Publications

Microbiome organisms can degrade environmental xenobiotics including pesticides, conferring resistance to most types of pests. Some cases of pesticide resistance in insects, nematodes and weeds are now documented to be due to microbiome detoxification, and is a demonstrated possibility with rodents. Some cases of metabolic resistance may have been misattributed to pest metabolism, and not to organisms in the microbiome, because few researchers use axenic pests in studying pesticide metabolism. Instances of microbiomes evolving pesticide resistance contributing to resistance of their hosts may become more common due the erratic nature of climate change, as microbiome populations typically increase and evolve faster in stressful conditions. Conversely, microbiome organisms can be engineered to provide crops and beneficial insects with needed resistance to herbicides and insecticides, respectively, but there has not been sufficient efficacy to achieve commercial products useful at the field level, even with genetically engineered microbiome organisms.

Strip is a major constraint to food production in Africa. Most technologies developed for the eradication of Striga asiatica from the United States are not adaptable to Africa. Imazapyr and pyrithiobac coated imidazolinone-resistant (IR)-resistant maize seed prior to planting at rates of 30 to 45 g ha(-1) provide near season long control of Striga and can increase maize yields three- to fourfold if supplied with fertilizer. Slow release seed coatings reduce maize injury when post-planting rains are sparse and improve Striga control when there is excessive rainfall early in the season. Models suggest that herbicide resistance may not be a significant threat in short season maize, but vigilance in removing flowering Striga plants that are not controlled is recommended due to the known risk of evolution of resistance to these herbicides. Stacking the IR gene with glyphosate resistance and using imazapyr treated seed and applying glyphosate mid-season would provide season long Strip control and delay the evolution of resistance to both herbicides. To date, adoption of this technology has been limited by a number of factors. However, it should be included as one component of a multi-factor approach to increasing maize productivity in areas of Africa where Striga is problematic. Nomenclature: imazapyr; pyrithiobac; Strip, Striga hermonthica (Del.) Benth. STRHE; Zea mays L.

Keywords: Algae; Biofuel; Fishmeal substitute; Transgenic mitigation

Imidazolinone resistant maize seed dressed with imazapyr has been successfully used to control the parasitic weed Striga in many locations, and has begun to be commercially successful in Africa. Despite this, occasionally poor effectiveness of control had been documented in some sites that required explanation. Analysis of the data against rainfall patterns suggested that: 1. poor maize emergence occurred when there was limited rainfall at germination, possibly due to inhibition by a very high herbicide concentration too near the maize seed; 2. there was poor Striga control in seasons of very high rainfall, possibly due to herbicide washout away from the maize root zone where Striga germinates and attaches. These field assessments were matched by experiments suggesting that slow release formulations might alleviate the problems. A series of slow release formulations were synthesized based on binding imazapyr to high capacity anion exchangers and using them to coat maize seed. The best seems to be a polyethyleneimine gel. Epidemiological field data from a multitude of sites support the conclusion that the slow release formulations increased stand establishment across sites and seasons compared to the control where there was low rainfall. (C) 2009 Elsevier Ltd. All rights reserved.

Abutilon theophrasti (Malvaceae) is a widely distributed weed that infests a variety of agricultural crops. Cotton is in the same botanical family, precluding the use of selective chemical herbicides. An Abutilon-specific pathovar of Colletotrichum coccodes has been considered as a potential bioherbicide for the selective control of A. theophrasti. An evolutionary balance between microorganism and weed maintains a relatively low virulence of the microorganism, such that it does not destroy its food supply by rapidly killing its host. We overexpressed the oahA (oxaloacetate acetylhydrolase) gene in C. coccodes to increase oxalate production, as oxalate inhibits plant-defensive callose synthase by chelating calcium, an obligate co-factor of this enzyme. C. coccodes oahA transformants acidified the surrounding medium, an indication of acid production by hydrolysis of oxaloacetate. Less callose was seen visually around lesions caused by C. coccodes oahA transformants than the wild type in preliminary experiments, but this was not biochemically quantifiable. Further optimization might be obtained through gene stacking, or the addition of multiple virulence genes to this potential biocontrol agent. (C) 2009 Elsevier Ltd. All rights reserved.

