few chlorotic local lesions in B—lo days when isolates MSF3, 6 and 10 were used as inoculum. Lycopersicon chilense: Systemic symptoms only: leaf-curling, stunting and necrosis in 10—15 days depending on the isolate (Fig. 24). No necrosis was caused by the isolates MSFI, 2,5, 12 and 15; partial vein necrosis was caused by the isolates MSF3, 4,6, 9 andl3 and severe leaf and top necrosis by the isolates MSF7, 8, 10, 11 and 14. Nicotiana dehneyi: Non-uniform large necrotic local lesions in 9—12 days were caused by the isolates MSFS, 6 and 11. Phaseolus vulgaris cv. Red Kidney: Some faint necrotic local lesions were initiated in 5—7 days from all isolates. Solarium rostratum: Systemic leaf-curling, stunting and vein necrosis in 12—18 days followed by leaf and top necrosis. The severity of the symptoms caused by the different isolates were comparable to those seen in L. chilense (Fig. 21). For further studies isolates ofL. chilense and S. rostratum were classified into the following categories according to their symptomology: I Mild isolates: MSFI, Hja's Tuomas, Hankkija Plant Breeding Institute 1976 MSF2, Pito, The University Farm, Viikki 1976 MSFS, Veto, Plant Breeding Institute, A.R.C. 1976 MSFI2, Prevalent, Pyhtää 1980 MSFIS, Provita, Liljendal 1982 II Moderate isolates: MSF3, Kaptah, Danish imported seed 1976 MSF4, Hja's Timo, Hankkija Plant Breeding Institute 1976 MSF6, Pito, Maaninka 1977 MSF9, Pito, The University Farm, Viikki 1978 MSFI3, Hja's Tuomas, The University Farm, Viikki 1979 111 Severe isolates: MSF7, Saturna, Prestoperuna Ltd 1977 MSFB, Sanna, Plant Breeding Institute, A.R.C. 1978 MSFIO, Record, Loppi 1979 MSFII, Bintje, The University Farm, Viikki 1980 MSFI4, Sanna, Plant Breeding Institute, A.R.C. 1980 3.3. Sap properties and virus purification Thermal inactivation point and the dilution end point values for PVM isolates were determined using a host species (L. esculentum cv. Kotitomaatti) that developed a latent systemic infection because no locally infected host showed clear symptoms. The assay plants were tested serologi- 220 Table 13. The thermal inactivation point (TIP) and the dilution end point (DEP) values for 5 PVM isolates from infected sap of Lycopersicon esculentum cv. Kotitomaatti plants. Virus isolates MSFI MSF3 MSFS MSF7 MSFB TIP C 64 67 64 67 67 DEP 10 2 10"' 10 2 10'3 10 3 cally 3 weeks after being mechanically inoculated with treated plant sap. The variation in TIP and DEP values was not great; the former being between 64 and 67 C and the latter between 10‘2 and 10‘3 (Table 13). Numerous virus particles were seen in electron micrographs of negatively stained preparates made from systemically infected potato and tomato sap. Most of the straight or slightly curved particles were equal in size at c. 650 X 12nm (Fig. 61, page 251). In purified virus EM preparates the same particle properties could be seen and c. 70 % of the particles had kept their normal length. A virus preparate made from frozen leaves of L. esculentum cv. Kotitomaatti, moved to one narrow band in linear sucrose gradients with density gradient centrifugation. The purified virus preparate remained infec- tive for at least 2 years in 0.05 M borate buffer containing 0.02 % NaN 3 at pH 8.2. The highest yields of purified virus were obtained from the following isolates as follows: MSFI = 30 mg, MSF3 = 42 mg, MSFS = 35 mg, MSF7 = 48 mg and MSFB = 35 mg /kg leaves. 3.4. Serological properties 3.4.1. Homologous titers of the antisera The PVM isolates used for antiserum production were found to be good immunogens. The titers of the antisera reached a level high enough for serological tests in 39 weeks (Table 14) but variation among the titers was found. The titers remained at the same level for several weeks after the last injection. The non-specific titer in the microprecipitin and in the chloroplast agglutination tests was 1/8 or lower. 3.4.2. Heterologous titers of the antisera All of the PVM isolates were serologically closely related so that no serological strains could be detected (Table 15). The isolates MSFI and MSF3 and similarly MSF7 and MSFB were serologically almost identical. The highest titers were obtained when an antigen concentration of 30—120 pg/ml was used. The titers of foreign antisera in reactions with Finnish PVM isolates were lower than those obtained with domestic antisera. However, all foreign antisera reacted strongly to all of the isolates studied (Table 15). 221 Table 14. Immunization and sampling schedules, and the homologous titers of PVM isolates as obtained with the microprecipitin test. —* = injection. Virus isolate and titer Time MSFI MSF3 MSF7 MSFB in weeks 0 -» -» 4 5 1/1024 1/2048 6 -»1/2048 -»1/512 7 1/1024 1/2048 9 10 -»1/512 -»1/1024 -»1/2048 -»1/256 11 12 1/1024 1/2048 1/4096 1/1024 13 14 -»1/1024 -»1/1024 1/4096 1/1024 15 16 1/2048 1/1024 17 1/2048 1/2048 18 -»1/2048 —l/512 19 1/1024 1/2048 20 1/4096 1/1024 21 22 —l/1024 1/4096 1/1024 23 24 1/1024 1/2048 1/512 25 26 1/1024 Table 15. Heterologous titers of Finnish and foreign antisera tested against 4 PVM isolates as determined with the microprecipitin test. Antigen Antisera and their titers MSFI MSF3 MSF7 MSFB Danish Estonian Polish Hungarian MSFI 1/1024 1/1024 1/2048 1/512 1/512 1/512 1/128 1/128 MSF3 1/1024 1/2048 1/2048 1/512 1/512 1/512 1/128 1/256 MSF7 1/1024 1/512 1/4096 1/1024 1/1024 1/512 1/256 1/256 MSFB 1/512 1/1024 1/4096 1/1024 1/1024 1/1024 1/512 1/512 3.4.3. Immunogenesis of the degraded antigen A pyrrolidine degraded antigen (MSF7) gave rise to antibody formation in rabbits. The titer of the antiserum rose slowly, however, and repeated injections were needed to keep it at the highest level. The highest titer obtained, as determined by an agar gel double diffusion test, was 1/64 and it 222 initially occurred 5 weeks after the first injection. The same titer level was also reached later in the immunization procedure, however some of the reaction specificity was lost in the course of the program (Fig. 18). The antiserum reacted similarly against all of the other PVM isolates studied. The most suitable antigen concentration for titer determination was c. 0.25 mg/ ml. A higher concentration resulted in broad or double precipitin lines, and a lower concentration led to a weakly visible line. Figs, 19 and 22. Stunting and "leaf rolling” symptoms in potato cvs. Ffja’s Tuomas (Fig. 19) and Bintje (Fig. 22) infected with PVM. Fig. 20. Rugosity and chlorotic lesions in potato cv. Pito infected with PVM. Fig. 21. Stunting and systemic vein necrosis in Solarium rostratum infected with PVM isolate MSFB. Fig. 23. Mixed infection of PVM and PVY° in potato cv. Ffja’s Tuomas. Symptoms typical of both the viruses are clearly visible. Fig. 24. Stunting, leaf-curling and leaf necrosis in Lycopersicon chilense infected with PVM isolate MSFIO. Fig. 18. PVM-D-antiserum production procedure and the titer of the antiserum as determined by the double diffusion test during the course of programme. 223 Fig. 19 Fig. 22 Fig. 20 Fig. 23 Fig. 21 Fig. 24 224 4. Potato virus Y (PVY) Potato virus Y, R/l :3/6:E/E:S/Ve/Ap, first described by SMITH (1931), is distributed worldwide in cultivated potatoes. The following selected synonyms have been used for the virus or the diseases it induces in potatoes: Potato rugose mosaic (SCHULTZ & FOLSOM 1923), Potato acropetal necrosis virus, Potato severe mosaic virus, Marmor upsilon, Solanum virus 2, Tobacco vein-banding virus (de BOKX & HUTTINGA 1981). The names ’’Potato virus C” (BALD & NORRIS 1945) and ’’Tobacco veinal necrosis” (SMITH & DENNIS 1940, RICHARDSON 1958) refer to the strains Yc and Yn , respectively. The virus has long flexous particles c. 730 X 11 nm. It is easily transmitted mechanically to a narrow range of hosts and it is transmitted by many aphid species non-persistently (KENNEDY et al. 1962, van HOOF 1980, de BOKX & HUTTINGA 1981). In addition to potatoes, the virus causes economically important diseases in peppers, tobacco and tomatoes (de BOKX & HUTTINGA 1981). In potatoes, the differences between primary and secondary symptoms are often indistinct. Typical mild primary symptoms include necrotic rings and lesions, and leaf yellowing followed by mottle or mosaic. The virus may also induce severe primary symptoms, such as necrotic spots and stripes, vein necrosis, ’’leaf drop streak” and sometimes even premature death. Secondary symptoms vary from mild mottling to premature death of the plants but the most typical symptoms are rugosity, chlorosis, vein and leaf necrosis and ’’leaf drop streak”. The degree of symptom severity in potatoes is primarily a result of the interaction between the virus strains and the potato cultivars. The PVY strain Yn is milder than the Y° strain, which often induces necrosis (DARBY et al. 1951, BEEMSTER & ROZENDAAL 1972, O’BRIEN & RICH 1976, WEIDEMANN 1981). Potato virus Y decreases the yield of infected potatoes from 10 to 80 %, depending on the virus strain, the potato cultivar and the time of infection (BALD 1945, ARENZ & HUNNIUS 1959, REESTMAN 1970, van der ZAAG 1977). When the tuber yield is about 40 tn/ha, every 1 % of infected plants causes a reduction in yield from between 0.54 and 0.61 % (BORCHART et al. 1964). The virus is transmitted to the tubers of all the plants that have secondary infections (de BOKX & HUTTINGA 1981). PVY° strains are distributed worlwide: Yn strains occur in Europe including the USSR, parts of Africa and South America; Yc strains are rather rare and probably occur in Australia, India and some parts of Europe (BAWDEN 1936, SMITH & DENNIS 1940, RICHARDSON 1958, KAHN & MON- ROE 1963, de BOKX & HUTTINGA 1981). The host range of about 60 species is mainly limited to the Solanaceae but some members of the Amaranthaceae, Chenopodiaceae and Leguminosae are also susceptible (MUNRO 1955, HORVATH 1964, THORNBERRY 1966, SCHMELZER 1967). The most suitable local lesion hosts are A6-hybrid (KÖHLER 1953), S. chacoense (de BOKX 1974) and S. demissum Y (CHRZANOWSKA et al. 1977). TV. tabacum cvs. Samsun and White Burley are useful in differentiating Y° and Yn strains (KLINKOWSKI & SCHMELZER 225 1957, 1960). Moreover these test plants are suitable hosts for virus purifica- tion (HUTTINGA 1973). Potato virus Y could be identified serologically as early as the 1950’s (CREMER 1952) but the identification results were unreliable (BARTELS 1957) until the late 1970’s when the ELISA test began to be used (GUGERLI 1978, MAAT & de BOKX 1978). 4.1. Potato symptomology and virus transmission Potato virus Y was very common and the cause of severe viral diseases in Finnish potatoes until the end of the 1970’5, when healthy seed potatoes became commonly available and they started to be used by many farmers (BRUMMER 1946, POHJAKALLIO et ai. 1961, SEPPÄNEN 1972, YLLÖ 1975). Also recent important Finnish potato cultivars such as Pito have rapidly lost their high yielding capacity when infected with PVY (SEPPÄNEN 1974, YLLÖ 1975) and this has maintained infection sources for the virus. PVY was rather common in commercial seed lots tested at the Depart- ment of Plant Pathology in 1975 and 1976 (Table 6). The symptoms of secondary infection by PVY were visible in all potato cultivars. However, the symptoms were indistinct and depended on the interaction between a particular virus strain and a particular potato cultivar. It was therefore impossible to be certain whether a cultivar was susceptible or not. Severe necrosis, which often led to the death of the plant, commonly occurred in the cultivars Ostara and Hja’s Timo (Figs. 27 and 28, page 234). Severe leaf necrosis (’’leaf drop streak”) was often found in the cultivars Norstern, Isabell and Carina (Fig. 30). Similar symptoms, although milder, were obvious in the cultivars Hja’s Tuomas and Hja’s Tanu. Dwarfing, mottling and leaf-curling were found in the following cultivars: Bintje, Jaakko, Pito, Puikelo, Record and Sanna. Milder symptoms occurred in the cultivars Frila, Posmo, Prevalent, Prumex, Sabina and Sieglinde. The symptoms in the cultivars Saturna and Veto always remained mild. Greenhouse experiments showed that symptom severity depended more on the virus strain than the potato cultivar. All five isolates tested, which belonged to the Y° strains, caused severe vein and leaf necrosis as primary and secondary symptoms in the cultivars Ostara and Record (Fig. 31). The isolates belonging to the Yn strains caused mottle, mosaic and rugosity but no necrosis was found (Fig. 29). Natural transmission of PVY from outside of the experimental area to the field plots was rapid. Most potato cultivars became severely infected with naturally occurring PVY during the three year experimental period (Fig. 25). Significant differences in infection susceptibility were found between the cultivars. At the end of the third successive experimental year some cultivars were totally infected with virus Y when tubers from the previous crop were used as the seed material. In some cultivars the PVY % of infected tubers was less than 10. In potato cultivars showing leaf or top necrosis the virus infection percentage was lower than the mean infection rate. All of the PYY isolates studied were readily transmitted by the aphid species Myzus persicae, Aphis nasturtii-frangulae and Aulacorthum solani from potato plants to potatoes or other susceptible test plants when an acquisition time of I—2 mins and an inoculation time of 4 18 hours was used. All the attempts to transmit the virus with the aphid species Rhopalosiphum padi were unsuccessful. 