Franck E. Dayan

Investigating the Mode of Action of Natural Phytotoxins

The potential use of natural phytotoxins (including allelochemicals) to develop novel tools for weed management is enhanced by the elucidation of their modes of action. This approach has not been emphasized by the agrochemical industry, although the possibility of discovering new target sites may be promising, since natural products tend to have modes of action different from synthetic herbicides. The approach of testing a compound on all known herbicide molecular target sites for commercial herbicides and other potent phytotoxins is feasible. However, this would preclude the discovery of new mechanisms of action. Discovering new target sites requires more challenging holistic approaches, initiated with physiological and biochemical studies that use whole plant assays. Studying basic plant responses to a compound may yield important clues to the specific physiological processes affected by the compounds and uncover novel mechanisms of action.

Allelopathic effects of volatile cineoles on two weedy plant species.

The volatile monoterpene analogs, 1,4-cineole and 1,8-cineole, have been identified as components of many plant essential oils, but relatively little is known about their biological activities. We compared the effects of 1,4- and 1,8-cineole on two weedy plant species by monitoring germination, mitosis, root and shoot growth, chlorophyll content, and photosynthetic efficiency. 1,4-Cineole severely inhibited growth of roots and shoots, causing cork-screw shaped morphological distortion, whereas 1,8-cineole caused a decrease in root growth and germination rates. Chlorophyll fluorescence data (yield and Fv / Fm) indicated that 1,4-cineole caused significantly higher stress (P ≤ 0.001) to photosynthesis when compared to controls. Mitotic index data showed that 1,8-cineole severely decreased (P ≤ 0.001) all stages of mitosis when compared with controls, while 1,4-cineole only caused a decrease in the prophase stage (P ≤ 0.05). Although superficially similar in structure, these two cineoles appear to have different modes of action.

Natural products as sources for new pesticides.

Natural products as pesticides have been reviewed from several perspectives in the past, but no prior treatment has examined the impact of natural product and natural product-based pesticides on the U.S. market, as a function of new active ingredient registrations with the Environmental Protection Agency (EPA). Thus, EPA registration details of new active ingredients for all conventional pesticide registrations and biopesticide registrations were compiled from the years 1997-2010. Conventional pesticide registrations and biopesticide registrations were examined both collectively and independently for all 277 new active ingredients (NAI) and subsequently categorized and sorted into four types: biological (B), natural product (NP), synthetic (S), and synthetic natural derived (SND). When examining conventional pesticides alone, the S category accounted for the majority of NAI registrations, with 78.0%, followed by SND with 14.7%, NP with 6.4%, and B with 0.9%. Biopesticides alone were dominated by NPs with 54.8%, followed by B with 44.6%, SND with 0.6%, and 0% for S. When examining conventional pesticides and biopesticides combined, NPs accounted for the majority of NAI registrations, with 35.7%, followed by S with 30.7%, B with 27.4%, and SND with 6.1%. Despite the common perception that natural products may not be the best sources for NAI as pesticides, when both conventional and biopesticides are examined collectively, and considering that NP, SND, and B all have origins from natural product research, it can be argued that their combined impact with the EPA from 1997 to 2010 accounted for 69.3% of all NAI registrations.

Invited Paper: Chemicals from nature for weed management

Abstract Natural products represent a vast repository of materials and compounds with evolved biological activity, including phytotoxicity. Some of these compounds can be used directly or as templates for herbicides. The molecular target sites of these compounds are often unique. Strategies for the discovery of these materials and compounds are outlined. Numerous examples of individual phytotoxins and crude preparations with weed management potential are provided. An example of research to find a natural product solution of a unique pest management problem (blue-green algae in aquaculture) is described. Finally, the problems associated with natural products for pest control are discussed.

