0-8493-1885-8/99/$0.00+$.50 © 1999 by CRC Press LLC 20 Exploring the Potential of Biologically Active Compounds from Plants and Fungi Donna M. Gibson and Stuart B. Krasnoff CONTENTS 20.1 Introduction 20.2 Fungi as Sources of Novel Chemistries 20.2.1 Entomopathogenic Fungi 20.2.2 Screening Entomopathogenic Fungi for Novel Metabolites 20.2.3 An Example from the Entomopathogenic Fungi: New Destruxins from Aschersonia sp 20.2.3.1 Purification of Active Principles 20.2.3.2 Biological Activity of Destruxins 20.3 Plant-Derived Biopesticides 20.3.1 An Example from Plants: Oat Roots as a Source of Active Principles 20.3.1.1 Extraction of Avenacins 20.3.1.2 Comparison of Avenacin Profiles among Varieties 20.3.1.3 Developmental Expression of Avenacins 20.4 Summary References ABSTRACT Our research concentrates on screening extracts from plants and insect- pathogenic fungi for novel pesticidal chemistries with high target selectivity and environ- mental compatibility. Our search is directed to narrowly defined and relatively untapped biological organisms. We also are interested in improving techniques for extracting and quantifying known secondary metabolites to refine our understanding of the roles these compounds play in nature and agroecosystems, and their potential roles in managing crop pests and diseases. Extracts of culture broth from the fungal genus Aschersonia sp., exhib- ited insecticidal activity in a per os assay against Drosophila melanogaster. Two new cyclic depsipeptides, Destruxins A4 and A5, were isolated by bioassay-guided fractionation. These are the first biologically active metabolites reported from an Aschersonia. Concentra- tions which resulted in 50% mortality of the population of D. melanogaster (LC 50 ) were esti- mated at 41 (A4) and 52 (A5) ppm. Avenacins, triterpenoid saponins from oats, may be important determinants of disease resistance and a source of pathogen-suppressive activ- ity in soils. Analytical protocols developed to quantify avenacins were used to show inter- varietal differences in production of these compounds, their location in the root zone, and their susceptibility to turnover. © 1999 by CRC Press LLC 20.1 Introduction Growing public concerns of undesirable effects of pesticides on human health and the envi- ronment have led to the withdrawal of some commercial pesticides, increasing the vulner- ability of crops to pests and diseases. In the scientific community, concern also extends to the increasing difficulty of managing pesticide resistance. These concerns have spurred efforts to develop crop-protection methods that do not depend on chemical pesticides alone. Furthermore, these efforts include a search for new pesticides that will (1) be effica- cious, (2) have minimal environmental impact, and (3) support efforts to manage pesticide resistance. We seek compounds with high target selectivity and novel modes of action to serve these goals. The rate of discovery of new agrochemicals has declined from 1 in every 5000 com- pounds tested to 1 in every 20,000 over the past two decades. 1 This decline highlights the need to identify new sources of pesticidal chemistries. Narrow, heretofore overlooked bio- logical niches, such as entomopathogenic fungi or suppressive cover crops and soils, may be extremely useful in providing leads for biologically effective compounds. Mining these unique niches for useful natural products offers the added benefit of identifying new ways to use renewable natural resources. 20.2 Fungi as Sources of Novel Chemistries Microorganisms produce a chemically diverse array of secondary metabolites, some of which have been developed as therapeutic agents, with antibiotics ranking as premier among these metabolites. 2,3 Since the mid-1940s, over 5000 antibiotic agents have been identified, primarily from bacteria and actinomycetes. 4 Fungi are the second largest micro- bial producers of antibiotics (ca. 1600 known compounds), leading bacterial producers (ca. 950), but trailing behind the actinomycetes (ca. 4600). Of the 10 fungal antibiotics com- mercially produced, the penicillins, cephalosporins, griseofulvin, and fusidic acid have a majority of their use in clinical settings. 4 More recently fungi have been looked to as sources of novel secondary metabolites, 5 although no fungal-derived products have yet been developed as biorational pesticides. Fungal biodiversity (an estimated 1.5 million fungal species worldwide), of which only 65,000 species (or 5%) of this biota have been described 6,7 far outstrips that of the bacteria (estimated 40,000 species), 2 so fungi will likely prove to be the richest microbial source of useful natural products. The vast pool of unde- scribed fungal species represents an immense untapped resource for novel metabolites of agricultural and biomedical importance. 