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Two of the most devastating pests in agricultural fields, mainly in Africa, the middle east and southern Europe, are the broomrapes (Orobanche spp.) and the witchweeds (Striga spp.). These flowering plants parasitize roots of many crops, exploiting photosynthate and water and significantly reducing and damaging the yields (Joel et al. 1995).

Crop domestication took place mainly through the selection for mutations that are advantageous for agriculture. In parallel, an unconscious process of selection for undesirable mutants is taking place, such as the selection for pests (weeds, insects, and pathogens) and more recently for pests that are resistant to pesticides. Experiments for selection of exceedingly rare events cannot be performed in the laboratory due to time and space constraints. Farmers, who enjoy larger facilities than scientists, are demonstrating time and again that they are capable of selecting for mutations that researchers cannot find. We are used to reports of single-nucleotide point mutations that change an amino acid, but in the report by Patzoldt et al. (1) in this issue of PNAS, we are regaled with a mutant that has lost a whole amino acid in the target protein of a xenobiotic herbicide as well as lost the gene encoding an isozyme of the target of the herbicide. People have easily made artificial deletion mutations in the laboratory by using DNA synthesizers, but naturally occurring mutations with internal deleted amino acids have not been reported. Single-nucleotide point mutations typically occur at a frequency of approximately one in a million per gene and approximately one per billion per specific nucleotide. It is thus conceivable to obtain herbicide resistance due to a single nucleotide substitution. If two independent mutations are necessary to obtain the resistance phenotype, e.g., in both alleles for recessive mutations or if two amino acids must be substituted, the frequencies go lower and jump to the square (e.g., 10−18 instead of 10−9 per nucleotide). Taking into account the possibility of gene conversion, or of partial resistance of the single mutant, the actual frequency for a double mutant might be higher. Still, these frequencies are extremely low and are probably in a range where the odds for such mutations in any mammalian species are almost nil.

Oxidant stress resistance in Conyza bonariensis and wheat (Triticum aestivum) has been correlated with high levels of antioxidant enzyme activities. Additionally, external oxidant stresses can increase a plant's levels of the enzymes of polyamine biosynthesis and polyamines, especially putrescine. We investigated the constitutive relationships between putrescine, putrescine-generating enzymes, and oxidant stress resistance in wheat and C. bonariensis. Putrescine was Constitutively elevated (2.5- to 5.7-fold) in 2-week-old-resistant wheat and C. bonariensis biotypes, which correlated with a 10- to 15-fold increase in paraquat oxidant resistance. Arginine and ornithine decarboxylase activities doubled, along with higher putrescine levels in resistant C. bonariensis. The variations in levels of putrescine and arginine and ornithine decarboxylase activities paralleled the constitutive variation of antioxidant enzymes, as well as oxidant resistance. Higher levels of both putrescine and antioxidant enzyme activities occurred during a peak of oxidant resistance at 10 weeks, when paraquat resistance in C. bonariensis plants is >50-fold greater than in the sensitive biotype. Application of 100 [mu]M putrescine can double oxidant-stress resistance in the resistant C. bonariensis. Putrescine may play an important role in contributing to the base level of oxidant resistance found at the nonpeak period.

The parasitic flowering plants Orobanche spp. (broomrapes) and Striga spp. (witchweeds) parasitize the roots of important crops, heavily reducing yields. The development of genetically-engineered herbicide-resistant crops was tested as solution to this intractable problem. Here we show that the use of three target-site resistances decimated Orobanche,demonstrating the potential of transgenic herbicide-resistant crops in the control of parasitic weeds.

