PHlYSIOLOGY September 1963

(1)

PLANT

Vol.

38

No.

5

PHlYSIOLOGY

September

1963

Analytical Study

of

Umbelliferone

and

Scopoletin

Synthesis

in

Sweet Potato Roots Infected

by Ceratocystis fimbriata

T. Minamikawa

3,

T. Akazawa

3,

and I. Uritani

Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University, Anjo, Aichi, Japan

Introduction

It has been reported by several workers (3, 4,5,

6, 8) that coumarins are produced in plant tissues in response to pathogenic infection and it has been hypothesized that they are concerned with disease resistance. In

1953,

Uritani and Hoshiya (9) re-ported that sweet potato roots infected by the black rot fungus,

CeratocYstis

fimlbriata,

produced

umbelli-ferone (7-hydroxv coumarin) and scopoletin

(6-methoxy-7-hydroxycoumarin) along with some other phenolic substances such as chlorogenic,

isochloro-genic,

and caffeic acids (10). They felt that the 2 coumarin compounds may act in the prevention of fungus penetration into the host tissue. The tvpical metabolic change in black-rotted sweet potato root is the production of polyphenols, such as chlorogenic

acid, in the healthy tissue adjacent to the infected tissue and of

furanoterpenoids,

such as ipomea-marone, in the infected tissue (2). A comparison of the patterns of synthesis of coumarin compounds with polyphenols and furanoterpenoids would appear

to be worthwhile to better understand the nature of the host-parasite relationship. The biosynthesis of coumarin compounds is also of biochemical interest because the carbohydrate metabolism of the diseased host tissue might presumably be altered to form such aromatic compounds having a possible role in the host resistance.

Materials

and Methods

Fungus Inoculation and Preparation of

Samtple:

Sweet potato (Ipomea batatas) roots harvested at the Toyohashi Farm of the Aichi Agricultural Ex-periment Station in October, 1961, were stored at

'Received Jan. 3, 1963.

2This paper constitutes Part 35 of the

phytopatho-logical chemistry of sweet potato with black rot. 3Present address: The International Rice Research Institute, Los Banos, Laguna, The Philippines.

100

until used. In most experiments. roots of Norin No. 1, a typical resistant

variety,

were used. Norin No. 10 and Norin No. 4, resistant and suscep-tible varieties respectively, were

employed

for

com-parison. The basic procedures of the fungus inocu-lation of root tissues and the subsequent

handling

were as reported previously (2). Individual roots

were cut into 1.5 to 2.0 cm thick slices which were

inoculated with a spore suspension of C.

fimnbriata.

Uninoculated control slices of equal size from

cor-responding parts of the same r3ots were used. Both samples were then incubated at 280. At 24-hour intervals, diseased and control tissues were harvested

for the chemical analysis of the coumarin compounds. In the analysis of Norin No. 1, 10 discs

measuring

10 mm in diameter were first cut out with a cork

borer; then 5 slices of 1.0 mm thickness were ob-tained from each disc with a hand

microtome, by

cutting from the surface to the inner

portions

of the discs. In experiments using Norin No. 10 and No. 4, 3 layers (0.5 mm in thickness) were harvested fromthe surface of each disc.

Corresponding layers

from 10 discs were combined and subjected to

analy-sis. The averaged fresh weight of the 10 layers

when combinedwas0.95 gfor Norin No. 1 and 0.48 g for Norin No. 10 and 4. The weights did not

fluc-tuate much throughout the experimental

perioxl

of

4 to 9 days following fungus infection.

Analysis of

Coumlarins:

The plant samples were

first homogenized with 5 ml of 95% methanol in a

Potter-Elvehjem glass homogenizer and then gently

boiled in a hot water bath for 30 minutes. After

cooling the mixture was filtered and concentrated in

a Rinco rotary flash evaporator. The concentrated

material was dissolved in a small volume of methanol

and was quantitatively applied to a thin layer silica gel chromatoplate

(6-8

cm X 15 cm), prepared

ac-cording tothe method of Kirchner et al. (7) as

modi-fied by Akazawa and Wada (1,2). The chromato-plate was developed in ethyl-acetate/n-hexane

