Download - Variation in sensitivity to tomatine and rishitin among isolates of Fusarium oxysporumf.sp. lycopersici, and strains not pathogenic on tomato

Physiological and Molecular Plant Pathology (1996) 48, 131–144

Variation in sensitivity to tomatine and rishitin among

isolates of Fusarium oxysporum f.sp. lycopersici, and

strains not pathogenic on tomato

P. S", A. M. T#, A. A. S#, M. A. M#

and D C. S"*

"Department of Plant Biology, and the Maryland Agriculture Experiment Station, Uni�ersity of Maryland, CollegePark, MD 20742, U.S.A. and #Agricultural Genetic Engineering Research Institute, Agricultural Research Center,9 Gamaa st, Giza, Egypt

(Accepted for publication September 1995)

Studies of several fungal plant pathogens have found an association between greater virulence andincreased tolerance of the host’s defense compounds among different isolates of that pathogenspecies. This study examined 17 Fusarium oxysporum isolates to determine if tolerance of either oftwo fungitoxic compounds produced by tomato, rishitin and tomatine, would correlate withvirulence or pathogenicity on tomato. Among the 12 pathogenic isolates (forma specialislycopersici), quantitative levels of virulence were significantly correlated with rishitin tolerance and,in more limited circumstances, with tomatine tolerance. A group of four highly virulent isolatesdisplayed a relatively high tolerance of both tomatine and rishitin compared to the other isolates.When these pathogenic isolates were compared to five F. oxysporum isolates nonpathogenic ontomato, the nonpathogens generally displayed the highest sensitivity to tomatine, but not torishitin. Although these results do not prove a role for rishitin or tomatine tolerance in virulenceor pathogenicity, they do indicate that sufficient natural variation in these traits exists for themto contribute to an isolate’s disease potential on tomato. # 1996 Academic Press Limited

INTRODUCTION

Fusarium oxysporum (Sacc.) Snyder & Hans. causes wilt disease in a number of crops.

Individual strains normally show a high degree of host specificity and are grouped into

formae speciales according to the ability to cause disease on certain hosts.

Tomato (Lycopersicon esculentum Mill.) is a host to two formae speciales, F. oxysporum f.sp.

lycopersici, which causes a vascular wilt, and F. oxysporum f.sp. radicis-lycopersici, which

causes a crown rot [11]. F. oxysporum isolates which are not pathogenic to tomato have

also been isolated from tomato roots [8]. It is not known what traits are unique to the

pathogenic F. oxysporum which allow infection of tomato, or which traits produce more

virulent pathogens within a forma specialis.

One trait that may potentially determine either pathogenicity or virulence on a

specific host is the ability of the fungus to tolerate fungistatic or toxic compounds

produced by the host, such as the constitutively produced phytoanticipins [21] or the

*To whom correspondence should be addressed.Abbreviations used in text : CR, Castle Rock II-PVP; IPa-VF, Improved Pearson-VF; PDA, potato

dextrose agar ; PDB, potato dextose broth; PI, Pearson A-1 Improved; SM, Super Marmand.

0885–5765}96}02013114 $12.00}0 # 1996 Academic Press Limited

132 P. Suleman et al.

induced phytoalexins [22]. Such tolerance may result from either degradation of the

compound or nondegradative mechanisms [22]. Studies of other pathogen-plant

systems have identified variation in host phytoalexin tolerance among different isolates

of a pathogen. In these studies, higher tolerance to the host phytoalexin or

phytoanticipin was correlated with higher virulence on that host [4, 6, 14, 16, 18, 20].

Two fungitoxic compounds produced in tomato are tomatine, a saponin, and rishitin,

a terpenoid. Previous studies have shown that levels of tomatine and rishitin produced

in tomato stems and roots approach or exceed inhibitory levels (ED&!

) for F. oxysporum

f.sp. lycopersici [15]. This suggests that these compounds have the potential to limit

growth of this fungus within the plant and so allow faster growth of isolates possessing

greater tolerance of these compounds.

Tolerance of rishitin or tomatine has been found to correlate with increased

virulence in two other plant-pathogen systems. Rishitin tolerance has been associated

with high virulence of Gibberella pulicaris (Fries) Sacc. (anamorph: Fusarium sambucinum

Fuckel) on potato among both field isolates and progeny of a sexual cross [6].

Tomatine tolerance has been associated with the ability of Fusarium solani mutants to

cause rot on green tomato fruits [5]. F. oxysporum f.sp. lycopersici has been shown to

enzymatically detoxify tomatine [9], and, like a number of other tomato pathogens, is

more tolerant of tomatine than other fungi which are not pathogenic on tomato [1].

Thus, by analogy to the other pathogen systems, a variation in virulence among

different isolates of F. oxysporum f.sp. lycopersici could possibly result from differences

among the isolates in their tolerance of either rishitin or tomatine. Differences in host

specificity among F. oxysporum, in particular the ability to be pathogenic on tomato,

could also arise in part from an isolate’s tolerance of these tomato compounds. Since

the relative tolerance of different F. oxysporum isolates, pathogenic or nonpathogenic,

have not been directly compared, evidence for or against such relationships is lacking.

