ABSTRACT The Effect of Latent Virus Diseases on the Growth, Yield and Quality of Sebago Potatoes By Sung Han Lim The effects of various virus diseases upon the symptomatology, vigor, yield and quality of Sebago variety, Solanum tuberosum L. were observed over a two year period. Serologicai techniques tested by bioassay methods were applied to greenhouse grown plants to determine the absence or presence of the viruses. The plants were classified as healthy or infected with virus 5, x or the combinaé tion of viruses 5 and x. For field plantings young potato plants were separated from the mother tubers with a sterilized knife and transplanted into the flats according to the serologicai classifi- cations. Ali plants classified as virus infected were inoculated with specific viruses to insure infection with single viruses or com- binations of the viruses. Since virus Y had not been found in the tested plants. healthypiants were inoculated with virus Y. virus 5 or virus X to give all combinations of the virus. Three field experiments were planted at the Lake City Experiment Station. Ten replicated hills, each comprised of four plants. were space planted fIVe feet apart in,a randomized complete block design. The number of plants infected with each virus changed from the original numbers due to the absence of virus Y and its combinations with the other viruses-infected plants. Many of these subsequently Sung Man Lim became infected with virus x and the combination of viruses S and x. The number of plants was, therefore, regrouped and data were analyzed on the basis of the results of the agglutination tests at different dates by combining the three field experiments. in this experiment, both viruses 5 and x were mild viruses and the symptoms were not severe. A somewhat lighter green with slight mottling was observed on the leaves of virus S-infected plants. but these symptoms were not consistently present or clearly visible. A mottling, light yellowish areas and a number of small necrotic lesions on the leaves of virus x and the combination of viruses S and X-infected plants were observed. The combination of viruses S and x did not appear to be a new complex symptom although the small necrotic lesions seemed to be more pronounced on the leaves of the plants infected with both viruses 5 and x. The healthy plants exhibited a more luxuriant growth and were larger throughout the growing season than all virus-infected plants except those infected with virus 5. There was no difference in plant growth between healthy and virus S-infected plants in the early growing season. However, the size of the healthy plants exceeded that of the virus S infected plants later in the season. The reductions in tuber yield varied with the various potato viruses. The virus infections affected the large tubers, those above 5 cm in diameter, for both number and weight. Virus S did not affect the number of tubers but it seemed to affect tuber weight. Sung Han Lim Both virus X and the combination of viruses S and x reduced both the number and weight of tubers. The percentage reduction in tuber'yield was l7.9% for virus x, l5.5% for the combination of viruses S and X, and 6% for virus 5 respectively. Apparently virus infection in this experiment did not affect the specific gravity and was not associated with the color of the potato chips. Foliage weight and tuber yield were positively correlated at a highly significant level. The greatest tuber yield and foliage weight were obtained from the healthy plants. Correlation of dry matter of foliage and tuber yield was negative but there were no significant differences. The dry matter of the potato foliage of the healthy plants was not greater than that of the virus X or virus S-infected plants. The combination of viruses 5 and x infected plants reduced the dry matter of the foliage. but differences were very small. In this experiment. the viruseinfected plants could have had a reduced rate of photosynthesis and difturbed carbohydrate metabolism causing interference with the translocating of reserves from diseased leaves to the tubers. It is concluded that the effect of virus X appeared early in growing stage. The effect of virus S appeared after the period of tuber set and resulted in a decreased number of large tubers. Finally, it has been shown that the agglutination test is a technique applicable to the identification of mild infections of viruses, and that plants in which the virus was not detected produced the greatest yields of marketable tubers. It can be utilized in a seed program to eliminate many plants which are a potential source of infection. THE EFFECT OF LATENT VIRUS DISEASES ON THE GROWTH, YIELD AND QUALITY OF SEBAGO POTATOES BY Sung Man Lim A THESIS -Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop Science 1966 ACKNOWLEDGEMENT The author wishes to extend his sincere appreciation to Dr. N. R. Thompson, Professor of Crop Science. for his valuable counsel and guidance in the execution and completion of this study. and to Dr. w. J. Hooker, Professor of Botany and Path- ology. for his helpful discussions and critical appraisal of the manuscript. He also wishes to thank Dr. S. T. Dexter, Dr. J. E. Crafius and Dr. C. M. Harrison, Professors of Crop Science. for their advice and guidance throughout the course of these studies at Michigan State University. Sincere thanks are also extended to Dr. L. N. Wise and Dr. H. D. Bunch, Mississippi State University, for their help and encouragement in facilitating my studies in the United States. Grateful acknowledgements are extended to my parents and Professor in Kwon Kim in Korea without whose understanding and moral support this study would not have been possible. To the Department of Crop Science, Michigan State University, grateful acknowledgement is made for an assistantship during the period in which these investigations were carried out. TABLE OF CONTENTS 'NTRODUCT'ON. O O O O O O O O O O O O O O O O O O O O O REVEEW 0F L'TERATUREO O I O O O O O O O O O O O O 0 O O MATERIALSANDMETHODS................. EXPERIMENTAL RESULTS AND DISCUSSION . . . . . . . . . . IV. V. VI- Observations on the spread of potato viruses . . Influence of virus infections upon plant growth and symptomatology. . . . . . . . . . . . PIaNt height. a e e e e a a e a e e e a 0 Influence of virus infections upon tuber yield. a. Number of potato tubers . . . . . . . . . be Height 0f pct‘to tUberS e e e a a a e a a influence of virus infections upon the specific graVIty Of POtatOCSO a e e a e e e e e a e e a e Influence of virus infections upon the color Of Pptato Chips 0 a a a a a e e a e e e a e e e a e influence of virus infections upon the potato f0".ge . 0 0 O O O O O O O C O O O O O O O O O a. Fresh weight of the potato foliage . . . . b. Percentage dry matter of potato foliage. . sumARY AND CONCLUS IO" C O I O O O O O O O O O O C O O O BIBLIOGRAPHY . C O O O O O O O O O O O O O O O O O O C 0 Lane. lb 20 20 2i 27 3i BI 39 50 52 52 58 6i Iable l. 2. h—b. S-ao S'b e 6. 7-a- LIST OF TABLES Number of clones infected with various diseases determined by the agglutination test. . . . . . . . . The number of potato hills either free of virus or infected with virus on three different dates. . . . . Analysis of variance of plant height measured on July l5. The data were grouped according to the reaction in the first agglutination test on AugUStI7...................... Analysis of Variance of plant height meaSured on August ID. The data were grouped according to the reaction in the first agglutination test on August i7.'. . . . . . .1. . . . . . . . . . . . . . Analysis of variance of plant height measured on August l0. The data were grouped according to the reaction in the third and final agglutination test on september '7 O O O O O O O O O O O O O O O Q 0 0 I Analysis of variance of plant height measured on September l2. The data were grouped according to_the reaction in the_first agglutination test on August l7. . . . . . . . . . . . . . . . . . . . . . . Analysis of variance of plant height measured on September l2. The data were grouped according to the reaction in the third and final agglutination test on September l7. . . . . . . . . . . . . . . . . Average number of potato tubers from virus free and virus-infected plants grouped according to the reaction in the agglutination test on three dates. 