ERET-ANCE OP RESlSTANCE lNH NTERNAL BROWNING MOSMC V‘RUS INDUCED \ TO TfifiACCO 1N TOMATOES Thesis for the gag!» :25 Ph. D. MKCHEGAN STATE UNIVERSH‘Y Michaafi fiahn Phimp 73.964» THESE; This is to certify that the thesis entitled The Inheritance of Resistance to Tobacco Mosaic Virus Induced Internal Browning in Tomatoes presented bg Michael John Phillip has been accepted towards fulfillment of the requirements for the Ph.D. degree in HortiCU1ture Major professor Date JUne 8, 1964 LIBRARY Michigan State University ABSTRACT INHERITANCE or RESISTAnCE TO TUJACCU mOSAIC VIRUS INDUCED IETERKAL Basswtna IN TOMATOES by Michael John Phillip Internal browning is a disease of tomato fruit caused by late infection of the plant with Tobacco mosaic Virus (TNV). Studies of the inheritance of resistance to this disorder and to the causal factor TMV, the effects of the virus syndrome va and Iotato Virus X (PVX), and virus build up in tomato plants infected with TMV, contributed the scope of the present study. A hybridization program with Fireball as the susceptible parent, and Plant Introuuction 23567} as the resistant parent, was initiated in the spring of 1962. Internal browning was induced in the various populations using the methods of Boyle and hharton (7); and seedlings were inoculated with TMV to induce mosaic. Ratios of resistant to susceptible plants were obtained from the segregat- ing populations. Seedlings were also inoculated with TMV and PVX to study the effect of the virus syndrome on (a) induction of internal browning (b) days to flowering (c) days to first fruit set (d) weight of vines (e) number of marketable fruits produced. The local lesion method of Holmes (24) was used for the virus build up in susceptible plants. P.I. 235673 was resistant to internal browning, but a few plants showed symptoms of mosaic disease with age. Although Fireball was 100 per cent susceptible to THV, penetrance was not complete in this variety for the genes controlling the expression of internal browning. The data suggest that internal browning is controlled by two genes showing complementary recessive epistasis, with levels of penetrance governed by the genotype of the plant. Resistance to- TMV appeared to be polygenic. Seedling inoculations with TMV and PVX (l) appeared to immunize susceptible plants against internal browning (2) had no effect on days to first bloom and days to first fruit set (3) re- duced the weight of vines and number of marketable fruit produced by plants of all the populations. Virus assay of susceptible plants suggests that in P.I. 235673 and resistant offspring, TMV build up is slow. This delays symptom expression. In susceptible plants rapid virus multiplication appears to be correlated with the early appearance of mosaic symptoms. Approved by Advisor IhthITAHCE OF RESISTANCE To TubACCU “OSAIC VIflJS INDUCLD ILTJKLAL bRoLKING IE TOMATOES By Michael John Phillip A THESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree DUJTOR OF PhlLUSUEHY Department of Horticulture 1964 ACMJU‘». LEDGfiubl‘lT S The author wishes to express his sincere appreciation to Dr. S. Honma for suggestion of the problem and for his advice and criticism during the preparation of this thesis. Grateful acknowledgement is tendered to Dr. H. Murakishi, Dr. D. Markarian, Dr. E. Everson and Dr. S. hittwer for suggestions during the investigation and for criticism in the preparation of the manuscript. In addition the author wishes to convey thanks to Dr. h. h. Adams for his advice and suggestions in analyzing and interpreting the data. TABLE OF CONTENTS Page IntrOductj-on...0.00000...O...00.0.0000...OOOOOOOOOOOOOO l ReView Of LiteratureOOOO0..0.0..OOOOOOOOOOOOOOOOOOOOOOO 7 Materials and hethods.................................. 14 Results and Interpretation............................. 23 Internal Browning................................. 23 Effect of T.A.V.+P.V.X. Syndrome on Tomato Plants. 32 T.M.V. Tests...................................... 46 Virus Assay of Susceptible tlants................. 50 DiscuSSionOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 58 Summary and Conclusion................................. 61 Literature CitedOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.00.00.00 62 I N T R O D U C T I O N The cultivated tomato Lyc0persicon esculentum Mill,iis susceptible to numerous diseases, among which are those caused by viruses. Three of these viral diseases which are conspicuously damaging and have assumed major importance are: Internal Browning, Tobacco mosaic Disease, and Streak or Stripe. Internal browning, a disease of the fruit was first reported in 1946 (22) (12) (69). It usually appears during the third or fourth picking and causes serious losses. Wharton and Boyle (67) and Conover (12) described this disorder as a browning which is usually confined to the outer pericarp. In severe cases the browning extends into the septa and central column. The discolorat- ion usually radiates from the stem end of the fruit and is often accompanied by a collapse of the large thin-walled parenchyma cells. External symptoms of fruit affected with internal browning vary with the stage of maturity. The disease is not usually evident in immature fruit unless they are cut open. Symptoms appear in ripe fruit as large brown to brownish grey areas which radiate from the sten end and are most prominent near the shoulder. (Fig. 1). Contrasted to this disease and similar in appearance is Greywall. This disease affects the vascular region of the fruit causing it to become necrotic and upon sectioning of the affected regions, brown or brownish grey streaks can be noted. The damage is usually confined to the outer walls and is seldom found in the central column as in the case of internal browning (Fig. 2). Greywall can appear anywhere on the fruit, in isolated patches, and the affected areas may vary in size from a single spot, to the entire surface of the tomato. Whereas, internal browning has been shown to be the result of late infection of the plant by Tobacco hosaic Virus (TMV), with the subsequent invasion and accumulation of the virus in the fruit (67), Greywall on the other hand seems to Figure 1. Fruits showing internal browning at different stages of maturity compared with normal fruits. A nature ripe uncut fruits showing internal browning. B Immature green cut fruit showing internal browning. Mature green cut fruit showing internal browning. U mature ripe cut fruit showing internal browning. E Normal immature green cut fruit F hormal mature green cut fruit G Normal mature ripe cut fruit Figure 1 Figure 2. -4- Comparison between fruits showing internal browning and Greywall. A & C Mature fruits showing ureywall. B, Mature fruit showing internal browning. Figure 2 -6- be independent of ThV infection, and caused by environmental factors (37). Tobacco Mosaic is probably the most destructive disease affecting tomatoes throughout the world. Several strains of tobacco virus have been reported. All cause mosaic symptoms on the foliage. These symptoms vary from a flecking or minute scattered chlorotic spots, to chlorotic patches and severe mottling. Severely infected plants grow abnormally and the yields are greatly reduced. The Streak Disease of tomatoes, which is caused by the synergistic effect of TMV and PVX (18), also induces serious losses. This disease is marked by the presence of necrotic lesions on the stem, petioles, leaves and fruits. The lesions on the stem form dark longitudinal streaks extending through the cortex and into the pith beneath. The leaves also show necrotic spots which enlarge and blight the foliage. Immature fruits may develop irregularly, and have sunken, necrotic blotches. Virus diseases are usually devastating once the tomato fields become infected. Resistant varieties offer the best practical means of control. A study of the mode of inheritance of resistance to internal browning in the tomato along with factors which appeared to be related to the disease was the objective of this investigation. These included: (a) Induction of internal browning in plants affected with TuV and PVX in the seedling stage (b) The use of the Streak syndrome to evaluate the performance of populations of similar genetic constitutions (0) Further study on the Inheritance of Resistance to TmV disease (d) Viral assays to determine if any relationship exist between virus build up and symptom expression of plants susceptible to TmV. REVIEW OF LI‘SRATURE Internal Browning The disease was first recorded in Dade County Florida in 1927, and has been seen annually in that county since 1937 (12) but not until 1949 were large losses suffered by growers. In 1946 Young (69) reported a similar disease in Texas and suggested that the browning could be due to the same causal agent as that of blossom-end rot. The disease was reported in naryland in 1946 by Cox and Weaver (1?). naenseler_(22) described a similar disorder in hew Jersey, and characterized it as an unexplained ripening disorder of tomatoes which occurred sporadically in tomato producing areas in the Eastern U.S.A. In 1949 Friedman (21) reported that fruits with symptoms identical to those described by Cox and Weaver (1?) and haenseler (22) were Obtained from crops grown in Pennsylvania, hew York, Florida and hew Jersey. Initially there was no explanation for the cause of the disease. Conover (12) reported