Cowpea chlorotic mottle virus local lesion area and infectivity increased by 2-thiouracil

VIROLOGY

43,

lo&-l(@

Cowpea

(1971)

Chlorotic

Mottle

Virus

Increased

Local

Lesion

Area

and

Infectivity

by 2-Thiouracil’

C. W. KUHN Department

of Plant Pathology

and Plant Genetics, University of Georgia, Athens, Georgia 30601 Accepted September

18, 1970

Treatment with 2-thiouracil caused cowpea chlorotic mottle virus (CCMV) lesion area on hypersensitive soybean to be enlarged 8-75 times. Lesion enlargement was detected 48 hr after inoculation, and lesions increased in size rapidly for 6-8 days. Although soybean becomes more resistant (fewer and smaller lesions) with increasing age, thiouracil caused lesions to reach a similar size regardless of host age. Compared to controls, lesion area increased 2, 13, and 4 times at 21, 27, and 32”, respectively. Increased lesion area was noted when thiouracil treatment began at 0, 12, and 24 hr after inoculation, but not at 48 hr. The full effect, however, occurred when treatment began between 0 and 12 hr. When lesion area was increased lo-13 times, the sap infectivity was increased 33-38 times. The increase in lesion size could be prevented when uracil (lo+ M) was added to thiouracil (l&4 M). Thiouracil treatment caused CCMV to move from one leaf half to another, but no systemic movement from one leaf to another was detected. CCMV sap infectivity was enhanced 3 times whereas tobacco mosaic virus sap infectivity was inhibited 7 times by thiouracil in Chenopodium amaranticolor, a local-lesion host for both viruses. INTRODUCTION

One type of resistance to plant viruses is evidenced by the production of necrot’ic local lesions (hypersensitivity). Alteration of the lesion size is generally regarded as an increase or decrease in the degree of resistance, and in some cases (Ross, 1961; Kuhn and Teas, 196s) the virus infectivity associated wit,h a local lesion is roughly proportional to t,he lesion area. Both physical and chemical treatments can alter lesion size. Preinoculation t’reatment with low light intensity (Bawden and Roberts, 1948) or high temperatures (Kassanis, 1957) causes plants to be more susceptible. When temperatures of 30-36” are applied continuously to infected plants, necrosis is prevented wit)h tobacco mosaic virus (TJIV) (Samuel, 1931) and southern bean mosaic virus (Brantley and Kuhn, 1970) which move systemically in their hosts in that 1 Journal Series Paper No. 839, University of Georgia College of Agriculture Experiment Stations, College Station, Athens.

temperature range. Furthermore, a brief hot water treatment (50-60”) causes TMV local lesions and virus infectivity to be increased ,5-6 times (Yarwood, 1958; Wu et al., 1969). 2-Thiouracil is recognized as a potent inhibitor of plant viruses; it reduces viral biosynthesis in systemic hosts (Commoner and >Iercer, 1951, 1952; Bawden and Kassanis, 1954) and decreases lesion size in hypersensitive hosts (Holmes, 1955; Francki, 1962). In contrast, our studies (Dawson and Kuhn, 1969) established that thiouracil treatment enhanced cowpea chlorotic mottle virus (CCMV) infectivity in thiouracil-treated plants. In the susceptible host cowpea, CCMV sap infectivit,y was increased 1.5-S times, and specific infectivity was increased 5-24 times, strongly suggesting a stimulation of CCMV biosynthesis. In this paper data are presented which show that thiouracil increases both the sap infectivity and the size of local lesions of CCMV in the resistant, hyper101

102

KU ‘HN

sensitive Merr .

host soybean, Glycine max (L.)

MATERIALS

AND

Havana 423 was used as a local lesion assay host for TMV.