0 O O O O O O O O O O O O 0 O O a 0 Analysis of variance on the number of total potato tubers per potato hill classified according to the reaction in the first agglutination test on August l7. Analysis of variance on the number of large potato tubers (larger than 5 cm) per potato hill classified according to the reaction in the first agglutination test on August l7. . . . . . . . . . . . . . . . . . . 20 27 28 28 29 30 32 33 8-a. Analysis of variance on the number of total potato tubers per potato hill classified according to the reaction in the second agglutination test on September 80 a e a e e a e a a a e a e e a a e e e a . a 0 3h 8-b. Analysis of variance on the number of large potato tubers per potato hill classified according to the reaction in the second agglutination test on September 8. . . . . . 35 9-a. Analysis of variance on the number of total potato tubers per potato hill classified according to the reactions in the third and final agglutination test on September '7. a a e a a a a a a a a a a a a e e e a a a 36 9-b. Analysis of variance on the number of large potato tubers per potato hill classified according to the reactions in the third and final agglutination test on September ‘7 e e a e a a a a a e e a a e a e a a e a a 36 lo. Average weights of large and small potato tubers from healthy and virus-infected potato hills grouped by the serological reactions on three test dates . . . . . . . . hi ll-a. Analysis of variance of the total weight of potato tubers grouped according to the reaction in the first agglutination tCSt 0n AUQUSE '7 a e a e a e a e e a e a a “2 ll-b. Analysis of variance of the weight of large potato tubers grouped according to the reaction in the first agglutination test on August l7 . . . . . . . . . . . . . 42 l2-a. Analysis of variance of the total weight of potato tubers grouped according to the reaction in the second agglutination test on September 8 . . . . . . . . . . . . #3 l2-b. Analysis of variance of the weight of large potato tubers grouped according to the reaction in the second agglutination test on September 8. . . . . . . . . . . . Ah i3-a. Analysis of variance of the total weight of potato tubers grouped according to the reaCtion in the third agglutination test on September l7. . . . . . . . . . . . #5 l3-b. Analysis of variance of the weight of‘large potato tubers grouped according to the reaction in the third and final agglutination test on September l2. . . . . . . #5 I5. l6. I7. 18. 19‘8e '9-be 20. 2|. The average specific gravity of Sebago potato tubers from various viruses-infected plants grouped by serological reactions on three test dates . . . . . . . . Analysis of variance of specific gravity data of Sebago tubers grouped according to the reaction in the first agglutination test on August l7 . . . . . . . . . . influence of virus infections upon chip color of potatoes from healthy and virus-infected potatoes grouped according to serological reactions on three tCSt dateSe a a a e a e a a e e a e e a a a a e e a e a 0 Analysis of variance of potato chip data of Sebago variety grouped according to the reaction in the first agglutination test on August l7 . . . . . . . . . . . . . Fresh weight of the potato foliage per hill (grams) . . Analysis of variance of fresh weight of the potato foliage grouped according to the reaction in the first agglutination EOSE on AUQUSE 17*. e e e a e e e e e e e 0 Analysis of variance of fresh weight of the potato foliage grouped according to the reaction in the third and final agglutination test on September i7. . . . . . . Means of the percentage dry matter of the potato foliage of healthy and virus-infected plants grouped by serological reactions on three test dates . . . . . . . . Analysis of variance of dry matter data of the potato foliage grouped according to the reaction in the first BQQIUtInatIOH tCSt AUQUSt '70 a e a e e e e a a e a a a 0 vi 29.92. #9 SO 52 52 53 53 5h 58 58 LIST OF FIGURES F i gu re _£2_P 3 l-a. Leaflet of a healthy Sebago. . . . . . . . . . . . . 2h l-b. Leaflet of virus S-infected Sebago showing a somewhat lighter green with slight mottling and VCIH'C'earIng e e a e e e e e a a a e a a a e a e 0 2h l-c. Leaflet of virus X-infected Sebago showing mild mottling and small necrotic lesions . . . . . . . . 25 l-d. Leaflet of the combination of viruses 5 and x- lnfected Sebago showing similar symptoms of virus x. 25 2-a. Leaves from a single plant of Nicotiana debneyi inoculated with virus S. inoculated basal leaf (left) is symptomless while middle and upper leaves Show VCLH'C'earInge a e a a a a a a a a e a e e e a 26 2-b. A vein-clearing and somewhat mottling show on the leaves of Nicotiana debneyi inoculated with the combinfitlon Of VIruses S aNd X- e e a e e a a a e a 26 3. Number of tubers from virus-infected potato hills expressed as percent of the weight of tubers from healthy hIIlS e a e a a a a a a e e a e e e a a a a “0 h. Weight of tubers from virus-infected potato hills expressed as percent of the weight of tubers from healthy hIIIS O O O O O O O O O I C O O O O O O I O “8 5. Relationship between fresh weight of potato foliage and number of tubers grouped according to disease reading on each of three agglutination tests. . . . 56 6. Relationship between fresh weight of potato foliage and weight of tubers grouped according to disease reading on each of three agglutination teStS a a a a a a a e e a a a a a a a e a a a a a e 57 vii INTRODUCTION in modern potato production a high yield of marketable potatoes is synonomous with efficiency and profit. To attain high yields, disease free seed is essential. Some diseases may be readily identified by symptomatology and are easily removed from seed stocks. Others are latent and difficult to diagnose. Their effects are not immediately obvious and their importance has been questioned. Although there has been some work on problems concerning the effect of viruses upon the growth and yield of potatoes. the results are somewhat inconsistent so cannot be applied universally. For instance, the reduction in yield from virus-infected potatoes re-' ported by several investigators varies from a very small amount to as high as 30 percent. Roguing or early lifting of visibly diseased plants is general practice in seed programs. However, the viruses that are practically symptomless and difficult to diagnose remain. Only since the develop- ment of serological methods. whereby antisera are mass produced, has a tool been available to attempt to remove the latent viruSes. The present study was undertaken to obtain information on the growth. yield and quality of potatoes infected with virus diseases. Serological and bioassay techniques were used to determine the absence or presence of the viruses. REVIEW OF LITERATURE Since Schultz and Folsom (37) in the United States and Quanjer (3k) in Holland, described a number of virus diseases of the potato Solgnum tuberosum L. in i923, a great amount of research has been conducted and many papers tublished. The reduction in yield from virus x-infected potatoes reported by several investigators varies from a very small amount to as high as 30%. The reduction varies with the potato variety and the strain of virus x (29. 32. 38. 