4.2. Symptoms in test plants and strain classification PYY isolates induced the following symptoms in the six test plant species which were used for virus isolation and classification: A 6 cut leaf: - necrotic rings; the isolates YSFS, 10, 13, 15, 17, 19, 20, 21, 22, 24, 25, 27, 29, 30, 31, 32, 36, 39, 40, 41, 42, 44, 48 - necrotic rings and vein necrosis; the isolates YSFI, 3,4, 5,6, 7,8, 9, 11, 12, 14, 16, 18, 23, 33, 34, 35, 38 - necrotic spots and vein necrosis; the isolates YSFI, 2,3, 4,6, 8,9, 11, 12, 14, 18, 22, 26, 27, 28, 33, 37, 45, 46, 47 Solarium demissum A (SdA) (Fig. 34): systemic infection, latent or mild crinkling in the top leaves; the isolates YSFI, 2, 3,4, 5,6, 7,8, 9, 11, 14, 16, 17, 18, 24, 25, 26,28,34, 35, 41, 45, 46, 47 - systemic crinkling and chlorosis; the isolates YSF 10, 12, 13, 15, 19, 20, 21, 22, 23, 27, 29, 30, 31, 32, 33, 36, 37, 38, 39, 40, 42, 43, 44, 48 S. demissum Y (SdY) (Fig. 32): - necrotic local lesions caused by all isolates Fig. 25. Natural transmission of PVY in field experiments at Viikki during three successive growing seasons using seed material from the previous crop. 226 227 - systemic leaf necrosis from all isolates - top necrosis followed by premature death of the plants from all isolates excluding YSFI, 11, 17 and 35 Nicotiana glutinosa (Fig. 33): - systemic vein-clearing and mild crinkling; the isolates YSFI, 3,4, 6,7, 8,9, 14, 17, 18, 26, 28, 30, 31, 34, 35, 39, 40, 41, 44, 45, 46 - severe systemic vein-clearing and crinkle; the isolates YSF2, 5, 10, 11, 12, 13, 15, 16, 19, 20, 21, 22, 23, 24, 25, 27, 29, 32, 33, 36, 37, 38, 42, 43, 47, 48 N. tabacum cv. Samsun (Figs. 35—38 and 40): - chlorotic or necrotic local rings followed by systemic vein-clearing; the isolates YSF4, 6, 26, 46 - systemic vein-clearing and vein mosaic; the isolates YSFI,2, 3,5, 7,8, 9, 11, 12, 14, 16, 18, 24, 28, 45, 47 - systemic vein necrosis followed by leaf-drop; the isolates YSFIO, 13, 15, 17, 19, 20, 21, 22, 23, 25, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 48 Physails floridana (Fig. 39): - some necrotic local lesions induced by all isolates - systemic crinkling and chlorosis; the isolates YSFIS, 17, 19, 20, 23, 25, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 48 - systemic chlorosis and stunting; the isolates YSFIO, 13, 16, 21, 22, 29, 33, 43 - systemic chlorosis, stunting and leaf necrosis; the isolates YSFI, 2,5, 8, 12, 18, 24, 27, 28, 45, 47 - systemic leaf and top necrosis followed by premature death of the plants; the isolates YSF3, 4,6, 7,9, 11, 14, 26, 28, 46 The PYY isolates were classified according to the following symptoms in the test plants: S. demissum Y; necrotic local lesions in 3—5 days followed by systemic leaf or top necrosis A 6 cut leaves: necrotic rings or lesions, often also veinal necrosis in 4—6 days The isolates were classified into strains according to their symptomology in N. tabacum cv. Samsun. The isolates inducing vein necrosis were classified into the PVYn strain and the rest into PVY° strain. The following test plant symptoms also gave further information about the isolates: - in A6: necrotic rings were caused by 86 % of the Yn isolates and necrotic lesions by 80 % of the Y° isolates - in S. demissum A: mild symptoms without chlorosis were caused by 95 % of the PVY° isolates and crinkling with chlorosis by 82 % of Yn isolates - in P. floridana: systemic necrosis was caused by all of the PVY° isolates and chlorosis and stunting without necrosis by 97 % of the PVYn isolates. Of 48 PVY isolates, 28 were classified into the strain Yn and 20 into into the strain Y°. The strain Yn of PVY was found more commonly than the strain Y° in seed potatoes produced in Finland in 1981. Of 36 randomly selected tuber lots 28 were infected with Yn isolates and 8 with Y° isolates. Both isolates were found in seed lots from southern Finland but only Yn isolates were found in tuber lots from northwestern Finland, which is where high class seed potato is mainly produced in this country. When early isolations of PVY were made from field potatoes the severe symptoms induced by the Y° isolates may have led to more samples being taken of plants infected with this isolate rather than from plants showing the mild symptoms of other the isolates. The origin of the isolates was as follows: Isolates of the PVY° strain: YSFI, Pito, Seed Testing Institute 1975 YSF2, Pito, The University Farm, Viikki 1975 YSF3, Pito -"- 1976 YSF4, Ostara, Dutch imported seed 1976 YSFS, Record, Hankkija Plant Breeding Institute 1976 YSF6, Isabell, Dutch imported seed 1976 YSF7, Puikelo, Espoo 1977 YSFB, Prevalent, Prestoperuna Ltd 1977 YSF9, Hja's Timo, Hankkija Plant Breeding Institute 1980 YSFII, Pito, Espoo 1981 YSFI2, Record, Viikki 1981 YSFI4, Puikelo, Inari 1981 YSFI6, Record, Raisio 1981 YSFIB, Ostara, Porvoo 1981 YSF24, Sabina, Kitee 1981 YSF26, Prevalent, Alajärvi 1981 YSF2B, Eigenheimer, Porvoo 1981 YSF4S, Bintje, Ahvenanmaa 1981 YSF46, "Black local", Viikki 1982 YSF47, Record, Lammi 1982 Isolates of the PVYn strain: YSFIO, Bintje, The University Farm, Viikki 1979 YSFI3, Pito, Ahvenanmaa 1979 YSFIS, Record, Tyrnävä 1980 YSFI7, Record, Liminka 1980 YSFI9, Record, Liminka 1981 YSF2O, Sanna, Tyrnävä 1981 YSF2I, Pito, Tyrnävä 1981 YSF22, Sanna, Liminka 1981 YSF23, Record, Tyrnävä 1981 YSF2S, Record, Liminka 1981 YSF27, Record, Kitee 1981 228 229 Table 16. The symptoms induced by PVY° isolates (YSF4 and YSFII) and PVY" isolates (YSFIO and YSFIS) in various test plants. If any differences within a strain were found they are mentioned in the table. ”L” indicates local symptoms and ”S” systemic symptoms. Symptoms Test plant PVY° isolates PVY" isolates A 6 C. amaranticolor C. cjuinoa G. globosa L. chilense L. esculentum L. pimpinellifolium N. physaloides N. clevelandii N. debneyi N. glutinosa N. tabacum cv. Samsun P. flondana S. chacoense S. demissum A 5. demissum Y Necr. L lesions or rings in 4—6 d. Chlor. and necr. L lesions in 10—12 d. Few diffusive chlor. L lesions in 10-12 d. Not infected S vein-clearing (YSF11); S mottle and mosaic (YSF4) in 12-16 d. Latent S infection Latent S infection Latent S infection S chlorosis followed by necrosis and premature death in 20—28 d. S interveinai chlorosis (YSF11); chlor. spots (YSF4) in 6—lo d. S vein-clearing and crinkling in 6-10 d. Chlor. and necr. L rings in s—B d. (YSF4), S vein-clearing and inter- veinal mosaic in 7—lo d. Necr. L lesions in 4—6 d. followed by leaf and top necrosis in B—ls d. and premature death in 20—30 d. Necr. L lesions in 4—6 d. followed by vein and top necrosis in 10—15 d. and premature death in 20-35 d. S latent infection (YSF4) or mild crinkling in 10—14 d. Necr. L lesions in 3—6 d. followed by vein and top necrosis; premature death by YSF4 Necr. L rings and vein necrosis in 4-6 d. Chlor. L lesions in 10—12 d. Few chlor. L lesions in 10—12 d. Not infected S vein-clearing and vein mosaic in 12-16 d. Latent S infection S vein-clearing and mosaic in 14-16 d. (fig. 41) Latent S infection S vein-clearing and mosaic followed by necrosis and premature death in 20-28 d. S interveinai chlorosis and yellow mosaic in 6—B d. S vein-clearing and severe crinkling in 6-10 d. S vein necrosis in 10—14 d. fol- lowed by leaf-drop Chlor. and necr. L lesions in 4-6 d. followed by S chlorosis and stunt- ing in 10—15 d. Necr. L lesions in 4—6 d. followed by vein necrosis (YSFIO) or top necrosis (YSFIS) in 12-16 d. S chlorosis and crinkling in B—l 2 d. Necr. L lesions in 3—6 d. followed by vein and top necrosis and pre- mature death in 20—35 d. YSF29, Record, Tyrnävä 1981 YSF3O, Record, Ylistaro 1981 YSF3I, Record, Hämeenlinna 1981 YSF32, Sabina, Lumijoki 1981 YSF33, Bintje, Porvoo 1981 YSF34, Provita, Ahvenanmaa 1981 YSF3S, Bintje, Ahvenanmaa 1981 YSF36, Record, Porvoo 1981 YSF37, Bintje, Porvoo 1981 YSF3B, Bintje, Porvoo 1981 YSF39, Record, Tyrnävä 1981 YSF4O, Bintje, Porvoo 1981 YSF4I, Record, Tyrnävä 1981 YSF42, Record, Tyrnävä 1981 YSF43, Pito, Tyrnävä 1981 YSF44, Record, Porvoo 1981 YSF4B, Record, Lammi 1982 When the symptoms of the 4 selected type isolates of PVY were studied further (with the use of wider host ranges) some further information about the differences between the isolates were obtained, but no new features important in strain classification were found (Table 16). 4.3. Sap properties The thermal inactivation point (TIP) values obtained for the PVY isolates were between 48 and 57 C. The TIP value of the isolate YSFIS was remarkably higher than that of the other isolates. The virus greatly lost its infectivity near the inactivation temperature (Table 17). All of the test plants that showed local symptoms also became systemi- cally infected. The dilution end point (DIP) values were between 10~2 and 10~3 and were higher for the Yn strain isolates than they were for the Y° isolates. The infectivity was markedly reduced near the dilution end point (Table 18). All of the test plants that showed local symptoms also became systemi- cally infected. Table 17. The thermal inactivation point (TIP) values for 4 PVY isolates determined with Nicotiana glutinosa sap inoculated into Solarium demissum Y. The mean number of lesions/leaf PVY strain and isolate PVY° PVY" Temperature C YSF4 YSFII YSFIO YSFIS room temperature 21.0 24.7 21.3 34.0 45 6.0 16.3 9.7 71.7 48 2.3 4.0 2.0 23.0 51 1.0 1.3 0.3 14.7 54 0.0 0.0 0.0 4.0 57 0.0 0.0 0.0 0.3 60 0.0 0.0 0.0 0.0 63 0.0 0.0 0.0 0.0 230 Table 18. The dilution end (DIP) point values of 4 PVY isolates determined by inoculating Nicotiana glutinosa sap into Solanum demissum Y. The mean number of lesions/leaf In negatively stained EM preparates made from potato or test plant sap the virus particles had a typical length of c. 730 nm. The particle density was higher in the PVY" preparates than in the PVY° preparates. In EM micro- graphs PVY particles appeared more flexous than those of PVS or PVM and were significantly longer than PVX particles and could easily be separated from them if several particles were compared. The only difference between PVY and PVA was the higher particle density of PVY, however this character cannot be used to reliably separate these two viruses. 4.4. Virus purification The purification method of STAGE-SMITH & TREMAINE (1970) was initially used as the standard purification method but it gave relatively low yields of purified virus. The highest yields obtained with this method were from the isolates YSFI and YSF4 (in N. glutinosa) and they were 7 mg and 4 mg/kg respectively. Later, a method based on chloroform + buthanol clarification and differential centrifugations was used and it gave the following yields of purified virus: YSF4 9 mg, YSFII 12 mg, YSFIO 21 mg and YSFIS 27 mg/kg leaves. This purification method had some destructive effects on the coat protein of the virus particles and it interfered with virus movement during density gradient centrifugation as well as reducing the stability of the virus. Also relatively high amounts of low molecular weight plant proteins existed in the virus preparates when they were ready for density gradient centrifuga- tion. The third purification method used, which was a slight modification of the method described by LEISER & RICETTER (1978), was found to be the most suitable for PVY. The virus containing preparates were almost clean even before density gradient centrifugation (Fig. 62, page 251) and the virus particles retained their typical properties at the end of the purification proce- dure. In density gradient centrifugation the virus moved to form a narrow band and very little particle aggregation was observed (Fig. 26). After one cycle of density gradient centrifugation the virus preparates were clean and the value 4 231 232 for the absorbance relation A 254/A 280 was 1.21. The maximum yields obtained of purified virus isolates YSFII (N. glutinosa) and YSFIS (N. tabacum cv. Samsun) were 16 mg and 26 mg/kg leaf matter respectively. The virus preparates could be preserved for at least 6 months in 0.005 M borate buffer containing 0.03 M NaCl and 0.003 M Na-citrate at pH 8.0. 4.5. Serological properties 4.5.1. Homologous titers of the antisera The four PVY isolates were moderate to good immunogens when injected intramusculary with Freund’s adjuvant into rabbits. The immune response of PVY" isolates was better than that of PVY° isolates. A titer high enough for use in the serological tests was attained within 3 to 6 weeks after the first injection (Table 19). The non-spesific titers of the antisera as determined with the microprecipitin and the chloroplast agglutination test were 1/4—l/16. 4.5.2. Heterologous titers of the antisera Serological variation between the PVY isolates was found and the heterologous titers were 2 8 times lower than the homologous ones. The titers of foreign antisera against Finnish PVY isolates were low. The foreign antisera were more closely related to Y° isolates than to Yn isolates (Table 20). Comparable information about the serological relationships between the PVY isolates was also obtained from EM-serological AVM tests. Good Fig. 26. Scanning pattern obtained in the ISCO density-gradient fractionator after one cycle of density- gradient centrifugation for 160 min at 24 000 rpm (Beckman SW-27 rotor) in 5—35 % w/v linear sucrose gradients. a = normal virus movement as indicated by a narrow single band b = broad band due to particle aggregation problems c = band showing both particle aggregation and distortion agreement with the results from the microprecipitin tests was found but the titers determined for the antisera were somewhat higher (Table 21). Table 19. The injection schedule and the homologous titers of PVY antisera as determined with the microprecipitin test. —> = injection Time Virus isolates and their homologous titers in weeks YSFI YSF4 YSFIO YSFTI l/128 -* 1/256 10 the rabbit died 1/2048 1/512 14 1/2048 1/1024 Table 20, The heterologous titers of PVY antisera against 3 Finnish PVY isolates as determined with the microprecipitin test. Antigen Virus isolates and their heterologous titers YSFI YSF4 YSFIO YSFII Estonian Polish Hungarian YSF4 1/256 1/512 1/256 1/512 1/128 1/32 1/32 YSFIO 1/128 1/64 1/2048 1/128 1/32 1/16 1/16 YSFII 1/512 1/256 1/512 1/1024 1/256 1/32 1/64 ,: " The isolate YSFI was lost in the course of the study. Table 21. The homologous and heterologous titers of four PVY antisera as determined with an EM- serological test. Antigen Antisera and their titers YSFI YSF4 YSFIO YSFII YSF4 1/512 1/1024 1/512 1/2048 YSFIO 1/256 1/256 1/4096 1/512 233 Fig. 27. Secondarily infected potato plant cv. Ostara killed by PVY". Fig. 30. Rugosity, stunting and leaf drop symptoms in potato cv. Isabell infected with PVY° isolate YSF6. Fig. 28. Severe vein and top necrosis in potato cv. Hja's Timo secondarily infected by PVY". The disease leads to the death of the plant. Fig. 31. Local and systemic necrosis in potato cv. Record 4 weeks after mechanical inoculation with PVY° isolate YSFII. Fig. 29. Primary symptoms, mosaic and faint chlorotic spots in potato cv. Record infected with PVY" isolate YSFIO. 