increased disease resistance enzyme activity induced ...plant physiol. (1966) 41, 1505-1512...

Plant Physiol. (1966) 41, 1505-1512

Increased Disease Resistance and Enzyme Activity Inducedby Ethylene and Ethylene Production by Black Rot

Infected Sweet Potato Tissue'M. A. Stahmann, B. G. Clare,2 and W. Woodbury

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin

Received May 2, 1966.

Summiary. Exposuire of root tissuie from a susceptible varietj- of sweet potato tolow concentrations of ethylene induiced a resistance to infection by Ceraitocvstisfimlbriaita and an increase in the activity of peroxidase and polyphenoloxidase in thetissuie. Susceptible tissuie that was inocLulated with a pathogenlic strain of C. fimzbri(ataor a nonpathogenic strain that can indtuce resistance liberated more ethylene intoclosed chambers than tissuie inocuilated wvith strains that di(d not induice resistance. Itis stuggested that ethylene may be a stimuluis that diffulses from infected areas intoadjoining tissue to initiate metabolic changes which may lead to disease resistance.Polyphenol oxidase bult not peroxidase activity was increased in slices of potato ttubersand parsnip roots treated with ethylene. The activity of these enzymes in root tissuleof carrot, radish or tuirnip was not altered by ethylene treatment.

The disease of sweet potato roots known asblack rot is cauised by certain isolates of the ftungusCeratocystis fimitbriaita Ell. and Halst. The suirfaceof root tissue which is normally suisceptible to blackrot can be made resistant to infection by prior in-ocuilation with nonpathogenic isolates of C. fittt-briata (16, 17). Root tissuie with this induicedl re-sistance has increased peroxidase and polvphenoloxidase activitv (16, 17) similar to that fouindl ininoculated natuirally resistant tissuie. V'olatile ma-terials from infected root tissute increased theactivity of these enzvmes and increased the resist-ance of uninfected suisceptible root tissuie (5).Preliminary experiments showed that volatile ma-terials from apples are able to induice similar in-creases in enzymic activity and resistance in nor-mally stusceptible tissue (5). Since apples areknown to produice ethylene (2) these resuilts gavesuipport to the view that ethylene may be involvedin the changes noted in sweet potato tissuie.Ethylene is produced b.' a variety of injtured andinfected plant tissuies (12) and it was consi(leredthat it may be the active volatile material produticedby diseased sweet potato tissule.

This paper reports the effect of ethylene on theresistance of sweet potato roots anid its effect oIn

I Sup)orted by funds from the Research Comiimitteeof the Graduate School, University of Wisconsin fromfunds supplied by the Wisconsin Alumni Research Foun-dation and by funds from the Herman Frasch Foundationfor Agricultural Research. Second author in receipt ofFulbright Travel Grant.

- Present address: Waite Agricultural Research In-stitute, University of Adelaide, Ade!aide, South Auistralia.

the enzymatic activity of roots of sweet potato,parsnip, carrot, tuirnip, radish and white potatotuibers. The produiction of ethylene by sweet potatoroot tissuie inoctulated with pathogenic and non-pathogeniic isolates of Cer(atocystis is also reported.

Materials and Methods

Preparatioi of Plait Tissiue. Tissuies uised werewhite potato tuLbers (Solanumm tuberosuim) and rootsof sweet potato [Iptiloeoa b(at(itas (L) Lam.], carrot[Dattucus cairotai (L) var. sativa], parsnip [Paistinalcasativa (L)], tuirnip [Brassica rapa (L)] and radish[Raphlinu(s satizvis (L)]. The sweet potato varietieswere jtulian (sutsceptible to black rot) and( Suinny-side (resistant to black rot). WVhole roots or tuberswere sutrface sterilized with sodiutm hypochloritesoluttion and ctut into slices 2 cm thick. These sliceswere inocuilated or treated with ethylene.

Inoculation of Szweet Potaito Tissute. Ctlltuiresof a pathogenic isolate of C. fimiibriat(a Ell. andHalst. (isolate U-6), another isolate of C. fimibriata(F-38) which is nonpathogenic and induLices resist-ance and a nonpathogeni, C. miliinor (Hedg.) Huintwhich does not indul1ce resistance, were grown oInpotato dextrose agar suipplemented with thiamine(14) at 250 for 1 week. Spores from these cultureswere suispen(le(l in sterile distilled water aindl uisedlas inoculuim. The cuit suirfaces of sweet potatoslices were inocuilated with 1 ml of spore suispelnsion.Control slices were treated with sterile distilledwater. The slices were placed on stainless steelscreens above w-et filter paper at the bottom ofcrystallizing dlishes (15 cm X /7.5 cm). Another

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PLANT PHYSIOLOGY

crystallizing dish of the same size was invertedover each (lish containinig root tissute. The jtunctionlhetween each set of dishes was covered with a stripof alutminutm foil and seale(d with adhesive tape.Approximately 200 g of tissute was place(d in eachchamber which ha(l a volume of 2.6 liters. Thetisstie was incutbated at room temperatuire for tipto 6 days.