This study evaluates the variation of rishitin and tomatine tolerance in 17 isolates of

F. oxysporum in order to determine if differences correlate with pathogenicity or virulence

on race 1-susceptible tomato. Recently collected isolates were the focus of the study in

order to be certain that the virulence of these strains was not altered by long-term

storage and subculture. Additional F. oxysporum isolates which were collected from

tomato or associated soils and found not to be pathogenic on tomato were also

evaluated. Tolerance of rishitin and tomatine was tested for three states of growth

(spore germination, germ tube elongation, and growth of mature mycelia) so as to

detect differential expression of tolerance if it were developmentally regulated. Wilt

pathogens would be expected to pass through all three stages during spread in the

xylem. Understanding the variation intolerance of host chemicals among different

isolates of this asexually-reproducing pathogen could explain the underlying

determinants of virulence.

MATERIALS AND METHODS

Plant material

The tomato cultivars used in the studies included Pearson A-1 Improved (PI),

Improved Pearson VF (IPa-VF), (Peto Seed Co., Woodland, CA, U.S.A.) ; Super

Marmand (SM) and Castle Rock II-PVP (CR), (Sun Seed Co., Hollster, CA, U.S.A.).

The cultivars SM, and PI are susceptible to races 1, 2 and 3. The cultivar IPa-vf is

Virulence and chemical tolerance of Fusarium oxysporum 133

resistant to race 1 and susceptible to races 2 and 3. The cultivar CR is resistant to races

1 and 2 but susceptible to race 3. All plants were grown in sterilized sand}top soil mix

(1:1) in the greenhouse. Soil temperature was maintained with bottom heat set to

27³3 °C.

Isolates of Fusarium oxysporum

Isolates from Egypt and Maryland were collected from either suspected infected plants

or from soil in fields with a history of continuous tomato cultivation. Isolates were

obtained from surface-disinfested stem sections and plated on Komada’s Fusarium-

selective medium [12] or from soil by dilution-plating soil suspensions on Komada’s

medium and selected from the plates by characteristic growth and morphology after

7–10 days. Single-spore cultures of each isolate were sub-cultured on potato dextrose

Agar (PDA) slants for further use. All isolates described here were identified as F.

oxysporum from morphological features [3]. Reference strains of F. oxysporum f.sp.

lycopersici were obtained from the American Type Culture Collection: race 1 g16417

(abbreviated to ATCC 417) and race 2 g16605 (ATCC 605). Fusarium oxysporum f.sp.

pisi, strain 247, was kindly provided by Dr Hans D. VanEtten of the University of

Arizona.

Plant inoculation

Microconidia of all isolates were harvested from seven-day old PDA cultures grown at

26 °C. Aqueous suspensions of microconidia were filtered through two layers of

miracloth to remove mycelium before dilution to a final concentration of

1¬10' microconidia ml−" with additional sterile water. Five-leaf stage seedlings were

used. Plants were uprooted from flats and the root system gently washed to remove soil

particles. Three millimetres of tissue was excised from the tip of the tap root and the

root system was dipped into the spore suspension for 30 s and immediately transplanted.

In order to determine pathogenicity, four plants of cultivar SM were tested for each

isolate with two plants per 4 inch pot. Isolates were considered nonpathogenic if no wilt

symptoms occurred after 45 days in all four plants inoculated with that isolate.

Characterization by race and �irulence

Tomato cultivars susceptible and resistant to race 1 or to race 2 of F. oxysporum f.sp.

lycopersici (SM, PI, IPa-VF, CR) were used to characterize isolates by race and

virulence. Plants at the five-leaf stage were inoculated as described above. Plants were

evaluated at 45 days after inoculation by a disease index rating of 0–10, a modification

of Wellman’s rating [23] of 1–15. Evaluations were based on number of plants infected,

number of internodes from the bottom of the plant at which leaves display symptoms

and the extent of vascular discoloration (to the nearest internode from the bottom of

the plant). Inoculated plants which displayed neither wilt symptoms nor vascular

discoloration were considered to be uninfected and excluded from the disease rating so

as to evaluate virulence independent from the efficiency of establishing infection.

Generally, a plant receiving a score of 7–8 was defoliated in all but the topmost 3–4

leaves, which showed some chlorosis. Plants with scores of 4–5 generally displayed little

defoliation but half the leaves were chlorotic. Plants with scores of 2–3 displayed

chlorosis on only the lowest two to three leaves and displayed vascular discoloration

between the roots and the soil line.

Plants were also evaluated by culturing 5 mm pieces of surface disinfested stem tissue


at each internode to determine the extent of vertical spread of the pathogen. Plants at

45 days post-inoculation generally had 10–11 internodes. One experiment tested 36

plants of each cultivar for each isolate while a replicate experiment tested 10 plants of

each cultivar for each isolate. Results of both experiments were combined. Data on

disease index and stem colonization were analyzed separately by analysis of variance

(ANOVA) and mean separation by Duncan’s multiple range test (DMRT) at the level

of P¯ 0±05.

Preparation of rishitin and tomatine

Rishitin was elicited in slices of potato with arachidonic acid and prepared by a

modified protocol described by Desjardins and Gardner [6]. Partial purification of

rishitin was achieved by column chromatography of the residue obtained from ethyl

ether extract of the treated potato slices. Rishitin was eluted between 600–900 ml and

the fractions were reduced by vacuum evaporation to about 1±0 ml. The residue was

resuspended in hexane:ethyl acetate (7:3) and the rishitin was detected by TLC (silica

gel 60A, 20¬20 cm) using 20 µl aliquots of fractions and plates developed in

hexane:acetone (4:1). Rishitin was visualized, after spraying the plates with 0±5%

(w}v) vanillin in sulphuric acid, as red spots which turn blue within minutes.

