VITAL STAINING OF PLANT CELL SUSPENSION CULTURES: EVALUATION OF THE PHYTOTOXIC ACTIVITY OF FUNGAL METABOLITES
M. S. C. Pedras, C. J. Biesenthal, and Y. Jiang
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon SK S7N 5C9, Canada
ABSTRACT
It is well-documented that penetration and colonization of plant tissues by pathogenic fungi can involve fungal production and release of host-selective phytotoxic compounds. We have been evaluating the role of host-selective phytotoxins as part of a research program aimed at understanding mechanisms of plant disease resistance. Host-selective phytotoxins are, by definition, fungal/bacterial metabolites toxic only to plants that host the pathogen. Nonetheless, it is difficult to determine whether a particular metabolite displays host-selective phytotoxic activity specially because: (1) most of the simple bioassays involve somewhat subjective evaluation of damaged plant tissue, and (2) the pathogen delivers the toxin to the plant cell in a complex process difficult to mimic.
To determine phytotoxicity of fungal metabolites we have developed a reproducible bioassay which involves vital staining of hypocotyl suspension cell cultures incubated with toxic metabolites. The effect of each metabolite is evaluated by determining the % viability of cell cultures.
KEYWORD: Alternaria brassicae; destruxin B; Brassica napus; B. juncea; Sinapis alba
It is well-documented that penetration and colonization of plant tissues by phytopathogenic fungi can involve fungal production and release of host-selective toxins (Graniti et al., 1991). Host-selective phytotoxins are, by definition, fungal or bacterial secondary metabolites toxic only to plants that host the pathogen. Nonetheless, it is difficult to determine whether a particular metabolite displays host-selective phytotoxicity, specially because the pathogen delivers the toxin to the plant cell in a complex process which depends on the host-pathogen system and is always difficult to mimic. Common and simple bioassays such as the application of a toxin solution to cotyledons, leaves, or stems of whole plants can only be used in qualitative evaluations. Quantitative bioassays need to be developed for each toxin-plant system before reliable conclusions regarding the selective phytotoxicity of a given toxin can be made.
We have been evaluating the role of host-selective phytotoxins as part of a research program aimed at understanding the chemical basis of plant disease resistance (Pedras, 1998). Because destruxin B is a phytotoxin produced (Ayer and Peña-Rodriguez, 1987; Bains and Tewari, 1987; Buchwaldt and Jensen, 1991; Pedras and Smith, 1997) by one of the most destructive (Saharan, 1993) fungal pathogens (Alternaria brassicae (Berk.) Sacc.) of the economically important oilseeds rapeseed (Brassica napus and B. rapa) and mustard (B. juncea), it is of great interest to establish its effect and fate on resistant and susceptible plant tissues. Thus, we have developed a quantitative and reproducible bioassay for the determination of destruxin B phytotoxicity which involves vital staining of plant cell suspension cultures of species resistant and susceptible to A. brassicae.
Destruxin B was synthesized and purified as previously reported (Ward et al., 1997). The spectroscopic and physical data obtained for synthetic destruxin B was identical in all respects to an authentic sample isolated from cultures of Alternaria brassica (Pedras and Smith 1997). Stock solutions of destruxin B (1 ´ 10-2 M) were prepared in CH3CN and stored at 4 °C.
Cell suspension assay: three concentrations (5 ´ 10-4 M, 5 ´ 10-5 M, and 1 ´ 10-5 M) were prepared by a serial dilution using 8p culture medium (Vamling and Glimelius, 1990) containing hormones at a quarter concentration, except for B. juncea where the original hormone concentration was used.
Cell cultures of S. alba, B. juncea, and B. napus cv. Westar were obtained from protoplasts prepared by a modification of previously reported work (Vamling and Glimelius, 1990).
In brief, S. alba and B. juncea seeds were surface sterilized, were rinsed, and were germinated on MS in the dark for 5 days at 20 ± 0.5 °C. The hypocotyls were sliced and incubated in plasmolysis medium for 30 minutes. After removal of plasmolysis medium, an enzyme solution of Cellulysin and Macerase in K3 medium containing 0.4 M glucose was added to hypocotyls of S. alba and B. juncea, followed by incubation at 25 ± 0.5 °C, in the dark for 12 hours and 8 hours, respectively; B. napus was incubated in 0.5% Cellulysin and 0.05% Macerase for 16 - 18 hours. The protoplasts were filtered through a nylon mesh and CPW 16 solution was added to the filtrate. A layer of the W5 medium was carefully added to the top of the CPW 16 / enzyme - protoplast mixture, keeping the layers distinct. This mixture was then centrifuged at 100 g for 20 minutes. 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 minutes. The pellet was rinsed again with W5 medium and centrifuged at 70 g for 10 minutes. The pelleted protoplasts were then diluted with 8p medium containing hormones and incubated in complete darkness at 25 ± 0.5 °C. S. alba and B. napus were initially cultured in medium containing the following hormones, 1.0 mg / L of 2,4-D, 0.1 mg / L of NAA and 0.1 mg / L of BAP then reduced to quarter strength after 7 days; B. juncea was cultured in medium containing 1.0 mg / L NAA and 0.4 mg / L BAP continuously (Kao and Séguin-Swartz, 1987) with no hormone reduction. The cell cultures were used for phytotoxicity assays after two weeks of incubation.