(50:50

v/v) containing 2% acetic acid as solvent. After

(2)

dlevelopnillent,

2 well seplarated nlajor fluorescent

bands w-ere (letected un(ler a UN" lamnp and their RF values were comparable with those of pure

sam-ples of unmbelliferone an(d scopoletin. The fornier

-wvas (leel) blue (RF:0.63 ) andl the latter greenish blue (

R,.:

0.32 ). Eaclh zone w-as illarke(l b1 a pencil and scratche(d ith a

slpatula

froni tIle

plate,

elutedl by liot nIethanol, and the eluate was concen-trate(l in a rotary flasll evaporator. The blank zones

corresponl(linlg to the position of umlbelliferone and scopoletinl were treate(l in the sanlle ilanliler, an(Il the

resultilng eluates serve(l as coIltrols. The concen-trate(l miaterial was further purified by

paper

chro-matography. Toyo-filter paper No. 51 was used Nitil 5 ajacetic aci(l as solxent. The

RF

values of

unilbelliferoile and

scopoletin

(letected 1w UIV lamp

were 0.57 aildl 0.44, respectively. Each fluorescent zone was cut out an(d extracte(l (luantitatively witll a nlixture of 0.2Mr

sodiumii

carbhonate (pH 10.0) and

etallanol (1: 1 v/v). Under these con(litions the

fluorescenice w-as perfectly stable for at least 24

liours. Siilce the UV

spectra

of the eluates thus

obtaine(i xwere found to be practically identical with

those of the pure sanmples ofuillbelliferone and

scopo-letini tile quantitative

estimiiation

of unibelliferone

ainli

scopoletin

was carl-ie(l out

)vy ineasuring

the

fluorescelnce intensitv of an

alppropriatel

(lilute(I eluate by a Sliilla(lzu fluorometer

eqiuipl)e(l

with filter

Fl-P,.

Quinine sulfate (10.0

uAg/nml

of 0.1 N

H.SO4) was used as a stan(lard fluorescent

sub-stance. The fluorescence intensity of the control

sanllIple

was practically negligible (about 0.9 %). Calibration curlVesxvere nmadewith pureulllbelliferone aInd

scopoletinl

under alkaline con(litions aind

w\ere

use(i for the calculation of the

amnount

of eacil

conm-pound in the tissue extracts. The recovery of these coumarins was deternline(l as follows: a known anmount of each pure

comipound

wvas

added to slices ofthe noniIlfected fresh root tissue andl the couillarins

xvere extracted and analyze(l for ill exactly the sanme imlanner as dlescribeci albove. Recoveries froml such a proceclure proved to be satisfactory. as slilowIl in

table 1.

Breakdo7un of

Couio(irin(

C)oinpounds by the

Fuin-guts: A spore suspensionl of C. fimitbriatta xvas added

to 50 l1 of culture me(lia [potato extract (250 g/liter

x-ater- ) colltaining 2 d(, sucrose an(l 0.4C, casein

Table I

Recoveries of

Unmbellifernce

antd Scopoletini

The values represent the average of duplicate analyses.

Experinleilt Compound added 1 Umbelliferone Scopoletin 2 Umbelliferone Scopoletin

hydrolx

sate] (11) in a Sakaguchi-flask,

andl

the

cul-ture

wtas

shaken for 2

days

at 280. A flask

wxhiclh

ha(l not been inoculate(d w%ith the fungus served as

control. After 2

days

10 mg each of

umlbelliferone

an(d

scopoletin (lissolvedl

in 5 ml of 10

'/%

ethalnol

were aseptically addledl to all the flasks. Five nml of 10 () etlianol were a(l(le(l to another culture flask sillmultaneously to (leterilline the effect of

ethalnol

on the fungal growtth. After shaking for another

4 days at

280,

thecontents of each flask were

brought

up to 100 nml wvith methanol. The mlycelia were

re-nIovedl by filtrationi. An aliquot of the filtrate was.

quanltitatively

applied

to a

silicca gel

cllronlatol)late

follow-ed by paper

chronlatograplly

for tile

aiCalvXsis

of counllarin conmpoun(ds. Cultures were

carriedl

out

in (luplicate.