Mode of Action, Localization of Production, Chemical Nature, and Activity of Sorgoleone: A Potent PSII Inhibitor in Sorghum spp. Root Exudates1

The root exudates produced by sorghums contain a biologically active constituent known as sorgoleone. Seven sorghum accessions were evaluated for their exudate components. Except for johnsongrass, which yielded 14.8 mg root exudate/g fresh root wt, sorghum accessions consistently yielded approximately 2 mg root exudate/g fresh root wt. Exudates contained four to six major components, with sorgoleone being the major component (> 85%). Three-dimensional structure analysis was performed to further characterize sorgoleones mode of action. These studies indicated that sorgoleone required about half the amount of free energy (493.8 kcal/mol) compared to plastoquinone (895.3 kcal/mol) to dock into the QB-binding site of the photosystem II complex of higher plants. Light, cryo-scanning, and transmission electron microscopy were utilized in an attempt to identify the cellular location of root exudate production. From the ultrastructure analysis, it is clear that exudate is being produced in the root hairs and being deposited between the plasmalemma and cell wall. The exact manufacturing and transport mechanism of the root exudate is still unclear. Studies were also conducted on sorgoleones soil persistence and soil activity. Soil impregnated with sorgoleone had activity against a number of plant species. Recovery rates of sorgoleone-impregnated soil ranged from 85% after 1 h to 45% after 24 h. Growth reduction of 9 14-d-old weed species was observed with foliar applications of sorgoleone. Nomenclature: Sorgoleone (2-hydroxy-5-methoxy-3-[(8′Z,11′Z)-8′,11′,14′-pentadecatriene]-p-hydroquinone); common purslane, Portulaca oleracea L. POROL; common ragweed, Ambrosia artemisiifolia L. AMBEL; cress, Lepidium sativum L. ;ns3 LEPSA; giant foxtail, Setaria faberi Herrm. SETFA; johnsongrass, Sorghum halepense (L.) Pers. SORHA; lambsquarters, Chenopodium album L. CHEAL; large crabgrass, Digitaria sanguinalis (L.) Scop. DIGSA; lettuce, Lactuca sativa L.; nightshade, Solanum spp.; purple photosynthetic bacterium, Rhodopseudomonas viridis; redroot pigweed, Amaranthus retroflexus L. AMARE; sicklepod, Cassia obtusifolia L. CASOB; spinach, Spinacea oleracea; shattercane, Sorghum bicolor (L.) Moensch SORVU; sorghum, S. bicolor (L.) Moensch SORVU; sudex, S. bicolor × Sorghum sudanense; sweet sorghum, S. bicolor ‘Della’; SX-15 and SX-17, S. bicolor × S. sudanense; 8446 and 855-F, S. bicolor (L.) Moensch SORVU; tomato, Lycopersicon esculentum L.; velvetleaf, Abutilon theophrasti Medicus ABUTH. Additional index words: Sorgoleone, root hairs, SORVU, SORHA. Abbreviations: ER, endoplasmic reticulum; HPLC, high-pressure liquid chromatography; PSII, photosystem II; QB, quinone binding; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TLC, thin-layer chromatography; 3D, three dimensional; UV, ultraviolet.

Chapter 81 – Protoporphyrinogen Oxidase-Inhibiting Herbicides

Publisher Summary Protoporphyrinogen oxidase-inhibiting herbicides, also referred to as Protox- or PPO-inhibiting herbicides, were commercialized in the 1960s. Nitrofen was the first Protox-inhibiting herbicide to be introduced for commercial use in 1964. This diphenyl ether (DPE) herbicide was eventually recognized as a relatively weak inhibitor of Protox, but it was a lead compound of an entire class of structurally related herbicides that were much more active. Several DPE herbicides have been successfully commercialized. Numerous nonoxygen-bridged compounds with this same site of action have also been discovered. Before glyphosate-resistant crops, there were several reasons for the high level of interest among agrochemical companies in the discovery and development of new Protox inhibitors. Protox is a particularly good target that can be inhibited by structurally diverse classes of herbicides, allowing for the development of a new chemistry not yet patented by other companies. These compounds are effective at very low application rates and have generally good ecotoxicology and human toxicology profiles at recommended application rates. Many are highly compatible with the trend toward no-tillage agriculture. Furthermore, unlike with some of the other herbicide classes, weeds do not readily evolve resistance at this particular site of action, although the discovery of a Protox inhibitor-resistant Amaranthus rudis biotype suggests that resistance can and will occur if the selection pressure is sufficient. The relatively low dose rates required for herbicidal activity may be far below the dose needed to adversely affect porphyrin metabolism in animals in field situations or in humans as a result of exposures in food, water, air, or during application.

