Natural and Engineered Pest Management Agents - ACS Publications

Chapter 19

Biorational Control of Weeds and Fungi with Peptides Downloaded by NORTH CAROLINA STATE UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch019

Alan R. Lax, John M. Bland, and Hurley S. Shepherd Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70179

Tentoxin is a cyclic tetrapeptide produced by thefungusAlternaria alternata that disrupts chloroplast development in most of the major weed species in soybean and johnsongrass in corn while having no effect on either of these crop species. The major impediment to the development of tentoxin as a herbicide is its limited availability because of low biosynthetic yields. Iturins are a family of cyclic octapeptides having a unique β-amino acid, iturinic acid, incorporated into the peptide backbone. Iturins have a broad antifungal activity, but low biosynthetic yields may limit their commercialization. Chemical synthesis of both molecules is possible and the production of analogues has led to meaningful structure/activity relationships. To date no analog has provided greater activity or specificity than the parent molecule for either of the two agents. Molecular genetic manipulation is considered the best strategy for the increase in production of both of these compounds for cost effective synthesis. Success of cloning of the biosynthetic genes may determine the eventual deployment of these biorational pesticides.

The cost of pest control in agriculture is staggering; direct costs of pesticides are estimated to be at least $20 billion annually, with unestimated indirect costs making the exact amount impossible to accurately state. Recent popular concern for the environment and legislation toward banning some of the more traditional pesticide compounds has led to the development of new strategies for the control of pests which include microbes as biological control organisms and the use of the biorational approach in which natural products provide a basis for the

This chapter not subject to U.S. copyright Published 1994 American Chemical Society In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

Downloaded by NORTH CAROLINA STATE UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch019

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development of highly effective compounds having great specificity and inherently lower effect on non-target populations. Herein we will focus on the biorational approach to the development of two peptide pesticides, one a herbicide based upon the fungal product, tentoxin, and the second a fungicide based on the bacterial product, iturin. Both of these molecules are highly active with the major constraint to their use in agriculture their limited availability. The commonly used herbicides today fall into relatively few chemical classes (7,2). The cost of the development of new chemistries is ever expanding in part because so many of the possible classes of chemicals have been exhausted and because of the increased cost for research and particularly registration for agricultural use. Secondary metabolites offer some particular advantage in this regard because many of the compounds produced in secondary metabolism are difficult syntheses which would likely not be tried in the more traditional "synthesize and spray" approach and also in part because of the potentially less stringent regulatory requirements facing biological and biorational compounds (7,2). Regulatory issues are increasingly a factor in the development of pesticides with future development likely to be more impacted because of public demand for low non-target toxicity, increased specificity and short environmental longevity. Although the total usage of fungicides is significantly lower than that of either herbicides or insecticides the potential for human health risks is most significant with fungicides because ninety percent (by weight) of those fungicides used are known to cause tumors in laboratory animals (5). While one cannot directly extrapolate these findings to human health, the significance of these data on possible future regulatory actions by EPA or FDA is alarming, given the increased reliance on such fungicides in the marketplace (5). Agricultural scientists must be ready to offer alternatives to traditional pesticides when these more toxic traditional pesticides are replaced. The biorational approach to pesticide design will be but one of several techniques which must be intergarted for successful pest control. Biologically active peptides represent tremendous potential as pesticides in the increasingly stringent development environment. Peptides are conspicuously under-represented as pesticides, while as a class they offer many advantages including the potential for exceptional specificity of effects, and low non-target activity (7,2). Because of their structure it is anticipated that their fate in the environment would be short lived, again environmentally advantageous, while reducing their potential for long term pest control via residual activity (4). Work in our laboratories has centered upon two cyclic peptides of diverse origin as model systems for the development of both herbicides and fungicides. The two will be discussed separately below with the emphasis on the development of iturin as a fungicide because of the work previously presented on the potential of tentoxin as a herbicide (7,2,5). However, some of the limitations to the development of tentoxin will be pointed out as illustrative and some of the potential solutions to the problems will presented. These molecules are important not only because of their anticipated use as pesticides but also because of their potential to identify new target sites in the host organisms through mode of action studies.

