Crop Protection 18 (1999) 397}405

Benzothiadiazole (BTH) induces resistance in cauli#ower (Brassica oleracea var botrytis) to downy mildew of crucifers caused by Peronospora parasitica Jean-Franc7 ois Godard, SmamK l Ziadi, Claudie Monot, Daniel Le Corre, Drissa SilueH * BBV, Bretagne Biotechnologie Ve& ge& tale, Penn ar Prat, F-29250 Saint Pol-de-Le& on, France Received 22 October 1998; received in revised form 10 May 1999; accepted 11 May 1999

Abstract Seedlings of Billabong, a downy mildew susceptible cauli#ower F1 hybrid, were screened for their ability to develop induced resistance following treatment with CGA 245704, also known as benzothiadiazole (BTH). The seedlings were sprayed with a water solution containing the following doses of the elicitor: 0.0015, 0.005, 0.01, 0.015, 0.045, 0.075, 0.15 and 0.25 mg active ingredient (a.i.) per ml. In the dose}response experiments, the treated seedlings were challenged four days later with inocula prepared from either frozen or fresh produced spores of the virulent downy mildew isolate FP06. Results obtained showed that up to a concentration of 0.45 mg a.i./ml, the e$cacies of the induced resistance was lower with fresh spores than with frozen ones. At 0.45 mg a.i./ml or higher, no di!erences were seen. Similar results were obtained on 30 day-old plants (young plants). In the time-course experiments, the treated seedlings were inoculated with isolate FP06 !1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 15 and 30 days later. Results obtained showed that no resistance was induced when the inoculation was done just after BTH treatment or one day before. The seedlings as well as the young plants exhibited induced resistance when challenged 1}30 days after treatment and this resistance was shown to be sytemic. Furthermore, it was shown that BTH solutions kept at 4}53C were still able to induce resistance 30 days after their preparation. In all experiments, growth reduction a!ecting seedling's height was noticed and its importance was dose-dependent. The reduction was of 5.9% for the lowest concentration (0.0015 mg a.i./ml) to 38.3 for the highest one (0.25 mg a.i./ml). At concentrations of 0.045 and 0.05 mg a.i./ml, induction of resistance was maximum and the height reduction was of approximatively 22%. On young plants, the growth reduction concerned only leaf length and width. At a concentration of 0.05 mg a.i./ml, the corresponding leaf growth reduction were of 14.1 for the width and 12.3% for the length. No direct e!ect of BTH on spore morphology or germination was shown.  1999 Elsevier Science Ltd. All rights reserved. Keywords: CGA 245 704; Induced resistance; Integrated pest management

1. Introduction Downy mildew of crucifers caused by Peronospora parasitica is one of the most serious diseases on Brassica crops. The disease is mostly controlled by fungicide applications, but mutant isolates with fungicide resistance have been reported (Crute et al., 1985; Brophy and Laing, 1992; Crute et al., 1994). Sources of resistance have been identi"ed at the cotyledon stage in several Brassica crops (Ohuguchi et al., 1990; Moss et al., 1991a,b; Dias et al.,

* Corresponding author. Tel.: #33-02-98-29-06-44; fax: #33-0298-69-24-26. E-mail address: [email protected]!.fr (D. SilueH )

1993; Nashaat and Rawlinson, 1994, Nashaat and Awasthi, 1995; SilueH et al., 1995,1996). Genetic analysis of resistance has shown that resistance at the cotyledon stage is generally controlled by single, mostly dominant genes (Lucas et al., 1988; Nashaat et al., 1997). Resistance conferred by these genes is often isolate speci"c, and the use of these genes may help reduce fungicide applications. An alternative way to control the disease is with integrated pest management (IPM) combining the use of resistance genes, low amounts of fungicides and appropriate agronomic practices. A new alternative way for protecting crops from diseases has arisen in the last few years and consists of the use of plant defense elicitors. In many pathosystems, induced resistance has been described and the

0261-2194/99/$ - see front matter  1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 9 9 ) 0 0 0 4 0 - X

