Publications

BACKGROUND: The widespread evolution of pesticide resistance poses a significant challenge to current agriculture, necessitating the discovery of molecules with new modes of action. Despite extensive efforts, no major molecules with new modes of action have been commercialized for decades. Most pesticides function by binding to specific pockets on target enzymes, enabling a single target site mutation to confer resistance. An alternative approach is the disruption of proteinprotein interactions (PPI), which require complementary mutations on both interacting partners for resistance to occur. Thus, our aim is the discovery and design of small-molecule inhibitors that target the interface of the PPI complex of O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT), key obligatory interacting plant enzymes involved in the biosynthesis of the amino acid cysteine. RESULTS: By employing in silico filtering techniques on a virtual library of 30 million small molecules, we identified initial hits capable of binding OASS and interfering with its interaction with a peptide derived from SAT with a half-maximal inhibitory concentration (IC₅₀) of 34 μm. Subsequently, we conducted molecular chemical optimizations, generating an early lead molecule (PJ4) with an IC₅₀ value of 4 μm. PJ4 successfully inhibited the germination of Arabidopsis thaliana seedlings and inhibited clover growth in a pre-emergence application at an effective concentration of 4.6 kg ha⁻¹. CONCLUSION: These new compounds described herein can serve as promising leads for further optimization as herbicides with a new mode-of-action. This technology can be used for discovering new modes of action chemicals inhibiting all pest groups.

Societal Impact Statement: Given the rapidly increasing drought and temperature stresses associated with climate change, innovative approaches for food security are imperative. One understudied opportunity is using feral cropsplants that have escaped and persisted without cultivationas a source of genetic diversity, which could build resilience in domesticated conspecifics. In some cases, however, feral plants vigorously compete with crops as weeds, challenging food security. By bridging historically siloed ecological, agronomic, and evolutionary lines of inquiry into feral crops, there is the opportunity to improve food security and understand this relatively understudied anthropogenic phenomenon. Summary: The phenomenon of feral crops, that is, free-living populations that have established outside cultivation, is understudied. Some researchers focus on the negative consequences of domestication, whereas others assert that feral populations may serve as useful pools of genetic diversity for future crop improvement. Although research on feral crops and the process of feralization has advanced rapidly in the last two decades, generalizable insights have been limited by a lack of comparative research across crop species and other factors. To improve international coordination of research on this topic, we summarize the current state of feralization research and chart a course for future study by consolidating outstanding questions in the field. These questions, which emerged from the colloquium \u201cDarwins' reversals: What we now know about Feralization and Crop Wild Relatives\u201d at the BOTANY 2021 conference, fall into seven categories that span both basic and applied research: (1) definitions and drivers of ferality, (2) genetic architecture and pathway, (3) evolutionary history and biogeography, (4) agronomy and breeding, (5) fundamental and applied ecology, (6) collecting and conservation, and (7) taxonomy and best practices. These questions serve as a basis for ferality researchers to coordinate research in these areas, potentially resulting in major contributions to food security in the face of climate change.

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.

The different levels of tolerance of crops and weeds to specific herbicides is the basis of herbicide selectivity; this is not the topic discussed in this chapter. Many weeds that were previously controlled by specific herbicides have evolved biotypes with increased levels of tolerance (Figure 1). ^{1 - 3} Some of these biotypes can have a seemingly absolute resistance; they can be treated with saturated solutions of herbicides that have little or no effect on the weed. Some resistant biotypes are controlled by concentrations of herbicides, but at application rates many times greater than the recommended agricultural dose of the herbicide. Biotypes with increased tolerance to herbicides are more common than resistant biotypes. ^{2 - 4} Tolerant biotypes are only partially affected by the herbicides: their yield is lowered but they complete a life cycle or there is only partial mortality. There can be many shapes of dose-response curve for tolerance (Figure 1) depending on the mode of tolerance and the number of sites of action and the proportion of tolerant individuals in the population. Unfortunately, the simple distinction between resistance and tolerance is often not understood and the literature is replete with references to resistance which are but minor increases in tolerance. True resistance has only appeared in the field to the s-triazines. to paraquat, to trifluralin. and to dichlofop-methyl. Selection for tolerance and resistance to many other herbicides has been possible in the laboratory, using algae ^{5 - 10} or higher plant tissue cultures. ^{11 - 13}.