234 Fig. 32. Systemic necrosis in Solanum demissum Y 10 days after aphid transmission with PVY' Fig. 35. Nicotiana tabacum cv. Samsun infected with PVY° isolate YSF4 showing local chlorotic and necrotic rings and systemic vein clearing. Fig. 33. Systemic vein clearing, wrinkling and stunting in Nicotiana glutinosa infected with PVY“ isolate YSFIS. Fig. 36. Nicotiana tabacum cv. Samsun infected with PVY° isolate YSFII showing faint chlorotic local spots, and severe systemic vein clearing and vein mosaic. Fig, 34. Solanum demissum A plants infected with PVY° isolate YSF4 (left) and PYY" isolate YSF29 (right). The latter shows systemic chlorosis and rugosity. 235 236 Figs. 37 and 40. Nicotiana tabacum cv. Samsun infected with any PVY" strain isolate first show systemic vein necrosis (Fig. 37) and then later leaf drop (Fig. 40). Fig. 38. Nicotiana tabacum cv. Samsun plants infected with PVY° isolate YSFI2 (left) and PVY" isolate YSFIO (right). The latter shows severe vein necrosis, the former mild vein clearing. Fig. 41. Severe systemic mosaic and chlorotic symptoms in Lycopersicon pimpinellifolium infected with PVY n isolate YSFIS. Fig. 39. Physalis floridana plants infected with PVYn isolate YSFI3 (left) and PVY° isolate YSFIB (right). The former shows chlorosis, wrinkling and stunting, the latter has been killed by the virus. 237 5. Potato virus A (PVA) Potato virus A, */*:*/*: E/E:S/Ap, first described by MURPHY & McKAY (1932) can be found all over the world wherever potatoes are grown. Selected synonyms for the virus are Marmor solani, Potato mild mosaic, Potato virus P and Solanum virus 3 (BARTELS 1971). The virus has long flexous particles of c. 730 X 11 nm. It is transmitted by several aphid species in a non persistent manner and can also be spread mechanically. The mechanical means of transmission, however, has no importance because the virus is rather unstable (Mac LAGHLAN et al. 1953, BARTELS 1971, O’BRIEN & RICH 1976). The narrow host range of this virus is restricted to the Solanaceae. The most suitable local lesion hosts are A6-hybrid (Solanum demissum X S. tuberosum cv. Aquila) and 5. demissum A. Systemic symptoms are seen in Nicotiana tabacum cvs. Samsun and White Burley, Nicandra physaloides and Lycopersicon pimpinellifolium (Mac LAGHLAN et al. 1953, BARTELS 1970, 1971, de BOKX 1970a, 1975). In potatoes the virus causes mild mosaic, chlorosis, mottling and mild crinkling. In some potato cultivars the virus is latent but on other hand it may cause top necrosis in hypersensitive cultivars (Mac LAGHLAN 1953, BARTELS 1971, de BOKX 1975, O’BRIEN & RICH 1976). Tuber yield losses caused by potato virus A are normally relatively low, from 2 to 15 % (de BOKX 1975), but they may be as high as 40 % (BORCHARDT et al. 1964, BARTELS 1971). In cases of mixed infection with viruses X, S, M or Y, PVA always causes significant tuber yield losses (BARTELS 1971). 5.1. Properties of the virus isolates that occur in Finland 5.1.1. Occurrence and symptomology in potatoes and test plants Potato virus A was relatively rarely found in diseased potato tubers or plants. Four isolates were successfullyrecovered from the following sources: ASFI, Pito, Viikki 1975 ASF2, Eigenheimer, Porvoo 1981 ASF3, Eigenheimer, Porvoo 1982 ASF4, "Black local", Viikki 1982 The virus isolate ASFI induced mild mosaic in its natural host, whereas the isolates ASF2 and ASF3 induced chlorosis and crinkling and the isolate ASF4 caused chlorosis and severe crinkling (Figs. 42 and 44). In the selected test plants all virus isolates induced the following symp- toms: A 6 cut leaf: Necrotic ’’starlike” local lesions in 45 days. S. demissum A: Numerous necrotic local lesions in 3—5 days followed by systemic vein necrosis in s—B days and premature death of the plant in 10—15 days (Fig. 43). 238 S. demissum Y: Occasional necrotic local lesions in 4—5 days followed by latent systemic infection. P. floridana: The plants did not become infected N. glutinosa: The plants did not become infected. L. pimpinellifolium: Systemic necrosis in 8— 12 days. The symptoms differed in the following test plants N. physaloides: Systemic vein-clearing was caused by all isolates, however the isolate ASF4 also caused leaf necrosis (Fig. 45). N. tabacum cv. Samsun: Systemic vein-clearing and diffuse mottling in 8 —lO days. The isolate ASF4 caused more severe symptoms than the other isolates. 5.1.2. Sap properties Electron micrographs of PVA particles look similar to those of PVY and no distinction between the particles of these two viruses can be seen. Very few PVA particles are seen in EM preparates made from either potato or N. physaloides leaf sap (Fig. 63, page 251). The thermal inactivation point values determined for the isolates ASF2 and ASF4 were 45 C and 48 C and the dilution end point values were 10'1 and 10‘2 respectively. At all these end point values N. physaloides and S. demis- sum A plants became systemically infected. According to host range and sap properties the PVA isolates can be classified into the mild (ASFI—3) or the moderate strains (ASF4). 5.1.3. Virus purification and serological identification In this study only a small amount of virus could be purified from systemically infected leaves of N. tabacum cv. Samsun. The method of LEISER & RICHTER (1978) was used with slight modifications and the yield of purified virus was 1.1 mg/100 g leaves. Several methods for the purification of PVA have been developed but all of them have difficulties with virus aggregation and have resulted in low yields of pure virus. Specific antisera to the virus have been prepared, however, because the virus is a good immuno- gen (FRIBOURG & ZOETEN 1970, MAAT & MIERZWA 1975, GUGERLI 1979, singh & McDonald 1981). Attempts at serological identification of the virus in plant samples were inconclusive until the ELISA method was employed (MAAT & de BOKX 1978, GUGERLI 1979). For routine identification of PVA the A 6 cut leaf test had previously been widely used (KOHLER 1953, de BOKX 1970a). The four PVA isolates tested could reliably be identified with the ELISA test in secondarily infected dormant tubers, sprouted tubers and leaves. The virus isolates did not give any significant reaction when tested with different PVY antiserum reagents in the ELISA test. High percentages of PVA infected tubers have been found in tuber samples from the Experimental Station of Central Finland in tests done 239 during the winter of 1983. Up to this point it is still unclear as to whether this virus is abnormally common in the vicinity of this station or not. Fig. 42. Chlorosis and rugosity in potato cv. Pito infected with PVA isolate ASFI Fig. 44. Severe chlorosis and wrinkling in potato cv. ’’Black local” infected with PVA isolate ASF4. Fig. 43. Local necrotic lesions in Solarium demissum A 5 days after mechanical inoculation with PVA. Fig. 45. Vein clearing and faint mosaic in Nicandra physaloides infected with PVA isolate ASF2. 240 6. Potato leaf roll virus (PLRV) Potato leaf roll virus, */*:*/* :S/S:S(I)/Ap, was first described (1916) by QUANJER, van der LEK and OORTWIJN BOTJES (PETERS 1970). The virus has also been named ’’Potato phloem necrosis virus” (QUANJER 1913). The virus particles are isometric and are about 24 nm in diameter. It is transmitted by about 10 aphid species in the persistant manner (MURPHY & McKAY 1929, MacCARTHY 1954, KENNEDY et al. 1962). The most effective vector is Myzus persicae Sulz. (ROBERT 1971, O’BRIEN & RICH 1976, KOSTIW 1981) but also the species Aulacorthum solani Kltb., Macrosiphum euphor- biae Thomas (KIRKPATRICK & ROSS 1952, ROBERT & MAURY 1970) and Aphis nasturtii Kltb. (BEEMSTER & ROZENDAAL 1972) may be important. Potato leaf roll virus is common wherever potatoes are grown (PETERS 1970, BEEMSTER & ROZENDAAL 1972). It causes rolling of the leaves and a stiff upright habit of the plant (PETERS 1970, O’BRIEN & RICH 1976). In the plant the virus is distributed in the phloem tissue, where it causes necrosis of the tissue and an accumulation of carbohydrates in the leaves (KOJIMA et al. 1969, PETERS 1970). Necrosis may also occur in the tubers of infected plants (O’BRIEN & RICH 1976). Potato leaf roll virus may cause yield losses in tubers of up to 70 % but normally the yield loss is between 10 and 40 % due to the compensatory abilities of the crop (TUTHILL & DECKER 1941, REESTMAN 1970). The virus has a narrow host range, mainly in the Solanaceae, but some non-solanaceus plants such as Amaranthus caudatus, Celosia argentea, Gom- phrena globosa and Nolana lanceolata are susceptible. Moreover, there are several solanaceus hosts, such as Atropa belladonna, Lycopersicon pimpinel- lifolium, Nicotiana glutinosa, N. rustica and Solanum nigrum, which can become systemically infected with the virus without showing symptoms (NÄTTI et ai. 1953). 6.1. Occurrence in Finland Potato leaf roll virus has been quite recently introduced into Finland and this has occurred naturally via aphids. Observations of the virus were made as early as the 1920’s (JAMALAINEN 1946) in some field experiments where imported seed material was used. Natural primary infection of the potato crop was recorded for the first time in Viikki in 1979 when PLRV appeared in a potato crop planted with healthy seed (KURPPA 1981). The symptoms were very clear in the cultivars Sieglinde and Record but also the cvs. Bintje and Pito showed symptoms. The primary symptoms were first seen in younger leaves at the end of July immediately after the time of blooming, later rolling of the leaves was seen in older leaves (Figs. 47 and 49, page 243). Bron- zing or red coloration was found particularly in the cvs. Bintje and Record. The following summer severe secondary symptoms occurred in potato plots planted with seed from plants that had been primarily infected during the previous growing season (Figs. 48 and 50). 241 The following PLRV isolates were identified from the virus outbreak in Viikki in 1979: PLRVSFI, Sieglinde PLRVSF2, Sieglinde PLRVSF3, Bintje PLRVSF4, Record PLRVSFS, Pito A new virus outbreak was observed in Sipoo in 1980, where primary and secondary symptoms occurred in abundance in a potato field of about 2 hectares. The PLRV isolate isolated from the field was called PLRVSF6, Record. During the 1981 growing season the virus was not significantly transmit- ted in the field because of exceptionally low aphid populations. However, several new virus outbreaks occurred in 1982 and this led to the identification of the following isolates: PLRVSF7, Prevalent, Renko PLRVSFB, Juliver, Pyhtää PLRVSF9, Sv 71118, Pyhtää PLRVSFIO, Provita, Liljendal PLRVSFII, Saturna, Loviisa The isolates PLRVSFB and 9 originated from experimental material but the other isolates came from large commercial potato fields. 6.2. Properties of the virus isolates All of the isolates induced easily visible symptoms in 14—20 days in Physails floridana and Datura stramonium when transmitted to them by M. percicae (Figs. 51 53). The isolates originally isolated in Viikki in 1979 caused severe or moderate stunting and interveinal chlorosis in the test plants and were classified into severe or moderate isolates according to WEBB et al. (1952) and MacCARTHY (1963). The isolates PLRVSF6, 7 and 11 were mild and 8, 9 and 10 moderate according to the same classification. In a field experiment the virus isolate PLRVSFI caused very significant losses in tuber yield in the three generations following primary infection (Table 22). When comparisons were made, tuber numbers and yields from the first secondarily infected plant generation were 29.2 %and 53.8 % lower than the tuber numbers and yields of the preceding crop. In the following generations the losses were the following: 43.5 % fewer tubers and 77.3 % lower yield in the 2 nd generation and 44.3 % fewer tubers and 77.6 % lower yield in the 3rd generation. 