In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Tentoxin Tentoxin is a cyclic tetrapeptide produced by the fungus, Alternaria alternata (6), having the structure depicted in Figure 1. Tentoxin affects a large number of weeds in both soybean and corn while having no discernible effect on either of these species (see mode of action below)(7,2,7). While the synthesis of tentoxin has been achieved in the laboratory (8-11), it is a difficult synthesis, achieving a limitedfinalyield because of the reaction at the cyclization step of the synthesis. While its fate in soil is as yet uncharacterized, tentoxin should be fairly readily broken down into its constituent amino acids by microbial action.(70)

A

Β

Figure 1. Structure of tentoxin [cyclo(N-methylalanyl-leucyl-'Nmethyldehydrophenylalanyl-glycyl)] . A) Molecular model, B) chemical structure The use of tentoxin as a herbicide has been hampered by its relatively low biosynthetic yield by even selected high producing strains of Alternaria (1,2,5,12), even with its potent biological activity in the micromolar range. This is probably the major obstacle to the development of tentoxin; because of their generally potent activity and consequently low biosynthesis this promises to be problematic with other, as yet undiscovered, microbial toxins as pesticides. Little is known concerning its biosynthesis, and it has yet to be determined whether tentoxin synthesis proceeds through thioester template typical of many cyclic peptides or through an as yet undiscovered mechanism. Data from our lab (5) and that of Iiebermann and Ihn (12) indicate a mechanism different from the thioester pathway, however, this pathway has not been ruled out. Strain selection, media composition and fermentation parameters have not yielded significantly higher production of the toxin. Further discussion concerning improving biosynthesis follows in a later section. As an alternative to higher natural production, we have undertaken extensive structure activity relationship studies utilizing computer modelling to guide synthetic efforts at simpler more readily synthesized molecules which retain both the potent activity and the desired specificity (75). In


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numerous analogues synthesized and tested none of the compounds has demonstrated greater activity than the parent molecule (8,9,13). Structure Activity Studies. Structure activity studies have led us to speculate that the composition and conformation of the leucyl-dehydophenylalanyl portion of tentoxin are essential for its activity (8-11,13). We have undertaken to produce simpler compounds whose conformations at this position match the parent compound. Using a computer modelling system we have identified several analogues, such as leucyl-dehydrophenylalanyl diketopiperazine, which have the predicted conformations of the dehydrophenylalanine, with the correct planar orientation of the phenyl ring. When tested in the lettuce seedling bioassay system we saw no chlorosis nor growth inhibitory effects. However, the products of this synthesis are extremely non-polar and have exceedingly low solubilities in water. We are attempting to formulate these compounds in such a way as to render them more available for uptake in this system and to evaluate the effects of sidechain modifications to improve solubility. One such analogue was the tripeptide diketopiperazine alanyl-leucyl-N-methyl-dehydrophenylalanyl diketopiperazine, containing the diketopiperazine of the analogue mentioned above, but with a third amino acid attached to the leucyl amine, thus adding a polar amino functionality. This compound, however, was also inactive. A large number of linear analogues have also been tested for chlorosis activity because of their increased water solubility, but with little if any positive results. Because we have not been successful in the synthesis of compounds having the required activity and ease of synthesis necessary for pesticidal application it is likely that the biosynthesis of tentoxin itself will have to be improved in order to make this compound a viable product unless formulations provide the required solubility to allow activity of previously synthesized compounds. Identification of Target Sites Through Mode of Action. Tentoxin is believed to affect the development of chloroplasts in one of two ways: 1) through disruption of import into the chloroplast of at least one protein, polyphenol oxidase (14-17), or 2) inhibition of chloroplast coupling factor 1 (CF1) (18,19). It has been demonstrated that tentoxin affects the relative quantities of several other proteins in addition to polyphenol oxidase in treated chloroplasts while these have not been identified (5). Tentoxin has also been shown to inhibit the activity of one of two chloroplast envelope ATPases presumably in a similar fashion to that of CF1 (20). The selective inhibition of one of the two envelope ATPases has been invoked to explain the more or less specific inhibition of polyphenol oxidase while other chloroplast proteins which are synthesized in the cytoplasm are unaffected by tentoxin treatment (5,20). Although tentoxin is known to inhibit chloroplast coupling factor 1, there are several compelling arguments to invoke another mechanism of chloroplast disruption (15,16). Recent work indicates that sensitive species have a CF1 having a 0-subunit with a single amino acid substitution compared to resistant species. Moreover transformation of Chlamydomonas CF1 to the sensitive amino acid composition yields tentoxin sensitivity not seen in the wild type (19). Further research is need to resolve this intriguing problem and determine if the two effects are in some way connected. Resolution of this



