J. Phytopathology 144, 131-134 (1996)(g 1996 Blackwell Wissenschafts-Verlag, BerlinISSN 0931-1785

Agriculture and Agri-Pood Canada, London, Ontario, Canada

A Rapid Tomato Seedling Assay for Verticillium Wilt

C. MADHOSINGH

Pest Management Research Center, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario N5V

4T3, Canada

Received June I, 1995; accepted November 29, 1995

Abstract

A rapid (!O-min) tomato seedling assay was developed for determiningthe wilt-capacity of cell-free culture filtrates of race 1 and race 2 isolatesof Verticillium dahliae. The assay also rapidly determined the dilTerencesin will resistance between tomato cultivars. Rapidity was attained bymanipulating incubation conditions to promote rapid wilting. Theseincluded inducing elongated stems in the susceptible cv. Bonny Bestseedlings by growth of plants in subdued light (2.2 x 10' lux) and byconcentrating two-fold the cell-free culture filtrates of the pathogen.Further, rapid uptake of the wilt factors in the culture filtrates wasfacilitated during incubation by increasing transpiration with brightlight (23.8 X 10' lux), a wind stream (125-150 metres/min) across theassay seedlings, iow relative humidity (28%) and a relatively high assaytemperature (30 C). When necessary, these conditions were altered toextend the assay times. This assay system was used to determine optimalincubation time, temperature and medium for obtaining culture filtrateswith increased wiit capacities. The assay also determined the relativewilt capacities of races 1 and 2 and the comparative resistance of fourtomato cultivars to wilt caused by races 1 and 2.

Ziisammenfassung

Ein schneller Test auf fcrticj/fiam-Weike an Tomatenjungpflanzen

An Tomatenjungpflanzen wurde ein schneller (10 Min. dauernder) Testentwickelt, mit dem die welkeauslosende Kapazitat zellfreier Kultur-flltrate von Verticillium-dahliae-lsolaten der Rassen I und 2 bestimmtwerden konnte. Der Test erlaubte auch eine schnelle Bestimmung derzwischen verschiedenen Tomatensorten bestehenden Welkeresistenz-Unterschiede. Die Schnelligkeit des Tests wurde erreicht, indem dieUmweltbedingungen so manipuliert wurden, daC die Pflanzen schnellwelkten. So wurde bei Jungpflanzen der anfalligen Sorte Bonny Besteine Bildung veriangerter Stengel induziert, indem die Pflanzen ingedampftem Licht (2,2 x 10' Lux) gehalten und einer doppelten Kon-zentration der zellfreien Kulturfiltrate des Pathogens ausgesetzt wur-den. Eine schnelie Aufnahme der in den Kulturfiltraten enthaltenenwelkeinduzierenden Faktoren wahrend der Inkubation wurde erreicht.indem die Transpiration dureh helles Licht (23,8 x 10' Lux), einen durehdie Testpfianzen geleiteten Luftstrom (125-150 Meter, Min.), eine nie-drige relative Luftfeuchtigkeit (28%) und eine relativ hohe Ver-suchstemperatur (30°C) erheht wurde. Bei Bedarf wurden dieseBedingungen geandert, um die Testdauer zu erhohen. Dieses Testsystemwurde verwendet, um die optimale Inkubationsdauer, die optimaleTemperatur und das optimale Medium ftir die Gewinnung von Kultur-flltraten mit erhohter welkeauslosender Kapazitat zu bestimmen. DerTest ermogiichte auch eine Bestimmung der relativen welkeauslosendenKapazitaten der Rassen 1 und 2 sowie einen Vergleich der Resistenz

von vier Tomatensorten gegenuber der von den F.-da/itoe-Rassen 1und 2 hervorgerufenen Welke.

Introduction

Verticillium dahliae Kleb. is the wilt pathogen of a wide range

of plant species including tomatoes, potatoes and cotton. The

diseases caused by races 1 and 2 of this pathogen have particular

economic significance in tomato and potato farms in Canada.

Resistant tomato cultivars have been developed for race 1 but

no resistant tomato cultivars are available for the more recent

race 2. The proteinaceous toxins of Verticiliium (Keen and

Long, 1972) have been the prime focus of research as the basis

of the wilt diseases caused by these pathogens (Buchner et al.,

1989). A recent report {Mansoori et al., 1995) and current

studies in my laboratory indicate the production in culture

of low molecular weight phytotoxic entities, some ninhydrin

positive, that are smaller than those described by Nachmias et

al. (1987).