39. #7). information regarding the effect of virus S on potato yield is scarce and no reports were found regarding the effect of viruses 5 and x- in combination on potato yields. in Maine. yield reductions of approximately l2 to 22% were obtained by Schultz et. al. (38). over a four-year period with potato virus x-infected stocks of the Chippewa and Katahdin varieties when compared with potato virus X-free stocks of the same varieties. Although a very few potato virus x-free stocks have been found among some of the old varieties by the above authors, they were not able to obtain a single tuber free from virus x when lOOO Green Mountain tubers from several potato regions were tested. They found it practically impossible to diagnose latent mosaic in the field. Seed stocks of the varieties Chippewa. Sebago and Teton. carrying a mild strain of potato virus x, were compared with virus X-free stock and commercial stock for each variety by Lombard (29). Both mild X and commercial seed produced reduced yields when compared to yields from virus x-free stock. Percentage reductions in yield for mild x were: Chippewa 6%. Sebago H%. and Teton 9%. Percentage reductions in yield for commercial stock were: Chippewa 6.8%, Sebago 9.8% and Teton 5.l%. Yield reduction in Chippewa and Katahdin was 6%.with virus x- infected plants in New‘York and reduced stands were observed in virus x-infected Sebago stock according to Wilkinson et. al. (M7). in the Sebago variety, there was a marked effect on the stands in addition to an effect on yields. in England, yield reduction in Majestic and Arran Banner ranged from 5 to 22% depending upon the severity of the virus X present, according to Bawden et. al. (5). Norris (32) found that potato virus x (latent mosaic) in the President and Up-to-Date varieties reduced the yield about 30% and that it was one of the chief causes of the reduction in yield of potatoes in Australia. its effects were evenly spread over the entire crop.l Scott (39) reported that in Scotland potato virus x (latent mosaic) was responsible for yield reductions of l6 to 25% and that similar yield losses resulted from potato virus A. Bald (A) has shown that the effect of a masked strain of virus x upon the yield of the Up-to-Date variety is due to the inability of diseased plants to transport hydrolyzable reserve leaf proteins to the tubers for late-season expansion. There was no difference between the yields of healthy and x-infected plants which were harvested lh to l5 weeks after emergence of the plants, but at maturity, diseased plants yielded about ll percent less than healthy ones. He suggested that final rapid tuber expansion at maturity was due to the translocation of reserve proteins from aging leaves in normal plants, and that virus infection interfered with the emptying of such reserves from diseased leaves. Transport and distribution of products of photosynthesis in potato plants were studied using Int and a starch test by Kosmakova (25). During the flowering phase, products of photosynthe- sis were assimilated chiefly into the tubers, with a small amount 1“c was found entering the roots and infloresences. After flowering only in the roots and tubers, but mostly in the latter. Later in the season, just before harvest, the products from the leaves was translocated only to the tubers. A starch test showed that in plants with mottling, teaf curl, and wrinkled mosaic, carbohydrate metabolism in the leaves was disturbed‘with less starch accumulating in them than in the leaves of healthy plants. Daniel et. al. (l5) reported that infection by a masked strain of virus x caused decreased photosynthesis in leaf tissue. Photo- synthetic rates were decreased in symptomless Y-infected leaves, and double infections resulted in the greatest decrease in rates of photo- synthesis. Systemic infections by viruses which invade leaf parenchyma apparently result in a decrease in the rate of photosynthesis, regardless of the symptoms produced. The reductions in yield caused by masked strains of virus x can be attributed, at least in part, to reduced photosynthesis in diseased plants. The effects upon photosynthesis of the several chronic infections closely parallel those upon yields. Yield reduction of virus S-infected Bintje has been observed in the Netherlands (36). in general the yield of virus S-infected clones was l0-l5% below the healthy clones. Virus S-infected clones produced a higher percentage‘of a smaller tuber and a smaller percentage of the larger tubers. The average reduction of the size larger than 50 mm in diameter was l5-20%. A similar reduction in yield was found in Switzerland, reported by Rozendaal (36). Clark (l2) counted the number of tubers per plant of the Rural New Yorker No. 2 variety, beginning at weekly intervals soon after the period of tuber fonmation and up to the time the plants were killed by frost. The range of growth period was from 6“ to l27 days. He found very little increase in tuber set after the blossom stage and concluded that small tubers at harvesttime are the result of an uneven growth rate rather than a late set. it indicates that most small potatoes are of the same chronological age as the large ones. Neither soil moisture nor loss of vines affected chip color at harvest (26). However, when potatoes were held at 60°F., differences between treatments developed. The tubers from saturated soil produced darker chips than tubers from dry soil. Het soil conditions produced an intermediate effect on chip color. Loss of vines caused consistently darker chips at all moisture levels after 2 weeks storage, but the effect 6 of vine removal on chip color was less than the effect of soil moisture. The problem of black discoloration after cooking was studied by Tottingham et. al. (#3). One of their first concerns in approaching this problem was the possibility that the abnormality had a pathological origin. However, their trials in two seasons on field plots at several locations failed to prove the transmissibility of a pathological con- dition to the succeeding crap. They concluded that the blackening condition was due to physiological factors in the growth of the plants. Akeley et. al. (l) observed that relatively large differences in density of tubers of the same variety occurred in the same field. The differences are still greater when the same variety is grown in different locations. Katahdin grown in 7 locations showed wide extremes in mean tuber-density class values from 3.l to 8.5, a highly significant difference. He concluded that environmental conditions affect tuber density to a high degree, a variéy grown in one location differing very greatly from the same variety grown in another place. Heinze et. al. (20) has shown that the average specifictgravity differs from year to year for the lots obtained from the same general location. Deviations indicate that these same lots of potato varieties were much more variable in some years than in others. The weighted average deviations for location show that potatoes tend to be more variable in specific gravity in some locations. Transmission of one or more viruses, mottle virus, from appar- ently healthy potatoes of common varieties was reported.by Johnson in l925 (2i). in Europe, this disease which shows a mild type of mottling in some varieties and has been described as simple mosaic or crinkle (3i). it is generally believed that Schultz and Folsom's rugose mosaic (37) is the same as the crinkle of Murphy and McKay (3i) and the common mosaic asldescribed by Quanjer (3h). In I931 Smith (#0) designated the virus causing this disease as X, which had been known in Europe as simple mosaic, and in the United States as latent mosaic or the healthy potato disease. Different strains of virus x have been found in some of the European and American potato varieties. The intensity of symptoms of such complexes causing crinkle and mild mosaic varied depending upon the strain of x present in the complex (l8). A mosaic of the Chippewa variety characterized by irregular chlorotic-mottle on upper leaves and small scattered necrotic-flecks on older leaves was observed in the field by Larson (27). The external and internal root, stem, and petiole tissues of affected plants were normal. The tubers were slightly smaller than normal, but showed no pathologic symptoms. The question of insect transmission of potato virus X has been investigated by many workers. Clinch (l3) stated that neither aphids nor leafhoppers need be considered as possible vectors of virus X. Thrips, flea beetles and various sucking insects have all been tested by Smith with negative results (hi). However, he observed that in carefully controlled experiments odd infections do turn up from time to time. He commented that these infections were due to casual spread of the virus by grasshoppers or to a mechanical transport of the virus on the boots or implements of workers. Halters (#6) demonstrated that grasshoppers transmitted the virus from tobacco to tobacco in 6 percent of greenhouse trials. Experiments on the spread of virus X in the field have been carried out by several workers. it is generally known that virus X is one of the few plant viruses which spreads in the field by contact between healthy and virus-infected plants (3i, #0). Virus X spreads through contact of sprouts in storage or foliage In the field (l3, 35). it is also spread by the cutting knife and there is evidence that it is spread by contact between roots (35). Observations were made by Roberts (35) on the spread of virus x in ho plots containing