Final Report for LNC99-150.1
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Kura clover was established as a ground cover in two apple orchards and tested for its ability to limit dispersal of Venturia inaequalis spores and thus inhibit apple scab primary infection. The hypothesis was not supported in data obtained by sampling airborne spore levels nor by assays of primary scab incidence on apple leaves. The efficacy of the environmentally benign compounds potassium bicarbonate and a solution of methionine and riboflavin was tested as sprays during the growing season to control apple scab and the sooty blotch/flyspeck complex. Disease suppression to approximately the level afforded by traditional sulfur sprays was observed.
The objectives of our research have focused on identification of integrated disease control strategies that might reduce the need for synthetic fungicide spray applications in commercial apple production. In orchards of the upper Midwest United States, spray campaigns typically begin in early spring to blunt the primary infection phase of apple scab disease, in which ascospores of Venturia inaequalis produced in decaying leaf litter on the orchard floor are the predominant source of infection. Over the course of the 5-6 month growing season, ten or more fungicide applications are commonly applied to control scab epidemics and late-season diseases caused by the sooty blotch/flyspeck (SBFS) complex. We reasoned that an orchard management strategy aimed at limiting initial scab infection in the spring, combined with use of environmentally benign antifungal spray compounds to control scab and other late-season diseases, might significantly reduce the reliance of apple growers on fungicides.
We hypothesized that the presence of a dense foliar canopy provided by a Kura clover (Trifolium ambiguum) ground cover crop would limit Venturia ascospore dispersal and thereby reduce the incidence of primary scab lesions. Accordingly, our approach included seeding Kura clover in portions of two Wisconsin apple orchards in the spring of 1997 and testing its impact on scab epidemiology in subsequent years. The effect of Kura on airborne ascospore levels was determined by placing spore samplers above the clover canopy and comparing spore capture rates there to those obtained by using samplers located in adjacent orchard sections that had not been seeded with clover (existing ground cover, EGC). In addition, the ability of a Kura clover ground cover to reduce initial scab infection was determined by comparing the incidence of primary scab lesions on leaves of apple trees growing in the Kura plots to that observed among leaves situated above the adjacent EGC plots.
Contrary to expectations, experiments conducted at both the Peninsular Research Station in Sturgeon Bay, WI, and the West Madison Agricultural Research Station (WMARS) in Madison, WI, indicated that airborne ascospore counts in Kura plots were not consistently different from counts obtained in control EGC plots.
Furthermore, at neither location did the presence of Kura clover consistently correlate with a significant decrease in incidence of primary scab lesions on young apple leaves. This might be attributable to several factors. One possibility is that the Kura foliar canopy, while obviously denser than that of the grassy EGC, nonetheless does not present a substantial impediment to ascospore dispersal. Alternatively, or in addition, it may be that the dispersal range of ascospores carried aloft from adjacent EGC plots is sufficient to obscure the anticipated effects of the Kura canopy on spore dispersal. Finally, it may also be possible that alternative sources of spring inoculum exist, such as spores that overwinter in trees, that would not be affected by the presence of Kura clover.
Results from experiments to assess the efficacy of environmentally-benign antifungal spray formulations were more promising. Two experimental sprays– a methionine-riboflavin mixture (M-R) and a potassium bicarbonate (KHCO3) formulation– were applied to apple trees in several Wisconsin orchards. Both compounds were consistently as effective as the Captan fungicide positive control regimen in reducing SBFS incidence. Furthermore, our preliminary results indicate that the M-R mixture, when applied in an early-season regimen, is as effective as sulfur treatments in controlling scab disease progression.
Based on those results, a commercial formulation of the KHCO3 spray (Kaligreen) has been selected for testing in USDA-sponsored multi-state trials for control of SBFS. The ability of the M-R spray to control apple scab—as well as SBFS– will be further evaluated.
1. Test the ability of the orchard floor cover crop Kura clover (Trifolium ambiguum) to break the life cycle of the apple scab pathogen (Venturia inaequalis).