Genes regularly move within species, to/from crops, as well as to their con- specific progenitors, feral and weedy forms (vertical' gene flow). Genes occasionally move to/from crops and their distantly related, hardly sexually interbreeding relatives, within a genus or among closely related genera (diagonal gene flow). Regulators have singled out transgene flow as an issue, yet non-transgenic herbicide resistance traits pose equal problems, which cannot be mitigated. The risks are quite different from genes flowing to natural (wild) ecosystems versus ruderal and agroecosystems. Transgenic herbicide resistance poses a major risk if introgressed into weedy relatives; disease and insect resistance less so. Technologies have been proposed to contain genes within crops (chloroplast transformation, male sterility) that imperfectly prevent gene flow by pollen to the wild. Containment does not prevent related weeds from pollinating crops. Repeated backcrossing with weeds as pollen parents results in gene establishment in the weeds. Transgenic mitigation relies on coupling crop protection traits in a tandem construct with traits that lower the fitness of the related weeds. Mitigation traits can be morphological (dwarfing, no seed shatter) or chemical (sensitivity to a chemical used later in a rotation). Tandem mitigation traits are genetically linked and will move together. Mitigation traits can also be spread by inserting them in multicopy transposons which disperse faster than the crop protection genes in related weeds. Thus, there are gene flow risks mainly to weeds from some crop protection traits; risks that can and should be dealt with. (c) 2014 Society of Chemical Industry

As there are industrial and societal interests in cultivating algae on a large scale, there inevitably will be spills of cultured algae into natural ecosystems. The assessment of environmental risks of such spills is especially hard for cultured, non-native microalgae species due to the "paradox of the plankton", the paradox that large numbers of species with varying degrees of fitness co-exist in natural ecosystems in an unpredictable, fluctuating species balance. The risk analysis may be more straightforward for special cases, e.g. transgenic or mutated strains of common, indigenous species because their behavior can be compared to their wild types. Risk assessment can be based on presently used Good Industrial Large Scale Practice considerations, particularly the use of mitigating traits that severely decrease fitness. Some desirable introduced genes may have some unfitness in natural ecosystems and can be coupled with antisense or RNAi suppressed genes to mitigate genes that increase fitness. The most stringent mitigation systems are needed especially for non-native species and can utilize deletion mutations (e.g. in carbon capture or nitrate utilization) that allow cultivation only in artificial systems and are lethal to the algae in nature.

Weedy Orobanchaceae often change host specificity at a rapid rate. These changes and the equally fast reversions to the previous host specificity are hard to explain by Mendelian mutations. The altered phenotype could be due to epigenetic reprogramming of gene expression. Conventional DNA analysis technologies can be used to ascertain whether epigenetic changes are involved. Epigenetic changes are caused by higher level alterations to the DNA structure such as histone modifications or cytosine methylation, but only the latter is maintained through meiosis in plants. Alternative splicing of mRNA could theoretically also be responsible for some cases. Methods for ascertaining changes in methylation patterns that could be adapted to determine whether changes are epigenetic are briefly described. Crosses can also be used to ascertain whether the adaptation to new hosts is genetic, although results may be ambiguous and could be confounded by reversion if the changed host specificity was epigenetic.

