Evaluation of Novel Chemical Methods for Verticillium Control – Annual Report March 2008
Annual Report to California Pepper Commission
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Evaluation of Novel Chemical Methods for Verticillium Control
Mike Coffey, Professor and Plant Pathologist,
Department of Plant Pathology and Microbiology,
University of California,
Riverside, CA 92521
E-Mail: coffey@ucr.edu
Location of Work: UC Riverside Campus
Budget Total: $15,000
Project Year: March 1, 2007 to February 28, 2008
Statement of the Problem and its Significance:
Verticillium wilt survives as dark-pigmented microsclerotia in the soil. As such it can survive indefinitely causing disease whenever susceptible peppers are planted. Many different crops such as tomato, strawberries, cotton, olives, etc and many common weeds can serve as alternate hosts. Thus the pathogen is persistent in soils and widely dispersed with many hosts. Verticillium Wilt is now widespread in pepper growing areas and no proven source of high resistance is commercially available. Fungicides, including the modern systemics, have proven ineffectual under field conditions. However, recent findings with two different cropping systems severely affected by Verticillium (1,2) indicate that control may be affected by the use of chemicals, often referred to as plant defense known to trigger natural resistance in plants. The one class triggers a general resistance response in plants and thus may have efficacy against a broader range of pathogenic fungi. The othergroup of chemicals, the phosphonates were primarily developed against Phytophthora but have long been known to have indirect effects in enhancing plant resistance mechanisms. Recently potassium phosphonate was found to be highly efficacious against Verticillium on olive trees in research done in Italy (2).
Objectives: The prime objectives in 2007 were to test two distinct classes of disease resistance activator with a selection of 5 commercially-grown bell pepper hybrids (, typical of those commonly used in California. Specifically, we tested Actigard and Phostrol, as both materials are registered in California.
Plans and Procedures: Fungal inoculum of an aggressive isolate of Verticillium was prepared by growing the pathogen in 100 ml liquid medium in 250-ml Erlenmeyer flasks at 24C under continuous shaking at 120 rpm for 10 days. After one week of growth, fungal propagules and mycelia will separated from the culture media via suction filtering through Whatman No.1 filter paper. The filtered biomass will be briefly homogenized in a Waring blender to disperse the spore clumps and spore concentration will be adjusted to 107 propagules/ml using a hemocytometer.
Commercially grown transplant seedlings at a 5-7 leaf stage will removed from their propagation trays and the lower section of the root balls trimmed to create the mechanical wounding necessary to facilitate entrance of the pathogen. Wounded transplants were root-dipped in the fungal inoculum for 15-20 minutes. Subsequently, they were carefully transplanted into clean UC soil mix amended with additional Verticillium inoculum in 4 inch square plastic pots.
Inoculated plants were kept at ~24 - 280C in the greenhouse under natural light Disease symptoms will be recorded every 7-10 days for up to 2 months after inoculation. Disease severity will be based on a disease rating scale .
All experiments were conducted on five different bell pepper hybrids. Nontreated control plants, both inoculated with Verticillium, and uninoculated (healthy, disease-free) plants will be included.
The experiments will be a replicated using an appropriate experimental design. Data will be analyzed by ANOVA (Analysis of Variance) using SUPERANOVA software package AND means separated by the Tukey's multiple range test.
Objectives and Procedures: The specific aims of the project were to evaluate the different concentrations of Phostrol, Actigard and combination of both to control Verticillium wilt of peppers. Five varieties of peppers were used to evaluate the efficacy of different concentrations of fungicides (TABLE 1). Tests were conducted in the greenhouse using five different varieties of seedlings.