20.2.1 Entomopathogenic Fungi Pathogens of insects and other invertebrates have been identified in more than 100 fungal genera with >700 species recognized that attack insect hosts. 8,9 These pathogens produce a variety of biologically active secondary metabolites. For the most thoroughly investigated of these metabolites, the cyclic depsipeptidal destruxins from Metarhizium anisopliae and beauvericin from Beauveria bassiana, a wide range of biological effects have been demon- strated from suppression of insect immune responses 10 to acute toxicity. 11,12 The mitochon- drial ATPase inhibitors, leucinostatins from Paecilomyces 13 and the efrapeptins from © 1999 by CRC Press LLC Tolypocladium spp., 14 are peptides composed of unusual amino acids and terminal blocking groups. Evidence that these compounds are toxic to insects 15 suggests that they may be determinants of virulence for the entomopathogenic fungi that produce them. Metarhizium flavoviride produces viridoxins, novel diterpine derivatives of polysubstituted gamma-pyrones that exhibit insecticidal activity against Colorado potato beetle. 16 These compounds are closely related chemically to phytotoxins produced by plant pathogenic fungi in the genus Colletotrichum. One of these phytotoxins, colletochin, which differs from viridoxins by the absence of an α-hydroxy acid moiety, is much less toxic to insects than the viridoxins. 16 This preliminary observation of a structure/activity relationship suggests that M. flavoviride, an insect pathogen, produces a toxin with more directed activity against insects. This is an encouraging example of the potential for finding insecticidal compounds with high target selectivity among the toxins of entomopathogenic fungi. The extant literature on toxins of entomopathogenic fungi, although substantial, deals with relatively few species. Virtually nothing is known of the secondary chemistry of many important fungal pathogens of invertebrates, even those that have demonstrated toxicity for which toxins may play a role in pathogenesis. Conversely, where mechanisms of patho- genesis are well understood for some species, 9 the role of toxins in the infection process is still largely a matter of speculation. 17-19 It is not yet possible to draw convincing parallels to the clear-cut roles that host-selective toxins play as determinants of pathogenicity and/or virulence in some plant disease systems. 20 20.2.2 Screening Entomopathogenic Fungi for Novel Metabolites The USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) housed in Ith- aca, NY has the world’s largest and most diverse germplasm repository for fungal patho- gens of insects and other invertebrate pests affecting agriculture, encompassing nearly 5500 accessions of more than 300 fungal taxa from nearly 900 hosts and 1200 locations worldwide. 21 For our screening purposes, stock mycelial cultures are maintained on solid media. Mycelial plugs are taken to inoculate several liquid media to optimize growth and production of active extracts. Cultures are harvested for extraction usually after 10 to 30 days of growth. When sufficient biomass has been produced, fungal mycelium and culture broth are sep- arated and extracted to yield polar and nonpolar fractions for a total of four extracts from each fungal strain. Crude extracts that show sufficient activity are then purified using appropriate bioassay-guided separation techniques. In some assays biological activity can be rapidly assessed, e.g., fungicidal activity can be visualized directly on TLC plates sprayed with a fungal spore suspension in dilute agar. Once a biologically active com- pound is purified in sufficient yield (usually >1 mg), structural data can be acquired by var- ious spectrometric methods. 20.2.3 An Example from the Entomopathogenic Fungi: New Destruxins from Aschersonia sp. Members of the genus Aschersonia (Coelomomycetes, Deuteromycotina) have been inves- tigated as potential biological control agents of important aleyrodid homopteran pest spe- cies such as the citrus whitefly, Dialeurodes citri, greenhouse whitefly, Trialeurodes vaporarum, and the sweet potato whitefly, Bemisia tabaci. 22 Some species of Aschersonia have been linked to their perfect stages in the genus Hypocrella. 23 Hypocrella bambusea is known to produce a group of biologically active compounds, hypocrellins, which are photoactive perylenequinones under evaluation as potential anticancer and antiviral agents. 