In the process of examining chromatographed low molecular

weight phytotoxic metabolites of V. dahliae associated with the

wilt, it became necessary to develop a rapid assay for the wilt

capacity of separated fractions from culture filtrates of the

pathogen. In the most recent paper on Verticillium toxins (Man-

soori et al.. 1995), the assay period was 7 days, and this was

less than those reported earlier. In order to develop a more rapid

assay, procedures were incorporated to increase the uptake of

solutes (by accelerating transpiration) and to induce earlier

wilting of tomato seedlings (by increasing the susceptibility of

the assay seedlings to wilt). A similar procedure (Madhosingh,

1995) was successful in assaying Pusarium toxins.

Materials and Methods

Organisms. Isolates of V. dahliae races ! and 2 were obtained from DrK. Dobinson at this Center. Single spore isolates were maintained on aCzapek-Dox agar medium in replicate cultures at 4"C.

Assay seedlings

Four tomato cultivars were used. Cv. Bonny Best, a susceptible variety,was used routinely for assaying pathogen culture filtrates and fractionsof active filtrates for wilt activity. The seedlings were grown in Promix,a mixture of peat and Vermiculite (Premier Peat Moss Inc., Reviere duLoup, Quebec). Growth under 12 h diffused fluorescent light (2.2 x 10'lux) and 12 h dark, induced elongated stems which reduced the wilt

«,nen.: 0931-1785/96/4403^131 $ 11.50/0

132 MADHOSINGH

time further. Ten-day-old seedlings of tomato cvs 9230, HI350, and9478, ail relatively resistant to race 1 of the pathogen, were used also.These cultivars were obtained from Dr K. Dobinson at this Center.

Medium for wilt cidture flitrate prodnctioo

The Czapek-Dox (CD) formulation in water amended with 2% caes-amino acids (CDCa) produced the most wilt-eflective culture filtrate.Cultures of both race 1 and race 2 were grown in 500 ml sterile mediumcontained m 1.5 litre flasks at 24-C and 100 r.p.m. After 20 days ofgrowth, all cells were removed from the culture filtrates by cen-trifugation and filtration. The filtrate was then concentrated t̂ s'o-foldby freeze drying. The sterility of the filtrates was ascertained by exam-ination of filtrate samples microscopically and by inoculation on CDCaagar medium incubated at 24 C for 7 days.

Assay

The basic rapid assay incubation conditions were 3O'C. 28% relativehumidity attained with activated Drierite (Mallinckrodt), 23.8 x 10' luxincandescent light and wind across the experimental seedlings at a speedof 125-150 metres min. The wind was produced by a battery operatedportable fan in the assay incubator. The specially grown cv. Bonny Bestseedlings with elongated stems were removed carefully from the Promixsubstrate retaining the root system as intact as possible. They wereselected for uniformity in size, leaf number and root systems. The rootswere washed in sterile water to remove substrate debris and driedbetween paper towels. Each seedling was then placed immediately into1.5 ml culture filtrate contained in individual micro tubes and incubatedas described above. Under these conditions, wilt induced by race 1culture filtrate occurred within 4 min, and in 10 min the seedlingswilted and collapsed. The rapid assay was useful particularly for thedetermination of wilt activity of numerous column chromatographyfractions of the culture filtrates. However, these basic conditions werealtered when it was necessary to increase the assay time in order toobtain better comparative data. Altered conditions are indicated in thedata tables. Twenty seedlings of each of the four tomato cultivars wereincubated with each cell-free culture filtrate of race 1 and race 2. Instudies examining variation in race 1 resistance within tomato cultivarpopulations, a minimum of 100 seedlings of each cultivar was incubatedwith the race 1 culture filtrate.

Results and DiscussionThe data in Table 1 show the comparative wilt capacities of theculture filtrates of race 1 and race 2 of V. dahliae grown indifferent media. Under the assay conditions described, race Iculture filtrate, form the Czapek-Dox medium supplementedwith 2% caesamino acids (CDCa) medium, wilted the lO-day-old Bonny Best seedlings in 10 min. This medium produced themost eifective wilt filtrate. The Czapek-Dox culture filtrate was

next in wilt effectiveness and the less chemically-defined yeastand malt extract produced the least effective filtrates. The dataalso demonstrate that the culture filtrate of race 1 invariablyhad a greater wilt capacity than that of race 2. Table 2 indicatesthat 20-day filtrates from race 1 and race 2 CDCa culturesinduced seedling wilt in 10 and 14 min, respectively. These werefaster wilt times than those obtained for the filtrates from theother incubation periods which ranged from 15 days to 30 days.These data indicate, therefore, that 20-day cultures of bothraces of the pathogen induced the most wilt-effective culturefiltrates. Table 3 shows that CDCa cultures of both race 1 andrace 2, incubated at 24 C, produced filtrates which had higherwilt capacities than filtrates from cultures incubated at 18 Cand 30 C. These data indicate that incubation at 24°C was the