In years gone by mixed farming was practiced, and weeds were kept at bay with a modicum of success by crop rotation with lots of cultivation. Crop varieties then available were far more competitive with the weeds than those presently grown. Yields were low because the crop competitiveness has a high cost in yield (cf. Herms and Mattson, 1992). Energy costs either as fuel for tractors, fodder for animals, or food for laborers were high. The advent of high yield-index crops and their greater relative responsiveness to fertilizer and water than their grandparents brought weeds, with a vengeance. A lower, noncompetitive stature is a direct outcome of high yield indices. Herbicides were and are a necessity to keep the crop yields up and the weeds down in the new, wimpy crops. Such herbicide use is wholly justified when one considers the alternative: famine. We don’t eat the straw, just the grain, so why grow straw? The herbicides made it too easy. The fields were clean, so why not grow just the high value cash crops; no need to rotate, and with no-till and herbicides, why ever cultivate and cause compaction and erosion? All this followed a scientific “razor” (principle), widely found in text books, by the 14th century English cleric Richard of Occam. As often stated Occam’s Razor is: “The simplest solution to a problem is most likely to be the best one”. Monoculture of major cash crops using a single herbicide over wide areas was the simplest solution to weed problems in crop food production. Industry, extension, and academia used Occam’s Razor and exhorted farmers to KISS, where KISS stood for “keep it simple, stupid”. My high school agronomy instructor 40 years ago ranted against monoculture, always stating that in the long run, rotation always pays. To him KISS meant: “keep it sophisticated, smarty” and time and again this has been proven right.

Despite the large number of excellent herbicides available, there are many cases where there are needs to control single weed species. These needs include: a) Weeds that have evolved resistance to a still favored herbicide. Typically, only a single weed (initially) evolves resistance under monoherbicide monoculture, and the herbicide still adequately controls all other weeds. b) There are weeds that are naturally resistant to the herbicide, and no selectivity is available (except by genetically engineering new resistance to the crop). This is typical when the weeds are within the same botanical family as the crops: e.g. Cassia in soybeans, Abutilon in cotton, barleys and bromes in wheat, etc. Parasitic weeds are often in this class; few selective herbicides control them. Recently, acetolactate synthase (ALS) inhibitors were shown to control parasites in legumes where there is a modicum of effective natural herbicide resistance (Gacia-Torres and Lopez-Granados, 1991), as well as where there is genetically-engineered herbicide resistance in crops (Joel et al., 1995). In such instances the parasitic weeds might join group (a), as weeds, including parasites rapidly evolve resistance to this herbicide group (Sibony et al., 1995). c) There are many cases where an especially high rate of a herbicide is needed to control one weed species and the rest of the weed spectrum can be controlled by lower rates. If this single weed spp. could be controlled by non-chemical means, then lower, more economical and less potentially polluting rates of the herbicide could be used. d) There are often problems with single weed spp., where herbicides cannot economically or easily be used. These include: (i) pasture weeds such as spurge and bracken, where herbicides can be uneconomical; (ii) urban situations with allergenic weeds, but spraying witht chemicals is unacceptable for any of a variety of reasons; (iii) narcotic weeds (that fit the definition of being deleterious to humanity), which need aerial treatment without damage to nearby crops.

A large body of evidence has accumulated showing that plant strains that are tolerant to a particular oxidant stress have a modicum of cross-tolerance to other oxidant stresses, whether caused by transient heat, drought, cold or oxidizing air pollutants or herbicides. We have studied a multienzyme scavenging system associated with oxidant tolerance at the metabolic and molecular levels in the model systems of pea and Conyza. Data from our experimental systems suggest that both development and subcellular compartmentalization play important roles in stress tolerance. The behavior of the chloroplast may differ from that of the cytosol. Further study of these controls is needed to acquire the understanding needed to generate oxidant stress tolerant field crops.

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.

The technologies of isolating genes conferring resistance and engineering them into crops have been worked out, and a number of field trials are under way. There seem to be few new initiatives. There are three major threats to the world food supply that are not being addressed where engineering will help: Parasitic weeds, for which there are no selective herbicides, halve yields in the third world. Engineering certain target-site resistances into the crops should allow selective control of these parasites; (2) Weeds have evolved cross resistances to all previously usable wheat selective herbicides, which can be alleviated by engineering new resistances into wheat; (3) Major maize herbicides in the triazine and chloroacetamide groups have been banned in various countries. Triazine-resistant weeds are also problems on 3 million hectares. Thus, new selectivities may have to be engineered into maize.