Results

Ju,,,e Cour-se Alnalasis of (c01Jola-(ri11 .vnwtliesis: Tlle amllounts of botlh uillbelliferoile (fig 1) ani(l

sco-E E ,III IIV V C 3 6

TIME- DAYS AFTER FUNGUS INOCULATION

FIG. 1. Time course of umbelliferone synthesis in Norin No. 1. (I) to (V) correspond to the layers (1

mm thick) starting from the inoculated surface to the Re- Re- innermost layer; e.g., (I) : outside layer of inoculated

covered covery sample, (II) : adjacent inner layer etc. (c): outside

Ag ,ug

c/

layer of uninoculated control

sample.

Arrow

(t)

de-notes tissue samples infected by fungus more than half

3.02 2.50 82.9 anarea. (fig 1-A).

1.51 1.30 85.8 FIG. 2. Time course of scopoletin syntiesis in Norim

35.1 25.8 85.2 No. 1. (I), (II), (III), (IV), (V), and (c) the sanme as for figure 1.

(3)

poletin (fig 2) in the 5 layers (1 mm in thickness) from diseased and control tissues of Norin No. 1 were analyzed continually throughout the infection

period. The time course of synthesis in the outside layer (I in figs 1 and 2) is particularly noteworthy. The synthesis of umbelliferone started only 12 hours

after fungus inoculation and continued until the 4th

day. Afterwards, quite a sharp decline started which levelled off after the 6th day of infection. In thecase of scopoletin synthesis, there was a lagphase

of about 1 day, and then a steady increase was

ob-served. The maximum period of scopoletin forma-tion occurred on the 4th

day,

similar to that of

umbelliferone, and after this period, a decrease was observed. The reason for the unexpected increase

of the scopoletin after the 7th (lay was not clear (I in fig 2). In both cases a marked synthesis of the

conmpounds seenmed to parallel the progress of fungus penetration into the host tissue. The amount of umbelliferone in the outside layer on the 4th day was about twice the amount of scopoletin. The rate of umbelliferone synthesis in the inner tissue was less marked as compared to the outside layer. The 2nd and 3rd layers gave a somewhat similar pattern (II and III in fig 1). In these 2 layers, a

measur-able increase was not observed until the 4th

lay,

and

synthesis

reached maximum on the 6th to the

3

7th day and levelled off afterwards. This fact may

indicate the inward movement of the site of umbelli-ferone synthesis in the diseased tissue after about the 4th day. The amount of umbelliferone in the next

inner layers (IV and V in fig 1) was very little,

though therewas also some increaseatthe end of the 9th day. The synthesis of scopoletin in the inner layers showed a pattern different from that of

um-belliferone; namely, it increased linearly until reach-ing its maximum, and the movement of the site of synthesis into the inner parts was also quite notice-able. The amount of scopoletin in the innermost region was small. The formation of the 2 coumarin compounds in the outside layer of the uninoculated control tissue was detected, but there was little

syn-thesized in comparison to that fornmed in the infected

root. Furthermore, in both the freshly cut and the inner parts of the control tissues, the formation was

negligible. Themaximum amount of the 2 coumarin compounds in the outside layer of the dliseased tissue was about 27-fold (umbelliferone) and 20-fold (sco-poletin) over that in the same region of the unin-fected control tissue.

Comiparative

Analysis

in Different Varieties:

The synthesis of the two major coumarins was in-vestigated in Norin No. 10, resistant, and Norin No. 4, susceptible varieties, and the results are shown in

4

I 2 3 4

TIME-DAYS AFTER FUNGUS INOCULATION

TIME- DAYS AFTER FUNGUS INOCULATION

FIG. 3. Synthesis of umbelliferone in Norin No. 10 and No. 4. (I), (II), and (III) correspond to the layers

(0.5 mm thick) starting from the inoculated surface, respectively. (c) is the first layer of the uninoculated control. FIG. 4. Synthesis of scopoletin in Norin No. 10 and No. 4. (I), (II), (III), and (c): the same as for

figure 3.