that repeated microscopic examinations and attempts at isolation failed to reveal the presence of any organisms, and suggested that the cause was either physiological or viral in nature. In 1949 Holmes (29) deduced that the disease was associated with strains of Tobacco Mosaic Virus which was isolated from a species of Plantago. However, the findings were inconclusive since the disease was not reproducable. numerous ideas had been advanced to explain internal browning and similarly appearing disorders. Rank vine growth, deep shade, high humidity, high soil moisture levels, soil compaction, excessive levels of fertility and nutritional imbalances were a few of the factors that were suggested (37) (23). Field observations however, did not consistently support these hypotheses. In 1957 Doyle and hharton (7) isolated strains of TmV from internally browned fruit and from the foliage of affected plants. Attempts to reproduce the disease by inoculating young tomato plants with these isolates of the virus were unsuccessful. when however, large tomato plants were inoculated at the time the first fruits were beginning to ripen, up to 93 per cent of the plants produced internally browned fruit. These workers attributed the cause of internal browning to a "shock” reaction as a result of virus invasion of the fruit followed by a hypersensitive res- ponse of the host. Streak or Stripe of Tomatoes A Syndrome Caused by Tobacco mosaic Virus and }otato Virus X howitt and Stone (53) observed this disease in entario in 1915. Jackson (55) then reported/ffiafgfge streak disease could be induced in plants after inoculating them with the juice extracts from diseased plants. In 1925 Dickson (18) found that the disease could be caused by inoculating tomato plants with a mixture of TMV and EVX. Vanterpool (63), nacNeil and Isuen (40) and murakishi and honma (45) have all agreed generally with Dickson (lo). kurakishi and Honma (45) deveIOped an inoculation procedure using both TMV and PVX as an aid in evaluating resistance to TMV. In their early studies, a large number of TMV inoculated seedlings of known susceptible varieties failed to show symptoms of the disease three weeks after inoculation. This meant that susceptible plants appeared constantly as the plants grew older and thus required a large pOpulation for the studies. however, when the double virus method of inoculating was used, they obtained 100 per cent necrotic streak and stunt followed by death of susceptible seedlings, as compared to 65 to 66 per cent systemic symptoms when TMV alone was used. By this method a large population could be handled in the seedling stage and thus the probability for the recovery of resistant segregates was increased. ho inheritance study on this syndrome has been found. Tobacco Mosaic Virus Tobacco Mosaic Virus (harmor tabaci Holmes) is prevalent where ever tomatoes are grown. It causes greater losses in tomatoes than is generally realized and the use of resistant varieties seems to be the only practicable means of control. most commercial varieties of tomatoes are susceptible to infection by TMV. Sources of resistance however, to TMV have been found in the tomato species. Doolittle, et a1 (19) reported a high degree of resistance in the species E. hirsutum, and Alexander and hoover (3) found resistance approaching immunity in segregates from hybridizing domestic tomatoes with the L. peruvianum species. Holmes (25) has reported that plants derived from crosses of E. esculentum with L. chilense tend to escape natural infection with TMV. He also reported that plants of g. pimpinellifolium were tolerant to infection. Frazier and Bennett (20) reported that in crosses involving L. esculentum, E. hirsutum, E. peruvianum and L. pimpinellifolium certain lines showed a delay in the development of mosaic symptom. They suggested that there may be more than one gene or gene modifier, or both involved, but it was difficult to determine whether resistance to infection, slow multiplication, or masking, delayed the movement of the virus in the plant. Literature on the inheritance of resistance to TMV is limited. Holmes (31) working with the species £° chilense found that resistance was associated with a single dominant gene. Watson, heinrich and Harvey (66) studied hybrids between £° esculentum and £° hirsutum and reported that two dominant genes were affecting disease response. All plants in their experiments contained virus after repeated inoculations, but plants with severe symptoms had more virus than those with masked symptoms. halter (64) studied the F2 segregating generation of a three way cross, (Rutgers x L. hirsutum) x Indian Baltimore and concluded that nonsymptomatic tolerance derived from E. hirsutum depended on the presence of three recessive pairs of genes. Soost (58) in 1963 obtained -10- inherited resistance to TMV from a complex hybrid of L. esculentum, E. hirsutum, g. peruvianum and L. pimpinellifolium. He used a virescent recessive gene as a marker and concluded that resistance was determined by a single dominant gene which was linked to the recessive virescent. Soost (55) also reported that since the resistance was derived from the same source used by Frazier and Bennett (20) the gene or genes involved could be the same. holmes (27) studied the inheritance of resistance to va in tobacco and he reported that the ability to produce primary necrotic lesions was due to a single dominant gene, and the ability to produce systemic chlorosis was from a recessive allele. holla (46) found that in tobacco, resistance to TmV was controlled by double recessive genes. It is evident that these workers have been incorporating in their breeding programs, resistance to ThV from diverse sources. It is suggested therefore that the nature of the resistance and the mode of inheritance will vary depending on the source of the resistance and the source of the virus. Although various workers (51) (6o) (ok) (58) have reported on the inheritance of resistance to TAV, no correlation has been made between the genetics of TmV resistance and internal browning. Resistance to TmV has also been studied in pepper Capsicum frutescens. nolmes (26) reported four types of responses to the distorting strain of PMV and attributed these responses to a series of allelomorphic genes. Cook (14) found a resistant plant of Capsicum annuum which remained symptomless after repeated inoculations with TmV. He crossed this plant with varieties homozygous for each of three members of the multiple allelic series which control response in pepper to TmV infection and he concluded that native resistance in g. annuum was associated with a homozygous dominant gene. -11- Resistance to Potato Virus X Three well defined types of resistance to PVX have been identified. The first type is displayed by plants which carry the virus with symptoms ranging from complete masking to a strong mottling. This may be accompanied by necrosis depending on the strains of virus X. The second type is a field immunity or hypersensitivity with plants responding to graft inoculations by well developed tOp necrosis. The third type is an immunity where the virus fails to survive in infected plants. Cockernam (9) determined field immunity by means of the top necrotic reaction, and suggested that resistance was associated with extreme susceptibility or hypersensitivity to the virus. Cadman (8) snowed that hypersensitivity to EVX is deter— mined by a single dominant gene inherited in a manner typical of an autotetraploid. Cockerham (10) (ll) also reported the occurrence of dominant genes which condition field immunity to the B strain of PVX. Potato seedlings showing immunity to PVX were first described by Schultz and Raleigh (54). Stevenson, Schultz and Clark (60) later investigated the inheritance of immunity and postulated tetrasomic inheritance characteristic of autotetraploids. They concluded that immunity was based upon the presence of both a dominant gene A and a dominangene 5, and that immune types were apparently of the genotype AAaaBBbb whereas, the susceptible genotype was aaaabbbb. Hutton and wark (34) suggested that the recessive allelomorph of a gene in the nulliplex condition resulted in immunity, whereas, the dominant gene in the quadruplex condition gave complete suscept- ibility. The simplex and duplex conditions could have given pheno- types with localized reactions. Thompson and Hooker (62) suggested that immunity to PVX is conditioned by two complementary genes and in the varieties Tawa and Saco which they studied the genes were quadruplex or triplex for one gene and simplex for the other. -12- Quantitative Assay of Plant Viruses Smith (57) reported that there are two reliable methods for obtaining quantitative assays of plant viruses. bne is the sero- logical method in which the precipitin test is used. The second method is the local lesion method which was develOped by Holmes (24) for measuring the biological activity of TnV. Spencer and Price (59) have reported that this method has been employed in various types of experiments and it has been adapted for use with other plant viruses. The number of lesions which develop on Licotiana glutinosa has been shown by Holmes (24) to be largely determined by the concentration of the virus in the inoculum. This local lesion tecnnique has become the basis of a method for estimating