398

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405

elicitors were either abiotic or biotic ones. Among the abiotic compounds, salicylic acid is the most widely known, and it was found to induce resistance in many plants to fungal, bacterial and viral pathogens (for review, Ryals et al., 1996; Schneider et al., 1996). Hijwegen and Verhaar (1993) have shown that 2,6-dichloisonitinic acid and three fungal culture "ltrates were able to induce resistance in red cabbage to P. parasitica; disease was reduced to less than 52%. Recently, CGA 245704, also known as benzothiadiazole (BTH), was shown to be a potent systemic acquired resistance (SAR) inducer (Friedrich et al., 1996; GoK rlach et al., 1996; Lawton et al., 1996) providing protection against a wide spectrum of plant pathogens. Reduction of the diseases caused by Cercospora nicotianae, Peronospora tabacina, Phytophthora parasitica and Pseudomonas syringae on tobacco reached 80% (Friedrich et al., 1996). Jensen et al. (1998) have shown that when used as a seed treatment, this compound is able to induce resistance in oilseed rape to the soil pathogen Rhizoctonia solani. The authors also showed that seed treatment helps protect cabbage seedlings against downy mildew caused by P. parasitica. BTH is now commercialized in Germany and Switzerland under the trade name Bion威, and good protection is obtained against di!erent crop diseases. The aims of this study were to test the ability of CGA 245704 to induce resistance in cauli#ower (Brassica oleracea var. botrytis) seedlings and young plants to downy mildew caused by P. parasitica and to assess whether it can help develop a new IPM strategy against this disease.

2. Material and methods 2.1. Plant material used Seeds of the cauli#ower F1 hybrid Billabong (Yates, Australia) were used in this study. This accession is susceptible to most of our P. parasitica isolates (SilueH et al., 1995,1996) and is now used as our susceptible check. Seeds were sown in `Ji!y potsa "lled with peat (De Baat, France) and grown in a greenhouse for eight days where temperature was maintained at 23}253C day, 16}183C night. Other experiments were conducted on 30 day-old plants. For these, the 8 day-old seedlings were therefore transferred to 16 cm;16 cm;16 cm plots. Supplementary light was applied to maintain an 18-h photoperiod. 2.2. Seedling treatment with BTH and inoculum preparation BTH was kindly supplied by Novartis (France) and contained 50% active ingredient (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester). It was dissol-

ved in distilled water to obtain active ingredient (a.i.) concentrations ranging from 0.0015 to 0.075 mg/ml and then either deposited (2;20 ll droplets per cotyledon) or sprayed on whole eight-day-old seedlings (ca 200 ll per seedling). In other experiments, a BTH solution were prepared and stored at 43C for one month and then tested for its stability over time to induce resistance in plants. After treatment with BTH, seedlings were maintained in the growth cabinet (163C night and 203C day, 12 h light) in propagators that were sealed. A propagator consists of a tray containing `je!y potsa in which seedlings were grown in peat and a translucient couvercle. After 1}30 days the seedlings were sprayed with a downy mildew conidial suspension adjusted to 20,000 spores per ml. The isolate used for dose-response, time-course response, and protection duration studies was FP 06 previously described by SilueH et al. (1995,1996). For inoculum preparation, sporulating cotyledons were agitated in a tube containing sterile distillated water to dislodge the conidia. To compare inoculum quality, the conidia used for inoculum preparation were either harvested from susceptible living plants just before inoculation (fresh inocula) or from the freezer at !203C (frozen inocula) for at least 24 h. 2.3. Inoculation of the seedlings Approximately 200 ll of the downy mildew spore suspension was sprayed per seedling using compressed air (0.5 bar). At least 60 seedlings per replicate were inoculated for each treatment (dose-response-, time course-, induction on seedlings and young plants-, comparison of frozen versus fresh spores-, systemic induction-, treatment methods tests) described in this paper and at least three replicates were done. We also compared spray versus drop inoculation (four droplets of 20 ll per plant, i.e. two droplets per cotyledon) inoculation. After inoculation, plants were kept in a growth cabinet (16 h light; 203C day; 163C night) for seven day for seedlings or 15-day for 30-dayold plants. After this incubation period, susceptible plants exhibited an intense sporulation on the cotyledons. 2.4. Assessmment of the induced disease resistance Seven days after inoculation, seedlings were assessed for disease resistance using a 6-point scale (Williams, 1985) already used by SilueH et al. (1996) in which six interaction phenotypes (IP) are de"ned (0, 1, 3, 5, 7, 9). Rating 0 was assigned to plants showing no visible symptoms and the rating 9 to those showing heavy sporulation and collapsed cotyledons. A disease index (DI) was then calculated using a formula already used by Williams