Preparative plates were sprayed only on the edges and the adjacent unsprayed material

collected by scraping the silica from the plate. Rishitin was eluted from the silica with

hexane:ethyl acetate (7:3) and the TLC was repeated. Chemical purity, as determined

by gas chromatography and mass spectrum analysis indicated a minimum of 95±6%

rishitin. Mass spectroscopy was provided by the University of Maryland spectroscopy

facility. Tomatine was obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Phytoalexin tolerance

Isolates were tested in potato dextrose broth (PDB) media amended with rishitin or

tomatine to determine the sensitivity of spore germination and germ tube elongation.

Both compounds were used at concentrations of 25, 50, and 100 µg ml−" in media.

Tomatine was dissolved in 0±5% acetic acid and then citrate-phosphate buffer pH 6

was added. Rishitin was dissolved in ethanol. Controls were amended with the volume

of solvent alone equivalent to a 50 µg ml−" treatment. The PDB medium had a pH of

5±0³0±1 with or without amendments and is close to that of tomato xylem fluid (5±3to 6±4, [15]). Approximately 400 microconidia ml−" were pipetted into each of three

replicate tubes of 2±0 ml of amended PDB. Spore germination counts were taken every

3 h for 18 h, while germ tube growth measurements were taken 12 and 24 h after

addition of spores.

Hyphal growth was evaluated from radial growth rates on PDA amended with

tomatine or rishitin, as described above. For each isolate, a 2 mm plug taken from

5-day-old cultures was placed upside down on PDA medium. All treatments were

maintained at 26}21 °C³1 (light and dark temp. in a growth chamber). Hyphal

growth measurements were taken daily for 6 days and the growth rate of each isolate

determined by regression of three replicates together. This rate was compared to the

radial growth rate on unamended PDA (three replicates) to express the percentage

reduction in growth rate. Data for spore germination, germ tube length and radial

growth were analyzed individually by ANOVA and by a means separation by


Duncan’s multiple range test (DMRT), at P¯ 0±05, using MSTATC (Michigan State

University). Correlative analyses were performed with MSTATC between data and

using replicate virulence measurements.

RESULTS

Characterization of pathogenicity and race

A total of 25 isolates of F. oxysporum were collected in Egypt and 41 isolates were

collected from Maryland. Ten of these isolates were able to cause wilt symptoms on

tomato plants susceptible to all three races of F. oxysporum f.sp. lycopersici and so were

identified as pathogens of this forma specialis. The race of each isolate, as determined

by pathogenicity displayed on differential cultivars, is shown in Table 1.

T 1Race and relati�e �irulence of Fusarium oxysporum f.sp. lycopersici isolates

Measures of virulence on tomato cultivar*

Pearson Improved Super Marmand

Isolate Origin RaceDiseaseindex

Diseaseincidence

Diseaseindex

Diseaseincidence Virulence†

ATCC 605 US 2 8±0a 87 8±5a 100 HATCC 417 US 1 8±0a 92 8±0a 100 HEY-101 Egypt 1 7±0ab 92 7±5ab 100 HEY-102 Egypt 2 7±0ab 90 7±0abc 94 HEY-103 Egypt 1 5±5bc 62 4±8d 53 LMD-L3 US 1 5±0c 54 6±5bcd 69 LMD-C7 US 1 4±5c 72 6±8bcd 100 LEY-104 Egypt 1 4±5c 49 5±0cd 44 LMD-S2 US 1 4±0c 45 5±5bcd 58 LMD-B2 US 1 4±0c 53 5±2cd 41 LEY-105 Egypt 1 4±0c 43 4±4d 50 LEY-20 Egypt 1 3±8c 54 5±8bcd 49 LControl 0d 0 0e 0

*Disease index based upon the extent of foliar symptoms (leaf chlorosis and wilting) and theextent of vascular browning along the stem as delineated by internodes. Scale is from 0 to 10,with 0 representing the lack of symptoms. Disease incidence is the percentage of plantsdisplaying a score of 1 or above. Plants which were inoculated with a fungal strain butproduced a score of 0 were not included in the average disease index score. Letters representgroupings by DMRT at the level of P¯ 0±05. Control was uninoculated plants.†Virulence designation of high virulence (H) and lower virulence (L) based upon highranking with both tomato cultivars.

E�aluation of relati�e �irulence on race 1 susceptible tomato

Virulence of the isolates was determined by measuring both disease severity and stem

colonization. Highly significant differences in disease severity (P! 0±01), as well as

differences in disease incidence, were observed among the various pathogenic isolates

(Table 1). Although the relative ranking of disease index was not the same on both race

1 susceptible cultivars tested, four isolates consistently caused the most severe symptoms,

and the highest incidence of infection on both cultivars. The same four isolates also


displayed the highest levels of colonization of tomato stem on SM (not shown). Thus,

for comparative purposes, we designated a group of four highly virulent F. oxysporum

f.sp. lycopersici isolates, and a group of eight lower virulence isolates (Table 1), although

there was continuous variation among isolates in the scores.

Spore germination

Microconidia germinated in unamended PDB within 12 to 24 h. The maximum

percentage germination after 24 h varied from 78% to 92% among the different

isolates. Addition of either tomatine or rishitin did not significantly alter the kinetics

of germination, and so the germination efficiency with treatments of each isolate were

compared at 24 h. These compounds only slightly reduced (! 8%) the germination of

all isolates, even at the highest concentration of rishitin or tomatine (100 µg ml−").