relative virus concentration. Samuel and Bald (52), Youden and Beale (66) and best a.d Bald (53) have all shown that age and position of leaves influence the number of necrotic lesions that develop. however, the influence of these factors have been largely overcome by employing appropriate experimental designs of the test plants and by the use of statistical analyses in evaluating the data obtained. Samuel and Bald (52) suggested the comparison of virus pre- parations on opposite half leaves of the same leaf, to eliminate the variation due to differences in the reaction of individual leaves and plants. Statistically analysing their results by Students "T" method, these workers found the half-leaf method to be reliable. Similar analyses by Beale (6) and Loring (39) confirmed their results. fouden and neale (68) employed variance analysis in the treatment of their data and found equal reliability in this method. deale (6) showed that the half-leaf technique was reliable for distinguishing between extracts wnich differed by 50 per cent, wnile Loring (39) reported that differences in virus protein concentration of 10 per cent or greater could be detected by the half-leaf method on Phaseolus vulgaris. Loring (39) however, pointed out that the use of g. glutinosa as a test plant was not reliable for distinguish- ing between extracts that differed by less than 20 per cent in -13- concentration. Spencer and Price (59) have pointed out that, although con- siderable work has been done on the isolation and purification of TMV protein, chemical determinations of virus-protein are not reliable as a measure of absolute amounts of active virus. They (59) demonstrated that chemical, physical, and serological tests which have been applied to the virus protein lack sufficient sensitivity to distinguish between an active and an inactive virus. Lith the local lesion technique, however, only active virus particles will produce lesions. MATERIALS AND mmfhpub hybridization Lrocedure In the spring of lao2 a tomato hybridization prOgram was started at the michigan Agricultural Experimental station, to study the mode of inheritance of resistance to internal browning and TMV, and the effect of the Thv and PvX syndrome on tomato populations of different genetic constitutions. The general plan was to make reciprocal crosses in the field during the summer, and grow the F 8 during the fall of lace and the winter of 1963. 1 These Fl hybrids were to be back crossed to both parents to produce seed for the back-cross populations, and to be selfed for the F2 populations. Plant Introduction 235673 was chosen as the resistant parent since it possesses the gene or genes for resistance to TMV in a homozygous condition, and has also escaped repeated outbreaks of internal browning (Holmes (32)). P.I. 235673 was selected by holmes (32) from another Plant Introduction line. It is indeter- minate and late in maturity. The vines assume fairly large preportions under suitable conditions, the foliage is dark green and the fruit are medium in size. The variety Fireball was chosen as the susceptible parent, because of its susceptibility to infection of TMV. It shows chlorosis and mottling two weeks after inoculation. The variety has never been known to escape internal browning if the disease occurs in the area where it is grown. Fireball is determinate, early maturing, medium to small in plant size. The foliage is light green, and fruits are medium to large in size. Original seeds of Fireball and }.I. 235673 were Obtained from single naturally selfed plants maintained for several generations at the nichigan Agricultural Experimental Station. For the initial hybridization, seeds of both varieties were sown in vermiculite and were transplanted into four-inch peat pots filled with soil. Prior to moving to the field, the plants were transferred to a coldframe -15- T1 for hardening. might plants each of Fireball and r.1. 235673, selected at random, were planted in the field, spaced five feet between plants in rows five feet apart. Reciprocal crosses were made between one plant of 1.1. 235673 and one Fireball. Jrosses were made as frequently as possible and were labelled, Flowers were also allowed to self naturally to obtain seeds for use in determining parental reactions to the virus. Seeds from the crosses and selfs were processed and sown in vermiculite in the late summer of 1962 for the production of F2 and back-cross progenies. All plants were transplanted into four- inch peat pots two weeks after sowing and were later transferred to the ground bed in the green house. Eight plants of each reciprocal cross and each parent were spaced two feet in the rows and rows were three feet apart. The plants were given 12 hours of light by supplementing the natural day light with four hours of artificial light. They were trellised and pruned periodically. As the plants flowered, the F s were crossed with both parental plants to l obtain the back-crosses. Flowers for these crosses were randomly selected, and seed obtained from each specific back-cross was bulked at harvest. To obtain seed for the F, generation, blossoms were a selfed by gently tapping the flowers at time of dehiscence. Inoculation Technique The Tobacco Mosaic Virus used in this study was provided by Dr. h. H. murakishi of the michigan Agricultural Experimental Station. This virus which was increased and maintained in Turkish tobacco for a number of years was originally Obtained from Dr. J. S. Doyle of the iennsylvania Agricultural Bx}erimental Station, who isolated it from an internally browned tomato fruit. The inoculum was pre- pared as described by Smith (57). A 1:10 dilution of crude juice was used for all inoculations. where a double inoculum was needed equal volumes of a 1:10 TmV solution and a 1:10 PVX solution were mixed prior to inoculation. To induce internal browning and obtain mosaic symptoms on susceptible plants three methods of inoculation -16- were used. The following technique was used for induction of internal browning. Prepared/ihggalum with Carborundum added was applied to the plants with foam pads. The pads were rubbed briskly on the young parts of the plants and above and below the first fruit cluster. Inoculated plants were labeled with the date of inoculation. Since the material was segregating for maturity four different inoculations were made on July 19, July 24, August 1 and August 13. The following method was employed for studies on direct seeded plants since the methods described by Smith (57) and other workers (7) (42) were unsatisfactory and laborious where working with small plants. A small atomizer operated by compressed air was used. Carborundum was mixed with the prepared inoculum and the mixture was applied to the leaves of the plant at a pressure of 50 p.s.i. Each plant was held and sprayed individually until a water soaked mark appeared on the underside of the young leaves. The third method of inoculation was employed on seedlings grown in the green house. This technique was a modification of a method described by Murakishi (43). Wooden holders with small rectangular foam rubber swabs at the ends were used, in applying the inoculum. These swabs were dipped into the inoculum and rubbed briskly onto the stem and leaves of the young plants. Internal Browning Test Seeds of Fl's, Fa's, back-crosses and parents were sown on April 26, 1963 in vermiculite. Three weeks later the seedlings were transplanted into flats, at a spacing of one and one-half inches apart. The flats were removed to a coldframe on may 30, 1963 and the plants were transplanted to the field on June 10, 1963. Seedling samples of each progeny was taken by using plants as they occurred in the seedling row. Planting was on land where Solanaceous plants had not been grown for two years. The design was a randomized complete block with six replications. Plants were spaced four feet in rows four feet apart. Each replication -17- consisted of : twenty plants each of the Fl'S, back-cross and parents and 40 F2 plants. Thus a complete stand comprised of 1440 plants of the following populations: two Fl's (including reciprocal) 120 plants each, four back-crosses 120 plants each, two parents 120 plants each and two Fa's (including reciprocal) 240 plants each. The final number of plants did not always reflect the initial number due to losses during the growing season. Precaution was taken to prevent viral infection of the plants prior to inoculation. when the fruit on the first cluster turned pink each plant was inoculated with the tobacco mosaic virus. Records were taken on days to flowering and days to first fruit set, and number of days for the first fruit to ripen. Data on individual mature plant reaction to the virus were recorded when internal browning symptoms were noted on the Fireball variety. All plants were harvested when internal browning was noted on Fireball. height of vines and the number of marketable fruit were also recorded. A random sample of 50 fruit (where possible) was cut to check for internal browning and data on the number of diseased fruit recorded. The families of the reciprocal crosses were tested for heterogeneity using the method of kanse and Sukhatme (47), prior to pooling the data for analysis. /TE% Eggdeoh°the segregation of susceptible and resistant plants were statistically interpreted by means of Chi square