242 Table 22. The effect of secondary PLRV infection on tuber number and yield of the cv. Sieglinde in the three generations following primary infection by isolate PLRVSFI in a field experiment in Viikki. Tuber number/plant Tuber yield/plant Treatment mean variation mean g variation g Healthy control 19.95 14-25 2055 1620-2460 Ist generation 14.03 8-20 950 370-1680 2nd " 11.28 6-18 466 100-880 3rd " 11.12 6-16 457 190-820 F tuber number = 76.8 X“ LSD[0 05 = 1.30 F tuber yield = 390“’' LSDt0 = 107 (g) 6.3. Virus identification The identification of PLRV in potatoes was initially based on symptom observation and ’’the tuber indexing method”, which was completed with the use of P. floridana as the test plant (WEBB et al. 1952, HEPP & de ZOETEN 1978). The callose test (Igel-Lange test), which showed abnormal starch accumulation in the infected tubers, became a routine test for PLRV identifi- cation in the 1950’s (IGEL & LANGE 1953, SPRAU 1957). The test was widely used until the end of the 1970’s (de BOKX 1967, ZAMBONI et al. 1977, MAAT & de BOKX 1978) when the ELISA method (CLARK & ADAMS 1977) started to supersede it (CASPER 1977a, MAAT & de BOKX 1978, GUGERLI 1979, TAMADA & HARRISON 1980, CLARKE 1981, MARCO 1981). The possibilities for using electron microscopy in the rapid identification of PLRV are restricted but promising attempts have been made by SARKAR (1975). Virus particles can be recognized in thin section preparates made from infected plant tissue (Figs. 64 and 65, page 252) but the method is unsuitable for routine work. To separate purified virus particles from other similar particles or to demonstrate serological relationships between the particles ’’the immunosorbent electron microscopy” and ’’antibody-virus mixture” methods (ROBERTS & HARRISON 1979, ROBERTS et al. 1979) have been used. All of the PLRV isolates tested with the ELISA method can be reliably identified in secondarily infected dormant and sprouted tuber and potato leaf tissue. For identification two reagents were used; a commercial one from the Boehringer Mannheim Ltd and an indigenous one prepared from unpure antiserum obtained from the Scottish Crop Research Institute (SCRI) (TAMADA & HARRISON 1980). Some serological variation between the iso- lates was found although the reagents were closely related to each other. However, the results obtained with the commercial reagent showed higher specificity because of lower background readings with this test. This could be demonstrated by value of the relation Av/A Ho which is a ratio between virus infected and healthy plant samples (Table 23). 243 Table 23. The absorbance values obtained with the ELISA test in undilutedand diluted healthy or PLRV infected potato leaf sap. For the identification reagents from the Boehringer Mannheim (I) and from the SCRI (II) were used. Substrate incubation was carried out at 20 C for 1 h. PLRVSFI 1.420 1.139 .560 .346 .104 .109 .030 .037 11 1.430 1.230 .599 .495 .097 .088 .024 .038 Mean value for healthy samples .051 .157 .031 .072 .016 .040 .012 .031 A V/A H„ 27.25 7.63 10.25 2.82 2.54 .65 .67 .27 6.4. Virus purification and particle properties Several methods have been developed for the purification of potato leaf roll virus (PETERS 1967, KOJIMA et al. 1969, MAAT & de BOKX 1978, HEPP & de ZOETEN 1978, ROWHANI & STAGE-SMITH 1979, TAKANAMI & KUBO 1979, CLARKE 1981). MURAYAMA and KOJIMA (1974) were the first to prepare PLRV antiserum. The virus could be purified from infected P. floridana leaves by the method of TAKANAMI & KUBO (1979) and from potato leaves by a method slightly modified from the method described by CLARKE (1981). The former method was discontinued, however, because of the high costs of the driselase enzyme which is needed in the course of the purification procedure. For higher yields of purified virus, the CLARKE (1981) method was modified in that the ELISA test was used following all of the steps in the procedure. The critical steps in the method were the polyethylene glygol precipitation and the release of the virus from the precipitate into the buffer solution. The yields of purified virus could be gradually increased and the highest yield obtained was 0.7 mg/kg leaf matter compared to 1.1 mg/kg leaf matter with the method of TAKANAMI & KUBO (1979). In linear gradients of sucrose (10—40 % w/v) the virus moved to one narrow band about 35 mm from the meniscus after 4 hs of centrifugation at 24 000 rpm in a Beckman SW-27 rotor (Fig. 46). The A 260/280 value determined for the virus was 1.75. In electron micrographs the purified virus particles were hexagonal with a diameter of about 25 nm (Fig. 66, page 252). 244 Fig. 46. Scanning pattern of partially purified PLRV obtained in the ISCO density-gradient fractionator after one cycle of density-gradientcentrifugation for 4 h at 24 000 rpm (Beckman SW-27 rotor) in 10-40 % w/v linear sucrose gradients. 245 Figs, 47 and 49. Primary symptoms of PLRV in potato cv. Pito (Fig. 47) and cv. Saturna (Fig. 49). Figs. 48 and 50. Secondary symptoms of PLRV in potato cv. Record (Fig. 48) and cv. Sieglinde (Fig. 50). 246 7. Tobacco rattle virus (TRY) Tobacco rattle virus, R/1:2.3 + 0.6 to 1.3/S:E/E:S/Ne, was first described by QUANJER (1943) as Tobacco partridge virus. Other synonyms found in the literature are Aster ringspot virus, Belladonna mosaic virus, Potato corky ringspot virus, Potato stem mottle virus, Ratel virus, Tabakmauche Virus, Tabak Streifen und Kräuselkrankheit Virus (HARRISON 1970), Potato spra- Fig. 51. Interveinal chlorosis in Datura stramonium 4 weeks after infection with the PLRV isolate SF2, Fig. 52. PLRV, isolate SFI, symptoms in Physalis floridana 3 weeks after aphid transmission. Fig. 53. Stunting and interveinal chlorosis in Physalis floridana 6 weeks after infection with the PLRV isolate SFI. ing virus (EDDINS et al. 1946), Nicotiana virus 5 (SMITH 1957) and Tobacco virus 11 (JOHNSON 1936). The virus particles are straight tubes of two predominant lenghts, the longer being c. 190 nm and the shorter being c. 45 —ll5 nm depending on the isolate. The fraction of shorter particles consists of particles of I—3 different lengths. The diameter of all of the particles is c. 25 nm. The virus is transmitted by Trichodorid nematodes (SOL 8c SEINHORST 1961, van HOOF 1968, TAYLOR 8c CADMAN 1969) and some of the isolates are also readily transmitted by inoculation with plant sap (HARRISON 1970). Tobacco rattle virus occurs in Europe, U.S.A., Brazil, Japan (HARRISON 1970) and in New Zealand (JONES 8c YOUNG 1978). Many strains are described which differ from each other in the symptoms they induce in host plants and also in their serological properties (CADMAN 8c HARRISON 1959, HARRISON & WOODS 1966, LISTER & BRACKER 1969, KURPPA et ai. 1981). In potato tubers TRY causes ’’spraing” or ’’corky ringspot”. This consists of rings of corky tissue in the tuber flesh which can make the crop unsaleable for human consumption (HARRISON & ROBINSON 1978). In potato plants the virus causes ’’stem mottle” and mottling of the leaves. Yellow areas may appear in the leaves resembling the symptoms caused by Potato aucuba virus. Some potato cvs. also show stunting, leaf deformation and even necrosis (HARRISON 8c ROBINSON 1978). The tubers produced by the diseased plants are only partially infected. According to BEEMSTER 8c ROZENDAAL (1972) about half of the crop consists of TRY infected tubers when the seed potatoes were all affected with ’’stem mottle” disease as compared to o—3o % diseased tubers produced by seed potatoes affected with ’’spraing”. In addition to potatoes TRY causes economically important damage to tobacco, peppers, sugar beets and several ornamental plants, particularly bulbous ornamentals (HARRISON 1970, HARRISON 8c ROBINSON 1978). The host range of TRY is very wide. More than 400 species in dicoty- ledonous and monocotyledonous families can be infected (SCHMELZER 1957), including a wide range of weed species which are able to maintain the virus in the soil (DAVIS 8c ALLEN 1975). The virus can be transmitted to several plant species mechanically or by nematodes. Necrotic local lesions are produced by TRY in many hosts, but only about half become infected systemically. Suitable local assay hosts are Chenopodium amaranticolor, C. quinoa and Phaseolus vulgaris. TRY infects systemically several species in the family Nicotiana, of which N. clevelandii is the most suitable for maintaining the virus and for virus purification (SCHMELZER 1957). Transmission from potato tubers showing ’’spraing” symptoms to susceptible host species is often difficult (HARRISON & ROBIN- SON 1978). Severe difficulties have been found with the serological identification of TRY. The virus concentration in potato tubers is very low. Also the wide serological variability and the occurence of NM-isolates (these consist of infective RNA only) makes serological testing unreliable (HARRISON 8c ROBINSON 1982). 5 247 248 7.1. Symptoms caused by the isolates occurring in Finland Tobacco rattle virus was isolated from three tuber lots and transferred to C. amaranticolor, C. quinoa and N. clevelandii plants via mechanical inocu- lation with tuber sap. The origin of the isolates were as follows: TRVSFI Pito,Jurva 1980 TRVSF2 Bintje, Renko 1981 TRVSF3 Record, Loppi 1981 Eight tuber lots showing ’’spraing” symptoms were studied but TRY could not be isolated from the rest of the possibly infected samples using the normal method of mechnical inoculation to test plants. The virus caused only mild mottling symptoms in plants grown from virus infected tubers or else no symptoms were found. Virus particles were seen in electron micrographs made from C. quinoa sap where numerous (> 10) local lesions had developed in inoculated leaves. In the C. quinoa leaves where only a few local lesions (< 3) existed no virus particles were found. The particle-forming isolates (3) caused the following similar symptoms in selected test plants: Chenopodium amaranticolor: Necrotic local pinpoint lesions in 3—5 days; not systemic (Fig. 54). C. quinoa: Necrotic local lesions in 3—5 days; not systemic (Fig. 56). Phaseolus vulgaris cv. Stella: Necrotic local pinpoint lesions in 2—4 days; not systemic (Fig. 58). Pisum sativum cv. English sword: Necrotic local lesions I—4 mm in diameter in 5—7 days; not systemic. Vida faba cv. Pirhonen: Necrotic local lesions in 57 days; not systemic (Fig. 57). Nicotiana clevelandii: Occasional chlorotic or necrotic lesions in 3—5 days followed by faint systemic mottle in 5 7 days. N. glutinosa: Large necrotic local lesions in 3—4 days followed by systemic necrotic spots and leaf distortion in 10—14 days. N. tabacum cv. Samsun: Large necrotic local lesions in 3—5 days followed by systemic mottling and leaf distortion in B—l 4 days (Fig. 55). The symptoms in the test plants were similar to those caused by the PRN isolate (CADMAN & HARRISON 1959, HARRISON & NIXON 1959). 7.2. Sap properties and virus purification The thermal inactivation point and the dilution end point values were the same for all of the isolates. The virus lost its infectivity when heated to 85 C or diluted to 10'7. When the virus was purified from systemically infected leaves of N. clevelandii by the method of KURPPA et ai. (1981) yields of between 65 —B5 249 mg/kg leaf material of purified virus were obtained. In density gradient centrifugation in linear sucrose gradients of 10—40 w/v sucrose the partially purified virus moved to two dominant bands. In electron micrographs most virus particles had a length of v. 190 or c. 75 nm and a diameter of 24 nm (Fig. 67, page 252). 7.3. Serological properties No antisera were prepared for the virus isolates but the reactions of all purified virus preparates were tested against 4 known and one unknown TRV antisera and a PEBV (Pea early browning virus, British isolate) antiserum according to the EM-serological AMV method (Table 24, Figs. 68 and 69). Table 24. Heterologous reaction of 5 TRV antiseraand a PEBV antiserum to 3 indigenous TRV isolates in the EM-serological AMV test. Virus concentrations of 10 ng/ml were used. Antigen Antisera and their titers CAM ORE PRN SYM 1627 PEBV TRVSFI 64 16 512 32 16 < 16 TRVSF2 128 32 1024 32 32 2 > 2 > 2 > 2 > 2 .857 .224 .126 .063 .029 XSFI3 >2 >2 >2 >2 1.641 .630 .209 .112 .044 .019 XSFI4 >2 >2 >2 >2 1.776 .706 .198 .113 .066 .024 XSF2 >2 >2 >2 >2 >2 1.041 .316 .171 .092 .033 PVY°(YSFII) .063 .042 .028 .019 .014 .015 .011 .009 .008 .006 Fig. 77. The effect of substrate incubation time on' the specificity of the reaction. Ratio Av/Aho = the absorb- ance value of (diluted) virus infected sample / the ab- sorbance mean value of un- diluted healthy samples. The virus is PVM in tomato leaf sap. 264 265 Substrate incubation time was not very critical if the immunoglobulin and conjugate reaction was virus-specific. If the reaction was highly virus-specific an incubation time of c. 30—180 min gave similar specific absorbtion values (Fig. 77). The higher reaction specificity obtained the longer incubation time could be used. An incubation time of 1 h at 22 C was normally satisfactory but in several cases the color reaction was too intense for the plate reader and so shorter incubation times were necessary. 