from 8 to 99 healthy potato plants and a single infector plant in the central area of each plot during a period of five years. From the ho infected sources 3h transmissions were recorded as the possible result of direct contact between healthy and infected plants. in l5 plots there was no spread of virus X within the season; in l7 plots only a single plant was infected; in 7 plots two plants adjacent to the infective source were infected; and in l plot infection spread to 3 adjacent plants. Studies on the spread of potato virus X were carried out at lO different locations by Hansen (l9). The average of three years results showed that l8% of the original virus free Bintje plants were infected with potato virus X. During each of the three seasons the infection was l9, 20 and l“% respectively. The percentage for an individual location varied from O to 60%. The experiments comprised altogether 335 Primary infected plants. The progency of these, about 3000,plants was tested individually. About “0% of the tubers from these plants were virus X- infected. Only lO% of the primary infected plants gave completely infected progeny. Cockerham (lh) reported l9.7% infection in a stock of the Majestic variety grown adjacent to virus X-infected stocks, 5h.2% in a stock which had been grown between infected Arran Banner, and 82.8% in a stock which had been grown throughout virus X-lnfected surroundings. Beemster (8) studied the translocation of potato viruses in the potato plant from the inoculated leaf to the tubers. The results showed that very few tubers were infected with virus X when the inocu- lation was made on mature plants. On the other hand, a higher percentage of infected tubers was found when the tubers were harvested five weeks after inoculation than when harvested three weeks after inoculation. From these results he concluded that the translocation of virus X from the leaf to the tubers continued frdm the third to the fifth week even when the plants had been inoculated in a later stage. A survey conducted by Todd (#2) indicated that: l) the spread of potato virus X is not normally rapid,\2) usually,infected plants could not be found until the second growing season but the content of infected plants would increase roughly at the rate of doubling every year, 3) extensive and rapid infection occurred in the stocks comprising a mixture of potato virus X and virus X-free potatoes and in plots which were surrounded by a great mass of infective materials, A) infection did not depend on the stock being next to a virus-infected stock and some grown in fields by themselves become infected, 5) Virus X is rapidly transmiss-. ible on the person, on clothing and on animals. IO Bawden et. al. (6) reported that when certain potato varieties, already infected with virus X, were inoculated with virus Y, symptoms of current-season infections were no more severe than those caused by virus Y alone. Daniel at. al. (l5) reported that chronic infections by both virus X and Y invariably resulted in diagnostic symptoms of rugose mosaic and leaf-drop streak in Placid potatoes. Symptoms were easily recognizable at all times during the seaSOn. Masked virus X in single infections caused no symptoms. Symptoms caused by virus Y alone were detected only during a short period about 6 weeks after planting, when diseased plants were smaller and lighter green than healthy ones. Plants infected by virus Y recovered rapidly, and were undistlnguishable from healthy or X- infected ones by midseason. However, mixed infections by virus X and Y in the variety Placid can be readily diagnosed in the field, even though each virus may be carried symptomlessly in single infections. Potato virus S was detected first by serological techniques at the Laboratory for Flower Bulb Research at Lisse, The Netherlands under the direction of E. van Slogteren (l6, 36, Ah). An attempt was made to prepare an antiserum against potato virus A by infecting rabbits with extracts from virus A-infected potato leaves. The antiserum he prepared did not react against virus A but, against a quite different antigen which had been obtained from Bintje as well from Light and Dark lndustrie. Transmisslbility of this virus antigen was proved (l6, 36), by tuber plug II graftings and sap inoculations. it was named as virus 5 from the first letter of E. van Siogteren's family name. Bagnall stated (2) that potato virus 5 was very wide spread in North American potatoes. symptoms incited on potatoes usually were so slight that they passed for normal effects of maturity. However, virus S could be distinguished from other potato viruses by serological tests and by thd reactions of a number of differential test plants: WM, Nicotigng debug" i'e'tc. Rozendaal (36) stated that the virus S infected plants are‘ generally characterized by a'lighter green color, a deepening of the veins (rugosity) a slight bending downward of the tip of the leaves, a more open plant growth and by more or less drooping of the haulm of older plants. The symptoms frequently are inconspicuous and in many transition forms. He found the virus to be easily transmitted by stem grafting or sap inoculation. A contaminated knife or needle infected 29% or more plants of susceptible varieties when the tubers were cut or when the eyes or young sprouts were picked. These results did not agree with Levieil's who reported that sap transmission of virus 5 is comparatively difficult. Kassanis (22) was unable to transmit virus S by‘flyggg persicae from infected to virus free plants of the King Edward variety. This is in agreement with Rozendaal (36). However, both workers stated that if aphids play a part in the transmission of virus S it Can only be a very minor one. According to Rozendaal (36) the combination of virus 5 with other viruses does net give rise to a new complex disease, but only to more pronounced symptoms. in contrast, the combination of viruses X and A I2 in many varieties causes a complex disease with symptoms different from those of the separate viruses. The variety Light Red Star, when infected with stipple streak virus is much more mottled and rugose when virus S is also present. The first serological investigations in connection with plant viruses were carried out in l927 by Dvorak (l7). She showed evidence that mosaic disease altered the serologic specificity of the globulin reaction of the cell sap and cytoplasm of the potato plant. Beale (8) also showed that tomato and tobacco plants infected with mosaic contained an antigenic substance which was associated with the virus. Several investigators have confirmed and extended these findings. The results of serological reactions, specific for a virus, may be arbitrarily summarized as follows: lL,virus-infected plants contain an antigenic substance which is not present in healthy plants (1.7),, 2).. The serologic titre for the antigenic substance is correlated with the concentration of virus {079,213.} . Virus-containing plant juice stimulates the production of antibodies which are specific for the virus (an), #9. Purified preparation of several viruses give positive reactions only with homologous virus antigens (Gill) .5.) .Antisera prepared against a plant virus can be absorbed with a healthy plant antigen. The absorbed serum contained reactive substances (l0). From these studies the first antisera, for routine analysis, was mass produced and used in the potato industry. Van Slogteren (#5) stated that the serological diagnosis is: l) independent of the symptoms, 2) quick and objective, 3) makes possible the identification and classification of many viruses, #) can help to l3 determine the concentration of a virus together with its localization and transport in the plant. He stressed the fact that its simplicity made it possible to test many plants to identify virus-free potatoes. Furthermore, the serological diagnosis can be helpful for l) speeding up the breeding of immune varieties, 2) the disentangling of complex virus diseases, 3) the I discovery of unknown viruses. As a routine testing method, the agglutination test was applied to identify potato virus X by Kristensen of Denmark (2#). Potato plants selected in the field were serologically tested twice during the growing season. These two serological tests showed corresponding~ results in 9#m5% of the tests. The same potato plants were tested by inoculation to.