University of New Hampshire University of New Hampshire

University of New Hampshire Scholars' Repository University of New Hampshire Scholars' Repository

Master's Theses and Capstones Student Scholarship

Spring 1978

TOl\1ATINE: ROLE IN RESISTANCE OF TOMATO TO FUSARIUM TOl\1ATINE: ROLE IN RESISTANCE OF TOMATO TO FUSARIUM

OXYSPORUM F, SP. LYCOPERSICI RACE 1 AND RACE 2 OXYSPORUM F, SP. LYCOPERSICI RACE 1 AND RACE 2

Cheryl A. Smith University of New Hampshire

Follow this and additional works at: https://scholars.unh.edu/thesis

Recommended Citation Recommended Citation Smith, Cheryl A., "TOl\1ATINE: ROLE IN RESISTANCE OF TOMATO TO FUSARIUM OXYSPORUM F, SP. LYCOPERSICI RACE 1 AND RACE 2" (1978). Master's Theses and Capstones. 1316. https://scholars.unh.edu/thesis/1316

This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact nicole.hentz@unh.edu.

II 111111111111111 ii 11111111111 ll 3 4600 00284 1366

/t

TOl\1ATINE: ROLE IN RESISTANCE

OF TOMATO TO FUSARIUM OXYSPORUM F, SP. LYCOPERSIC I

RACE 1 AND RACE 2

by

C:{E RYL A. SMITH

B, s., University of New Hampshire, 1974

A THESIS

Submitted t,o the Uni ve:csi·sy of New Ha.J:psh ire

In Partial Fulfillment of

The Requirements for the Degree of

Y1ast2 r of Sc i encA

Graduats School

Department of 3o~a- y and Plan~ Pathciogy

This thesis has been examined and approved,

Thes i s d i rector , William E, MacHardy, Assoc. Prof . of Pl ant Pathology

Avery E. Rich , Prof. of Plant Pathology

-~ -- -r✓ ,,,,

L -.,,--✓ /l / (_(.,(__ ..... ,,: .... ,.-L::", C ------~ -- -"---- ·'-___ ..... _--__________ _

Lincoln C, Peirce, Prof, of Plant Science and Genetics

Dat e

ACK NOWLEDGEMENTS

I am deeply grateful to Dr. William MacHar dy fo r

his most generous financial support, continued encour agement ,

valuable suggestions and ideas, and so much more .

I am also sincerely grateful to: Dr . Aver y Ri ch

and Dr. Lincoln Peirce for their guidanc e a nd experti se;

William Conway for his kind as s i s t a nc e wi th t he t echni ca l

aspects of this study; Van Cotter f or help with t he

statistical analysis, for his enthusiasm a nd interes t i n my

research, and for being such an inspira tion t o me ; Stuart

Blanchard for his willingness to transla te a German journal

article into English; Ro-oin Eifert and Le e Anne Cha ney f or

their friendship, Robin for her c onf i denc e i n me and Lee ArL'1e

for prayerful support; and to my parents I wish to ext end

a special word of appreci ati on for their pat ience and

underst anding .

. ii

TABLE OF CONTE NTS

LIST OF TABLES

LIST OF ILLUSTRATIONS ................................. ABSTRACT

SECTION I. THE SIGNIFICANCE OF TOMATINt IN THE HOST RESPONSE OF SUSCEPTIBLE AND RESISTANT TOMATO ISOLINES INFECTED WITH FUSARIUM OXYSPORUM F. SP. LYCOPERSICI RACE 1 OR RACE 2 , , ••••• , , , , , • , •. , , . , , • , .

Introduction ........ ~ ............................ . Materials and Methods ••••••••• •••••••.•••••..

Cultural Procedures .•••••• • ••....•.. Host'"'••·•••••••••• •••••· Pathogen . . . . . . • . . . . ......... . Inoculation of J-iost •••• , • • . ••••

Quantitative Determination of Tomatine in Xylem Fluid ,,, ••..•••••••••••

Obtaining Xylem Fluid , ,,, ,,, T_hin Layer Chromatography • • • • , •.•.

Effect of Tomatine on Colony Growth and Spore Production ••••.••••.•• ,,., •••

Colony Growth • • • • • • , ••.••••• , ••.• , Spore Production .••••. ,,,. Spore Viability •••••.•••.••.•.

Effect of Tomatine on Spore Germination and Hyphal Extension • , .••••••• , ..••• ,

Results ................... ,.,o•••••••••·•• · · • •· Quantitative Determinat i on of Tomatine i n

Xylem Fluid ... ~ .... . .... . ............ . Effect of Tomatine on Colony Growt h and

Spore Production •.•• ,.. •. Colony Growth ••• , , • • , •. Spore Production • , • • • • • , Spore Viabil i t y • ••••

Effect of Tomat i ~e on Spor e Ge r minat ion and Hyphal Extension

Spore Germi a t ion Hypha l Ext e s io ..

...

Discuss ion ··~···················· ················

iv

v i

vii

ix

1

1

1 0 1 0 1 0 1 0 1 0

11

11 13

15 15 16 16

17

19

19

21 21 25 31

Jl 31 JJ

35

SECTION II . THE EFFECT OF FUSAR IUM OXYSPOR - F . S? . LYCOPERSICI rtACE 1 AND RACE 2 ON THE ANTI !"UNGAL ACTIVITY OF TOMATINE

Introduction ....... ........... .. .... ... ...... .... Materials and Methods ••••.• ••• . •• . ..•.•.• •..•. ...

Effect of Successive Spore Populations on Germination •...........................

Effect of Snore Concentration on GerminatiOn . ti a •••••••••••• '1 .....

Slide Bioassay ••• •••• ·• •..• • Shaker Table Bioassay • •••• •

Results ..............•..•..•........ , .......... . . Effect of Successive Spore Populations

on Germination .................. . ...... .. . . Effect of Spore Concentration on

Germination • . • • • • • • • • • • • • . ••• Slide Bioassay . . . . . . . . . ...... ., ...... . Shaker Table Bioassay •••

Discussion ••••• .. • • o•• •••• ••o•~•••• •• •• • ••• • ••••••

LITERATURE CITED •e••••••••••••a•••••••••••••••••• • ••••

-+9

so 50 so 51

52

52

52 52 55

60

63

LIST OF TABLES

1. Tomatine content (moles ) of xylem f l uid exudate from stems of Imnroved Pea r son and Pearson VF- 11 tomato near-isollnes at selected time intervals , Plants we r e nonwounde d- noninocul a ted, wounded-noninoculate d , inoculated with Fusarium oxysporum f. sp. lycopersici r ace 1 or inoculated with Fusar ium oxysporum f, sp . lycopers i ci r ace ,2 • • • • • • • . • • . • • . . • • • . . . • • . . • . . • . . 20

2. Tomatine content (mg produced/plant) of xylem fluid exudate from stems of Improved Pearson and Pearson VF-11 tomato nea r-i solines at se l ected time i ntervals . Plant s were nonwounded ­noninocul ated , wounded- noninoculated , inoculated with Fusarium oxysporum f. sp . l yc ope r sici r a ce 1 or inocul ated wi th Fu sarium oxysporum f. sp. l ycopers ici race 2 •• • •.• . •••• • • •. . .• •. .. . . 22

LIST OF ILLU STRATI S

1, The structur a l f or mu l a f or alpha- tomatine ..•••• . •. 4

2. Method for collecting and ext r a c t i ng xyl em fluid exudate from decapit a ted t omato plant s ••••.•...... 12

J. Standard curve of va rious concentrati ons of commercia l a l pha-tomati ne plotted against the percent transmittance of the t omatine - H2so4 chromagen mea sured a t J25 nm ••.. , .•••••.••.• •. . . . , 14

4. Colony di ameters of Fusarium oxysuor um f , sp. lycopersici r a ce 1 and rac e 2 cultu r e s g r own on buffered Pfltat o dext r ose agar containi ng A) 10- J M, B) 2 X 10- · M, C) 4 X 10-5 M or D ) 8 X 1 0- 6 M alpha-tomatine . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5, Mycelial dry we i ght s of Fusar ium oxyspor um f. sp. lycopers ici rac e 1 and r a ce 2 cultur es gr own on buffered pp t ato dext ros e agar cont a ining A) 10- J M, B) 2 X 1 0- 4 M, C) 4 X 1 0- 5 M or D) 8 X 1 0- b M al:pha-ton1atine . . ...... fl ••••••••• .,..... . . .. ......... 27

6.

7.

8 .

9 .

1 0 .

Microconidial pr oduc t ion of Fusarium oxysporum f, sp, lycopersici r ac e 1 a nd r ac e 2 cul tur es grown on buf f e r ed pot tt o dextro se agar containing A) 1 0-J M,

6B) 2 X 10- M, C ) 4 X 1 0- 5 M or

D) 8 X 10- M a l pha-tomat ine ••••.•••.••..... •• . • . .

Effect of various c oncentrations of buffered (pH 4,6) alpha- tornatine on the germination of Fusa r ium oxysporum f , sp . l vcope r sici race 1 and rac e 2 microconid i a .... " . . ..... .. ,, ..... " . .... . . . " . .

Effect of vari ous concent r ations of buffered (pH 4. 6) a lpha- tomatine on the germ tube length o: Fusarium oxyspor um f , sp. l ycope r sici r a ce 1 and r ac e 2 microconidia ..... .. .. .. ..... , . . . . . .. . . . .

1 he effe ct of var ious spore suspension seque~ces on the ge r minat i on of m· cr oconi ia o: Fusari urn oxys por um f . sp . l vcope r sici race l or race G

i n A) an 8 X 10- 4 M toma~ine solut ion or a) a bu f f e r control solutio~ •• •. .. . .. •.•. .. . ..... • . ... .

The effect of selected spore cor.ce ~ratios or. the germination of rnicro~onidia of ?us~riu~ oxysporL m f . sp. l ycopersici r ace l i ~cubatEd ~0~ 24 , ou r s on mic r osc one slicies ........... . . . .... . . .

vii

JO

J2

CL:.

57

11, The effect of sel e c ted suor e conc ent r ations on the germination o f mic roconidia of Fu s a r ium oxysporu m f, s p . l y c ope r s ici r a c e 1 i ncubated fo r 24 h ours i n f l ask s on a shake r table .•....•... 58

viii

ABSTRACT

TO!VI.ATINE: ROLE IN RESISTA NCE

OF TOMATO TO FUSARIUM OXYSPORUM F, SP, LYCOPERSICI

RACE 1 AND RACE 2

by

CHERYL A. SMITH

Previous workers have suggested that the host­

produced fungitoxicant, alpha- tomatine, may play a key

role in the resistance of tomato plants to the vascula r

wilt pathogen, Fusarium oxysnorum f, sp. lycopersici

(Sac c.) Hans. & Snyd. race 1, The in ,ivo and in vi t ro

studies reported herein were undertaken to examine that

hypothesis and to investigate the effect of tomatine on

Fusarium oxysporum f. sp . lycopersici (Fol) race 2 .

~eterminations of the concentration of tomat ine :n

the xylem fluid of Improved Pearson (IP) and Pearson VF-11

(VF) near-isolines of tomato were made using cr.ro~atograp: i

and spectrophotometric techniques , Plants we re samp:ed at

selected time intervals following a severeri - t roo~

inoculation with Fol race 1 e r race 2 . ': is r esi -"ant

race 1 but susceutible to the no e virule~~ r ace: . ~

susceptible to both r ac es. An other g r o~p c~ ~la~"s wa~

i::

wounded, but not inoculated .

plants served as controls ,

onwounded-non·nocula-;;ed

There was no significant difference in the to. atine

content of VF and IP plants prior to inoculat ion . The

t omatine c oncentration of IP plants remained at pre­

inoculation levels (lo-4 M) regardless of treatment . The

concentration of tomatine in the VF isoline increased to

fungitoxic levels (10- J M) 2 days after inoculation in both

the resistant VF- race 1 and the susceptible VF-race 2

combi nations . Tomatine also increased to 10- J M i n VF

plants 2 days after wounding.