There was significant variation among isolates in their tolerance of either compound

(P! 0±05) with respect to germination; however the highly virulent isolates were not

significantly more tolerant than the isolates displaying lower virulence or the

nonpathogenic isolates (Table 2).

T 2Percentage reduction in germinaton efficiency of F. oxysporum isolates in the presence of rishitin or

tomatine*

Rishitin TomatineIsolate Virulence† 100 µg ml−" 100 µg ml−"

ATCC-605 H 2±0fg 0±0i

ATCC-417 H 0±6hi 3±4def

EY-101 H 4±1c 2±5efg

EY-102 H 2±6ef 0±0i

EY-103 L 1±0hi 2±0fgh

MD-L3 L 3±4cde 5±5bc

MD-C7 L 5±3b 3±8de

EY-104 L 3±6cd 5±7bc

MD-S2 L 3±8cd 7±3a

MD-B2 L 0±0i 0±0i

EY-105 L 0±0i 2±5efg

EY-20 L 6±8a 4±5cd

EY-3 N 3±2cde 4±3cd

EY-10 N 0±0i 0±6hi

EY-9 N 2±9def 6±9ab

EY-19 N 0±1i 1±3ghi

f.sp. pisi N 1±2gh 2±9defg

*Percentage reduction in germination efficiency of microconidia are shown for each isolateand each treatment, compared to unamended medium. Similar letters in each column indicatevalues which are not significantly different in the same column using DMRT (P¯ 0±05).†Designation of isolate as high virulence (H) or low to moderate virulence (L), or non-pathogenic on tomato (N). The f.sp. pisi isolate is F. oxysporum f.sp. pisi, which causes wilt onpea but not on tomato.

Germ tube elongation

Since spore germination was not greatly reduced by rishitin or tomatine treatment, it

was possible to measure germ tube length from the elongating germ tubes of germinating


microconidia. The mean lengths in unamended PDB ranged from 189 to 262 µm

among different isolates after 24 h. The presence of tomatine or rishitin significantly

reduced germ tube length (P% 0±01) after 24 h and was concentration dependent

(Table 3). The maximum reduction in length was 84±0% and 87±4% at 100 µg ml−"

T 3Percentage reduction in germ tube length of F. oxysporum isolates after growth for 24 h in medium with

rishitin or tomatine*

Rishitin Tomatine

Isolate Virulence† 25 50 100 µg ml−" 25 50 100 µg ml−"

ATCC-605 H 58±0bcd 65±5cd 84±0a 58±5abc 70±6cde 87±4a

ATCC-417 H 53±6bcdefg 62±7de 76±8bcd 42±3g 68±2efg 81±8abc

EY-101 H 48±1g 64±4d 74±7d 51±5de 64±4fgh 79±8bc

EY-102 H 52±2defg 62±1de 81±9abc 51±9cde 60±1hi 72±4de

EY-103 L 65±6a 76±4a 74±0d 60±0ab 74±8bcd 82±4abc

MD-L3 L 51±0efg 62±0de 74±0d 58±0abcd 69±5def 82±5abc

MD-C7 L 58±7bc 71±8ab 81±8abc 52±1cde 68±5efg 77±9cd

EY-104 L 50±5fg 61±4de 77±1bcd 53±8bcde 62±9gh 85±7a

MD-S2 L 50±3fg 57±1ef 64±0e 40±7g 56±1i 71±4e

MD-B2 L 53±1cdefg 58±2ef 74±7d 49±8ef 67±1efg 77±8bc

EY-105 L 54±9bcdef 67±1bcd 76±5cd 43±7fg 59±6hi 83±1abc

EY-20 L 53±4bcdefg 52±9f 64±6e 51±3de 65±1efgh 77±8cd

EY-3 N 50±9efg 62±1de 83±2d 49±1ef 78±0ab 86±2a

EY-10 N 51±2efg 65±0d 82±1abc 54±5bcde 82±5a 85±4ab

EY-9 N 59±6ab 71±5abc 82±3ab 62±3a 82±3a 84±6ab

EY-19 N 54±0bcdefg 64±3d 82±1abc 51±1de 76±6abc 87±2a

f.sp. pisi N 57±1bcde 63±0de 79±5abcd 59±8ab 75±6bcd 82±3abc

*Percentage reduction in germ tube length from germinated microconidia after 24 h ofincubation are shown for each isolate and each treatment, compared to unamended medium.Similar letters in each column indicate values which are not significantly different usingDMRT (P¯ 0±05) in that column.†Designation of isolate as high virulence (H) or low to moderate virulence (L), or non-pathogenic on tomato (N). The f.sp. pisi isolate is F. oxysporum f.sp. pisi, which causes wilt onpea but not on tomato.

for tomatine and rishitin, respectively. Although the reduction in germ tube length

varied significantly among isolates (a difference of 25–47%, depending on treatment),

the highly virulent isolates generally did not display less reduction (greater tolerance)

than the low virulence isolates. Virulence was not correlated with either rishitin or

tomatine tolerance (Fig. 1B). Nonpathogenic isolates were not more sensitive to rishitin

than the pathogenic isolates. In contrast, the nonpathogenic isolates were among the

most sensitive to tomatine at 25 and 50 µg ml−" tomatine (Table 3, Fig. 1).

The reduction in length of the germ tubes in either the tomatine or rishitin amended

medium was accompanied by a significant change in growth pattern. The germ tubes

were much reduced in length compared to unamended medium controls, and

produced short branched hyphae. Microconidia were produced from the branched

hyphae.