§gmgh£ggg globosa and when these tests were compared with the serological test the results corresponded in 92.2% of the cases. He stated that partial infection occurred~frequently in the field and caused some variation in the different tests on the same individual plant. in most cases when the tested plant was virus-infected the agglutination reaction occurred within a few seconds and was performed much more quickly than by any other method. MATERIALS AND METHODS Variety: The Sebago potato variety was used in this experiment. The initial seed was obtained from a Michigan Foundation seed grower. The perfection of serological techniques as a tool to detect the presence of viruses permitted the classification of adequate numbers of plants for agronomic studies. Serological tests: As a modification of the agglutination test, flat-bottomed petri dishes about lO cm. in diameter were coated with a thin film of polyvinyl formal. A l‘% solution of "formvar" in chloroform was poured into the dry petri dishes and poured out again immediately. A hydrophobiclfilm forms on drying. Small drops of antisera were placed in rows on the petri dish with one end of a toothpick and mixed with a drop of the antigen (plant juice) with the other end. The toothpick was then discarded. Drops were covered with mineral oilttp prevent evaporation and spontaneous flocculation at their edges. Later, flat-bottomed plastic dishes about-IO cm. square were used instead of petri dishes. it was found that drops of antiserum can be mixed with drops of antigen on the plastic dishes without the coating of polyvinyl. The antisera used in this test were normal serum, anti-S-serum, antl-M-serum, antidY-serum and anti-x-serum. These _ were obtained from the Laboratorium Voor Bloembollenanderzoek, Lisse, The Netherlands. A press was designed ahd built to extract plant juice from the leaf samples of test potato plants. Each leaf sample was placed between two 5 cm. square metal plates and pressed. A few drops of extracted juice from the press were collected. The metal plates were i# I5 washed and sterilized between each leaf sample. Bioassay Method: Bioassay was carried out in the greenhouse where the temperature was usually held at 20-25°C to verify the use of serological techniques as a tool for screening large numbers of plants in a seed program. The indicator plants to be inoculated were sprayed uniformly with carborundum on the upper surface of the leaves. The plant leaves were inoculated by rubbing the extracted crude-sap on the upper surfaces of the leaves with a tongue depressor. A new tongue depressor was used for each individual inoculation. The leaves were washed with water after the inoculation was completed. Gggphreng globosg plants were used as the indicator plant of potato virus X. The half-leaf method was applied on Ggmphreng globosa plants. Local necrotic lesions surrounded by reddish borders developed within 5 days on the inoculated leaves when test potato plants were carrying virus X. ulcogigg. debeneyi plants were used as the indicator plant for potato virus S. The whole plant method was used. .fle debeneyi plants were inoculated in the young 2 or 3-leaf stage. Potato virus 5 incited no local symptoms on the inoculated leaves but about 25-30 days after inoculation, a vein-clearing appeared on the first leaf above the base of the inoculated leaf when test potato plants were carrying virus 5. Gree ouse e eri nt in the winter of i963, ZOO tubers from individual clones of the Sebago variety were planted in pets lO x l3 cm and placed on benches in the greenhouse. Three leaflets were collected from each stem of the 200 plants when they were 25-30 cm high. Extracted plant juice from these leaf samples l6 was then used in the agglutination tests in order to detect virus diseases which might be present. Three weeks later the plants were tested again. All tested plants*were then classified into different groups according to their reactions in the agglutination tests. Viruses S, X and the combination of S and X were detected. No positive reactions were obtained with antisera of virus M and virus Y. The number of virus diseased Sebago clones determined by the agglutination test in the winter of l963 are presented in Table l. Table l. Number of clones infected with various virus diseases determined by the agglutination test. Virus Vi rus Virus Virus 5 pTusTT yirgsgs frggj § 1L: Virus X First test l63 8 l7 l2 Second test l57 ll l8 l# Since there was a slight variation between the two agglutination tests, only clones which reacted similarily in both tests were saved: Virus S, 7: Virus X, l7; Virus 5 plus Virus X, ii and Virus free, l57. Tubers from these clones were space-planted 5 feet apart in a randomized complete block design with lO replications in l96#. Hashing, caused by excessive rainfall, created great variations in emergence and stand. it was not possible to evaluate the effect of virus diseases on growth and yield. The experiment was repeated in i965. The procedures of l963 were used to identify the potato virus diseases in the i96# plantings. Bioassay was also carried out on each plant which showed a positive reaction in the agglutination test. During the winter of l96#-65 a tuber from each hill grown in the field in l96# I7 was grown in the greenhouse and test by both methods for the presence of virus. Tubers, free of virus and with specific viruses were planted in sterilized sand. The agglutination test provided a technique for positive identification of plantsiinfected with a virus. Ten plants for each of the potato viruses: 5, X, the combination of S and X, and virus free plants were grown in the greenhouse for observation on their growth end for the expression of virus symptoms. Plants for the field experiments of l965 were started in the greenhouse to be transplanted to the field in order to obtain a perfect stand and an equal number of stems in each potato hill. Tubers, free of virus and with specific viruses were planted in sterilized sand. When the young potato plants were about 7-lO cm. high, they were separated from the mother tubers with a sterilized knife and transplanted into flats according to their classification. Approximately l50 plants for each category, i.e., infected with virus 5, X, or the combination of viruses S and X and virus free plants, were started in flats. An additional l50 virus free plants were started to be inoculated with virus Y since it had not been found in the original 200 tubers. Virus M was not detected in any tubers tested so was not included in field experiments. The transplanted plants were inoculated when they were about is cm high to insure that they were infected with the virus diseases. Also, the combination of viruses S and Y, viruses S and X, viruses Y and X and viruses S, Y and X were inoculated on Sebago plants by individual inoculum of virus S,vlrus Y and virus X. The inoculum for virus 5 was prepared from virus 5 - infected N, debene i; the inoculum for the virus X l8 from virus X - infected Sebago plants and inoculum for virus Y from the virus Y - Infected'N. b cco. Twenty-five grams of 600 size grit of carborundum mixed*with one hundred cc. of prepared inoculum for each of the virus groups was diluted with distilled water in the ratio of l to 50. The diluted inoculum was sprayed on the leaves of the transplanted potato plants with 65 pounds of air pressure. The atomizer was sterilized each time inocula were changed. field experiment: Three field experiments were planted at the Lake City Experiment Station. Each experiment consisted of virus free plants or plants carrying and inoculated with either virus 5, virus Y or virus X to provide plants infected with a single virus, or combinations of viruses 5 and Y, viruses 5 and X, viruses Y and X and viruses S, Y and X. Four plants were transplanted together to make each hill on June l5, l965. These hills were space-planted 5 feet apart in a randomized complete block experiment with W replications. Each replication consisted of 9 hills, 7 diseased hills and 2 virus-free hills. Two weeks after transplanting, any missing plants in each hill were replaced. Since the agglutination tests showed that the viruses had spread during the growing season, all the data were grouped and analysed according to the results of agglutination tests at different dates by combining analysis of variance for the three experiments. Duncan's multiple-range test was used to compare each mean with every other meanr obtained in the results of this experiment. Normal cultural practices were followed during the growing season. The field was irrigated and sprayed with fungicides plus insecticides l9 throughout the growing season. Weeds were controlled by hand hoeing. Later, a small hand cultivator was used instead of ahhoe. The agglutination test was carried out at three different times: August l9, September l8 and September l7. The following observations were recorded: l) Growth: a) observations were made weekly throughout the growing season to record the expression of any disease symptoms on the potato plants in each individual hill. b) the plant height was measured three times. c) flowering and maturity were also recorded. d) vines of #5 hills from each of three experiments were harvested and the fresh weight and dry weight were obtained. 