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problem will allow further development of compounds having the desirable specificity shown by tentoxin and will allow the development of crops resistant (if they are not already immune) to the pesticide when it is introduced into the marketplace. Furthermore discovery of a target different from CF1 whose disruption has such effects on chloroplast development would provide a new molecular target for pesticide design having the specificity inherent to tentoxin. Because tentoxin specifically inhibits chloroplast development while having no discernable effect on other plant organelles or on mitochondrial coupling factor, it can be assumed that tentoxin would have no non-target effects. Even within the same genus, different species have different sensitivities to tentoxin indicating a very specific site of action with limited effect. This is one of the more compelling reasons to develop tentoxin as a herbicide given the increasing public support for safer pesticides. Iturin as a Fungicide Bacillus subtilis, the organism which produces iturin(s) has been registered for use in the prevention of storage losses of peach to brown rot and has been suggested for the protection ofricefrom infection by Aspergillus sp and thus protection from aflatoxin (21,22). This organism moreover has been suggested for the protection of soil-borne diseases of greenhouse crops (23). Because of the often different environmental windows for growth of fungi and this potential biocontrol bacterium, we have begun investigations into the use of the antibiotic iturin when the producing organism is either unable to compete or when neither bacterial nor fungal growth is desirable (24). Little is known about the mode of action of the iturins. Unlike tentoxin's unique specificities iturin affects virtually all fungi tested and we will not discuss mode of action for this compound in detail. Chemistry of the Iturins. The iturins are a family of cyclic lipopeptides having seven α-amino acids and an unusual β-amino fatty acid, iturinic acid as depicted in Figure 2. Unlike tentoxin which is composed solely of L-amino acids the iturins contain both D-and L-forms. Members of the iturin family include the iturins, bacillomycins, and mycosubtilin. In all cases the LDDLLDL sequence of the α-amino acids is kept constant, as well as the β-amino acid, D-Tyr, and DAsn . Within the family of iturins there can be amino acid substitutions which change the character of the activity of the compound. Even within a single class of iturin the β-amino fatty acid can vary in its length (13 to 17 carbons) and branching (n-, iso-, or a/ite£so-configuration) to provide a number of different compounds having the same amino acid complement but differing only in the aliphatic side chain (25,26). The structure-activity relationships of the various naturally occurring iturins and several chemically modified analogs have been partially investigated and yield some insight into the portions of the molecule which are required forfiingicidalactivity. Much research remains to fully elucidate the exact functional moieties and spatial relationships required for their antifungal properties. The naturally occurring iturins contain a D-tyrosyl moiety; chemical modification of the residue by methylation or acetylation completely abolishes the antifungal activity of the iturins. However, similar modification of 3