Table 2The effect of incubation period on the wilt capacity of 24 C CDCaculture filtrates of Verticillium dahliae races 1 and 2 on cv. Bonny Bestseedlings

Wilt time (min)

(days)

15202530

Race 1

20101518

Race 2

25142124

Wilt time differences between periods and between races 1 and 2 aresignificant at P = 0.05. Assay conditions as m Table 1. Wilt capacity ofculture filtrates increase as wilt times decrease.

Table 3Effect of incubation temperature on the wilt capacity of 20-day CDCaculture filtrates of Verticillium dahliae races 1 and 2 on cv. Bonny Bestseedlings

Incubationperiod( C )

Wilt time" (min)

Race i Race 2

182430

151020

221528

"Decrease in wilt time indicates increase in wilt capacity. Differences inwilt times between incubation temperatures and between races 1 and 2are significant at P = 0.01. Assay conditions as described in Table 1.

Table IThe effect of culture media on thewilt capacity of 20-day culture fil-trates of race 1 and race 2 Ver-ticitlium dahliae grown at 24 C and100 r.p.m.

Culture filtrate(media)

CDCaCDNachmias et al. (1987)Yeast extract (Difco)Malt extract (Difco)Controls—sterile water

media cone, two-fold

Wilt

Race 1

1015192528——

time (min)

Race 2

1418253035——

Comparativewilt capacityof filtrates

12345

N/AN/A

Twenty 10-day-o!d cv. Bonny Best seedhngs were used in each treatment. Differences in wilt timesbetween media culture filtrates and between race 1 and race 2 are significant at P = 0.05. Media: CD,Czapek-Dox (Difco); CDCa, CD supplemented with 2% caesamino acids (Difco); Nachmias, 100 mlcontained: 2 g glucose, 0.2 g asparagine, 0.15 g KH2PO4 • T H A 1 mg FeSC • 5H2O, 0.6 mg CaSOj, Img thiamine-HCl, 0.5 mg pyridoxine. pH 6.7. Wilt capacity increases as wilt time decreases. See textfor assay conditions.

A Rapid Tomato Seedling Assay for Verticillium Wilt 133

Tomatocultivar

9230H135O9478Bonny Best

Wilt time"

Race 1

3 h 30 min51 min40 min25 min

Race 2

4 h 15 min64 min53 min36 min

Resistance- of tomato

cultivar*

1234

Comment

smallest plantlargest plant, 1st wilt signsintermediate size plantsusceptible cv.

Assay conditions were: light, 19.4x10' lux; humidity, 28%; wind, nil; temperature, 30C; age ofseedlings, 20 day; filtrates, celt-free from 20-day cultures of races 1 and 2. Twenty replicate seed-iings/assay." Wilt time differences between tomato cultivars are significant at P = 0.01. Wilt time differences betweenpathogen Race 1 and Race 2 are significant at P = 0.05.'Cultivar resistance increases as wilt time increases.

Table 4Comparative resistance to wilt oftomato cvs H1350, 9230, 9484 (cul-tivars relatively resistant to race 1),and cv. Bonny Best determined bythe seedling assay using culture fil-trates of Verticillium dahliae race Iand race 2

best temperature of those examined for the production of themost wilt-effective culture filtrates.

The assay was also used to determine the comparative resist-ance of four tomato cultivars, to wilt induced by 20-day 24 CCDCa culture filtrates of race 1 and race 2. The assay conditionswere modified by reducing the light to 19.4 x 10' lux and omit-ting the wind, as described in Table 4, to obtain longer wilttimes which produced better comparative resistance data. Whenspores of race 1 and race 2 were used to induce seedling diseasein the four tomato cultivars, wilt results (Table 5) were similarto those obtained with the cell-free culture filtrates. In bothassay systems, cv. 9230 was the most resistant and cv. BonnyBest the least resistant to both race I and race 2. However, thecell-free culture filtrate assay produced more significant datamore quickly. Further, the flexibility of the culture filtrate assayconditions facilitated the more definitive determination of com-parative resistance of cultivars. The data of Tables 4 and 5support the inference reported by Nachmias and Krikun (1986)that the symptoms induced by toxins in culture filtrates are thesame as those caused by the pathogens. The data in Table 5demonstrate also that the race 2 pathogen was isolated earlierthan the race 1 pathogen from the stem of the inoculated seed-lings, results indicating a faster invasion of the seedlings' stemsby the race 2 organism. Nevertheless, the race 1 pathogencaused earher wilting in all the cultivars examined.