24 © 1999 by CRC Press LLC 20.2.3.1 Purification of Active Principles A 2 mg/ml solution in 10% sucrose of a methylene chloride extract of culture broth from an undescribed Aschersonia sp. collected as a pathogen of D. citri in the Phillipines pro- duced 80% mortality against fruit flies, Rhagoletis pomonella and Drosophila melanogaster. 25 The extract was flash chromatographed on silica gel using a methylene chloride-methanol gradient. Activity was detected in the fraction eluting in 2% MeOH in methylene chloride. This was then purified via semi-preparative reversed-phase HPLC using a 5 µm C18 (250 × 10 mm) column, eluted with acetonitrile:water (50:50). Two major components accounting for all the biological activity were isolated (Figure 20.1); a third major component also was purified, but displayed no activity. The molecular formulae C 30 H 49 N 5 O 7 , C 31 H 51 N 5 O 7 , and C 31 H 53 N 5 O 7 assigned to com- pounds 1, 2, and 3, respectively, were deduced from high resolution mass spectra. Based on proton, C13, and HMBC, and HMQC NMR experiments, structures for two novel com- pounds, destruxins A4 and A5, were assigned to compounds 1 and 2, respectively. 25 Com- pound 3 was identified as homodestruxin B which was previously reported from the plant pathogenic fungus Alternaria brassicae. 26 20.2.3.2 Biological Activity of Destruxins LC 50 values for the new destruxins in the Drosophilia melanogaster bioassay were estimated using the probit model (Figure 20.2). Destruxin A4 was more active than Destruxin A5, with an LC 50 of 41 ppm (95% confidence interval: 32 to 50 ppm) vs. 52 ppm (95% confidence interval: 44 to 63 ppm). Homodestruxin B was inactive at 400 ppm. These data are consis- tent with previous structure/activity work on the known destruxins, 27 suggesting that the olefinic side chain in the α-hydroxy acid moiety of A group of destruxins confers greater biological activity than the saturated side chain seen in the B family of destruxins. Destruxins A4 and A5 are the 28th and 29th destruxins to be characterized, and the first biologically active metabolites reported from the genus Aschersonia. 25 Various biological activities have been reported for destruxins, including phytotoxicity, 28 antitumor, 29 and antiviral 30 activity, as well as toxicity to insects. 11 Insects injected with destruxins exhibit a tetanic paralysis thought to be due to an effect on calcium channels in muscle. 18 Others have reported that destruxins degranulate insect hemocytes in vitro and, thus, may have an immunosuppressive effect, 10 although it is not clear whether destruxins mediate this pro- cess during infection in vivo. The potential role of destruxins as virulence factors for Ascher- sonia fungi should be explored in light of the economic importance of their insect hosts. FIGURE 20.1 Structures of destruxins isolated from the entomopatho- genic fungus Aschersonia sp. (From Krasnoff, S. B. et al., J. Nat. Prod. , 59, 485, 1996. With permission.) © 1999 by CRC Press LLC Due to regulatory considerations, toxigenic strains have been avoided up to now by those who seek to develop entomopathogenic fungi as biocontrol agents. These strains, however, may hold promise as pinpoint delivery systems for insect selective toxins (cf. Bt toxin from Bacillus thuringiensis) and should be evaluated in this context. 20.3 Plant-Derived Biopesticides There is a plethora of current and ethnobotanical literature listing plants with known pest control properties. Over 2000 plant species are known to have insecticidal properties, 31 and many of these plants are still used by natives in developing countries. It is estimated, how- ever, that only 5 to 15% of the 250,000 to 500,000 known plant species have been assessed for biological activity, and there is little information concerning biological properties in the vast majority of tropical plant species. 32 Potentially useful biological compounds remain undiscovered, uninvestigated, undeveloped, or underutilized from this reservoir of plant material. 33 Likewise, knowledge of production and location of these compounds during plant development may aid in effective strategies to incorporate their use for crop protec- tion, either for screening germplasm for resistance or deployment to generate suppressive soils in crop rotation strategies. Roots perform many vital functions for plants and constitute 3 to 40% of the total biomass. They share the subterranean environment with fungi, bacteria, and nematode parasites that include some of the major pests of crop plants. Plant roots are known to be rich sources of alle- lochemicals; 34 these chemicals are plant secondary compounds that exhibit biological effects on other organisms and are thought to function