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405

(1985): ι (i;j) DI"  n where n are total plants, i the interaction phenotype class and j the number of plants per class. With the DI values, six classes resistance phenotypes were de"ned: Very resistant (DI)1), resistant (1(DI)3), moderately resistant (3.1)DI(4), moderately susceptible (4.1)DI(5), susceptible (5.1)DI(6) and very susceptible (DI*6.1). On young plants, the disease was assessed 12}15 days after inoculation, based on sporulation intensity and the percentage of diseased leaf area. Four classes were assigned, ranging from resistant R: resistant (no visible symptoms or presence of hypersensitive necrotic #ecks), MR: sparse sporulation and presence of yellow necrotic #ecks on up to 10% of the whole leaf area diseased, MS: medium sporulation and large yellow necrotic #ecks, 11}20% of the whole leaf area diseased, S: intense sporulation and more than 50% whole leaves diseased and sometimes collapsed. To address whether BTH can induce systemic resistance on 30-day-old plants, one fully developed leaf was randomly treated with BTH, and four days later the whole plant was challenged with the pathogen by spraying 3 ml/plant of a frozen spore suspension adjusted to 20,000 conidia/ml. Disease rating was done 12}15 days later as described above. The elicitor-treated leaf was always resistant. The other leaves were either R, MR, MS or S; and a mean disease category was assigned to the non-treated leaves. Four couples of interaction phenotypes were therefore made: R/R, R/MR, R/MS and R/S. In each couple, the "rst category concerns the treated leaves and the second the untreated leaves. Whole control plants were rated and mean disease categories were assigned. In some experiences, resistance induction e$cacies were calculated as follows: rating of the control minus the treatment. The data obtained were divided by the rating of the control and multiplied by 100. 2.5. Test of the systemic resistance induction by BTH Experiments to test whether BTH could induce a systemic resistance were carried out on both 8-day-old seedlings and 30-day-old plants. On seedlings, only one cotyledon/seedling was treated (two droplets of 20 ll BTH solution) and the whole seedling was challenged with the pathogen. On adult plants (30-day-old) only one fully developed and randomly selected leaf/plant was treated (approximatively 100 ll/leaf of BTH solution), and the whole plant was challenged with the pathogen. Two concentrations were tested in each experiment: 0.015 and 0.075 mg a.i./ml on seedlings and 0.03 and 0.075 mg a.i./ml on young plants.

399

2.6. Assessment of direct ewect of BTH on the pathogen To assess whether the elicitor has a direct e!ect on the downy mildew pathogen, fresh conidia were suspended in either water or a 0.075 mg a.i./ml solution. One hundred ll of the suspension (conidia in BTH at 0.075 mg a.i./ml or water, and 100,000 conidia/ml) were incubated at 20}223C in the dark on glass slides kept in Petri dishes for 12 h. Spore morphology was then observed and percentage of germinating conidia assessed.

3. Results 3.1. Dose}response for induction of resistance Six concentrations of BTH were tested on 8-day-old seedlings: 0.0015, 0.005, 0.01; 0.015, 0.045 and 0.075 mg a.i./ml (Table 1). Resistance was induced at and above a concentration of 0.0015 when challenged with frozen inocula. It was interesting that at *0.045 mg a.i./ml, the corresponding e$ciency was at least 69% (Table 1) with both types of conidia (fresh or frozen). These results also show that when fresh conidia were used as inoculum the induced resistance was less e$cient up to a concentration of 0.045 mg a.i./ml (Tables 1 and 2). When higher BTH concentrations were tested, no signi"cant di!erences were noticed. On 30-day-old plants, two elicitor concentrations (0.03 and 0.075 mg a.i./ml) were tested for their ability to induce resistance. Results (Fig. 1) obtained showed that the plants exhibited a dose-dependent resistance to downy mildew. In fact, when a 0.075 mg a.i./ml was applied, most of the plants (19 out of 22) were scored resistant (R). None of them scored susceptible (S). On the opposite, application of the lower concentration led to lower number of R plants (12 out of 23) and a higher number of MR (11 out of 23). Control plants were mostly S (17 out of 21). Results obtained on 8-day-old seedlings are similar to those obtained on 30-day-old plants. We also showed that 8-day-old seedlings treated with the elicitor were still resistant when challenged with the pathogen 15 (data not shown) and 30 days later (Fig. 2). Again, the higher concentration gave the higher e$cacy. The induced resistance was always characterized by the presence of brown, small necrotic spots which were visible during the disease ratings. These symptoms were not present on non-downy mildew inoculated seedlings treated with the elicitor. The observed tissue necrosis is similar to hypersensitive reactions (HR) obtained in incompatible cultivar-isolate combinations. 3.2. Time course response In another study we conducted, we showed that when challenged with the pathogen just after treatment with