It is not hard to learn from pesticide use history how to prevent or delay resistance. Resistance has predominantly evolved where a single herbicide chemical or group was used annually with high selection pressure herbicides having high residual activities, or when ephemeral herbicides were repeatedly used. Resistances have not evolved where meaningful rotations or mixtures were used, despite multiple treatments. Analysis of this history allows modelling potential strategies to delay or prevent resistance. Three factors have powerful resistance delaying effects: selection pressure, fitness, and the use of herbicides exerting ‘negative cross-resistance’ (i.e. that control resistant biotypes at lower rates than the wild types). The broad cross- resistances to wheat selective herbicides that evolved in two grasses are disturbing as these metabolically mimic wheat in degrading the herbicides. The overlap of herbicide spectra precludes using negative cross-resistance for delaying resistance in monoculture wheat. Herbicide mixtures or rotations cannot be designed (without crop rotation) precluding metabolic cross-resistance. Using low selection pressure herbicides should help. It is imperative to engineer new modes of resistance into wheat to prevent major problems.

Buzzwordby Walton CookPublic Policy Press, Boalsburg, PA, USA, 2001, US$ 15.95, pbkISBN 0 9709832 0 4Until 11 September, the daily terror of existence was the possibility of being robbed by an unpredictable drug-addled individual who has no care for human life, seeking the wherewithal to support addiction. Few realize the relationship between the drug problem and the international terror groups that perpetrated the mass destruction in the USA and terror elsewhere. A tin of heroin will support a terrorist for a year, including pilot training. The Hizbolla ‘protects’ Lebanese hashish growers, the old IRA ran drugs for revenue, poppy growth in Afghanistan supports bin-Laden and the Taliban. Illicit drugs have replaced oil as a source of income for international terror, as described in this book with an emphasis on South America. If one puts aside the novelistic trimmings in this book, one has an excellent description of the problems, and the solutions of using aerial applications of biological control agents against weeds.This ‘biothriller’ is different from The Andromeda Strain 1xSee all References1 type bioscience; it deals with doable biotechnology, the thriller aspect is in the applications. The book follows the US government's decision against deploying coca-specific Fusarium oxysporum developed with federal support. It is of credit to science that the possibilities of transgenically enhancing virulence alluded to by the author is well documented (for other targets) in the various chapters of Enhancing Biocontrol Agents and Handling Risks 2xSee all References2. In the novel, a potato pathologist working on the side on diseases of coca, publicly proposes biocontrol of coca after his wife is murdered by narco-maniacs. The novel effectively treats the innate selectivity of enhanced biocontrol agents so that they specifically kill drug plants and not legitimate crops. The interactions with officialdom to obtain support, the philosophies of acceptance of modern biotechnology during the debates among the protagonist government officials, scientists and the disseminators of discrediting disinformation about biotechnology, are lessons in how to get scientific information to the uninitiated. The ‘thriller’ parts include the requisite sex, espionage, the geo-biodiversity of narco producers, traffickers, and purveyors, the purported intellectual and physical tricks of their trades, chase scenes, and paramilitary operations. Even this has scientific messages, for example, the Mafia chief explains the importance of getting incorrect information about biotechnology published before the facts: “The winner's edge goes to whoever shapes the public perception first. Establish him as a rogue scientist, willing to break all the principles of civilized society.”Biological or chemical warfare implies human targets, using organisms or chemicals that directly cause human casualties, which is far cry from chemical or biological control of plants. Herbicide use to deprive cover and food supply is not chemical warfare (although in Vietnam the herbicide was contaminated with dioxins). Biological control of plants poisonous to humanity (coca, poppies or even tobacco) indirectly deprives farmers, terrorists and traffickers of ill-gained income, but does not affect the food supply and is thus not biological warfare. The land can return to productive agriculture or forestry.Narcotic plants should be easier to control than agricultural weeds. Biocontrol agents, even augmented transgenically, are best at debilitating a target plant, severely limiting its growth. An 80% reduction of leaf area would make coca, poppy, or marijuana uneconomical; this is not good enough for crop weeds. The author's fictional approach of enhancing the level of biocontrol by using a rust fungus and another fungus that enters through the rust pustules on the leaf is known to work. The use of solid chopped grain as a slow release substrate, as well as using insect vectors “that do not brush their teeth” for inoculation of biocontrol microorganisms, are feasible. The discourses on the short- and long-term effects and implications of biocontrol, as well as ethical issues are well wrought.Hopefully, the new US-led coalition will use aerial deployment of biocontrol agents against narcotic species in the war against international terror, with the possible benefit of reducing the terror of drug-related crime. There should also be considerable peaceful fallout from drug biocontrol for deployment against other pests.