2) Number of tubers: At harvesttime the tubers from each hill were divided into two groups and counted on the basis of size; larger or smaller than 5 cm in diameter. 3) Height of tubers: Heights of tubers per hill were obtained for each of the two size groups. #) Potato chips: Three slices approximately 2 mm thick were cut from the center of 2 or 3 tubers from each individual potato hill. Slices were rinsed in water to remove excess starch, blotted with paper towels, and cooked in a commercial grade of homogenized vegetable shortening at a temperature I 3#5°F. They were removed from the cooker when bubbling stopped. All the chips were placed in cellophane bags and kept at room temperature, 20-25°C. Three readers evaluated the chip color according to the color reference standard of the Proctor and Gamble Company. There are ten classes ranging from a very light, almost white, to very dark brown in the color reference standard. The scores were averaged. RESULTS AND DISCUSSION l. Observations on the Spread of potato viruses. inoculations with potato virus Y and the various combinations of virus Y with the other viruses were not successful. Consequently, no data on virus Y could be included in this experiment. The trends in virus infections are illustrated in Table 2. Table 2. The number of potato hills either free of virus or infected with virus on three different dates. Date of Virus Virus Virus Virus 5 +r Total Agglutination Tests free 5 X Virus X lst test Exp. l 2h lo 33 23 90 August l7 Exp. 2 2i lo 32 27 90 Total .72 28 99 71 270 2nd test Exp. 1 2i 8 2h 37 90 September 8 Exp. 2 22 7 22 39 90 fl. 3 32.; 9 ill 33 90 Total __ 6k 2k 23 lg? 270 3rd test Exp. l 2h 3 l3 50 90 September l7 Exp. 2 ‘ l9 9 l6 1+6 90 Total 57' 26 459, 137 A220 The result of the third and final agglutination test showed a decrease in the number of virus free potato plants and plants infected with either virus 5 or virus X and an increase in the number of hills infected with the combination of virus 5 and virus X. The greater than anticipated number of potato plants infected with virus X and the combination of viruses S and X.in the first agglutination test 20 2| was due to the absence of virus Y-infected plants which subsequently become infected with virus x or the combination of viruses 5 and x. The reduced number of virus X-infected plants in the final test may be largely due to the appearance of virus 5, which had previously existed in the virus X-lnfected plants but could not be detected on the first test. The total number of virus S-lnfectedihilis was fairly constant throughout all the agglutination tests. ThereIwas a slight decrease in the number of infected plants in the second test. However, at the time of the third test there was a marked decrease in Experiment l and a sizeable increase in Experiment 3. Some of these plants accounted for the increase in the number of plants infected with the combination of virus S and virus x. it may be that virus S required a long time to become estab- lished and the virus concentration was not high enough to show a positive serologic reaction in the first agglutination test. This is in agreement with Rozendaal et. al. (36) who was unable to observe primary symptoms on potato leaves inoculated with virus 5. in his work the infection was detected by the agglutination test, but occasionally it was detected only by the micro-precipitation test. Frequently both tests were negative, yet the progeny proved to be infected. Potato virus 5 has been shown to be very*widespread in many potato varieties since the application of the serological test. ii. Influence of virus infections upon plant growth and. symptomatology. All the potato plants in the field were slow to recover from 22 transplanting. Growth during the early growing season, i.e., in late June was slow. The average temperature in June was l6.5°c., the weather often cloudy. After frequent irrigation and an application of ammonium nitrate fertilizer, the plants gradually recovered from transplanting. By early July, the plants had fully recovered and the color of the leaves was changing from light yellowish green to dark green. The average temperature in July was 18.00c. and total rainfall was quite low, 23.6 mm. The departure from nonmal was -56.6 mm. A light yellowish mottling and small necrotic lesions were observed on the leaves of virus X and the combination of viruses 5 and X- infected plants. All these symptoms were mild and no severe symptoms were observed during this period. in late July, all plants were growing vigorously and were fairly uniform. in August, the average temperature was l8.5°C. and the total rainfall was l29.8 mm. Very little irrigation was required during this period. By this time, the plants were blossoming profusely. The mild mottling, the light yellowish areas and the number of small necrotic lesions were more evident on the leaves of virus X and the combination of virus S and virus X-infected plants. The small necrotic lesions seemed to be more pronounced on the leaves of plants infected with the combination of viruses S and virus X. The combination of virus S and X did not appear to be a new complex symptom. A somewhat lighter green with slight mottling was observed on the leaves of virus 5 infected plants. But these symptoms were not consistently present and were not clearly distinguishable. 2.3. in September, the average temperature dropped down to lh.0°c. from the l8.5°C. in August. The total rainfall was l6l.3 mm and the departure from normal was +82.0 mm. Rainfall was frequent. The plant foliage was killed by a heavy frost on September 23. The symptoms of various viruses are shown in Figure l and 2. Rozendaal et. al. (36) pointed out that the combination of_ virus S with other viruses, i.e. virus A, virus X and a strain of virus Y, does not give rise to a new complex disease, but only to more pronounted.symptoms, depending upon the potato variety. The symptoms of virus S are very mild and variable depending upon the strain of the virus, the weather conditions and the soil. in addition to a deepening of the veins causing Egggsity of the leaves, they observed a distinct mottle on the leaves of virus S- lnfected some potato varieties, i.e. Gloria and Benelander, and small necrotic spots in the Profijt. in general symptoms of VTFfis 5 appear later than those of virus X and virus A. Figure l-a. Leaflet of a healthy Sebago. (3-88) Figure l-b. Leaflet of virus S-infected Sebago showing a somewhat lighter green with slight mottling and vein-clearing. (2-86) 3‘88 Figure l-c. Leaflet of virus X-infected Sebago showing mild mottling and small necrotic lesions. (2-33) Figure l-d. Leaflet of the combination of viruses S and X-infected Sebago showing similar symptoms of virus x. (2-2) 2" 33 Figure 2-a. Leaves from a single plant of Nicotiana debneyi inoculated with virus S. inoculated basal leaf (left) is symptomless while middle and upper leaves show vein-clearing. Figure 2-b. A vein-clearing and somewhat mottling show on the leaves of Nicotiana debneyi inoculated with the combination of viruses S and X. 27 Plant height The height of all plants was measured on July l5, August l0 and September l2. Plants here classified and grouped according to theirreaction to the agglutination tests on August l7, September 8 and September l7. Sinee the groupings on September 8 and September l7 were similar only the September l7 grouping is shown. No statistically significant difference in plant height was found between the healthy plants and those infected‘with virus S_ (table 3). However, both these groups exceeded the height of plants infected‘with virus x and the combination of viruses X and S. Table 3. Analysis of variance of plant height measured on Ith l5. The data were grouped according to the reaction in the first agglutination test on August l7. swrce 0f VCrILM-g ~ dofe JeSe Experiments 2 90.h00* Virus diseases 3 h3h.l97** Experiments x virus diseases 6 36. 