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the serine residue in iturin A only slightly diminishes the activity. Modification at both of the residues likewise destroys the activity. It can thus be concluded that the tyrosyl moiety is essential for fungal inhibition (27). Other naturally occurring modifications in the iturin structure are amino acid substitutions. These substitutions also provide insight into the conformational constraints on the cyclic peptide required for activity. Iturin C differs from iturin A only in the substitution of L-aspartic acid for L-asparagine at position 1, and is totally inactive against fungi. Because this residue is between the lipophilic β-amino acid and the essential tyrosine (position 2), it might be assumed that ionic interference results in loss of activity. However, bacillomycin L also contains an analogous Laspartyl residue at position 2 yet retains antifungal activity. It is therefore uncertain at this time which amino acid residue(s) are required for antifungal activity. Although naturally occurring iturins contain a mixture of eight methylene homologs and structural isomers of the iturinic acid, it had not been shown whether the aliphatic moiety is required for antifungal activity. We have previously reported the synthesis of iturin analogs with iturinic acid substituted by both β-alanine and α-ηοηνΐ-β-aspartate and demonstrated the loss of antifungal activity when the side chain was not present or included a polar functionality. HPLC separation of the natural mixture of iturin A, containing iturinic acid with n-C , anteiso-C , iso-C^, n-C , wo-C , n-C , and anteiso-C configurations, enabled the effect of β-amino acid side chain length and type of branching on antifungal activity to be determined. The relative activity of the homologues was determined on Aspergillus and Pénicillium sp. showing an increase in activity proportional to chain length and for branching type giving an order of ho > normal > anteiso. Recently we have synthesized iturin A2 and shown that its activity is identical to that of the natural compound (28). 14

15

15

16

16

17

A

Β

Figure 2. Structure of iturin A2 [cyclo(ituryl-asparagyl-D-tyrosyl-Dasparagyl-glutamyl-prolyl-D-asparagyl-seryl)]. A) Molecular model, B) chemical structure. Rationale for Development. A partial list of the fungi which are affected by iturin includes numerous species of Aspergillus, Pénicillium, and Fusarium; among them are some of the most troublesome fungi from a food safety standpoint because



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of the potent mycotoxins which they produce (24). It was originally hoped that iturin may prove to protect stored grains from aflatoxin contamination caused by species of Aspergillusflavusor parasiticus. While some of the aflatoxin producing strains in our laboratory proved to be sensitive to high concentrations of iturin, we have found that some of the producing strains are not sensitive to the toxin and that in some cases the synthesis of the aflatoxin in liquid culture at least is increased by the iturin (29). It is questionable whether iturin can be used to control all species of Aspergilli and their toxins. The most compelling arguments for using iturin per se for the protection of stored grain are ecological. Many of the fungal organisms which are associated with stored grains are more or less xerophytic organisms which thrive in dry conditions associated with stored agricultural commodities. B. subtilis however is not active under these conditions, so we felt that the compound responsible for the fungal inhibition may be better suited to the protection of such stored grains than the organism itself. We have not as yet explored the possibility that iturins, under these conditions, which may be less ideal than the liquid culture media employed, do in fact protect from infestations by the aflatoxin producing organisms. We are currently exploring this still intriguing possibility. Notwithstanding the possible failure of iturin at protecting against aflatoxin production by all isolates of the fungus, the potential for development of itruin as a fungicide in other agricultural settings is significant. Onlyfivenew fungicides introduced into the marketplace between 1975 and 1988 have gained greater than five percent of the market for any major crop (3). Increased presence of pesticides in groundwater (3) and potential health problems previously alluded to with continued use of current fungicides, create a potentially bright future for the use of iturin or its derivatives as a fungicide. Delivery of Iturin. Bacillus subtilis has been used for the protection ofricefrom aflatoxin contamination and B. subtilis suspensions have been registered for use in controlling brown rot of stone fruits (peach) during storage. In neither of these cases has the iturin itself been isolated as the active compound, but rather has been delivered through the concomitant infestation of the commodity with the producing organism itself. Whether this is the best strategy for delivery again appears to depend on ecological factors associated with the organs needing protection. Seed treatments with living spores of the bacterium may prove adequate for protecting the rhizosphere of emerging seedlings from damping off fungi which require moist conditions; however/organisms which infect the aerial portions of the plant or phylloplane are often inhibited by just those conditions in which Bacillus would thrive, while the fungi are activated under conditions in which the bacterium would sporulate and go dormant. In these cases it may prove beneficial to have a ready source of the active compound to be applied in a more traditional manner. Antibiotic Production Production of iturin in culture has been more studied than that of tentoxin, while none of the biochemical steps per se has been identified. Optimal production of