400

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405

Table 1 Induction of resistance in 8-day-old cauli#ower seedlings treated with di!erent doses of BTH and inoculated four days later with downy mildew spore suspensions derived from either fresh or frozen conidia Concentration (m g a.i./ml)

0 (Control) 0.0015 0.005 0.01 0.015 0.045 0.075 0.15 0.25

Growth reduction in % (height reduction)

* 5.9 7.7 13.1 ! 20.8!" 21.3!" 24.7" 37.2# 38.3#

Inocula of fresh conidia

Inocula of frozen conidia

Mean disease indexes

Mean e$cacies (%)

Mean disease indexes

Mean e$cacies (%)

9.0
9.0 9.0 8.3! 8.4 2.8$ 1.8' 1.0, 1.0,

/

8.6 7.8" 5.8# 2.7% 2.0& 1.2'( 1.1( 1.2 1.0

/ 12$7 35$3 69$6 77$1 86$4 87$2 86$2 88$2

0$0 1$1 8$6 6$10 69$15 80$5 * *

Growth reduction was calculated based on plant size in the water control. Statistical analysis was made on plants size in each treatment and treatments with the same letter are not di!erent.
Statistical analysis was done on disease index values and treatments with the same letter are not di!erent. Values are the mean e$cacies expressed as percentages $ the standard deviations. Mean e$cacies are based on the di!erence of disease indexes between treatment and control divided by the control and multiplied by 100. These means e$cacies were calculated from data obtained in three replicates and 60 seedlings were analyzed in each replicate. Not included in the statistical analysis.

Table 2 Comparison of two methods (drop vs. spray) of treating cauli#ower seedlings with BTH on induction of resistance four days after inoculation with spore suspensions derived from either fresh produced or frozen conidia Type of plant treatment Spray Drop Concentrations (mg a.i./ml)

Fresh conidia

Frozen conidia

Fresh conidia

Frozen conidia

0 (control) 0.015 0.075

9.0
6.2 1.6

9.0 1.9 0.5

9.0 3.7 1.1

9.0 1.3 1.0

In the spray method, each seedling was sprayed with approximately 200 ll of a BTH solution using compressed air. In the drop treatment method two 20 ll droplets were deposited on each cotyledon (four droplets/seedling) using a automatic dispenser.
Disease rating was done using the six-points scale (0-1-3-5-7-9) described by Williams (1985) in which 0"no visible symptoms and 9"intense sporulation on both sides of the cotyledons that usually die. Data in this table are means of three replicates and 60 seedlings were rated in each replicate. Mean disease indexes are then calculated and seedlings sorted in categories according to the mean DI: Very resistant (DI)1), resistant (1(DI)3), moderately resistant (3.1) DI(4), moderately susceptible (4.1) DI(5), susceptible (5.1) DI(6) and very susceptible (DI*6.1).

the elicitor, the seedlings were not resistant. No curative e!ect was obtained when the seedlings were inoculated 24 h before their treatment with BTH. They were all susceptible to downy mildew. Other experiments we

Fig. 1. Induction of resistance in 30-day-old cauli#ower plants treated with two concentrations of BTH and challenged two days later with frozen conidia of the downy mildew isolate FP 06.

carried out showed that when seedlings were challenged with the pathogen from 1}8 days after treatment with the elicitor, resistance was induced from one day and seemed to be maximum two days after treatment (Figs. 3 and 4). However, comparison of the two inocula (fresh vs. frozen) showed that the induced resistance was more consistent and reliable with the higher BTH concentration (0.075 mg a.i./ml, Fig. 4) than with the lower one (0.015 mg a.i./ml). In fact, resistance induced with a 0.015 mg a.i./ml elicitor concentration showed variation within the replicates and was less e$cient against fresh inocula (Fig. 3).