60h Error 258 25 350 ’ 19.311 m e * P < .05 **P < .0l By comparingfnthe following results were obtained: .j Virus 5 Healthy Viruses S and X m;[g pint height (cm) 21.3 - W”? .2211 A Zlo-I “3941- w a...— a .A» - w..." v ._ r -tM-. \ Wavwai Any two means not underscored by the same line are significantly different. when the data were regrouped by the reactions on September l7, there were‘no significant differences in plant height between plants infected with viruses and healthy plants. 28 The plant height measured on August l0 is presented in TableL-a and h-b. Table h—a. Analysis of variance of plant height measured on August l0. The data were grouped according to the reaction in the first agglutination test on August l7. Source of Variance Experiments 2 80h.9l5** Virus diseases 3 226.h85* Experiments x virus diseases’ 6 135.592 Error 258 63.7l3 Total 269 w *P < 005 “P < 00' lllfls By comparing. the following results were obtained. Health! Virus 5 Viruses S and x giggg x Plflflt he'ght (CM) 5506 21:06 206 51.8 Any two means not underscored by the same line are significantly different. Table hfb. Analysis of variance of plant height measured on August l0. The data were grouped according to the reactIOn in the third and final agglutinationxtest on September l7. Source of Variance d.f. ' 3;”. .1135? Experimentsd 2 803.579** Virus diseases 3 i63.282* Experiments x virus diseases 6 , 138.h29 Error 258 6i.797 Total 269, *P < .05 ”P < 00] By comparing means. the following results were obtained: -..— vv‘ ‘ ,n _ S'WF m ”E s 3“ Eli § § 33" l lim’l P'ENt height (CM) 550% SEJ r e530L I 5202 29 Any two means not underscored by the same line are significantly different. The healthy plants were significantly taller than the plants infected with virus x and the combination of viruses S and X. There was again no significant difference between virus free and virus S- infected plantsf‘Table h—a). in Table Heb, the healthy and virus S-infected plants were significantly taller than the plants infected with virus x. Results of the final measurement of the plant height were presented in Table S-a and 5-b. Table S-a. Analysis of variance of plant height measured on September 12. The data were grouped according to the reaction in the first agglutination test on August l7. Son!“ Of !:rl.nce iafe Hd§a ‘ Experiments 2 80.772 Virus diseases 3 h93.8l8** Experiments x virus diseases 6 393.62hte Error 258 65.306 Leo I 3.62 : a ‘*P '<.05 **P <.Oi By comparing means, the following results were obtained. . Healthy Viruses S and x gm Virus x Plant height (cm) 73.h ‘62.8 68.8 67.6 Any two means not underscored by the same line were significantly different. 30 Table 5-b. Analysis of variance of plant height measured on September l2. The data were grouped according to the reaction in the third and final agglutination test on September i7. Source of Variance d.f. H.s. Experiments 2 80.05l Virus diseases 3 383.562** Experiments x virus diseases 6 229.6l3** Error 258 69.099 Total 269 *P < .05 **P < .Ol By comparing means. the following results were obtained. Healthy y_i_lrus Viruses S and X Virus S Plant height (cm) 730 42.2 $4 f 33.] Any two means not underscored by the same lihe were significantly different. The measurements of September l2 showed that the height of healthy plants exceeded that of,the virus-infected plants. when grouped according to reactions of both August and September agglutination tests. The influence of virus S‘on plant height did not appear in the early part of the growing season but had a pronounced effect late in the growth cycle.. 0n the contrary virus x reduced the plant growth early in the season and this retardation remained throughout. it should be pointed out that the number of virus free plants and plants infected with viruses varied between the first and final agglutination tests. The number of plants infected with the combination of viruses 5 and X detected in the final test was increased by the appearance of virus S into virus X-infected plants or by a current season infection of both viruses S and x. The late infection apparently did not influence the plant height whereas the plants infected with both viruses at the time of the first agglutination test were affected. Ill) influence of virusxinfections upon tuber yield. a) Number of Potato Tubers The number of tubers above and below 5 cm in diameter was determined at harvesttime. The average number of sized tubers per hill for virus free and virus-infected tubers is pfsented in Table 6. The virus free plants produced the-largest total number of tubers in all cases when the plants were grouped according to their virus reactions on different test dates. There was a gradual decline in the total number of tubers between virus free and virus 5, virus S and the combination of viruses 5 and x and between the combination and virus X alone. (Table 7-a and 7-b). Virus S did not affect the number of large tubers per hill but the hills infected with virus X and the combination of viruses X and 5 produced fewer large tubers. The agglutination test of September l7 detected current season infection and showed that viruses had spread. Some healthy plants became infected either with a single virus or the combination of viruses 5 and X. Regrouped according to the findings of the third test portrays the same trends but a slightly different picture. Plants that were virus free for the first period of growth had acquired a virus and 32 ma 9. We o.» u; nd in 01.. Jné 3: _.~ _. :_ N. .3533 m.m m.~ N.» m.m ~._ w.o o.o_ m.~ _.n m.m m.~ a.“ __ one» can 5.5 :.~ 3 m.~ fim m. .N m6 . .axu m.~ n; :6 a; m. oé m.~ :. w coneoumom mé mé mé o; 5.. mi o6. 5m «.0 9m ~.~ :é : 33 van ~& 3 7m in» oé m5 m6 o.~ m6 m.» 5... NS . .9.“ “.m1 o._ _.o ~.w m._ a.» o.m o._ ouNi o.o_ w.~ muN .44 n. uwema< :5 mé .é oé m.. Tm 9m in me Tm m.~ :é __ $3 a... m m m._ o m m s a N p m n. m.~ N m o.m .o . .exu .euok __mEm , e04 .muOH __e5m cm; _mu0h __ m cm; _mu0h __m5m 0 cm; ow_m women , Lonny ocm co_umc_u:_mmm x oce m momac_> x u:c_> m m:c_> poem m:c_> mum:e_> mo puma , econome— .mouee omega :0 money co_umc_u:_mme ecu c. co_uomoc on» on mc_ocouom o0e30cm mace—e ouuoomc_um:c_> ocn oocmum:c_> 20cm nausea obeuoe mo cease: ommco>< .m u_nmh 3.3 consequently were placed in another category. Table 7-I- Analysis of variance on the total number of potato tubers per potato hill classified according to the reaction in the first agglutination test on August i7. Source of Variance A; yd.f. H.S. Experiments 2 29.58l* Virus diseases ‘ 3 55.17o** Experiments x virus diseases 6 i0.767 Error 258 6.335 Iotal _;§a *P " < .05 V “P < 00] By comparing means. the following results were obtained: Healthy Virus 5 Viruses S and X Virus x Total number of 9.6 8.8 ' 8.l 2.6 tubers Any two means not underscored by the same line were significantly different. Table 7-b. Analysis of variance on the number of large potato tubers (larger than S cm.) per potato hill classified according to the reaction in the first agglutination test on August l7. Source of Variance d.f. 53-S- Experiments .2 37.3l5** Virus diseases .3 25.0i6** Experiments x virus diseases '6 5.8k2 Error 258 3.9l6 Iotal lg? *P < .05 **P ‘<.Ol By comparing means. the following results were obtained. Tia . i3. ., 3h Healthy Virus S Viruses S and X Virus X Number of large tubers 7.l 6.5 6.i 5.7 Any two means not underscored by the same line were significantly different. There were no significant differences in the number of small potato tubers. The means of small tubers for healthy and viruses infected plants were as follows: Healthy Virus 5 Viruses S and X Virus X Number of small tubers 2.5 2.3 2.0 l.9 Table 8-a. Analysis of variance on the total number of potato tubers per potato hill classified according to the reaction in the second agglutination test on September 8. Source of Variance ' d.f. M.S. Experiments 2 33.858** Virus diseases 3 h8.6l5** Experiments x virus diseases 6 5.662 Error 258 6.832 Total 44269 *P < .05 ”P < 00] By comparing means, the following results were obtained: Healthy Virus 5 Viruses S and X Virus X Total number of tubers 9.5g gg9.3gg 8.0 _7.5 Any two means not underscored by the same line were significantly different. 