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iturin is afforded by the inclusion of aspartic acid into the medium, but the highest reported synthesis of iturin in culture to date is reported to be around 60 mg per liter after 40 hours of fermentation (30). Tentoxin biosynthesis has been similarly shown to be affected by medium composition, and culture conditions but to date the highest expected biosynthetic yields are around 90 mg per liter (31). Tentoxin production is found to be maximal after 6 to 14 days depending on the isolate of the fungus used. Tentoxin biosynthesis on a large scale is also hampered by the fact that still culture of the fungus is required. Aeration provided by swirling or bubbling air inhibits the production of the toxin making commercial fermentations of this toxin seem prohibitive at this time (31). The biosynthesis of tentoxin has been correlated with the presence of a fungal virus; any cultural manipulation which reduces the presence of the virus similarly affects the production of the toxin, while those cultural conditions which were reported to prevent the production of the toxin reduce the presence of the fungal viruses. Purification of the virus followed by electrophoretic separation of the coat proteins and western blotting using antibody produced against tentoxin showed antigenic similarities (5). We have been unable to show directly the relationship between the virus and tentoxin production since the virus has so far shown no infectivity toward non-virus containing strains, nor have we been able to cure the fungus of the virus; after removal of the conditions which reduce it the virus returns to previous levels (5,31). While there is precedence in the literature for the production of cyclic peptides from longer precursors which are subsequently cleaved and cyclized, the majority of cyclic peptides are synthesized using thioester templates such as that involved in the biosynthesis of tyrocidine and gramicidin by Bacillus brevis (32). The enzymes for a large number of these multifunctional peptide synthetase have been isolated and characterized. There are regions of homology in many of these peptide syntetases (32) which may make them amenable to molecular genetic probing for analogous enzymes in both tentoxin and itruin production. Because of the low biosynthetic potential of both of these important biologically active peptides we have begun an intensive search in our laboratories for analogous genes for their production with the hope of increasing the production of these compounds through genetic manipulation. Work has already begun on the identification of the genes for the production of iturin with promising results (34). Whether success in this area provides sufficient material for pesticide production remains to be seen. Conclusions Much research remains to be conducted prior to the release of either of these potential products into the marketplace. Because of their potent activity and specificity which should provide good control of the target pests with fewer side effects seen with more traditional pesticides there is justification for continued investigations. Research into the genetics of the production of these compounds may permit economical production while at the same time offers the possibility to determine the role of tentoxin in the phytopathogenicity of Alternaria alternata and the role of iturin in control of such diseases by Bacillus isolates. Development of these promising compounds would surely lead the way to


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discovery of other potentially useful peptide toxins and further biorational pest control research. It is our hope that this work stimulates such discovery and in some small way guides the development of such products in the near future. Literature Cited


1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. la 17. 18. 19. 20. 21. 22. 23. 24.