It is necessary to control root parasitic weeds before or as they attach to the crop. This can only be easily achieved chemically with herbicides that are systemic, or with herbicides that are active in soil. Long-term control can only be attained if the crops do not metabolise the herbicide, i.e. have target-site resistance. Such target-site resistances have allowed foliar applications of herbicides inhibiting enol-pyruvylshikimate phosphate synthase (EPSPS) (glyphosate), acetolactate synthase (ALS) (e.g. chlorsulfuron, imazapyr) and dihydropteroate synthase (asulam) for Orobanche control in experimental conditions with various crops. Large-scale use of imazapyr as a seed dressing of imidazolinone-resistant maize has been commercialised for Striga control. Crops with two target-site resistances will be more resilient to the evolution of resistance in the parasite, if well managed. (C) 2009 Society of Chemical Industry

Genes may introgress from transgenic crops into sexually compatible wild relatives via pollen flow. This could potentially enhance the ecological expansion of the introgressed hybrids and their progeny at the cost of other plant species, or affect health of humans and animals, depending on the novel trait engineered into the crop. To prevent generating such competitive transgenic progeny, we previously used tobacco (Nicotiana tabacum L.) as a model for validating a transgenic mitigation (TM) mechanism using tandem constructs where a gene of choice is linked to mitigating genes that are positive or neutral to the crop, but deleterious to a recipient when in competition with the wild type. In the present study, attempts were made to achieve interspecific sexual hybridization between transgenic TM allotetraploid N. tabacum (pollen donor, representing a crop bearing novel traits) into one of its progenitors, diploid wild type Nicotiana sylvestris (representing a wild relative as well as a progenitor). N. sylvestris plants were manually pollinated by transgenic tobacco. The F, interspecific sexual hybrids had > 75% pollen sterility and produced no seeds. When the F, was backcrossed as the pollen donor to N. sylvestris, the progeny produced almost no germinable seeds. With such low risk of gene flow, transgenic tobacco bearing novel traits could be Cultivated with minimal concern where N. sylvestris is a native or ornamental species. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Models related to plant protection can give an appreciation of a phenomenon, and provide ideas for priorities in research, as well as suggest management strategies. The problem with models is that mistakes can be huge when inaccurate assumptions are made about key parameters, as described with three sets of models: (1) our own model predicting that five herbicide-resistant Striga plants would appear per hectare per season was based on an inaccurate assumption that heterozygotes would be selected, and a heterozygous mutation frequency was used, while a recessive mutant frequency should have been used. A revised model with a recessive mutation would predict five resistant plants per million hectares per season; (2) the model predicting that Bt resistant insects would quickly evolve in transgenic cotton and maize unless massive refuges were instituted, assuming a single binding site for the toxin and minor unfitness of resistant individuals, not realizing that resistant individuals may be extremely unfit or that Bt may have multiple targets; (3) a model that claims that unfit transgenes from crops would decimate wild relatives by swamping. The model assumed animal-type low progeny numbers, and did not consider competition for replacement, nor the infrequency that crop pollen could reach wild relatives. Models must be retrospectively critiqued, based on field data and new knowledge, and not be allowed to become accepted as being axiomatic. (c) 2005 Elsevier Ltd. All rights reserved.

Transgenic crops can interbreed with other crop cultivars or with related weeds, increasing the potential of the hybrid progeny for competition. To prevent generating competitive hybrids, we previously tested tobacco (Nicotiana tabacum L.) as a model for validating the transgenic mitigation (TM) concept using tandem constructs where a gene of choice is linked to mitigating genes that are positive or neutral to the crop, but deleterious to a recipient under competition. Here, we examine the efficacy of the TM concept at various ratios of transgenically mitigated tobacco in competition with the wild type tobacco in an ecological replacement series. The dwarf/herbicide-resistant TM transgenic plants cultivated alone under self-competition grew well and formed many more flowers than the tall wild type, which is an indication of greater reproductivity. In contrast to the wild type, TM flowering was almost completely suppressed in mixed cultures at most TM/wild type ratios up to 75% transgenic, as the TM plants were extremely unfit to reproduce. In addition, homozygous TM progeny had an even lower competitive fitness against the wild type than hemizygous/homozygous TM segregants. Thus, the TM technology was effective in reducing the risk of transgene establishment of intraspecific transgenic hybrids at different competitive levels, at the close spacing typical of weed populations.

