Differential response of prunus necrotic ringspot and Tulare apple mosaic viruses to stabilizing agents

VIROLOGY

46,

Differential

613-W

(1971)

Response Mosaic

of Pronws Necrotic Viruses

Ringspot

to Stabifizing

0. W. BARNETT

AS-D

and

Tulare

Apple

Agents

R. W. FULTOS

Department oj Pkmt Pathology and Fhyriiology, Clemson Unl.‘vsrsily, Clemson, South, Carolinct $9631; and Department of Plan,t Pathology, L’nivewit~J oj W’iscon,nin,, Madison, Wisconsin

53706

Prunes rlecrotic ringspot (NRSV) and Tulare apple mosaic (TAMV] viruses axe rapidly inactivated in sap of infected cucumber. The h&-life of NRSV-G was lo;! min and that of TAMV 114 min. TAMV was stabilized by 0.02 & 2-mercaptoethanol (Z-ME) but not by 0.02 M sodium diethyldithiocarbamate (DIECA) while NRSV was stabilized by DIECA, but not by Z-ME. This response to st*abilizing agents was the same when either purified virus was added to sap from plants infected with the other virus. Purified TAMV was inactivated by I)IECA and purified NRSV was inrtctivated by 2-ME. Purified TAW and NRSV were inactivated rapidly in 0.01 M pchlorumcrcuribenzoat,e but NRSV was not inactivated as rapidly as TAMV by 0.02 1% iodoaeetamide. The inactivating .._svstem in healthy or infected cucumber was I sedimented by eentrifu~ation. INTRODUCTION

disks of infected cucumber cotyledons in 0.03 M phosphate buffer, pH 8 (15 disks in 1 ml buffer L- 1: 5 dilution). The imxtivator concentration in a 1: 5 dilution was sufficient. to inactivate most of the virus in 4 hr of aging. Immediately and after aging, wuall~ for I, 2, and 4 hr aftrr grinding? samples w~rf: sufficiently dilutc>d with buffer so Chat numbers of lesions upon inoculaGon would bt: wuntablc. This t nchniyue permit,tcd aging the virus at onr concentration of inhibiting or stabilizing substance: but inoculabing at :I lower c~)~~ce~~,rati~~r~ and had the cffoct of decreasing any effect additives might. hnw on infection. Final dilutions usrd for inorxlations varied with the season, but ctou~~t.nhtc numbers of lesions could usually bc?obtainwl \vith final dilutions of 1:40 for NRSV and MATERIALS A?;D METHODS 1: 120 for TM/IV. When purified virus Virus idates und inoculation p~~~c~~;ures. (Barnctt and E’ult,on, 1969) was aged in addiThe TAMY, NRSV-N (Barnett and F&ton, tives or plant, sap, tho virus was added at 1969) and NRSV-G (Fufton, 1957) isolat,es S-8 times the ~oi~~~en~ra~~onnrtded for were periodically transferred from &her countable fcsiorrs and diluted to an esstimatrxf desiccated cucumbrr tissue or Prwzus w&nnzso = 0.01”-0.05 for iI~~)culatio~~. Purific*d virus was added to the aging solution just 2eb L. to Cucumisl sativus L. cv. Lemon. Inoculum was propared by grinding &mm prior t 0 tlir! xcso timck inoculation or, if plant Some labile viruses are st,abilized in plant sap by reducing Bgents, such as 2-mercaptoethanol @-ME); whereas others require a chelating agent, such as sodium diethyldithiocarbamate (DIECA), for stabilization. We have used Tulare apple mosaic virus (TAJIV), a virus stabilized by 2-M& and Prunus necrotic ringspot virus (NRSV), a virus stabilized by DIECA, in cucumber extracts to show that t’he host is not rcsponsible for the differential response to stabilizing agc2nt.s.Some of the intrinsic virus properties responsible for this differential stabilization were also determined. A preliminary report has been publislwd (Bamett, and F&ton, 1970).