(...). The inoculum threshold of thousands of spores per square centimeter of leaf surface or tens to hundreds of spores per droplet has been expected and accepted. Evidence is presented that the high threshold concept does not hold with Alternaria cassiae and A. crassa, as one spore per 2-μl droplet was sufficient to infect plants of Cassia obtusifolia and Datura stramonium, respectively, when the droplet was an invert emulsion containig a 1μl mixture of oils and waxes on the outside and 1 μl of water, sodium alginate, and conidia on the inside. The intensity of infection always was enhanced by the emulsion (...)

We have presented data evaluating and comparing three rapid methods of estimating phytotoxicity (precursor incorporation into macromolecules, membrane permeability to fluorescein and reduction of triphenyltetrazolium chloride) with three slower methods of estimating growth (packed volume, dry wt. and cell number). We have described the advantages and drawbacks of each technique, as well as cases of a specific sensitivity of a particular technique to some toxic compounds. Despite all the differences, there was an overall correlation among all the techniques and the choice of technique depends on a desire to assay for a particular type of toxicity. As with other biological systems, these data demonstrate the problems in establishing both the time as well as the occurrence of death.

The rapid effects of the herbicide EPTC ( S -ethyl dipropylthiocarbamate) and the protectant DDCA ( N,N -diallyl-2,2-dichloroacetamide) on [2- 14 C]acetate incorporation into lipids of maize cell cultures were studied in order to determine whether they act at similar sites of lipid synthesis. DDCA, at 0.05 m M and 0.1 m M , increased the incorporation of [2- 14 C]acetate into neutral lipids of a total lipid extract within 2 h. It had very little effect on the major polar lipid constituents. DDCA altered neither the distribution of label within the major lipid classes, nor turnover of the major lipids within 2 h. EPTC (0.1 m M ) inhibited overall uptake of [2- 14 C]acetate into both neutral and polar lipids by about 30% after a 2-h incubation. The major polar lipid affected was an unidentified glycolipid. In addition to reducing the quantity of lipids synthesized, EPTC changed the lipid profile, altering the distribution of label, mainly within the neutral lipid fraction. A crude membrane fraction from maize cells contained both polar lipids and some neutral lipids. DDCA stimulated [2- 14 C]acetate incorporation into different lipid species. EPTC inhibited incorporation of [2- 14 C]acetate into both neutral and polar membrane lipids but altered significantly only its distribution into neutral lipids. DDCA (0.1 m M ) given together with EPTC (0.2 m M ) partially counteracted the effect of EPTC within the neutral lipid fraction. It is suggested that DDCA has a rapid effect on lipid synthesis, but it is probably not sufficient to account for the entire mode of action of the protectant.