The plants used for phytoremediation pose special biological risks, whether transgenic or not, as most of the species: (a) are semi-domesticated; (b) are introduced from other habitats; (c) can become established in the contaminated site; (d) can spread and displace native species, and/or; (e) may introgress transgenes into related species. The addition of transgenes can reduce the risks, e.g. to sterilize or render the species and hybrid offspring hypersensitive to environmental effects (heat, cold), or to a chemical that will cull the species. Various measures can contain transgenes used in phytoremediation species to prevent gene flow, but most containment technologies are both uni-directional (prevent either outflow or influx), and are inherently leaky, even a concept specifically utilizable for phytoremediation - grafting non-transgenic scions on bioremediating transgenic rootstocks. Containment mechanisms should be either stacked with each other or with "mitigator" genes. Transgenic mitigation (TM) has mitigator genes added in tandem to the desired primary transgene (genetically linked) and the mitigator genes confer traits that are positive or neutral to the desired species but are deleterious to hybrids, keeping them at very low frequencies. The concept was demonstrated in tobacco and oilseed rape with a dwarfing mitigator gene that enhanced the reproductive productivity (harvest index) when cultured alone, but eliminated it from mixed populations. Besides the mitigator genes previously proposed for crop species (sterility, no seed shattering, dwarfing, no secondary dormancy) there are genes especially appropriate for phytoremediation, e.g. overexpression of cytokinin oxidase (reduces cytokinin levels) conferring reduced shoot systems (unfitness to compete) with a more extensive root system that is better for extracting toxic wastes as well as no-flowering for vegetatively propagated species. Thus, biotechnology can be harnessed to reduce risks from both

The input costs of pesticides to control biotic constraints are often prohibitive to the subsistence farmers of Africa and seed based solutions to biotic stresses are more appropriate. Plant breeding has been highly successful in dealing with many pest problems in Africa, especially diseases, but its limited to the genes available within the crop genome. Years of breeding and studying cultural practices have not always been successful in alleviating many problems that biotechnology may be able to solve. We pinpoint the major intractable regional problems as: (1) weeds: parasitic weeds (Striga and Orobanche spp.) throughout Africa; grass weeds of wheat (Bromus and Lolium) intractable to herbicides in North Africa; (2) insect and diseases: stem borers and post-harvest grain weevils in sub-Saharan Africa; Bemesia tabaci (white fly) as the vector of the tomato leaf curl virus complex on vegetable crops in North Africa; and (3) the mycotoxins: fumonisins and aflatoxins in stored grains. Abiotic stresses may exacerbate many of these problems, and biotechnological alleviations of abiotic stress could partially allay some predicaments. Some of these constraints are already under study using biotechnological procedures, but others may require longer-term research and development to alleviate the problems. Despite the huge impacts of post-harvest weevils and of mycotoxins in grains, these issues had not been given high priority in national biotechnological programs, possibly due to a lack of knowledge of their immensity. The need for public sector involvement is accentuated for cases where immediate profits are not perceived (e.g. lowering mycotoxin levels in farmer utilized grain, which does not increase yield) but where the public weal will gain, and will be invaluable, especially where the private sector supplies genes already isolated. (C) 2004 Elsevier Ltd. All rights reserved.

Future biocontrol preparations will likely carry specific hypervirulence and other genes to keep the agents on target, overcome host defenses.

Three different methodologies have been evaluated to estimate the octanol-water partition coefficient (P-ow) of amphiphilic dithiocarbamates (DTC) (sodium salts of dithiocarbamic acids) with different substituents. The conventional shake-flask method has been used to measure P-ow coefficients. This method gave good results for dithiocarbamates with short aliphatic substituents, but it presents difficulties for measuring P-ow of DTC with long-chain substituents. The fragment contribution method PROLOGP, which uses theoretical calculations, has the advantage of its speed but it is unable to distinguish between isomeric compounds. Indirect methods show some advantages for screening purposes. This is the case of chromatographic and electrophoretic methods. Preliminary HPLC experiments showed that it was not useful for the determination of P-ow for DTC. So, micellar electrokinetic capillary chromatography (MECC) was used to estimate P-ow. Two different micellar systems, sodium dodecyl sulphate (SDS) and sodium deoxycholate (NaDCh) were tested. NaDCh was the most suitable surfactant for the analysis of DTCs. Retention measurements, at the same experimental conditions, for some compounds with known P-ow values were used to describe the correlation between P-ow and the MECC capacity factor (k'). Results showed a linear relation, which allowed the estimation of the P-ow for dithiocarbamates. The obtained data were compared with the measured P-ow coefficients obtained by the conventional shake-flask method and those calculated with the fragment contribution method PROLOGP. (C) 2002 Elsevier Science B.V. All rights reserved.