614

BARHETT

AND FULTON

material was included, just prior to grinding C~~~c~~ used. A commercial ~re~~at~on the plant material. of catechof oxidase {m~hroom ty~~~~~e, Assays of virus infeeti~rity were made on ~ort~mgton Bio~hen~eals) was used to local lesion hosts; ~~~~~~~~cu~~s~~~~~ L, oxidize ~-coumari~ acid, a phenol found in for NRSV-G or H, ~olic~os bi$O?%8 L. for cucumbers (Engelsma and Meijer, 1965). NRSV-H, and Phaseolus vu$garis L. cv. The reaction was carried out using high conBountiful for TAMV. Inoculations were centrations of phenol and enByrne to assure made to the halves of primary leaves, lightly virus inactivation in a short time. dusted with corundum. Four aging times of R*ESULTS the same treatment were compared on the same plant and each aging time appeared an equal number of times on left and right halfNRSV and TAMV were propagated in leaves of four plants. Calculation of ~al~-l~~eof ~33s ~~~ect~~~~. cucumber cotyledons and aged in the diluted The half-lives were calculated using the sap of infected tissue with or ~thout stabilize equation suggested by Yarwood and Syl- ing agents. ~itbont stabi~zing agents, t&e vester (1959). The number of local lesions infectivity of both viruses was almost nil was pIotted against Gme, and the line of best after aging 4 hr (Table 1). When 0.02 X fit wa.~,determined by linear regression (Steel Z-ME was added prior to grinding the inand Torrie, 1960). The zero and 2-hr values fected tissue, the half-life for TAMV more than doubled. The infectivity of NRSV was were used to calculate half-lives. S~~~s~~~ u~~~~~s. Lesion counts plus 1 not changed si~i~~a,ntly by adding 2-ME, were analyzed by a statistical analysis sys- although there appeared to be a consistent tem developed by A. J. Barr and J. H. Cood- tendency for more infectivity after 4 hr when 2-ME was present than when it was not. Innight (North Carolina St,ate University) using the transformations suggested by creasing the concentration of 2-ME to 0.05 Kleczkowski (1955), His transformation for or 0.1 M gave no better stabilization of small means was used on all lesion counts TAMV or NRSV. When 0.02 X DIECA was except those for Table I, where the transfor- added prior to ~r~~di~g tissue the infectivity mation for means greater than 10 was used. of TAMV was lost faster than when aging Differences between means were determined was in bu.Ger alone. NRSV, on the other by Duncan’s multiple range test (Steel and hand, was st,abilized by DIECA and the halflife was more tha,n twice that of the virus Torrie, 1960). TABLE 1 EFFECTS OF 2-~~ERCAPTOETHANOL (2-M%) AND SODIUM DIETHYLD~TWIOCARBAMATE(DIECA) ON TElE Loss OF INFECTIVITY OF NECROTIC RXNQSPOT(NRSV-G) AND TULARF: AP~,E MOSAIC (TAMV) VIRUSES

.~ Virus

Aged at i :S dilution in8

NRSV-G

Buffer 2-ME DIECA

TAMV

Buffer Z-ME I3IECA

XN SAP

OF fiiFi2C~ED

CUCUMBER

Average number lesions per half-leaf after aging and further d&&ion* -~ -.---0 4Rr 1 Hr 2Hr 104 d 92 d 84 d

85d 85 d 87 d

23 f 50 de 65 d

O.l& 5g 31 ef

Half-life (in) 102 132 222

389 a 21 c 316 a 114 108 ab 413 a 282a 284 a 185 ah 266 334 a 9c Oc 84 88 b __..“_~_ --““~---” ”,.,--.. * Four trials with four half-leaves per aging time. Local lesion hosts were ~~~~rd~~u ~a~~u~~~a for NRSV-O and Bountiful bean for TAMV. Letters after each figure indicate significance at the 1%level; figures followed by the same letter are not si~ui~ea~~Iy diEerent. The data for each virus were analyeed separately. ~ee~~~eu~ of variation = 1O70 for NRSV, 17% for TAMV. @Phosphate buffer, 0.03 M, pII 8; 0.02 J& 2-&m or 0.01 H DIECA in km&z.

DIFFERENTIAL

RESPONSE

(il.5

TO STABILIZ$TION

from cucumbers infected with the other virus (Table 2). Both viruses also responded to the addition of 2-ME and DIECA in the same manner as when aged in sap from cucumber tissue infected with the virus being assayrld, i.e., as in Table 1. There was no indication in these experiments of a specific virus-induced inactivator; the next experiments were designed to determine whether the differential response to stabilizing agents was a result. of a difference in the intrinsic nat,ure of 1he viruses. Both viruses were aged in a synthetic inactivating system composed of catechol oxidase and p-coumaric acid. Catechol oxidase alone reduced the infectivit,y of both viruses after 24 hr, but only TAMV infectivity was significantly reduced by p-coumaric acid. The combination of catechol oxidase and p-coumaric acid completely inactivated both TAMV and NRSV in 30 min (Table 3), When either DIECA or 2-ME was added most of the infectivity of TAMV and KRSV survived 30 min aging except when 2-&IE was added to NRSV. After 24 hr aging, hoivever, NRSV infectivity remained only when DIECA was added and TAMV infectivity remained only when 2-ME was added. Differences in the intrinsic nature of the viruses seemed the most likely reason for 111~