It was postulated that if tomatine plays a role

in the resistance of VF to race 1, then race 2 may be l ess

sens i tive than race 1 to hi gh l evels of tomatine presen~ in

VF following infection . The effect of tomatine on mycelial

dr y weight, colony diameter, spore germinatior. and germ

tube length of race 1 and race 2 was compared , To~atine

was inhibitory to both rac es , but no important differences

in sensitivity we r e observed. It was furthe r postulated

that tomatine may contribute to resistance by iY:.hi bi ti .. g

sporulation of Fol, but spore produc~ion of bo--::1 races wa_,

stimulated, not inhibited .

It was conclude d -;;h~t toma-;;ine does no"'.; pay a

primary role in resistance , jut · t my be

sequential resista~ce process , such a pr ocess , vasc~-~r

occlusi n ould ace· r firs~ to ::mit tte ~pva r ~ spr eac o:

? o_, ':'omatine ace mulatio.. el w occ:i.uce ;:cr-;;.:..ons 0:

X

vessels would then inhibit fungal vegetative growth, thus,

limiting the lateral spread of the pathogen to adjacent

vessels.

Two additional studies were conducted to examine

the effect of Fol race 1 and race 2 microconidia on

tomatine. In the first study, it was hypothesized t hat Fol

may detoxify tomatine, If a given population of germinating

spores did detoxify tomatine, the percent germination of

subsequent populations of spores placed in the tomatine

solution would i nc rease . In the second study, it was

hypothesized that increasing the spore load in a given

concentration of tomatine would reduce the effectiveness of

the fungitoxicant, resulting in an increase in spore

germination. In both bioassays, percent germination

decreased in the buffer control solutions as well as in

tomatine. Although conclusions could not be made r egarding

the original hypotheses , the data provided evidence that

the ge r minating Fol conidia produced a self-inhibitory

substance.

xi

SECTION I

THE SIGNIFICANCE OF TOMATINE

IN THE HOST RESPONSE OF SUSCEPTIBLE AND RESISTANT

TOMATO ISOLI~~S INFECTED WITH FUSARIUM

OXYSPORUM F. SP, LYCOPERSICI

RACE 1 OR RACE 2

Introduction

The use of tomato cultivars bred for resistance

to Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd. &

Hans. has long been the most practical means of controlling

this vascular wilt pathogen. However, efforts to discern

the nature of the host resistance mechanism have led to

conflicting conclusions . Phenolic compounds (Matta et al.,

1969) and vascular occlusion (Beckman et al., 1972) have

been proposed as determinants of resistance. The possible

involvement of host-produced fungitoxicants in resistance

to Fusarium oxysporum f. SD, fycopersici (Fol) rac e 1 has

also been examined by several investigators . Tomatine is

one such fungitoxicant implicated in Fusarium wilt

resistance.

Irving et al. (1945) reported that tomato plant

extracts conta ined an inhibit ory agent exhibiting marked

fungistatic action against Fol race 1. The partially

purified antibiotic was designated "tomatin", Although

some investigators failed to de-~ect any inhibitor in tissue

1

extracts (Heinze and Andrus, 1945) or xylem fluid (Snyder

et al., 1946), further experimentation confirmed the

presence of tomatin in tomato tissue (Irving, 1947; Irving

et al., 1946; Little and Grubaugh, 1946).

Fontaine et al. (1947) suggested t hat tomatin

might be either wholly or partially responsible for resist­

ance. Kern (1952), however, disagreed with that t heor y .

2

He measured tomatin levels in roots, stems and vascular

fluids of tomato plants, and found that the concentrations

in these critical sites were insufficient to iri~hi bit spore

germination or mycelial growth. Kern concluded that

tomatin could not be responsible for resistance to Fusarium

wilt.

Efforts were made to isolate and purify the anti­

biotic present in the crude tomatin extracts. From tomatin

concentrates, Fontaine et al. (1948) were able to isolate

crystalline alpha-tomatine. The purified antifungal agent

was characterized chemically as a glycosidal alkaloid

(Fig. 1), Further chemical and physical data established

the steroidal nature of the alkaloid (Fontaine et al .,

1951; Uhle and Moore, 1954).

The extent of involvement of tomatine in the plant's

defense mechan~sm was generally ignored until Arneson and

Durbin (1968) tested the sensitivity of JO fungal species

to commercially purified tomatine . The test organisms

included fungi which we re pathogenic, non- pathogenic or

saprophytic on tomato plants . The study demonst rated the

Fig. 1. The structural fo rmula for alpha-tomatine. The molecule consists of a

tetrasacc ha rid e moiety (two molecul es of g lucose and one each of xylose and galactose) and

an a g lycone moiety (tomat i dine ). Beta2-tomat ine is a form of tomatine lacking one glucose

unit .

::c

(")

::c u

, I

(")

:c u

I.LI

z .... < ~ 0 I­

I C

..c: _g. 0

:I: I

I

0 ::c

~06 uy J: 0 £ u

0

::c

w z C

····-·•-•: -I

>­V)

9 ~ _,

~ .!J c:; 0 u :, -I t, I ~ >-0 u ::::::> -I

..$2.. .1 >­V)

9 >­x ---------;

4

inhibitory effect of tomatine on a broad range of fungi.

Generally, tomato pathogens, including Fol race 1, were

found to be less sensitive to tomatine than were

non-pathogens. Arneson and Durbin suggested that the

association between tomatine and resistance should be

re-evaluated .

Continued efforts to resolve questions regarding

the role of tomatine in Fusarium wilt resistance have

5

failed to provide clear-cut answers. Mace and Veech (1971)

cited evidence that spore germination or mycelial growth

is retarded in the stems of resistant tomato plants inocu­

lated with Fol race 1, suggesting a chemical basis for

resistance. Langcake et al. (1972) published a detailed

study of an inhibitor produced in response to infection.

Based on chromatographic evidence, the investigators

identified the compound as tomatine. They reported a sub­

stantial increase in the tomatine concentration of tomato

stems and roots immediately following infection. However,

this increase did not substantiate the involvement of

tomatine in resistance because similar increases occurred in

both resistant and susceptible cultivars,

Mccance and Drysdale (1975) estimated the levels of

tomatine in the vascular tissue of race 1-inoculated and

noninoculated tomato plants. An increase in tomatine

levels occurred in both the resistant and suscepti ble

cultivars following inoculation, confirming the data

obtained previously by Langcake et al. (1972). Mccance and

Drysdale (1975) disputed Kern's earlier claim that the

concentration of - tomatine in vivo was far too low to

inhibit Fol. They found that tomatine levels in the

taproots of resistant and susceptible cultivars were

considerably higher following inoculation than would be

required to completely inhibit spore germination or hyphal

extension in vitro.

6

Hammerschlag and Mace (1975) provided further

evidence that host-produced fungitoxicants could account

for resistance. The antifungal activity of root extracts

from wounded-noninoculated or wounded-inoculated tomato

plants resistant to Fol race 1 was nearly twice as great as

that of extracts from susceptible plants similarly treated.

The inhibitor was identified as tomatine on the basis of

chromatographic and bioassay data.

Stromberg and Carden (1974, 1976, 1977) have

recently reported that acetone ext r acts of xylem vessel s i n

resistant and susceptible hosts are fungitoxic to Fol

race 1 and race 2~ Within the infected susceptible

cultivar, the race 1 fungal populat ion began to decrease

J days after inoculation, while the toxicity of the xylem

extract decreased. However, in the resistant cultivar the

fungal population remained low and the xyl em extracts

became highly fungitoxic.

Few tomatine studies have utilized resist a nt ~nd

susceptible isogeni c lines (Hammerschlag and Mace, 1 975 ;

Mace and Veech, 1 971). A comparative study using

near-isolines would greatly increase the probability that

differences between susceptible and resistant host

responses to infection could be attributed to the resist­

ance mechanism. The tomato isolines chosen for this study

were Improved Pearson (IP) and Pearson VF-11 (VF), VF

carries the dominant 1 gene for resistance to Fol race 1,

whereas IP lacks this gene. Race 2, another pathogenic

race of Fol, represents a one-step transition from race 1

(Gerdemann and Finley, 1951). Both VF and IP are suscep­

tible to race 2.

The two isolines, in combination with the two

pathogenic races, result in one resistant host response

(VF-race 1) and three susceptible responses (VF-race 2,

IP-race 1 and IP-race 2), Observations can be made on a

susceptible and resistant host reaction in two separate

cultivars, both inoculated with the same pathogenic race,

as well as a susceptible and resistant reaction in a single

cultivar inoculated with two different races. A compar­

ative study of this type would provide a valuable tool in

furthering our understanding of wilt-resistant and

wilt-susceptible responses.

To date, most researchers have used whole stem or

root extracts to determine tomatine levels, However,

because Fol confines itself to the host vessels, the

concentration of tomatirein the xylem fluid, rathe r than

whole tissue, must be considered in evaluating the rol e of

tomatine in resistance. One of the thre e objectives of

7

this investigation was to determine the levels of tomatine

in the xylem fluid of near-isogenic tomato lines treated

as follows, i) nonwounded-noninoculated, ii) wounded­

noninoculated, iii) Fol race 1 inoculated and iv) Fol

race 2 inoculated.

The toxicity of crude plant extracts (Fisher, 1935;

Gottlieb, 1943; Irving et al., 1945; Langcake et al., 1972)

and purified tomatine (Arneson and Durbin, 1968; Fontaine

et al., 1948) has been examined as one means of evaluating

the role of tomatine in resistance. Determinations of the

ability of tomatine to inhibit spore germination and

myceJial growth of Fol race 1 have been the focus of these

fungitoxicity assays. The assumption has been that if

tomatine is a determinant of resistance, the mode of

operation within the plant is the inhibition of vegetative

growth or spore germination. However, the rapid rate of

host colonization by vascular pathogens depends not on

mycelial growth but on the ability of the fungus to

sporulate within the xylem vessels (Beckman et al,, 1 961) .

Spores are transported in the transpiration stream until

they become trapped on the end walls of a vessel, Conidia

germinate, penetrate the obstruction and sporulate in the

newly invaded vessel. The new generations of spores are

transported in the transpiration stream until they reach

the next barrier, and the process is repeated, A

host-produced substance which could effectively inhibit

sporulation within t he xylem vessels could seriously limi t

8

9

the ability of the pathogen to colonize the host and, thus,

contribute to resistance. Host colonization by vegetative

growth alone would be slow, allowing more time for the host

to respond with other defense mechanisms. To date, no

attempt has been made to investigate how tomatine affects

sporulation. The second objective of this study was to

determine the effect of tomatine on microconidial produc­

tion of Fol race 1 and race 2.

Fol race 2 is able to overcome the resistance

mechanism of tomato plants carrying the single dominant I

gene for resistance to race 1. It was hypothesized that,

if tomatine determines resistance by inhibiting vegetative

growth of the pathogen, then the more virulent race 2

should be less sensitive to tomatine than race 1.

Previously, growth inhibition studies have been concerned

with race 1, but not with race 2. The third objective of

this investigation was to compare the effect of tomatine on

race land race 2 with respect to colony growth, germ tube

length and spore germination.

1 0

Materials and Methods

Cultural Procedures

Host. Near-isogenic lines of tomato (Lyconersicon

esculentum Mill.) served as host plants. The isolines IP

and VF are, respectively, susceptible and resistant to Fol

race 1. Both cultivars are susceptible to the more

virulent Fol race 2. Tomato seeds were sown in Jiffy Mi x

and transplanted at the two-leaf stage to 10-cm-diameter

pots containing a 1:1:1 soil:peat:Perlite mixture. Plants

were maintained under greenhouse conditions, and daylight

was supplemented with cool-white Sylvania fluorescent

lighting to provide a 15-hr photoperiod. Soluble 16-J2-16

fertilizer was applied weekly.