(4)

figures 3 (umibelliferone) andcl 4 (scopoletin). The

synthesis of umlbelliferone in the outside layer of

Norin 10 was

surprisingly

high and continued rapid-lr without a lag phase until the 3rd to the 4th day. The pattern of

synthesis

in the 2nd an(d 3rd layers

seemle(d to be somiiewhat simliilar to that of the outside

layer

though the amountwas nmuch less. Inthe case

of Norin No. 4, synthesis continued until the 2nd

(lay, though the magnitu(le was evidently snmaller than that of Norin No. 10, and stopped abruptly after this perio(l. Apparently, movemiient of the site of the coumarin synthesis dloes not occur in this

variety. As in the varietv Norin No. 1, the

forma-tion of umlbelliferonie in the noninfected cut tissue

was slight in both Norim No. 10 andl Norin No. 4

throughout the incubationi period. Thus, it can be

calculate(d that about a 45-fold increase of

umbelli-ferone was induced in the outsidle

lay)er

of Norin No. 10 and about 14-fol(d increase in that of Norin 4 over that in the uninifecte(d contrel tissue (luring 4

(lays infection perio(l. In the case of

scopoletin.

the

synthesis curve showed a steadlv increase until 1 to 2

days

after infection in both varieties, similar to

the results obtainedl in Norim No. 1. In Norin No. 10,a mlarkedl synthesis was observedl afterthis period,

whereas in Norim No. 4, the svrnthesis apparently

stoppe(l. The ratio of the

scopoletin

content in the outsi(le layer to that of noninfectedl control tissue

oni the 4th (lay was about 27 in Normii No. 10, but only 3.5 in the case of No. 4.

Fuingal Breakdowin of

titbelliferotic

ani(d

Sco-polctini:

During the culture period of 2 (lays. the

fungus grew vigorously aln(l the mle(lia becamle turbidl

with mycelia. After another 4 days of shaking the

licquid mie(lia becamiie dlensely black, irrespective of

the addition of counmariln conmpounds. Added

um-l)elliferone or scopoletin did not seem to retarci the

fungal growth at a concenitration of about 200 mg

per liter. The control

experiment

showedl

that the

fungus

prodluced

neithel- umlbelliferone nor scopoletin in li(qui(d culture medlia and none of the fluorescent

mlaterial was formle(l. It is striking that the fungus

legradle(d a imieasurable amiount of scopoletin and uim-belliferone as showrn in table II, an(d the imiagnitudle

of scopoletin breakdown was nmore renmarkalle tlhain

that of umbelliferone. It was shown that 90 % of

the added scopoletin was broken clown (luring the

same period. Interconversion of the 2 cDunmariln

compounds was not observed and the level of

cou-marins added to the media didl not (lecrease in the absence of the fungus.

Discussion

In our previous study (2) it has been established that, in addition to the formation of coumarin

com-poundls. large amnounts of polyphenols and

ipomea-marone are prodluced in sweet potato roots in response

to the black rot infection. Thus, 3 types of

com-pounds

are

typical

mletabolites

of this disease of

sweet potato. The present

stu(ly

indicates, however,

that the synthesis of 2 major counmarin

compounds,

umbelliferone an(d scopoletin, occurs on a much smaller scale than that of other 2

types

ofcompounds. Furthernmore, the bound( fornms of such coumarin com-poundls have been foun(d to be negligibly low (Mina-mikada. Akazawa, and Uritani, unpubl. data). Hence, froml the

pathological

view point, it is rather hard to beliexve that these coumiairin conipoundls

might have a

potent

antipathogenic function in the lisease(d sweet potato root.

Syntlhesis of coumiiarin compounds, in particular that of umlbelliferone, occurs miiost

actively

in the

out-si(le

layer

of the fungus-infected tissues, and this

picture is

very

nmuch similar to that of ipomeaiiiarone

synthesis. although there is a lag phase of abnut 1 (lay in the latter case (2). Furtlhernmore, it shoul l be notedl that there is an apparently close

parallel

between the amiount of post-infection coumiiarin

pro-(luction and the (legree of host resistance against

the pathogen; a similar relationship was observed in the case of ipinmeamarone synthesis. Thus, the

pattern of synthesis of these coumarins after infec-tion seemiis to be (lifferent fromii that of polyphenolic

colilpounId(s, e.g. clhlor-ogenic aci(l. This was indee(l

verified wvhen the ratio of the amlounlt of chlorogenic acicd in the inner layer to that of the outer inoculate(d

laver w-asfound( to be very mutch higher than the

cor-Table II

Fitfl(Jal BrIrakdozwi of

l

1i1)CllifeioJlc (1)i(d Scopoeltil

The figures represenit total amount present in the media.