Fishmeal had been a major feed source, mainly for monogastric animals, as well as dairy cattle, and is required for many fish species in aquaculture, and fish oil is a major source of omega-3 fatty acids. Due to diminishing fish stocks, it is being replaced by vegetable protein supplemented with synthetic amino acids. Transgenically domesticated algae could be engineered to be enriched in specific amino acids, have improved digestibility, and contain replacements for expensive feed additives, becoming a cost-effective replacement for fishmeal and fish oil. The enrichment must be in algae engineered to obtain stable, contamination-free cultures that are transgenically mitigated to be unable to survive outside of cultivation. If culture conditions can be rendered cost-effective, such cultures could compete with soybeans/oil palm and be the next technological breakthrough to overcome Malthus' predictions of insufficient food for humanity, as the feed material can be cultivated on desert lands or floating on bodies of water, using seawater and thus augmenting agriculture without competing for land and freshwater.

Keywords: Algae; Biofuel; Fishmeal substitute; Transgenic mitigation

A greater number of, and more varied, modes of resistance have evolved in weeds than in other pests because the usage of herbicides is far more extensive than the usage of other pesticides, and because weed seed output is so great. The discovery and development of selective herbicides are more problematic than those of insecticides and fungicides, as these must only differentiate between plant and insect or pathogen. Herbicides are typically selective between plants, meaning that before deployment there are already some crops possessing natural herbicide resistance that weeds could evolve. The concepts of the evolution of resistance and the mechanisms of delaying resistance have evolved as nature has continually evolved new types of resistance. Major gene target-site mutations were the first types to evolve, with initial consideration devoted mainly to them, but slowly 'creeping' resistance, gradually accruing increasing levels of resistance, has become a major force owing to an incremental accumulation of genetic changes in weed populations. Weeds have evolved mechanisms unknown even in antibiotic as well as other drug and pesticide resistances. It is even possible that cases of epigenetic 'remembered' resistances may have appeared.

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.

Transgenic herbicide-resistant rice is needed to control weeds that have evolved herbicide resistance, as well as for the weedy (feral, red) rice problem, which has been exacerbated by shifting to direct seeding throughout the world-firstly in Europe and the Americas, and now in Asia, as well as in parts of Africa. Transplanting had been the major method of weedy rice control. Experience with imidazolinone-resistant rice shows that gene flow to weedy rice is rapid, negating the utility of the technology. Transgenic technologies are available that can contain herbicide resistance within the crop (cleistogamy, male sterility, targeting to chloroplast genome, etc.), but such technologies are leaky. Mitigation technologies tandemly couple (genetically link) the gene of choice (herbicide resistance) with mitigation genes that are neutral or good for the crop, but render hybrids with weedy rice and their offspring unfit to compete. Mitigation genes confer traits such as non-shattering, dwarfism, no secondary dormancy and herbicide sensitivity. It is proposed to use glyphosate and glufosinate resistances separately as genes of choice, and glufosinate, glyphosate and bentazone susceptibilities as mitigating genes, with a six-season rotation where each stage kills transgenic crop volunteers and transgenic crop x weed hybrids from the previous season. (C) 2009 Society of Chemical Industry

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 conditionswith 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.

The greatest cost to the farmer in time and variable costs is control of weeds (Highly Invasive Vegetation = HIV). Despite farmer efforts, weeds still result in the greatest crop losses of all biotic constraints. The costs due to this HIV are greatest in the parts of the developing world where manual labor (more typically \u201cfemanual\u201d) is used, and the populations are becoming more aged (due to youth abandoning agriculture), or more feeble due to debilitating diseases such as malaria and HIV-AIDS. Two case studies are presented: the previously intractable problems with Striga (witchweed) species in Africa; and rice cultivation and the emerging problems with the labor-saving shift to direct seeding, which has resulted in outbreaks of a weedy/feral form of rice, as well as herbicide-resistant Echinochloa spp. Biotechnology has much to offer as part of the solution to these major HIV problems, whether as transgenic herbicide resistant crops, or as weed resistant crops or through transgenically enhanced weed-specific biocontrol agents. Where neces-sary, transgenic tricks will also be needed in many cases to prevent transgene flow from crop to related weedy relatives, and possible technologies are described. Ever since the advent of agriculture, solutions to crop protection problems have been effective but ephemeral, and new solutions will be needed in the future. Using mixed solutions leads to solutions lasting synergistically longer, so efforts should be made to use mixed technologies to extend the lifetime of the next generation of sorely needed solutions to the problems of HIV.