Pathogen. Isolates of Fusarium oxyspor um f. sp.

lycopersici races 1 and 2 were obtained from Dr. G, M.

Armstrong (Georgia Agricultural Experiment St ati on ,

Experiment, Georgia J0212), Race 1 and rac e 2 cultures

were grown on potato dextrose agar (PDA ) and i ncubated at

28 C in the dark. Microconidia for inoculum were washed

from the cultures with distilled water and then filtered

through lens paper. The spore concentra tion was adjusted

with t he aid of a hemacytomete r.

Inoculation of host. Plants grown to the seven-

to eight-lea f stage we re used for i noculation. The plants

were uproot ed and the roots washed free of soil, Taproots

11

were severed in the region where they were approximatel y

2 mm in diameter~ 7-10 cm below the cotyledons. The root

systems were submerged in a spore suspension of 106

micro­

conidia/ml distilled water for JO min, Plants were then

repotted. Another group of plants was wounded by severing

the taproot and submerging the root system in distilled

water in place of the inoculum, Nonwounded-noninoculated

plants served as controls.

Quantitative Determination of Tomatine

in Xylem Fluid

Obtaining xylem fluid. Xylem fluid was obtained

using a modification of the root pressure technique

described by Schnathorst (1970). Twenty-five plants of

each treatment were decapitated 2, 7, 12 or 17 days after

inoculation. The lower stem of each plant was thoroughly

cleansed with distilled water and then disinfested with

70% eth2.nol, An oblique cu t was made between t he first

and second nodes using a r a zor blade rinsed in 70% ethanol .

Alcohol-sterilized t ygon tubing was fitted ove r t he s tump

and an autoclaved 9" Pasteur pipet was inserted i nto t he

tubing. A bend in the tip of the pipet served as a trap

for air-borne contaminants. Xylem fluid whi ch exuded i nt o

the collecting tube by root pressure was wi thdr awn by

ins erting a 22 G needle wi th a 5- ml syri nge into t he tygon

tubing (Fig , 2). Collections we r e made fo r 2 days

following decapitation . The exudate was f ilte r ed through

a Millipore filter ( 0 . 45 um pore s iz e) and frozen at - 17 c.

Fig. 2. Method for collecting and extracting xylem

fluid exudate from decapitated tomato plants.

12

l J

Thin layer chromatography. Tomatine was separated

from the xylem fluid exudate using thin layer chroma­

tography (TLC). Xylem fluid (5 ml) was evaporated to

dryness under reduced pressure at 45 C. Flask contents

were taken up in 10 ml of 50% methanol acidified with two

drops of SO% H2so4 • Aliquots (500 ul) were applied as a

14-cm band on 20 X 20 cm pre-coated silica gel TLC plates.

Chromatograms were developed in a solvent system consisting

of iso- propanol1formic acid:distilled water (IFW, 73:J:24).

After the solvent front had advanced 10 cm to a pre-scored

line, plates were removed from the tanlrn and air dried at

room temperature. Tomatine was located at Rf o.66 either

by spraying a plate with modified Dragendorff reagent

(Cromwell, 1955 , p. 506) or by placing a plate in an iodine

chamber. A bright orange color reaction indicated the

presence of tomatine. The tomatine zone was scraped from

unsprayed chromatograms and eluted in 6 ml of 50% methanol

acidified with one drop of SO% H2so4 • The silica ge l

particles were concentrated by centrifuging at 1000 rpm

for? min. The supernatant was then collected and evapo­

rated to dryness. Concentrated H2so4 (10 ml) was added to

the residue and the mixture incubated at 40 C for 24 hours.

The percent transmittance of the resulting chromagen was

measured at J25 nm on a Beckman DB-G grat i ng spectrophoto­

meter. The molar concentration of tomatine present was

determined by reference to a s tandar d curve (Fig, J )

developed using known concentrations of comme rcial

14

80

w u 60 z

\x < I-I-

~ V)

z 0 < 40 ~ I-

I- ~ ESTIMATED z w x OBSERVED u Qi:! 20 w 0..

0

10-7 10-6 10··5 10 -4 10-3

TOMATINE CONCENTRATION (M)

Fig, J Standard curve of various concentrations of

comme rcial alpha- tomatine plotted against t~e percen~

transmittance of t he tomatine - H2sou chromager. meas~red a~

J25 nm . Estima ted values were derived f r o:n a regression

analysis of the observed va l ues (correlation coef::c · er.t=

0 . 988) .

alpha-tomatine (ICN Life Sciences Group, Cleveland,

Ohio 44128). Total mg tomatine produced per plant was

calculated on the basis of the average amount of xylem

fluid exuded per plant for each treatment. The data were

analyzed statistically using Duncan's multiple-range test

(P=O. 05).

Effect of Tomatine on Colony Growth

and Spore Production

15

Solutions of tomatine were prepared to giv e molar

concentrations of 10-J, 2 X 10-4 , 4 X 10-5 and 8 X l □- 6

when added to the test media. Pure alpha-tomatine was

dissolved in 10 mM HCl and the pH adjusted to 4,6 wi t h a

phosphate-citrate buffer. Solutions were sterilized by

filtration through a Millipore filter (0.2 um pore size)

and added to autoclaved, rehydrated Difeo FDA, Buffered

PDA lacking tomatine ser~ed as a control. Aliquots ( 8 ml )

of media were poured into sterile polyurethane petri

dishes (60 X 15 mm).

Test and control media were seeded with a s ingl e

4-mm-diameter plug taken from the margin of 4 - da y- old

colonies of Fol race land race 2. All cultur e s were

incubated at 27 C in the dark . Radial growth , dry weight

and spore production (microconidia/mg dr y wt) of each

treatment were determined ever y 48 hours f or t wo weeks ,

The data were analyzed s tatist i cal ly using analysis o:

va rianc e (P=0.01 ) .

Colony growth, Radi a l growth was deter~i-ec by

16

making two perpendicular measurements of the diameter of

each of three colonies per treatment . colonies were fre ed

from the media for dry weight determinations. First, the

agar containing the colonies was removed from the plates

and autoclaved. Distilled water was used to dilute the

agar and, thus, prevent re-solidifying. The d i luted

medium was then filtered through Whatman #1 filter paper

and the remaining fungal materi al was rinsed with hot

distilled water. Colonies were oven-dried at 110 C for 24

hours and then weighed . Each determination was replicat ed

three times.

Spore production. A separate set of three culture

plates per treatment was used to measure spore production .

Mi croconidia were washed from each plate with distilled

water and then filtered through lens paper. The result ing

spore suspension was brought up to a constant volume

(20 ml) . The total number of spores prod1..;.c ed per plate

was determined with the aid of a hemacytomete r (spor es/ml X

20 ml) , The three dry we ight measurements were randomly

mat ched with the three total spore counts of the same

treatment, and spore production was det er mined on t he basis

of microconidia/mg dry wt.

Spore viability. Conidia f rom fou r- and eight - day­

old colonies grown on the control media , 10- J Mand

2 X 1 0- 4 M tomatine were tested !or viability usi g a

germination bi oa ssay . The s p ore susper.s~or.s sec i~

spore

determini ng microconi di a l produc t i on we r e adjasted to a

17

concentration of 2 X 105 spores/ml, One drop of the

suspension was added to one drop of phosphate-citrate

buffer (pH 4.6) on a microscope slide coated with 2,5%

cellulose nitrate. Slides were incubated for 24 hours at

26 C. Following incubation, the spores were stained and

fixed with 1% cotton blue in lactophenol. Percent

germination was determined by counting the number of spores

out of 100 whi ch had germinated. Three rand om fields per

replicate were examined for each treatment,

Effect of Tomatine on Spore Germination

and Hyphal Extension

Microconidia were harvested from 10-day-old stock

cultures of Fol race land race 2 and filtered through lens

paper. The spore concentration was adjusted to 2 X 105

conidia/ml distilled water with the aid of a hemacytometer.

Solutions of t omatine were prepared to give molar concen­

trations of 10- 3 , 8 X 10-4 , 4 X 10-4 , 2 X 1 0- 4 , 4 X 10- 5,

8 X 10-6 and 16 X 10- 7 when added to the spore suspensi on.

Pure commercial alpha-tomatine was dissolved in 1 0 mlY! ECl

and the pH adjusted to 4.6 with a phosphate-citrate buffe r.

A buffer-HCl solution lacking tomatine served as t~e

control. One drop of the test solution was add ed to one

drop of the spore suspension on a mi croscope s lide coated

with 2.5% cellulose nitrate . Ea ch treatment was r eplicated

six times. Sl i des were placed i n ste rile mo i s t petri

dishes a nd incubated for 24 hour s a t 26 c . Aft er

incubation, spores were sta ined and ~i xed wi~ h 1% cottor.

blue in lactophenol.

Percent germination was determined by counting

the number of spores out of 100 which had germinated.

Spores were counted as ge r minated if the germ tube was at

least the length of the spore. Three random fields ner

replicate were examined for each treatment. Germ tube

lengths were measured using a microscope with an

eye-piece micrometer. Twenty-five spores pe r replicate

were measured for each treatment . The data were analyzed

statistically using analysis of variance (P=0,01).

18

Results

Quantitative Determination of Tomatine

in Xylem Fluid

19

There was no significant difference in the molarity

of tomatine in the xylem fluid of IP and VF controls

(Table 1), There was, however, a significant difference

in the response of the two cultivars to the various treat­

ments. The VF isoline consistently produced mo r e tomati n e

than IP following inoculation or wounding, The molarity

of tomatine present in the re sistant VF-race 1 and i n the

susceptible VF-race 2 host-pathogen combinations t wo days

after i n oculation was ten time s hi gher than the l evel

present in the control, This maximum mo lar c oncentration

was maintain ed until the seventh day after inoculation .

From the twelfth day to t he seventeenth day, the co1cen­

trations of tomatine in VF-race land VF-race 2 plants

were compara ble to the controls , Wound ing also induced a

ten-fold increase in the molarity of tomatine in the VF

cultivar . The maximum level of tomati~e was r eached on the

second day after wounding , t . en decreasing to the level of

the cont rol by the seventh day ,

Inoculat ion or wounding fa iled to ·nduce any

increase in the molarity of tomatine present in the I?

isoline (Table 1). The tomatine concent r ati0n in the

xy l em fluid of the two susceptible IP - inocula~ed c mbi~a-

TABLE 1. Tornatine content (moles) of xylem fluid exudate from stems of Improved Pearson (IP) and Pearson VF-11 (VF) tomato near-isolines at selected time intervals. Plants were nonwounded-noninoculated (CK), wounded-noninoculated (W), ino~ulated with Fusarium oxysporum f. sp. lycopersici race 1 (Rl) or inoculated with Fusarium oxysporum f. sp, ~ycopers ici r a ce 2 (R2). Host-pathogen combinations resulted in either a susceptible (S) or resistant (R) reaction,

Tomatine concentration (moles)*

Days after treatment

Host Treatme nt reaction 2 7 12 17

VF-Rl R 1. 9 X 1 0-J d 1.6 X 10-J b 2.J X 10-5 a J.4 X 10- a

VF-R2 s 1.4 X 10- J cd 1.5 X 10-J b -4 8.1 X 10 a 7.1 X 10-4 a

VF-W 1. 2 X 10-J bed 6 -4 -4 7.8 X 10- 5 a - .OX 10 a 1.1 X 10 a

VF-CK - 1.L~ X 10-4 a 1.4 X 10-4 a 1.4 X 10- 4 a 1.4 X 10-4 a

-4 L~.2 X 10-4 a _L~ C:

IP-Rl s 9 .0 X 10 abc 1. 0 X 10 a 7.8 X 10-.J a

n,-H2 s 4 _L~ . 9 X 10 a bc 1.J X 10-4 a L~.l X 10-4 a 5.6 X 10_/.J. a

JP-VI - 1.9 X 1 0- 4 a 2.2 X 10-4 a 4.0 X 10- 5 a 4 -4 .1 X 10 a

JP-CK - Lj - 4 J . · X 10 a 4 -4 J. X 10 a 4 -4 J. X 10 a J . L~ X lO_L~ a

-lfEo.ch value represents the mean of three observations . Means within a column not followed hy tl1e same letter are signi ficantly different, P=0,05, ac cording to Dunc an ' s nrultipJe-ranr c test .