in plant defense. However, few ecological and FIGURE 20.2 Insecticidal effect of destruxins A4 and A5 against Drosophila melanogaster. Percentage of mortality is plotted on a probability scale and concentration of formulated destruxins on a log 10 scale. Each point represents a sample of 40 to 65 insects. Control mortality (response to 4.75% EtOH/10% sucrose solution) was zero in this experiment. The probit regression lines shown (probit mortality = 3.51 (log 10 dosage –0.66; probit mortality = 2.73 (log 10 dosage) + 0.31) were used to estimate effective doses for destruxins A4 and A5, respectively. (From Krasnoff, S. B. et al., J. Nat. Prod. , 59, 485, 1996. With permission.) © 1999 by CRC Press LLC physiological studies of the effects of allelochemicals on root parasites have been published to date, although the root zone may be the arena determining resistance or suppression. Since much of the chemical domain of plant roots remains unexplored, work in this area holds promise for the identification and characterization of natural compounds with high target selectivity and environmental compatibility for agricultural use. 20.3.1 An Example from Plants: Oat Roots as a Source of Active Principles Field studies have shown that using oats as a rotation crop can suppress parasitic infections on primary crop species. 35-37 Although susceptible to Gaeumannomyces graminis var. avenae, the form of takeall that affects oats, wheat, and barley, 38 oats are resistant to G. graminis var. tritici. Consequently oat rotations have been used successfully to reduce the incidence of var. tritici in wheat and barley. 36,39 Oats produce fungitoxins, including steroidal and triterpenoid saponins and phytoalex- ins which may play a role in their disease-resistance properties. 40-43 The avenacins, triterpe- noid saponins, are thought to account for the resistance of oats to G. graminis var. triciti. 43- 45 Avenacins were initially isolated as general antimicrobials by Maizel et al., 46 and the com- plete structural characterization of the avenacins and their relationship to takeall disease was reported by Crombie’s group. 43,44,47 Of the four structurally related avenacins, A-1 is the most fungitoxic and predominant avenacin produced by oats. Avenacins are produced in oat roots with highest concentrations in young root tips. Levels decline gradually from 12 µg/mg (1.2%) dry weight in 3-day-old roots to 0.005% in 77-day-old root tissue. 48 No appreciable differences in avenacin content in root tissue were detected in a study of 30 oat varieties, 49 although an oat species, Avena longiglumis (lacking detectable levels of avenacin and highly susceptible to G. graminis var. tritici), has been reported. 38 Since oat varieties vary in their disease resistance to a number of plant pathogens, 49-53 it was of interest to determine whether avenacins are the major biological metabolites confer- ring resistance. Our work in this system illustrates the need to carefully prepare biological materials in order to quantify differences in secondary metabolites as well as to understand the ecological and physiological relevance of active compounds at their site of action. 20.3.1.1 Extraction of Avenacins Initial results using traditional methanol extractions of root material produced highly vari- able amounts of avenacins in replicated samples, although ranges were similar to those reported previously. 44,48 A preliminary evaluation of these methanolic extracts by HPLC revealed a high level of mono-deglucosyl avenacin A-1 (mono-dG-A1), indicating that extensive hydrolysis was taking place, even under cold storage, possibly via endogenous glycosidase activity. Also, extracts were highly colored due in part to the presence of fla- vonoids and polyphenols. Immediate blanching of the harvested roots in hot 20 mM potassium metabisulfite, fol- lowed by dilution to 50% methanol, and boiling for an additional 5 min, minimized endog- enous glucosidase and polyphenol oxidase activities, as detected by reduction in enzymatic activity (data not shown), increased undeglucosylated avenacin concentrations, and a decrease in mono-dG-A1 (Figure 20.3). Yields of A-1 and A-2 are approximately 10 times higher with the improved extraction procedure, while yields of B-1 are approxi- mately 2 times higher. Decreased B-2 levels in the 20 mM potassium bisulfate/methanol (PBM) extracts may indicate that B-2 exists in the plant as a hydrolysable complex that can be released via glycosidase activity in the 50% methanol extract. © 1999 by CRC Press LLC 20.3.1.2 Comparison of Avenacin Profiles among Varieties A selected group of oat varieties was chosen to represent a range of cultivars, each with known resistance or susceptibility to soil fungi or nematodes. Oat variety Newdak is resis- tant to crown and stem rust 52 while Porter displays moderate resistance. 