2.3.3. Relationships between the viruses and the virus isolates as determined with the ELISA test The PVX antibody preparates did not noticeably react with potato viruses S, M (Table 37) or Y (Table 34). The serological variability of the PVX isolates as tested with the ELISA test was insignificant, however the homologous reaction was the strongest (Table 34). The PVS reagents were weakly serologically related to the PVM isolates, and similarily the PVM reagents were somewhat related to the PVS isolates, but no serological relationship to virus X was detected by either of these reagents (Tables 35 and 37). The antibody preparates of PVS SSFI and SSF4 reacted similarly against a selected isolate from the group, isolate SSF6, but the preparates of isolate SSFI4 were distantly related to the same isolate (SSF6). The antibody preparates made for PVM were similarly serologically closely related to a given PVM antigen but some differences in their relation- ship to PVS isolates were found. Marked serological variation between the PVY isolates was detected with the ELISA test. When diluted leaf sap samples of potato and N. glutinosa sap infected with PVY° and PVYn strain isolates were simultaneously tested with the antibody preparates produced against each virus strain the strain-specific homologous reaction was always significantly stronger than the non-strain- Table 35. Serological relationships between potato viruses S and M as detected in cross reaction tests done with the ELISA method. Immunoglobulins (Ig) at a concentration of 1 ug/ml and enzyme conjugates (E Ig) at a concentration of 2.5 pg/ml were used. Three replicates were done and substrate incubation was carried out for 40 min at 22 C. Absorbance at 405 nm and its dilution SSFI SSF4 SSFI4 MSFI MSFB PVM, tomato 10"' .066 .057 .089 1.771 1.804 10~ 2 .030 .036 .055 .884 1.006 " potato 10"' .057 .066 .087 1.691 1.470 Healthy tomato 10_l .047 .017 .077 .027 .031 PVS, tomato 10 ' 1.331 1.762 .960 .049 .067 Healthy potato 10"' .061 .034 .081 .031 .036 266 specific (heterologous) reaction. Strain specific samples of antigen preparates diluted ten times gave higher absorbance values than ten times less diluted non-strainspecific samples (eg. the absorbance values for PVY° samples diluted to 10~2 , were higher than for 10-1 diluted PVYn samples and vice versa) (Figs. 71 and 76). Variation in the absorbance values could be decreased with the use of mixed (1:1) serum preparates of both virus strains (Table 36). The non- specific reaction to plant protein remained low even in these cases. Fig. 71. The mean absorbance values obtained from the samples of N. glutinosa infected with different PVY-isolates using antiserum preparates for PVY° and PVY". Ig = 1 pg/ml, E Ig 2.5 pg/ml. The substrate was incubated for 1 h at 22 C and 3 replications were done. The bar represents a 10“' diluted sample and the darkened part a 10-2 diluted sample. Most of the PVY isolates could be typically included in the strains Y° or Yn but some isolates, such as YSFIS and YSFI9, have the properties of both strains (Fig. 71). Potato viruses X, S and M could be individually identified in virus mixtures without any specificity problems or noticeable loss of sensitivity when the ELISA test was used. All of the viruses could be reliably identified in mixtures of systemically infected test plant leaf sap diluted to 1/300 Table 37, Fig. 74). Table 36. Mean absorbance values obtained from primarily infected potato leaf samples with the ELISA test. The serum preparates were as follows: 1 = Y°, 2 = Y", 3 = Y° +n . The concentration of the immunoglobulins (Ig) was 1 pg/ml and that of the conjugates (E Ig) c. 2.5 pg/ml. Substrate incubation was carried out for 1 h at 20 C and three replicates were done. Serum preparates Absorbance at 405 nm for and samples tested PVY isolates tested (1) YSF4 YSFIO YSFII YSFI2 YSFIS Virus infected sap 10 ' .449 .177 .511 .420 .299 10~2 .288 .128 .297 .202 .183 Healthy sap ICT 1 .048 .033 .047 .031 .037 Virus infected sap 10"' .179 .650 .198 .244 .534 (3) Virus infected sap 10_1 .311 .418 .371 .317 .521 10"2 .119 .211 .162 .151 .217 Healthy sap 10_1 .041 .037 .035 .041 .026 Table 37. Absorbance values obtained for potato viruses X, S and M tested individually or as a mixture in diluted plant sap using specific antibodies for each virus or antibody mixtures. Plate coating and conjugate dilution was 1 pg Ig/ml and the substrate incubation time was 40 min at 22 C. Test sample Antibody preparates and the mean absorbance and its dilution values at 405 nm PVX PVS PVM Mixture PVX N. glutinosa 1/10 1.792 .023 .020 1.932 PVS N. debneyi 1/10 .006 1.536 .026 1.546 PVM L. esculentum 1/10 .005 .027 1.496 1.318 Virus mixture 1/30 1.611 1.209 1.209 1.930 1/300 1.135 .464 .794 1.529 Healthy plant sap mixture 1/30 .015 .015 .009 .013 267 Figs. 72 and 73. Double diffusion tests in agar gel. The sharp precipitin line indicates a specific reaction of optimal concentrations of antibodies and antigens (Fig. 72, lower precipitin line). During the course of the immunization program the antiserum loses its specificity and the precipitin lines are broader and diffuse (Fig. 73). Fig. 75. Serial dilutions of PVX-D antigen in a single diffusion test. Fig. 76. ELISA test of different PVY isolates. The same samples and the same dilutions are seen in both plates. Antiserum reagents for PVY° were used in the upper plate and reagents for PVY" were used in the lower plate. Fig. 74. The identification of potato viruses X, S and M individually or as a mixture using specific antibodies for each virus (vertical rows 1, 2 = PVX; 3, 4 = PVS; 5, 6 = PVM) or antibody mixtures (vertical rows 7—10). Horizontal rows: 1 = PBS-Tween, 2 = PVX 10“' diluted sap, 3 = PVS 10~l diluted sap, 4 = PVM 10 diluted sap, 5 = 1/30 mixture, 6 = 1/300 mixture, 7 = PVY° 10 diluted sap. Plate coating and conjugate dilution was 1 pg/ Ig/ml and the substrate incubation time was 40 min at 22 C. 268 2.3.4. Virus identification in potato samples at different developmental stages 2.3.4.1. Identification of known isolates of PVX, PVM, PVS and PVY with indigenous test reagents Potato viruses X and M could be equally reliably identified in comparable tuber and leaf samples because of their relatively high concentrations in the Table 38. The mean absorbance values obtained from comparable tests of secondarily infected potato tuber and leaf samples for PVX with the ELISA test. The plate coating and conjugate dilution was 1 pg Ig/ml and substrate incubation was carried out for 40 min at 22 C. Ten virus infected (V) and ten healthy (H) comparable samples at the same developmental stage were tested and three replicates were done. Sample dilution and absorbance values at 405 nm Test sample 10° 10M 10~ 2 10"' 10~4 10~5 10~ 6 10"7 10" 8 Dormant tubers V >2 1.538 .838 .277 .124 .068 .034 .024 .015 H .076 .064 .041 .034 .027 .022 .014 .011 .010 Sprouted tubers V >2 >2 1.303 .645 .326 .128 .064 .036 .022 H .046 .031 .022 .016 .009 .007 .005 .005 .007 Leaf samples V >2 >2 >2 .977 .404 .198 .100 .060 .027 H .063 .044 .036 .024 .020 .024 .023 .016 .014 Fig. 78. The ratio (A„/Aho ) of absorbance values obtained from diluted, infected sap and undiluted, healthy potato sap. The diseased samples were from potatoes secondarily in- fected with PVX and the samples were taken at diffe- rent developmental stages. • • • = dormant tuber * * * = sprouted tuber 000 = leaf sample Substrate incubation was carried out for 40 min at 22 C. 269 samples (Tables 38 and 39). PYX could be identified in sap from secondarily infected dormant tubers diluted to 10“3 , in sap from sprouted tubers diluted Table 39. The mean absorbance values obtained from comparable tests of secondarily infected potato tuber and leaf samples for PVM with the ELISA test. Tl)e plate coating and conjugate dilution was 1 pg Ig/ml and substrate incubation was carried out for 40 min at 22 C. Ten virus infected (V) and ten healthy (H) comparable samples at the same developmental stage were tested and three replicates were done. Sample dilution and absorbance values at 405 nm Test sample 10° 10_l 10~ 2 10~3 lO" 4 10~5 10~6 10~7 Dormant tubers V .943 .377 .125 .061 .026 .014 .009 .008 H .087 .047 .027 .018 .014 .008 .006 .006 Sprouted tubers V >2 .690 .244 .105 .038 .020 .011 .007 H .066 .038 .028 .017 .011 .007 .006 .005 Leaf samples V >2 >2 .777 .215 .073 .028 .012 .009 H .046 .031 .019 .011 .008 .006 .004 .003 Fig. 79. The ratio (A m/Ah„) of absorbance values obtained from undiluted and diluted, infected sap and undiluted, healthy potato sap. The diseased samples were from potatoes secondarily in- fected with PVM and the samples were taken at diffe- rent developmental stages. • • • = dormant tuber * * * = sprouted tuber 000 = leaf sample Substrate incubation was carried out for 40 min at 22 C. 270 to 10 4 and in leaf sap diluted to 10 3 (Table 38, Fig. 78). The non-specific reaction was low in all tests. The variation among the absorbance values obtained for the critical sample dilution was as follows: dormant tubers (1CT 3 ) = .217 - .370, sprouted tubers (10“ 4 ) = .259 - .442 and leaf samples (10“ 5 ) = .170 .241. PVM identification in dormant tubers was still reliable in 10-1 diluted sap, in sprouted tubers in 1CT2 diluted sap and in secondarily infected leaves in 1CT3 diluted sap. During sprouting the virus concentration increased markedly and the non-specific reaction decreased becoming insignificantly low (Table 39, Fig. 79). Fig. 80. The ratio (A/A,,,,) of absorbance values obtained from undiluted and diluted, infected sap and undiluted, healthy potato sap. The diseased samples were from potatoes secondarily in- fected with PVS and the samples were taken at diffe- rent developmental stages. • • • = dormant tuber • * * = sprouted tuber 000 = leaf sample- Substrate incubation was carried out for 40 min at 22 271 Table 40. The mean absorbance values obtained from comparable tests of secondarily infected potato tubers and leaf samples for PVS with the ELISA test. The plate coating and conjugate dilution was 1 pg Ig/ml and the substrate incubation was carried out for 40 min at 22 C. Ten virus infected (V) and ten healthy (H) comparable samples at the same developmental stage were tested and three replicates were done. Sample dilution and absorbance values at 405 nm Test sample 10° 10" 10" 10" 10" 10" Dormant tubers V .447 .233 .122 .046 .021 .011 H .111 .064 .043 .025 .011 .008 Sprouted tubers V 1.020 .508 .161 .072 .031 .018 H .094 .057 .028 .019 .016 .011 Leaf samples V >2 1.336 .397 .149 .051 .021 H .071 .046 .024 .020 .016 .011 Table 41. The mean absorbance values obtained from comparable tests of secondarily infected potato cv. Record tuber and leaf samples for PVY with the ELISA test. The plate coating and conjugate dilution was 1.4 pg Ig /ml and substrate incubation was carried out for 1 h at 22C. Five samples of each PVY isolate and ten healthy samples at the same developmental stage were tested, a = dormant tubers, b = sprouted tubers, c = leaves. Test sample Sample dilutions and absorbance values at 405 nm (and the isolate) a. 10° 10"' 10" 10" 10" 10" (YSFII) 1 .359 .171 .074 .039 .022 .015 (YSF4) 2 .281 .147 .087 .044 .024 .014 (YSF 10) 3 .373 .190 .101 .059 .027 .019 (YSF 15) 4 .391 .194 .122 .064 .031 .019 (YSF 17) 5 .554 .261 .149 .071 .030 .021 Mean value for infected samples .391 .216 .107 .054 .027 .017 Mean value for healthy samples .131 .076 .057 .038 .019 .014 b. 1 .404 .264 .092 .050 .032 .016 2 .419 .279 .107 .054 .037 .015 3 .647 .427 .164 .087 .044 .020 4 .612 .434 .198 .098 .045 .020 5 .843 .459 .271 .132 .076 .037 Mean value for infected samples .585 .373 .166 .084 .047 .021 Mean value for healthy samples .076 .054 .038 .026 .021 .015 c. 1 .717 .671 .392 .140 .059 .023 2 .784 .713 .403 .161 .072 .034 3 .912 1.014 .549 .213 .092 .040 4 .871 .999 .447 .244 .117 .051 5 1.166 1.304 .714 .381 .171 .074 Mean value for infected samples .890 .940 .507 .228 .102 .044 Mean value for healthy samples .069 .052 .039 .029 .021 .014 272 The variation between the absorbance values obtained from the undiluted virus infected samples was as follows: dormant tubers = .761 1.201; sprouted tubers = 1.116 - > 2.0; leaves, all values more than 2.0. The identification of PVS was not always reliable in dormant secondarily infected tubers but with sprouted tubers no difficulties existed. The virus concentration greatly increased during sprouting and when finally tested in leaves the virus could be reliably detected in 10“3 diluted sap (Table 40, Fig. 80). The variation between the absorbance values obtained from the undiluted virus infected samples was as follows: dormant tubers = .375—.636, sprouted tubers = .779 1.475 and leaves = 1.761 —>.2.0. Potato virus Y could be reliable detected in secondarily infected sap from sprouted tubers or leaves (Table 41, Fig. 81). In sap from dormant tubers PVY could be also detected but the level of non-specific reactions was too high for the test to be useful. The variation between the absorbance values obtained from the undiluted virus infected samples was as follows: dormant tubers = .214—.582, sprouted Fig. 81. The ratio (A v/A ho) of absorbance values obtained from undiluted and diluted, infected sap and undiluted, healthy potato sap. The diseased samples were from potatoes secondarily in- fected with PVY and the samples were taken at diffe- rent developmental stages. • • • = dormant tuber * * * = sprouted tuber 000 = leaf sample Substrate incubation was carried out for 1 h at 22 C. 273 tubers = .361 —.966 and leaves = .482 1.417. The values for the healthy controls were as follows: .088 —.157, .049—.094 and .046—.083 respectively. 2.3.4.2. Identification of known isolates of PVY, PVA and PLRV with foreign test reagents A commercial reagent for PVY made by the Boehringer-Mannheim Ltd gave rather specific reaction absorbance values when tuber or leaf samples were tested. The absorbance values for the Y° strain isolates were markedly higher than those for the Yn strain isolates. The specific absorbance values (A y/Aho) calculated from the absorbance values of the leaf material tested were high (Fig. 82) particularly for the Y° strain isolates. The specific Fig. 82. Specific reaction ratios for PVY-ELISA -re- agent from the Boehringer- Mannheim Ltd calculated from the absorbance values of undiluted and diluted PVY infected (A y ) and healthy undiluted (A|,o) potato samples at different developmental stage. Subs- trate incubation was carried out for 1 h at 22 C. 274 absorbance values calculated from the readings of the tuber material tested were noticeably lower because of the moderately high non-specific absorb- ance values (from .028 to .071) obtained for healthy tubers. Potato virus A could be detected in secondarily infected tubers or leaves with a commercial reagent made by the Boehringer-Mannheim Ltd. The reactions showed high virus specificity with low values for the background and healthy plant material. The absorbance values for healthy leaves were .043 —.063 and for healthy tubers .024—.042. The virus concentration in all samples was very low which resulted in a rapid decrease in the readings and the specific absorbance values (A a/Aho ) when the samples were diluted (Fig. 83). Fig. 83. Specific reaction ratios for PVA-ELISA-rea- gent from the Boehringer- Mannheim Ltd calculated from the absorbance values of undiluted and diluted potato tuber and leaf sam- ples with secondary PVA infection (AJ and healthy undiluted samples (A|,o). Substrate incubation was carried out for 1 h at 22 C. 275 276 Potato leaf roll virus could be reliably identified in secondarily infected leaves or tubers with a commercial reagent made by Boehringer-Mannheim Ltd and with a reagent made indigenously from an antiserum