TABLE 1. Treatments used in the experiments
| 1 (Phos 20) | Phostrol 20 ml in 1000ml |
| 2 (Phos 15) | Phostrol 15 ml in 1000ml |
| 3 (Phos 10) | Phostrol 10 ml in 1000ml |
| 4 (Phos 20 + Acti 0.1g) | Phostrol 20 ml + Actigard 0.1g (a.i.) in 1000ml |
| 5 (Phos 15 + Acti 0.1g) | Phostrol 15 ml + Actigard 0.1g (a.i.) in 1000ml |
| 6 (Phos 10 + Acti 0.1g) | Phostrol 10 ml + Actigard 0.1g (a.i.) in 1000ml |
| 7 (Phos 20 + Acti 0.15g) | Phostrol 20 ml + Actigard 0.15g (a.i.) in 1000ml |
| 8 (Phos 15 + Acti 0.15g) | Phostrol 15 ml + Actigard 0.15g (a.i.) in 1000ml |
| 9 (Phos 10 + Acti 0.15g) | Phostrol10 ml + Actigard 0.15g (a.i.) in 1000ml |
| 10 (Acti 0.1g) | Actigard 0.1g (a.i.) |
| 11 (Acti 0.15g) | Actigard 0.15g (a.i.) |
| 12 (Water infe.) | Water infected control |
| 13 (Water noninfec.) | Water noninfected control |
TABLE 2. Different varieties of peppers used in the experiments
| 1. Zarco (Z) | Yellow variety |
| 2. Valiant (V) | Green variety |
| 3. Maccabi (M) | Red variety |
| 4.Samson (S) | Red variety |
| 5. Super Beitar (B) | Red variety |
Five varieties of peppers were treated with thirteen treatments. Eight plants of each pepper variety were dipped in 100 ml of each treatment in the food storage container on September 13, 2007. On September 17, 2007 the plants were treated with the Verticillium inoculum.
Some plants showed phytotoxicity symptoms after four days of treatment with different fungicides. The plants were
B4, M4, Z4
M5, Z5
M7, Z7, B7, S7, V7
Z8, B8, V8
Z9, M9
Observations: The plants were observed for Verticillium wilt after three weeks. The plants were scored on the scale of 1 to 6.
- Healthy plant
- Small chlorosis or slight yellowing of leaves
- Chlorosis , stunted and leaf tips turning necrotic
- Wilting, stunting and leaf necrosis
- All leaves necrotic and wilted
- Dead
Isolation of Verticillium
Isolations from infected pepper plants were also done. 5-7cm long stem was taken from the infected plants. The stems were cut into 1cm longitudinal sections; surface sterilized with 70% ethanol for 10 seconds, were immersed in sterile distilled water and plated on water agar medium. After a week incubation period at room temperature, conidiophores slender branched confirmed as Verticillium by microscopic examination.
Results
Data analysis was done by SAS software using ANOVA.
Experiment 1.
1. Zarco
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 3.75 | abc |
| 2 (Phos 15) | 4.25 | abc |
| 3 (Phos 10) | 4.00 | abc |
| 4 (Phos 20 + Acti 0.1g) | 5.00 | ab |
| 5 (Phos 15 + Acti 0.1g) | 5.00 | ab |
| 6 (Phos 10 + Acti 0.1g) | 2.75 | c |
| 7 (Phos 20 + Acti 0.15g) | 5.50 | a |
| 8 (Phos 15 + Acti 0.15g) | 4.00 | abc |
| 9 (Phos 10 + Acti 0.15g) | 4.75 | ab |
| 10 (Acti 0.1g) | 4.00 | abc |
| 11 (Acti 0.15g) | 3.75 | abc |
| 12 (Water infe.) | 3.50 | bc |
| 13 (Water noninfec.) | 1.00 | d |
* Means with the same letter are not significantly different.
2. Valiant
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 3.75 | abcd |
| 2 (Phos 15) | 4.25 | abc |
| 3 (Phos 10) | 3.75 | abcd |
| 4 (Phos 20 + Acti 0.1g) | 3.50 | abcd |
| 5 (Phos 15 + Acti 0.1g) | 3.75 | abcd |
| 6 (Phos 10 + Acti 0.1g) | 2.75 | bcde |
| 7 (Phos 20 + Acti 0.15g) | 5.00 | a |
| 8 (Phos 15 + Acti 0.15g) | 4.50 | ab |
| 9 (Phos 10 + Acti 0.15g) | 2.00 | de |
| 10 (Acti 0.1g) | 2.50 | cde |
| 11 (Acti 0.15g) | 2.75 | bcde |
| 12 (Water infe.) | 3.75 | abcd |
| 13 (Water noninfec.) | 1.00 | e |
* Means with the same letter are not significantly different.
3. Macabi
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 4.00 | a |
| 2 (Phos 15) | 3.50 | abc |
| 3 (Phos 10) | 3.75 | ab |
| 4 (Phos 20 + Acti 0.1g) | 2.25 | cd |
| 5 (Phos 15 + Acti 0.1g) | 2.50 | bc |
| 6 (Phos 10 + Acti 0.1g) | 3.25 | abc |
| 7 (Phos 20 + Acti 0.15g) | 4.25 | a |
| 8 (Phos 15 + Acti 0.15g) | 3.75 | ab |
| 9 (Phos 10 + Acti 0.15g) | 3.25 | abc |
| 10 (Acti 0.1g) | 3.25 | abc |
| 11 (Acti 0.15g) | 4.50 | a |
| 12 (Water infe.) | 3.50 | abc |
| 13 (Water noninfec.) | 1.00 | d |
* Means with the same letter are not significantly different.