Parasitic Orobanche spp are major constraints to vegetable crop production in the Mediterranean basin (to eastern Europe) and in localized places in India, China and the USA. Transgenic target-site herbicide resistance (eg, to acetolactate synthase inhibitors) allows for movement of unmetabolized herbicide through the crop to the photosynthate sink in the parasite, as well as through the soil. We report the successful engineering of a mutant acetolactate synthase (ALS) gene into carrot, allowing control of broomrape already in heterozygotes of the first back-crossed generation, by imazapyr, an imidazolinone ALS inhibitor. It is expected that homozygotes will have higher levels of resistance. (C) 2002 Society of Chemical Industry.

Agents proposed for biocontrol of major weeds in arable row-crop agriculture have not met expectations because an evolutionary balance has developed between microorganism and weed, even when the mycoherbicide is used inundatively at very high levels (> 10(4) spores/cm(2)). Sufficient virulence can be achieved by transferring genes to the microorganism, tipping the evolutionary balance. Virulence was increased ninefold and was more rapidly effected; furthermore, the requirement for a long duration at high humidity was decreased by introducing NEP1 encoding a phytotoxic protein, to an Abutilon theophrasti-specific, weakly mycoherbicidal strain of Colletotrichum coccodes. The parent strain was at best infective on juvenile cotyledons of this intransigent weed. The transgenic strain was lethal through the three-leaf stage, a sufficient time window to control this asynchronously germinating weed. Strategies of coupling virulence genes with fail-safe mechanisms to prevent spread (due to broadened host range) and to mitigate transgene introgression into crop pathogens could be very useful in the biocontrol of major weeds in row crops.

Imazapyr and pyrithiobac dressed to seeds of imidazolinone-resistant maize effectively control Striga hermonthica (witchweed). The effects of the movement of these herbicides in the soil and in maize plants was measured on Striga germination and on legumes intercropped with maize. Striga seeds were killed when high rates of either herbicide percolated through simulated soil columns; almost no viable Striga seeds remained in the upper 10 cm, and >80% were killed at 30 cm. The herbicides applied to maize leaf whorls moved systemically out of roots, killing attached and germinating Striga. Sensitive crops (beans, cowpea, and yellow gram) were unaffected when planted at >15 cm. from maize coated with 0.4 mg a.i. pyrithiobac or 0.84 mg a.i. imazapyr seed(-1), but were severely inhibited when planted within 12 cm. Simple herbicide seed coatings are thus compatible with commonly used African intercropping systems, while facilitating maize growth and depleting the Striga seed bank. (C) 2002 Published by Elsevier Science Ltd.

Controlled inhibition of the enzyme Cu/Zn superoxide dismutase (SOD), one of the most important enzymes of the antioxidant defense system of aerobic organisms, is the subject of this paper. It is desirable in several noted medical and agricultural applications. This paper describes the synthesis of a very limited "sublibrary" of bifunctional metal chelators. The first function is the metal chelator DTC (sodium diethyldithiocarbamate (Et2DTC)), which has the sole chemical function of specific metal binding. An amphiphile (more precisely, an oligoether) is the second function and has a sole physical function: changing the physical properties of the molecule. The paper discusses novel copper chelators with variable amphiphilic properties: disubstituted dithiocarbamates (DTCs, R-1(CH2CH2O)(2)(NRCS2Na)-C-2) with one alkyl (R-2 = hexyl, octyl, decyl, or dodecyl) and one oligoether (R-1(CH2CH2O)(2), where RI = Et or Bu) substituent. The octanol-water distribution ratios of the dithiocarbamates (partition measurements by the shake-flask method) and their penetration through the liposome bilayer were measured to predict their transport behavior through biological membranes. The comparative copper binding constants and the stepwise transformation of Fe(DTC)(3) to Cu(DTC)(2) were measured and show the selectivity of the ligands for copper over iron. Differential effects of dithiocarbamates on the rates of copper removal from SOD are shown. The influence of DTCs on SOD superoxide dismutation activity was measured by the cytochrome C/xanthine/xanthine oxidase assay. The SOD dismutation activity was recovered after incubation of inactivated SOD with copper. Inhibition of peroxidase activity of SOD by different DTCs was determined using electron paramagnetic resonance spectra of the DMPO-(OH)-O-. adduct formed in solutions containing H2O2 and CuZnSOD and in the presence of the spin trap compound. The conclusions are that the addition of an oligoether chain of up to eight alipha