prepared by TAMADA and HARRISON (1980). The virus could be detected also in primar- ily infected tubers but large variation and unclear low readings were obtained. The low virus concentration in all samples could be seen from the rapidly decreasing specific absorbance values (A v/Aho ) calculated from comparable absorbance values for virus infected and healthy samples. In diluted tuber sap the virus cannot be reliably detected after being diluted to more than 10-1 (Fig. 84). The absorbance values obtained for healthy tuber samples varied from .059 to .108 and those for leaf samples from .041 to .046. Fig. 84. Absorbance values obtained for potato tubers with primary and secon- dary PLRV infections and leaf samples with secon- dary PLRV infections. The ratio of absorbance values for infected samples (A v ) and healthy samples (A hl,) shows the specificity of the reaction. Substrate incuba- tion was carried out for 1 h at 22 C. • • • = tuber samples with primary infection * * * = tuber samples with secondary infection o o o = leaf samples with secondary infection 277 2.3.4.3. Identification of unknown viruses in naturally infected potatoes In the routine testing of potatoes with the ELISA method, the results obtained for PVX and PVM were satisfactory at all stages of development but for PVS and PVY a significantly higher number of infected samples were detected from sprouts or leaves than from dormant tubers (Table 42). The concentrations of the same viruses in different cultivars at the same developmental stage were similar according to the readings obtained. Reliable comparisons between the readings obtained from dormant tubers are not possible because primarily infected tubers infected with variable virus con- centrations also existed. The concentrations of all viruses greatly increased after tuber dormancy was broken. Due to this the non-specific reaction became insignificant and the test reliability improved (Fig. 85). Table 42. The percentage of virus infected potatoes as detected with the ELISA test from comparable samples of three potato cultivars at different developmental stages, a = dormant tubers, b = sprouted tubers, c = leaves. 4- indicates the number of infected samples/number of tested samples, % indicates the agreement with the results from c. Potato cultivar and test resultsViruses and samples tested Ostara Saturna Hja’s Tuomas 4- % 4- % + % X % PVX a. 3/138 100.0 71/106 100.0 48/150 100.0 100.0 b. 3 100.0 71 100.0 48 100.0 100.0 c. 3 (100.0) 71 (100.0) 48 (100.0) PVS a. 16/138 76.2 49/106 74.2 79/150 72.5 74.3 b. 21 100.0 63 95.5 106 97.3 97.3 c. 21 (100.0) 66 (100.0) 109 (100.0) PVM a. 10/138 100.0 24/106 100.0 36/150 97.3 99.1 b. 10 100.0 24 100.0 37 100.0 100.0 c. 10 (100.0) 24 (100.0) 37 (100.0) PVY a. 5/138 41.7 39/106 47.0 45/150 50.0 46.3 b. 11 91.6 76 91.5 26 92.9 92.0 c. 12 (100.0) 83 (100.0) 28 (100.0) F values for PVS and PVY: developmental stages PVS = 275.9"', PVY = 477.5'" cultivar PVS = 2.22, PVY = 1.45 278 2.3.5. Comparisons between the ELISA test, the chloroplast agglutination test and the A 6 cut leaf test The test results obtained from comparable sprouted tuber samples and leaf samples with the ELISA test and from the same leaf samples with the chloroplast agglutination test and with the A 6 test (PVY) showed good agreement for PVX but as for the other viruses, great variation existed (Tables 43—45). The detection of PVS with the chloroplast agglutination test was particularly a problem and numerous incorrect identifications were found to be caused by the non-specificity of the test and the relatively low virus concentration in the samples (Table 43). According to the results from the chloroplast agglutination test the mean % of infected tubers was 43.5 for PVS and 20.8 for PVM. Comparative results from the ELISA test were 49.5 % for PVS infected and 28.3 % for PVM infected tubers. The chloroplast agglutination test gave incorrect positive reactions 7.4 % of the time for PVS and 1.8 % of the time for PVM. Fig. 85. Mean absorbance val- ues from ELISA tests car- ried out on 399 samples of three potato cultivars at different developmental stages. The mean values of PVX and the mean values of PVM leaf samples were higher than 2.0. The non- specific reaction of PVX was similar to PVM. Subs- trate incubation was carried out for 40 min at 22 C. a = dormant tuber b = tubers with sprouts c = leaf samples Tabic 43. Comparative identification results for potato viruses X, S and M obtained from potato leaf sap with the chloroplast agglutination test (A) and the ELISA test (E). + = reaction observed, - = no reaction observed Cultivar and Number of Result comparisons PVX 0 0 0 0 0 Jaakko (66 samples) Pito (70 samples) PVX 2 2 2 0 0 Veto (90 samples) PVX 2 2 2 0 0 PVS 75 77 64 11 13 PVM 39 47 35 4 12 The chloroplast agglutination test failed to detect 25.0 % of PVS infected and 22.8 % of PVM infected samples. PVY could be reliably detected with the A 6 cut leaf test in potato leaf samples but the cut leaves also reacted with necrotic lesions to some other viruses (Table 44). Satifactory results were also obtained in later tests for PVX and PVM when tested with the chloroplast agglutination test and the ELISA test, but for PVS the lack of agreement between tests was noticeable. Some inconsis- tency between test results was also observed in the identification of PVY (Table 45). The ELISA test results from potato leaf sap were more reliable than the results from the sap of sprouted tubers from the same material, the difference in incidence between the 2 sap sources being as follows: PVX = 0.00 %, PVS = 8.04 %, PVM = 3.41 % and PVY = 10.20 %. 7 279 280 Table 44. Results from comparative identifications of potato viruses X, S, M and Y first in sprouted tubers with the ELISA test and later in seedling leaves grown from the same tested tubers with the chloroplast agglutination test, the A 6 cut leaf test and the ELISA test. Thirty samples from each potato cultivar were tested at both developmental stages. A = chloroplast agglutination test or A 6 leaf test for PVY E, = ELISA test from sprouted tubers E 2 = ELISA test from leaves + = reaction observed = no reaction observed Cultivar and Number of Comparison results virus tested reactions observed A E, E 2 A+ A+ A- A+ A+ A- E,+ E,+ E,- (orA6) E,+ E,- E,+ E,+ E,- E2+ E2+ E,- E 2+ Bintje PVX 100 010010000 PVS 711 12 7 0 4 7 0 511 0 1 PVM 466 402402600 PVY 23 20 22 20 3 0 20 3 2 20 0 2 Ostara PVX 111 100100100 PVS 17 23 23 16 1 7 16 1 8 23 0 0 PVM 18 10 11 10 8 0 11 7 0 10 0 1 PVY 967 630720601 Saturna PVX 16 18 18 15 1 3 15 1 3 18 0 0 PVS 6 12 13 6 0 6 6 0 7 12 0 1 PVM 699 603603900 PVY 20 13 14 11 9 2 12 8 2 13 0 1 Hja's Timo PVX 000 000000000 PVS 489 315316801 PVM 333 300300300 PVY 655 421511500 281 Table 45. The % infected samples obtained for 4 potato cultivars (2x523 samples) tested comparably first in sprouted tubers with the ELISA test (F,.) and later in leaf material grown from the tested tubers with the ELISA test (E 2) and the chloroplast agglutination (A) or the A 6 cut leaf test (A6). The incorrect results obtained with the chloroplast agglutination test and the A 6 cut leaf test are compared to the results from the simultaneous ELISA test (E 2). Test method Mean % of infected samples PVX PVS PVM PVY Incorrect negative results with A or A 6 15.8 46.5 17.2 12.5 D. Discussion 1. Properties of the viruses and the virus isolates Knowledge of the properties of virus strains and isolates has become increasing important since the introduction of highly sensitive and specific methods for virus identification. The practicality of these new principles, particularly for the identification of potato viruses, has emphasized the need for a better biological understanding of the viruses. Prior to this study no systematic information about the strains or isolates of potato viruses occurring in Finland was available and similarly the special problems associated with their identification were unknown. The materials for this study were therefore carefully selected to represent the viral and viral isolate diversity present in Finland. The seven potato viruses which occur in Finland are all widely known. However, potato leaf roll virus has only recently been transmitted here via its natural vectors, probably over the sea from the Baltic countries. In addition to the viral diseases discussed here, the following viral or similar potato diseases may occur in Finland: ’’Potato mop top” (CALVERT & HARRISON 1966, HARRISON & JONES 1970), Potato aucuba mosaic (KAS- SANIS & GOVIER 1970), ’’Potato spindle tuber” disease (SCHULZ & FOLSOM 1923, DIENER & RAYMER 1971) and ’’Potato yellow dwarf” (BARRUS & CHUPP 1922, BLACK 1970). Among the potato viruses found in Finland only PYX and PYY had clearly detectable strains. The ring spot strain of PYX does not commonly occur according to LAGEBURG et al. (1950), however isolates including this strain were found in several lots of imported seed potato from countries in Central Europe. Although PYY strains Y° and Yn are commonly found in Europe, as reported by BODE (1959) and de BOKX and PIRON (1977), no 282 reliable information about strain distribution and strain interrelationships exists and thus observations of strain occurrence could not be compared with any similar results. Yc strain isolates were not found in the course of this study, which together with the observations of LINUSTEN (1981), indicates that the strain does not occur in Scandinavia. The strain classification of PVY should be revised. Suitable criteria exist for Yn strain isolate classification, but the criteria for the Y° and Yc strains is unclear. Also isolates not included in any of these strains are found, as reported by de BOKX et al. (1975) and WEIDEMANN and KOENIG (1979). The most important of the potato viruses which occur in Finland is PVY which in several domestic and foreign field experiments has been shown to be a serious disease causing virus. The symptoms and the disease severity, however, vary greatly depending on the virus isolate and potato cultivar as also shown by WEIDEMANN (1981). Observations of severe disease caused by potato leaf roll virus and virus M agree with those reported by REESTMAN (1970) and CHRZANOWSKA (1976). Although PVX and PVS were still very common in seed potatoes produced in Finland in the 1970’5, their true importance is unclear because in only a few cases were they found to induce severe disease. Little information is available about PVA and TRV. The former is probably more common and important, as is believed in Finland. The identification methods used before the ELISA test was established were unsuitable for the detection of PVA as an individual virus. TRV may be of local importance as TAPIO (1972b) and KURPPA (1982) have reported rich Trichodorid nematode populations occurring in field and plant nursery soils, and SCHMELZER (1957) and KRISTENSEN (1962) have reported the presence of common weeds, which can maintain the virus in similar soils. According to GUGERLI (1977), TRV transmission potential is high because of many sources for infection and the high transmission capacity of the Trichodorid nematodes found in the soil. No explanation was found for the rapid transmission potential of the ring spot strain isolates of PVX in the field experiments. However, all the isolates were found to be only mechanically transmittable. No data is available to be able to compare the similar behaviour of PVS and PVM in certain susceptible and resistant potato cultivars. All the viruses studied could be detected with susceptible test plant species. However, the reaction of certain viruses to certain test plants differed from the literature descriptions. Lycopersicon esculentum cv. Nevskij showed severe systemic symptoms after inoculation with PVS, unlike the symptoms KOWALSKA et al. (1976) have reported. No reliable local hosts were found for PVM although HIRUKI (1970) has reported Phaseolus vulgaris cv. Red Kidney and KAHN and MONROE (1970) Datura metel as being such hosts. Little information is available about the local reaction of PVX strains to A6 and Solarium demissum Y, which showed necrotic lesions after inoculation with the ring spot strain isolates. The most suitable test plant for PVY strain classification was Nicotiana 283 tabacum cv. Samsun as reported by BAWDEN and KASSANIS (1951) and RICHARDSON (1958). Further information about PVY classification, obtained with the use ofPhysalis floridana and Solanum chacoence, have been reported by EASTON et al. (1958) and de BOKX (1974) but no information about the comparative use of S. demissum A for PVY strain separation is available. Symptom variation between the isolates of the other viruses was less clear so no strain classification was carried out. However, variation in aphid transmissibility and symptoms in Nicotiana debneyi similar to that reported by BODE & WEIDEMANN (1971) and MacKINNON (1974) was found between the PVS isolates. The symptom variation observed between the PVM isolates in Solanum rostratum and Lycopersicon chilense agreed with that described by ROSS (1968), KOWALSKA and WAS (1976) and KOWALSKA (1978). The physical values obtained for domestic virus isolates differed slightly from those published in the literature. The thermal inactivation point values (66—69 C) for the ring spot strain isolates of PVX agreed with those of LAGEBURG et al. (1950) but the values (63 —66 C) for the mild mosaic strain isolates were lower than those reported by KÖHLER (1962). FRIBOURG (1975) has, however, reported even lower values for the mild mosaic strain of PVX. The thermal inactivation point values (54 —57 C) and the dilution end point values (10‘ 2 ) for PVS were lower than those reported by WETTER and BRANDES (1956). The thermal inactivation point value (51 C) for the PVY° strain isolates was low compared to those of DARBY et