METHODS

Virus inoculum. Cowpea chlorotic mottle virus was cultured in cowpea, Vigna sinensis (Torner) Savi cv Early Ramshorn. Sap inoculum was prepared by selecting leaves with high infect’ivity levels (5-10 day infection) and grinding them in 0.01 M neutral potassium phosphate buffer containing 1% Celite. Incubation procedure. Primary leaves of soybean (cv Bragg) were inoculated with CCMV. Immediately thereafter, one-half of each leaf was floated on 15-18 ml of thiouracil (0.5 X 10d3 M) in a petri dish. The ot’her half was floated on water. Each petri dish had 3-4 leaf halves, and each treatment consisted of 6-12 leaf halves, each from a different plant. The dishes were incubated in a growth chamber at 27”, with 800-1200 ft.-c. from fluorescent lights, and with a photoperiod of 18 hr. Lesion measurement. The diameter of local lesions was measured to the nearest 0.25 mm with a dial calipers. All lesions on a half-leaf were measured if they numbered fewer than 50. If numerous lesions developed, 50 randomized lesions were measured. An estimate of lesion area was made based on the observation that most lesions were circular. Measurements were made 5-7 days aft,er inoculation. Infectivity assay. After incubation for the appropriate period, the half-leaves of each treat’ment were rinsed with running water and dried with paper towels. The tissue was ground in a mortar with 9 ml of phosphate buffer containing Celite per gram fresh weight. Double dilutions of each treatment were prepared which caused fewer than 100 lesions per half-leaf. The local lesion assay host was Bragg soybean, and 6-12 replications of each treatment were used in a randomized block design. When CCMV and TMV (AC 1 of the American Type Culture Collection) were cultured in Chenopodium amaranticolor L., the leaf tissue was ground in buffer containing 1% bentonite which aided in overcoming natural inhibitors of infection (Yarwood, 1966). Nicotiana tabacum L. cv.

RESULTS

E$ect of Thiouracil Treatment on Lesion Area and Number In initial tests with thiouracil, more CCMV local lesions were produced and the lesions were obviously larger on the chemically treated half-leaves of soybean than on opposite half-leaves treated with water (Fig. 1). Thiouracil never failed to cause an increase in t,he size of CCMV local lesions

FIG. 1. CCMV local lesions on soybean treated with thiouracil (left) and with water (right).

TABLE EFFECT

OF

1

THIOURACIL (0.5 X 10-S M) ON CCMV LOCAL LESIONS ON SOYBEANS

Xumber of lesions

Area/lesion (mm21

Test

Area ratio (thiouracil/

water)

- --1 2 3 4 5 Average a Counts inoculation.

1

113 80 67 65 32

90 66 62 44 24

4.02 7.32 3.45 6.32 10.24

0.37 0.32 0.40 0.58 0.30

10.9 22.9 8.6 10.9 34.1

71

1 57

6.27

0.39

17.5

and measurements

made 6 days after

INCREASE TABLE

IN

CCMV

INFECTIVITY

2

COMPARISON OF THE COEFFICIENT OF \7a~~~~~o~ OF CCMV LOCAL LESIONSONLE.~VESFLOATED ON THIOURACIL AND WATER

1

Test

1 2 3

Thiouracil

Lesion diameter (mm)

1.68 f 0.76” 1.44 f 0.50 1.94 z!z 0.64

a Standard

Water

Lesion diameter (mm)

0 .56 f 0 .66 f 0 .56 f

0.24” 0.27 0.33

deviation.

in 65 tests and five representative tests are shown in Table 1. The amount of increase varied from test to test, but the lesion area increased at least 8 times and a few tests were as high as 75 times. Lesion size on thiouracil-treated leaves was relatively much less variable than on control leaves, as suggested by a coefficient of variation test (Table 2). Although 50-75 % of the lesions were visible 24 hr after inoculation (incubation at 27’), no differences in lesion size could be detected between leaves floated on thiouracil and water. At 48 hr, lesion enlargement caused by thiouracil was observed and lesions continued to enlarge and could be measured for at least 6 days (Fig. 2). Lesion growth rate after the sixth day was difficult to determine because of coalescence. When inoculated leaves were floated on water or when left on plants, the lesions reached a maximum size between the second and fourth days, in contrast to lesions on treated leaves which had a maximum enlargement rate between the third and eighth days. Lesion appearance was also affected by thiouracil t’reatment’. On the third day after inoculation, a yellowish zone was observed around t’he brown necrotic lesion on thiouracil-treated leaves. The yellowish zone continued to enlarge for 3-5 more days and frequently, the entire zone became necrotic. No similar yellowish zone was observed on control tissue.