35 Table 8-b. Analysis of variance on the number of large potato tubers per potato hill classified according to the reaction in the second agglutination test on September 8. Source of Variance 4_g:f. H.S. Experiments 2 h2.68l** Virus diseases 3 26.202** Experiments x virus diseases 6 2.h73 Error 258 3.76“ Total 269 *P .05 **P .01 By comparing means. the following results were obtained: Healthy Virus S Viruses S and X Virus X NUMber Of large Ala] 700 600 A597 tubers Any two means not underscored by the same line was significantly different. No significant differences were found in the number of small potato tubers. Healthy yirus S Viruseg S and X yirus X Number of small tUberS . 20“ 203 200 '08 The number of tubers obtained when the data were grouped according to the reaction in the second agglutination test show the same trend as the results in Table 7-a except the virus S infected plants which are significantly different from the plants infected with the combination of viruses S and X for both the total number and the large size tubers (Table 8-a and 8-b). The analysed data on the number of potato tubers according to the reactions in the third and final agglutination test are presented in Table 9-a and 9-b. 36 Table 9-a. Analysis of variance on the total number of potato tubers per potato hill classified according to the reactions in .the third and final agglutination test on September l7. Source of Vartgnge d.f. H.S. Experiments 2 33.06l** Virus diseases 3 37.707** Experiments x virus diseases 6 7.68l Error 258 6.700 Total eggs *P < .05 **P ‘<.Ol By comparing means. the following results were obtained: Healthy Virus S Viruses S and X Virus X Total number oftubers 9.9 8.9 8.l 7.6 Any two means not underscored by the same line were significantly different. Table 9-b. Analysis of variance on the number of large potato tubers per potato hill classified according to the reactions in the third and final agglutination test on September l7. Source of Variance d.f. H.S. Experiments 2 h2.233** Virus diseases 3 l7.887** Experiment x virus diseases 6 h.2hi Error 258 3.8l3 Total v 369 *P <.05 “P (00' By comparing means. the following results were obtained: Healthy Virus S Viruses S and X Virus X Number of large 7.0 ‘ 6.6 ‘fifiy 6.0 75.9 tubers Any two means not underscored by the same line were significantly different. 37 No significant differences were found in the number of small potato tubers. Healthy Virus S Viruses S and x Virus X Number of small 2.h 2.3 2.l l.6 tubers Results similar to Table 7-a are shown in Table 9-a except that the virus S-infected plants were not significantly different from the other viruses-infected plants for the number of large tubers (Table 9-b). Results of statistical analysis would indicate that virus S did not affect the number of tubers set during the growing season. Since there is evidence of very little increase in the number of tubers set after the blossom stage (l2), most small potatoes are of the same chronological age as the large ones. The effect of virus S did not appear until the blossom stage of the potato plants. This was also true in plant height where the effect of virus S did not appear until August l0. However. the height of virus S-infected plants was less than that of the virus free plants in the final measurement of September l2. There was a great decrease in the total number of. potato tubers due to virus X infection of the plants when compared to virus free plants and virus S-lnfected plants. It would indicate that the effect of virus X appeared in the earlier growing stage. The plants infected with the combination of viruses S and X set a lesser number of tubers than the virus free plants. However, there was 38 no difference between the combination of viruses S and X. and virus X-infected plants. it would indicate that the combination of viruses S with X did not give rise to a new complex disease and their effect on the number of tuber set is somewhat between the effect of virus S and virus X. This is in agreement with previous works (22. 36). The percentage reduction in the number of tubers set. due to infection with specific viruses is summarized in Figure 3. Percentage reduction in the total number of tubers of virus X- infected plants and of the plants infected with the combination of viruses 5 and X was fairly constant. 20% and l5% respectively. On the other hand. the number of tubers from virus S-infected plants depended upon the groupings of the reactions in the different agglutination tests; 8.3% in the first. 2.l% in the second and 5.3% in the third groups respectively. This variation resulted from the changing of a plant from one classification to another. Alterna- tively. there might be more variation within virus S-infected plants since fewer plants were available in this category. The trends of percentage reduction in the number of large tubers were very similar to the number of total tubers. Although there were no significant differences among means of the small tubers of the plants infected with various viruses. there was a difference among them when expressed as a percent of the healthy plants. There was a little. about 5.5%. reduction in the number of 39 small tubers of virus S-infected plants when compared to virus free plants. But a great reduction in the number of small tubers occurred in virus X and the combination of viruses S and x-infected plants: l7.fl% and 27.“% respectively. Although Brust (36) stated that the average reduction in the size 50 mm and larger was between is or 20% in’the variety Bintje. he also observed very great differences between plant families. For the variety Bintje. it has been shown that the virus S-infected families produced a higher percentage of small tubers. ~There is a great variation among the different varieties since very great differences in the tuber size per family have been observed in previous works (36). in this experiment. virus S-infected plants produced more tubers both large and small size than the other virus-infected plants.‘ There was no statistical difference between the number of tubers from virus S-infected plants and the virus free plants. b) Height of Potato Tubers Data of the tuber weight were grouped and analyzed by the pro- cedures applied to tuber number. Means of the tuber weight from individual experiments are.pre- sented in Table l0. In Table ll-a total weights of potato tubers for the healthy and virus S-infected plants were highly significantly different from the weights of both virus X and the combination of viruses S and X-infected plants. The results of large size tubers were similar to the total D Healthy - Virus x l'ir‘l; b [:3 Coufiin.tiuu of Viruses S and i 100 - \‘ 5;, 90 _ V g s S s as: \ \ ES 70 r 3 § § a 2 § § § :8 a? 60 - § h b By \ h h h .\ .\ August 17 September 8 Seotember 17 100 - V t 5E. -: R .- 5 8 3: \ ‘1' 05;, 7. - 3. s 2;: ”5; '2 \ L. . \ ._. 3 60 ' .1 § 3 3' \ '1‘ . N. August 17 September 8 September 17 Figure 3. Number of tubers from virus infected notato hills expressed as percent of the weight of tubers from healthy hills. iii 3K 93 He. eéwu . a... .N 0.. 2. N. .3838 aim, ~..-.. 93 333 mém 08.... HQ 3.3.. .. :3 En «.me 3.8.. 3.. «£3 98 93.... ~65 mémm; .ém... was imam; .13.. 1... H.211. hem 98.... :4 33.82% {R 3:... in» 93... .éu. 9%... was .63.. .. :3 e5 o~.~e :.m.o.. . n.mm m.m.m ..mm m.~.:.. .~.om m.m-.. . .axu . 1mm 72... 3.0 3.8 .68 93. 1mm... .2 t 3% Q8 «.me .18 1.3... 92. 3.2.. Tom 9...... ._ 33 an. ~.ne ..m.o.. ..sm m..mm o.m... oo..~.. ..m. m.am~.. _ .axm 22$ :25 .2... :25. o... :3 x one m wom:._> x ma._> m a:..> willuuuulwaqqa. co.oee.u:_mm< AmEmem c. oommoeexov . .moueo one» open» :0 mco.uumu. .eo.mo_ocom ogu xn panacea m__.: cannon oouoome. ma..> pee xsu_eo: seem m.p¢:u.0ueuoe __eEm ecu omen. mo usm.oz omeco>< .o— o_nmh #2 weight of potato tubers. Analysis of variance is presented in Table il-b. The comparison of the means are presented. Table ll-a. Analysis of variance of the total weight of potato tubers grouped according to the reaction in the first agglutination test on August l7. Source of Variance