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2006 Plant Management Network.
Accepted for publication 23 February 2006. Published 6 April 2006.


Low-Level Copper Plus Chitosan Applications Provide Protection Against Late Blight of Potato


Lee A. Hadwiger, Professor of Plant Pathology, and Pamela O. McBride, Murdock Foundation Fellow, Department of Plant Pathology, Washington State University, Pullman 99164


Corresponding author: Lee A. Hadwiger. chitosan@wsu.edu


Hadwiger, L. A., and McBride, P. O. 2006. Low-level copper plus chitosan applications provide protection against late blight of potato. Online. Plant Health Progress doi:10.1094/PHP-2006-0406-01-RS.


Abstract

The organic grower can manage potato late blight, caused by Phytophthora infestans (Mont.) de Bary, with natural copper compounds such as copper sulfate pentahydrate, copper oxide, or copper hydroxide. However, recent environmental concerns about copper residues by the USDA's National Organic Program indicate a need to control late blight with reduced copper levels. This report describes a strategy for disease control using lower levels of copper sulfate pentahydrate in combination with a chitosan sticker and complexing agent. In excised leaf assays, this combination provided moderate control of late blight and protection against copper-related leaf yellowing at copper levels approximately 40 fold lower than those recommended for a commercial fungicide with copper hydroxide as the active ingredient.


Introduction

Potato late blight, caused by P. infestans, can be controlled with multiple applications of commercially-available synthetic-chemical fungicides (1), even in years with conditions optimal for infection. A degree of late blight suppression has been realized due to the development of new potato varieties that have inherent tolerance to the disease and in the employment of cultural procedures such as planting disease-free seed, volunteer control, cull pile, and irrigation management. With the uncertainties that accompany epidemics, disease management measures that directly kill the pathogen are often essential. The organic grower, however, is restricted to cultural controls, the use of some copper-containing fungicides or other methods that utilize natural ingredients (3). An ingredient currently approved by the USDA's National Organics Program (7) is copper sulfate pentahydrate. Also, the inert ingredient chitosan is EPA-exempt. Our initial field observations on the efficacy of a copper sulfate pentahydrate preparation designed to keep the copper in solution (CT-100, Bontech, Muncie, Indiana) were encouraging; however, the residual copper from multiple applications led to potato leaf yellowing (Fig. 1). Preliminary electron microscopy (X-ray analysis) results designed to detect copper transport into potato leaves indicated that when CT-100 was combined with chitosan, the detectable copper was less prevalent inside the cytoplasm than when CT-100 was applied alone. This observation suggested that the chitosan/copper complex retained a presence on the leaf surface where infection by P. infestans occurs.


 

Fig. 1. Potato leaflets taken from plants treated with (A) water, (B) a combination of the copper sulfate pentahydrate solution (CT-100) at 14 g/ml plus crab-shell chitosan at 25 g/ml, and (C) CT-100-only at 14 g/ml. After 2 days the leaflets were removed from the plant and inoculated with genotype US 8 of P. infestans (25,000 sporangia/ml within a 25 l volume per leaflet). The photo was taken 8 days following inoculation and incubation at 16C, with an 8-h light/16-h dark day cycle.

 

Chitosan is a large, natural polymer of the amino sugar glucosamine. The chelating properties of chitosan are based primarily on the availability of the amino groups on the glucosamine residue when in solution with a pH less than 6.5 (5). The orientation of the positive charges from the amino group of glucosamine enable chitosan to attach both to the negative charges on the surface of the potato leaf and to the CT-100, giving it a "sticker" function (4,6). Copper sulfate has long been utilized for its fungicidal properties, however at high concentrations it can become phytotoxic to the point of being herbicidal. This report documents research from excised leaf trials and one field trial evaluating the efficacy of CT-100 plus chitosan treatments, with the purpose of providing concentration ranges that constitute the window between effective fungicidal action and potential copper toxicity. The efficacy of this combination was compared with selected synthetic fungicides and reagent grade copper sulfate pentahydrate.