53 Saia shows field resistance to Pratylenchus species, especially P. penetrans (root lesion nematode). 49 Variety Pennuda is an older variety with low to moderate resistance to crown and stem rust. Five replicate extracts were prepared from 7-day-old root samples from 4 oat varieties, using both extraction procedures, and subsequently analyzed for avenacins via HPLC (Tables 20.1 and 20.2). Total active avenacins included A-2, B-2, A-1, and B-1, while total avenacins included active avenacins and mono-dG-A-1 which has some residual fungi- cidal activity. The concentrations of avenacins A-1, B-1, and A-2 were greater in all cases with the PBM extraction, and in some cases, over 10 times greater levels were detected. For example, with the variety Pennuda, 50% methanol extracts contained only 16.8 µg A-1/g fresh weight, while the PBM extract contained 704.6 µg A-1/g fresh weight. A pairwise comparison test between extraction methods showed significant differences (p = 0.05) between concentrations of individual avenacins and total active avenacins, although total avenacins did not differ between extraction methods or variety. Active avenacin concentra- tions ranged from 0.6 to 1.1 mg/g fresh weight (12 to 22 mg/g dry weight) and total avenacins ranged from 0.8 to 1.2 mg/g fresh weight (16 to 24 mg/g dry weight), or approximately 5 to FIGURE 20.3 HPLC analysis of avenacins extracted from oat roots using either 50% methanol (- - -) or heated 20 mM potassium bisulfate/methanol (PBM) (—-). Samples were analyzed using a C18 reversed phase column, mobile phase of (0.1% (NH 4 ) 2 CO 3 in H 2 O: acetonitrile (61:39), flow rate of 1 ml/min, column temperature of 21°C, and detection at both 226 and 283 nm. Avenacins are eluted in the following order: A-2, 7.89 min; B-2, 10.59 min; A-1, 12.49 min; B-1, 15.73 min; monodeglucosyl A-1, 18.71 min; and bisdeglucosyl A-1, 27.9 min. © 1999 by CRC Press LLC 8 times higher than levels reported previously (3.13 mg/g dry weight in 10-day-old roots). 48 20.3.1.3 Developmental Expression of Avenacins Utilizing the fluorescent qualities of the avenacins, several varieties were monitored by direct autofluorescence of root tissue up to 6 weeks in age. Bright zones of autofluorescence were visible at the growing root tips in all varieties examined. The most notable overall dif- ference among the varieties examined, however, was in the amount of lateral root develop- ment over time. Roots of the oat variety Saia were more highly branched with a larger number of autofluorescent root tips, compared with varieties Pennuda or Porter, indicative of greater levels of avenacins per unit area of root surface, especially at the root tips. As a preliminary test of whether avenacins were released from root surfaces, we exam- ined whether fungal growth was supported on the paper toweling used to produce oat roots for extraction. Inhibition of fungal growth was evident adjacent to the root zone; this area also exhibited blue fluorescence under UV light, indicative of avenacins and consistent with an earlier report. 54 To determine whether avenacins were present in exudates from growing roots, varieties Saia, Pennuda, and Porter were grown in a greenhouse root growth system for up to 6 weeks. Agreenhouse growth system for root exudate collection 55,56 was set up in triplicate for each variety; exudate collections were twice weekly via elution of individual chambers onto Amberlite XAD-4™ resin, which was subsequently flushed with 100 ml of distilled water to remove polar materials, then flushed with 100 ml of methanol to elute avenacins. The methanol solutions of exudates for each oat variety were pooled, evaporated to dry- ness, and yields recorded. Components of exudates from weeks 1 through 6 were separated by TLC and assessed for biological activity (Figure 20.4). Root exudates collected from TABLE 20.1 Avenacin Content of Oat Varieties (µg/gfw Roots) Prepared Under Minimal Hydrolytic Conditions Avenacin (µg/gfw Root) Variety A-1 Active Avenacins Total Avenacins Newdak 534.2 ± 151.2ab 815.0 ± 212.1ab 845.5 ± 213.0a Pennuda 704.6 ± 177.2ab 1074.4 ± 254.9a 1226.9 ± 331.7a Porter 688.1 ± 176.5a 965.1 ± 259.9ab 1204.5 ± 257.6a Saia 349.5 ± 146.6b 594.9 ± 181.4b 1018.8 ± 150.7a Note: Concentrations were determined by HPLC. TABLE 20.2 Avenacin Content of Oat Varieties (µg/gfw Roots) Prepared Under Conditions Allowing Hydrolysis Avenacin (µg/gfw Root) Variety A-1 Active Avenacins Total Avenacins Newdak 80.5 ± 13.3a 