aged in buffer alone. Increasing the concentration of DIECA to 0.05 M hastened the inactivation of TAMV and did not increase the stability of NRSV. At 0.1 M DIECA infectivity of NRSV decreased. Thus the differential response to stabilizing agents found by Hampton and Fulton (1961) and Fulton (1967) with the viruses in different hosts occurs when t,he viruses are propagated in the same host. Diferential Response of the Viruses to Stabilizing A gents There appeared to be at least two possible explanations for the response of the viruses to the stabilizing agents. Either the viruses induced the host to produce different inactivators which respond differently to stabilizing agents or differences in the viruses were involved in the differential response to stabilizing agents. The possibility of virus-induced inactivators was tested by aging purified virus in the sap of plants infected by the other virus. For these t,rials a different strain of NRSV, NRSV-H, was used. NRSV-H does not produce lesions on Bountiful bean while TAMV produces only a few very tiny lesions on D. biflorus, a local lesion host for NRSV-H. Both NRSV-H and TAMV were inactivated rapidly when aged, without addibives, in sap TABLE

2

EFFECTS OF %MERCAPTOETHANOL (2-ME) AND SODIUM DIETHYLDITHIOCARBAMATE (DIECA) ON IsFECTIVITY OF PURIFIED NECROTIC RINGSPOT (NRSV-H) OR TULAR~ APPLE Mos.~c (TAMV) VIRIJSI~X IN EXTRaCTS OF CUCUMBER INFECTED WITH THE OTHER VIHUS

-

Purified virus __--_

Aged at 1: 5 dilution of infected cucumber tissue inb

__-.

Average number lesions per half-leaf and dilution0

after aging

0

1 Hr

2 Hr

1 Hr

NRSV-H

Buffer 2-ME DIECA

3c 4-c 13 a

Od Id 9 ah

Od Od 6b

0 d 0 d 7 1)

TAMV

Buffer 2-ME DIECA

11-l A 152 A 72 A

56 BB 79 .4B 46 H

51 AB 127 .4 13 c

11 C’ 98 AH 0.2 1)

a Four trials with four half-leaves per aging time for TAMV; three trials with three half-leaves per aging time for NRSV-H. Local-lesion hosts were DoZichos bijlorus for NRSV and Bountiful bean for of NRSV-H, TAMV. Concentration after 1:s dilution of TAMV, A 260 = 0.01-0.02; after 1:5 dilution at the 1% level; capital letters indicate the A 260 = 0.04. Small letters after figures indicate significance 5% level; figures followed by the same letter are not significantly different. The data for each virus were analyzed separately. Coefficient of variation = lUyO for NRSV, 25y0 for TAMV. b Phosphate buffer, 0.03 M, pH 8; 0.02 M 2-ME or 0.01 M DIECA in buffer.

616

BARNETT

AND FULTON TABLE

3

EFFECTS OF ~-~EW.XPT~ETHANOL ('L-ME) AND SODIUM DIETHYLUITHIOCARRAMATE (DIECA) ON INF~CTWITY OF PURIFIED NECROTIC RINGSPOT (NRSV-H) AND TU~ARE APPLE MOSAIC (TAMV) VIRUSES IN .4 p-COUMAMC hcru CATWHOL OXIDASIG-INACTIVATXNO SYSTEM --

Average number lesions on 12 half-leaves from purified virus, aged in different treatments diluted 1:8 with 0.03 &f phosphate buffer, pH 80 Treatment

Phosphate buffer, 0.03 M, pH 6.5 Catechol oxidase, 0.05 mg/ml buffer p-Coumaric acid, 0.001 M in buffer Catechol oxidase + p-coumaric acid in buffer Cateehol oxidase + p-coumaric acid $ 0.02 M DIECA in buffer Catetechol oxidase -I- p-coumaric acid + 0.02 M 2-ME in buffer