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405

401

Fig. 2. Induction of resistance in 8-day-old cauli#ower seedlings treated with BTH (0.03 and 0.075 mg a.i./ml) and challenged 30 days later with the downy mildew isolate FP 06. Disease assessment consisted in classifying plants as R (resistant), MR (moderately resistant), MS (moderately susceptible) and S (susceptible).

Fig. 3. Time}course responses for protection ef"ciency of two concentrations of BTH (0.015 and 0.075 mg a.i./ml) treatment on 8-day-old cauli#ower seedlings challenged with fresh produced conidia 1}8 day after treatment. Bar show standard deviations of disease indexes.

3.3. Comparison of two plant treatment methods on resistance induction Results we obtained (Table 2) showed that drop or spray treatment gave comparable results when the higher a.i. concentration (0.075) was tested, and the treated plants were challenged with both types of conidia. In fact, the respective e$cacies were, respectively, 82 and 94 for the spray/fresh-frozen conidia combinations, and 88 and

88% for the drop/fresh-frozen conidia combinations. With the lower concentration, plants challenged with fresh conidia exhibited variable levels of resistance. In fact, 31 and 59% e$cacies were shown with, respectively, the spray and the drop inoculation method. With frozen conidia, the di!erences were less (79 vs. 86%). At higher a.i. concentration no di!erences were seen between methods. To save time, we adopted the spray method and the higher BTH concentration in our routine tests.

402

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405

Fig. 4. Time}course responses for protection ef"ciency of two concentrations of BTH (0.015 and 0.075 mg a.i./ml) treatment on 8 day-old cauli#ower seedlings challenged with frozen conidia 1}8 days after treatment. Bar show standard deviations of disease indexes.

Table 3 Systemic induction of resistance in 8-day-old cauli#ower seedlings treated with BTH challenged 4 days later with inocula obtained from either frozen or fresh produced conidia. Only one or two (control) cotyledons of each seedling were treated with the elicitor and ratings were made separately on the treated and non treated cotyledons Means disease indexes on cotyledons Concentrations (mg a.i./ml)

Control (water)

Both cotyledons treated (control)

Treated cotyledon


Nontreated cotyledon


Fresh conidia

0.015

9.0

3.7

4.3

6.9

Frozen conidia

0.015 0.075

9.0 9.0

1.0 1.1

0.7 0.7

6.4 1.6

Disease rating was done using the six-points scale (0-1-3-5-7-9) described by Williams (1985) in which 0 corresponds to no visible symptoms and 9 to intense sporulation on both sides of the cotyledons which most of the time die. Data in this table are means of three replicates and 60 seedlings were rated in each replicate. Mean disease indexes then calculated and seedlings sorted in categories according to the mean DI: Very resistant (DI)1), resistant (1(DI)3), moderately resistant (3.1)DI(4), moderately susceptible (4.1)DI(5), susceptible (5.1)DI(6) and very susceptible (DI*6.1).
In column No. 5 are summarized disease indexes obtained on the treated cotyledons challenged with the downy mildew isolate FP 06. In the last column are summarized mean disease indexes obtained on the nontreated cotyledons (control) of the same seedlings.

3.4. Stability of the elicitor solution

3.5. Test of the systemic induction by BTH

To test whether the elicitor solution was stable with time, a 30-day-old solution (0.075 mg a.i./ml) kept at #43C was compared with a freshly prepared solution for its ability to induce resistance. Results obtained (data not shown) showed that the induced resistances did not di!er and reached an e$cacy higher than 80%, whatever the type of conidia (fresh or frozen) was used. It can therefore be concluded that BTH solutions kept at #43C are highly stable.

Results obtained (Tables 3 and 4) showed that resistance was induced in the untreated plant organs and was dosedependent. Again, the induced resistance with the 0.015 mg a.i./ml concentration was less e$cient when the plant material was challenged with inocula prepared with fresh produced inocula (Table 3). Similar results were obtained on 30-day-old plants (Fig. 1). Based on results summarized in Tables 3 and 4, it can be assumed that BTH induces systemic resistance in both cauli#ower seedlings and adult plants.