103

BY 2-THIOURACIL

Thiouracil treated leaves generally, but not always, produced more lesions than control leaves (Table 1). In 15 % of the t’ests, the water control had 10% more lesions than the t’reated leaves and in 28%, there was no difference. In 18, 24, and 15% of the tests, the lesion numbers on treated leaves were 10, 25, and 50% greater, respectively, than on the control leaves. Correlation oj Lesion Size and Virus Infectivity in Lea! Extracts The infectivity of CCMV in leaves with local lesions was determined by local lesion assay. Virus could be detected in inoculated leaves left on plants or floated on water, particularly if t’here were 10 or more lesions per half-leaf. Since lesion numbers were decreased by sap diluted less than 1: 10 (1 g of leaf tissue + 9 ml of buffer), all assays were made with sap diluted more than 1: 10. Although quite variable and dependent on the size of the lesions, untreated leaves with 30-75 lesions per halfleaf caused an average of about 25, 5, and 1 lesions per half-leaf when the sap was diluted 1: 10, 1: 100, and 1: 1000, respectively. In the same assays and with similar lesion numbers (30-75), sap from thiouraciltreated leaves diluted 1: 10, caused 200-400 lesions per half-leaf (not countable with accuracy). At dilutions of 1: 100 and 1: 1000, the lesion counts were in the range of 100200 and 10-60, respectively. Untreated

HOURS AFTER INOCULATION FIG. 2. Growth of CCMV leaves treated with thiouracil

lesions on soybean and water.

104

KUHN TABLE

CORRELATION OF LESION SIZE AND CCMV

3

SAP INFECTIVITY

AS DETERMINED

BY LOCXL LESION ASSAY Infectivity ratio (thiouracil/ water)

Treatment

Number of lesion per half-leaf

Lesion diameter (mm)

Total lesion area (sq mm)

Thiouracil Water

14 14

2.45 0.67

65.9 4.9

13.4

75 2

37.5 -

Thiouracil Water Thiouracil Water

37 41 65 64

2.59 0.70 2.31 0.75

193.5 15.6 272.4 28.2

12.4 9.7

98 3 176 5

32.7

Area ratio (thiouracill water)

Sap infectivity” (No. of lesions)

a Leaves with lesions noted in column 2 were ground and used as inoculum Assays for each thiouracil-water combination were independent of each other.

susceptible cowpea plants at the peak of their infectivity produce lo-50 lesions per half-leaf at a dilution of 1: 1000. Thus, the amount of infective CCMV in thiouraciltreated, hypersensitive soybeans, with an average of 50 lesions per half-leaf, was similar to the amount in untreated cowpeas. In several tests, the sap infectivity was compared with the lesion area on both thiouracil-treated and untreated tissue. It appeared t’hat t,he difference in infectivity between treated and untreated was greater than the lesion area difference (Table 3). The reliability of determining precise quantitative relationships with local lesion assays with lesion number differences as great as those in Table 3 is questionable. However, reliable estimates of the differences can be made, particularly when tests are repeated numerous times. For direct local lesion assay comparisons between treated and untreated tissue (Table 3), the sap from bot(h treatments was diluted the same amount. Under this condition, the lesion number differences were so great that usually one treatment only occurred on t’he proportional part (E-S5 lesions per halfleaf) of the dilution curve (Gay, 1967). In other tests, sap from treated tissue diluted 1: 1000 produced about 10 times as many lesions as sap from untreated tissue at a dilution of 1: 200. This suggests t’hat thiouracil caused an infectivity increase of 50 times as compared to a lesion area increase of lo-13 times (Table 3). Although the exact infectivity differences have not been established, it seems clear that infec-

in local

35.2

lesion

assays.

tivity differences were great’er than lesion area differences. The number of lesions per half-leaf apparently did not affect the lesion size or the ratio of the sap infectivity between treated and untreated tissue (Table 3). The lesion diameter was about the same even though t’he lesion numbers varied as much as 4.5 times. The lesion diameter and the lesion area ratio (treated:untreated) were slightly less on the leaves with the largest numbers; however, this difference was probably insignificant because lesions coalesced and were difficult to measure. Effect of Temperature and Thiouracil centration on Lesion Size