NRSV-H aging time

TAMV aging time

30 Min

24 Hr

34 bed

15 de

21 cde

3 fs

36 bed 0.3g 20 de

5 efg

3oMii

24

Hr

156 a

11 de

15%a 136 a 129 a

og 6 efg

og 182 a

oa: Og

0g

135 a

118 a

51 bc 61 b

0 Three trials with four half-leaves per aging time. Local lesion hosts were ~o~ord~~~ ~a~~~rn~n~for NRSV and Bountiful bean for TAMV. Concentration after dilution of TAMV, Atso = 0.01402; for NRSV-II, Aleo = 0.05. Letters after figures indioat,e significance at the 1% level; figures followed by the same letter are not significantly different.

differential response of TAMV and NRSV to the stabilizing agents. In separate trials with only 0.02 M DIECA in 0.03 M phosphate buffer, infeetivity of TAMV did not decrease for 4 hr at pH 6.5, although it was low after 24 hr. At pH 8.0 infectivity was completely lost after 4 hr. In sodium ethylene ~a~netetraa~et,at~ (EDTA) TAMV was still infective even after 3 days at room temperature. Purified TAMV has been kept at 2” in 0.01 M EDT& pH 6.0, for many months wit,hout detectable loss of infectivity. Infectivity of NRSV-H in 0.03 M phosphate buffer, pH 8, with added 2-ME (0.02 ti) was less after 30 min and t,he virus was not infective after 24 hr aging. In other trials in 0.02 M Z-ME, NRSV-H infectivity was ~changed after 4 hr at pH 6.5 but was very low after 4 hr at pH 8.0. Thus, TAMV was inactivated by DIECA and NRSV by 2-ME and purified TAMV in DIECA and NRSV-H in 2-ME were more labile at pH 8.0 where infectivit,y of NRSV was greatest (Fuhon, 1957). To determine whether the host systems inactivating TAMV and NRSV were similar, tissue containing each virus was ground and cent~fuged (105,O~ g, 96 min) under nitrogen. Purified TAMV was aged 4 hr in the

TABLE

4

EFFECT OF CENTRIFUUATION OF CUCUMBER EXTRACTS ON THE INFECTIVITY OF TULARE

APPLE MOSASCViRUS

Fraction obtained by

cen~~fu~tion=

1 Average local lesions per ha&leaf I from 3 trials of 4

haIf-Ieavesper trial after 4 Ix in the fractionsb

Pellet, 15 min at 1000 g Pellet, 15 min at 6500 g Pellet, 15 min at 10,300 g Pellet, 90 min at 105,O~ g Supernatant fluid, 90 min at 105,009 g Phosphate buffer, 0.03 JK, pH 3

31 b 18 b lc 12 b 100 a 9oa

a Successive fractions of healthy cucumber extracts in 0.03 M phosphate buffer, pH 8, obtained by differential oentrifugation and mixed with purified TAMV (ASSO ca. 0.02). b Local lesion host was Bountiful bean, Letters after figures indicate significance at the 1% level; figures foIlowred by the same Ietter are not significantly different.

supernatant fluid from TAMV-infected cucumber and purified NRSV-H in the supernatant fluid from Neal-H-infested cucumber. No inactivation occurred (two trials on

I~IFFERENTIAL

RESPONSE TABLE

617

TO PTABTI~IZATION 5

EFFICCT OF SULFHYDRYL REAGENTS, IODOACETAMIDE AND p-CHLOXOMERCURIBF,NZOATE (PCMB), ox INFECTIVITY OF NECROTIC HIN~SPOT (NRSV-H) AND TGLARX APPLE MOSAIC (TAMV) V~nrs~s

Average number locallesions perhalf leaf after aging” __-_ -4 Hr I Hr 2 Hr 0

Virus aged in

Virus

Phosphate buffer, 0.03 IM, pH 8

NRSV TAMV

37 cde 184 :t

32 rde 88 b

34 cdc 87 c

23 det’ 4T2et

13uffer-j- 0.02 M iodoacetamide

NRSV TAMV

36 cd 75 b

22 def 3 11

14 fg Lh

9g 0 11

Buffer -f- 0.01 iM PCMB

NRSV TAMV

14 fg 2h

0.4 h Oh

_--.