al. (1951) but the values (51 —57 C) for the PVYn strain isolates were similar to those reported by DELGADO-SANCHEZ and GROGAN (1970). The dilution end point values for all PVY isolates agreed with published values. The purification method of SHEPARD (1972) used routinely for the purification of PVX, PVS and PVM, gave satisfactory results although some particle aggregation was found after PEG-concentration of the viruses. The yields of purified PVS and PVM were slightly greater than those reported by PROLE and RICHTER (1979). Lycopersicon esculentum cv. Nevskij was a more suitable host for PVS purification than Nicotiana debneyi. For the purification of PVY the method of STAGE-SMITH and TREMAINE (1970) gave low yields of purified virus and was too laborious to use as a routine method. Large losses were caused by the many repeated steps in the procedure and the aggregation of the long flexous particles, as SHEPHERD and POUND (1960) and HUTTINGA (1973) have reported. The slightly modified method of LEISER and RICHTER (1978) contained essential improvements for PVY purification. The virus particles could be fairly easily separated out of almost all plant material within two days. The yields of purified virus were higher from fresh infected plant material than from frozen material. The higher yields of purified virus obtained for the PVYn isolates corresponded with the values of their physical properties. For the purification of PLRV several methods published are suitable because of the stability of the virus, as shown by PETERS (1970). Purification problems caused by low virus concentration and particle accumulation in the phloem have been overcome by the enzymatic digestion of plant tissue, as 284 suggested by TAKANAMI and KUBO (1979). This principle has many advan- tages over normal procedures but due to the high costs of the enzyme the method was abandonned. A method modified from those of ROWHANI and STAGE-SMITH (1979) and CLARKE (1981) finally gave yields of purified virus almost comparable to those obtained with the method of TAKANAMI and KUBO (1979). All viruses and virus isolates studied were found to be good or moderate immunogens although titers as high as those reported by BAGNALL et al. (1959), WETTER (1960) and BERCKS (1970) were not achieved. It should be pointed out, however, that the microprecipitin test, which was used for the titer determinations, gives ’’relative values” which are not always totally comparable. The dosages of purified virus widely suggested for each injection are higher than needed and may increase the non-specificity of the antisera. High titer antisera have been produced by RICHTER et al. (1979) and CLARKE (1981) using very low dosages of immunogens. Also the number of injections can be minimized in order to obtain more specific antibody responses in the rabbits. In spite of great biological variation amongst the isolates of PYX, their serological relationships were fairly close, as earlier reported by LARSON (1943), LADEBURG et al. (1950) and FRIBOURG (1975). On the other hand marked serological differences between the isolates has been found by CHESTER (1936) and particularly MATTHEWS (1949) whose strain classifica- tion was based on serological properties. The serological properties of PVS and PVM are in accord with those reported by BAGNALL et al. (1959). According to BAWDEN and KASSANIS (1951) the isolates of all PYY strains are serologically closely related. Significant serological variation was, however, found between the Y° and Yn strain isolates, as is also reported by BARTELS (1957) and MAAT and de BOKX (1978). Little information is available about the serological properties of some of the other potato viruses which occur in Finland. However, the TRY isolates were closely related to the PRN isolate described by HARRISON and NIXON (1959) and the PVA and PLRV isolates included strains which occur in Europe. Of the foreign antisera, only the Danish ones were comparable to indigenous antisera. The others were stored in a freeze-dried form and were diluted to the original volyme before use. It is therefore uncertain if the dilutions were correct and whether some loss of antibody activity had occurred during storage. No difficulties were encountered in modifying the gel diffusion methods for the identification of long flexous particles. Antisera with the same properties as reported by SHEPARD and SECOR (1969), SHEPARD (1970 b) and SHEPARD et al. (1971) could be readily prepared. 285 2. Identification methods The traditional drop-agglutination technique can be reliably used for the serological detection of potato viruses X, S and M in potato leaf sap as reported by de BOKX and MOOI (1974). The chloroplast agglutination method and its modifications have, however, several disadvantages such as low sensitivity, low specificity and the requirement of leaf sap for the test. Reliable results from clarified tuber sap with the microprecipitin test have been obtained by STASZEWICZ (1977) and with the bentonite flocculation or the latex agglutination test by KAHN et al. (1967), BERCKS (1967) and KRYLOV and GNUTOVA (1974). Some new modifications of the latex aggluti- nation test are very sensitive, as shown by KOENIG and BODE (1978), KHAN and SLACK (1978) and TORRANCE (1980a). The unreliable identification of PYY with the chloroplast agglutination test confirms the observations of BARTELS (1957) and BLOTSKAYA (1972). For PYY identification with the microprecipitin test, 10—20 pg/ml of purified virus was needed in order to obtain a clear reaction. According to these values the chloroplast agglutination test cannot be sensitive enough for the identification of this virus. Furthermore, the physiological variation between the test samples is likely to decrease the test reliability (VULIC & ARENZ 1963 and GHENA & VIORIGA 1970). Little information is available about the reaction of the ring spot isolates of PYX to A 6 cut leaves, which was the main problem with this biological test. De BOKX (1970 a), however, has mentioned local lesions on A 6 cut leaves induced by PYX, and KACZMAREK (1976) has reported symptoms caused by PVS and PVM on A 6 leaves. According to de BOKX and CHRZANOWSKA (1972) PAMV and TRY may also cause local lesions on A 6 leaves. The cut leaves of Solarium chacoense reacted to all PYY isolates but they were too small for reliable virus detection, as has also been shown by de BOKX (1974). The leaves did not show reliable PVA infection and physiologi- cally old leaves tended to show non-specific reactions. S. chacoense did not improve the detection of PYY and PVA with the cut leaf test. The agar gel double diffusion test was not sensitive enough to be used as a routine test for identifying potato viruses. The lowest concentration of degraded virus protein detected, 10 yag/ml, agrees with the values obtained by SHEPARD (1972) and indicates that the test is only suitable for the detection of PYX. The single diffusion test reached the same detection sensitivity, 1 pg/ml, as SHEPARD and SECOR (1969) and SHEPARD (1972) have reported. Higher virus concentrations were needed, however, for the identification from plant sap and the values obtained, 2.5 10 pg virus/ml, correspond to those reported by RICHTER et al. (1977). The single diffusion test is sensitive enough for the identification of PYX, PVS and PVM in potato leaves with secondary infection and is even valid for the reliable identification of PYY from potato leaves as reported by SHEPARD et al. (1974) and RICHTER et al. (1979). The test is not suitable for the detection of viruses in tuber sap because of insufficient sensitivity and because of the non-specific reactions 286 caused by lectins present in tuber sap, as has also been shown by SHEPARD (1970a). The single diffusion test was found to be a very simple and practical method to test a low number of samples. It also indicates the approximate virus concentration if a test standard is used. Until 1979, virus purifications were carried out without the final density gradient centrifugation procedure. This is why some antisera produced before that point may have contained too many antibodies to host plant protein. However, after host antibody absorption these antisera became suitable for use in the preparation of reagents for the ELISA test. The PVY antisera were not particularly highly titered and so were not optimal material for the preparation of reagents for the ELISA test. Antisera for the Y° and Yn strains of PVY with high titers and very good specificity, however, have since been produced by KURPPA and KORHONEN (unpub- lished). The values Av/A(, for the absorbance ratios of virus infected test samples and healthy comparable samples, PVX max. >,lOO, PVS max. 94, and PVM max. 114 were high and are highly virus-specific, except for that of PVY which was only 23 max. When calculated from the absorbance readings from tuber samples the ratio values were somewhat lower but still noticeably higher than those that can be calculated from the comparable figures pub- lished by de BOKX et al. (1980), CLARKE et al. (1980) and BANTTARI and FRANK (1982). Test results showing more virus-specificity for PVY than published in this paper have been reported by MAAT and de BOKX (1978) and DANIEL and HUNNIUS (1980). A commercially available ELISA reagent for PVY also gave high values for the ratio of absorbances but was too specific for the Y° strain. The low reaction specificity of some of the indigenous reagents was due to the low amount of enzyme present in the conjugate or to incomplete conjugation between the antibodies and the enzyme. The most favourable concentrations of reagents for the tests, 1 pg/ml of immunoglobu- lin and 1—2.5 pg/ml of its conjugate, are similar to the values suggested by CLARK and ADAMS (1977). The substrate incubation time was not very critical if the test reagents were highly virus-specific; one hour at room temperature was normally suitable. The titration of the test reagents against a series of dilutions of virus infected plant sap and against non-diluted healthy sap of the same species clearly illustrated the specific function of the reagents. A straight declining line following the dilutions indicates a high virus specificity and a high percentage of virus particles reacting with the antibodies of all of the differentially diluted samples. The ratio of absorbances calculated from the values above also clearly shows the critical dilutions of different samples from which virus detection is reliable. The lowest concentrations of purified virus (PVX 0.1 ng/ml) and the relative dilutions (PVX 10"6) in plant sap detected belong to the lowest values ever reported using the direct ELISA test. The serological relationship between PVS and PVM found by BAGNALL et al. (1959) was nearly undetectable with the ELISA test. The serological differences between the isolates of the PVY° and the PVY" strains were significant as tested with the ELISA method. The absorb- ance readings obtained from heterologously tested undiluted samples were equal to those of ten times diluted homologously tested samples. Similar results have been reported by MAAT and de BOKX (1978), who concluded from this fact that two strain specific reagents are needed for the reliable identification of PVY. The serological variations between the isolates of PVX, PVS and PVM were not critical with regard to identification reliability. That no marked serological variation was found between the PVA and the PLRV isolates could possibly depend on the low number of samples tested. In any case, LIU and DUFFUS (1982) have reported three serotypes of PLRV which are so distantly related that strain-specific antiserum is needed for their identifica- tion. The lack of increase in the non-specific reaction found when potato viruses X, S and M were identified as a mixture using specific antibodies for each virus agree with the observations of BANTTARI and FRANK (1982). Comparable ELISA tests from sprouted tubers and leaves gave similar results. The total agreement for PVX and the 90 % or higher agreement for the other viruses tested is comparable to the results published by DANIEL and MUNZERT (1980). The remarkable increase in the specific absorbance values during tubersprouting, particularly in the identification of PVS and PVY, has also been reported (DANIEL & HUNNIUS 1980, and BANTTARI & FRANK 1982). The observations of low increase in the specific absorbance values of PLRV confirm the results of CLARKE et al. (1980). Unlike the findings of MAAT and de BOKX (1978), de BOKX et al. (1980) and TAMADA and HARRISON (1980), no noticeable variation in non-specific absorbance values between potato cultivars was found. The ELISA test was more reliable than the chloroplast agglutination test for the identification of PVX, PVS and PVM. The increase in correct detection of these viruses with the ELISA test was greater than that found by DANIEL and HUNNIUS (1980) when they compared the ELISA test to the microprecipitin test. The test results obtained for PVY with the ELISA test and the A 6 cut leaf test are not totally comparable because the latter also includes the possible reactions of PVA to the leaves. For the identification of PVY, the ELISA test increased the test reliability mainly by eliminating the incorrect positive reactions caused by several other viruses to A 6 cut leaves. To determine reliable positive readings, a flexible standard ratio of the absorbance values obtained from virus infected (A v) and comparable healthy (Ah) samples (that is, A v > 3 Ah) is a precise and better measure than a given exact positive value (for example, 0.100) for each test reagent. In cases of abnormally high background values wrong determinations easily occur if inflexible standard values are used. For the identification of PLRV the ELISA test is far superior to the older methods. In this study the ELISA test was satisfactory in the detection of PLRV in potato tubers and leaves, and this agrees with the observations of CASPER (1977a), GUGERLI (1979) and DANIEL and MUNZERT (1982). 