N 0

tions and the IP-wounded treatment were comparable to t he

control on al l four sampling dates .

21

The average yield of exudate per plant r ang ed from

2- 7 ml. Recognizing that variations i n y ield c ould

conceivably influence quantitative determinations based on

molarity, compari sons of the vari ous treatments were also

made on the basis of total mg t omatine produced per plant

(mg/plant). These data a re presented in Table 2.

Generally, an examination of tomatine concentrations in

mg/plant reveale d the same trends observed when tomatine

concentrations were expressed as molarity . In the VF

isoline, tomatine i ncreased to a leve l above the control

following i noculation or wounding. However, while toma t ine

molarity within VF-race 1, VF-race 2 and VF - wounded plants

increased above the control by a factor of t en, tomatine

in mg/plant increased only three- to five- fold for the

same treatment s. The conc entrati ons of tomatine in

mg/pl ant we re comparable for all IP-inoculated, - wound ed

and control plants, r e ga rdle s s of sampling time .

Effect of Tomatine on Colony Growth

and Spore Production

Colony growth. Tomatine signific a ntly i~h i b i ted

the radial g r ow~h of race 1 and r a ce 2 , bu t r a c e l was

inhibited more than r ace 2 ( ~i g . 4 - A, 3) . Di:fe r ences i~

t he sensitivity of race l and race 2 t o t he i n r. ioi t ory

effect of tornatine bec ame more evide nt wi~h ~he ~roar es io~

of time . These differe;1c e s were observable a:-: er 6 - 8 da: _

22

TABLE 2. Tomatine content (mg produced/plant) of xylem fluid exudate from stems of Imnroved Pearson (I P ) and Pearson VF-11 (VF) near-isolin~s at selected time intervals. Plants were nonwounded-noninoculated (CK), wounded-noninoculated (W) , inoculated with Fusarium oxysporum f, sp, lyconersici race 1 (Rl) or inoculated with Fusarium oxysporum f, sp. lycopersici race 2 (R2). Host-pathogen combinations resulted in either a susceptible (S) or resistant (R) reaction.

Tomatine concentration (mg/plant)*

Days afte r treatment

Host Treatment reaction 2 7 12 17

VF-Rl R o.49 d 0.61 C 0,01 a 0.02

VF'-R2 s O,JO bed 0 .52 be 0,11 a 0.11

a

a

VF-W O.J4 cd 0.24 abc 0,06 a 0.04 a

VF-CK 0,07 a 0,07 a 0.07 a 0,07 a

IP-Rl s 0.19 abc 0 .22 abc 0.06 a O,OJ a

IP-R2 s 0,07 a 0 . 06 a 0 ,1 9 a 0 . 21 a

IP-W 0.05 a 0.06 a O.lJ a O.l J a

IP-CK 0 ,11 ab 0.11 ab 0 .11 a 0,11 a

*Each value represents the mean of three observations. Means within a column not followed by the same lette a~e significantly different, ~= 0 , 05, according to Duncan ' s multiple -range test.

Fig. 4. Colony diameters of Fusarium oxvsnorum f. sp,

lycopersici race 1 and race 2 cultures grown on buffered

potato dextrose agar containing A) 10- 3 M, B) 2 X 10-4 M,

C) 4 X 10-5 Mor D) 8 X 10- 6 M alpha-tomatine. Control

cultures were grown on buffered media lacking tomatine.

>­z 0

0 u

"'

>­z g 0 u

50

40

30

20

10

0

50

40

30

20

10

0

C

·---• · --• · --•--➔ A / _,,/

•✓/ /.

/ I~ o-----------0

/ /// o 0/ / Jo / .{I

/ /

0

BUFFER (p H 4 .6 ) I {/ • - -• RACE 1 or 2

/ ~e TOMATINE (10·3 M ) .~ o--o RACE I • •-o RACE 2

0

C

2

4

DAYS

6 OF

8 10

GROWTH

12

SUFFER (pH 4 6\ •--• RACE 1or2

TOM A TINE 4 X 10· 5 \.\ 1 o--o RACE

•--• RACE 2

4 6 8 1() '2

DAYS O < GR W TH

I 4

1.;

>­z 0

0 u

50

40

20

10

0

so

40

"' 30

~ <(

0

>­z g 0 u

20

0

I •

B

D

4

BUFFER (pH 4 6) •--<> RACE 1 or 2

TOMATINE (2 X 10·4 M ) o--o R.11.CE 1 •--• RACE 2

6 10 12

DAYS OF GROWTH

BUFFER pH J 6

•--• RACE c.r ro .-.. A-INE s x ·0· 0 y

o--o RACE •--o RACE

4 6

CAYS OF

,.4

14

of growth on 10-J M tomati ne and afte r 2 - 4 days of

growth on 2 X 10- 4 M tomatine . Race 1 and r ace 2 buffer

controls grew to the limits of the petri plates after 6

days. Race 2 took 14 days on 1 0- 3 M tomatine and 10 days

on 2 X 10- 4 M tomatine to grow to the limits of the plates .

Even after 14 days of growth, c olony diameters of r ace 1

-3 2 - 4 · t· 1 2-,:1_ on 10 Mand X 10 M toma tine were , respec ive y , J~

and 7% less than the control (F ig. 4 -A, B) , Race 1

appeared to be slightly inhibited on 4 X 10- 5 M tomatine

after 2 and 4 days of growth, while r ace 2 was unaffe cted

at that concentration (Fig , 4-C), Neither r ace was

inhibited at the l owest concentration i nc luded in this

experiment (Fig , 4-D), Colony morphology was also influ ­

enced by tomatine. Levels of tomatine whi ch we re

inhibitory to the r adial g r owth of race 1 or race 2

colonies caused an increase in ae ri a l g rowth.

Tomatine also significantly re duced t he d r y weights

of race 1 and race 2 colonie s compar ed to the c ontrols

(Fig. 5-A, B, C, D), but there was no significant differ­

ence between the two races. Race l and r ace 2 co _ ony

growth, as determined by dry weight , was inhibi ted on

10- 3 Mand 2 X 10- 4 M tomatine (Fig . 5 - A, 3) , Only race 1

was inhibited on 4 X 1 0- 5 'vl tomatine (Fig . 5 - C) . _;ei't;her

r a ce was affected on the l owe st conce n :ration (?~g . : - ) ,

Spore produ cti on , ~ omatine c aused a s:gr.· _ icant

increase in the spore p r oduc ion (nicrocon~ ia/ x 0 dry ·y~ }

of Fol r ace 1 and r a ce 2 , b t r ace 1 was af~e ted ~ere

Fig. 5. Mycelial dry weights of Fusarium oxysporum

f. sp. lycopersici race land race 2 cultures grown on

buffered potato dextrose agar containing A) 10- 3 M,

B) 2 X 10-4 M, C) 4 X 10-S M or D) 8 X 10- 6 M alpha­

tomatine. Control cultures we r e grown on buffered med ia

lacking tomatine,

50

40

.... 3 30

► 0: 0

20 ': w u ► ::,;

....

10

0

50

40

3 30

>­"' 0

< 20

w u >-:;

10

0

0

0

,o, / '

BUFFE R (pH 4 .6 ) o--o RAC E I

•--" RACE 2 TOMATINE i l0"3M '

o-o RACE I •-• RAC E 2

/ '-o /

0

0 /

,, ' ' I / '

/ I 'o---"· ~, I I I

I I I I

I o I /

-·

/ / // -----◊ :, /0 /:,__.

I / // I / !l:,"'"

Io' ;/ / / _//

// /o !/ 0~ /

e' /,,o/ ---•1/ e---o

6 10

DAYS OF GROWTH

BUFFER (pH 4 .6) o- -o RACE I .,_. -• RACE 2

TOMATINE (4 X I0.5 M ) o--o RACE I •-o RACE 2

4

DAYS

6

OF

8 10 GROWTH

12

12

A

14

C

14

50

40

3: 30

>­"' 0

w u >-::,;

10

0

50

40

3: 30

>-0: 0

~ 20 ~

w u >-::,;

10

C

BUFFER ' pH -1 6 1 o- - o RACE I •--• RACE 2

T0MAT INE (2 X I0.4

M \ o--o RACE l &-• RACE 2

0 4 6

DAYS OF

BUFFER (pH 4 6 )

0

o - -o RACE I o--~ RACE 2

TOMA T !N E \8 X 10·6

M l o-o RACE l ..-c RAC E 2

2 4

DAYS

6

OF

27

B

8 10 12 14 GROWTH

D

.,

28

than r ace 2 (Fig, 6) . An initial inhibit ion of sporulat:o,

- J - 4 . in colonies grown on 10 Mor 2 X 10 M tomatine was

followed by a dramatic increase in the number of spores

produced/mg dry wt, reaching a peak after 4 days of g r owth .

Culture s grown on the buffer control, on 4 X 10- 5 M

toma tine or on 8 X 10-6 M tomatine re a ched thei r maximum

levels of spore production after 2 days of growth

(Fig . 6-C, D). At the peak levels of spore production on

10-J Mand 2 X 10-4 M tomat i ne, rac e 1 p r oduced three times

more spores/mg dry wt and race 2 produced two times more

spores/mg dry wt tha n their respective cont rol s , Race 1

colonies grown on 4 X 10- 5 M tomatine r eached a l evel of

spore product ion twic e as high as the peak l evel observed

in the control. Race 2 g rowing on 4 X 1 0- 5 or 8 X 1 0- 6 M

tomatine and race 1 g rowing on 8 X 10- 6 M t omatine r eached

l evel s of spore production comparabl e to t heir respective

control s , Once spore p roduction r eached a maximum in

race 1 and race 2 buffer controls, spore production quickly

dropped to a near-minimum and then l eveled off . Mic ro ­

conidial production in cultures grown on tomatine

generally remained at maximum or near- maximum levels for

sev eral days aft e r reaching the peak in spore production.

Aft 6 8 d f th " 0-J Mt . . er - ays o grow on i oma~_ne , rac e 'was

producing 2J time s more spores/mg dry wt and race 2 was

producing 11 times more spores/mg d ~y w~ than ~heir

respective cont rol s (Fig 6- A), uring ~he sa e ~i~e I

period on 2 X 1 0- ~ M tomatine , race 1 was rod cing 1 6

Fig, 6. Microconidial production of Fusarium oxysporum

f. sp. lycopersici race 1 and race 2 cultures grown on

buffered potato dextrose agar conta ining A) 10- 3 M,

B) 2 X 10-4 M, C) 4 X 10-S or D) 8 X 10-6 M alpha-tomatine.

Control cultures were grown on buffered media lacking

tomatine.