Excised Leaf and Field Trials

Crab-shell chitosan was obtained from Vanson/Halo Source, Redmond, WA. The dried flakes of crab-shell chitosan (200 g) are not directly water-soluble. To solubilize the chitosan, the dried flakes were wetted with 50 ml of glacial acetic acid overnight. The mix was progressively taken to a volume of 12 liters with de-ionized water. The solubilized chitosan was filtered to remove any particles that could clog spray nozzles and was adjusted to pH 5.2 with 5 N NaOH. The final stock solution consisted of approximately 7 mg of chitosan per ml. Alternately, commercially available crab-shell chitosan-lactate was prepared and supplied by Vanson/Halo Source. The crab-shell chitosan had been solubilized in dilute lactic acid, pH adjusted, and desiccated to a water-soluble powder. A copper sulfate pentahydrate (53 mg/ml) formulation, CT-100, was labeled by Bond Tech and was manufactured by Chemtech Inc., Muncie, IN. Other copper treatments consisted of Baker Analyzed reagent-grade copper sulfate pentahydrate (Cu) (100% active ingredient) and a commercial fungicide with 53.8% copper hydroxide, Kocide 2000, obtained from Griffin LLC, Valdosta, GA. Additionally, Bravo WS, a synthetic fungicide with 72% chlorothalonil as the active ingredient, was obtained from ISK Biosciences Corp., Mentor, OH.

Russet Burbank potato plants were grown to the 12-leaf stage in a greenhouse in potting soil for the excised leaf trials and to at least the 12-leaf stage for the field trial in Pullman, WA. In the field, each treatment (200 ml of spray solution) was applied to two randomly-assigned rows of 10 plants (20 ml per plant) within field plots of Russet Norkota. In the greenhouse each of 3 individually-potted (15-cm pots) Russet Burbank potatoes plants was sprayed with a 25-ml volume of each treatment solution. For excised leaf assays the plants were sprayed 3 to 5 days prior to inoculation. In the field trial, experimental plots within plant rows were sprayed approximately weekly from July until September. Three to five days after treatment, seven sets of three leaflets or typically seven complete leaves (replications) of uniform size (seven leaflets each) were removed from each treatment of potato plants from the field or from greenhouse plants. The leaves were briefly washed on each side with a water atomizer and organized, upper surface up, in 27- 40-cm casserole dishes that contained three layers of presoaked paper towels under three layers of plastic door screen. Following inoculation of the center of each leaflet with a 25 l droplet of a spore suspension of P. infestans containing 1.5 to 2.5 104 sporangia per ml, the dishes were covered with 3 layers of gas-permeable plastic wrap. The virulence of the P. infestans (genotype US 8) inoculum was maintained on excised potato leaves. Virulent sporangia used for inoculum were harvested from infected potato leaves 5 to 10 days after inoculation. The inoculated leaves were held in casserole dishes at 16C, with 8 h of light and 100% humidity and with daily rotation within the incubator for at least 5 days prior to analysis. Disease intensity was measured as the percent surface area of each leaflet showing visible symptoms. Leaf yellowing associated with copper toxicity was assessed visually.

Treatments were established in a randomized complete block design, typically with six to ten treatments in each trial. The "percent surface area of the leaflet infected" was determined for each of seven leaflets per leaf and there were seven to eight leaves per treatment. Data was processed with the ANOVA procedure of SAS 6.12 (SAS Institute Inc., Cary, NC). The LSD values are noted in the figure legends.

For the field trial, Ranger Russet potatoes were grown in Pullman, WA (an area isolated from commercial potato production) in three-row plots, 12 ft long, with 3-ft spacing between rows and 10-inch spacing between plants. A row bordering each of the plots was inoculated with P. infestans, US 8, on July 19 and 23, 2004. The secondary spread of late blight by spores, which developed within 7 days after inoculation, was encouraged by sprinkler irrigation at night. Five applications of the chitosan-lactate (90 mg) plus CT-100 (2.16 ml) and control treatments (water) were applied August 3, 13, 20, 26, and September 10 to four replications (36 plants per replicate) per treatment by a backpack CO2 sprayer at a total (for 4 reps) volume of 2.7 liters per treatment. This treatment, along with a water treatment that served as the control, were arranged in a randomized complete block design. Plant symptoms in each replication were visually analyzed for both late blight and early blight by percent of disease symptoms for each plot on August 3, 10, and 21. The efficacy of the fungicide was determined by comparing the area under the disease progress curve (AUDPC). A protected LSD statistical procedure (PROC GLM, SAS) was used to compare AUDPC means between treatments.