F. 1. Virulence of pathogenic and nonpathogenic isolates compared to their sensitivity totomatine and rishitin. The rishitin or tomatine sensitivity of different isolates are plotted as eitherreduction in the rate of mature mycelial growth (A), or germ tube length after 24 h growth (B).Growth medium was amended with the amount of tomatine or rishitin shown in each graph.Virulence of each F. oxysporum f.sp. lycopersici isolate on the race 1-susceptible cultivar Pearson A-1 Improved (_) is plotted on the y-axis. Virulence on the race 1-susceptible cultivar SuperMarmand (D) is shown for mycelial growth only. Nonpathogenic isolates (*) were assigned avirulence of 0 to reflect the lack of symptoms caused in similar assays. Reduction in fungal growthare plotted as a mean of values from three replicates. Correlation coefficients for the graphs areshown in Table 5.


Hyphal growth

The growth of mature mycelium was measured on PDA amended with either rishitin

or tomatine, and was compared to the growth on unamended PDA. Mean hyphal

growth rates on unamended medium ranged from 4±8 to 5±5 mm day−". The radial

growth rates of all isolates on PDA with rishitin and tomatine were significantly

reduced (P% 0±01), and were concentration dependent (Table 4). Radial growth was

T 4Percentage reduction in radial growth rate of F. oxysporum isolates in the presence of rishitin or tomatine*

Rishitin Tomatine

Isolate Virulence† 25 50 100 µg ml−" 25 50 100 µg ml−"

ATCC-605 H 8±6e 31±5efgh 67±5abc 22±1fg 37±5g 62±8bc

ATCC-417 H 9±4de 26±4h 73±7a 16±3ghij 35±4g 57±5c

EY-101 H 9±2de 28±5gh 71±3ab 14±7hij 37±8g 65±3bc

EY-102 H 11±5cde 35±6defg 67±0abcd 12±1ij 40±6fg 58±2c

EY-103 L 8±5e 41±7bcd 65±0bcd 19±3fgh 45±7defg 62±4bc

MD-L3 L 11±8bcde 46±1ab 66±2abcd 12±5ij 42±5efg 64±6bc

MD-C7 L 15±1ab 31±8efgh 70±0abc 10±4j 43±6defg 64±8bc

EY-104 L 12±4bcd 52±1a 49±4f 19±8fgh 50±6cdef 61±1bc

MD-S2 L 18±6a 46±2ab 62±5cd 24±4ef 37±1g 57±0c

MD-B2 L 14±4bc 42±1bc 72±0ab 18±1fghi 36±9g 62±3bc

EY-105 L 14±6bc 38±4cde 62±4cd 15±8ghij 53±9bcd 76±0a

EY-20 L 10±2de 40±5bcd 53±6ef 12±5ij 36±4g 56±9c

EY-3 N nt 36±4cdef nt 29±8a 59±4abc 66±6abc

EF-10 N nt 40±0bcd nt 32±0a 68±7a 76±3ab

EY-9 N nt 30±4fgh nt 21±4fg 63±0ab 66±7abc

EY-19 N nt 28±1gh nt 31±8d 52±8bcde 66±5abc

MD-C4 N nt nt nt 46±0a 62±0ab ntMD-S4 N nt nt nt 41±6ab 54±3bcd ntMD-B1 N nt nt nt 38±6bc 50±3cdef ntMD-L1 N nt nt nt 33±4cd 54±2bcd ntMD-C10 N nt nt nt 32±0d 58±5abc ntf.sp. pisi N 10±5de 29±6fgh 60±0de 30±6de 52±1cde 67±1abc

*Percentage reduction in radial growth rate of myceliumon PDA are shown for each isolate andeach treatment, compared to unamended medium. Similar letters in each column indicatevalues which are not significantly different in the same column using DMRT (P¯ 0±05).Conditions not tested are indicated by nt.†Designation of isolate as high virulence (H) or low to moderate virulence (L), or non-pathogenic on tomato (N). The f.sp. pisi isolate is F. oxysporum f.sp. pisi, which causes wilt onpea but not on tomato.

linear with time on both treatments and controls. This indicates that induction of

tolerance, if present, would be on a time scale either shorter than 1 day or longer than

6 days. Again, there was significant variation among isolates in the amount of

reduction with treatment (a difference of 34–207%, depending upon treatment). This

variation among isolates generally decreased with higher concentrations of either

compound. At 50 µg ml−" of tomatine or rishitin, the highly virulent isolates scored

among the most tolerant (least reduction in growth) of the pathogenic isolates (Table

4). Lower virulence isolates displayed a moderate or low tolerance to at least one of


these compounds. For example, at 50 µg ml−" of either compound, the lower virulence

isolate EY-104 was very sensitive to both tomatine and rishitin. Three other lower

virulence isolates, MD-S2, MD-B2 and EY-20, were as tolerant to tomatine as the high

virulence isolates, but displayed only low to moderate tolerance for rishitin. Conversely,

the lower virulence isolate EY-105 was relatively tolerant of rishitin but sensitive to

tomatine. Thus, at this concentration, there appeared to be an association of relatively

high tolerance to both compounds with only the group of highly virulent pathogens.

However, such a relationship was not strictly observed at lower (25 µg ml−") or higher

(100 µg ml−") concentrations of either compound.

In order to extend the above analysis, the correlation of tolerance for each

compound with quantitative virulence measurements (disease index) was tested.

