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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 [email protected].
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
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, I
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:c u
I.LI
z .... < ~ 0 I
I C
..c: _g. 0
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I
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~06 uy J: 0 £ u
0
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w z C
····-·•-•: -I
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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
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z w V
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z 0
< z
100
80
60
40
20
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80
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54
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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
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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.
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