Parasitic broomrapes (Orobanche spp.) are major uncontrolled weeds in the Mediterranean regions of Europe and the Near East causing major losses to vegetable, grain legume, and sunflower crops. Selective herbicides alone cannot provide persistent, season-long control of these parasites, and much methyl bromide is used for their control, where affordable. Thus they are excellent targets for biocontrol. The recent progress by the COST 816 Orobanche working group in this area is reviewed herein. Natural infestation by the fly Phytomyza orobanchia of seed capsules of Orobanche crenata parasitising faba bean halved Orobanche seed production while inundative releases of adults reduced it to 5% of viable seeds. The fungi Fusarium arthrosporioides E4a and F. oxysporum E1d, as well as strains of bacteria were isolated from diseased, juvenile, Orobanche flower stalks. They are pathogenic to O. aegyptiaca, O. crenata and O. ramosa on most vegetable crops. A F. oxysporum f. sp. orthoceras was specifically pathogenic to O. cumana on sunflowers. All were used in various experiments with a modicum of success. Methods were developed to formulate isolated mycelia, which could eventually allow the use of transgenic hypervirulent pathogens in asporogenic (deletion) mutants (as a failsafe against spread). Mycotoxins were also isolated from different Fusarium and other fungal species that kill Orobanche, and are being considered for direct use, or to augment other strategies. All three Fusarium spp. used have been transformed with gus and/or gfp genes allowing tracing their movement in the environment, and opening the way to future transformations to hypervirulence.

Whole-plant, negative cross-resistance was studied in Conyza canadensis and Echinochloa crus-galli, important global weeds. Negative cross-resistance can be a most useful preemptive, cost-effective tool for delaying the evolution of resistance, as well as for resistance management, after resistant populations evolve. Seeds of triazine-resistant and -susceptible biotypes were collected in or near orchards that had been continuously treated with atrazine for more than 10 yr. Plants grown from the seeds were treated, in a greenhouse, with herbicides from the following chemical families: triazine, benzothiadiazole, phenyl-pyridazine, arylophenoxy-propionate, cyclohexanedione, phenoxycarboxylic acid, pyridine carboxylic acid, phosphinic acid, glycine phosphate, chloroacetamide, sulfonylurea, and bipyridylium. Eleven of the 18 herbicides tested exerted significant negative cross-resistance against atrazine-resistant weeds, ranging from 0.03 to 0.67 of the concentration required to affect the triazine-sensitive type. No synergism was found between bentazon and fluroxypyr in mixture on Conyza, even though both separately exerted negative cross-resistance. Using a mixture with half the amount of each component lowers the environmental effect of each component while controlling a broader spectrum of other weeds.

The production of large amounts of fungal spores for preservation and formulation are considered constraints to effective use of fungal biocontrol agents. Few successful attempts have been made to store fungal mycelia alone. Late log-phase liquid fermenter cultured isolated mycelia of two Fusarium spp. specific to the parasitic broomrapes (Orobanche spp.) from fermenters were formulated in alginate beads or in 'Stabileze' (starch, sucrose, corn oil, and silica) and air-dried. 'Stabileze' formulations exhibited 9 months and retained pathogenicity to the weed for over a year, while mycelia harvested earlier, and conidia from liquid culture exhibited >40% loss of viability. Mycelia from liquid culture yielded >20 times more colony forming units (cfu) of F. arthrosporioides and >2 times more cfu of F. oxysporum than spores at late log phase. Efficient formulation of mycelia should significantly change the economics of biocontrol. (C) 2000 Elsevier Science Ltd. All rights reserved.

