Final Report for ANC93-016
[Note to online version: The report for this project includes graphical figures that could not be included here. The regional SARE office will mail a hard copy of the entire report at your request. Just contact North Central SARE at (402) 472-7081 or email@example.com.]
Aqueous extracts of anaerobically fermented spent mushroom substrate (SMS) were applied during the growing seasons of 1993, 1994, and 1995 to apple trees at two locations (university experimental orchard and commercial orchard) for control of the apple scab disease caused by the fungus Venturia inaequalis. Extracts significantly reduced the disease but were not as effective as a fungicide sprayed at the same intervals. Evaluation of the extract potency, based on laboratory assays for inhibition of germination of the pathogen's spores, suggested that the major active principle was a small, heat-stable, non-protein metabolite produced by microorganisms in the compost. In studies conducted on various composts to examine the effect of compost sample size (50 to 5,000 grams) on the precision of estimates of extract potency, we found that samples of at least 500 g were needed to avoid large errors related to sample heterogeneity and efficacy. Experiments to test the effects of aging and storage conditions (indoors vs outdoors) of compost on efficacy of extracts showed that the effects of these factors vary with compost and may be negligible or significant. Similarly, storage conditions of extracts (5 months at 24°C, 4°C, or -20°C) influenced efficacy of extracts from one source tested but not the other.
i)To test water extracts of composts for seasonal and overwintering control of the apple scab disease.
ii)To determine mechanism of action as direct vs. indirect, and microbiologically-based or chemically-based.
iii)To determine how mass of compost samples for bioassays relates to predictability and reproducibility of assay results.
iv)To determine optimal formulation of compost or compost extract for storage.
Objective i) Efficacy of water extracts.
To control the apple scab disease caused by Venturia inaequalis, aqueous extracts from two sources of spent mushroom substrate (SMS), anaerobically fermented for seven days and amended with spreader-sticker, were applied at weekly intervals to apple trees (cv. McIntosh) from green tip to petal fall and biweekly thereafter. Trials were conducted for three seasons at two locations in Wisconsin. Both extracts significantly reduced (α = 0.05) the leaf area affected by scab relative to water and spreader-sticker controls as evaluated on the Horsfall-Barratt scale (Figs. 1-2). Disease incidence was similarly decreased but to a lesser extent (Fig. 3). Extracts were not as effective in inhibiting disease as captan sprayed at the same intervals. No difference was detected between extracts with and without spreader-sticker. Higher populations of bacteria, which persisted for at least one month after the final spray, were detected on leaves treated with the extracts (Fig. 4). No differences were found in total numbers of fungi (Fig. 5). Inhibitory activity of extracts, assessed as in vitro inhibition of Venturia conidia germination, was monitored over time for extracts prepared from SMS stored under different conditions (Fig. 6). For one source of SMS, neither time nor storage conditions (outdoors uncovered, indoors in sheds) affected inhibitory activity of extracts. Decline in efficacy of the other source was apparent by 13 weeks relative to unstored compost, though not between storage regimens.
Research with other crop-pathogen systems gave mixed results on compost efficacy. SMS extracts applied for control of tomato leaf and fruit blights (caused by the fungi Alternaria and Septoria) or early blight of potato (Alternaria solani) were ineffective, whereas extracts inhibited conidial germination of Sphaeropsis sapinea (causal agent of red pine shoot blight) in vitro and gave significant reduction in disease on pine seedlings.
Objective ii) Mechanism of action and inhibitory principle.
Clarified water extracts of slurries of SMS inhibited in vitro germination of conidia of V. inaequalis by up to 98% relative to germination in water controls (Fig. 7). Inhibition of conidial germination increased with incubation time of slurries over five to seven days and persisted at least 14 days, at which time experiments were terminated. Compost slurries became anaerobic within 1 hour when incubated without aeration (Fig. 8). There was no difference in efficacy between filtered (0.1 μm) and untreated extracts (Fig. 9). Autoclaved extracts were less effective than untreated extracts but retained most of their efficacy (Fig. 9). Passage of filtrate through microconcentrators with molecular weight cut-off limits ranging from 100 to 3 kDa did not diminish activity of the extracts (Fig. 10). Aeration of slurries decreased efficacy of the resulting extracts compared to non-aerated controls. When aerated slurries were allowed to incubate without further aeration, extracts regained efficacy, becoming not significantly different from non-aerated controls (Fig. 11). Extracts produced from sterile SMS were virtually ineffective compared with those from nonsterile SMS. When small volumes of slurry from raw SMS were added to slurries of sterile SMS and incubated for an additional period, efficacy of the inoculated SMS was enhanced compared to uninoculated controls (Fig. 12). Thus, a major inhibitory principle of the SMS extract is a low molecular weight, heat-stable, non-protein metabolite produced by anaerobic microorganisms in the compost. The implications of this part of the research are that now that the mode of action is tentatively identified, it should be possible to isolate and characterize the compounds responsible and to increase production in composts or in vitro, and retention and stability on leaf surfaces.