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405 Table 4 Systemic induction of resistance in 30-day-old cauli#ower plants treated with a 0.075 mg a.i./ml solution or water and challenged 4 days later with inocula derived from frozen conidia. One fully developed and randomly selected leaf per plant was treated with elicitor and ratings made separately on non treated leaves Disease resistance categories BTH treatment

Water treatment (control)

R

MR

MS

S R MR

MS

S

Nb of plants per resistance category

14

26

16

1 0 11

16

14

Percentage/total

24

46

28

2 0 27

39

34

The elicitor-treated leaf was always resistant and the other leaves were either R (resistant), MR (moderately resistant), MS (moderately susceptible) or S (susceptible). Whole control plants were rated and mean disease categories were assigned.

3.6. Ewect of BTH on plant growth In all these experiments, plant growth reduction was noticed and was shown to be dose dependent (Table 1). On seedlings, the growth reduction concerned plant height and on young plants (30-day-old), only the width and length of the leaves were reduced. On seedlings, the plant height reduction varied from 5.9% for the lowest concentration (0.0015 a.i./ml) to 38.3% for the highest one (0.25 mg a.i./ml). At concentrations of 0.045 and 0.05 mg/ml which we currently use in the laboratory, maximum protection against downy mildew was obtained. The corresponding seedlings-height reduction was approximately 22% (Table 1). On 30-day-old plants, the corresponding leaf growth reductions were of 14.1 for the width and 12.3% for the length. 3.7. Ewect of BTH on the pathogen Because P. parasitica is an obligate pathogen, the direct e!ects of the elicitor on the pathogen were assessed by observing conidia morphology and in the presence of the elicitor. Results obtained (data not shown) showed no di!erences in conidia morphology and germination between conidia suspended in either water or the elicitor solution.

4. Discussion Our study has shown that fresh-prepared or 30-dayold BTH solutions (kept at 43C) were able to induce downy mildew (caused by P. parasitica) resistance in both cauli#ower seedlings and 30-day-old plants. This was

403

true with both application methods of BTH (droplets or spray); the spray method is therefore prefered for practical reasons. In another study (Godard et al., 1998), the e$cacy of the induced resistance (IR) in cauli#ower seedlings was tested against 14 downy mildew isolates from di!erent origins (host, country) and exhibiting di!erent virulence patterns. Results obtained showed that none of the tested isolates was able to overcome the IR. It is therefore likely that the IR is not isolate-speci"c. This study also showed that the induction of resistance by BTH in cauli#ower to P. parasitica was dose-dependent, the most reliable and e$cient induction being obtained with a.i. concentrations of 0.45}0.5 mg/ml. In this research, it was also clearly shown that when plants were treated with a.i. concentration lower than 0.45 mg/ml and challenged with fresh conidia, the IR was less e$cient and reliable than with frozen conidia. With concentrations *0.45 mg a.i./ml, no di!erences in e$cacies of the IR were observed anymore. To date, no satisfactory explanation is available but some hypothesis drawn: (i) it is likely that cell wall fragments released from conidia or plant tissues during the freezing/defreezing process may also induce resistance in the plant tested and amplify the resistance induced by BTH. (ii) freezing the conidia leads to reduced pathogenicity. This is unlikely since both types of conidia were fully pathogenic on accession Billabong in our tests. It is also possible that this accession is too susceptible so that di!erences between both types of conidia for pathogenicity cannot be shown. Testing other moderate susceptible accessions will help conclude this question. Time course resistance induction studies clearly showed that maximum induction is reached two days after treatment, with either low concentrations (0.015 mg a.i./ml) or high concentrations (*0.045 mg a.i./ml). A later challenge with the pathogen do not show greater gains of induced resistance. No curative e!ect was shown in our test. These results are comparable to those of GoK rlach et al. (1996) in the wheat-powdery mildew pathosystem. In fact, the authors (GoK rlach et al., 1996) did not observe any resistance when plants were inoculated 1}2 days before their treatment with BTH. The protection obtained with BTH seems to be durable, since when the challenge occurred 15}30 days after treatment, the plants were still resistant. It was not possible to test longer periods between treatment and inoculation with our equipment. GoK rlach et al. (1996) reported that a single application of 30 g of BTH/ha was enough to protect wheat "elds from powdery mildew for the entire season. Even if the same duration of protection is not applicable to cauli#ower culture in the "eld, the 30-day protection we recorded would be of great value especially in short-cycle crucifer cultures (e.g. Romanesco and autumn cauli#ower) since 2}3 applications would be enough to give season-long protection.