Excised leaf greenhouse experiments designed to compare CT-100 only, chitosan only, and the CT-100-plus-chitosan combination are presented in Figures 1 and 2. The combination of CT-100 plus chitosan sticker consistently retained the green leaf color (Fig. 2) and was more effective in limiting the area of the leaf with symptoms when compared with leaves treated with CT-100 only. Subsequently, two commercial fungicides, chlorothalonil (Bravo) and copper hydroxide (Kocide 2000), were included in these comparisons (Fig. 3). Compared to application of water alone, the combination of chitosan (25 g/ml) plus CT-100 (14 g/ml copper sulfate pentahydrate) significantly reduced symptoms of late blight. Copper hydroxide (600 g/ml) and chlorothalonil (3200 g/ml) were significantly more efficacious than the CT-100 plus chitosan treatment, however, this higher suppression of disease involved the use of much higher concentrations of each fungicide. Treatments with increasing concentrations of CT-100 (from 4 to 8) resulted in increased leaf symptoms (Fig. 4); however, the symptoms changed at high concentrations from the pathogen-associated lesion to tissue blackening which appeared to be due to copper toxicity. A gradual increase in toxicity was also observed with 4- to 8-fold concentrations of CT-100 only and with similar CT-100 concentrations applied to non-inoculated tissue (not shown). The data in Fig. 4 also show that chitosan's sticker properties provided no enhancement of efficacy when combined with chlorothalonil and copper hydroxide. This observation held true over a range of concentrations (not shown). Thus, any beneficial effect of chitosan appears to be non-existent at the higher levels of copper hydroxide. Surprisingly, the chitosan-only treatment often resulted in an increase in the symptomatic leaf area. The positive charges present in these low concentrations of chitosan may have assisted the pathogen in attaching to the leaf surface. The concentration of chitosan (25 g/ml) utilized as a sticker in these trials is well below those concentrations (250 to 1000 g/ml) capable of inducing plant defense responses in peas (2).


 

Fig. 2. A comparison of CT-100 (processed form of copper sulfate pentahydrate) with and without chitosan 25 g/ml (Chit) as a sticker on potato late blight. Triplicate plants were sprayed (25 ml/plant) with CT-100 (14 g/ml), chitosan (25 g/ml), or CT-100 (14 g/ml) plus chitosan (25 g/ml). Each leaflet of the excised leaves was inoculated with 25 l of a 20,000-sporangia-per-ml suspension of P. infestans and incubated 12 days at 16C. Values for percent of leaf surface area infected are plotted. Yellowing of leaflets was observed only with the CT-100-only treatment. Values of percent symptomatic leaflet area with the same letter are not significantly different, based on an LSD 0.05 = 9.60.

 

 

Fig. 3. A comparison of symptom development in potato plants (two plants/treatment) sprayed (25 ml/plant) with chitosan (25 g/ml), CT-100 (14 g/ml), CT-100 (14 g/ml) plus chitosan (25 g/ml), and the commercial fungicides copper hydroxide (600 g/ml) and chlorothalonil (3.2 mg/ml). Sprayed leaves were excised and each of 49 leaflets per treatment inoculated with 25 l of a 20,000-sporangia-per-ml suspension of P. infestans. Leaves were incubated 12 days at 16C. Values of percent symptomatic leaflet area with the same letter are not significantly different based on LSD 0.05 = 10.66. Symptoms of the CT-100 only treatment included yellowing of the total leaf.

 

 

Fig. 4. Effect of increasing concentrations of CT-100 on the symptom development 8 days post inoculation. Seven leaves (49 leaflets/treatment) were harvested from greenhouse-grown Russet Burbank plants and inoculated with P. infestans (25 l of a 20,000-sporangia-per-ml suspension per leaflet) 4 days after the following treatments were applied in a volume of 25 ml per plant (two plants/treatment): water alone, chitosan (50 g/ml), copper hydroxide (600g/ml), copper hydroxide (600 g/ml) plus chitosan (50 g/ml), chlorothalonil (3.2 mg/ml), chlorothalonil (3.2 mg/ml) plus chitosan (50 g/ml), chitosan (50 g/ml) plus CT-100 2X (28 g/ml), chitosan (50 g/ml) plus CT-100 4X (56 g/ml), chitosan (50 g/ml) plus CT-100 8X (112 g/ml), CT-100 2X (28 g/ml), CT-100 4X (56 g/ml), or CT-100 8X (112 g/ml ). Leaves receiving no treatment also were inoculated. Values of percent symptomatic leaflet area with the same letter are not significantly different, based on an LSD value of 10.27. (The severity of symptoms following the CT-100, 8-fold treatments was primarily related to copper toxicity in addition to the infection-related symptoms.)