Gas Anattlysis. Samples wvere withdrawnv fromthe chambers described above usling a Hamilton gassyringe. Analyses were carried outt in an Aero-graph 1520 gas chromatography apparatuts eqltippedwith a hydrogen flame ionization (letector. .A stain-less steel colutmn (152 cm X 3 mm) packe(d with15 % diethylene glycol sutccinate (D)EGS) on hexa-methyldisosilazaine (HMDS) treatedI ChromosorbW\ (80-100 mesh) was tused at 240. Nitrogen car-rier gas was sutpplied at 15 ml per minutte. Hydro-getn flow wvas 32 ml per minulte.

Treatmnent of Tissues with Etlhylentc. Slices oftissute aboutt 2 cm thick were placed in 20 1 con-tainers. Ethylene mixtuires of from 8 ppm to 220ppm were introdLuced into these chambers lsling theevacuation methodl (13). The contaiiners weresealed anid inculbatedl at 250 for either 2 or 3 days.Tisstues incubated in similar contaiiners in the ab-seince of added ethylene serveed as controls. Theslices were then removed and were either tusedl forthe preparatioin of extracts or were inoctilate(l withspore suispensionis of C. fimntbri(ata and incubated at250 for 3 wveeks. Resistance to infectionl wtasassessed after this perio(l.

Preparation of Extr(cts. Cylinlders ('18 mm(iam) were cutt from treated root ant(I tul)er slices.D)iscs I mm thick were cutt transversely from thesecylinders anid( extracte(l under nitrogen in an equtalvolume of 0.1 zs Tris-hydrochloride buffer, pH 7.4,containiing 0.36 xm sutcrose (6), aind 0.06 Mf ascorbicacid. Extractions were (loie at 40 in a mo(lifie(lsodiutm press. The extracts were centrifulge(d at100,000 X ql for 15; minuLtes aindI the suiperinatanitfractioins were collecte(d and storedI at -15 .

Gel Electrophoresis. Extracts were subjected toelectrophoresis in polyacrylami(le gels (10). Gelsobtained with extracts of sweet potato were staine(dto cletect amylase, phosphatase, peroxidase anid( poly-phenol oxidase activity. Acid anid alkaliine phos-phatase activity was detected by incubating the gelsin filtered soltutions of a-naphthyl phosphate (1mg/ml) diazo blute B (I mg/ml) and 'MgC., (1mg/ml) in either 0.1 M acetate buiffer pH 4.6 or0.1 -i Tris-HCI btuffer pH1 9.0 (4). The gels wereimmersed in the appropriate buffer for 30 minuitesbefore being inctlibated in the staining soluitions.Amylase was detectedl by incorporating 1 % starchin the gels before electrophoresis. After electro-phoresis the gels were washed with 0.2 M acetatebtl)ffer pH 4.6 for 30 minuites and(I theni iniciubatedin a soltutionl of iodinle (0.04 mg/ml ), KI ( 10mg/ml) in 0.2 M\ acetate buffer pH 4.6. Peroxidlasewas tletecte(l tusing 20 m_u guaiacol anid 0.3 % H,2,

( 11 ). Polyphenol oxidlase was detected in gelswhich ha(l been washed for 30 mintutes in 0.1 MNphosphate buiffer pH 6.5 utsing a soltution of catechol(1 mg/ml) and proline (I mg/ml) in the samebuffer (7).

Enzyiic Assays. Peroxidlase activity in extractswas measutred spectrophotometrically by followingthe oxidation of pyrogallol at 420 mju or catecholat 390 mn (8) at 15 second intervals in the presenceof H..O.,. Polyphenol oxidase was meastured spec-trophotometrically or manlometrically utsing catecholas substrate (1).