analyses. -18- Table 1. Chi square test for heterogeneity among the reciprocal populations of F's and back- -crosses from a cross between Plant In%roduc%ion 235673 and Fireball. NO. Of n0. Of Populations Res. Pl. Sus. kl. Chi so. P (Pl x P2) Fl 98 13 (:2 x Pl) r1 95 lo .31 .3o-.50 (Pl x P2) F2 166 58 (P: x P2) Fl x P1 93 2; (r2 x :1) El x L1 65 26 1.35 .O5-.3O (Pl x P2) Fl x P2 106 9 (P2 x Pl) Fl x P2 100 13 .65 .30-.5O -19- EFFECT OF TOBACCO “panic VIRUS Ann kLTATU VIRUS X UN TQMATU kLANTS Internal drowning Seedlings of reciprocal F 's, back—crosses and parents 1'5’ F2 were grown as described above. At the first true leaf stage, the plants were inoculated with TMV and PVX using the method described by hurakishi and honma (45). Plants showing severe necrotic streak and stunt were periodically discarded. healthy plants were planted in the field, in a randomized complete block, with three replications. The experiment with a complete stand had 360 plants comprised of the following pOpulations: two Fl's (including reciprocal) 30 plants each; four back-crosses 30 plants each; two parents 30 plants each; and two Fa's (including reciprocal) 60 plants each. when the fruit on the first cluster turned pink each plant was inoculated with TMV as performed in the previous experiment. Records were taken on the number of days to flowering and days to fruit set, days to maturity of fruit on the first cluster, weight of vines, number of marketable fruit, and number of internally browned fruit per plant, from a random sample of 50 fruit (where possible). Data on plant performance in this and in the previous experiment were statistically interpreted by means of analyses of variance as a split plot arrangement. "T" tests were computed to compare the performance of populations of similar genetic constitu— tions between treatments. Tobacco Mosaic Virus Tests This study was divided into two phases. The initial phase was to study the inheritance of resistance to 25V. The second phase was to obtain information on the virus build up in suscept- ible plants of the various populations when symptoms were detected. Progeny obtained from the hybridization prOgram together with their parents were field seeded in twenty foot rows. As the seed- lings emerged they were thinned to obtain the following populations: P.£. 235673, 48 plants; Fireball, 82 plants; reciprocal Fl'S. 105 -20- plants; reciprocal F 's, 225 plants; reciprocal back-crosses to 2 Fireball, 65 plants; and reciprocal back-crosses to }.I. 235673, 76 plants. Plants were inoculated at the first true leaf stage with virus using the atomizer. Data on the appearance of symptoms were recorded at seven day intervals. Plants classified as susceptible were removed after leaf samples were Obtained. These samples provided material for the second phase of study, the determination of virus build up in susceptible plants. Virus Assay Technique leaf samples from THU infected plants were placed in a plastic bag and frozen for virus assay. Virus build up in the plant samples collected was determined by the necrotic lesion technique as devised by holmes (24). The method suggested by Samuel, Best and fiald (53) was employed since it was reported by Bawden (5) as a convenient way to compare different virus pre- parations. The method requires the inoculating with a standard preparation on one-half of every tobacco leaf of the test plant and inoculating the other half with the unknown so that each unknown could be compared directly with the standard and indirectly through the standard with any unknown sample. Nicotiana tabacum var. Xanthi n c, a hybrid between E. tobacum x E. glutinosa, was selected as the test plant due to its special advantages of reduced genetic variability, better leaf surface, size and quality than fl. glutinosa. The test plants were grown in four- inch clay pots in a uniform mixture of sterilized soil and in an 80°F temperature room. Plants with 12 - 15 large leaves, were selected for uniformity and the growing points removed prior to assay. As reported by Holmes (24) the number of lesions on the test plant varied with the virus content of the inoculum. leaves inoculated with undiluted sap produced hundreds of lesions while leaves inoculated with a 1:1,000 dilution produced only a few, thus -21- allowing for comparative estimates of virus concentration. Pre- liminary trials, similar to those done by Spencer and Price (59) were made to obtain a workable virus concentration. The various preparations, both from tobacco and tomato leaf samples were tested at different dilutions ranging from the crude juice to l:l0,000. A dilution of 1:100 was most suitable. Variance analysis (Table 2) between and within samples suggests that this dilution gave consistent results between leaves. For the assay, ten gram samples of frozen tomato leaves and leaves of the check (Turkish tobacco) were used. Inoculum pre- parations as described by Smith (57) were diluted 100 fold. These inocula were stored in phials and frozen until needed. Every precaution was taken to prevent errors in handling the inocula from the tomato samples and during the inoculating on the tobacco plants. Prior to inoculation the tobacco plants were uniformly dusted with Carborundum and each leaf was labeled with the sample number. Two drOps of the sample inoculum were then placed on the right half of each leaf and was spread evenly with a glass spatula. To the other half of the leaf two drops of the standard inoculum were placed and spread using another spatula. Each tomato sample was inoculated on randomly selected leaves and replicated ten times. necrotic lesions were counted 72 hours after inoculation using an electric counter. The data were recorded as a percentage of the lesions caused by the unknown sample over the control. -22- Analysis variance of necrotic lesions between and Table 2. within tobacco leaves sampled with a 1:100 dilution of extract from susceptible tomato plants. D.F S.S M.S F F Exgt. Between 9 1,666 185 .45 2.80 within 50 20,342 407 Total 59 22,008 RESULTS AhD INTERPRETATION Internal drowning A random sample of 50 fruits was cut from each plant to observe for internal browning. Plants were classified as suscept- ible when one or more internally browned fruit was found. Symptom expression of the diseased fruits varied with the stage of fruit maturity. Fig. 3 shows a random sample of leaf and internally browned fruit symptoms from individual plants. These samples suggest that there is no consistent leaf symptom pattern associated with the disease. These observations agree with those made by haenseler (22). however, Boyle and Wharton (7) reported that a definite leaf symptom pattern was associated with internal browning. Differences in leaf symptom expression commonly associated with internal browning were noted. Some plants which appeared free of mosaic symptoms showed internally browned fruit, while some plants with severe leaf mottling did not have any infected fruit. This Observation suggests that there was no relationship between leaf symptom and internal browning. Perhaps some of the plants were mildly infected prior to inoculation since the virus is easily transmitted and the tomato is susceptible to it. This may have been responsible for the appearance of mosaic symptoms without internal browning. Table 3 shows the results from the various populations studied prior to and after pooling the data as was suggested by the heterogeneity test in Taole l. The data suggest no cytoplasmic effect on the inheritance of this character. Approximately 50 per cent of the Fireball parent was found to be susceptible. nurakishi (unpublished) obtained up to 90 per cent susceptibility in previous experiments, suggesting that in Fireball penetrance of the genes controlling the expression of this character is variable. no }.I. 235673 plants were found with internally browned fruit. In the F1 generation 87 per cent of the plants were resistant to the disease and 13 per cent susceptible. in the F2's 71.5 per -24- Figure 3. Random samples of internally browned fruits and leaf samples from the same infected plants. A Mature fruit at the pink stage. 8 Immature green fruit. C mature green fruit. D hature ripe fruit. Flame 3 Table 3. Classification of plants for resistance to internal browning in parents, reciprocal F 's, F,'s, and back-crosses from the cross between rlant Introduction 235673 and Fireball. Total no. LO. of res. Lo. of sus. Generations of_plants _p1ants #plants Fireball (Pl) 106 51 55 1.1. 