This bioassay was adapted from previously published studies (Widholm, 1972; Li et al., 1997). Experiments were carried out in triplicate in 4-well Nunclon plates, with each well containing 500 µL of destruxin B solution at the various concentrations and 100 µL of 2-week-old cell cultures. The plates were incubated in complete darkness at 25 ± 0.5 °C for 10 days. The cell viability was determined after adding 10 µL of 0.1% (w/v) phenosafranin to each well and counting cells directly in the 4-well plates using a Hund Wilovert S inverted microscope (100 x). Dead cells stained red or pink and were clearly differentiated from nonstained live cells. The percent viability was determine by counting random fields of view for each replicate (at least 300 cells).
Data was analyzed using Microsoft Excel and Minitab. An ANOVA test and the Tukey HSD test were used to compare the cell viability of different plant species after incubation with destruxin B. Results are presented as percent viability with the standard error.
The effect of destruxin B on the cell viability of S. alba, B. juncea, and B. napus are shown in tables 1-3. These results indicate that, at any of the three concentrations tested, cells of S. alba and B. juncea are less affected by destruxin B than cell cultures of B. napus, after four days of incubation. After six days of incubation with destruxin B at 1 ´ 10-5 M or 5 ´ 10-5 M, the viability of cells of S. alba and B. juncea is significantly higher than the viability of cells of B. napus. Interestingly, however, at the highest concentration destruxin B is more toxic to S. alba and B. napus than to B. juncea.
Table 1. Effect of destruxin B on percent viability of 2-week-old cell cultures of Sinapis alba cv. Ochre.
|
Days in culturea Concentration 2 4 6 |
|||
|
Control |
85 ± 2 |
75 ± 1 |
68 ± 2 |
|
1 ´ 10-5 M |
81 ± 1 |
72 ± 2 |
67 ± 2 |
|
5 ´ 10-5 M |
77 ± 2 |
70 ± 1 |
63 ± 2 |
|
5 ´ 10-4 M |
71 ± 2 |
62 ± 2 |
36 ± 5 |
a Results are the means of at least four independent experiments; mean ± standard error.
b Time in culture following addition of destruxin B.
Table 2. Effect of destruxin B on percent viabilitya of 2-week-old cell cultures of Brassica juncea cv. Cutlass.
|
Days in cultureb Concentration 2 4 6 |
|
|||
|
Control |
71 ± 2 |
68 ± 3 |
67 ± 3 |
|
|
1 ´ 10-5 M |
72 ± 2 |
66 ± 2 |
65 ± 4 |
|
|
5 ´ 10-5 M |
69 ± 2 |
66 ± 3 |
61 ± 4 |
|
|
5 ´ 10-4 M |
68 ± 3 |
61 ± 5 |
53 ± 5 |
|
a Results are the means of at least four independent experiments; mean ± standard error.
b Time in culture following addition of destruxin B.
Table 3. Effect of destruxin B on percent viabilitya of 2-week-old cell cultures of Brassica napus cv. Westar.
|
Days in cultureb Concentration 2 4 6 |
|||
|
Control |
67 ± 1 |
67 ± 1 |
64 ± 1 |
|
1 ´ 10-5 M |
55 ± 3 |
53 ± 3 |
47 ± 10 |
|
5 ´ 10-5 M |
52 ± 4 |
47 ± 3 |
39 ± 10 |
|
5 ´ 10-4 M |
39 ± 3 |
33 ± 3 |
29 ± 6 |
a Results are the means of at least four independent experiments; mean ± standard error.
b Time in culture following addition of destruxin B.
The determination of phytotoxicity of fungal metabolites can be assessed utilizing cell suspension cultures and a simple staining method. The major disadvantage of this bioassay is the time required to routinely prepare 2-week-old cell suspension cultures. The “fresh” cells are required because it is difficult to differentiate between viable and nonviable cells in older cell cultures.
We would like to thank Professor D. E. Ward and Dr. Y. Gai, Department of Chemistry, University of Saskatchewan for synthesizing destruxin B. We gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (strategic grant to M. S. C. P.) and the University of Saskatchewan.
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