Results

Sweet potato tissute treatedl with ethylene showeda markedl increase in resistance to black rot (fig 1).Extracts of treated tissute showed a marked increasein peroxidase (fig 2) and polyphenol oxi(lase (fig3) activity only after electrophoresis in polyacr)yl-amide gels. Polyphenol oxidlase activity couldl notbe detected when criu(le extracts were assayed mano-metrically or spectrophotometrically. This apparentlack of activity was foutnd to be duIte to the presenceof inhibitory stl)stances in the extracts. The in-hibitory stubstances colkd lbe remove(l 1h electro-phoresis or precipitate(d from extracts b)y a(ljulstingthem to 1.5 M with ammoniitm suilfate. Polyphenoloxidase activity was then detectedl in the fractionwhich was precipitate(d with satuirate(d amniuomili11istlfate. After fractionation with ammonlilum sill-fate, it was foutnd( that increases in polvphenoloxidase activity in extracts from ethylene treate(dtissute cotuldI be detected spectrophotometricallv(table I) and manometrically. The restults obtained1w these 2 methods were similar.

Peroxidlase activity in crll(le extracts ani(l in ex-tracts fractionate(l with ammoniiuim sutlfate wereessentially the same when assayed spectrophoto-metrically. There was an approximate 10-foldc in-crease in peroxidase activity in sw,eet potato tissuiestreated with ethylene (table I).

Extracts from ethylene treated tissiue wheni suIb-jectedI to gel electrophoresis showveed increase(l anm-ylase activity but no appreciable alterationi in aci(or alkaline phosphatase activity.

In extracts from white potato treated withethylene, increased polyphenol oxidase activity wasfotund whereas peroxidase activity decrease(l (tableI).

Polyphenol oxidase activity w,as higher in ex-tracts from parsnip roots treated with ethylene thanin extracts from uintreated roots (fig 3). Polv-phenol oxidase activity was (letected in extractsonly after electrophoresis in polyacrylamide gels.No activity cotuld he dletected spectrophotometricallyin either crudcle parsnip extracts or in extracts frac-tionate(l with ammoniutm suilfate. No changes inlperoxidlase activity of parsniip extracts followinlgethylene treatment were dletecte(l.

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STAHMANN ET AL.-ETHYLENE INITIATED RESISTANCE AND OXIDASE INCREASE

Resistance Increased by Ethylene Treatment

Sweet potato slices treated with ethylene for two daysand then inoculated with C. fimbriata.

Julian

0 ppm 8 ppm 150 ppm

FIG. 1. Resistance increased by ethylene treatment. Sweet potato slices were exposed to ethylene-air mixtures for2 days at the concentrations indicated and then inoculated with C. fimbriata.

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8;

a_

ti40. I

__aimNc

/ ..t

I

/*4

FIG. 2. Peroxidase activity of discs cut 2 mm belowthe surface of susceptible sweet potato tissue treated with0 (2), 24 (5), and 220 (8) ppm ethylene. Extracts ofdiscs were subjected to electrophoresis on acrylamide gelsand stained for enzyme activity.

r

4 K l

9

FIG. 3. Polyphenol oxidase activity of discs cut 2 mmbelow the surface of susceptible sweet potato tissue treatedwith 0 (4), 8 (5) and 150 (6) ppm ethylene. Extractsof discs were subjected to electrophoresis onl acrylamidegels and stained for enzyme activity.

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i1.

If




Table I. Peroxidase and Polvplicnol Oxidase Activity in Extracts of Swcet Potato Root Tissue and White PotatoTuber Tissue Treated with Ethylene

Enzyme activity is expressed as change in optical density at 390 mu/ml of extract/minute.

Swseet potato (Julian)Ethvlene (ppm) Peroxidase Polyphenol oxidase

08

150

1.820.526.8

0.060.130.13

White potatoPeroxidase Polyphenol oxidase

0.200.160.08

0.361.500.78

Table IT. E!hylene Productioni bl,y Szceet Potato Tissue Inoculated with Fungal Spores Or Left Uninoculated andIncubated for Three Days

Fungus

None (unwinoculated)Ceratocvstis rtinor*C. fimbriata F38**C. fimbriata U6***

Ethylene produced (ppm)Julian (susceptible) Sunnyside (resistant)

1.54.09.0

15.0

1.52.013.075.0

* Nonpathogenic to sweet potato, does not induce resistance to black rot.** Nonpatlogenic to sweet potato, induces resistance to black rot.

*** Produces black rot in susceptible sweet potato.

Ethylene treatment had no apparent effect uiponthe peroxidase or polyphenol oxidase activity ofcarrot, radish or tturnip root tisstue. Very littlepolyphenol oxidase activity was detected in un-treated and ethylene treated radish and tturnip ex-tracts.