235673 (P2) 115 115 0 (r1 x P2) r1 111 98 13 / - F . " ' (l2 x Pl) 1 114 98 lo Pooled ratio 87:13 (hi x P2) r2 234 100 68 (P2 x r1) r2 251 166 05 fooled ratio 71.5:26.5 (Pl x P2) 11 x :1 116 95 a} 1 P _ fl 1 (r2 x l)Fl x r1 111 05 26 tooled ratio 78.5:21.6 (e1 x P2) 11 x £2 115 100 9 (P2 x Ll) Fl x P2 113 100 13 Pooled ratio 90.3519-65 -27- cent were resistant and 28.5 per cent were susceptible. With the back-cross to the resistant parent 90.35 per cent were resistant and 9.65 per cent susceptible and with the back-cross to the susceptible 76.5 per cent were resistant and 21.6 per cent suscept- ible. Since the ratios of the F2 and back-cross populations did not fit the classical genetical models it appeared that penetrance level varied with the genotypes of these generations. The hypothesis that the parents were differentiated by two recessive gene pairs operating in an epistatic manner and showing different levels of penetrance depending on the genotype of the plant is proposed. In £.I. 235673 these genes are symbolized as AABB and in the Fireball aaob, with the genes being in a complementary recessive epistatic state. The levels of penetrance were determined as suggested by Allard (2) from the observed percentage of resistant and suscept- ible plants of the parents and the F1 generation. P.l. 235673 with a genetic constitution of Ahab was assigned 100 per cent since it was resistant to internal browning. llants having the genotype of the F1 AaBb or carrying the genes in a heterozygous condition at either the a or b locus were assigned a penetrance level of 87 per cent. llants having the genotypes of aah— or A—bb were assigned a penetrance level of 50 per cent. Table # presents the theoretical expected ratios for resistance and susceptibility to the disease when the above hypothesis of penetrance is considered. Genetic analysis of the data presented in Table 5 suggests 's, F 's and the back-crosses to the resistant 1 2 parent fit the hypothesis. however, in the back-cross to Fireball that the parents, F the probability of the Chi square value is less than .01 per cent. This was the only family to deviate significantly from expectation. The deviation was towards an excess of dominant phenotypes and is probably attributed to the failure of a portion of the genotypically susceptible plants to develop the disease. As reported by Allard (1), penetrance is determined by the genotype of the plant and the manner of expression is related to the genotype. This back-cross contained 75 per cent of Fireball's -23- Table 4. Theoretical genotypic and phenotypic ratios of different populations derived from hybridizing Plant introduction 255o75 x Fireball based on the hypothesis that flant Introduction 255o75 and Fireball were differentiated by two major gene pairs showing comple- mentary recessive epistasis for internal browning, with different levels of penetrance. Fireball (Pl) F P.I. 255673 (P2) aabb Aabb AAdB Back-crosses to £1 res. sus. % AaBb resistant with 57 per cent penetrance 21.75 5.25 K-Aabb susceptible with 50 per cent " 12.50 12.50 %.aaBb " " 50 “ " " 12.50 12.50 % aabb " " 50 " " " 12.50 12.50 59.25 40.75 Expected ratio 59.25:40.75 Back-crosses to P2 M AAnB resistant with 100 per cent penetrance 25.00 00.00 AI‘ AAbb H II 0'7 H n H 21.75 5.25 )4 AabB n n C7 u n H 21.75 3.25 K AaBb n n 87 u n H 21.75 3.25 90025 . 9075 Expected ratio 90.25:9.75 F2 AAbB resistant with 100 per cent penetrance 6.25 00.00 AaBB AABb u n 57 n n H 43.50 6.5 AaBb AAbb Aabb aass susceptible with 50 per cent ” 21.90 21.90 aabb aabb — W 71.05 20.40 Expected ratio 71.6:26.4 -29- Table 5. Chi square test for the theoretical and observed ratios for resistance and susceptibility of the parents and the various generations derived from crossing Plant Introduction 255675 and Fireball to internal browning. Ubserved Theoretical Chi Generations ratio Expt. ratio sq. P Fireball (Pl) 51:55 55:55 .14 .7o-.95 l.1. 255673 (P2) 115:0 115:0 .oo Pooled Fl. 196:29 196:29 .00 Pooled F2 552:135 555:152 .01 .70-.95 Pooled back-crosses to Fireball 176:49 155:92 55.8 .01 Pooled back-crosses to P.I. 255675 206:22 206:22 .00 -30- germplasm and it would appear that this back-ground genotype of Fireball influenced the ability of the a and b loci to reproduce the disease. This causes the expression to fall below some critical threshold level. Thus, the back-ground of Fireball influenced penetrance to such an extent that although the genes were present for the expression of the disease the threshold of expressivity was not reached. In this back-cross there are two new genotypes Aebb and aaBb which previously were lacking in the Fireball back-ground. It would appear that these two genotypes nave a different level of penetrance because they contain a large A or a large B. This altered level of penetrance appears to be responsible for the deviation observed. The hypothesis that a large A or a large B in the Fireball baca—ground will increase the number of resistant plants is prOposed. Table 6 presents the thebretical ratios for this back-cross. These ratios suggest that the back-ground genotype of Fireball influenced the level of penetrance for the two genes. It seems possible that these new genotypes may carry the virus and may be potentially susceptible to the disease; however, the presence of the large A or the large 5 prevented the virus from multiplying to the level necessary for necrosis to appear. This theory in part explains the erratic behavior of the back-crosses to the Fireball parent. In plants with the r.I. 255675 back-ground and also in the Fl's and the F2's where the back-ground genotype is less predominantly Fireball, it seems that the performance of the plant fit the pattern as postulated. The latter hypothesis can be tested by assaying fruits from the back-crosses to Fireball and recovery of the virus would suggest that the threshold necessary for the development of the disease probably had not been reached, and thus, although the genotype was present for susceptibility to internal browning expressivity would be lacking. Since hurakishi (unpublished) had obtained up to 90 per cent penetrance with Fireball and the penetrance level in this experiment -31- Table 6. Theoretical and observed ratios for res'fitance and susceptibility to internal browning of/back-cross to Fireball. Genotypes Res. Sus. %.Aadb resistant with 07 per cent penetrance 21.75 5.25 K Aabb susceptible with 18 per cent ” 20.50 4.50 )‘aah‘b " " 18 H " " 20.50 4.50 %-aabb " " SO " " H 12.50 12.50 75.25 3 25.75 75.25:24.75 70.#:21.6 Theoretical ratio Observed ratio -52- was only 50 per cent, it would suggest that variability is evident for the Fireball parent. This data suggest that the parents are differentiated by two major gene pairs which show complementary recessive epistasis, and control various levels of penetrance depending on the genotype of the individual plants. bFFhCT OF TOBACCO deALQ VIRUS Ahu rUTnTD VIRUS A SYhudOuh 0h TOMATO PLANTS Internal browning In this stud arents, F 's and the segregatin- generations 5 0 8b were inoculated in the seedliig stage with TMV and PVX. Plants which survived the inoculation were transplanted to the field and were later inoculated with TMV at the time when fruit were turning color on the first cluster. A 50 fruit sample from each plant was checked for internal browning as in the previous study. ho internally browned fruit was found as shown in Table 7, and the results are in agreement with the report of Boyle and Wharton (7). boyle and Wharton (7) however, used only ThV while in this study a double inoculum of TMV and PVX was used. The results of this experiment suggest that plants susceptible to internal browning which have been infected with a virus during the early part of their growth probably build up immunity, so that future inoculations failed to produce the virus build up and shock necessary to indfice internal browning. Days From Seeding to First bloom Plants from the various populations which survived the inoculation with TMV and PVX in the seedling stage were compared with non—inoculated seedling populations of similar genetic back- ground. Plants were classified as having bloomed on the day the corolla of the first flower opened. The Analysis of variance computed for days to first bloom is shown in Table 8. The F- values for families and interaction between families and treatment are significant as would be expected since Fireball is an early parent Table 7. Internal browning in parents, F_‘s and segregating generations from the cross between Plant Introduction 255075 and Fireball inoculated with tobacco mosaic virus and potato virus X in the seedling stage. ho. of ho. with Generations gplants internal browning Fireball (P1) 50 O v.1. 235675 (P2) 50 0 Pooled F1 60 0 Pooled F2 120 0 Pooled back-crosses to Fireball 60 0 Pooled back-crosses to P.I. 255675 60 0 -3h- Table 8. Analysis of variance as split plot for days from seeding to first bloom of treated and untreated families from the cross between flant Introduction 255675 and Fireball. Variance due to D.F 5.3 m S F main plots 5 60.55 12.06 Replications 2 5.69 1.64 1.59 Treatments 1 54.00 54.00 40.90 Error (a) 2 2.64 1.52 Sub plots 35 329-99 9-4fi Families 5 133.29 26.66 10.15" Treatment x Families 5 65.77 16.75 6.57“"I Error (b) 20 52.60 2.65 “Significant at l per cent level and P.I. 255675 is late. Comparison among populations by use of the "T” test are shown in Table 9. It is interesting to note that Fireball was the only population in which flowering was delayed by the seedling inoculation with TmV and PVX. The number of days to flowering of the other populations were not altered. This suggests that plants of the other populations are resistant in the seedling stage and the complex of viruses was unable to disturb the normal formation and development of/fggwer. It is also suggested that the £.I. 255675 parent in the seedling stage carried the resistance to the virus syndrome. Seeding to First Fruit Set First fruit set was determined by noting the development of the ovary at the time of the shedding of the corolla. The analysis of variance from days of seeding to first fruit set is presented in Table 10. "T" tests comparing the p0pulations are given in Table 11. With