2. Test the efficacy of environmentally benign compounds as sprays during the growing season to control apple scab disease.
Objective 1: To test the ability of the Kura clover canopy to restrict ascospore dispersal and primary infection.
a. Plot establishment.
In anticipation of possible funding by SARE, we seeded Kura clover in spring 1997 in orchards at two locations. Two adjoining 90 ft x 240 ft (27 m x 73 m) plots, each containing approximately 80 trees (cultivar/rootstock: Empire/M.26 in plot 1; Redmax/M.26 in plot 2) were established at the university’s Peninsular Agricultural Research Station (PARS), Sturgeon Bay, WI (site 1). One 60 ft x 160 ft (18 m x 48 m) plot containing 24, 15-year-old McIntosh trees was established at the West Madison ARS, Madison, WI (WMARS, site 2). Floor treatments were (1) seeded Kura clover; or (2) the orchard floor maintained as existing ground cover (EGC), primarily grass. Each treatment was performed twice at Sturgeon Bay and once at Madison. Kura clover (Treatment 1) was inoculated with commercial rhizobia (Liphatech, Inc.), and seeded at 8 kg/ha following standard site preparation recommendations. Existing ground cover (EGC, Treatment 2), consisting of mixed grasses and weeds, was left unaltered.
b. Quantification of airborne ascospores.
We hypothesized that the Kura clover canopy could reduce dispersal of V. inaequalis ascospores by imposing a barrier that prevents escape in the spring of ascospores from leaf litter on the orchard floor. To test that hypothesis, as in 2000, two Rotorod spore samplers were placed 40 cm above ground level in the center of each subplot (Treatment 1 [Kura] and Treatment 2 [EGC]). Samplers were installed when apple buds were approximately 1/2″ green–on April 13, 2001 at the WMARS orchard and on April 21, 2001 at the PARS orchard. Samplers were activated manually, or by use of electronic leaf wetness sensors, upon rain events when ascospores are expected to be released during the primary scab infection cycle. Grease-coated Plexiglas rods (2 per unit), spinning at approximately 2400 rpm, were used to capture airborne spores by impaction. Trapped V. inaequalis ascospores were enumerated by microscopic observation of rods stained with toluidine blue. Two modifications were made to the protocols used previously for quantification of ascospores. First, in previous years the samplers were activated upon rainfall events and allowed to run continuously for 8 to 12 hrs. While longer run-times afford the opportunity to sample a greater cross-sectional volume of the atmosphere, buildup of debris (abiotic airborne particles, pollen, other spores, insects) makes visualization of Venturia ascospores difficult or impossible. Thus, sampling conducted in spring 2001 limited runs to successive 4 hour periods, between which the rods were removed and replaced. While visibility was substantially enhanced by that modification, it also doubled the number of sampling rods (from 8 to 16) generated upon each rain event. Previous experience suggested that only a small number of those events would prompt release and capture of a statistically significant number of ascospores. In contrast to past practice, then, in which all rods generated during ascospore season were examined in their entirety, in 2001 we conducted a prescreen in which 1/3 of the total surface capture area of two rods per run, per rain event, were inspected for the presence of ascospores. In those cases in which the preliminary screen indicated the presence of significant numbers of captured spores, the surfaces of all rods of those sets were subsequently evaluated for total number of ascospores trapped.
c. Disease quantification.
To determine the ability of Kura clover ground cover to reduce initial scab infection, leaves of mature orchard trees and of potted trees obtained from a commercial nursery were evaluated with respect to primary scab lesion incidence on leaves situated above Kura and adjacent EGC subplots. Redmax saplings (½”- caliper) were obtained from Hilltop Nursery, Hartford, MI and transplanted in mid-March into 14” deep, 12” diameter pots containing a 2:1 ratio of composted soil mix:MetroMix potting soil. Potted trees were maintained in the outdoor cold frames of the UW Walnut Street Green house until early April, at which time they were relocated to the WMARS (April 10, 2001) and PARS (April 21, 2001) orchards (20 total in each orchard distributed evenly between Kura and EGC plots). Scab incidence was subsequently evaluated on the basis of lesions per leaf for all leaves on potted trees (May 31, 2001 in WMARS) or on leaves emanating from 10 clusters (June 13, 2001 in PARS; approximately 75 leaves/tree). Incidence on leaves of planted (mature) trees in both orchards was evaluated at the same dates by inspection of equal numbers (50) of spur and terminal leaves on each of four branches of four trees in each subplot. A similar protocol had been employed in experiments of spring, 2000.