Parasitic witchweeds inflict most of their damage while still underground and attached to crop roots. Most selective translocated herbicides are detoxified by crops such as corn and thus cannot reach the attached parasites. Corn with target site resistance to acetolactate synthase (ALS)-inhibiting herbicides was tested to ascertain whether these herbicides could control witchweeds, assuming that witchweeds do not obtain amino acids from the crop. Postemergence directed sprays of 27 g ae ha(-1) imazapyr 54 d after planting (DAP) delayed Striga asiatica emergence on corn in South Carolina from 3 wk (control) to 7 wk and to 11 wk when mixed with 45 g ae ha(-1) AC 263 222. Treatments with up to 71 g ae ha(-1) imazamox, and up to 71 g ae ha(-1) AC 263 222 only delayed Striga emergence by 1 wk, and 71 g ae ha(-1) imazethapyr was ineffective. ALS-inhibiting herbicides were far more effective when applied in 1-ml drenches above the seed at planting. Chlorsulfuron (10 g ai ha(-1)) and sulfometuron (50 g ai ha(-1)) were somewhat phytotoxic to Pioneer 3245IR. Rimsulfuron (30 g ai ha(-1)), metsulfuron (10 g ai ha(-1)), halosulfuron (120 g ai ha(-1)), and imazethapyr (140 g ae ha(-1)) were marginally active in Kenya, with some mature Striga hermonthica seed.-bearing capsules appearing at harvest (12 wk). Imazapyr at 15 g ae ha(-1) gave 70 to 95% suppression of capsule formation, whereas no capsules appeared at 30 g ae ha(-1). The use of imazapyr in Kenya increased the harvest index by 17% when corn plants in Striga-infested soils were kept insect and disease free by using insecticides and fungicides. Thus, complete control can be achieved at affordable cost by farmers in subsistence conditions.

A new method is described that allows the selective staining and quantification of glutathione reductases (EC 1.6.4.2) in cell extracts following acrylamide gel electrophoresis. The method is based on modifications of two previous procedures; it uses DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] to develop a yellow color on reaction with GSH formed from the NADPH-dependent reduction of oxidized glutathione. This is followed by specific counterstaining of glutathione reductase with dichlorophenolindophenol/nitroblue tetrazolium. The use of DTNB in the initial staining step inhibits enzymes other than glutathione reductase that could be stained with the dichlorphenolindophenol/nitroblue tetrazolium counterstain. Enzymes such as thioredoxin reductase, which can directly reduce DTNB with NADPH, may be nonselectively stained by this new procedure. Plant ferredoxin-thioredoxin reductase is not reduced by NADPH and therefore does not appear. Glutathione reductase stains much quicker with DTNB in the presence of GSSG than with thioredoxin reductase, allowing them to be distinguished, if parallel gels are run without GSSG, where the two enzymes react at the same rate. The sequential use of two staining procedures results in distinct, sharp permanent bands that can be used to quantify the activity of glutathione reductase while precluding artifacts generated by the previous methods. (C) 1997 Academic Press.

Sweeping generalizations promulgating dangers of herbicide-resistant crops and ''superweeds'' rarely meet scrutiny. Engineering resistance to little used, and especially multi-site herbicides can relieve selection pressures for evolution of resistance to the currently sometimes overused herbicide groups (e.g. inhibitors of ALS and ACCase). Engineering resistance to widely-used, single site herbicides into crops, increases evolutionary pressure for resistance. Such transfers could be justified for the control of parasitic weeds, where the alternatives are worse. Engineering resistance into crops that easily interbreed with weeds can sometimes be unwise. The rates of movements of such genes to weeds are unknown but gene transfer could be learnt using innocuous cases. There could still be advantages to resistant crops, without weed danger to other cropping situations (rice/red rice). Rational, case by case risk/cost/benefit analysis must replace irrational fear-striking generalizations, as agriculture can gain from many herbicide-resistant crops.