Objective iii) Compost sample size vs predictability of efficacy.
We examined the effect of compost sample size (50 to 5,000 g) on the precision of estimates of inhibition of V. inaequalis conidial germination induced by extracts of various anaerobically-incubated composts. Composts were prepared from vegetable material; some were amended with manure. Variability due to sample size was considered as a function of extract efficacy (high, medium or low) and compost heterogeneity (high or low). Extract efficacy was characterized as ability to inhibit more than 75% of the conidia (high), 75% to 40% (medium), or fewer than 40% (low). Heterogeneity was characterized as visual or tactile presence (high) or absence (low) of parent material in the compost. To address the question of sample size-related variability, we partitioned individual extract incubations (samples) into aliquots (sub-samples), and aliquots into microtiter plate wells (sub-sub-samples). For this nested design, the largest component of variation was consistently found to be that associated with wells; aliquots were a negligible source of variation (Fig. 13). Sample size over the range examined was generally of small importance for extracts of medium and high efficacy, but not low efficacy, independent of compost heterogeneity. For previously untested composts, particularly those of high heterogeneity, statistical analyses of our data suggest that samples of at least 500 g circumvent potentially large errors and consequent difficulties to detect differences among composts or effects of experimental variables.
The importance of this objective relates to the broader issue of whether effective composts can be distinguished reliably from ineffective composts based on screening small samples. While it has long been known that sectors of a compost pile can differ substantially in physical, chemical, and biological properties, there is essentially no information on how sample size influences results. This information is necessary for researchers to efficiently and accurately screen prospective sources.
Objective iv) Formulation and storage.
To test whether there was retention of efficacy over time, the extracts from two sources of compost were assayed periodically during storage for five months at 24°C, 4°C, and -20°C. One source (SMS) lost significant efficacy after two months storage at 24°C. Inhibitory potential of the other source (cow manure/wood chips/soil) did not decline appreciably with time at any temperature, but was less inhibitory overall than SMS.
The broader context of these findings is that if composts are to be used widely in practice for disease control, it will be necessary to: (i) store them prior to application; (ii) transport them conveniently (e.g. in lyophilized form); and (iii) ensure maximum retention and efficacy on foliar surfaces.
Economic losses due to foliar disease induced biotically or abiotically are difficult to estimate, but are on the order of $1 billion annually in the United States for frost injury alone. Moreover, any direct costs must be viewed within a broader context that includes environmental costs and constraints, increasing cost of pesticide research, and the increasingly frequent revocation of registered (or failure to reregister) pesticides by regulatory agencies.
Apples are among the top five of the major food commodities in the United States. This country ranks second internationally in apple production (about eight billion pounds valued at $1 billion). The apple scab disease is distributed worldwide. In moist, temperate areas it is considered to be the most important pest problem of the crop. Growers in Wisconsin and climatically similar regions routinely apply 11-15 fungicide sprays each season. Apples rank third nationally in percentage of acres treated with fungicide (78%) and third in total fungicide expenditures ($23.5 million). These sprays thus represent an appreciable input cost to growers and, additionally, they can have substantial indirect costs in impact on the environment. Fungicides also pose the threat of adverse effects on health. For example, about 90% of all fungicides used in agriculture are animal oncogens. Although these chemicals constitute only ca. 10% of all pesticides applied to food crops, they account for roughly 75% of the oncogenic risk associated with consumption of processed foods and nearly 60% overall (versus 27% from herbicides and 13% from insecticides).
If compost extracts can be used successfully they will provide, for conventional farmers, a needed alternative to fungicides and, for organic farmers, a nonchemical means to control disease. For all farmers, compost would facilitate the movement away from high input, synthetic chemical practices towards a sustainable philosophy that emphasizes alternative, low cost inputs, alternative cultural practices, and use of recycled on-farm wastes. It is even possible that home owners in urban areas could use compost extracts prepared from food and yard wastes to control disease in their gardens. If so, this would reduce a significant pesticide source to the environment and one often associated with a misuse.