A volatile compouindl with the same retentionitime as ethylene on the gas chromatographic coltumnused in these experiments was demonstrated in theatmosphere sturrotunding sweet potato tissue inctu-bated in closed chambers after being treated withwater or being inoculated with C. finmbriata or C.minor (table II). The retention time for bothethylene and the material from sweet potato was20 seconds. In table II the approximate amotuntsof this compouind produtced by the variouisly treatedtisstues are expressed as ppm ethylene. The valueswere obtained bv comparing areas tin(ler peaks onthe chromatograms at a retention time of 20 secondswith areas uinder peaks produiced by known con-centrations of ethylene at the Fame retention time.However, these values and the identification of thecompouind as ethylene mutst be regarded as tentative,for oinly 1 type of column was used. Uninocuilatedresistant or susceptible tissue produiced very littledetectable ethylene. Produiction of ethylene in-creased following inoculation with all 3 fuingi butthe increase was larger when the nonpathogenicstrain of C. finbriata (F-38) which induice(d re-sistance w-as used than when the nonpathogenicstrain of C. imlitnor which did not induice resistancewas uised. It was largest when the pathogenic strainwas tused. Under the experimental conditions tused,ethylene produiction decreased after 4 days incluba-tion. This decrease may have been duie to decreasedO., and increased CO., levels withiin the seale(dconttitiners.

Discussion

The results of these experiments indicate thatethylene can increase resistance of sweet potatotissute to infection by C. fimlbriat(o. They also in-dicate that such indtuced resistance is associated withan increase in the peroxidase and polyphenol oxi(laseactivity of ethylene treated tissuies. Ethylene wasnot detected above cultures of C. fimtbriatia grownon potato dextrose agar btut was detected aboveinfected sweet potato tisstues at concentrations ap-proximating those used in ou r experimental treat-ment of utninoctulated tissue. It is therefore prob-able that ethylene is an active principle in volatilematerials from infected tisstues which cauise theincreased resistance and peroxidase activity in tin-inocuilated sweet potato tissule (5)).

Higher levels of ethylene were observed abovesweet potato tissue inoculated Nvith a nQnpathogenicstrain of C. fimlbriatat (induicer, F-38) which in-duices resistance to black rot than above uninocuilatedtissuie or tissue inocuilated with nonpathogenic C.mtinor (noninducer) which does not indtuce resist-ance to pathogenic strains of C. fimitbriata. Thissuiggests that ethylene may be involved in the induic-tion of resistance to black rot in suisceptible tissuieinocuilated with inducers (16,17). Peroxidase andpolyphenol oxidase activity were also increase(d intissuie with indtuced resistance to black rot (16, 17).

It is possible that ethylene is one of the stimuiliwhich move from areas of C. fimibriaita infection insweet potato into adjoining tissue to initiate themetabolic changes which lead to resistance to fuir-ther penetration by the pothogen. Since ethylenecan increase peroxidase and polyphenol oxidaseactivity in sweet potato tissule it appears that theseenzymes may be involved in the resistance mecha-

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PLANT IPHYSIOLOGY

nism. However, as was previou sly noted (17),increases in the activity of these enlzymes alonecainniot lie solely responsible for resistanice. Otherfactors are involved an(l these may inclul(le the rateof synthesis or transport of substrates to the areasof increase(d polyphenol oxidlase activity. It is notknown whether the inhibitor of polyphenol oxidasefoulnd( in sweet potato extracts is effective in vivoand hence howN mulch of the increase in activity ofthis en7zyme is effective in the tissule. However,there is a growving body of ev%i-idence that perox-i(lases act in oxi(latioin reactions requiiring onlycatalytic amotunits of hy-drogen peroxide (18). In-crease(l activity of these 2 enzymes in sweet potatotissuie stimulate(d by ethylene couldk therefore verylikely give rise to abnormal or increase(d aromaticbiosynthesis resulltinig in the formation of 1)othphysical andcl chemical barriers to infectioln (9, 15).

Little change in peroxidase or polyphenol oxidlaseactivity was note(l in eth,Aene treate(d carrot, tulrInipor radlish tissule. Polyphenol oxidase buit not perox-i(lase activity was inlcrease(l in ethylenie treatedpotato and(I parsnip tissuie. It is possible that thechanges note( iin ethylene tr.-ated sweet potatotissule are peculliar to that species. Ho\wev er, itseems probable that ethylene has a more genieralrole in the (lefense mechanisms of plailts. It mayact as a stimu1lLls of localized metabolic changeslea(dinig to necrotic and(1 hypersensitive reactions inp'anits following infectioln. Since ethYlene is capa-ble of produicinig chlor -phyll degra(lationi in fruiits(2), it is also possible that ethylene pro(duiction ininfecte(d greeni planit tissuie may caulse the chlorosisso commonly observed inl sulchi tissu1es.