"o X

:i:

320

280

240

>- 200

"' 0

<.:> ~ 160

-----0: 0

z 120 0 u 0 "' u i 80

40

0

280

24 0

X

,-.. 200

3: >-~ 160

<.:> ;E

';;;- 120 0

z 0 0 80

"' ~ ;E

4

A SUFFE R (p H 4 .6 )

o--o RACE I •-_. RACE 2

TOMATINE (10 -J M ) o-- o RACE I •--• RACE 2

~ \ 0

,/\/ 0

/\_,-e -'\ " ~ ,~[/ \ -ty:-.., \ •

It ' \ // 0 •, \

¥ ' ' '.'./

.. ' o, I , ' o- ·--o--- • Y o---•__:--:3---o- --o

0

C

0

2 4 6 DAYS O F

8 JO 12 14

GROW TH

BUFFER (pH 4 . 6 ) o--o RACE •--o RACE 2

TOMATINE (4 X 10-5M ) o--o RAC E I e--• RACE 2

o'---..__

~o

\ --0 0--0

6 10 12 14

DAYS OF GROWT H

320

2S0

~ 2 40 X

:i: >- 200 "' 0

<.:> :l': 160 '-<t

0

~ 120 V

0 "' v

X

80

4 0

0

280

- 200 3:

0

z 0

0 ac :x v

B BU FFE R pH 4 6 j

o -- o RACE I

• - -• R E 7 TO MAI I E 7 X '0 'M

o----o ij ACE • - • SlAC E ~ o-_,_-\

0

,:./ \ . 0

11:1, \ .............. . • 01' \ " /

I•' \ " . •, \ .--' o _

' --.._ o_ - • ·- -• · --• •--- • . -==-- o- - - 0 -- 0

~

0

0

,l 6

DAYS O F

D

<>--- o

j --0

o -l • , \ \

/I

1I ' ti I •

'

0

8 10 12

GROWTH

BUFFER p>-i 4 b o - -o RA CE • - -• RA CE 7

IOMAl l°'E 9 X '()., '-\ o--o ~ACE I • --• R,>.CE 2

I 1

I

times more spores/mg and race 2 was producir.g 7 times , or e

spor es/mg than the controls (Fig. 6- B) . Race 1 spore

production on 4 X 10- 5 M tomatine was 7 ti es greater tha

t he buffer cont r ol (Fig . 6-C). The difference between

r a ce 1 cu l tures on 8 X 10- 6 M tomatine and the control was

l es s drastic (Fig. 6- D). Race 2 spore production on the

two l owest concentrations of tomatine (4 X 10- 5 and

8 X 1 0- 6 M) was comparabl e to the controls (Fig . 6-C, D).

Spore viability . The viability of rac e land

r a ce 2 rnic r oconidia produced on 10- 3 Mand 2 X 10- 4 ~

t oma t i ne was compared to the viability of conidia produced

on the control media . The percent ge r minati on of r ace 1

and r ace 2 sp ores was comparable (85- 95%) , whether the

sp ores were from colonies grown on tomatine or from

c olonies grown on the bu:fer control.

Effe c t of Tomatine on Spore Germination

and Hyphal Extension

Spore germination . Tomatine significantly

r e s tricted spore germinat ion of both r aces. There wa s no

difference in the sensitivity of race 1 and r ace 2 , except

at 10- J M torr.atine (Fig. 7) . A-c t .at concent r a tio. 1, the

more virulent race 2 was surprisingly more s ensit i ve than

race 1 . While spore ge rm:nation of r a c e was oO~ l ess

than the control, race 2 s:i:,ore ge m: :. .a-:.o:: was ir.r.i i -ced

by 80% . Spore germin2.:tion wa only slie:;r.tl. iJ:J1i oi 1:e

(12 - 24%) at 1 _ iJ. • M~-'-; _ !_. X , . "tO ... C.v- e , 3e ow - X _c !: ,

spore ger ination i n torr.a~ine was - ~ ~v

32

fa--~ I ~:r--~ ] 100 <t •-IID'a.--. o - -- ... - o-<l-"' -------- ke ...... ,-. --

~ -.._,/ m...., -.,.J ~

"'o z 80 0 -

0 \v I- ~ <!

~ z ~ cc 60 w 0

w ~ O--O RACE l 0 a. 40 G.....,. @ RACE 2 V\

I-z UJ u Ci:: 20 w a.

0 -

TOMATINE CO NCE N TRATIO N (M)

Fi g . 7 , E.c-fec-;; o va r io ·s concer.-;;rai;.:.cnr c: ;_,L,~:-_r ec

(pH 4 . 6) al ha- tornatine or~ &er:i.i_ atio!i o: :\.:s-_ri '!': ox·.-~.,.cr -

f . sp . lycouersici r ace 1 and r a ce: ~ic r cccnici~ . ~c~~rc_

sol 1 · ons lac: -- ed to:na~i r_e . .....,o-r.:...:ia ~.,;e !:" e :__ c;;::c . .-:ei: -r

hou r s ~t o

cont rols . The ED50 for the i~~:.bit · on of spore ;er~ir.ation

was 8 X 10- 4 M,

HyPhal extensi on . The inhibitory e:fec~ o:

tomatine on germ tube length was comparable for both race l

and race 2 (Fig . 8) , Tomatine concentra ti ons of 10- 3 ~ and

8 X 10- 4 M were highl y inhibitory to Fol, resulting :.n 2.n

8 0-90% r eduction in ge r m tube length compared to the

controls. The ge rm tubes of microconidia in 4 X 10- 4 j '. or

2 X 10- 4 M tomatine were less t han one-half the length of

the control g erm tubes. The t hree lowest molar concen­

trations (4 X 1 0- 5 M, 8 X 10- 6 Mand 16 X 1 0- 6 M) r es~l ed

in l ess than a 25% inhibition of germ tube length. The

ED50 for the inhibition of hyphal extension was c :< 10- 5 M

tomat ine .

J4

80

@-..., - - - - o ....... --70 o ... - - ....... ......

V') --• e z ..... ~ 0 f)' 0:: 60 u .,/e> :::E 0 .

50 :I: I-

~ (!)

4 0 z w ....,j

30 0 --0 RA CE l w 0

'° o.,.,..e RA CE 2 \ :::, 0 I- @~\ 20 ~

~a a::: w (!) 10 c\

0

0 ~-7 10-6 10 -s 10-4 10-3

TO MATINE CONCEN TRA TlON (M )

Fig . 8 . Effect of various concentra~ions of buffered

(pH 4 . 6) alpha- tomatine on the g erm tube leng th of Fusariurn

oxysporum f . sp. lycopersici race land race 2 rnicroconidia.

Control solutions lac ked tomatine . Conidia we re incubat ed

for 24 hours at 26 c .

;_,

Discussion

Two types of host re sistance mechanis, s are kr.ow ..

to oper ate i n vascular wilt diseases (Ta l boys , 1 964 , 1972) .

"Extra - vascular determinants" are those host respo .ses

which act to limit cortical invasion and , thus, Freet

pathoge n entry into the vascular system . Lignituber

f ormat i on and cortical cell suberization- can function as

def ense mechanisms in the pre - vascular stage of pathorene~is

(Tal boys , 1958) , Resistance in pre - vascular tissue hao

a lso been a t t ri buted t o increased synthesis of antifu. ~al

c omp ounds i n r esponse to infection (Bell , 1 969) . It is

conc e i vable that tomatine could similarly act as an

ext r a -vas cular determinant of resistance to Fol , but this

has not yet been investigated .

Root damage may enable many wilt patho[e s,

including Fol, to by- pass extr a - vascular determinants . ?he

preset study was designed to examine ~he . a~u r e of the

host resistance r esponse once v sc lar inf c~io. has

occurred . The pathogen was introduced directl' in o the

xyl em vessels via a severed - t proot inoc la~ior. -:cch~iq· e .

As such , the main c oncern here is with he seco .d :• ~ O!

mechanism , " a scula r determina ,t -" o: r s: -:::. ce . Va.:cu:. .r

deter i .ants a::e host resFonse- wn · ch or r3. :e .. ,.:_ :~. ~~ - ."1.C

va "c l a r t issue o imi: sys e.,.:.c c.:.s:r:!._ 11-::0:-. o:-: .e

athoe;en . Pheno_c:,

com ounds h ·e bee. i:n1=-ica--.: c c.

- ' .. ~

36

tomato resistance to Fol race 1.

Matta et al. (1969) demonstrated in resistant

tomato plants that phenols increased to a maximum concen­

tration only 3 days after inoculation. Phenols increased

gradually in susceptible plants, not reaching a maximum

concentration until 18 days after infection. Matta et al.

suggested that phenols could either act directly to inhibit

fungal growth or aid in the formation of physical defense

barriers.

Vascular occlusion is a general mechanism by which

plants, once infected, can restrict systemic invasion

(Beckman, 1964, 1966, 1968; Beckman and Halmos, 1962).

Tyloses act as a physical barrier which the pathogin is

unable to penetrate. Beckman et al. (1972) compared

resistant VF-race 1 and susceptible IP-race l host-pathogen

interactions. Resistant plants were characterized by a

rapid host response, resulting in maximum vascular

occlusion 2 days after inoculation. Further occlusion was

maintained, effectively sealing off infection. Susceptible

plants also were initially characterized by a rapid host

response 1-2 days after inoculation; however, further

tylose development was retarded. Final occlusion did not

occur in the susceptible plants until the pathogen had

already become systemic.

Sinha and Wood (1967) suggested that tyloses may

not be the only factor involved in the resistance of tomato

plants to vascular wilt organisms. They presented evidence

that an antifungal compound may also be operating to

confirm resistance in Verticillium wilt of tomato.

J7

Early investigators of Fusarium wilt (Irving, 1947;

Irving et al., 1945) postulated that certain tomato

cultivars were resistant because of the presence of anti­

biotics inhibitory to the fungus. These substances would

be absent in susceptible cultivars. Tomatine was originally

thought to be such a compound. Further investigation

revealed that tomatine was present in both susceptible and

resistant tomato cultivars (Irving, 1947), This was also

found to be true in the present study, The concentration

of tomatine in the xylem fluid prior to infection (lo- 4 M)

was comparable in the VF and IP isolines, resistant and

susceptible, respectively, to Fol race 1,

Following the discovery of tomatine in resistant

and susceptible plants, researchers turned next to

investigate the effect of infection on tomatine levels

(Irving, 1947; Langcake et al., 1972; McCance and Drysdale,

1975), It is apparent from the discussion of tyloses and

phenolics as determinants of resistance that the critical

factor is the rate at which the plant is able to respond to

infection. Timing is also critical in any considerat ion of

tomatine as a vascular determinant, Since localization

occurs within 2-4 days after infection, any other mechanism

which is a primary factor in resistance should be evident

within that period. An increase in tomat ine concentration

immediately following infection in a resistant plant could

act to inhibit the pathogen until vascular occlusion has

been completed.

J8

The results presented herein indicate that the

concentration of tomatine in the resistant host-pathogen

combination did increase to fungitoxic level s during the

critical period following vascular infection. The data

also indicate a significant difference in the resistant

VF-race 1 and susceptible IP-race land IP-race 2 reactions.

Maximum tomatine production (approximately 10-J M) within

VF infected with race 1 was reached 2 days after inocu­

lation and was maintained until the seventh day. By the

twelfth day after inoculation, tomatine decreased to the

level of the control (approximately 10-4 M), The molarity

of tomatine in the susceptible IP-race 1 and IP-race 2

combinations did not increase significantly above the

pre-infection level at any time during the course of the

experiment. The differences observed here apparently

support the view that tomatine plays a primary role in

resistance to Fusarium wilt.

These results are supported by Stromberg and Gorden

(1974, 1977) and Hanunerschlag and Mace (1975) who also

demonstrated differences in tomatine levels in resistant

and susceptible hosts following infection. In contrast,

Langcake et al. (1972) and Mccance and Drysda l e (1975)

reported that comparable increases in the concentration of

tomatine occurred in both the resistant and susceptible

cultivars 2-4 days after inoculation.

If post-infection tomatine levels are critical to

the resistance of VF to race 1, then the susceptible

VF-race 2 reaction should yield results comparable to

39

those of the susceptible IP-race 1 and IP-race 2 reactions,

However, this was not the case here. Rather, tomatine in

VF-race 2 plants increased to a level comparable to that

of the resistant VF-race 1 combination. This is in

disagreement with Stromberg and Corden (1976) who suggest

that susceptibility to race 2 is due to failure of fungi­

toxicants to accumulate in response to infection,

Although rapid increases of tomatine in both

susceptible- and resistant-type reactions would suggest

that tomatine cannot be a primary factor in the resistance

of VF to Fol race 1, there is an alternative explanation.