Natural products as sources for new mechanisms of herbicidal action.

Abstract New mechanisms of action for herbicides are highly desirable to fight evolution of resistance in weeds, to create or exploit unique market niches, and to cope with new regulatory legislation. Comparison of the known molecular target sites of synthetic herbicides and natural phytotoxins reveals that there is little redundancy. Comparatively little effort has been expended on determination of the sites of action of phytotoxins from natural sources, suggesting that intensive study of these molecules will reveal many more novel mechanisms of action. Examples of natural products that inhibit unexploited steps in the amino acid, nucleic acid, and other biosynthetic pathways are given. AAL-toxin, hydantocidin, and various plant-derived terpenoids are discussed. Strategies and potential pitfalls of using natural products as leads for new herbicide classes are summarized.

The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase.

The inhibitory activity of 34 natural products of various structural classes on hydroxyphenylpyruvate dioxygenase (HPPD), the target site for triketone herbicides, and the mode of interaction of selected natural products were investigated. Recombinant HPPD from arabidopsis is sensitive to several classes of natural compounds including, in decreasing order of sensitivity, triketones, benzoquinones, naphthoquinones and anthraquinones. The triketone natural products acted as competitive tight-binding inhibitors, whereas the benzoquinones and naphthoquinones did not appear to bind tightly to HPPD. While these natural products may not have optimal structural features required for in vivo herbicidal activity, the differences in their kinetic behavior suggest that novel classes of HPPD inhibitors may be developed based on their structural backbones.

EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas.

BACKGROUND Resistance to glyphosate in weed species is a major challenge for the sustainability of glyphosate use in crop and non-crop systems. A glyphosate-resistant Italian ryegrass population has been identified in Arkansas. This research was conducted to elucidate its resistance mechanism. RESULTS The investigation was conducted on resistant and susceptible plants from a population in Desha County, Arkansas (Des03). The amounts of glyphosate that caused 50% overall visual injury were 7 to 13 times greater than those for susceptible plants from the same population. The EPSPS gene did not contain any point mutation that has previously been associated with resistance to glyphosate, nor were there any other mutations on the EPSPS gene unique to the Des03 resistant plants. The resistant plants had 6-fold higher basal EPSPS enzyme activities than the susceptible plants, but their I(50) values in response to glyphosate were similar. The resistant plants contained up to 25 more copies of EPSPS gene than the susceptible plants. The level of resistance to glyphosate correlated with increases in EPSPS enzyme activity and EPSPS copy number. CONCLUSION Increased EPSPS gene amplification and EPSPS enzyme activity confer resistance to glyphosate in the Des03 population. This is the first report of EPSPS gene amplification in glyphosate-resistant Italian ryegrass. Other resistance mechanism(s) may also be involved.

Somatic mutation‐mediated evolution of herbicide resistance in the nonindigenous invasive plant hydrilla (Hydrilla verticillata)

Hydrilla (Hydrilla verticillata L.f. Royle) was introduced to the surface water of Florida in the 1950s and is today one of the most serious aquatic weed problems in the USA. As a result of concerns associated with the applications of pesticides to aquatic systems, fluridone is the only USEPA‐approved chemical that provides systemic control of hydrilla. After a decrease in fluridones efficacy at controlling hydrilla, 200 Florida water bodies were sampled to determine the extent of the problem and the biological basis for the reduced efficacy. Our studies revealed that hydrilla phenotypes with two‐ to six‐fold higher fluridone resistance were present in 20 water bodies. Since fluridone is an inhibitor of the enzyme phytoene desaturase (PDS), the gene for PDS (pds) was cloned from herbicide‐susceptible and ‐resistant hydrilla plants. We report for the first time in higher plants three independent herbicide‐resistant hydrilla biotypes arising from the selection of somatic mutations at the arginine 304 codon of pds. The three PDS variants had specific activities similar to the wild‐type enzyme but were two to five times less sensitive to fluridone. In vitro activity levels of the enzymes correlated with in vivo resistance of the corresponding biotypes. As hydrilla spread rapidly to lakes across the southern United States in the past, the expansion of resistant biotypes is likely to pose significant environmental challenges in the future.