Con-

As temperat’ure increased from 21” to 32”, the lesion size increased on leaves floated on thiouracil or water (Fig. 3). The greatest lesion size difference between t,hiouracil treated and untreated occurred at 27”. In general, lesion area increased with an increase in t’hiouracil concentration (Fig. 4). Small but significant increases were demonstrated at 10m4 and 10v5 M; the greatest increase occurred at lop3 M when leaves were incubated at 27”. Temperature exerted an influence on the effect, of thiouracil concentration on lesion size, particularly at 32”, where the lesion increase was less with 1O-3 M than with lo-* M (Fig. 4). Att,empts to use thiouracil at lo-” &’ were unsuccessful because it was necessary to dissolve t,he thiouracil in a

INCREASE

IN

CCMV

INFECTIVITY

105

BY 2-THIOURACIL TABLE

4

EFFECT OF HOST AGF, ON CCMV LESION DEVELOPMENT WHEN LEXVES WERE FLOATED ON THIOURXIL OR WATER Number of lesions

Age of tissue (dw)

I’hio1xacil

Area/lesion (mm2)

Area ratio (thiouracil/ water)

vater

l-

l*........................ 210 TEMPERATURE

210 FIG. 3. Effect area on soybean water.

I

of temperature leaves floated

’ l&i

on CCMV lesion on thiouracil and

1 16%

1PM THlOURACll

1 3F

MOLARITY

FIG. 4. Effect, of different concentrations of thiouracil on CCMI’ lesion area on soybean leaves kept at different temperatures. Relative lesion increase = lesion area ratio (thiouracil/water).

basic solution, and t,he combination a deteriorat,ion of leaf tissue.

caused

12 14 18 21 25

-

35 34 38 26 26

30 26 24 20 19

8.39 11.46 11.57 9.29 10.72

0.58 0.58 0.20 0.16 0.16

14.5 19.8 57.8 58.1 67.2

days old (Table 4). With thiouracil treatment, however, the lesions appeared to reach a similar size regardless of host age (Table 4). It appears, therefore, that older plants are more resistant because of factors induced after viral infection rather than factors that developed naturally with age. Ross (1966) made a similar proposal that lesion size was limit,ed because of development’ of immunity in advance of the virus, but later (Ross and Israel, 1970) suggested that events leading to cell collapse are responsible for virus localization. Lesion numbers also decreased when older plants (25 days as compared to 12 days) were inoculated (Table 4). Plant tissue 18-25 days old was used in tests where maximal differences were desired. Erratic result)s were obtained when t’rifoliolate soybean leaves were used. Enhancement of lesion size and increased sap infectivit)y caused by thiouracil was obvious; however, this leaf tissue was apparently more susceptible than primary leaf tissue since the lesions were larger, and a few lesions continued to enlarge, when leaves were floated on water.

E$ect of Host Age

Timing and Length of Thiouracil

The variation from t,est to test in relative lesion size and lesion number caused by thiouracil could be partially explained by the effect of host age on CCMV lesion development. When inoculated leaves were Aoat,ed on water, the lesion area was 3 times great’er on primary leaves from plants 12-14 days old t’han on those from plants 21-25

Half-leaves were removed from plants at various intervals after inoculation and Aoat’ed on thiouracil and water. Lesion size was enhanced when thiouracil treatment began at 0, 12, and 24 hr after inoculat#ion (Fig. 5). The increase was 13-14 times at 0 and 12 hr and 6 times at 24 hr. No enlargement was detected when treat,ment was

Application

106

KUHN

initiated 48 or 72 hours after inoculat,ion (Fig. 5). In some experiments, leaves placed on water at 0 and 12 hr after inoculation had lesions that increased 2-3 times when compared to lesions on leaves left on plants or removed at 48 and 72 hr (Fig. 5). The increase in lesion size on the control tissue was greater when young tissue (12-15 days) was used rather than old (20-25 days). In a second series of tests, inoculated half-leaves were first floated on thiouracil, rinsed thoroughly, and t’hen maintained on water. Lesion area increase was great,est when tissue was continuously kept on thiouracil for 5 days (Table 5). The increase was less, but also significant, when t,hiouracil treatment was terminated after 6, 12, 24, or 48 hr. Yo attempt was made to measure residual thiouracil in leaves when they were transferred to water. These data indicate t.hat the factors that control lesion size can be altered during the 24-hour period following inoculation. After 48 hours, however, factors limiting lesion size cannot be reversed by t’hiouracil treatment . Virus Movement Normally, CCMV cannot be isolated from interlesionary tissue, and the virus does not move into uninoculated plant tissue. Since lesions coalesced and the virus was generally distributed throughout t’he leaf when treated with t.hiouracil, attempts were made to determine movement into S-