404

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405

Treatments of either one cotyledon per seedling or one leaf per plant and challenging the whole plant with downy mildew isolates demonstrated systemic protection. This protection was not maximal probably due to the quantity of elicitor applied to each plant organ. We only applied 40 ll on one cotyledon per seedling or 100 ll on one leaf of each young plant and this quantity is probably not su$cient to induce maximal protection of the whole plant. Further tests involving more BTH quantities applied on either one cotyledon (seedling test) or/and more on more leaves (young plant test) are needed. To address whether BTH has a direct action on P. parasitica, we checked if conidia morphology or their germination was a!ected, this because the fungus is an obligate pathogen. Our results showed that the tested solution had no visible direct e!ect on the conidia. Our studies clearly showed that BTH application reduced growth of plants of the cauli#ower accession Billabong. The reduction was less important on the 30day-old plants (leaf 's width and length reduction of, respectively, 14.1 and 12.3%) than on the seedlings (height reduction of 22%). Test of other cauli#ower accessions showed that these growth reductions were genotype-dependent. In fact, seedlings and young plants of some cauli#ower accessions showed no growth reduction after BTH treatment. The growth reduction we noticed after BTH treatment were also noticed when salicylic acid (SA)- or jasmonatederived compounds were tested in our laboratory (unpublished data). Because the chemical structure of BTH has similarities with SA, it can be hypothesized that SA and BTH act in a comparable manner on growth of cauli#ower plants. BTH induces SAR in cauli#ower to downy mildew as already shown in other pathosystems (Friedrich et al., 1996; GoK rlach et al., 1996; Lawton et al., 1996). Our results strengthen those of Jensen et al. (1998) who showed that the cabbage cultivar Scanner could be protected from downy mildew by means of seed treatment with BTH. It would be interesting to test whether combination of seed and foliar treatment is more e$cient in the control of the disaese in the "eld. Crop protection by BTH has been described to be associated with activation of defense genes [pathogenesis-related (PR) proteins (GoK rlach et al., 1996; Lawton et al., 1996; Friedrich et al., 1996)] and phenolic compounds as well as b-glucoside residues (Benhamou and BeH langer, 1998). Crucifer crops contain glucosinolates which a!ect crop quality and are thought to be involved in disease resistance. Furthermore, the glucosinolatebreakdown products, the isothiocyantes, are suspected to be the precursors of phytoalexins (Monde et al., 1994). Although to date no clear evidence has been shown between crucifer crop resistance to diseases and their

glucosinolate or phytoalexin contents, it may be interesting to investigate the action of BTH on accumulation of these products. If con"rmed in "eld tests, results we obtained may be useful for farmers in controling diseases by either using the compound alone or integrating it in a new IPM strategy combining resistance genes, fungicides and appropriate agronomic practices.

Acknowledgements We thank Novartis, France and Switzerland, for providing samples of BTH. Special thanks to Ms. Tanne M.-N. (Novartis, France) and Dr. Ruess (Novartis, Switzerland).

References Benhamou, N., BeH langer, R., 1998. Induction of systemic acquired resistance to Pythium damping-o! in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response. The Plant Journal 14, 13}21. Brophy, T.F., Laing, M.D., 1992. Screening of fungicides for the control of downy mildew on container-grown cabbage seeedlings. Crop Protection 11, 160}164. Crute, I.R., Norwood, J.M., Gordon, D.L., 1985. Resistance to phenylamide fungicides lettuce and Brassica downy mildew. Proc. Mixture Centenary Meeting, Bordeaux. British Crop Protection Council Monograph No 31, pp. 311}314. Crute, I.R., Gordon, P.I., Moss, N.A., 1994. Variation for responses to phenylamides in UK populations of Bremia lactucae (lettuce downy mildew) and Peronospora parasitica (Brassica downy mildew). In: Heaney, S., Slawson, D., Hollomon, D.W., Smith, M., Russel, P.E., Parry, D.W., (Eds.), BCPC Monograph No. 60: Fungicide resistance, BCPC, 1994, pp. 155}162. Dias, J.S., Ferreira, M.E., Williams, P.H., 1993. Screening of Portuguese cale Landraces (Brassica oleracea L.) with Peronospora parasitica and Plasmodiophora brassicae. Euphytica 67, 135}141. Friedrich, L., Lawton, K., Dincher, S., Winter, A., Staub, T., Uknes, S., Kessmann, H., Ryals, J., 1996. Benzothiadazole induces systemic acquired resistance in tobacco. Plant Journal 10, 6170. Jensen, B.D., Latunde-Dada, A.O., Hudson, D., Lucas, J.A., 1998. Protection of Brassica seedlings against downy mildew and damping-o! by seed treatment with CGA 245704, an activator of systemic acquired resistance. Pesticide Science 52, 63}69. Hijwegen, T., Verhaar, M.A., 1993. Induced resistance to Peronospora parasitica in red cabbage. Netherland Journal Plant Pathology 99 (Suppl. 3), 103}107. Godard, J.F., Ziadi, S., Monot, C., Le Corre, D., SilueH , D., 1998. CGA 245704, a benzothiadiazole-derived compound induces resistance in cauli#ower (Brassica oleracea var botrytis) to downy mildew of crucifers caused by Peronospora parasitica. In: Modern fungicides and antifungal compounds. 12th International Reinhardsbrunn Symposium, Friedrichroda, Germanay, May 24}29, 1998, in press. GoK rlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., Ryals, J., 1996. Benzothiadazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8, 629}643.