d. Comparison of scab incidence between naïve and previously infected potted trees
In previous years, levels of airborne ascospores in the WMARS orchard were low and leaf litter was essentially nonexistent. Nonetheless, trees in that orchard consistently experience substantial scab disease pressure. We hypothesized that an alternative source of inoculum could be spores or mycelial fragments that survive winter in tissues (e.g. bud scales) of the trees. To test that hypothesis we placed in the WMARS orchard a cohort of potted trees, equal in number to the previously-uninfected (naive) bioassay trees, that had in the year previous experienced serious scab disease. Lesion incidence (lesions per leaf) on those trees was evaluated by visual inspection of upper and lower surfaces of all leaves.
Objective 2. Treatments Applied to Trees to Control Apple Scab Disease
a. Treatment Application
The hypothesis being tested is that environmentally benign compounds can provide commercially acceptable suppression of fungal diseases of apple. In 2000 and in previous years our focus was on control of SBFS. In experiments of 2001 we tested their ability to control scab. Treatments were applied in 2001 to trees in the WMARS orchard. Whole-tree treatments were applied to a total of 12 Cortland trees: 3 were sprayed with water only (negative control); 3 were sprayed with Captan (positive control); 3 were sprayed with potassium bicarbonate (0.5% weight/volume; Kaligreen)-Ultrafine Sunspray Oil (5mL/L); and 3 were sprayed with a methionine-riboflavin (M-R)formulation (1mM methionine, 26.6µg/L riboflavin, 1 mM copper sulfate, 1% sodium dodecyl sulfate. Spray applications were performed at 10-day intervals from 4/14/01 to 6/7/01 and at biweekly intervals from 6/19/01 to 7/19/01, totaling nine applications. Treatments were applied to runoff using hand-held Hudson Perfection sprayers.
b. Disease Quantification
Scab lesion incidence was determined at the end of the ascospore release period (6/13/01) and again during late summer (8/20/01) to evaluate the ability of the various spray regimens to control primary and secondary infection cycles, respectively. Incidence (presence or absence) of lesions was determined by visual inspection of upper and lower leaf surfaces.
Objective 1. Orchard Floor Treatments
The data reported here are from the 2001 growing season. The annual report from 2000 presents a detailed discussion of data gathered during the first year of this project. We consolidate and contrast the results, where apprpriate, in the following section. Formal integration of the two years is not possible because of year to year adjustments in experimental design.
a. Ascospore Quantification
The first airborne ascospores were detected on April 21, 2001 at the WMARS orchard (Fig. 1A) and on May 1, 2001 at the Sturgeon Bay orchard (Fig. 1B). As witnessed in previous years, we did not consistently observe the hypothesized reduction in numbers of airborne ascospores over Kura clover plots as compared to EGC plots. In those instances in which statistically significant numbers of spores were recovered, capture rates from the air above the Kura plot were slightly lower than the rates recorded over the EGC plot at WMARS in 2001; in spring 2000 more ascospores were detected over Kura plot than captured above the EGC plot. At the PARS orchard in Sturgeon Bay, ascospore numbers were generally higher in EGC plots as compared to Kura, but in 2001 the opposite was observed.