in preliminary studies, this assay system was used to examine

the variation of race 1 wilt resistance within the four tomatocultivars. In these studies lots of 100 seedlings of each cultivarwere examined. The data showed that ca. 1 % of the seedlingswithin a cultivar demonstrated resistance at least !0% abovethe mean wilt. These preliminary data suggest that the assaycould be useful for identifying and selecting individual plantswhich are more resistant within a population.

The recent studies of Cerezine and Kurozawa (1992) andMansoori et al. (1995) employed assays which extended for 28days and 7 days respectively. The assay reported here is quickerand has the advantage of easy modifications and flexibility forobtaining significant comparative data on seedling resistanceand for identifying separated phytotoxic fractions of the cell-free culture filtrates. The assay, which has shown significantdifferences in the wilt capacity between race 1 and race 2 undera variety of conditions (Tables 1-4), could also be used to assessthe wilt capacity of individual pathogen isolates from widerpopulations of V. dahliae.

Acknowledgement

The personal assistance of Professor Alan Davenport is acknowledgedwith appreciation and thanks. Dr Davenport is Director of the Boun-dary Layer Wind Tunnel, Faculty of Engineering Sciences. Universityof Western Ontario. London, Canada. He assisted by determiningthe wind speeds generated by the portable electric fan used in theseexperiments.

Tomatocultivar

9230HI 3509478Bonny Best

Wiit time (days)Racel

27242320

Race 2

29262522

Pathogen isolation' (day)Race 1

22171412

Race 2

20161210

Resistance*

1234

Incubation conditions were: temperature, 30 C; light/dark cycle, 12 h; light. 23.8 x 10' lux; relative humidity.68%; replicates, 80 seedlings/assay. Seedling inoculation; 30 min in 5 x 10' spores/ml from race 1 and race2 grown on PDA at 24°C for 20 days."Period after inoculation when V. dahliae was isolated from seedling stem location 4 cm above groundlevel, isolations were made daily (day 5 to day 30) from two seedhngs from each treatment and culturedon antibacterial potato dextrose agar (PDA) medium (Difco). Isolations were made from all remainingseedlings in the experiment and cultured on day 30.* Decrease in wilt time indicates decrease in seedling resistance.Controls: four seedlings for each cuitivar, were dipped for 30 min in sterile water and replanted similarlyto the spore-treated seedlings. Wilt time figures based on data on 20 seedlings/treatment. Wilt timesdifferences between tomato cultivars and between race I and race 2 are significant at P = 0.5. The wilt timedifferences between cv. Bonny Best and other cultivars significant at P = 0.05. All spore-treated seedlingswilted and isolations produced Verticillium cultures. Controls remained healthy and isolations from themproduced no fungi in culture.

Table 5Comparative resistance of tomatocvs 9230. 9478, HI350 and BonnyBest to disease developed by inocu-lation with Verticitlium dahliae race1 and race 2 spores

134 MADHOStNGH

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ticillium dahliae-pTodviced phytotoxic peptides purified from culturefluids and infected potato stems. Physiol. Moi. Plant Pathol. 35,235-269.

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Keen, N. T., M. Long (1972): Isolation of a phytotoxic protein-iipo-polysaccharide complex from Verticitlium alho-atrum wilt of cotton.Physiot. Plant Pathol. 2, 307-315.

Madhosingh. C. (1995): Rapid tomato seedling assay for virulent iso-

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Mansoori, B., J. M. Milton, C. J. Smith (1995): Isolation and partialpurification ofaphytotoxin related to pathogenic Verticillium species..j . Phytopathol. 143, 33-36.

Nachmias, A., J. Krikun (1986); Screening for Verticillium resistance:can bioassay in vitro replace field trials. Potato Res. 26, 404.

Nachmias, A., V. Buchner, L. Tsror. Y. Burstein, N. Keen (1987):Differential phytotoxicity of peptides from culture fluids of Ver-ticillium dahliae races 1 and 2 and their relationship to pathogenicityof the fungi on tomato. Phytopathology 77, 506-510.