Virulence of the different isolates was significantly (P% 0±05) correlated with their

tolerance of rishitin, at 25 or 50 µg ml−" rishitin for cultivar PI and at 50 µg ml−"

rishitin for cultivar SM (Table 5). Rishitin tolerance was also significantly correlated

T 5Correlation analyses between �irulence of Fusarium oxysporum f.sp. lycopersici isolates on two tomato

culti�ars and their tolerance of rishitin and tomatine

Conc.Rishitin Tomatine

Cultivar µg ml−" r* P† r P

Pearson Improved 25 ®0±666 ** ®0±032 NS50 ®0±701 ** ®0±378 NS

100 ®0±534 NS ®0±193 NSSuper Marmand 25 ®0±464 NS ®0±164 NS

50 ®0±701 ** ®0±610 *100 ®0±515 NS ®0±287 NS

*Coefficient of correlation.†Significance of correlation coefficient. * and ** indicate significance at P¯ 0±05 and 0±01respectively. NS indicates not significant at P¯ 0±05.

to the disease incidence under similar circumstances (not shown). There was no

significant correlation of rishitin tolerance at 100 µg ml−" rishitin to either virulence or

disease incidence, apparently due to the more uniform sensitivity of the isolates at this

concentration. Virulence on the cultivar PI was not significantly correlated to tomatine

tolerance at any concentration (Fig. 1A, Table 5). However, virulence on another

cultivar, SM, was significantly correlated with tomatine tolerance at the 50 µg ml−"

concentration (P! 0±05).

Comparison of pathogenic and nonpathogenic isolates indicated that all non-

pathogenic isolates were among the most sensitive to tomatine. In order to further test

this result, five additional nonpathogenic F. oxysporum strains were evaluated for

tomatine tolerance in radial growth assays (Table 4). At 25 and 50 µg ml−" tomatine,

growth of most nonpathogenic isolates was significantly more reduced (less tolerant)

than was any pathogenic strain (Table 4, Fig. 1A). For example, at 25 µg ml−"

tomatine, eight out of 10 nonpathogenic isolates were significantly more sensitive than


any pathogenic isolates (P! 0±05), displaying a 1±5 to 4-fold greater reduction in

growth rate in response to tomatine. In contrast, the nonpathogenic isolates showed no

such association with rishitin sensitivity. All nonpathogenic isolates were as tolerant to

rishitin as the pathogenic isolates, including, in some cases, those in the high virulence

group.

DISCUSSION

Variation in virulence among isolates of a pathogen can provide the basis for further

genetic and biochemical studies to identify virulence determinants. Variation in the

virulence of F. oxysporum f.sp. lycopersici has not been studied much beyond the original

observations by Wellman [23]. The 12 isolates of F. oxysporum f.sp. lycopersici used in the

present study varied significantly with respect to both their ability to incite disease and

the severity of symptoms that they caused on infected plants. Here, we focused upon

symptom severity (vertical extent of foliar symptoms and vascular discoloration) as a

measure of virulence. Although the variation in virulence among isolates was

continuous, with the relative ranking dependent upon the tomato cultivar used, one

group of four pathogenic isolates colonized tomato stems more aggressively than the

other isolates tested, causing more severe symptoms and higher disease incidence, on

both cultivars of race 1-susceptible tomato. This group of high virulence isolates

included examples of both race 1 and race 2 genotypes, indicating that the virulence

of these isolates is independent of their race. Relative ranking among the lower

virulence isolates was more dependent upon the cultivar of tomato used.

Evaluation of the growth of these F. oxysporum isolates in the presence of rishitin and

tomatine demonstrated levels of tolerance which are consistent with a previous report

using a single F. oxysporum f.sp. lycopersici isolate (e.g. ED&!

of 8 µg ml−" for germ tube

growth with either compound [15]), considering the pH dependence of toxicity with

these compounds and the different media used in each study [1]. Different stages of

growth of these F. oxysporum isolates displayed different levels of sensitivity to these

compounds and differences in correlation of sensitivity to virulence. Spore germination

was largely unaffected by either compound, even at 100 µg ml−", and so the variation

measured between isolates would presumably have little consequence in secondary

germination within the plant. Germ tube elongation was the most sensitive stage to

both compounds but the degree of sensitivity among isolates did not correlate with

virulence. Radial growth rate, however, displayed the widest variation among isolates

and was correlated with virulence and pathogenicity under certain conditions.

Evaluation of a possible association of tolerance with virulence focused upon the

pathogenic isolates. Mycelial growth in the presence of rishitin was strongly and

significantly correlated with virulence under several of the experimental conditions

evaluated: at 25 and 50 µg ml−" rishitin in the tolerance assays, and when virulence on

either tomato cultivar was considered. Since rishitin has been found to be present at

concentrations ranging from 8 to 44 µg g−" fresh wt. in vascular and stem tissue

respectively [7, 15], there may be circumstances where this could lead to selective

growth of more tolerant isolates within the host. The lack of correlation of virulence

with rishitin tolerance at 100 µg ml−" rishitin does not detract from this conclusion

since rishitin has not been reported to accumulate to such high concentrations in


tomato. Correlation of virulence with tomatine tolerance was only found to be

significant under limited experimental conditions : only when virulence on the SM

cultivar was considered, and only at one tomatine concentration (50 µg ml−") tested in

the tolerance assay. Although this concentration is similar to that found in tomato

(50 µg g−" fresh wt. in root and stem tissue [13], 100 µg g−" fresh wt. in stem vascular

tissue [15]), tomatine tolerance may affect F. oxysporum virulence in more narrow

circumstances. The cultivar-dependence of the correlations could be attributed to the

higher virulence of five moderately virulent isolates on SM than on PI. Four of these

isolates were found to be highly tolerant of tomatine, but relatively more sensitive to

rishitin. Since tomatine or rishitin levels have been reported to vary by 2 to 6-fold

among different tomato cultivars [7, 17], the relative contributions of tolerance of each

compound to virulence could depend upon the levels of each compound in the cultivar

tested.