8 287 288 The ELISA test helps to identify PVA as an individual virus apart from PVY. It is also possible to identify TRV in potatoes with the ELISA test although according to GUGERLI (1979) a rather high virus concentration is needed for reliable detection. As well the large serological variability and the occurence of NM-isolates with TRV, as reported by HARRISON and ROBIN- SON (1978, 1982), causes serious problems. The ELISA method is a great improvement in potato virus identification because all of the important viruses can be detected simultaneously with this method. Potato tubers are always unevenly infected by viruses and tubers with late primary infections may be infected with viruses in extremely low concentrations. In addition to coping with these conditions, the ELISA test can be used to reliably detect late primary infections in sprouted tubers, as BANTTARI and FRANK (1982) have also shown. The ELISA test is being continually improved as a tool for intensive research and thus new modifications of it will be established yearly. The main goals of further research might be the minimization of non-specific reactions and the production of non-strain-specific reagents for routine work. E. Summary The aim of this study was to improve the knowledge of the viruses and the virus strains which occur in potatoes in Finland in order to solve the problems associated with their reliable identification. To achieve this purpose many viruses were isolated between 1975 and 1982 from various selected sources. In the course of this study the antisera required for the serological studies were prepared for some isolates found typical of a given virus but also antisera of foreign origin, including commercially available reagents for the ELISA test, were used. The following seven potato viruses were identified: potato virus X (PYX), potato virus S (PVS), potato virus M (PVM), potato virus Y (PVY), potato virus A (PVA), potato leaf roll virus (PLRV) and tobacco rattle virus (TRY). PYX isolates occurring in Finland include two type strains, which are the normal (or the ’’mild mosaic” strain) and the ring spot strain. Similarly PVY isolates include the strains Y° and Yn . Both strains of PVY occur in the southern parts of Finland but in the northwestern areas, where seed potato is mainly produced, only Yn strain isolates were found. The variability among the isolates of the other viruses was less noticeable and so they were not classified into distinct strains. All of the viruses and their isolates could be detected and also identified according to their symptomology in test plants but some of the symptom observations disagreed markedly with those reported in the literature. Potato viruses X, S, M and Y were very common in Finnish commercial seed potatoes in the middle of the 1970’s but the occurrence of viruses X, S 289 and M has since decreased as increasing quantities of healthy seed stock have become available for seed potato production. PYY and PLRV were found to be highly virulent in all of the potato cultivars tested. Isolates of the PVY° strain killed potato plants of the cvs. Ostara and Hja’s Timo and induced necrosis in several other cultivars. Isolates of the PYY" strain induced milder symptoms in potatoes without necrosis. PLRV induced leaf-rolling of the upper leaves as primary symp- toms and similar, but more severe, symptoms starting from the basal leaves as secondary symptoms. In addition red coloration or bronzing of the leaves was typical of this virus. Secondary infection of PLRV caused a decrease of 77 % in tuber yield of individual plants of the cv. Sieglinde grown In field experiments. PVM and PVA induced severe disease in certain potato cul- tivars. Typical of the symptoms caused by PVM was dwarfing and spoon- like leaf formations. The tuber yield reduction of individual plants could be as high as 50 %. PVA induced crinkle, chlorosis and mosaic in potato leaves. The remaining viruses caused generally mild symptoms in potatoes. All of the aphid-borne viruses were easily transmitted if sources for infection were available. The highest infection potential was shown by PYY. Significant differences in aphid transmissibility between the PVS isolates were found. The ring spot strain isolates of PYX were more readily transmit- ted in the field than the ’’mild mosaic” strain isolates. However, only the mechanical means of transmission was demonstrated for both strains. Large variability was found in the infection sensitivity of the potato cultivars and several of them were simultaneously either susceptible or resistant to PVS and PVM. All of the viruses studied could be purified from infected plant material using published methods or improved modifications of them. The highest amounts of virus purified from 1 kg of infected plant material were as follows: PYX 640 mg, PVS 31 mg, PVM 48 mg, PYY 26 mg, PVA 12 mg, PLRV 1.1 mg and TRY 85 mg. The antisera produced for the selected isolates of PYX, PVS, PVM and PYY reached titers of between 1/1024 and 1/8192 as determined with the microprecipitin test. The titers of the foreign antisera to the Finnish virus isolates were lower than those of the indigenous antisera. The titers of the antisera produced for the agar gel diffusion tests were 1/128 (PYX), 1/32 (PVS) and 1/64 (PVM), as determined with the double diffusion test. The lowest indentifiable PYX D-protein concentrations detected were 10 ixg/ml with the double diffusion test and 1 /zg/ml with the single diffusion test. In potato leaf sap PYX could be identified in dilutions of 1/128 and 1/1024 respectively. The lowest identifiable concentration of the same virus with the ELISA test was 0.1 ng/ml. In potato leaf sap PYX could be detected in dilutions of between 10"6 and 10“7 , which is equal to the dilution end point of the virus. The absorbance values of the ELISA test obtained from virus infected plant samples were more than 100 times higher than those obtained from comparable healthy samples when the absorbance values given by a freshly made substrate solution was used as the 0 standard for the readings. The serological differences between the isolates of the PVY° and the 290 PVY" strains are very important when the ELISA test is used. The serological variation between the isolates of the rest of the viruses studied was insignific- antly low with regard to the test reliability. The chloroplast agglutination test and the A 6 cut leaf test frequently gave incorrect reactions because of the low sensitivity and the non-specific nature of the test. A 6 leaves reacted to the ring spot isolates of PVX with symptoms similar to those caused by PVY. The agar gel double diffusion test was not sensitive enough to detect potato viruses on a routine basis but the single diffusion method could be used for the identification of PVX, PVS and PVM in secondarily infected potato leaf sap. With the ELISA method, PYX, PVS, PVM and PVY could be reliably identified in potato leaves and sprouted tubers. The correspondence between correct positive readings from leaf sap and sap from sprouted tubers for the viruses was as follows: PYX 100 %, PVM 97 %, PVS 92 % and PVY 90 %. 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Potato J. 54: 475—476. 300 SELOSTUS Suomessa esiintyvät perunavirukset ja niiden määrittäminen Aarne Kurppa Helsingin yliopiston kasvipatologian laitos Tämän tutkimuksen tarkoituksena oli selvittää, mitä perunan tautiviruksia esiintyy maas- samme, sekä erityisesti soveltaa tai kehittää menetelmiä niiden luotettavaksi määrittämiseksi. Viruksia eristettiin vuosina 1975 1982 kasvinjalostuslaitoksillamme ja siemenperunaviljelys- sämme tuotetusta perunasta, maahan tuodusta siemenperunasta sekä eri puolilta maatamme viljelyksiltä tavatusta viroottisesta perunasta. Serologisissa määrityksissä tarvitut virusantiseerumit valmistettiin virusrotututkimusten perusteella valituille isolaateille. Vertailuaineistona käytettiin myös ulkomaisia antiseerumeita sekä ELISA-menetelmässä myös valmisreagensseja. Tutkimuksessa todettiin seuraavat 7 perunan virusta: perunan X-virus (PVX), perunan S-virus (PVS), perunan M-virus (PVM), perunan Y-virus (PVY), perunan A-virus (PVA), perunan kierrelehtisyysvirus (PLRV) sekä tupakan rattle virus (TRV). Perunan X-viruksella todettiin kaksi päärotua, normaalirotu ja rengaslaikkurotu. Myös Y-viruksen päärotuja esiintyi kaksi, Y°- ja Y"-rodut. Maan eteläosissa tavattiin molempia rotuja. Pohjanmaan siementuotantoalueella esiintyi lähinnä vain Yn-rotua. Molemmissa roduissa todettiin lisäksi isolaattieroja. Muiden virusten biologiset ominaisuudet vaihtelivat vähemmän, eikä niiden isolaatteja luokiteltu eri rotuihin kuuluviksi. Kaikki virukset ja virusisolaatit voitiin osoittaa ja osin myös tunnistaa testikasvioireiden perusteella. Joidenkin virusten ja virusisolaattien oireet testikasveissa poikkesivat olennaisesti julkaistuista. Perunan X-, S-, M- ja Y-virukset olivat yleisiä luokitellussa siemenperunassa 1970-luvun puolivälissä, mutta erityisesti X-, S- jaM-virusten esiintyminen ja merkitys on vähentynyt sitä mukaa, kun tervettä perusaineistoa on saatu siemenperunan alkutuotantoon. Taudinaiheuttajina Y-virus ja kierrelehtisyysvirus todettiin erittäin haitallisiksi kaikissa koelajikkeissa. Y-viruksen Y°-rotu esiintyi tappavana Ostara- ja Hankkijan Timo -lajikkeissa ja esti näissä sadonmuodostuksen lähes täysin. Rotu aiheutti useimmissa lajikkeissa nek- roosioireita. Yn-rotu aiheutti eri asteisia kurttu- ja mosaiikkioireita lajikkeesta riippuen, mutta nekroosia ei esiintynyt. Perunan kierrelehtisyysvirus aiheutti primäärioireina latvaosista alkavaa lehtien kiertymistä, sekundäärioireet ilmestyivät ensimmäiseksi vanhoihin alalehtiin. Kierreoireiden lisäksi taudinkuvaan kuului antosyaanimuodostus useimmissa lajikkeissa. Sekundäärisen kierreviroosin todettiin alentavan yksilösatoa Siikli-lajikkeella 77 %. Myös M- ja A-virukset aiheuttivat eräissä lajikkeissa voimakkaan taudin. M-viruksen aiheuttamiin oireisiin kuului selvä kitukasvuisuus, pensastuminen sekä lehtien kiertyminen lusikkamaisiksi. Yksilösatoa virus saattoi alentaa noin 50 %. A-virus aiheutti kurttuisuutta, kloroosia sekä mosaiikkioireita. Muiden virusten aiheuttamat oireet olivat lieviä. Kaikki kirvalevintäiset virukset levisivät infektiolähteistä herkästi, herkimmin leviävä oli Y-virus. S-virusisolaattien välillä esiintyi huomattavia levintäeroja. X-viruksen rengaslaik- kurotu levisi kasvustossa normaalirotua herkemmin, vaikka sekin osoitettiin pelkästään mehulevintäiseksi. Perunalajikkeiden infektoitumisherkkyydessä todettiin huomattavia eroja. Useat lajikkeet olivat yhdenmukaisesti alttiita tai kestäviä S- ja M-viruksille. Kaikki tutkitut virukset voitiin puhdistaa kasvimateriaalista käyttämällä julkaistuja menetelmiä tai niistä parannettuja muunnoksia. Suurimmat puhdistetut virussaaliit olivat: PVX 640 mg, PVS 31 mg, PVM 48 mg, PVY 26 mg, PVA 12 mg, TRV 85 mg jaPLRV 1.1 mg/ kg tuoreita tai pakastettuja lehtiä. X-, S-, M- jaY-virusten isolaateillevalmistettujen antiseeru- mien tiitterit olivat mikropresipitaatiomenetelmän avulla määritettynä 1/1024—1/8192. Ulkomaiset antiseerumit reagoivat tutkittuihin antigeeneihin heikommin kuin itse valmistetut seerumit. Geelidiffuusiomenetelmiin soveltuvien, pilkotuilla X-, S- ja M-virusproteiineilla 301 tuotettujen antiseerumien tiitterit olivat 1/128, 1/32 ja 1/64 kaksoisdiffuusiomenetelmällä määritettynä. Pienin kaksoisdiffuusiomenetelmän avulla määritetty virusproteiinikonsentraatio (PVX) oli 10 jug/ml, yksivaihemenetelmällä vastaavasti 1 /jl g/ml. Perunan lehtimehusta määritetyt laimennokset olivat 1/128 ja 1/1024. ELISA-menetelmän avulla määritetty alhaisin viruskon- sentraatio (PVX) oli 0.1 ng/ml. Perunan lehtimehusta PVX voitiin määrittää 7 -laimennoksista, mikä vastaa viruksen laimennosrajaa infektiokokeissa. ELISA-testissä saatiin X- ja M-viroottisista kasvinäytteistä parhaimmillaan yli 100-kertainen, S-viroottisesta 94- kertainen ja Y-viroottisesta 46-kertainen absorbanssiarvo vastaavaan terveeseen näytteeseen verrattaessa, kun mittauksen 0-tasona käytettiin tuoreen substraatin antamaa absorbans- siarvoa. Y-viruksen Y°- ja Y"-rotujen serologinen eroavuus osoittautui ELISA-testissä merkit- täväksi, heterologisessa reaktiossa mitattu absorbanssiarvo vastasi kymmenkertaisesti laimen- netusta homologisesta näytteestä saatua arvoa. Muiden virusten isolaattien väliset serologiset erot olivat määritysluotettavuuden kannalta merkityksettömän vähäisiä. Agglutinaatio- ja A6-irtolehtimenetelmämäärityksissä todettiin runsaasti virheitä, joiden syynä oli menetelmien vähäinen herkkyys tai epäspesifisyys. Agglutinaatiotestin herkkyys ei riittänyt Y-viruksen määrittämiseen perunan lehtimehusta. A6-irtolehti reagoi Y-virusta muistuttavin oirein X-viruksen rengaslaikkurotuun. Kaksoisdiffuusiomenetelmä ei ollut riittävän herkkä perunavirusten rutiinimääritykseen, mutta yksivaihemenetelmän avulla saatiin sekundääri-infektoituneiden perunoiden lehtimehusta X-, S- ja M-viruksista luotettavat määritystulokset. Menetelmä ei kuitenkaan soveltunut rutiinikäyttöön. ELISA-menetelmän avulla X-, S-, M- ja Y-virukset voitiin määrittää täysin luotettavasti perunan lehtimehusta ja lähes yhtä luotettavasti myös idätetystä mukulasta. Tulosten vas- taavuus oli: PVX 100 %, PVM 97 %, PVS 92 % ja PYY 90 %. Myös A-virus ja kierrelehti- syysvirus voitiin määrittää luotettavasti idätetystä mukulasta. Primääri-infektoituneen itämättömän mukulan viruskonsentraatio saattaa olla niin alhainen, ettei siitä saada luotettavaa määritystulosta. Mukulan vähäinen idättäminen kohottaa viruskonsentraatiota, jolloin virukset voidaan määrittää ELISA-testin avulla riittävän luotet- tavasti idullisesta mukulan silmupalasta.