L-

0.2 h Oh

0.2 h 0 h

= Three trials with four half-leaves per aging time. Local lesion hosts were Mormordica balsamina fw SRSV and IBountiful beanfor TAMV. Letters after figuresindicate significanceat the 1% level; figures followed by the samelett,er are not significantly different,.

four half-leaves per trial for each virus). This experiment indicated that both viruses were inactivated by a system sedimented by the centrifugation. In further experiments with TAMV, substances in the pellet from healthy cucumber tissue ground in air inactivated the virus (Table 4). After 4 hr aging, infectivit,y of TAI\!lV in the supernatant fluid did not differ from the infectivity in phosphate buffer.

trials with 0.05 M histidine, lysine, asparagine, or glutamine indicated that the amides might have a slight stabilizing effect. In two subsequent,trials with infected cucumber sap neither TAMV nor NRSV was stabilized by asparagine or glutaminc. L)ISCUSSION

The stabilization of TAMV by a reducing agent and NRSV by a chelating agent was confirmed even ivhen the viruses were produced in the same host and aged in concenIodoacetamide and p-chloromercuriben- trated sap. The inacbivating system of known aoate (PCMB), which react specifically with components further confirmed the differensulfhydryl groups, were tested for their effect tial response of the viruses to stabilizing on the purified viruses. Purified TAMV and agents and also demonstrated that the virus, NRSV were inactivated rapidly in 0.01 M not the inactivating system, was t,he basis of IYXB (Table 5). TAMV was also inacti- t*he differenbial response. Both Z-ME and vated rapidly in 0.02 ,W iodoacetamide, but DIECA should prevent virus inact’ivation SRSV was not inactivated as rapidly by by the $7..coumaric acid-catechol oxidasr! iodoacetamide as by PCMB. Regression system; 2-ME because the sulfhydryl group analysis showed that, the slopes of infectivity would either react,with the quinone or reducti loss wit,h time were signi~cantly di~erent be- t,he quinone back to ~-~ouxnarie acid and tween viruses in each treatment and between DIECA because the topper prosthct.ic group treatments for each virus of the enzyme would be removed by chclr\tion (Pierpoint, 1966). Fffecl of A mim7 A cirls Inactivation of TAMV by DIECA was greater at pH S than at pH 6.5. The half-life Quinones not only react with sulfhydryl of DIECA is much longer at, pH S than pH groups, but also rvith amine groups (&son, 19%). There are also reports of enzyme sys- 6.5 (Hallaway, 1959) so the brcakdo~~n tems being protected by amino acids when products, dirthyIamin~ a’nd carbon disulfi&, glutat,hione or cysteine had no protective are probably not involvrd in the inactivaticm of TANV. The :Icstion of DIECA on ThJI\Y ef!fec+(l’at,il and Dimond, 1967). Preliminary

618

BARNETT

AND FULTON

may not be the same in sap as in buffer. In sap an enzymatic breakdown of DIECA, as suggestedby Mink (1967), might be involved in virus inact#ivation. Inactivation of NRSV by 2-ME was unexpected since in plant sap infectivity has usually been slightly higher with 2-ME added than without 2-ME (Table 1). This is probably the result of inhibition of the inactivating system more rapidly than the virus was affected. The inactivation of both NRSV and TAMV by PCMB may be due to different reasons if our earlier interpretation of the reactions with 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) was correct. The sulfhydryl groups of NRSV reacted slowly with DTNB while those of TAMV reacted rapidly. This data indicabed that the cysteine residues of NRSV were probably located between morphological units while the cysteine of TAMV was not (Barnett and Fulton, 1969). PCMB could react with the shielded sulfhydryl groups of NRSV and cause inactivation due to interference with the conformation of the viral protein coat. Boyer (1959) postulated that PCMB reacted with sulfhydryl groups that were temporarily exposed by small configurational changes in the protein. Since the sulfhydryl groups of TAMV do not appear to be shielded, PCMB might inactivate the virus by hindering the infection process. Mink et al. (1963), however, found no inactivation of TAMV by PCMB at a lower pH. Iodoacetamide, although supposedly also reacting specifically with sulfhydryl groups at pH 8 (Cecil, 1963), inactivated NRSV more slowly than it did TAMV. Reichmann and Hatt (1961) proposed that small radicals, such as acetyls, did not degrade potato virus X but that bulky radicals, such as mercuribensoate interfered with linkage sites resulting in degradation of the virus particle. The effects of PCMB and iodoacetamide on inactivation of TAMV and NRSV support our interpretation of the involvement of sulfhydryl groups in the intersubunit, bonding of NRSV (Barnett and Fulton, 1969). The inactivating system in cucumber resembles that found by Bawden and Pirie (1957) in tobacco, which also sedimented at low centrifugal force. The inactivating system was present, in healthy as well as in in-