Inundative mycoherbicides have not been successful in weed control in row crops, probably due to evolutionary barriers, and adding virulence factors was considered essential. Exogenous addition of the products of various geneswas used to ascertain synergy as a prelude to adding them transgenically. Transgenically over-expressing single "soft" genes (host lytic enzymes such as pectinase, cellulase and expansins, or natural hormones such as IAA), or "hard" genes encoding toxins such as NEP1 and CP1, has enhanced virulence, but not enough. Gene stacking to obtain synergies among the various genes is considered a top priority, both to achieve sufficient virulence and to delay the evolution of weed resistance to the fungal pathogens.

Parasitic weeds are major contributors to hunger, malnutrition, and food insecurity across sub-Saharan and northern Africa by reducing crop yields in half. Over 20 million hectares of cereal grains in sub-Saharan Africa are infested with Striga (witchweed). Food production losses due to Striga in African countries range from 20% to 90%, amounting to over 10 million tons of food lost annually. The control options for Striga are currently ineffective and management possibilities for these weeds are urgently needed. The research progress with a specific forma speciales of Fusarium oxysporum as a biological control for Striga in Africa illustrates the potential to positively impact many lives and improve the health and livelihood of rural and urban poor. Can F. oxysporum wild type be the Achilles heel of Striga, or do we need enhanced biocontrol to achieve rapid, safe, cost-effective solutions for this major biotic constraint to food production in Africa?

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.

Introgressive hybridization has played a crucial role in the evolution of many plant species, especially polyploids. The duplicated genetic material and wide geographical distribution facilitate hybridization and introgression among polyploid species having either homologous or homoeologous genomes. Such introgression may lead to the production of recombinant genomes that are more difficult to form at the diploid level. Crop genes that have introgressed into wild relatives can increase the capability of the wild relatives to adapt to agricultural environments and compete with crops or to compete with other wild species. Although the transfer of genes from crops into their conspecific immediate wild progenitors has been reported, little is known about spontaneous gene movement from crops to more distantly related species. We describe recent spontaneous DNA introgression from domesticated polyploid wheat into distantly related, wild tetraploid Aegilops peregrina (syn. Aegilops variabilis) and the stabilization of this sequence in wild populations despite not having homologous chromosomes. Our results show that DNA can spontaneously introgress between homoeologous genomes of species of the tribe Triticeae and, in the case of crop-wild relatives, possibly enrich the wild population. These results also emphasize the need for fail-safe mechanisms in transgenic crops to prevent gene flow where there may be ecological risks.

At a time when much of humanity is already but one failed harvest removed from starvation, we cannot afford to ignore any potential danger to food security, especially when that danger poses a threat to rice, the staff of life for so much of the world. Crop Ferality and Volunteerism brings together research pioneers from various disciplines including the crop, plant, and weed sciences to discuss crop ferality and volunteerism. The book provides thorough coverage of crop and plant molecular biology and genetics as it pertains to ferality and weeds. In an exhaustive effort to provide complete and highly useful coverage of this impending crisis, the authors go beyond the science of the problem to discuss the potential economic and social impact of crop ferality, particularly in relationship to rice. Readers will discover a wealth of well-organized and well-written material about the overall biology and management of weeds and weedy crops. Many examples of ferality are considered, because, as the editor states, readers will discover that there is no unified theory of ferality. Thanks to the incredible diversity of the plant kingdom, \u201cSurprises abound in every chapter.\u201d.

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 non-transgenic and transgenic phytoremediation species.

