Final Report for ANC95-032
Water extracts of slurries of spent mushroom substrate compost inhibit the apple scab pathogen Venturia inaequalis in the laboratory, and reduce severity of scab disease in the field. This project was an attempt to isolate the compound(s) responsible based on the polarity, charge, and size of the bioactive molecule(s). The methods used included extraction (partitioning) into nonpolar solvents at acidic, neutral, or basic pH; cation exchange column chromatography; paper electrophoresis; and ultrafiltration through membrane filters (microconcentrators) of known porosity or Sephadex G-10 and G-25 columns. Almost all of the activity was associated with polar (water-soluble) molecules and at least some of it was less than 3 kilodaltons in molecular weight.
To continue determination of the pathogen-and disease-inhibitory principle(s) in the spent mushroom subtrate (SMS) extracts.
We attempted to separate the compound(s) of interest from the crude spent mushroom substrate (compost) extract by using standard organic chemistry methods detailed elsewhere (Cooper 1977; Cronin et al. 1996; Sunshine, 1969). In overview, such methods (summarized Fig. 1) essentially are based on size (ethanol precipitation, column chromatography, ultrafiltration), or polarity (differential solubility in polar/nonpolar solvents), or charge (electrophoresis, cation exchange chromatography). We also developed bioassay methods and a standardized quantification scheme for assessing efficacy of the chemical fraction of interest.
Compost filtrates for assay. Incubation conditions of SMS with water and general preparative procedures are described in Cronin et al. (1996). The SMS slurry was passed through cheesecloth (four layers), centrifuged, and the supernant filtered successively through coarse filter paper (Reeve Angel #202), glass fiber (1.0 µm) and membrane (0.45 and 0.20 µm) membrane filters. The final product, termed “SMS filtrate,” was tested for sterility and stored at 4°C for up to two weeks without appreciable loss of activity (Fig. 2) pending use. Biochemical procedures were not conducted aseptically, but the resulting fractions were passed through a 0.2 µm filter prior to assay.
Bioassay based on Venturia spore gemination in microtitre plates. The procedure is described in Cronin et al. (1996). To monitor change in activity of fractions during the purification process, a common basis and established reference point for expressing activity was needed. In plots of percent inhibition vs. filtrate concentration, we took this to be the LD50 value, i.e. the concentration corresponding to 50% inhibition of germination of Venturia spores. This level of activity was assigned an arbitrary value of 100 units per ml. In Figure 3 the actual filtrate concentration is 0.12 which means that the original (undiluted) filtrate at 0.12X has 100 units per ml or 833 units per ml if undiluted. Since we processed routinely 500 ml, this amount contained 416,650 total units.
Colorimetric assay. An alternative to assaying viability based on microscopy (laborious counts of percent germinated spores) is to use a colorimetric procedure based on vital strains such as tetrazolium (Levitz and Diamond 1985). The reduction of this compound to a colored formazan product occurs only in living cells. Thus, relative cell numbers can be estimated indirectly by determining the color change in an automated assay with a 96-well microtitre plate reader. Venturia spores were harvested, added to the wells at various concentrations and incubated for 48 hours, following which the standard reagents (Promega Corp, 1996) were added, the system incubated for a further four hours, and absorbance at 490nm was read and recorded.
Fractionation and purification of active compound(s). Separations based on polarity involved mixing the (aqueous) SMS filtrate with organic (non-polar) solvents by sequential extraction in hexane followed by chloroform. Hexane and chloroform were distilled off and collected in a rotary evaporator. The dry residue was re-dissolved in DMSO and diluted for assay. The water layer was reduced in volume to ensure that all solvents had been removed. To improve separation, in some experiments the large molecules were first removed by cold ethanol precipitation. SMS filtrate (500 ml) was mixed with 1500 ml cold 95% ethanol, and the resulting precipitate removed by centrifugation at 13,000 rpm for 20 min. Ethanol was removed from the supernatant by evaporation at 40°C and the remaining aqueous supernatant partitioned as above, under neutral (pH7), acidic (pH4), or basic (pH10) conditions.
To separate based on charge, cation exchange chromatography and paper electrophoresis were used. For the former, a column was prepared from Amberlite IRC-50, stored in 1M NH4OH, and neutralized with ammonium phosphate buffer before use. The SMS filtrate (300-1,000ml) was loaded and allowed to run through. Neutral buffer was used to rinse the column and this eluant was collected and bioassayed. Cations stuck to the column were eluted with 1M NH4OH (pH8.0). All fractions collected were dried to remove ammonium ions, redissolved in water, and bioassayed. For paper electrophoresis, we clarified the SMS filtrate with ethanol as above, filtered it through a 3kD microconcentrator (Cronin et al. 1996), and applied a small quantity to Whatman #1 chromatography paper with orange G as a standard anion. The paper was soaked in ammonium bicarbonate buffer (pH 9.2) and electrophoresis conducted for 50 min. These conditions would impart a negative charge to most polar compounds so they would migrate toward the anode [(+) electrode]. The paper was dried, portions checked for bands with reagents following standard methods, and strips eluted in water and the eluant bioassayed.