the exception of Fireball and the back-crosses to Fireball there are no significant differences in the mean number of days to fruit set among the various families. It would appear that in the seedling stage, plants which showed resistance to the viruses functioned normally and their physiological development was not impaired. Weight of Vines weight of vines and yield of marketable fruit data were obtained from plants which were inoculated with TMV at ripening of the first fruit. A comparison was made between a population inoculated with TnV and PVx in the seedling stage and TmV at fruit ripening, and a population inoculated with Tfiv at fruit ripening alone. See Fig. 4. The vine weight was determined by weighing the entire plant. The analysis of variance for this data are presented in Table 12. There were significant differences between families and between treatments. The "T” test at Table 15 comparing the populations of -36- Table 9. "T" test for days from seeding to first bloom between treated and untreated populations of similar genetic constitution resulting from crossing Plant Introduction 255675 and Fireball. Not treated Treated Populations seedlings seedlings t. t.O5 P1 6103 6603 -400‘ 20776 P2 ' 7102 7006 096 207'26 (Pl x P2) x Pl bo.5 05.0 -1.5 2.220 (Pl x P2) x P2 66.5 75.0 -2.5 2.220 (Pl x P2) Fl 60.0 00.6 -l.b 2.220 I J. f' o ‘1'“. V o o 6 (Pl x P2) r2 0? o b/ o 15 2 22 *Significant at 5 per cent level Table 10. -37- Analysis of variance as split plot for days to first fruit set of treated and untreated families from the cross between flant Introduction 855675 and Fireball. Variance due to D.F S.S M.S F Main plots 5 66.65 15.55 Replications 2 16.15 6.06 .65 Treatments 1 51.17 51.17 5.22 Error (a) .2 19.35 9.67 Sub plots 55 #75.07 15.52 Families 5 156.92 51.58 5.24’ Treatment x Families 5 56.50 11.26 1.16 Error (b) 20 193.20 9.66 ‘Significant at 5 per cent level -38- Table 11. "T" tests of days to first fruit set between treated and untreated pepulations of similar genetic con- stitution resulting from crossing llant Introduction 255675 and Fireball. hon-treated Treated Populations seedlings seedlings t. t.05 Pl 600.]. '7qu "307‘ 20776 L 78.4 76.3 1.5 2.776 (P P2) P2 7#.# 79.0 -5.5‘ 2.228 (2 r2) 73.9 75.1 -1.1 2.228 ‘Significant at 5 per cent level Figure 4. -39- Fireball plants showing the effects of the two types of inoculation. A Inoculated with tobacco mosaic virus at fruit ripening. ' B Inoculated with tobacco mosaic virus and potato virus X in the seedling stage and with tobacco mosaic virus at fruit ripening. -41- Table 12. Analysis of variance as split plot for mean weight of vines of treated and untreated families from the cross between flant Introuuction 255675 and Fireball. Variance due to n.‘ S.S m.S F main plots 5 5604.71 720.94 Replications 2 54.97 27.46 .99 Treatments 1 5494.75 5494.75 127.15‘ Error (a) 2 54.99 27.49 Suo plots 55 6716.02 191.85 Families ' 5 2550.14 466.05 105.67H Treatment x Families 5 695.20 156.64 51.44H Error (0) 20 66.15 4.41 ‘Significant at 5 per cent level HSignificant at l per cent level -42- Table 15. "T" tests of tne mean weight of vines between treated and untreated p0pu1ations of similar genetic consti- tution derived from crossing Elant Introduction 255075 and Fireball. hon-treated Treated Pogulaticns seedlings seedlings t. t.05 Pl 9.51 3.9 11.8 2.776 P2 47.4 10.5 12.5* 2.776 1 _ , .2 .. (r1 x F2) x P1 20.9 7.1 7.01 2.228 .. _, . ‘ u - i A. ’ (Pl x P2) x 12 33.9 o.0 14.3 2.22o , .- , _ -* - (Pl x P2) Fl 55.0 9.5 18.6 2.220 (P x r ) F 20.0 5.9 4.4‘ 2.228 *Significant at 5 per cent level -43- similar genetic constitution show that there are significant differences between all families. It is interesting to note that }.I. 255675 as well as Fireball was adversely affected by the double inoculum. This suggests that there may be two levels of resistance to the viruses. one level of resistance Operating during the seedling stage when the plant does not appear to be affected, and the other level at the mature stage when presumably the virus haS‘ been able to build up to hinder the normal growth and development of the plant. Mean Number of marketable Fruit The number of marketable fruit was determined by counting the fruit with a diameter of two or more inches from each plant. Analysis of variance for the data is presented in Table 14. F- values are significant for treatments and treatment x families. It would appear that with maturity resistance decreased allowing for the virus to increase thereby, reducing the plant's efficiency. Comparison between seedlings treated with TMV and PVX and untreated seedlings by use of ”T" tests as shown in Table 15 suggest that there are significant differences among all families. When plants of the various families which were inoculated at the seedling stage begin to fruit, they seem to have very little resistance to the virus build up. This results in considerable reduction in the production of marketable fruit. -44- Table 14. Analysis of variance as split plot for mean number of fruits produced by treated and untreated families from the cross between flant Introduction 255075 and Fireball. Variance due to Q.F 5.5 5.3 F main plots 5 46,405 9,296 Replications 2 206 104 4.16 Treatments 1 46,225 46,225 1649.0*‘ Error (a) 2 50 25 Sub plots 35 75.494 2.157 Families 5 17,545 5,467 57.27“ Treatment x Families 5 10,001 2,160 25.25“ Error (o) 20 1,oo5 93 *‘Significant at l per cent level -45- Table 15. "T" tests of the mean number of fruit produced by ' treated and untreated populations of similar genetic constitution derived from crossing rlant Introduction 255675 and Fireball. non-treated Treated Populations seedlings seedlings t. t.05 P1 37 6 7.6* 2.776 P2 150 26 21.01"I 2.776 (Pl x P2) x P1 61 27 5.0* 2.226 (P1 x P2) x P2 125 25 22.7‘ 2.228 (Pl x P2) F1 116 29 17.1* 2.226 (Pl x P2) F2 76 22 12.4‘ 2.226 ‘Significant at 5 per cent level Tobacco Mosaic Virus Tests Plants were classified as susceptible to ThV when mottling, and/or severe chlorosis or distortion were noted. Table 16 and Fig. 5 summarise the data for resistance and susceptibility to TMV of the parents and the various populations derived from hybridization. The data suggest that the parents differed in their ability to resist infection by TMV. The means and standard errors indicate that the F was intermediate in resistance between 1 both parents. The F data suggest resistance to TmV to be polygenic. Fireball used a: the susceptible variety showed 100 per cent infection two weeks after inoculation, while }.I. 255675 was free of infected plants. Resistance to TMV was found to be non- symptomatic during the early growth of the plant. The delay in symptom expression suggest that resistance to the disease changes with the growth of the plant. The results of this study are not in agreement with the reports of earlier investigators who reported that resistance was controlled by one or two dominant genes (51) (58) (66) or by three recessive genes (64). This study however, differed from those of the earlier investigators with the exception of Walter (64), since the study was not terminalized at any growth period but was carried through— out the growing season. Although £.I. 255675 was reported to be resistant, mosaic symptoms began to appear with age. This was reflected in the populations derived from hybridization. To understand the mechanism of inheritance it might be essential to distinguish between tvu) developmental stages of the plant's growth (1) seedling phase (2) fruiting phase. In the first phase, perhaps a few genes may be responsible for the delay in development of mottling and/or distortion of tomato leaves, and at the second phase, a large number of genes or modifiers may be involved in the control of the virus symptoms. Table 16. -47- Frequency distribution of the various populations derived from the cross between 255675 and Fireball for resistance to tobacco mosaic virus. blant Introluction Days from inoculation and number of susceptible plants Total no. Populations of plants 14 21 28 35 42 49 56 63 x 3.2 C.V Fireball (P1) 82 02 14 1.1. 235673 (P2) 48 20 4 1 36 .56 .7.7 (Pl x P2) Fl 56 8 13 12 3 25 1.2 27.6 (P2 x Pl) Fl 49 8 18 4 20 .94 25.5 Pooled 105 16 31 16 3 22 .70 25.9 (Pl x P2) 109 37 20 15 4 3 1 21 .92 39.2 (P2 x Pl) 116 33 21 18 3 4 l 21 .93 39.9 Pooled 225 70 41 33 7 i 2 21 .66 39.5 (P2 x Pl) P ,34 20 9 5 18 .88 28.3 (Pl x P2) P 51 26 13 9 3 19 .89 33.8 Pooled 85 46 22 14 3 19 .66 31.9 (P2 x Pl) P 51 10 32 2 2 2 2 21 .85 28.0 (Pl x P2) P 25 3 6 6 1 2 25 1.9 32.5 Pooled 76 13 33 8 3 4 22 .86 31.9 -48- in, e..- -. Figure 5. Frequency distribution of tomato plants from the various populations resulting from the cross between I.I. 235673 and Fireball, showing susceptibility to tobacco mosaic virus. Number of Plants Infected T..wr:\m\ OI _oo- 0 IIIIIIO I.||I T \..IIII mo- \ ./ \\ 383: m0! \ NO! \\\\.:// \\ x/ m _ a oo a men \ /\\ m o . / mo: \Smomlxj / \ / A0]. \ ‘I/// :o x n V x n //// UOI \ _ N/ N I,” .1/ \\ I, // \ . x / Nou\ / / .\.O/. 