TABLE 5 EFFECT OF LENGTH OF APPLICITION OF ON CCMV LOC.
Hz0 Thiouracil Thiouracil Thiouracil Thiouracil Thiouracil 120 hr

6 hr, then 12 hr, then 24 hr, then 48 hr, then continuously

H,O Hz0 Hz0 Hz0 for

Lesion area (mm”)

_

Area ratio (thiouracil/ water)

0.24

-

2.83

11.8 bimes. 11.5 17.3 25.3 28.1

2.77 4.15 6.07 6.74

-I

other tissues. When inoculated F,l:snts were excised at the soil line and placed in tubes. with thiouracil, the new trifoliolate growth became distorted and necrotic, but virus. could not be isolated from the tissue. Another test, however, demonstrated that CCRIV could move from one leaf half to the other when floated on t’hiouracil. One leaf half was inoculated, and no lesions, developed within 5 mm of t,he midvein. After 13 days, yellowed and necrotic tissue was observed in the 5 mm zone and in the l-3 mm zone across the midvein. Tests for infectious CCMV were made in three areas: (a) inoculated half-leaf, 2 mm strip along midvein; (b) uninoculated half leaf, O-8 mm from midvein; and (c) uninoculated half-leaf, 9-20 mm from midvein. Virus was isolated from the first two areas, but not the last. It appeared, therefore, that the virus was moving in nonvascular tissue since it was not recovered from the outer portion of the uninoculated leaf half. Counteraction by cTracil

l . . .. . ... . .*

0

. . . . . . .._.

48 12 24 NOURS AFTER INOCULATION

FIG. 5. Effect of initiating thiouracil at different times after inoculation lesion area on soybean.

72

treatment on CCMV

The increase in lesion size could be prevented when uracil was added to the thiouracil solution (Table 6). When equal molarities (10-3) of the two chemicals were used, the relative increase was reduced from 14.7 times for thiouracil alone to 3.1. Excess uracil however, was needed to completely count)eract the effect of thiouracil. Light vs. Dark Incubation Since the inhibitory action of thiouracil on TMV in tobacco was much greater in

INCREASE TABLE EFFFXT

IN

CCMV

6

OF THIOURMXL PLUS URACIL LFXION ARE.& ON SOYBUN

ON

CCMV

Chemical :oncentration GW

Treatment

Thiouracil Thiollracil Uracil Thiouracil uracil Thiouracil uracil Hz0

INFECTIVITY

10-4 10-S 10-S +

10-4 + la-3

+

10-a + 10-a

44 51 35 42

4.99 8.54 0.69 D.46

8.6 14.7 1.2 0.8

1.77 41

0.58

-

daylight than in darkness (Bawden and Iiassanis, 1954), the effect of light and dark incubation was checked for CCMV in soybean. Lesions were produced when leaves were incubated in total darkness, and in three tests, t’hiouracil caused the sap infectivity to be increased by an average of 50 t’imes. It was concluded, therefore, t’hat the effect of thiouracil on CCMV local lesions was independent of light. Other Methods oj Thiouracil

107

BY 2-THIOURACIL

viruses. Unfortunately, lesion size could not be measured because thiouracil caused a chlorosis of the host tissue and lesion margins could not be ascertained. Sap infectivity tests, however, showed that CCMV synthesis was enhanced and TJIV was inhibited in the same host (Table 7). Simultaneous tests with TMV on tobacco conGrmed Holmes (1955) results which established the inhibitory effect of thiouracil. DISCUSSION