Literature Cited

1. .\ALBiEG1INA, F. A. NI. 1964. Clilorogenic aci(doxidases fromil ip)tato tuber slices p)artial piurifi-cationl anid prop)erties. Phytochenilstry 3: 65-72.

_. ButV i<(J, S. P. 1962. Phx! siolog ot ethylene forma-tioIn. Ann. Rev. Plant Phvsiol. 13: 265-302.

3. AND F. A. BURG. 1965. EthvleneaIctiOln and the ripening of fruit. Scienice 148:1190-96.

4 BURSTONE, M. S. 1962. Enzvme histoclieniistrAca(lemilc Press. Nes Yorl5. e) 621.

5. CLARK, B. G., D. J. WVElER, A.\N M. A. STAHMANN.1966. Peroxidase activity aw(I resist.ance to C(rI-

t,c, stis filrialta in sw eet 1)otatto increas.e'1 hx vola-tiile mlaterials from iiifecte(d s\ eet 1otat() roots.Scielnce 153 : 02-63.

6. HEITEFUS.S, R. D. J. BUCHANAN-DAVIDSON. ANI)M. A. STAII.MANN. 1959. The stabi ization ofextracts of cabbage leaf proteins by eIlvhx1hdroxycompounds for electrophoretic anid immiluniologicalstudies. Arclh. Biochem. Biophys. 85: 200-08.

7. JOLLEYA, R. L. JR. AND H. S. IMASON. 1965. Thlemultiple forlms of mushroomil tyrosinase. J. Biol.Chemil. 240: PC 1489.

8. KANVASHIIMA, N. AND I. URITANI. 1965. SOtm1ep)rop)erties of pieroxidase in sweet pbotato inifected1b the black rot funigius. Plant Cell Ph'A-siol. 6:247-65.

9. Kuc, J. 1964. Plheniolic compounds and disease re-

sistanice in plants. In: Plheinolics in normal and(liseasedl fruiits and(I vegetables. V. C. Runeckles,cel. Planit Plheniolics Groul) of Northl AmericaSymposhimi,i NorAood, Massachliusetts. 1 63.

10. ORNSTEIN, L. AND B. J. DAvIS. 1960. Disc clec-trophoresis. Preprint. Distillation Products In-(htistries.

11. RUDOLPH, K. AND M1. A. STAHMANN. 1964. Inter-actioni of p)eroxidases anid catalases liets\-eeii Phasc-u/us vU/oari.s and(i PscudomIonas phas',4ir/,1a ( hlaloblight of beani). Natuire 204: 474-75.

12. SMIHTH, W. H., D. F. MEI;H, ANI) J. C. PARKIIKR1964. Effect of damage and fungal inifection onthe productionl of eth!vlene bv carnattioi)n. Natuire204: 92.

13. ULMBREIT, WV. XW., R. H. BURRIS, ANM) F. ST.UF-FER. 1957. IManometric techniques. Burg.ess Pulb-Iishing Cornpaiiy, Minneapolis, Minne,.sota.

14. URITANI, 1. AND MI. A. STAHMANN. 1961. Clangesill ilitrotgeii metabolisni in sweet p)otato ws ith blacl-rot. Plalnt Phvsliol. 36: 770-82.

15. TOmIYAfMA, K. 1963. Ph\ siologo-v adI liocheniist-vof (lisease resistance in plants. Ani. Rev PM Ot-)athololzY 1: 295-324.

16. \WEBER, I). J. ANI) MI. A. STAIHIMANN. 1964. C(ri-t(C5'./ti. infe. tion in sws eet potato: Its- -ffects olnproteins. isonziinies and acquilired inmilillitv. SScielice146: 929-31.

17. \VE-BER, D. J., B. G. ClARE, ANI) M1. A. STAHMANN.1966. Hiz mic chaniges asso( ciated \ ith in1duice(dand niaturi-al resistanice of sweet potato to Ccra/t-c's/ti-s ifim/)i'iaa. PhytopathologyV, ill ress.

18. YA.MAZAKI, ., K. YOKOTrA, AmN) R. NAKAJIMA.1965. A mechanism anid II1o(lel of erox()xidase-oxC;(lase reaction. In Oxidases and related redoxsc stemils. T. E. KLing, H. S. Mason, au 1 M. Mor-rison, e(ds. \iley and(l Sons, Inc., -Nes- Yorl; p485.

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increased disease resistance enzyme activity induced ...plant physiol. (1966) 41, 1505-1512...

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