The more virulent Fol race 2 could be less sensitive than

race 1 to the irJ1i bi tory effects of tomatine. If this were

the case, then race 2 could overcome the resistance

mechanism in VF merely by being able to tolerate levels of

tomatine that would otherwise be fungitoxic. To test this

hypothesis, the relative sensitivities of race 1 and race 2

were compared by examining the effect of tomat ine on colony

growth, germ tube length and spore germination, It has

already been established that tomatine is inhibitory to

mycelial growth and spore germination of Fol race 1 at

concentrations present in tomato plants (Mccance and

Drysdale, 1975).

The data presented here indicate that, as expected,

tomatine is more inhibitory to the colony growth of r ace 1

than race 2. This difference in sensitivity between the

40

two races argues for the involvement of tomatine in resist­

ance. However, two lines of evidence argue against this

involvement. When race 1 and race 2 colony diameters were

compared after 2 days of growth on 10-3 Mor 2 X 10-4 M

tomatine, there was no significant difference between the

two races. It was only after 4 days of growth that

differences in the diameters of race land rac e 2 became

apparent. Although race 2 was considerably more successful

than race 1 as the organism continued to colonize the media,

both races grew at the same r a te initially. It is not this

greater tolerance in the later stages of colonization, but

a more immediate one which would be i mp ortant to race 2 in

the VF isoline. The fact tha t tomatine was equally

inhibitory to race land race 2 during the initial period

of growth was further supported by slide bioassay data,

There was no significant difference in the sensitivity of

the two races to tomatine when germ tubes were measured

after 24 hours of growth . These data are supported by

Stromberg and Carden (1 976) who also conc l uded t hat race 1

and race 2 spores were equally sensitive to a fungitoxicant

in ac etone stem extracts.

Although the evi dence presented above makes one

question tomatine •s role i n resistance, it is true that

tomatine inhibits colony growth and germ tube length at

concentrations present in tomato tissue , The concsntra~ion

present in infected plants (l0- 3 M) was highly inhibitory

to growth, while the concentration found in healthy plants

(lo-4 M) was moderately inhibitory to growth.

Tomatine was more effective in inhibiting hyphal

extension than in restricting spore germination. Spore

germination was not inhibited at concentrations below

41

4 X 10-4 M tomatine, and even 8 X 10-4 M tomatine was only

moderately inhibitory to germination. This is in agreement

with a recent study by Mccance and Drysdale (1975) who also

presented evidence that tomatine is more inhibitory to

hyphal extension than to spore germination. In contrast,

Fontaine et al. (1947) indicated that tomatine was

fungicidal to spores of Fol race 1, but only partially

inhibitory to vegetative growth. The results reported here

show that race 2 is not less sensitive than race 1 to the

effect of tomatine on spore germination, Only at l □-3 M

tomatine was any difference between the races observed. At

that concentration, the more virulent race 2 was actually

more sensitive than race 1.

Thus, an examination of all the data from these

in vitro studies indicates that race 2 is actually not any

more tolerant than race 1 of fungitoxic levels of tomatine.

This argues against tomatine as the sole mechanism of

resistance.

It was hypothesized here that tomatine m2.y play a

key role in resistance by inhibiting spore production. The

importance of spore production in the rapid di stribution of

vascular pathogens within vessels of infected plants is

weil documented (Beckman, 1964; Beckman and Halmos, 1962;

Beckman et al., 1961; Dimond, 1955). However, data

presented here indicate that tomatine is a stimulant, not

an inhibitor, of sporulation. Tomatine stimulated spore

production at concentrations knovm to be present in vivo.

The molarity of tomatine in xylem fluid of healthy

. t . 1 1 t . ' 1 10-4 M resis ant or susceptib_e pans was approxima~e y •

42

Following infection, the level of tomatine increased to

10- J Min both a susceptible (VF-race 2) and a resistant

(VF-race 1) host-pathogen combination. The greatest

stimulation of race 1 and race 2 spore production was also

induced by 2 X 10-4 Mand 10-J M tomatine.

If tomatine causes a proliferation of spores in

vivo, the role of tomatine in resistance appears even more

doubtful. Pathogen build-up through massive spore

production is a characteristic feature of susceptible, not

resistant, host-pathogen interactions (Elgersma et al.,

1072). An in vivo stimulation of fungal propagules would

not be important to host colonization in resistant reactions

because vascular occlusion through rapid tylose formation

can physically localize wilt pathogens. Therefore 1 the

upward movement of conidia within the vessels would be

blocked in the resistant cultivar. In susceptible

cultivars, however, vascular occlusion is delayed, Tyloses

form too late to act as an effective barrier and are, thus,

by-passed by the fungus. Stimulation of spore production

in a susceptible plant would then be likely t o cont ribut e

to further host colonization by the pathogen. In light of

the data presented here, the hypothesis that tomatine

could play a role in resistance by inhibiting spore

production appears unlikely.

Sinha and Wood (1967) working with Verticillium

wilt, and Mace and Veech (1971) working with Fusarium wilt,

postulated that an inhibitory agent present in the upper

stem of resistant tomato plants may be responsible, at

least in part, for resistance. It seems unlikely, however,

that the presence of inhibitory levels of tomatine in the

upper stem would be of primary importance to Fusarium wilt

resistance. Elgersma et al, (1972) found that there was

almost no advance of Fol race 1 beyond the initial uptake

of inoculum in the resistant VF isoline , while extensiv e

secondary distribution beyond t he initial uptake was

observed in the sus ceptible IP isoline. Conway (1976)

concluded from his work that VF is resistant to race 1

because secondary distribution i s limited , but sus ceptible

to race 2 because secondary distribution is exte nsive. It

is clear from thes e t wo studies that re sistance result s

4J

only when Fol is confined to t he lower portion of the plant.

Vascular occlusion was cited as the mechanism r e spons i bl e

for restricting secondary distribu t i on , If tyl os e develop ­

ment is retarded, as in a susceptible hos t - pathogen inter­

action, colonization becomes extensive . Once t he pathogen

begins t o colonize the uppe r stem f oll owing natural

infections, resistance mechanisms are not effective and

susceptibility results. The presence of an inhibitor in

44

the upper plant axis would be insufficient to confirm

resistance once extensive colonization has taken place,

Nevertheless, an antifungal compound located in the upper

portion of a resistant plant could act to restrict the

growth of those few fungal propagules which manage to escape

the localization process,

It may be that tomatine is still contributing to

resistance, but not as the primary mechanism of host

defense. Tomatine may be effective in the lower stem as

part of a sequential resistance process. Vascular occlution

in resistant plants may not only be important in restricting

the distribution of the parasite, but may also impede the

flow of the transpiration stream, resulting in the further

accumulation of tomatine in the infected vessels of the

resistant plant. These localized concentrations of

tomatine may very well exceed l □- 3 M and approach the con­

centration required to completely inhibit mycel.ial growth

or spore germination. First, tylose development would form

a barrier restricting the upward spread of the pathogen,

and then the build-up of tomatine behind this stationary

front would effectively prevent the lateral spread of the

fungus from vessel to vessel. Clearly , occlusion is the

primary mechanism of resistance in such a system.

SECTION II

THE EFFECT OF FUSARIUM OXYSPORUM

F. SP. LYCOPERSICI RACE 1 AND RACE 2 ON THE ANTIFUNGAL

ACTIVITY OF TOMATI NE

Introduction

Fontaine et al. (1947) were the first to suggest

that the host-produced fungitoxicantj tomatine, may be

responsible for the resistance of certain tomato cultivars

to Fusarium ox:vsporum f. sp. lycopersici (Sacc.) Snyd. &

Hans. race 1. Tomatine was found in both resistant and

susceptible tomato plants prior to infection. After

infection, a high level of tomatine was maintained in

resistant plants, while tomatine gradually disappeared from

the susceptible cultivar, Tomatine was absent from wilted

and dying tomato plants (Irving, 1947), Kern (1952) late r

confirmed those results.

Arneson and Durbin (1967) suggested that the

decrease in tomatine content of infected plants indicated

that in susceptible tomato plants Fusarium oxysporum f. sp.

lycopersici (Fol) race 1 was either able to degrade

tomatine, or the fungus i nduced the host to degrade

tomatine. Recently Stromberg and Corden (1974) came to a

similar conclusion. They presented evidence that ?ol

race 1 fungal populations increased within an infected

susceptible cultivar three days after inoculation, while

45

46

the fungitoxicity of acetone xylem extracts decreased.

However, in the resistant cultivar, the fungal population

remained low, and the xylem extracts became highly

fungitoxic, The persistence of a high level of an

inhibitory substance in a resistant plant, in contrast to

a decrease in this substance within a susceptible plant

following infection, led the authors to conclude that

susceptibility was due to detoxification of the inhibitor.

Little research has been done on the microbial

transformation of tomatine. As a result, only a few fungi

have been shown to be capable of detoxifying tomatine.

Arneson and Durbin (1966) demonstrated that extracts from

heal thy tomato leaves iri .. hi bi ted spore germination and

mycelial growth of Septoria linicola and§, lactucae, both

non-pathogenic on tomato plants. However, extracts of

leaves heavily infected with the tomato leaf spot pathog e n ,

§, lycopersici, were not toxic to those fungi. The authors

suggested that§., gcopersici detoxified tomatine following

infection, This was supported in a later study (Arneson

and Durbin, 1967) which showed that§, lycopersici

detoxified alpha-tomatine by enzymatically hydroly zing one

glucose unit from the tomatine molecule, The result i ng

compound was beta2-tomatine (Fig, 1), Althoug h

alpha-tomatine occurred in healthy tomato leaves ,

beta2-tomatine was also present in leav es i n fect e d with

S. lycopersici.

These data, coupled with the f act tha t ot he r fungal

47

parasites of tomato have also been reported to be relatively

insensitive to tomatine, led Arneson and Durbin (1967) to

hypothesize that these parasites also may detoxify tomatine.

It was also suggested that the ability to overcome the

toxicity of alpha-tomatine may be necessary for the success

of tomato pathogens.

Botrytis cinerea, which causes a latent infection

in young, green tomato fruits, is capable of detoxifying

alpha-tornatine (Verhoeff and Liem, 1974). In this case,

tomatine was hydrolyzed to tomatidine, a derivative of

alpha-tomatine (Fig. 1) which is non-toxic to B, cinerea.

One of the two objectives of the present study was

to determine if Fol race 1 or race 2 was capable of

detoxifying tomatine. It is known that tomatine i s

inhibitory to spore germination of both Fol races. It

was hypothesized that if a given population of spores was

able to detoxify tomatine, then the percent germination of

subsequent populations of spores placed in the tomati ne

solution would increase. The percentage of spore germi­

nation would then continue to increase as more t omatine was

detoxified with each successive population of spores.

With the exception of Stromberg and Gorden (1977),

few researchers have considered toxicant concentration-~o­

fungal population ratios in their investigations of the

effect of tomatine on Fol , It was hypo~hesized that spore

concentration may be i mportant in deter~ining the effec­

tiveness of a given concentration of tornatine on spcre

48

germination. If this were the case, then as spore levels

increased and the tomatine concentration remained constant,

less tomatine would be available per spore and the

germination percentage would increase. Conversely, as

spore levels decreased, more toxicant would be available

per spore, and spore germination would decrease. The

second objective of this study was to determine the effect

of spore load on the percent germination of Fol race 1

subjected to a known concentration of tomatine.

