homodestruxin B 2, and desmethyldestruxin B 5 Fig. a presumed toxin produced also by Materials and methods

destruxin B 1 and homodestruxin B 2 [7]. Sub- sequently, to determine the usefulness and poten- tial application of these metabolic studies, it was essential to establish the phytotoxicity of destrux- ins 1 – 4 Fig. 1. Although the phytotoxic activity of destruxin B and homodestruxin B was previ- ously evaluated, there is no quantitative evaluation of their toxicity to the disease resistant species Sinapis alba. Consequently, we have developed a quantitative bioassay utilizing the staining of plant cell suspen- sion cultures of S. alba to establish a structure- phytotoxic activity correlation among destruxin B 1, homodestruxin B 2, and desmethyldestruxin B 5 Fig. 1, a presumed toxin produced also by A. brassicae. Next, we have compared the phyto- toxicity of destruxin B 1 and hydroxydestruxin B 3 in cell suspension cultures of resistant and susceptible plant species. Finally, destruxin B 1, homodestruxin B 2, hydroxydestruxin B 3, and hydroxyhomodestruxin B 4 Fig. 1, were tested on resistant and susceptible plant species utilizing leaf assays of whole plants and leaf uptake of toxin solutions.

2. Materials and methods

2 . 1 . Preparation of solutions of destruxins for bioassays All chemicals were purchased from Sigma – Aldrich Canada Ltd., Oakville, Ont. All solvents were HPLC grade. Destruxin B, [8] hydroxyde- struxin B and hydroxyhomodestruxin B [7] were synthesized and purified as previously reported; the syntheses of homodestruxin B and desmethyldestruxin B will be reported elsewhere Ward, Gai, and Pedras, unpublished. The spec- troscopic data, including NMR and HRMS, and physical data obtained for the synthetic de- struxins were identical in all respects to the natu- rally occurring compounds isolated either from cultures of A. brassicae destruxin B [9], homode- struxin B and desmethyldestruxin B [4] or from leaves of S. alba incubated with destruxin B or homodestruxin B hydroxydestruxin B and hy- droxyhomodestruxin B [7]. The purity of each compound was determined by TLC, HPLC, and HRMS to be higher than 99. Stock solutions of destruxins 1 × 10 − 2 M in acetonitrile were utilized to prepare lower concentrations for bioassays. 2 . 2 . Plant material Plants were grown in a growth chamber, with 16 h light fluorescent and incandescent, 450 – 530 mmol s − 1 m − 2 8 h dark, at 24 9 2°C. Three different species of known resistance to A. brassicae were employed in the bioassays: S. alba, cv. Ochre resistant, B. napus, cv. Westar susceptible and B. juncea, cv. Cutlass suscepti- ble. 2 . 3 . Preparation of cell suspension cultures Cell cultures of three different species of known resistance to A. brassicae blackspot were ob- tained from protoplasts prepared by a modifica- tion of previously reported work [10], as described below. S. alba and B. juncea seeds were surface sterilized with 70 vv ethanol for 1 min, 80 vv commercial bleach for 20 min, and then were rinsed three times with sterile water. B. napus seeds were surface sterilized similarly but were kept in 80 vv commercial bleach for 30 min. The sterilized seeds were germinated on MS Sigma medium 2.2 gl MS medium, 20 gl sucrose, 7.5 gl agar in the dark for 5 days at 20 9 0.5°C. The hypocotyls were sliced into 0.5 – 1 mm pieces and incubated in plasmolysis medium for 30 min. After removal of plasmolysis medium, an enzyme solu- tion of 1 Cellulysin and 0.1 Macerase Cal- biochem-Behring in K3 medium [10] containing 0.4 M glucose was added to hypocotyls of S. alba and B. juncea, followed by incubation at 25 9 0.5°C, in the dark for 12 and 8 h, respectively; B. napus was incubated in 0.5 cellulysin and 0.05 macerase for 16 – 18 h. Fig. 1. Chemical structures of destruxins: 1 destruxin B, 2 homodestruxin B, 3 hydroxydestruxin B, 4 hydroxyhomode- struxin B, 5 desmethyldestruxin B. The enzyme treated hypocotyls, i.e. protoplasts, were filtered through a nylon mesh of 100 mm to eliminate large pieces of plant tissue. To the filtrate an equal volume of CPW 16 solution [10] was added and mixed with the enzyme-protoplast solution. A layer of the W5 medium [10] was carefully added to the top of the CPW 16enzyme- protoplast mixture, keeping the layers distinct. This mixture was then centrifuged in a swing-out rotor at 100 g for 20 min at 25 9 0.5°C. Viable protoplasts with intact