3 muumuu \ m. 30.2. V/ A .0 \\\ ll // o \ \n/ ,Q/IIVx..1?ll// \\ / o o/ _ m u a u m u -50- Assay For Virus Build Up in Tomato Plants Showing Symptoms of Tobacco Mosaic Virus Infection Virus build up was determined by noting the necrotic lesions on the test plants caused by extracts from infected plants. The half-leaf method as described by Samuel and Bald (52), Youden and Beale (68) and Sawden (5) was used. The necrotic lesion method gives an approximation of the virus concentration measured by the development of necrotic spots on the tobacco. Figs. 6 and 7 show samples of necrotic lesions formed on the leaves of E. tobacum var. Xanthi n c inoculated with extracts from parent plants showing symptoms of TMV infection. Fig. 6 shows necrotic lesions which were induced by extracts from infected F2 plants. The frequency distribution for the mean percentages of necrotic lesions for parents, Fl's, F2's and back-cross generations are presented in Table 17 and 35118:. 9 o A definite difference between the parents in the number of lesions produced on the leaves of the test plants can be noted. The means and standard errors suggest that there are genetic differences among the families. The results suggest polygenic control. It appears that build up of TAV in the various families is quantitatively controlled. These findings support the hypothesis suggested that resistance to TMV is multigenic. It appears that the genes for resistance slow down the multiplication of the virus since two weeks after inoculation all Fireball plants were classified as susceptible with virus content of 93 per cent and maintained this throughout the growing season. P.I. 2356?} however, did not show symptoms until the fifth week and at that time the virus content was 74 per cent. This suggests that in Fireball genes are not present to hinder virus build up and subsequent symptom expression, while P.I. 235673 carries genes for slow build up of the virus and a prolonged tolerance to symptom expressions. Figure 6. Figure 7. -51.. Lesions induced on leaves of E. tabacum var. Xanthi by extracts from plants of P.I. 255075 sampled at weekly intervals. control inoculum on right-half of leaves. Lesions induced on leaves of E. tabacum var. Xanthi by extracts from plants of Fireball sampled at weekly intervals. Control inoculum 0n right—half of leaves. -_ ._a g -.—_.—- ‘0 ‘— _- _ _‘.—“- -53- Figure 8. Lesions induced on leaves of i. tabacum var. Xanthi by extracts from tobacco mosaic virus infected plants of the F generation of the cross between }.I. 235673 and Fireball, samiled at weekly intervals. Control inoculum on right-half of leaves. Figure 8 -55.. Table 17. Frequency distribution of necrotic lesions produced by extracts from samples of susceptible plants from the pOpulations derived from crossing Plant Introduct— ion 255073 and Fireball. Days from inoculation and mean percentage of lesions Generations 14 21 28 35 42 49 56 x S.E c.v Fireball (P1) 94 14 r.I. 235673 (P2) 74 40 30 40 .46 13.9 Pooled“?l 3. 88 66 63 55 50 30 .67 37.6 Pooled F2 89 6o 65 57 52 45 29 .61 40.9 Pooled Back-crosses to Fireball 77 57 59 60 24 .50 33.5 Pooled Back-crosses to 2.1. 235673 78 57 53 48 57 44 29 .66 42.1 h—fl Figure 9. -56- Frequency distribution of lesions caused by extracts from tobacco mosaic virus susceptible plants of the various populations derived from a cross between P.I. 235075 and Fireball. _— ——_——— - —_ _ - -_—_ M.‘ —_-* A mfiAfi‘- “A_A-‘-—~‘—A._A Percent Lesions \I\O\|\|.lo.\|m Ilellulelllno _oou \\ 133.. $0]: / \M lw/ / / mo: \ /// \ // T.» .323 J 63.3 //// /. V \.|O/. #01 I III #"“"I I ll \ /I mo: An. x F... x b. I l// / \ I/Ifil/ o . // I II / GOT AV_XUNvXVN I v /// I [ll \. yo’.’"' 31 II n /I/ 81 \T E 83.3 ./.:e c 81 N _O . _ _ __ _ r _ _ N u A u m .N $3.3. Internal Browning The behavior of the Fireball variety as a completely suscept- ible parent to internal browning made it necessary to analyse the the data by apportioning levels of penetrance to both of the parents. The results are similar to that reported by Allard (l) on Lima bean. In his studies the variety Ventura, although homozygous dominant for the gene which caused seedlings to become partially chlor0phyl deficient, rarely produced seedlings with more than 10 per cent showing the characteristic. Based on the level of incidence of the abnormality 10 per cent penetrance was assigned for the genes by Allard (l). Penetrance is a phenomenon which disturbs the gene character relationship, obscures the correspondence between genotype and phenotype and complicates the genetic analysis of data. In the inheritance study, levels of penetrance were assigned according to the genotype of the plants. In the back-crosses to Fireball, the theoretical expected ratios did not fit the observed ratios using the penetrance level assigned to the genotype, suggesting that the back-ground genotype of Fireball had influenced the penetrance of the gene or genes primarily concerned with virus reaction. Although Murakishi (unpublished) obtained up to 90 per cent penetrance for internal browning with Fireball, penetrance as assigned in this study was based on the performance of the populations. As pointed out by Allard (l) penetrance can be a certain percentage on the average for a particular character, since plants perform differently under certain environmental conditions. This could partly account for the variability as found in the Fireball variety. It appears that internal browning is the result of the inter- action of two gene pairs. These genes seem to be epistatic to each other in the recessive state, and control different levels of pene- trance depending on the genotype of the plant. This condition -59- might only be applicable to this particular cross between F.I. 235673 and Fireball, and the performance using other varieties might not be the same. Effect of Tobacco mosaic Virus and Potato Virus X Syndrome on the Tomato Plant The syndrome of both viruses was effective in immunizing some otherwise susceptible plants to internal browning. Plants which survived the double inoculation in the seedling stage flowered and set fruit equally as well as non-inoculated seedlings of similar genetic back-grounds. However, the complex of viruses did affect the weight of vines and the number of marketable fruits produced by the inoculated seedlings. It appears that }.I. 235673 is tolerant to the virus syndrome in the seedling stage, and perhaps slow multiplication and slow transport of the virus are responsible for the tolerance shown. Tobacco Mosaic Virus Tests Previous studies on the inheritance of resistance to ThV were not carried out to the end of the growing season. Holmes (31) in reporting that resistance was due to one dominant gene counted lesions on tomato plants five days after inoculation with the virus. Watson, et al (66) determined their ratios after two readings, the latter of which was taken one month after inoculation. Soost (58) failed to mention the time his data was taken. halter (64) however, made weekly readings. The fact that susceptibility was a function of age suggests that a terminal experiment would be inadequate to determine inheritance of this character. Data were taken at weekly intervals until the plants were killed by frost. The results presented in Table 16 and Fig. 5 suggest that a large number of genes govern resistance or susceptibility'b the disease. These data suggest that resistance depends on the suppression of virus multiplication in resistant lines. it is -60- possible that }.I. 235673 escaped infection with TMV in a similar manner as reported by Holmes (26) for E. chilense. This would probably account for the develOpment of symptoms on some }.I. 235673 plants with age. Virus assay of susceptible plants presented in Table 17 and Fig. 9 support the theory of polygenic inheritance. It would appear that in the P.I. 235673 parent and the resistant offspring there was a slow multiplication of the virus. In Fireball however, multiplication of the virus was rapid. Since there are other sources of resistanceeto TMV (13) (19) (20) (28), polygenic control may be applicable only to the cross between P.I. 235673 and Fireball. For further information on the total number of genes involved in the manifestation of mosaic symptoms it would be essential to record data at two to three days intervals during an extended growing season, and assay each progeny with symptoms as they become apparent. SUMHARY AND CONCLUSION l. The progenies of a reciprocal cross between P.I. 235673 (resistant) and Fireball (susceptible) were evaluated in a study of the mode of inheritance of resistance to internal browning, a disease of tomato fruit caused by late infection of the plant with TmV. Results from the segregating generations suggest that two genes showing complementary recessive epistasis, controlled the levels of penetrance of this character. 2. The Fireball parent did not perform as reported by other workers suggesting that there is variability in this variety. The level of penetrance was apparently influenced by the variable genotype. 3. The results of the experiment on the effect of the virus syndrome of TMV and PVX on the tomato plant, suggest that plants can build up an immunity to virus infection so that subsequent inoculations would prevent internal browning. There was no difference between families of similar genetic constitution for date of flower- ing and date to first fruit set, where one group was inoculated in the seedling stage with TMV and PVX while the other was not. The uninoculated seedlings however, produced vines of greater weight, and an increased number of marketable fruit suggesting that in the seedling stage some plants are tolerant to the virus complex, while in the mature stage tolerance breaks down and plants become affected. 