Although the analyses show separate correlations of virulence with rishitin tolerance,

and to a lesser extent with tomatine tolerance, an isolate’s tolerance of both compounds

together may provide a more accurate prediction of virulence since both compounds

occur in challenged tissues. Indicative of this possibility, the highly virulent isolates

generally displayed high tolerance of both tomatine and rishitin at 50 µg ml−". In

contrast, the lower virulence isolates displayed a similar high level of tolerance of only

one, or neither, of these compounds. Thus the correlations of virulence with tolerance

of one compound alone may underestimate the significance of its potential contribution

to virulence. Desjardins and Gardner [6] reported a similar relationship in G. pulicaris,

where tolerance of both potato phytoalexins rishitin and lubimin were necessary, but

not sufficient, for high virulence.

Despite the apparent association between the level of rishitin tolerance with

virulence, this association is only correlative and does not prove a role in virulence.

Two concerns might minimize such a role. The first concern is that the tolerance

displayed during spore germination and germ tube elongation did not correlate with

virulence. Any effect of differential tolerance on virulence could therefore function only

during the stage of growth of mature mycelia. However, the lack of perforation plates

in tomato xylem vessel elements has been used to argue that continued sporulation and

germination may not be necessary for spread in tomato xylem; fungal growth in the

xylem is mostly in the form of non-sporulating hyphae [19]. Therefore, limitation of

hyphal growth could indeed be expected to affect virulence more than a limitation of

germination or germ tube elongation. A second concern is that the variation in rishitin

tolerance among strains of F. oxysporium f.sp. lycopersici is much narrower than that

displayed by G. pulicaris, a pathogen of potato, to the same compound (18–88%

reduction in hyphal growth rate on 200 µg ml−" rishitin [6]). This suggests that

differences in rishitin tolerance may have a more limited role in the variation in its

virulence than in G. pulicaris. Part of this difference between pathogens may, however,

be related to the generally higher concentrations of rishitin in potato tubers (90 µg g−"

fresh wt. or more [10]) than in tomato.

When the pathogenic and nonpathogenic F. oxysporum isolates were compared, the

nonpathogens appeared to possess greater sensitivity to tomatine during germ tube

elongation and growth of mature mycelia at 25 and 50 µg ml−". Similar concentrations

of tomatine are generally thought to exist in healthy tomato tissue (50 µg g−" fresh wt.


in root and stem tissue [13], 100 µg g−" fresh wt. in stem vascular tissue [14]), and thus

may provide some selection against growth of more tomatine-sensitive strains. A

smaller growth differential between pathogens and nonpathogens would be expected

in the xylem fluids where concentrations of tomatine are higher (100–400 µg ml−"

[17]), but growth of nonpathogens is generally halted prior to this stage of infection [2].

Similar tolerance studies with F. oxysporum f.sp. radicis-lycopersici, which causes crown

and root rot in tomato, also indicate a greater tolerance of tomatine among isolates

pathogenic on tomato than the nonpathogenic isolates tested here. Sixteen isolates of

this forma specialis displayed only a 6–43% reduction in hyphal growth rate at

100 µg ml−" tomatine (Suleman, Straney and McGovern, unpublished). In the light of

the differences in tomatine sensitivity between the two tomato pathogenic formae

speciales and the nonpathogens, we could speculate that a certain level of tolerance of

tomatine may be required, but not in itself be sufficient, for certain types of pathogenic

growth on tomato. Although this study can not rule out a similar role for rishitin,

similar extremes in rishitin sensitivity do not appear to occur frequently in

nonpathogenic F. oxysporum associated with tomato.

Collectively, these results indicate that differences in virulence among pathogenic

isolates correlate most strongly to quantitative differences in tolerance of rishitin and

only more narrowly to differences in tolerance of tomatine. However, a certain

threshold level of tomatine tolerance is associated with pathogenicity on tomato since

growth of nonpathogens was generally more sensitive to tomatine than was that of

pathogenic isolates. Such different effects with the two compounds are consistent with

the different timing of rishitin and tomatine production in tomato. The preformed

nature of tomatine would allow inhibition of the establishment of an infection, while

the induced nature of rishitin would affect fungal growth only later during infection.

Although such differences in tolerance can not fully account for all differences in

virulence or pathogenicity of the different isolates, there are general trends which are

consistent with such relationships contributing to disease potential. An analogous

situation was observed with G. pulicaris, where tolerance to two different phytoalexins

in potato were necessary, but not sufficient, for high virulence in potato [6]. In this

case, sexual genetic analysis clarified the role of rishitin tolerance in G. pulicaris

virulence [6]. Similar manipulation of F. oxysporum, through parasexual genetics or

molecular cloning will be necessary to prove such role for tomatine or rishitin tolerance

in F. oxysporum f.sp. lycopersici. This study has characterized the variation among a set

of field isolates, and suggests possible roles for tomatine and rishitin tolerance, for

further detailed analysis.