The enhancement effect of thiouracil on CCMV in a hypersensitive host is clearly in contrast to the reduction in lesion size and number caused by the chemical with TMV (Holmes, 1955) and tobacco necrosis virus (Francki, 1962). All previous reports, in fact, indicate that thiouracil either inhibits virus multiplication and infectivity, or, in some host-virus combinations (Bawden and Kassanis, 1954), has no effect). In addition to the breakdown of resistance in soybean, CCMV sap infectivity was enhanced and T!MV sap infectivity was inhibited by thiouracil in t.he same host’, Chen,opodium amaranticolol-. Thus, the primary t,hiouracil effect may be on virus synt)hesis, not on the host or virus localizatJion. Thiouracil inhibits

Application

Two other methods of t’hiouracil application were attempted: (1) spraying intact plants and (2) inserting excised stems into tubes. Both methods caused CCMV lesion area increases similar to the petri dish method, but neither method was consistent because of the bhiouracil effect on the host. The petioles of sprayed plants became necrotic in 3-5 days, and the leaves frequently abscissed. With excised plants in tubes, the relative humidity of the incubation quarters (usually the greenhouse) determined thiouracil uptake and the chemical effect varied from test to test. Differential

E$ect of Two Viruses on the Same Host

Since the effect of thiouracil on CCMV in soybean is obviously different from that on TMV in tobacco (Holmes, 1955), a test was designed with Chenopodium amaranticolor L. which is a local lesion host for both

TABLE EFFECT OF THIOURXIL INFECTIVITY

Virus

TMV CCMV

IN

7

ON TM\’

Chenopodium AT 27"

SND CCMV

SAP

amaranticolor

Treatment

Thiouracil Water Thiouracil Water

20 118

16 110 25 9

0.15 2.78 -

a A combination of the deleterious effects of thiouracil on the host and small lesions made it impossible to measure the lesions or to count the CCMV lesions with accuracy. b Infected Chenopodium tissue was used to inoculate the appropriate assay hosts.

108

KUHN

TMV in both hypersensitive and susceptible hosts whereas CCMV is enhanced in both types of host (Dawson and Kuhn, 1969). The chemical, therefore, could inhibit the formation of localization factors for both viruses, but its inhibitory effect on TMV synthesis could be so great that lesions fail to develop or enlarge. On the other hand, CCMV synthesis appears to be stimulated by thiouracil and the blockage of localization factors would allow lesions to develop unimpeded. Other chemicals have been shown to increase the susceptibility of hypersensitive hosts to TMV. Actinomycin D causes T&IV lesion area to be increased about seven times in Phaseolus vulgaris (Loebenstein et al., 1969). In Nicotiana glutinosa L., chloramphenicol (Sela et al., 1969) increases TMV lesion size by 3.2 times, and six cytokinins (Milo and Srivastava, 1969) prevented TMV lesion formation, but stimulated virus synthesis by 1.2-3.8 times. Ascorbic acid reduces TMV lesion number, but it appears to stimulate virus synthesis and lesion size (Farkas et al., 1960; Parish et al., 1965). Although the biological reaction of these chemicals is unknown, it may be similar to the reaction caused by thiouracil with one major exception; thiouracil inhibits TMV multiplication, and the other chemicals enhance its biosynthesis. The fact that thiouracil caused an increase in lesion numbers is probably related more to an increase in lesion size than to a change in the number of infection sites. Helms et al. (1962) reported both macro and micro lesions of TMV on Pinto bean and suggested that lesions vary in size due to heterogeneity of lesion sites. A similar condition occurs with CCMV, where some necrotic areas can be observed with magnification only, and these probably enlarged to macro size under the influence of thiouracil. Furthermore, lesions vary less in relative size after t,hiouracil treatment than they do normally, indicating that factors controlling size have been overcome. There has been a general assumption that thiouracil interferes with ribonucleic acid (RNA) metabolism. This is true not only for plant viruses, but for various plant physiological reactions as well (Woodstock