Compound added Umbelliferone Umbelliferone Scopoletin Scopoletin Inoculation + Added Umbelliferone mg 11.5 11.5 11.5 11.5 Found Scopoletin U,mbelliferone mg mg 11.1 11.4 8.0 8.5 .. 12.1 12.1 12.1 12.1 Scopoletin mg .. 11.4 11.8 0.9 1.2

(5)

responding ratios of ipomeamarone or either

cou-marins, indicating a different site for polyphenol

synthesis.

The liquid culture experiment showed a rather

marked breakdown of coumarincompounds. Though

this type of observation might not be

directly

the breakdown of these compounds in the later stage

of infection may possibly be interpreted as due to

fungusaction. We cannot, of course,

deny

the possi-ble action of certain enzymes in the host tissue as

being responsible for the coumarin destruction.

Summary

A chromatographic metho(d was developed for the quantitative estimation of umbelliferone and

scopoletin in sweet potato (Ipomlea batatas) root tissues infected by the black rot fungus, Ceratocystis fimbriata. In the variety Norin No. 1, a miarked synthesis of umbelliferone occurred 12 hours after

inoculation, whereas scopoletin synthesis started after

a lag phase of 1 day. The amount of umbelliferone

at its maximum stage was about twice as much as that of scopoletin. The formation of these couimiarin compounds was very low in the uninoculated cut tissues, and thus, counmarin synthesis is believ-ed to occur after infection.

The

synthesis

of the 2 coumiiarins was

conmpared

in 2 varieties, Norin No. 10, resistant andI Norimi

No. 4,

susceptible.

Coumarin

synthesis

was miore conspicuous in the resistant root variety as

com-pared to the less mnarked synthesis in the susceptible

one.

The black rot fungus was foundl to (legrade

uni-belliferone and scopoletin during

4-day

incubation period in liquid culture. Scopoletin was degraded

more easily (about 90%) than unmbelliferone (about 20 %). Neither transformations betweein these

cou-marins nor endogenous formation by the fungus alone was detected.

Literature

Cited

1. AKAZAWA, T. AND K. WADA. 1960. Quantitative estimation of ipomeamarone by chromatostrip technique. Agr. Biol. Chem. 25: 30-3.

2. AKAZAWA, T. AND K. WADA. 1961. Analytical

study of ipomeamarone and chlorogenic acid

alter-ations in sweet potato roots infected by Ceratocystis

fimbriata. Plant Physiol. 36: 139-44.

3. ANDREAE, W. A. 1948. The isolation of blue fluo-rescent compound scopoletin, from green mountain potato tubers, infected with leaf roll virus. Can. J. Res. 26C: 31-4.

4. BEST, R. J. 1944. Studies onl a fluorescent sub-stance present in plants. II. Isolation of the sub-stance in a pure state anid its identification as

6-methoxy-7-hydroxy 1: 2 benzopyrone. Aust. J.

Exptl. Biol. Med. Sci. 22: 251-55.

5. FUCHS, W. H. 1956. Ein Beitrag zur patholog-ischen Physiologie. Angew. Bot. 30: 141-46.

6. HUGHES, J. C. AND T. SWAIN. 1960. Scopoletin production in potato tubers infected with

Phyto-phthora intfestanis. Phytopathology. 50: 389-401.

7. KIRCHNER, J. G., J. M. MILLER, AND G. J. KELLER. 1951. Separation and identification of some ter-penes by a new chromatographic technique. Anal. Chem. 23: 420-25.

8. TA'MARI, K. AND J. KAJI. 1959. Biochemical studies on the blast disease of rice plant. Part

XII. The isolation of coumarin from the blast disease rice plant. Agr. Chem. Soc. Japan 33:

178-80.

9. URITANI, I. AND HOSHIYA. 1953. Phytophatho-logical chemistry of the black-rotted sweet potato.

Part VI. Isolation of coumarin substances from sweet potato and their physiclogy. J. Agr. Chemn. Soc. Japan 27: 161-64.

10. URITANI, I. AND M1. MIYANO. 1955. Derivatives of caffeic acid in sweet potato attacked by black rot. Nature 175: 812.

11. URITANTI, I. AND M. A. STAHMIANN. 1961.

Pecto-lytic enzymes of Ceratocwstis fizbriata. Phyto-pathology. 51: 277-85.