4. Studies on inheritance of resistance to ThV showed that there was non-symptomatic tolerance in the seedling stage, however, symptoms appeared on plants with age. The data suggested that resistance to TMV was quantitatively inherited. 5. The results of the virus assay suggest that for the suscept- ible Fireball parent the virus multiplied rapidly and maintained a high level throughout the growing season, while in the resistant P.I. 235673 a low level of build up of the virus is noted. This resistance and/or tolerance was noted in testing the progenies of the cross. 10. ll. LITERATURE CITfiD Allard, R. W. 1960 Principles of Plant Breeding. New York, London. John Wiley & Sons Inc. , and H. L. Alder. 1960 The effect of incomplete penetrance on the estimation of recombination values. Heredity 15: Alexander, L. J. and M. u. hoover. 1953 Irogress report of national screening committee for disease resistance in 1952. U.S. Dept. Agr. Pl. Dis. Rptr. 37: 317—324. , RanK. Lincoln and Vedder Wright. l942 a survey of the genus chopersicon for resistance to the important tomato diseases occurring in Ohio and Indiana. U.S. Dept. Agr. Pl. Dis. Rptr. Sup. 136: 51-05. Bauden, F. C. 1950 Plant Viruses and Virus Diseases. 3rd ed. Waltham, Mass. Chronoca Sotanica Co. Beale, nelen P. 1934 The serum reactions as an aid in the study of filterable viruses of plants. Contr. Boyce Thompson Inst. 6: 407-435. noyle, J. S. and D. C. Wharton. 1957 The experimental reproduction of tomato internal browning by inoculation with strains of tobacco mosaic virus. rhytOpath. 47: 199-207. Cadman, C. a. -‘l942 Autotetraploid inheritance in the potato: some new evidence. Jour. Genetics 44: 33-52. Cockerham, G. 1943 The reactions of potato varieties to viruses X, A, B, and 0. Ann. Appl. 6101. 30: 330-344. l943 lotato breeding for virus resistance. ann. Appl. Biol. 30: lO5-lCo. 9 1945 Some genetical aspects of resistance to potato viruses. Ann. Appl. Biol. 32: 200—201. 12. 13. l5. 17. 19. 2l. 22. 23. -63- Conover, R.A. 1949 Vascular browning in Dade County Florida green wrap tomatoes. 0.6. Dept. Agr. bl. Dis. Rptr. 33: 203-204. COOK, A. A. 1960 Genetics of Capsicum anhuum to two virus diseases. rhytopath. 50 no. 5 304-907. 71961 Mutation for resistance to potato virus Y in pepper. ihytopath. 51 No. 8 550-553. 1963 Dominant resistance to tobacco mosaic virus in an exotic pepper. 0.6. Dept. Agr. r1. Dis. Rptr. VOlo 4? (9). , and C. h. Anderson. 1900 Inheritance of resistance to potato virus Y derived from two strains of Capsicum annuum. ihytopath. 50 ho. 1 73-75. Cox, J. E. and L. O. weaver. 1950 Internal browning of tomatoes in haryland (abs.). rhytopath. 40: 070. Dickson, B. T. 1925 Tobacco and tomato mosaic. Science 62: 396. Doolittle, S. P., u. S. forte and F. S. Beecner. 1946 high resistance to tooacco mosaic in certain lines of bycopersicon hirsutum (abs.). rhytopatn. 50: 065. Frazier, h. A. and R. K. Dennett. 1949 Tomato lines of Lyc0persicon esculentum type resistant to tobacco mosaic virus. broc. Amer. Soc. dort. Sci. 54: 205-271. Friedman, o. A- 1949 The occurrence of vascular browning or internal browning in tomatoes from Iennsylvania and hew lork. U.S. Dept. Agr. kl. Dis. Rptr. 33: 404. haenseler, C. n. 1949 Internal browning of tomatoes in Lew Jersey. U.S. Dept. Agr. El. Dis. Rptr. 33: 330-337, hal} ‘10 D. aLd Re A 8 Di 0r. . c 1 ' 955 nnV1ronmentaf Iacgors influenClng vascular browning of tomato fruits. Amer. Soc. nort. Sci. Iroc. '~- --~.~—./ 05: 579-590. 24. 25. 29. 30. 31. 53- 35- holmes, F. b. 1929 Local lesions in tooacco mosaic. Dot. daz. 07, 39. 1954 Inheritance of ability to localize tooacco mosaic virus. lhytopath. 24: 904-1602. 1937 Inheritance of resistance to tooacco mosaic disease in the pepper. .hytopath. 27: 037-642. 1930 inheritance of resistance to tobacco mosaic disease in tooacco. inytopath. 20: 553-561. 1943 A tendency to escape tobacco mosaic disease in derivatives from a hybrid tomato. bhytopath. 33: 1949 association of strains of tobacco mosaic virus with internal browning in tomatoes. 0.5. Dept. Agr. r1. 1950 Internal browning disease of tomato caused by strains of tooacco mosaic virus from Planta o. Phytopath. 40: 407-492. 1954 Inheritance of resistance to infection by tobacco mosaic virus in tomato. lhytopath. 44: 040-642. 1900 Control of important viral diseases of tomatoes. Troc. r1. Sci. Seminar, Camden, iew Jersey, Campbell Soup $0., 1-10. howitt, J. E. and R. E. Stone. 19lo A troublesome disease of winter tomatoes. rhytOpath. 6: 162-100. mutton, d. u. and D. C. hark. 1952 A relationship between immunity and localized reaction to virus X in the potato. Austral. Jour. Sci. Res. Ser. B 5: 237-243. Jackson, h. S. 1917 Tomato diseases. Ann. Rptr. Purdue Univ. Agr. Expt. Sta. No. 30: 23. 36. Jones, J. r. and L. J. Alexander. 1956 Studies on the etiology of blotchy ripening (abs.). Ihytopath. 46: 16. 37. hidson, E. S. and D. J. Stanton. 1953 Cloud or vascular browning in tomatoes. Lew Zeal. Jour. Sci. Tech. Sec. A34: 521—530. 30. hikuta, h. and L. A. Frazier. 1947 freliminary report on breeding tomatoes for resistance to tobacco mosaic virus. Proc. Amer. Soc. Hort. Sci. 49: 256-262. 39. Loring, h. S. 1937 Accuracy in the measurement of the activity of tobacco mosaic virus protein. Jour. Diol. Chem. 121: 637-647. 40. macNeil, D. d. and h. Ismen. 1950 A newly differentiated component of the tomato virus-streak complex. U.S. Dept. Agr. 11. Dis. Rptr. 42: 898. 41. . 1960 Studies on the virus-streak syndrome in tomatoes. can. Jouro not. 30: 9-200 42. hurakishi, a. H. 1959 Varietal response to factors influencing the expression of tomato internal browning symtoms (abs.). PhytOpath. 49: 546. 1+3. 0 O 1960 Present status of research on greywall and internal browning of tomato. Quart. Bull. Aich. Agro BAPto Uta. VUlo “2: 720-7320 44. . 1960 Comparative incidence of greywall and internal browning of tomato and sources of resistance. rnytopath. 50: 406-412. 45. , and S. honma. 1963 nesistance to tooacco mosaic virus in Lycgpersicon hybrids evaluated oy potato virus X synergy. .uphytica 12: 27-31. 46. 5011a, J. A. B. 1938 Inheritance in hicotiana III a study of the character for mosaic resistance in hicotiana tabacum L. Jour. Hered. 29: 42-40. 47. 49. 50. 51. 52. 54. 55- \n O‘\ o 57- 56. -66- Panse, V. G. and P. V. Sukhatme. 1961 Statiscal methods for Agricultural Research Workers. Indian Council of Agricultural Research, new Delhi, 2nd ed. Porter, R. H. 1930 The resistance of cucumbers to mosaic. Phytopath. 20: 114. 1932 The reaction of cucumbers to types of mosaic. Iowa State doll. uour. Sci. 6: 95-129. Pound, e. S. and l. J. Cheo. 1952 Studies on resistance to cucumber mosaic virus in Spinacn. Phytopath. 42: 301-306. naychaudnuri, S. P. 1953 Reproduction of symptoms resembling internal browning of tomato under green-house conditions. U.S. Dept. Agr. kl. Dis. sptr. 37: 220-221. Samuel, 3. and J. G. Bald. 19§3 0n the use of the primary lesions in quantitative work with two plant viruses. Ann. Appl. Biol. 20: 70-99. , and R. J. Best. 1935 studies on quantitative methods with two plant viruses. Ann. Appl. niol. 22: 506-523. Schultz, E. S. and L. P. Raleigh. 1933 Resistance of potato to latent mosaic. Phytopath. Shifriss, 0.,40, H. Myers and C. Chupp. 1942 Resistance to mosaic virus in the cucumber. Phytopatn. 32: 773-734. Sinclair, u. s. and J. 0. walker. 1955 Inheritance of resistance to cucumber mosaic virus in cowgea. xhytopath. 45: 563—564. Slnith , i; o 1". 1937 A text book of rlant Virus Diseases. Cnurcnill ltd. London. Soost, R. n. 1963 Hybrid tomato resistant to tobacco mosaic virus inheritance for resistance in derivatives of a COmplex species hybrid. Jour. hered. 54: 241-244. 59. 61. 62. 64. 66. 69. -57- U] *U‘ encer, E. L. and W. C. Price. 1943 Accuracy of the local lesion method for measuring virus activity in tobacco mosaic virus. Amer. dour. dot. 30: 260-269. Stevenson, F. J., E. S. Schultz and C. F. Clark. 1939 Inheritance of immunity from virus A (latent mosaic) in the potato. lhytopath. 29: 362-365. Taylor, G. A., C. B. Smith and R. E. Fletcher. 1956 Influence of some environmental and nutritional factors on the incidence of internal browning of tomato. Penn. State Univ. Agr. Expt. Sta. bull. 629.21. Thompson, n. R. and w. J. Hooker. 1962 Gametic sampling of two potato varieties for genetic analysis of immunity to virus X. XVIth International horticulture Congress - 1962, Srussels. Vanterpool, T. C. 1926 Streak or winter blight of tomato in Quebec. Phytopath. 20: 631-63). Walter, J. M. 1956 Hereditary resistance to tooacco mosaic virus in tomato. Phytopath. 46: 513-516. hasuwat, S. l. and J. C. Walker. 1961 Inheritance of resistance in cucumber to cucumber mosaic virus. Phytopath. 51: 423-426. Watson, R. 0., E. C. fieinrich and W. R. narvey. 1954 Inheritance of resistance to tobacco mosaic virus in an interspecific tomato cross. U. of Idaho, College of Agr. Research Bull. 27 (20 pages). Lharton, D. J. and J. S. Boyle. 1957 The pathological histology of the internal browning disease of the tomato. Phytopath. 47: 200—212. Iouden, W. F0 and Helen P. Beale. 1934 A statistical study of the local lesion method for estimating tooacco mosaic virus. Jontr. Boyce Thompson Inst. 6: 437. Young, P. A. 1946 Tomato diseases in Texas. Tex. Agr. Expt. Sta. Ciro. 113. 805%.”.- USE Bi‘éLY