Materials and Methods

Isolates of Fusarium oxysporum f, sp, lycopersici

races 1 and 2 were obtained from Dr, G. M, Armstrong

(Georgia Agricultural Experiment Station, Experiment,

Georgia 30212). Stock cultures were maintained on silica

gel (Perkins, 1962). Microconidia for spore suspensions

were harvested from 10-day-old cultures grown on pota to

dextrose agar. Spores were filtered through lens paper and

the concentration adjusted with the aid of a hemacytometer.

Two solutions were tested, one containing 8 X 1 0- 4 M

tomatine and one without tomatine. Pure alpha-tomatine

(ICN Life Sciences Group, Cleveland, Oh i o 44128) was

dissolved in 10 mM HCl and the pH brought up to 4.6 wi t h a

phosphate-citrate buffer . Buffered solution lacking

tomatine served as the control. Tomatine and buffer control

solutions were sterilized by filtration through a Millipor e

fil ter (0.2 um pore size). The pH of the solutions was

tested after spores had been incubated.

Percent germination was determined by count ing t he

number of spores out of 100 which had germinated. Spore s

were considered germinated if the germ tube was a ~ least

the length of the spore. Three random fie l ds pe r r epl i cate

were examined for each treatment. The da ta were ar.al yzed

statistically using Cunca n•s multiple - r ar.ge t es t (P= 0 , 05) ,

Effect of Successive Spore Populations

on Germination

50

Samples (2.5 ml) of tomatine or buffer control

solutions were added to 2.5 ml of a Fol r ace 1 or r a c e 2

spore suspension in sterile 25 ml Erlenmeyer flasks. Spore

suspensions were adjusted to give a concentration of 1 X 105

microconidia/ml when added to the two test solutions. Each

of the four combinations was replicated six times . Flasks

were placed on a shaker table (70 cycles/min) and incubated

at room temperature (25-26 C) for 24 hours. After

incubation, a drop was removed from each flask, and percent

spore germination was determined. Microconidia were r emo·red

from the flasl< contents by filtration through a Millipore

filter (0.2 um pore size). Flasks from each combination

were divided into two groups. Race 1 spores were a dded to

the first group of conditioned solutions; race 2 spores

were added to the second group. Percent germination was

determined after 24 hours on the shaker table. Solutions

were filtered free of spores and the process was repeated

twice. Each time, group one flasks received r ace 1 spore s

and group two fl asks received race 2 spores.

Effect of Spore Concentration

on Germination

Slide bioassay. Fol race 1 spore concentra tions of

1 X 104 , l X 105 , 1 X 1 06 and 3 X 1 06 microconidia/ ml we r e

tested. One drop of t he tomatine soluti on or buffer

solution was added to one drop of s pore suspensi on on a

51

microscope slide coated with 2.5% cellulose nitrate. ~ach

treatment was replicated six times. Slides were placed in

sterile, moist petri dishes and incubated at 26 C for 24

hours. Following incubation, spores were stained and

fixed with 1% cotton blue in lactophenol and the percent

germination determined.

Shaker table bioassay. Fol race 1 spore

concentrations of 1 X 105 and 1 X 106 mic rocon i dia/ml were

tested. Samples (2.5 ml) of tomatine or control solution

were added to 2.5 ml of spore suspension i n sterile 25 ml

Erlenmeyer flasks. Each treatment was replicated three

times. Flasks were placed on a shaker table (70 cycles/min)

and incubated at room temperature (25-26 C). After 24

hours, a drop was removed from each flask and placed on a

microscope slide. Spores were stained and fixed with 1%

cotton blue in lactophenol and the percent germination

determined.

Results

Effect of Successive Spore Populations

on Germination

Tomatine significantly inhibited ge r mination of

52

race 1 and race 2 spores in t he i ni tial suspens ion, but

there was no si gnificant difference in t he r esp ons e of t he

two races (Fi g , 9-A), Percentages of spore ge r mi nat ion of

race 1 in the initial buffer control soluti ons we r e a l s o

comparable to race 2 (Fig. 9- B), Spore germi nati on in

conditioned tomatine or buf f e r c ont rol solutions dec r eased

significantly compa red to ge r mination in the i nitial

suspension. Germination was reduced as muc h a s 59- 78% in

conditioned buffer solutions, while a 41- 65% r eduction in

germination was observed for spores in conditioned tornat i ne

solutions. Spore germination c onti nue d to decr ease with

each successive spore suspension . Genera lly , perc ent

germination of conidia in t he condit i oned t ornat i ne solutions

was not significantly dif f ere nt fr om ge r mi nat ion in the

conditioned buffer c ontrol solutions , when c omparing

similar spore suspe ns ion s equ enc es . The pH of a l l solut~ons

remained at 4. 6 .

Effect of Spore Concent r ation

on Germinat i on

Slide bioas say . The pe r cent gerrr.inatio. o: spores

in the buffer co ntrol or tow.a t ine sol utions decreased

Fig, 9. The effect of various spore suspension

sequences on the germination of microconidi~ of Fusarium

oxysporum f. sp. lycopersi ci race 1 (Rl) or r ac e 2 (R2) in

A) an 8 X l □- 4 M tomatine solution or B) a buffe r control

solution. Means labeled with the same letter are not

significantly different, P=0,05, according to Duncan's

multiple-range test.

z 9 ~

"' z =< "' " w

0 e;

z w V

"' ~

z 0

< z

100

80

60

40

20

0

100

80

~ 60

" 40

0

0

i 'I';'

'.

' I

RI

j

I ,,

~ ' '

(

'

~

,.

R l

elg

I " RI R I

~ ~ ;,;,

..

'

. R' Rl

b,d

RI RI RI

cde

~ ,.

RI RI RI

0

RI RI RI R I

I RI R: RI RI

:s

R I

j

I " ~ ,. w,

' ~1

~,

'

t RI

TOMATI N E (sx1o·'M)

d e f

~ b , d ..

lr ·'1

:~ ·;;~ RI R I RI R2 R2 R2 R2 R2 R2 R2 RI RI RI

R2 R2 Ill R I R2 RI

SPO RE SUS PE N SIO N SEQ UENCE

det

)~s-' 1

RI R2

BUFFER (pH 4 .6)

j

I I ~-

•

~ t ' ~

dol ,, .,

t '. ~ ~ :i

0 l RI <I R2 R2 R1 R2 Q2

R

r.dd ~ . ~

R2 RI

,de ,:i ,, r r;

R1 RI RI

SPORE SUSPENSION SEQUENCE

~

/) ~ ·' ;,;

>; ;'

~

~

111 RI RI RI

R2

j t',

[ J'j.

t;;: ,,, I

r.? ' ~ •,

·-

~-

:'

~

(i " R2

,de

R2 R2

tkf I":

;, ~

R2 R2

R2 R2 R2

h r:

~

[-'. ii

54

A

11 2 R2 R2 11 2

B

d•I .. o'>

I'•

" ji n ., R2 R7

significantly as spore concentra t i on increased (F i g . 1 0) .

Germination of conidia at concentrat ions of 1 X 104 and

55

1 X 105 microconi dia/ml was compara ble in the c ontrol

solutions. As the spore concentra tion was i nc r ea s ed

ten-fold _ from 1 X 105 to 1 X 106 microconidia/ml, ge r mi ­

nation was reduced by 2 9% , A three-fold incre a s e i n spor es

fro m 1 X 106 to J X 1 06 conidia/ml resulted in a 53%

reducti on in ge r mi nation . Germination of conidia in the

tomatine solution was significantly l ess than g erminat ion

in the buffer solution at the same spore concentrati on

(Fig. 1 0). I n creas ing t he spore level in tomatine fr om

1 X 104 to 1 X 1 05 microconidia/ml caused a 2 6% d rop i n

germination. An SL~% decrease in g ermination occu rre d when

the spore concentration increased from 1 X 1 05 to 1 X 1 06

conidia/ml . The l argest reduction (92%) in spore germi­

nation occurr ed when J X 1 06 conidia/ml was tested i n

tomatine. The pH of al l sol uti ons remained at 4 . 6 .

Shaker table bioassa y. Percent ge r mina tion of

spores at a concentration of 1 X 1 05 microconid i a/ml was

similar whether spor es were incubated on the s hake r t abl e

or on microscope sl i des (Fig. 1 0 , 11), Howe v er, the effect

o f spore load on germination was more ~ren ounc ed when the

shaker t a ble was used than when slides we r e used . When the

spore concentration i n the control soluti on was inc r eased

f 1 X 7 OS t 1 X 1 06 . . .. / 1 . ... . rom _ o microcon i a ia m , ge r mina - io~

decreased by 8 7% (Fig. 11 ) , The s a me inc r ease i~ spore

concentrat:on in the tomatine solution r es lted in a

Fig. 10. The effect of selected spore concentrations on the germination of micro­

conidia of Fusarium oxysporum f. sp. lycopersici race 1 incuba t e d for 24 h ours on

microscope slides. Means labeled with the letter~ a r e not s i gnificantly di f f erent,

P~0.05, according to Duncan's multiple-rang e test.

57

'°o >< M -~

-.:- ,0

'o 0 .... X >< --.J

co :E - ........_

w 1.,; < z 0 .-

I- >< 0 -< z :E 0 0 ~ u I- 0 0 .-

>< c, u ~ ..__.

z 0 I-

< 0:: r-

'°o z w

>< u (""') z

0 ~ ,0

u "¢ 0

.-J: >< UJ

C. 0:: - 0 0:: 0.. w lJ") V')

LL 0 LL .-:::::, >< ca

~ 0

C .... ><

0 0 0 0 0 0 0 co -0 ~ N

NOl1\1NIW~38 3~0dS 1N3)~3d

100

Z 80 0 r-<( z :E ~ 60 <.')

w ~

0 e; 40

1-

z w ~, o-: 20 UJ a.

0

5

BUFFER (pH 4 .6 ) TOM ATINE ( SX 104M)

1 x105

SPORE CONC EN TRA TI O N

(M lC RO CO Nl DI A/M L)

Fig . 11 . The ef::ec1; o: selecc:ed spore co:-:ce . . tr2.t.-:.o!":s

on t he ge rmin2.tio. of nic r oco n.-:. dia o:: ?usariu::i o::·.· O::'.''.l:

f . su . l yc ope r s.-:.ci r 2.ce 1 incubated for 2...- nou:-s .:.,. :l.::..~::.:

on a shake r t abl e .

c omp l ete (1 00%) inhibition of germinatio~ . The Hof all

solutions remained at 4 . 6 .

59

Discussion

The foc us of previous studies has been to investi ­

gate the effect of t omatine on spores (Langcake et al.,

1972; Mccance and Drysdale, 1975), It was the intent of

this experiment to determine what effect spores may have

on tomatine. In the first assay, the possibility that

oO

Fol r a ce land/or race 2 c ould detoxify tomatine was

examined . The possibility that increased spore load would

lessen the inhibitory effect of t omatine on spore ge rm i ­

nation was examined in the second set of assays . It was

expected , in both cases, that the effectiveness of tomat ine

would decrease, resulting in an increase in spore germi ­

nation , I nstead, a dramatic decrease in germination was

obs erved, but in the buffer solutions as well as :n the

tomatine solutions . Thus, the or'ginal hypothes es c an

neither be substantiated nor denied. Nevertheless , the

data do provide evidence that Fol race 1 and r a ce 2 spor es

produced a substance which was self - inhibitory.

Spores general ly do not germinate in the cult re:~

which they are produced ( Macko et al ,, 1976). This may be

due to dormancy, but more com..~only it is due ~o the pre e~ce

of inhibitory substances . Spor es of m~ny :ung~ germi~~te

poorly or not at all when they are excessively crowded o~

a surface or in a dense suspension (Coc:~ane, 1958; Yac~o

et al . , 1976). ~he crowding effect aloe s ~o~

sufficient evidence fo~ the pre ence of a se:~- i~hib~~or,

but it does suggest that an inhibitory s u bstance may be

present .