4. Samson
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 4.00 | ab |
| 2 (Phos 15) | 4.00 | ab |
| 3 (Phos 10) | 3.75 | ab |
| 4 (Phos 20 + Acti 0.1g) | 3.25 | ab |
| 5 (Phos 15 + Acti 0.1g) | 2.75 | b |
| 6 (Phos 10 + Acti 0.1g) | 2.75 | b |
| 7 (Phos 20 + Acti 0.15g) | 4.00 | ab |
| 8 (Phos 15 + Acti 0.15g) | 3.00 | ab |
| 9 (Phos 10 + Acti 0.15g) | 4.00 | ab |
| 10 (Acti 0.1g) | 2.50 | bc |
| 11 (Acti 0.15g) | 4.75 | a |
| 12 (Water infe.) | 3.75 | ab |
| 13 (Water noninfec.) | 1.00 | c |
* Means with the same letter are not significantly different.
5. Super Beitar
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 3.75 | ab |
| 2 (Phos 15) | 4.00 | a |
| 3 (Phos 10) | 4.00 | a |
| 4 (Phos 20 + Acti 0.1g) | 2.50 | cd |
| 5 (Phos 15 + Acti 0.1g) | 2.75 | bcd |
| 6 (Phos 10 + Acti 0.1g) | 2.25 | d |
| 7 (Phos 20 + Acti 0.15g) | 3.50 | abc |
| 8 (Phos 15 + Acti 0.15g) | 2.75 | bcd |
| 9 (Phos 10 + Acti 0.15g) | 3.75 | ab |
| 10 (Acti 0.1g) | 3.75 | ab |
| 11 (Acti 0.15g) | 3.75 | ab |
| 12 (Water infe.) | 3.50 | abc |
| 13 (Water noninfec.) | 1.00 | e |
* Means with the same letter are not significantly different.
Experiment No. 2
1. Zarco
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 4.50 | abcd |
| 2 (Phos 15) | 4.75 | abcd |
| 3 (Phos 10) | 3.50 | cd |
| 4 (Phos 20 + Acti 0.1g) | 4.75 | abcd |
| 5 (Phos 15 + Acti 0.1g) | 4.75 | abcd |
| 6 (Phos 10 + Acti 0.1g) | 3.00 | d |
| 7 (Phos 20 + Acti 0.15g) | 5.75 | ab |
| 8 (Phos 15 + Acti 0.15g) | 6.00 | a |
| 9 (Phos 10 + Acti 0.15g) | 3.50 | cd |
| 10 (Acti 0.1g) | 3.00 | d |
| 11 (Acti 0.15g) | 5.00 | abc |
| 12 (Water infe.) | 4.00 | bcd |
| 13 (Water noninfec.) | 1.00 | e |
* Means with the same letter are not significantly different.
2. Valiant
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 3.50 | ab |
| 2 (Phos 15) | 3.75 | ab |
| 3 (Phos 10) | 3.75 | ab |
| 4 (Phos 20 + Acti 0.1g) | 3.25 | ab |
| 5 (Phos 15 + Acti 0.1g) | 1.25 | c |
| 6 (Phos 10 + Acti 0.1g) | 2.00 | bc |
| 7 (Phos 20 + Acti 0.15g) | 4.75 | a |
| 8 (Phos 15 + Acti 0.15g) | 2.25 | bc |
| 9 (Phos 10 + Acti 0.15g) | 3.25 | ab |
| 10 (Acti 0.1g) | 3.25 | ab |
| 11 (Acti 0.15g) | 3.25 | ab |
| 12 (Water infe.) | 3.50 | ab |
| 13 (Water noninfec.) | 1.00 | c |
* Means with the same letter are not significantly different.
3. Macabi
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 4.00 | a |
| 2 (Phos 15) | 4.00 | a |
| 3 (Phos 10) | 4.25 | a |
| 4 (Phos 20 + Acti 0.1g) | 2.75 | a |
| 5 (Phos 15 + Acti 0.1g) | 2.75 | a |
| 6 (Phos 10 + Acti 0.1g) | 2.75 | a |
| 7 (Phos 20 + Acti 0.15g) | 2.75 | a |
| 8 (Phos 15 + Acti 0.15g) | 3.25 | a |
| 9 (Phos 10 + Acti 0.15g) | 3.25 | a |
| 10 (Acti 0.1g) | 3.25 | a |
| 11 (Acti 0.15g) | 3.00 | a |
| 12 (Water infe.) | 4.00 | a |
| 13 (Water noninfec.) | 1.00 | a |
* Means with the same letter are not significantly different.