J.-F. Godard et al. / Crop Protection 18 (1999) 397}405 Lawton, K., Friedrich, L., Hunt, L., Weymann, K., Staub, T., Kessmann, H., Ryals, J., 1996. Benzothiadazole induces disease resistance in Arabidopsis by activation of the systemeic acquired resistance signal transduction pathway. Plant Journal 10, 71}82. Lucas, J.A., Crute, I.R., Sheri!, C., Gordon, P.L., 1988. The identi"cation of a gene for race speci"c resistance to Peronospora parasitica (downy miuldew) in Brassica napus spp. oleifera (oilseed rape). Plant Pathology 37, 538}545. Monde, K., Takasugi, M., Ohnishi, T., 1994. Biosynthesis of cruciferous phytoalexins. Journal of American Chemical Society 116, 6650}6657. Moss, N.A., Lucas, J.A., Crute, I.R., 1991a. Evidence for di!erential response to isolates of Peronospora parasitica (downy mildew) in Brassica rapa. Annals of Applied Biology 118 (supplement), 96}97. Moss, N.A., Lucas, J.A., Crute, I.R., Gordon, P.L., 1991b. Sources of seedling resistance to Peronospora parasitica (downy mildew) in Brassica oleracea. Annals of Applied Biology 118 (supplement), 94}95. Nashaat, N.I., Rawlinson, C.J., 1994. The response of oilseed rape (Brassica napus spp. oleifera) accessions with di!erent glucosinolate and erucic acid contents to four isolates of Peronospora parasitica (downy mildew) and the identi"cation of new source of resistance. Plant Pathology 43, 278}283.

405

Nashaat, N.I., Awasthi, R.P., 1995. Evidence for di!erential resistance to Peronospora parasitica (downy mildew) in accessions of Brassica juncea (mustard) at the cotyledon stage. Journal of Phytopathology 143, 157}159. Nashaat, N.I., Heran, A., Mitchell, S.E., Awasthi, R.P., 1997. New genes for resistance to downy mildew (Peronospora parasitica) in oilseed rape (Brassica napus spp. oleifera). Plant Pathology 46, 964}968. Ohuguchi, T., Nagamatsu, S., Asada, Y., 1990. Races of Japanese radish downy mildew fungus. Memoirs of the College of Agriculture, Ehime University 34, 349}363. Ryals, J.A., Neuenschwander, U.H., Willits, M.G., Molina, A., Steiner, H.-Y., Hunt, M.D., 1996. Systemic acquired resistance. The Plant Cell 8, 1809}1819. SilueH , D., Laviec, C., Tirilly, Y., 1995. New sources of resistance in Brassica oleracea var botrytis to downy mildew caused by Peronospora parasitica. Journal of Phytopathology 143, 659}661. SilueH , D., Nashaat, N.I., Tirilly, Y., 1996. Di!erential responses of Brassica oleracea and B. rapa accessions to seven isolates of Peronospora parasitica at the cotyledon stage. Plant Disease 80, 142}144. Schneider, M., Schweizer, P., Meuwly, P., MeH traux, J.P., 1996. Systemic acquired resistance in plants. International Review of Cytology 168, 303}340. Williams, P.H., 1985. Crucifer Genetics Resource Book. Dept. of Plant pathology, Univ. of Wisconsin, Madison, USA.