cell membranes remained between the two layers and were removed with a Pasteur pipette. The protoplast suspension was diluted with W5 medium and was centrifuged at 70 g for 10 min 25 9 0.5°C. The pellet was rinsed again with W5 medium and centrifuged at 70 g for 10 min 25 9 0.5°C. The pelleted protoplasts were then diluted to a density of 2 × 10 4 ml with 8p medium casamino acid and coconut water were omitted from 8p medium, otherwise prepared as previously by Vamling and Glimelius [10] con- taining growth factors to a concentration of 2 – 5 × 10 4 ml and incubated in complete darkness at 25 9 0.5°C. S. alba and B. napus were initially cultured in medium containing the following growth factors, 1.0 mgl of 2,4-D, 0.1 mgl of NAA and 0.1 mgl of BAP then reduced to quar- ter strength after 7 days; B. juncea was cultured in medium containing 1.0 mgl NAA and 0.4 mgl BAP continuously [11] with no growth factor re- duction in four-well Nunclon plates with no shak- ing first cell division observed after 24 h. The cell cultures were used for phytotoxicity assays 100 m lwell after 2 weeks of incubation. 2 . 4 . Punctured leaf assay Three toxin concentrations 1 × 10 − 4 M, 5 × 10 − 5 M and 2 × 10 − 5 M in 2 vv aqueous acetonitrile were prepared by a serial dilution. Leaves were scratched on the left side with the tip of a glass pipette, and punctured on the right side with a needle; 10 ml droplets six droplets per leaf were applied on scratched and punctured sites. Control leaves were treated similarly employ- ing 2 aqueous acetonitrile instead of toxin solu- tion. Plants were incubated in a growth chamber 16 h light fluorescent and incandescent, 450 – 530 mmol s − 1 m − 2 8 h dark, at 24 9 2°C and the diameter of the lesions was measured after 7 days. 2 . 5 . Leaf uptake assay Two toxin concentrations 2 × 10 − 5 M and 1 × 10 − 5 M in 2 vv aqueous acetonitrile were prepared by a serial dilution. Leaves were cut at the base of their petiole and each leaf immediately placed in a 1.5-ml Eppendorf tube containing the phytotoxin solution 1 ml per tube per leaf. After the phytotoxin solution was taken up, an aqueous solution of BAP 1 × 10 − 5 M was added to each tube and leaves were incubated 16-h fluorescent light, 25 – 60 mmol s − 1 m − 2 8-h dark at 20 9 0.5°C for 7 days. Control leaves were treated similarly. 2 . 6 . Cell staining assay This bioassay was adapted from previously pub- lished reports [12,13]. Three toxin concentrations 5 × 10 − 4 M, 5 × 10 − 5 M, and 1 × 10 − 5 M were prepared by a serial dilution using 8p culture medium [10] containing growth factors at a quar- ter concentration, except for B. juncea where the original growth factors concentration was used. The final concentration of acetonitrile in the cell culture medium was 5. Experiments were carried out in triplicate in four-well Nunclon plates, with each well containing 500 ml of toxin solution at the various concentrations and 100 ml of 2-week-old cell cultures prepared as described above. The plates were incubated without shaking in complete darkness at 25 9 0.5°C for 10 days. The cell viabil- ity was determined after adding 10 ml of 0.1 wv phenosafranin Sigma – Aldrich Canada to each well and slightly shaking plates, and counting cells directly in the four-well plates using a Hund Wilovert S inverted microscope 100 × . Dead cells stained red or pink and could be clearly distinguished from non-stained live cells. The per- cent viability was determined, by counting random fields of view for each replicate at least 300 cells. 2 . 7 . Data analysis Data was analyzed using Microsoft Excel and Minitab. An ANOVA test and the Tukey HSD test were used to compare the differences between the phytotoxins at 95 confidence interval. Re- sults are presented as percent viability with the standard error. Table 1 Effect of destruxin B on percent viability a of 2-week-old cell cultures of S. alba cv. Ochre resistant Days after treatment b Concentration 4 2 6 8 10 75 9 1 68 9 2 Control c 65 9 1 85 9 2 68 9 2 1×10 − 5 M 81 9 1 72 9 2 67 9 2 65 9 1 55 9 4 77 9 2 5×10 − 5 M 70 9 1 63 9 2 54 9 4 43 9 4 62 9 2 36 9 5 21 9 3 71 9 2 15 9 2 5×10 − 4 M a Results are the means of at least four independent experi- ments; mean 9 standard error. b Time in culture following addition of destruxin B. c Control cell cultures were incubated in medium containing 5 acetonitrile.

3. Results and discussion