 

 

Fig. 5. Effect of processed copper sulfate pentahydrate (CT-100) and reagent grade copper sulfate pentahydrate (Cu) on late blight development. Duplicate sets of potato plants were untreated or sprayed (25 ml per plant) with: water, chitosan (25 g/ml); Cu (14 g/ml), Cu (14 g/ml) plus Chit (chitosan) (25 g/ml), CT-100 (14 g/ml), CT-100 (14 g/ml) plus Chit (25 g/ml), Cu 10X (140 g/ml), and Cu 10X (140 g/ml) plus Chit (25 g/ml). Four days following application of products, 49 leaflets from each treatment were inoculated with P. infestans, US 8 (25 l of a 20,000-sporangia-per-ml suspension) and observed for 10 days post inoculation. Yellowing of leaflets was observed for the Cu, Cu/chitosan, CT-100, Cu10X, and Cu10X/chit treatments. Values of percent symptomatic leaflet area with the same letter are not significantly different based on LSD0.05 = 8.73.

 

The processed copper sulfate pentahydrate (CT-100) plus chitosan treatment appeared to be more effective than reagent grade copper sulfate pentahydrate (Cu, Fig. 5) for control of late blight. The reagent grade copper sulfate pentahydrate plus chitosan protection was significantly more effective than no treatment; however, it is inadequate for commercial use. The data in Fig. 6 indicate that the preprocessed, water-soluble chitosan-lactate is also effective when combined with CT-100 in managing late blight in the excised leaf assay.


 

Fig. 6. Effect of two commercially available products, chitosan-lactate (Chit-Lac) and CT-100, at varying concentrations on the development of potato late blight. Duplicate plants were treated with a 25 ml volume of spray mixture per plant. The chitosan lactate 1X concentration was 25 g/ml and the CT-100 1X concentration was 18 g/ml. The standard chitosan dissolved in acetic acid and neutralized is indicated as Chit 2X (50 g/ml). Leaves were sprayed 3 days pre-inoculation. Seven sprayed leaves (49 leaflets) of each treatment were excised and each leaflet inoculated with P. infestans genotype US 8 (25 l of a 20,000-sporangia-per-ml suspension) and incubated in 100% humidity at 16C for 12 days. Values of percent symptomatic leaflet area with the same letter are not significantly different, based on a LSD0.05 = 7.01.

 

In the inoculated field plot trial, the chitosan-lactate plus CT-100 combination gave moderate control under environmental conditions that would favor the development of a full-fledged epidemic. The resulting severity of symptoms was recorded as AUDPC. The chitosan plus CT-100 treatment significantly reduced the symptoms of late blight (AUDPC = 935) compared to control plants treated only with water (AUDPC = 1151, LSD0.05 = 159). Symptoms were also recorded for a natural infection of early blight in the same plots. The severity of early blight was significantly reduced by the chitosan-lactate plus CT-100 treatment (AUDPC=74) compared to control plants (AUDPC=145, LSD0.05 = 45). The very low copper concentrations of the chitosan-lactate plus CT-100 treatment gave a level of management approaching that of fungicides currently available in the excised leaf study; however, as indicated in the field data, when conditions of maximal inoculum and optimal environment exist it was not possible to achieve complete control of potato late blight. The survival of plants in chitosan-lactate plus CT-100 treated field plots under these severe conditions indicates that this treatment will enable the organic grower to raise potatoes in the advent of greater restrictions on copper components. This treatment should help keep inoculum levels low in the periods prior to the development of the environmental conditions required for epidemics. Although the leaves from plants treated in the greenhouse were sprayed with water prior to inoculation, the conditions of driving rain sometimes existing in the field are likely to be more severe. Alternately, field conditions would seldom be as optimal as in the enclosed humid chamber that maintains a constant optimal infection temperature for the duration of the incubation period. Additional field trials under epidemic conditions are needed and will be continued in parts of the State of Washington where such conditions sometimes exist.


Acknowledgments

We thank the Murdock Foundation for the support of P. McBride, also for support by Washington Sea Grant Program grant R/B 38, Bondtech, and Vanson/Halo Source. We thank Hongyan Sheng and Katie Murray for data analysis, Tom Cummings and Dennis Johnson for conducting the field inoculation trials, and Sierra Hartney for the critical review.

PNNS no. 360, Department of Plant Pathology, Washington State University.


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