294.2 ± 53.7a 1117.8 ± 201.9a Pennuda 16.8 ± 24.6b 164.3 ± 67.1b 1041.1 ± 418.7a Porter 85.0 ± 40.3a 284.0 ± 98.9a 1484.9 ± 379.6a Saia 38.3 ± 12.9b 237.1 ± 25.3ab 944.06 ± 99.0a Note: Concentrations were determined by HPLC. © 1999 by CRC Press LLC week 2 through 4 from varieties Saia, Porter, and Pennudam produced TLC spots that co- eluted with avenacin standards A-1 and B-1. Week 1 exudate from Pennuda did not pro- duce an inhibition zone, although exudates from both Saia and Porter produced detectable inhibition zones. By week 3, exudates from all three varieties displayed strong inhibition zones at Rfs equal to those of avenacin A-1 and B-1. By weeks 5 and 6, only slight inhibition was seen with exudates from oat varieties Pennuda and Porter. Saia exudates, however, still produced a large fluorescent spot and a strong inhibition zone at the Rfs of A-1 and B-1 through week 6. FIGURE 20.4 TLC bioassay of root exudates collected at weeks 1 through 6. Exudate samples were concentrated to 20 mg/ml, then separated via TLC using chloroform/methanol/H 2 O, 13:6:1) and dried. Plate was then sprayed with 25 ml of B. sorokiniana spore suspension (4.0 to 6.0 × 10 5 spores/ml) in potato dextrose broth containing 0.1% agar and 0.1% Tween 80 and incubated in a moist container in the dark at 25°C. Inhibition was scored within 24 h. (A) Week 1 samples: lane 1, Saia; lane 2, Porter; lane 3, Pennuda; lane 4, avenacin standard; Week 2 samples: lane 5, Saia; lane 6, Porter; lane 7, Pennuda. (B) Week 3 and week 4 samples loaded as in A. (C) Week 5 and week 6 samples loaded as in A. © 1999 by CRC Press LLC Saponins, such as avenacins, may be important determinants of resistance and suppres- sive activity in crops due to their widespread occurrence in nature and reported antimicro- bial and antihelminthic activities. 57,58 Although saponins are biologically active compounds, a clear correlation between saponin content and cultivar resistance has yet to be established, as in the case of alfalfa resistance to downy mildew. 59,60 Our study indicates that the amount of saponin present in a sample may be extensively hydrolyzed during extraction. Earlier studies also are difficult to interpret because resistance may be linked to concentration or release of particular active saponins during the infection process rather than total saponin content. 61 We were able to detect avenacins in root exudates up to 6 weeks past germination in the oat variety Saia; other oat variety exudates contained ave- nacins up to 4 weeks after germination. The slow release of avenacins into the rhizosphere over an extended time should provide some measure of antimicrobial and/or antihelmin- thic protection for the plant as well as transfer suppressive activity to the soil, both during growth and from residues. Varietal differences in avenacins, the expression of avenacins along root surfaces, and the release of avenacins from the root zone during plant develop- ment, and perhaps during infections, may all contribute to overall plant resistance and sup- pression of fungal soil pathogens and nematodes in oats and related plants. 20.4 Summary The goal of our research is to complement the development of practical pest control strat- egies that minimize or eliminate chemical pesticides by discovery of novel, naturally occur- ring pesticidal chemistries. Both plants and fungi offer new opportunities as source materials in this search effort. Since much of the secondary metabolism of fungal patho- gens of invertebrates is unknown, there is great hope for successes in this endeavor, as in the case of the novel destruxins from Aschersonia. 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Thornquist, P.O., Vey, A., Johansson, M.W., and Soderhall, K., The effect of the fungal toxin destruxin E on isolated crayfish hemocytes, J Insect Physiol., 36, 785, 1990 11 Poprawski, T.J., Robert, P.-H., and Maniania, N.K., Susceptibility of the onion maggot, Delia antiqua, (Diptera: Anthomyiidae), to the mycotoxin destruxin E, Can J Entomol., 11, 801, 1985 12 Gupta, S., Krasnoff, S.B., Underwood, . A-2, B-2, A-1, and B-1, while total avenacins included active avenacins and mono-dG-A-1 which has some residual fungi- cidal activity. The concentrations of avenacins A-1, B-1, and A-2 were greater. mono-dG-A1 (Figure 20. 3). Yields of A-1 and A-2 are approximately 10 times higher with the improved extraction procedure, while yields of B-1 are approxi- mately 2 times higher. Decreased B-2. 0-8 49 3-1 88 5-8 /99/$0.00+$.50 © 1999 by CRC Press LLC 20 Exploring the Potential of Biologically Active Compounds from Plants and Fungi Donna M. Gibson and Stuart B. Krasnoff CONTENTS 20. 1