Duke, S.O. Rev. Weed Sci, 1986, 2, 15-44. Duke, S.O.; Lydon, J. Weed Technology, 1987,1,122-128. Anonymous, In Alternative Agriculture, National Research Council, 1989. National Academy Press, Washington, DC. 448pp. Edwards, J.V.; Dailey, O.D.; Bland, J.M.; Cutler, H.G. In American Chemical Society-Agrochemicals, Proceedings, 194th National Meeting, August 1987, New Orleans, Louisiana. Chapter 3, pp 35-56. Lax, A.R.; Shepherd, H.S., In American Chemical Society-Agrochemicals, Proceedings, 194th National Meeting, August 1987, New Orleans, Louisiana. Chapter 2, pp24-34. Templeton, G.E., In Microbial Toxins, Kadis, S.; Ciegler, A ; Ajl, S. J. eds.; Academic: New York, 1972; chapter 7, pp 169-192. Rich, D.H., In Toxins in Plant Disease. Mechanism of Action; Durbin, R.D., Ed.; Academic: New York, 1981. Physiological Ecology Series, Chapter 8. Rich, D. H.; Mathiaparanam, P., Tetrahedron Lett., 1974, 15, 4037-40. Rich, D. H.; Bhatnagar, P.; Mathiaparanam, P.; Tarn, J. P.J.Org. Chem., 1978, 43, 296-302. Edwards, J.V.; Lax, AR.; Lillehoj, E.B.; Boudreaux, G.J. Int. J. Peptide Protein Research, 1986, 28, 603-612. Jacquier, R.; Verducci, J. Tetrahedron Lett., 1984, 25, 2775-2778. Liebermann, B.; Inn, W. J. Basic Microbiol., 1988, 28, 63-70. Bland, J.M.; Edwards, J.V.; Eaton, S.R.; Lax, AR. Pesticide Science, 1993. in press. Lax, AR.; Vaughn, K.C., Sisson, V.A; Templeton, G.E. Photosynth Res., 1985, 6, 113-120. Lax, AR.; Vaughn, K.C. Physiol Plant., 1986, 68, 384-391. Vaughn, K.C.; Duke, S.O. Physiol Plant., 1981, 53, 421-428. Vaughn, K.C.; Duke, S.O. Physiol Plant., 1984, 60, 257-261. Steele, J.A; Uchytil, T.F.; Durbin, R.D.; Bhatnagar, P.; Rich, D.H. Proc. Nat. Acad Sci U.S.A., 1976, 73, 2245-2248. Avni, A ; Anderson, J.D.; Holland, N.; Rochaix, J-D., Gromet-Elhanan, Z.; Edelman, M. Science, 1992, 257, 1245-1247. Lax, AR. Plant Physiol, 1987, 83S, 53. Pusey, P.L.; Wilson, C.L. 1988 United States Patent, 4,764,371. Klich, M.A; Lax, AR.; Bland, J.M.; Scharfenstein, L.A Mycopathologia, 1993, in press. Bochow, H. Developments in Agricult. and Managed Forest Ecology, 1990, 23, 236-240. Peypoux, F.; Guinand, M.; Michel, G.; Delcambe, L.; Das, B.; Lederer, E. Biochemistry, 1978, 17, 3992-3996.


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Isogai, A, Takayama, S.; Murakoshi, S.; Suzuki, A Tetrahedron Lett, 1982, 23, 3065-3068. Besson, F.; Peypoux, F.; Michel, G. J. Antibiotics, 1979, 32, 828-833. Phae, C-G.; Shoda, M. J. Fermentation Bioengineering, 1991, 71, 118-121. Matsuno, Y.; Hiraoka, H.; Ano, T.; Shoda, M. FEMS Microbiol. Lett, 1990, 67, 227-230. Matsuno, Y.; Ano, T; Shoda, M. J. Gen.Appl.Microbiol, 1992, 38, 505-509. Bland, J.M.; Lax, AR.; Klich, M.A Peptides 1992: Proceedings, TwentySecond European Peptide Symposium, 1992, 332-333. Shepherd, H.S. J. Virol., 1988, 62, 3888-389. Krause, M.; Marahiel, M.A; von Dohren, H.; Kleinkauf, H.J.Bacteriol., 1985, 162, 1120-1125. Mittenhuber, G.; Weckermann, R.; Marahiel, M.A J. Bacteriol., 1989, 171, 4881-4887. Matsuno, Y.; Hitomi, T.; Ano, T.; Shoda, M. J. Gen.Appl.Microbiol, 1992, 38, 13-21.

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