b. Disease Quantification
i) Orchard Trees
In 2001, as in 2000, the incidence of primary scab lesions on the spur leaves of mature McIntosh orchard trees at the WMARS orchard was statistically indistinguishable from the incidence among spur leaves in the Kura plot; the same was true in 2000 with respect to terminal leaves. However, lesion incidence among terminal leaves was significantly less among leaves in the EGC plot as compared to those above the Kura clover canopy in 2001 (p<.05; Fig. 2). Among leaves of orchard trees in the PARS orchard, lesion incidence among all leaves of Redmax trees was significantly higher for trees in the Kura plot in 2000, while the opposite was true in 2001 (Fig. 3). Among Empire trees, no significant difference in lesion incidence was observed in either year. ii) Potted Bioassay Trees
In 2001, among potted trees placed in the WMARS orchard, we observed a significantly lower incidence of lesions on leaves in the Kura plot as compared to those in the EGC plot, a result similar to that observed in 2000. However, among an equal cohort of trees trees placed in Kura and EGC subplots of the PARS orchard, the incidence of primary scab lesions on the leaves of trees placed in the Kura plot was not significantly different from the incidence of lesions observed on trees located in the EGC subplot in 2001. The results in 2000 were mixed: the incidence of lesions on leaves of trees in the Redmax Kura plot was significantly higher than in the corresponding EGC plot, whereas there was no difference in incidence among trees in the Empire plot.
c. Comparison of scab incidence between naïve and previously-infected potted trees
To test the hypothesis that at least some initial scab lesions arise from spores that overwinter in tree tissues, lesion incidence was evaluated on leaves of previously infected potted trees that were interspersed among the previously uninfected (naïve) bioassay trees (item b, ii above) in the WMARS orchard. Scab incidence, both in terms of the average number of lesions per leaf (Fig. 5) as well as average number of infected leaves per cluster (data not shown) was significantly greater among previously infected trees than among naïve potted trees.
d. Overview and conclusions
In the assays of 2001, as in experiments conducted in prior years from 1998 to 2000, a consistent effect of a Kura clover ground cover crop on scab severity was not apparent. Neither airborne ascospore levels nor primary scab lesion incidence were consistently lowered in Kura plots as compared to results obtained in adjacent EGC plots. Failure to reduce airborne ascospore levels could indicate that the Kura canopy, although denser than vegetation in the EGC plots, nonetheless does not present a significant impediment to ascospore dispersal following rain events. Alternatively, or in addition, sufficient mixing of ascospores derived from the Kura and adjacent EGC plots may have occurred to obscure any expected effect on ambient ascospore levels.
The ultimate test of the utility of a Kura ground cover is its ability to reduce the incidence of primary scab lesions. However, assays in which previously uninfected potted trees were placed in Kura or EGC plots revealed no consistent beneficial effect of the Kura clover in 2001 or in previous years. Evaluation of the ability of Kura clover to protect leaves of mature, planted trees from scab infection was made less straightforward by results obtained in 2001. Experiments using previously infected potted trees suggest that a substantial proportion of primary scab lesions found on the leaves of previously infected trees originate not from ascospores from the orchard floor but instead from spores that overwinter in tree tissues.
Objective 2. Treatments Applied to Control Apple Scab Disease
a. Scab abatement through early-season spray regimens
The efficacy of relatively benign compounds to control fungal diseases of apple was investigated again in 2001. In prior years the focus of experiments was on control of late-season disease caused by the sooty blotch/fly speck (SBFS) disease complex; although assays of scab incidence were conducted and showed a modest beneficial effect, the spray schedule was dictated by appearance of SBFS symptoms, which typically occur later in the season. We hypothesized that a spray regimen of potassium bicarbonate or M-R formulations would prove more effective for control of scab disease if initiated early in the growing season, in order to target both the primary as well as secondary infection phases.
Evaluations of scab incidence among leaves treated with the M-R formulation indicate that it is as effective as a commercial sulfur-based (Captan) fungicide for control of apple scab when applied according to a spray regimen specifically targeted for that disease. Control was evident at both the initial infection stage and in late-season evaluations of scab disease progression (Fig. 6). The application of a potassium carbonate solution also provided a significant level of scab suppression as compared to the water-only control treatment, although significantly less than that afforded by either M-R or Captan sprays.
b. Overview and Conclusions
While research in 2000 had shown the utility of bicarbonate and M-R formulations for control of late season fungal diseases, principally SBFS, experiments conducted in 2001 were the first to rigorously test their ability to control scab. The M-R solution was shown to control scab disease progression as effectively as a commercial fungicide. These results indicate the utility of M-R in disease management programs for apple.