Finally, the urgency of finding alternative control measures is evident from the recent controversy regarding withdrawal of the ethylenebisdithiocarbamate (EBDC) fungicides. Because of concern about the carcinogenicity of ethylenethiourea, a breakdown product of these chemicals, the leading manufacturers decided in September 1989 to modify the registrations to eliminate use of EBDC fungicides on all but 13 of the 55 crops for which they are registered. Subsequently, the EPA has proposed to cancel use of EBDCs on 45 of the crops. One of the principal uses of these chemicals has been on apple, and the EBDCs (mancozeb, maneb, manzate, zineb, metiram) are now no longer available to orchardists. Although other fungicides are available for the time being, it may be only a matter of time before they are withdrawn, because most are oncogenic (see above). Those that are not, such as copper and sulfur, are either relatively poor protectants, or can be phytotoxic, or both. This situation is typical of the fungicide dilemma.
a. A primary biocontrol mechanism operating in compost extracts is antibiosis.
b. The scientific basis for the enhanced biocontrol efficacy achieved by incubating composts in water is production of a pathogen- and disease-inhibiting metabolite(s) by anaerobic fermentation. When the extract is applied to plants, this metabolite(s) inhibits the pathogen.
c. Compost samples of 500 g are needed to reliably predict efficacy of composts in disease suppression.
d. Compost extracts will work in practice to control foliar diseases if: (1) the inhibitory principle(s) is stable or can be stabilized and (2) the extracts adhere to leaf surfaces sufficiently to withstand weathering.
e. The best means to store composts to retain efficacy is to freeze-dry the extracts.
f. Efficacy can be enhanced by adding substrates to the compost slurry or antagonists to the extract.
g. The active principle of extracts can be isolated, identified, and either composts developed and stored to maximize efficacy, or the compound synthesized and applied in place of fungicides.
h. Compost efficacy does not decline appreciably with age up to six months and is not altered by weathering (i.e. composting can be done unsheltered outdoors).
This approach to disease control is still considered experimental and has not yet been promoted or adopted by farmers. Growers attending our meetings showed enthusiasm for the method and would likely adopt it if consistent efficacy were shown.
Educational & Outreach Activities
Yohalem, D. S., R. F. Harris, and J. H. Andrews. 1994. Aqueous extracts of spent mushroom substrate for foliar disease control. Compost Sci. Utiliz. 2:67-74.
Yohalem, D. S., C. M. Simmer, J. H. Andrews, and E. V. Nordheim. 1994. A method to distinguish bias from variation in evaluating spore germination. Phytopathology 64:1068 (Abstr.).
Yohalem, D. S., R. P. Voland, E. V. Nordheim, R. F. Harris, and J. H. Andrews. 1996. Sample size requirements to evaluate inhibition of spore germination by aqueous extracts of composts. Soil Biol. Biochem. (in press).
Cronin, M. J., D. S. Yohalem, R. F. Harris, and J. H. Andrews. Putative mechanism and dynamics of inhibition of the apple scab pathogen Venturia inaequalis by compost extracts. Soil Biol. Biochem. (submitted).
Yohalem, D. S., E. V. Nordheim, and J. H. Andrews. The effect of water extracts of spent mushroom compost on apple scab in the field. Phytopathology (submitted).
Yohalem, D. S., R. F. Harris, and J. H. Andrews. 1995. Aqueous extracts of spent mushroom substrate reduce apple scab infection. Phytopathology 85:1147 (Abstr.).
22 October 1993. Talk on compost extract research to growers at Michael Fields Agr. Inst. workshop. Attendance: About 20 persons.
22 February 1994. Talk on compost extracts at Wisconsin Fruit and Vegetable Conference, Stevens Point, Wisconsin. Attendance: approximately 100 persons.
11-14 March 1994. Talk on disease control by compost extracts at conference on "Nature and Uses for Spent Mushroom Substrate," Philadelphia, Pennsylvania. Attendance: approximately 70 persons.
26 July 1994. Talk and tour of orchard research involving compost extracts at Ela Orchard, Rochester, Wisconsin, as part of Michael Fields Agr. Inst. Orchard Field Day. Attendance: approximately 75 persons.
6-14 August 1994. Talk on methods to minimize error in laboratory assay of spore germination in compost extracts. Presented at Amer. Phytopathol. Soc. Meeting, Albuquerque, New Mexico. Attendance: approximately 40 persons.
12-16 August 1995. Talk on extracts to control apple scab. Amer. Phytopathol. Soc. Meeting, Pittsburgh, Pennsylvania. Attendance: about 70 persons.
11-15 September 1995. Talk on mode of action of extracts. 6th Internat. Conf. Microbiol. Phyllosphere, Bandol, France. Attendance: about 80 persons.