We tested a hypothesis that biotypes of various species that evolved resistance to one oxidant stress might have cross tolerance to oxidant stresses from other sources. A paraquat-resistant Conyza bonariensis biotype was tolerant to SO 2 , and to atrazine, acifluorfen, and two experimental pyrimidinedione derivatives, herbicides that generate or induce the generation of oxidants in the light. Its resistance was between 1.5 and 10 times greater than that of the sensitive biotype. Two strains of Lolium perenne , one paraquat tolerant and one SO 2 tolerant, were compared for their tolerances to paraquat. Both were at least 10 times more tolerant to paraquat than the sensitive strain. The paraquat- and SO 2 -tolerant Lolium strains both had 70% more superoxide dismutase and 20% more glutathione reductase than the sensitive strain. One of four ozone-tolerant Nicotiana tabacum cultivars was 16 times more tolerant to paraquat than the ozone-sensitive cultivar. The ozone-tolerant cultivar had 60% more of the three superoxide dismutase isoenzymes present than the sensitive biotype. These results suggest that tolerance in these cases is related to a general ability to more rapidly detoxify the generated oxygen species, as we have previously shown with C. bonariensis .

The homogeneity, density and axenic nature of cell culture system s have made them an amenable tool for studying many aspects of biocide research; screening, metabolism , m ode of action and resistance. Cell cultures are “multi-homogeneous” and each homogeneous state may be analogous to different plant parts. Cultures can be homogeneously green, homogeneously white in either a homogeneously exponential or stationary phase of growth. Examples are be presented showing why no single homogeneous state should be envisaged to be analogous to a whole plant in biocide studies. Because of the possibility of more uniform herbicide application, culture systems have a use in isolatin g resistant crop strains, at higher selection pressures and much higher “plant” densities than is possible in the field, with recent successes. Protoplast fusion techniques may allow the transfer of genetic resistance between related but genetically incompatible crop species.

In an effort to find photoreceptor mutants, we isolated mutants of the fungus Trichoderma defective in blue-light induced conidiation, but still inducible by stress, an alternate stimulus. This selection criterion was based on a physiological model in which both stimuli share a common pathway for expression (output).

A system is described for measuring thermal denaturation of nucleic acid fractions directly in polyacrylamide gels. Total nucleic acids were fractionated by disc gel electrophoresis. The buffer within the gel was then exchanged for one commonly used in denaturation studies. Thermal denaturation profiles of DNA and ribosomal RNA in the gel were determined using a specially constructed Gel Carriage to position the appropriate fraction during spectrophotometric measurements. These profiles were compared with denaturation patterns obtained by classical methods in free solution; the two methods yielded similar patterns. Thermal denaturation profiles were also obtained for chloroplast light ribosomal RNA resolved by gel electrophoresis of total plant nucleic acids. Thus, denaturation patterns of individual, minor components present in complex nucleic acid mixtures can be directly measured in gels.

A triazine-resistant (R) biotype of a Graminaceous weed,Brachypodium distachyon(= Trachynia distachya), was found in the lower Galilee area of Israel after repeated treatments of roadsides with simazine [2-chloro-4,6-bis (ethylamino)-s-triazine] and atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine]. It was not affected at levels up to 3.0 kg/ha of soil-incorporated atrazine or simazine. The (R) biotype was unaffected by atrazine, as ascertained by measuring whole-leaf and plastid fluorescences and the Hill reaction. The triazine-sensitive (S) biotype was inhibited in all these reactions. The data suggest that the (R) biotype has a plastid mode of resistance common to many weed species. The (R) biotype metabolized14C-(ring)-atrazine more rapidly to both water-soluble and to chloroform-soluble compounds than the (S) biotype did, but that could not explain the immediate resistance at the plastid level. Probably there was evolution towards increased tolerance by detoxification as well as plastid level resistance.

Dikegulac sodium 2,3:4,6 di-O-isopropylidene-2-keto-L-gulonate) is a translocatable agent used for chemically 'pinching' terminal buds to break apical dominance. It does not inhibit mature leaves. Cell culture systems were developed and used to ascertain whether or not it differentially inhibits green tissue and whether or not it differentially inhibits stationary cells at the concentrations used. A differential inhibition of dividing cells was found.