fected tissue but no attempt was made to determine relative amounts. Hampton and Fulton (1961) found higher catechol oxidase activity in NRSV-infected than in healthy cucumber tissue. Catechol oxidase activity may not be involved in the sedimentable inactivating system of cucumbers since the phenol-catechol oxidase system of tobacco remains in the supernatant, after low speed centrifugation (Harrison and Pierpoint, 1963). ACKNOWLEDGMENT The authors thank Dr. W. E. Johnston for help with statistical analysis by computer. Research supported by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, by Northeastern Regional Cooperative Project NE 14, and by Grant 13911 from the National Science Foundation. REFERENCES BARNETT, 0. W., and FULTON, R. W. (1969). Some chemical properties of Prunus necrotic ringspot and Tulare apple mosaic viruses. Virology 39, 556-561. BARNETT, 0. W., and FULTON, R. W. (1970). Differential stabilization of certain labile viruses. Phytopathology 60, 1283 (Abstr.). BAWDEN, F. C., and PIRIE, N. W. (1957). A virusinactivatingsystem from tobacco leaves. J. Gen. Microbial. 16, 696-710. BOYER, P. D. (1959). Sulfhydryl and disulfide groups of enzymes. In “The Enzymes,” P. D. Boyer, H. Lardy, and K. Myrback (eds.), 2nd ed., Vol. 1, pp. 511-588. Academic Press, New York. CECIL, R. (1963). Intramolecular bonds in proteins. I. The role of sulfur in proteins. In “The Proteins,” H. Neurath (ed.), 2nd ed, Vol. 1, pp. 379476. Academic Press, New York. ENQELSMA, G., and MEIJER, G. (1965). The influence of light of different spectral regions on the synthesis of phenolic compounds in gherkin seedlings in relation to photomorphogenesis. I. Biosynthesis of phenolic compounds. Acts Bat. Neer. 14, 5472. FULTON, R. W. (1957). Properties of certain mechanically transmitted viruses of prunus. Phytopathology

47, 683-687.

FULTON, R. W. (1967). Purification and some properties of tobacco streak and Tulare apple mosaic viruses. Vivirology 32, 153-162. HALLAWAY, M. (1959). The stability of sodium diethyldithiocarbamate in biochemical experiments. Biochim. Biophgs. Acta 36, 538-540.

DIFFERENTIAL

RESPOTSE

HAMPTON, R. E., and FULTON, R. W. (1961). The relation of polyphenol oxidase to instability in vitro of prune dwarf and sour cherry necrotic ringspot viruses. virology 13, 4452. HARRISON, B. D., and PIERPOINT, W. S. (1963). The relation of polyphenoloxidase in leaf extracts to the instability of cucumber mosaic and other plant viruses. J. Gen. Microbial. 32, 417427. KLECZ~OWSI~I, A. (1955). The statistical analysis of plant virus assays: a transformation to inrlrlde lesion numbers with small means. J. Gen. Microbial. 13, 91-98. MASON, H. S. (1955). Comparative biochemistry of the phenolase complex. Advan. Enzymol. 16, 105-184. MIXI<, G. I. (1967). An indirect role of cytochrome osidase in loss of prune dwarf virus infectivity in squash t,issue homogenates. Phytopathotogy 57, 797-798.

TO STABILIZATIOK

c119

MINK, G. I., N.~NCILOFI-, J. B., and NMAKAKKAREN, M. J. (1963). Properties of Tulare apple mosaic virus. Phytopathology 53, 973-9i8. PATIL, S. S., and DIMOND, A. E. (1967). Inhibition of Verticillium polygalacturonase by oxidation products of polyphenols. Phytopathology 37, 492-496. PIERPOINT, W. S. (1966). The enzymic oxidatiotl of chlorogenic acid and some reactions of the quinone produced. Biochem. J. 98, 567-580. REICHMANN, &I. E., and HATT, D. L. (1961). The effect of the substitlltion of sulfhydryl groups on the macromolecular structure of potato virus S. Biochim. Biophys. dcta 49, 153-159. STEEL, R. G. I).. and TOHRIE, J. H. (1960). “Principles and Procedures of Statistics.” McGraw Hill, New York. YARWOOD, C. IX.. and SYLVESTER, E. S. (1959). The half-life concept of longevity of plant, pathogens. Plant Dis. Rep. 43, 125-128.