4. Samson
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 4.50 | ab |
| 2 (Phos 15) | 4.00 | bc |
| 3 (Phos 10) | 4.00 | bc |
| 4 (Phos 20 + Acti 0.1g) | 5.50 | a |
| 5 (Phos 15 + Acti 0.1g) | 2.75 | d |
| 6 (Phos 10 + Acti 0.1g) | 2.50 | d |
| 7 (Phos 20 + Acti 0.15g) | 3.00 | cd |
| 8 (Phos 15 + Acti 0.15g) | 2.75 | d |
| 9 (Phos 10 + Acti 0.15g) | 2.50 | d |
| 10 (Acti 0.1g) | 2.25 | d |
| 11 (Acti 0.15g) | 5.00 | ab |
| 12 (Water infe.) | 4.00 | bc |
| 13 (Water noninfec.) | 1.00 | e |
* Means with the same letter are not significantly different.
5. Super Beitar
Tukey's Studentized Range (HSD) Test for scoring
| Treatment | Mean | Tukey grouping* |
| 1 (Phos 20) | 3.75 | abc |
| 2 (Phos 15) | 4.00 | ab |
| 3 (Phos 10) | 3.50 | abcd |
| 4 (Phos 20 + Acti 0.1g) | 3.25 | abcd |
| 5 (Phos 15 + Acti 0.1g) | 2.00 | de |
| 6 (Phos 10 + Acti 0.1g) | 2.25 | cde |
| 7 (Phos 20 + Acti 0.15g) | 4.50 | a |
| 8 (Phos 15 + Acti 0.15g) | 3.00 | abcd |
| 9 (Phos 10 + Acti 0.15g) | 2.75 | bcd |
| 10 (Acti 0.1g) | 4.50 | a |
| 11 (Acti 0.15g) | 4.00 | ab |
| 12 (Water infe.) | 4.00 | ab |
| 13 (Water noninfec.) | 1.00 | e |
* Means with the same letter are not significantly different.
Results and Discussion
The combination of specific concentrations of Actigard and Phostrol resulted in reduced disease symptoms in most instances. Occasionally, Actigard alone caused a reduction in symptoms. In all cases Verticillium was isolated from the the stems of inoculated plants. The enhanced activity of specific combinations of Phostrol and Actigard is presumably due to their ability to activate different plant defense mechanisms in pepper sufficient to reduce but not prevent infection.
Actigard (acibenzolar-S-methyl) when used at field rates of ~0.4 oz/acre or less, specifically activates the host's systemic acquired resistance (SAR) process in many crop plants. It offers broad protection against fungi, bacteria and viruses without having any direct activity on these pathogens. Actigard has performed best when incorporated into a program of chemical sprays, as the inherent level of disease control has seldom been sufficient when applied alone. This product has initiated a whole new field of research into utilizing peptides for controlling diseases, and other means of stimulating SAR and the Jasmonic pathway (JA) with chemicals and biological agents in plants.
Previous research has revealed that when certain species of Phytophthora infect certain plant species treated with phosphonate fungicides such as Phostrol, fungus-inhibiting chemicals called phytoalexins are produced. A recent study involving Eucalyptus showed that the concentration of phosphite ions in plants may determine the extent of host defense activation. When concentrations of phosphite ions in the roots were low, host defense enzymes were stimulated; but when concentrations of phosphite ions were high, host defense enzymes remained unchanged and the phosphite ions inhibited growth of the pathogen before it caused disease (3).
References:
- Australasian Plant Pathology 29(3) 170 – 177 Effect of 2,6-dichloroisonicotinic acid or benzothiadiazole on Alternaria leaf spot, bacterial blight and Verticillium wilt in cotton under field conditions. E.S. Colson-Hanks, S.J. Allen and B.J. Deverall
- ISHS Acta Horticulturae 586: IV International Symposium on Olive Growing
FOLIAR TREATMENTS WITH PHOSETYL-AL TO CONTROL VERTICILLIUM DAHLIAE IN OLIVE TREES
A.S. Fodale, R. MulÈ, A. Tucci, A. Cappello - Plant Pathology 49:147-154.
Action of the fungicide phosphite on Eucalyptus
Jackson, T.J., T. Burgess, I. Colquhoun, G.E.S. Hardy. 2000.