The authors wish to thank Ann Desjardins and Susan McCormick of the Mycotoxin

Research Unit, USDA}ARS, Peoria, IL for their assistance and kindly providing

rishitin standards. Peto Seed Co. kindly supplied tomato seed which is not distributed

in U.S. market. Research was supported by a collaborative grant from the National

Agricultural Research Program (NARP) and the U.S.AID}Cairo, and coordinated in

the U.S. by USDA}OICD. Further support was provided through a NARP fellowship

to A. A. Saleh and a Peace Fellowship from the Egyptian Embassy (U.S.) to A. M.

Tohamy for work at the University of Maryland. Scientific article number A7783 of

the Maryland Agricultural Experiment Station.


REFERENCES

1. Arneson PA, Durbin RD. 1968. The sensitivity of fungi to α-tomatine. Phytopathology 58 : 536–537.2. Beswetherick JT, Bishop CD. 1993. An ultrastructural study of tomato roots inoculated with

pathogenic and nonpathogenic necrotrophic fungi and a saprophytic fungus. Plant Pathology 42 :577–588.

3. Booth C. 1971. The genus Fusarium. Kew, England: Commonwealth Mycol. Inst.4. Bowyer P, Clarke BR, Lunness P, Daniels MJ, Osbourn AE. 1995. Host range of a plant

pathogenic fungus determined by a saponin detoxifying enzyme. Science 267 : 371–374.5. Defago G, Kern H. 1983. Induction of Fusarium solani mutants insensitive to tomatine, their

pathogenicity and aggressiveness to tomato fruits and pea plants. Physiological and Molecular PlantPathology 22 : 29–37.

6. Desjardins AE, Gardner HW. 1989. Genetic analysis in Gibberella pulicaris : Rishitin tolerance, rishitinmetabolism and virulence on potato tubers. Molecular Plant Microbe Interactions 2 : 26–34.

7. Elgersma DM, Liem JI. 1989. Accumulation of phytoalexins in susceptible and resistant near-isogeniclines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici. Physiological andMolecular Plant Pathology 34 : 545–555.

8. Elias KS, Schneider RW, Lear MM. 1991. Analysis of vegetative compatibility groups innonpathogenic populations of Fusarium oxysporum isolated from symptomless tomato roots. CanadianJournal of Botany 69 : 2089–2094.

9. Ford, JE, McCance DJ, Drysdale RB. 1979. The hydrolysis of tomatine, by an inducible extracellularenzyme from Fusarium oxysporum f.sp. lycopersici. In: Hawkes JG, Lester RN, Skelding AD, eds. Linn.Soc. Symp. Ser. �ol. 7; The Biology and Taxonomy of the Solanaceae. London: Academic Press.

10. Henfling JWDM, Kuc’ J. 1979. A semi-micro method for the quantitation of sesquiterpene stressmetabolites in potato tuber. Phytopathology 69 : 609–612.

11. Jarvis WR. 1988. Fusarium crown and root rot of tomatoes. Phytoprotection 69 : 49–69.12. Komada H. 1975. Development of a selective medium for quantitative isolation of Fusarium oxysporum

from natural soil. Re�iews of Plant Protection Research 8 : 114–125.13. Langcake P, Drysdale RB, Smith H. 1972. Post-infectional production of an inhibitor of Fusarium

oxysporum f.sp. lycopersici by tomato plants. Physiological Plant Pathology 2 : 17–25.14. Lucy MC, Matthews PS, VanEtten HD. 1988. Metabolic detoxification of the phytoalexins

maackiain and medicarpan by Nectria haematococca field isolates : relationship to virulence on chickpea.Physiological and Molecular Plant Pathology 33 : 187–199.

15. McChance DJ, Drysdale RB. 1975. Production of tomatine and rishitin in tomato plants inoculatedwith Fusarium oxysporum f.sp. lycopersici. Physiologial Plant Pathology 7 : 221–230.

16. Miao VPW, VanEtten HD. 1992. Genetic analysis of the role of phytoalexin detoxification in virulenceof fungus Nectria haematococca on chickpea (Cicer arietinum). Applied En�ironmental Microbiology 58 :809–814.

17. Smith CA, MacHardy WE. 1982. The significance of tomatine in the host response of susceptible andresistant tomato isolines infected with two races of Fusarium oxysporum f.sp. lycopersici Lycopersiconesculentum. Phytopathology 72 : 415–419.

18. Soledade M, Pedras C, Taylor J. 1993. Metabolism of the phytoalexin brassinin by the ‘‘Blackleg’’fungus. Journal of Natural Products 56 : 731–738.

19. Stromberg EL, Corden ME. 1980. Scanning electron microscopy of Fusarium oxysporum f.sp. lycopersiciin xylem vessels of wilt-resistant and susceptible tomato plants. Canadian Journal of Botany 58 :2360–2366.

20. Tegtmeier KJ, VanEtten HD. 1982. The role of pisatin tolerance and degradation in the virulence ofNectria haematococca on peas : A genetic analysis. Phytopathology 72 : 608–612.

21. VanEtten HD, Mansfield JW, Bailey JA, Farmer EE. 1994. Two classes of plant antibiotics :Phytoalexins versus ‘‘phytoanticipins ’’. Plant Cell 6 : 1191–1192.

22. VanEtten HD, Matthews DE, Matthews PS. 1989. Phytoalexin detoxification: Importance forpathogenicity and practical considerations. Annual Re�iew of Phytopathology 27 : 143–164.

23. Wellman FL. 1940. Differences in Growth Characters and Pathogenicity of Fusarium Wilt Isolations tested on ThreeTomato Varieties. Technical Bull. no. 705. Washington, DC; U.S. Department of Agriculture.