The orchard floor cover crop (Kura clover) portion of the work ultimately proved to be an ineffective strategy for managing scab disease incidence and will not be further studied. However, in the course of that research we have developed evidence that ascospores from the orchard floor may not be the sole, or even most important, source of spring scab inoculum. If those results are substantiated through additional studies, it would represent a dramatic break with dogma that governs scab control strategies and suggests additional or alternative strategies that may lead to more effective and sustainable practices.
The second portion of the work concerning the utility of various benign alternatives to synthetic fungicides has shown that two compounds, methionine-riboflavin mixtures and potassium bicarbonate, are effective in controlling the sooty blotch and flyspeck diseases; furthermore, the M-R formulation displayed outstanding control of scab in the 2001 field trials. This offers the possibility for growers, especially organic producers, to replace fungicides with less toxic substitutes.
Our research is broadly relevant as follows. The first rationale for the project derives from the importance of apples in the American diet. Apple is consistently among the top five commodities in the USA and, in terms of commercial production (about 11 billion pounds valued at approximately $1 billion), second only to oranges among tree fruits. Internationally, as a producer, the USA ranks behind only China. The second rationale relates to the mainstay of control, which remains fungicides. Orchardists in Wisconsin and climatically similar regions routinely apply 8-15 fungicide sprays each season, primarily to combat scab. In the U.S., over 90% of apple acreage is treated with fungicides. While apple is produced on less than 0.01% of total cropland treated with pesticides in the U.S., it accounts for 10% of all fungicides used. This represents an appreciable input cost to growers. For example, the price of Captan, used widely for control of scab and SBFS, increased 29% during the period 1991-1995. Fungicides also can have substantial indirect costs in impact on the environment resulting from toxicity to nontarget organisms and also pose the threat of adverse effects to human health. For example, about 90% of all fungicides used in agriculture are animal oncogens.
Educational & Outreach Activities
Since submission of the 2000 annual report the project was:
i) Presented before the Michigan State Organic Fruit School, March 5-6, 2001 (75 participants).
ii) Presented for Apple IPM Field Day, held July 11, 2001 at the Peninsular Agricultual Research Station, Sturgeon Bay, WI. (50 growers in attendance).
iii) Described in a SARE press release April, 2001.
iv) Publication Andrews, J.H., O’Mara, J.K., and McManus, P.S. (2001). Methionine-riboflavin and potassium bicarbonate-polymer sprays control apple flyspeck and sooty blotch. Plant Health Progress. Online at http://www.planthealthprogress.org/current/research/apple/article.htm.
Areas needing additional study
The potential of Venturia inaequalis spores to overwinter in tree tissues has received little, if any, consideration as a significant source of spring scab inoculum. If our further research confirms the results obtained in 2001 we expect that view to change. Methods to control that source of infection will have to be discovered and implemented to allow growers to target all potential sources of inoculum.
SARE-sponsored studies of the efficacy of a bicarbonate solution for control of SBFS have lead to its inclusion in a multiyear, multistate trial sponsored by the USDA Crops at Risk program (P. McManus, Co-PI) to evaluate its potential for control of SBFS. The ability of the M-R formulation to control apple scab will be assessed through field trials in spring-summer of 2002, if funding is available.
A “best-case” scenario is that application of the M-R formulation in late autumn and/or early spring, followed by periodic season-long applications, is sufficient to provide non-toxic control of apple scab and other apple diseases. Accordingly, we have initiated trials in which potted trees that suffered scab in the 2001 growing season were sprayed with the M-R cocktail in early November. A subsequent spray program will be initiated at about the time of bud-break in early spring, 2002 and continued through the growing season to assess the ability of the M-R formulation to control apple scab disease.