Evidence from the two bioassays performed here

indicated that physical crowding alone did not account for

the inhibition. Considering the spore load assay a lo ne ,

one could argue that decreasing germination percentages

61

with increasing spore conc ent r ation was due to a physical

crowding effect. However, the other bioassay technique

showed that the effect was really due to the p resenc e of an

inhibitory substance. In this assay , spore concentration

remained the same, while one population of spores was

followed in sequence by another population. The further

decrease in spore g ermination with each subsequent spore

suspension indicated that a chemical inhibitor p roduce d by

the spores remained in the so l ution when the population was

removed, As the compound built up in solution with each

successive population, the inhibitory effect a l so increased .

The total effect was similar to tha t of increas ing the

actual spore numbers in the solution,

Reduced spore germination due to the production of

self-inhibitory substances has been demons trated fo r a

number of other fungal species . Conidia of Glomere ll a

cingulata in liqu id shake cultures ge rmi nated at a concer.­

tration of 1 X 105 spores/ml, but l ess than 1% o the sno~es

germinated when the concentration was increased to 1 X 1 09

spores/ml (Lingappa and Lingappa, 1965 , 1966, . An i. hi bi­

tory fraction was extracted from t he conidia . The r s ~s,

particularly Puccinia graminis tritici (Allen, : 955) ,

provide a classic example of the self-inhibitory

phenomenon.

Solutions conditioned by race 1 or race 2 were

inhibitory to both race 1 and race 2, The toxic compound

was apparently produced by both r aces and exerted the same

in~ibitory effect on the two races. Spore ge rmi nation in

conditioned solutions was restric ted regardless of whe~her

spores were incubated in tomatine or buffer. In f act , any

initial differences in the germination of conidia in the

two solutions were later obscured,

The self-inhibiton phenomenon could have some

very interesting implications if it occurs in tomato

62

plants infected with Fol. Spores which build up behind the

physical barriers produced in response to infection may

fail to germinate due to the excretion a~d accumula~ion of

this inhibitory compound. The self-inh~bi tory factor,

along with tomatine accumulation, could contribute to

r esistance by r estricting lateral fungal spread n infected

vessels.

LITERATURE CITED

Allen, P. J . 1 955 . The role of a self- inhibitor in the germination of rust uredospor es . Phytopathology 45 1 259-266 .

Arneson, P .A., and R. D. Durbin . 1966 . De toxification of tomatine by Septor ia lycopers.:.ci. Phy,;opathology 56 : 870. (Abstr.

Arneson, P.A., and R. D. Durbin. 1967 , Hydrolysis of tomatine by Septoria l ycopersici 1 a detoxifica tion mechanism. Phytopathology 57 zlJ58- lJ60 .

Arne s on, p . A., and R, D. Durbin. 1968 . The sensitivity of fungi to alpha- tomatine. Phytopathology 58 z5J6- 5J7 ,

Beckman , C.H. 1964. Host responses to vascular infection . Annu. Rev. Phytopathol, 2 : 231- 252 .

Beckman, C. H, 1 966 , Cell irr itability and localization of vas cula r infect ions in plants . Phytopathology 56 : 821-824 .

Be c kman, c. H. 1 968 . An evaluation of possible resi stance mechanisms i n broccoli, cotton and tomato to vascular infection by Fusarium oxysporum . Phytopathology 58 : 429-4JJ .

Be ckman, C. H,, D, M. Elgersma , and W. E. MacHardy . 1972. The localizat i on of fusarial infections in the vascula~ tissue of single- dominant-gene re sistant tomatoes . Phytopathology 62 :1256- 1260.

Beckman, C, H,, and S. Halmos . 1962 , Relation of vascular occluding r eactions in banana roots to pathogenicity of r oot invading fungi. Phytopathology 52 :89J- 897 ,

Beckman, C, H. , M. E . Mace , s . Halmos, and~ - w. McGraha 1961. Physical barriers associated wi~h res is- ance in Fusarium wilt of bananas . Phytopathology 51 :507- 5-5 ,

Bell, A. A , 1 969 . Phytoalexin prod ction a:1d Veri;icill.:. ... wilt r esistance i n cotton . Phytopatholo y 59 :1119-11L7 ,

Cochrane, v. W. 1 958 , Physio~ogy of ?ungi , Wiley . 524 p.

6J

·ew :ork 1

Conway, w. s . 1976 . Colonization, distribution and phenolic content in susceptible and r esistant ~o~ato isolines infected by Fusarium oxysporum f . sp . lycopersi c i races 1 and 2 . Ph . D. Thesis . Unive r sity of New Hampshire . 64 p.

Cromwell, B. T . 1955, The alkaloids . Pages 367- 51 6 i n K. Paech and M. v. Tracey, eds. Modern Me thod s o f Plant Analysis, Vol. IV , Springer-Verlag , Berlin, 766p.

Dimond, A. E, 1955, Pathogenesis in the wilt diseases . Annu. Rev . Plant Physiol, 61329- 350,

Elgersma, D. M., w. E , MacHardy, and C , H. Beckman . 1 972 . Growth and distribution of Fusarium oxysporum f . lycopersici in near-i sog enic lines of tomat o resi s t ant or susceptible to wilt . Phytopathology 62 : 1232- 1 237 ,

Fisher, P, 1, 1 935, Physiological studies on the pathogenicity of Fusarium lycopersici Sacc. for the tomato plant. Md . Agric. Exp . Stn. Bull . ff3 74, 21 p .

Fontaine, T, D., J . s . Ard, and R, M, Ma , 1 951 , Tomat i dine , a steroidal secondary amine. J. Amer . Chem . Soc. 73 1 878 - 879.

Fontaine, T, D,, G, W. Irving , ands . P, Doolittle . 1947 . Partial purification and properties of tomatin, an antibiotic agent from the tomato plant. Arch . Biochem. Biophys, 1 2 1395- 404 ,

Fontaine, T, D., R, Ma , J, B. Poo l e , and S . P, Doolittl e , 1 948. Isolation and partial characte rizat ion of cryst alline tomatin , an antibiotic a~e nt from the t omato plant . Arch. Biochem . Biophys . 18 1407- 475 .

Ge r demann , J, W,, and A. M, Finley . 1 951, The pathogen­icity of Fusarium ox~sp orum f , lycopersici . Phytopathology 41:23 - 244 .

Gottlieb , D. 1943. Expressed sap of tomato plants ir. relation to wilt resista nce . Phytopathology 33 :1111, (Abstr . )

Hammerschlag , F ,, and M, E , 1:ace, 1 975 , Antifun6al activity of extracts from Fusar ium wilt - uscep~ible ar.a - resistant toma to plants . Phytopa~hology 05 193- 9 .

Heinze , P, H,, and c . F , Andrus, 1 9 5 , Apparen localization of Fu s 2.riu wilt r esista?1ce i. the = Ame rican toma o . Amer . J . aot . 32 162 - 66 .

Irving , G. VI ., Jr . 1947 . The significance of to.na tin _ plant and animal disease . J , Wash . Acad . Sci , J7 : 293- 296 ,

Irving , G , w., Jr ., T , D, Fontaine, and S , P , Doolittl e , 1945, Lycopersicin, a fungistatic a g ent from the tomato plant . Science 102 : 9- 11 ,

Irving , G, w., J r., T , D. Fontaine , and S , P , Doolittl e , 1946 . Partial antibiotic spectrum of tomatin, an antibiotic agent from the tomato plant, J , oa cteriol , 52:601-607.

Kern , H. 1952 , Uber die Beziehungen zwischen dem Alkaloidgehalt verschiedener Tomaten sorten und ihrer Resistenz gegen Fusarium l ycopersici . Phytopath ol. z. 19:351- 382.

Langc ake, P., R, B. Drysdale , and H, Smith. 1972 . Post ­infection production of an inhibitor of Fusarium oxysporum f . l ~cope rsici by tomato plants . Physi ol , Plant Pathol, :17-25.

Lingappa, B, T,, and Y. Lingappa , 1965 . Effects of nutrients on self-inhibition of germination of c on i d ia of Glomerella c ingulata. J, Gen. Microbiol , 41 : 67 - 75,

• c::

Lingappa , B, T., and Y, Lingappa . 1966 . The nature of self-inhibition of ge r mire.tion of conidia of Glome r el l a cingulata . J. Gen . Microbiol, 43:91- 100 .

Little, J , E., and K. K, Grubaug h , 1946. activity of some crude p l ant juices . 587- 591.

Antibiotic J . Bacte riol . 52 :

Mace, M. E ., and J . A . Veech , 1971. Fusarium wilt of suscepti ble and r esistant tomato isolin es 1 host colonization . Phytopathology 6l 18J4- 840 .

Macko, v., R, c . Staples , z. Yaniv , and R. R, Gra nado s . 1976 , Self- inhibitors o f fung al spore g ermination . Pages 73-1 00 in D, J. Weber and w. M, Eess, eds , The Fungal Spore : Form and Function . New York : Wiley . 895 P•

Mat ta, A,, r. Genti l e, and r. Giai . 1969 . Ac c u u l a-,;io. of phenols in tomato plants infected by dif f e e -,; :or ~s o: Fusarium oxysporum . Phytopa t ology 59 : 512- 513 ,

Mccance , D, J ,, and R, o, Drysdal e . 1975 , Fred c~io o: tomatine and r ishi tin in toma to plants i noc ·la1:e · w.:. -: .. Fusar i um oxysporum f , sp . l y cop ersic i . hysio _ , ? _a!1-c Pathol , 7 :221- 2J •

60

Perkins, D. D, 1962 . Preservation of :·e rospora stoc·· culture s with anhydrous silica gel . Can . J . ;,:icrobio:. . 8 1591-594 ,

Schnathorst , W, C. 1970 , Obtaining xylem :luid from Gossypium hirsutum and its uses in stua·es on vascular pathogens . Phytopathology 60 11 75-176 ,

Sinha , A, K,, and R, K, s. \'iood . 1967 . An analys·s of responses of r esistant and o: suscepti ble tomato ~lant to Verticill ium i nfection, Ann . Appl , Biol , 59:143-154 ,

Snyder , w. C., K, F , Baker, and H, N, Hansen . 1946 . Interpretation of r esistance to Fusarium wilt in tomat o . Science 103 17 07- 708 .

Stromberg , E, L,, and M. E . Gorden. 1974 , Fungitoxicity of xylem extracts f rom Fusarium wilt resis tant ad susceptible tomato plants. Proc . Amer . Phytopathol , Soc. 1:79, (Abstr .)

Stromberg, E, L,, and M, E, Gorden . 1976 . Fungitoxicity of xylem extracts from Jefferson tomato pl ants i noculated with Fusar ium oxysuoruM f , sp . lycopersici race 2 . Proc. Amer, Phytopathol . Soc , 3120-21, (Abstr.)

Stromberg , E , L,, and M, E , Gorde n . 1977 . Fungitoxicity of xylem extrac ts from tomato plants resistant or susceptible to Fusarium wilt , Phytopathology 67 1 693-697.

Talboys, P , W. 1958 , Some mec hanisms contribut ir.g to Verticillium-resistance in hop root. Trans . 3 . mycol, Soc , 411 227- 241.

Talboys, P , w. ·1964 . A concep~ of the host - parasite relationship in Verticillium wilt disease . Nature 02 1 J61-J64 .

Talboys, P, W, 1972 , Resis t ance to vascular Nilt fu.~i . Proc. R, Soc. Lond . B, Biol . Sci . 181 131 -JJ2 .

Uhle, F , C. , and J , A, Moore. 195~ . The a rt ial s r.the~:s of tomatidine. J , Ame r. Chem . Soc . 7 ~6~- - 6~1~ .

Verhoeff , K,, and J , I , em . 1975 , Toxici~y of tomatire to Botry~is c inerea , in r elation .. o 12. .. e .. c .,· . Phytopathol , z. 82 i JJ3 - JJ8 .