and Brown, 1963; Inouye, 1965). A few reports, however, have indicated that thiouracil causes a stimulation of specific reactions or chemicals in plants. The most nobable stimulation is the marked increase, in t’he production of turnip yellow mosaic virus (TYMV) protein shells (no RNA) when thiouracil treatment is initiated 5, days after inoculat,ion (Francki and Matthews, 1962). Ralph and Wojcik (1966) found that thiouracil stimulates (3H) ATP’ incorporation into double-stranded TYMVRNA in cell-free extracts. The synthesis of a specific enzyme, carbamyl phosphate synthetase-1, in tadpole liver cubes, is enhanced by thiouracil treatment (Cohen, 1970). Porter and Weinstein (1960) reported that thiouracil causes increases in the free amino acid and amide-nitrogen pool, but they attributed this to inhibition of protein synthesis. Although these various stimulatory effects of thiouracil cannot be related to enhanced CCMV activity at this time, they do indicate that thiouracil can affect protein and RNA metabolic reactions in extremely different ways. My results enhance the value of thiouracil as a research tool for studies in three significant areas. A comparison of the dissimilar action of thiouracil on biosynthesis of CCMV and TMV may lead to new insights into RNA metabolism. Second, the increased synthesis of CCMV will allow purification and charact,erization of a virus from a hypersensitive host, notwithstanding the possibility of virus alteration by thiouracil. Third, the exact mechanism of virus localization in a hypersensitive host remains obscure despite many reports, and thiouracil appears to alter the reaction more than ot,her treatments, chemical or physical. ACKNOWLEDGMENTS I wish to thank Miss Joan Council for assistance while participating in the National Science Foundation Secondary Science Training Program. REFERENCES BAWDEN, F. C., and KASSANIS, B. (1954). Some effects of thiouracil on virus-infected plants. J. Gen. Microbial. 10, 1fX-173. BAWDEN, F. C., and ROBERTS, F.M. (1948). Photosynthesis and predisposition of plants to infec-

INCREASE

IN

CCMV

INFECTIVITY

tion with certain viruses. Ann. Appl. BioE. 35, 418-428. BRANTLEY, B. B., and KUHN, C. W. (1970). Inheritance of resistance to southern bean mosaic virus in southern pea, Vigna sinensis. J. Amer. Sot. Hart. Sci. 95, 155-158. COHEN, P. P. (1970). Biochemical differentiations during amphibian metamorphosis. Science 168, 533-543. COMMONER, B., and MERCER, F. (1951). Inhibition of the biosynthesis of t,obacco mosaic virus by thiouracil. Nature (London) 168, 113-114. COMMONER, B., and MERCER, F. (1952). The effect of thiouracil on the rate of tobacco mosaic virus biosynthesis. Arch. Biochem. Biophys. 35, 278289. DAWSON, W. O., and KUHN, C. W. (1969). Enhancement of virus infectivity in plants treated with manganese or 2-thiouracil. Ga. Acad. Sci. 27, 82-83. FARKAS, G. L., KIRALY, Z., and SOLYMOSY, F. (1960). Role of oxidative metabolism in the localization of plant viruses. Virology 12, 408421. FR~NCKI, R. I. B. (1962). The inhibition of plant virus multiplication in two host species by 2-thiouracil. Virology 17, l-8. FRANCKI, R. I. B., and MATTHEWS, R. E. F. (1962). Some effects of 2-thiouracil on the multiplication of turnip yellow mosaic virus. Virology 17, 367-380. GAY, J. D. (1967). Specific infectivity of cowpea chlorotic mottle virus in different host. Ph.D. Thesis, Univ. of Georgia. HELMS, K., and MCINTYRE, G. A. (1962). Studies on size of lesions of tobacco mosaic virus on pinto bean. Virology 18, 535-545. HOLMES, F. 0. (1955). Preventive and curative effects of thiouracil treatments in mosaic-hypersensitive tobacco. Virology 1, l-9. INOUYE, J. (1965). Effects of chemicals on flower bud initiation in cereals. I. Effect of 2.thiouracil on rice plants grown under aseptic conditions. Plant Cell Physiol. 6, 131-134. KASSANIS, B. (1957). Effects of changing temperature on plant virus diseases. Advan. Virus Res. 4. 221-241. KUHN, C. W., and TEAS, H. J. (1968). Increased resistance to tobacco mosaic virus in gamma irradiated tobacco leaves. Radiat. Bot. 8,317-323.

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109

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