To separate based on size, the SMS filtrate, with or without subsequent ethanol clarification and passage through a 3kD filter, was loaded onto Sephadex G-10 (effective range
Colorimetric assay. Results are summarized (Fig. 4) for three concentrations of spores (5,000; 10,000; 50,000) with controls (spores killed with azide; spores without reagent), indicating that differences in absorbance were detectable. Despite these promising results, the color change assayed (yellow) was in the same range as the color of the filtrates. Hence this assay did not appear to be useful for our purposes.
Polarity. In partitioning experiments with organic solvents, no activity was recovered from the hexane or chloroform fractions. Inhibitory activity restricted to the aqueous phase indicated that the compound(s) is polar. Ethanol precipitation removed the large molecules rendering a clear supernatant which was much easier to use in subsequent steps, particularly filtration, but at the expense of substantial loss in activity (about 40% recovery of the initial level). This may have been caused by heating to 40°C, which is unlikely because we have shown (Cronin et al, 1996) that the principle is largely retained after autoclaving. Alternatively, either large as well as small molecules are involved in the activity, or some of the small molecules were entrapped and lost in the precipitate with the macromolecules.
Charge. No activity attributable to the cation fraction was obtained for cation electrophoresis of either the SMS filtrate or the < 3kD component. The only activity recovered was in the eluant at neutral pH of the crude filtrate that passed directly through the column. These results suggest that the active principle is not a cation. The paper electrophoresis results showed a broad band of UV absorption just to the positive side of the origin. Inhibitory activity was associated with this band (Fig. 5) but it was not further localized, and activity > 30% was not recovered.
Size. At least some of the active principle(s) passed through 3 kD filters (Table I), confirming the findings of Cronin et al., (1996). Treating the SMS filtrate with cold ethanol removed the large molecules and promoted filtration rate, but also resulted in appreciable loss of activity (Table I). Solutions subjected to sequential filtration (100 kD, 10 kD, 3 kD filters) had less activity than those filtered only once, suggesting that some of the bioactive molecules may have adhered to the filters (no residue was visible on the filters. The results of sizing experiments based on Sephadex G-10 and G-25 are summarized in Figs 6 and 7, respectively. Compounds that absorbed at 280 nm corresponded to the void volume (G-10) or the void plus one bed volume (G-25). All fractions showed some relatively low activity against Venturia (10-30% inhibition) and did not correlate with A280 readings. The results are consistent with dilution of the active compound(s), retention of the compound(s) on the column, or overloading of the columns with poor separation of fractions. Whatever the explanation, Sephadex chromatography, under these conditions, was an inappropriate separation method for the bioactive products of interest to us.
The importance of this work relates to finding, and understanding the mechanisms of, alternatives to fungicides. The SMS filtrate might be one such alternative and is attractive because it could be produced locally by farmers from natural waste products and it is low cost, sustainable, and potentially nonthreatening to the environment. 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. Despite various nonchemical options, including the planting of disease-resistant cultivars, the mainstay of control of the apple scab disease remains fungicides. Orchardists in Wisconsin and climatically similar regions routinely apply 8-15 fungicide sprays each season, primarily to combat scab. Apples rank third nationally in percentage of acres treated with fungicide (78%) and third in total fungicide expenditures (estimated in 1987 to be $23.5 million). These sprays represent an appreciable input cost to growers. They can have substantial indirect costs in impact on the environment resulting from toxicity to nontarget organisms. Extensive use of fungicides on apple has been associated with the evolution of fungicide resistance. Fungicide residues (90% of all fungicides used in agriculture are animal oncogeous) pose a threat to human health.
This project was a basic laboratory project in organic chemistry. As originally conceived, it included field components and farmer involvement. The project was reduced from three objectives budgeted at $92,833 to one objective (chemistry of the SMS filtrate) at $40,000.
Educational & Outreach Activities
No formal outreach presentations were made during the time frame of this award. Talks on this general subject were given earlier in 1995 as documented in our previous report. A general talk, “Ecological Essentials in Biocontrol,” was given at a joint symposium on biological control sponsored by the American Phytopathological Society and the Mycological Society of America at Indianapolis in July 1996.
Areas needing additional study
A start was made towards isolating and identifying the active fractions of the compost filtrate. This work needs to continue in order to realize this goal. Once the compound(s) is known, it may be possible to produce it in commercial quantities or, alternatively, to manipulate the composting conditions to promote its production.
Cooper, T.G. 1977. The tools of biochemistry. Wiley, N.Y.
Cronin, M.J., D.S. Yohalem, R.F. Harris, and J.H. Andrews. 1996. Putative mechanism and dynamics of inhibition of the apple scab pathogen Venturia inaequalis by compost extracts. Soil Biol. Biochem. 28:1241-1249.
Levitz, S.M. and R.D. Diamond. 1985. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J. Infect. Dis. 152(5):938-945.
Promega Corp. 1996. Cell titer 967 aqueous nonradioactive cell proliferation assay. Tech. Bull. No. 245. Promega Corp, Madison, WI.
Sunshine, I. (ed). 1969. Handbook of analytical toxicology. Chemical Rubber Co., Cleveland, OH.