Acetolactate synthase (ALS)-inhibiting imidazolinone and sulphonylurea herbicides have been found to be effective in selectively controlling the pernicious parasitic weeds Orobanche, Striga, and Alectra spp. in some crops. This control could be effected both as whole field applications and as seed dressings. Weeds rapidly evolve resistance to this single-target, high mutation frequency group of herbicides, which usually exert heavy selection pressure. This type of rapid evolution of resistant populations was previously predicted by models, and later validated in the field in other weed and cropping situations. The selection pressure of this herbicide group may be exceedingly strong with parasitic weeds, as they are controlled by very low dose rates and the doses used are in the 'overkill' range. A good management strategy with non-parasitic weeds was to lower selection pressure, but this may be less effective with parasitic weeds. Many of the areas of the world where parasitic weeds are a problem do not use mechanical harvesters that rapidly spread weed seed. The inhomogeneous seed spread with hand harvesting necessitated developing new models. With mechanical-harvester spread of parasitic weed seeds, the best strategy is to treat the resistant crop seed with high herbicide levels (that are low per hectare) and ensure the immediate removal of the rare resistant plants that appears, before they set seed, as well as to ensure that crop seed is free of resistant seed of the parasitic weeds. Modelling suggests that resistant parasitic weed populations will evolve within two to six seasons of use without resistance management, using mechanical crop harvesting. With hand harvesting of crop, delays of about eight seasons of effective herbicide use can be expected, much longer ii the mitigating strategies of roguing resistant stalks and using parasite-free crop seed are implemented.

''After the fact'' remedial strategies are often ineffective, especially where resistance is widespread and/or refuges are large. Good pesticides are too often lost. The ''it won't happen here'' view accounts for the rarity of area-wide management strategies. The successful national example of abolishing agricultural use of DDT in Sri Lanka in favor of its use only in mosquito control precluded resistance until now. Preventive strategies must be immediately cost-effective, as well as useful in delaying resistance, or they will not be implemented. The tendency to cut dose rates is increasing resistances due to multiple-cumulative events (polygenic, amplifications, or sequential mutations within a gene). We have modeled alternating low with intermediate dose rates to delay both major gene and multiple cumulative-resistances as part of IPM. Such novel. strategies must be verified with economic and pest control data to convince farmers that they can work.

Keywords: Agronomy; Plant Sciences

1-2-Rhamnosyltransferase catalyzes the production of disaccharide-flavonoids that accumulate to 75% of dry weight. Vast energy is expended in a short time span to produce these flavonoids. The highest rhamnosyltransferase activities and immunodetected concentrations were observed in early development of Citrus grandis (pummelo), coinciding with up to 13% of fresh weight as naringin. The concentration of naringin in leaves, petals, receptacles, filaments, albedo, and flavedo drops drastically during development and correlates directly with a decrease in the activity and amounts of 1-2-rhamnosyltransferase. Anthers had minute rhamnosyltransferase activities and low concentrations of naringin. Conversely, high 1-2-rhamnosyltransferase activity and naringin concentrations appeared in both young and mature ovaries, as well as in young fruits. The total amounts of naringin in mature leaves decreased without detectable in vitro degradation of naringin in leaves. There was still a net accumulation of naringin in the albedo and flavedo of older fruit even though these tissues had only traces of 1-2-rhamnosyltransferase. Traces of enzyme synthesis in fruits, or import of the product from leaves, may explain the net accumulation of naringin in growing fruits. Unlike the late-expressed genes for glycosyltransferases in anthocyanin biosynthesis, the rhamnosyltransferases from Citrus are active only in juvenile stages of development.

There is great need to reduce pesticide inputs to save costs, reduce environmental loads, and lower the selection pressures resulting in rapid evolution of resistance. This can often be done by judiciously chosen mixtures; with synergism given the economic definition of a mixture that gives more cost-effective pest control than the sum of the separate components. Pesticides are often used alone at high rates to control the most recalcitrant pests in the infestation spectrum, where a low rate of a second pesticide can be complimentary and even synergistic. Herbicide mixtures based on field weed spectra matched to the best available compounds are more prevalent in Europe than in the U.S.A. High use rates are often required to overcome endogenous degradation pathways in certain pests, and inhibitors of the offending pathways can be biorationally chosen to synergistically lower rates. This requires a better knowledge of the reasons for the lack-of-action of pesticides in target pests, an area insufficiently researched. There are already compounds known that enhance pesticide penetration, that block pesticide degradation by monooxygenases, glutathione transferases, as well as compounds that block detoxification of the active oxygen species generated by many herbicides.

The major effort in developing pathogenic fungi into potential mycoherbicides is aimed at increasing fungal virulence to weeds without affecting crop selectivity. Specific suppression of biosynthesis of a phytoalexin derived from the shikimate pathway in Cassia obtusifolia L. by a sublethal dose (50 micromolar) of glyphosate increased susceptibility to the mycoherbicide Alternaria cassiae Jurair & Khan. Glyphosate applied with conidia suppressed phytoalexin synthesis beginning at 12 hours, but not an earlier period 8 to 10 hours after inoculation. The phytoalexin synthesis elicited by fungal inoculation was also suppressed by darkness. The magnitudes of virulence of the mycoheribicide in the dark or with glyphosate in the light were both higher than after inoculation in the light with the same concentration of conidia in the absence of glyphosate. Five times less inoculum was needed to cause disease symptoms when applied with glyphosate than without. Glyphosate did not render A. cassiae virulent on soybean (Glycine max), a crop related to the host. These results suggest that a specific inhibition of a weed's elicited defense response can be a safe way to enhance virulence and improve the efficacy of the mycoherbicide.

Keywords: Biochemistry & Molecular Biology; Pharmacology & Pharmacy

Herbicide resistant populations have evolved only in monoculture and/or mono-herbicide conditions at the rates we have previously predicted. Contrary to our original model, no populations of atrazine-resistant weeds have appeared in corn where rotations of crops and herbicides or herbicide mixtures were used. This is due to the previous lack of information about the greatly reduced fitness of the resistant individuals, which could be expressed only during rotational cycles, and also to the greater sensitivity of resistant individuals to other herbicides, pests and control practices. Our new model, presented here, describes how these factors reduce the resistant individuals to extremely low frequencies during rotation.It is not yet clear whether such factors will delay the rate of appearance of other types of resistance; e.g. the multiple resistances to grass-killing herbicides in wheat, or to inhibitors of acetolactate synthase, where the fitness of resistant biotypes may be higher.

Gressel: In the laboratory, the triazine-resistant biotypes are more sensitive to another group of photosystem 11-inhibiting herbicides (Arntzen et a1 1982), which isn’t surprising. They are also more susceptible to thiocarbamates (Laval-Martin et a1 1983), and the reason for this is unknown. We are likely to see cases that are just the opposite of the huge cross-resistances that have been described for insecticides. Does cross-resistance occur with fungicides?

The first generation of species-selective phenoxy herbicides went into use at the same time as modern antibiotics, chlorinated hydrocarbon insecticides and new fungicides and rodenticides. No resistance has appeared to the still widely used phenoxy herbicides. However, resistance has developed. in many isolated areas around the world, to s-triazine herbicides and, in a few small areas, to bipyridillium herbicides. The parameters of population genetics that govern herbicide resistance and those that govern resistance of microorganisms and fungi to other xenobiotics are the same: generations, selection pressure and fitness. Even though generation times are longer with plants. a sufficient number has passed for resistance to be apparent. The only special factor that controls the development of herbicide resistance in weeds is the spaced germination of seed throughout many seasons. The reason that resistance has not developed to the phenoxy herbicides is probably because of a low effective selection pressure; germination of susceptible seeds occurs late in the season after the herbicide is biodegraded. The highly persistent triazines. and the monthly used, highly ephemeral bipyridillium, paraquat, have exerted much stronger selection pressures. Different modes of tolerance and resistance seem to have evolved in the same species. Crop and herbicide rotation can considerably delay the possibility of resistance development until it is effectively precluded.

Clarke: The soils of interest here are ones that have not been physically standardized. How much variation in the soils occurs over short distances? For example if a soil has patches with low as well as high aluminium concentrations, would one need more than one variety of a crop to produce a profitable maximum growth in such a field? [first paragraph]

Clarke: Have the bare rocks at the Sudbury smelters in Canada always been bare or did they result from erosion after the vegetation had gone? [first paragraph]

Clarke: If the attacking fungus primes the plant so that it is ready to produce the phytoalexin response when it is attacked again, why does the plant not get itself into that ‘ready’ state all the time? What is the evolutionary explanation of the need to be primed? [first paragraph]

Keywords: Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology

Wood: On the question of genes that give resistance to different insecticides being linked, I have a few comments. In Drosophila, Tsukamoto & Ogaki (1954) and Kikkawa (1964b) found a single locus (RZ) that gave a much wider spectrum of cross-resistance than had been found in other insects. They may have been dealing with a cluster of linked genes, although irradiation experiments suggested a single mutation site (Kikkawa 1964a). [first paragraph]