Keywords: HIGHER-PLANTS; PLASTID TRANSFORMATION; PATERNAL INHERITANCE; TANDEM CONSTRUCTS; TRANSGENIC PLANTS; WILD RELATIVES; INDIAN MUSTARD; EXPRESSION; MAIZE; PHYTOREMEDIATION

Some transgenic crops can introgress genes into other varieties of the crop, to related weeds or themselves remain as 'volunteer' weeds, potentially enhancing the invasiveness or weediness of the resulting offspring. The presently suggested mechanisms for transgene containment allow low frequency of gene release (leakage), requiring the mitigation of continued spread. Transgenic mitigation (TM), where a desired primary gene is tandemly coupled with mitigating genes that are positive or neutral to the crop but deleterious to hybrids and their progeny, was tested as a mechanism to mitigate transgene introgression. Dwarfism, which typically increases crop yield while decreasing the ability to compete, was used as a mitigator. A construct of a dominant ahas^R (acetohydroxy acid synthase) gene conferring herbicide resistance in tandem with the semidominant mitigator dwarfing Δgai (gibberellic acid-insensitive) gene was transformed into tobacco (Nicotiana tabacum). The integration and the phenotypic stability of the tandemly linked ahas^R and Δgai genomic inserts in later generations were confirmed by polymerase chain reaction. The hemizygous semidwarf imazapyr-resistant TM T₁ (= BC₁) transgenic plants were weak competitors when cocultivated with wild type segregants under greenhouse conditions and without using the herbicide. The competition was most intense at close spacings typical of weed offspring. Most dwarf plants interspersed with wild type died at 1-cm, > 70% at 2.5-cm and 45% at 5-cm spacing, and the dwarf survivors formed no flowers. At 10-cm spacing, where few TM plants died, only those TM plants growing at the periphery of the large cultivation containers formed flowers, after the wild type plants terminated growth. The highest reproductive TM fitness relative to the wild type was 17%. The results demonstrate the suppression of crop-weed hybrids when competing with wild type weeds, or such crops as volunteer weeds, in seasons when the selector (herbicide) is not used. The linked unfitness would be continuously manifested in future generations, keeping the transgene at a low frequency.

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

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.

Infection by many fungi activates a variety of calcium dependent defenses in the hosts, slowing or suppressing the attacker and limiting the efficacy of mycoherbicides. The calcium requirement for fungal growth is so low that it could only be implied based on fungi containing calcium-dependent signaling enzymes. Analytical grade media contain

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 10cm, and > 80% were killed at 30cm. 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 > 15cm from maize coated with 0.4mg a.i. pyrithiobac or 0.84mg a.i. imazapyr seed^-1, but were severely inhibited when planted within 12cm. Simple herbicide seed coatings are thus compatible with commonly used African intercropping systems, while facilitating maize growth and depleting the Striga seed bank.

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.

Although there are reports of isolation of mycoherbicidal pathogens attacking the widespread broomrapes (Orobanche spp.) that parasitize legumes and vegetables, none is in use or available. This is despite there being no good method of controlling broomrapes in most crops other than by preplant fumigation with methyl bromide. Two highly parasitic fungi, Fusarium arthrosporioides strain E4a (CNCM 1-164) and F. oxysporum strain Eld (CNCM 1-1622), were isolated from nearly 100 organisms found on diseased, juvenile, emerging Orobanche flower stalks. A near-axenic polyethylene envelope system for culturing broomrape on tomato roots was used to ascertain pathogenicity of these strains. Both organisms fulfilled Koch's postulates for being primary pathogens. Their DNAs were analyzed and fingerprinted by restriction fragment length polymorphism and random amplified polymorphic DNA, showing that they are indeed different from each other and from many other Fusarium spp. and other formae speciales of F. oxysporum including a strain that attacks O. cumana on sunflowers. Both strains infect O. aegyptiaca, O. cernua, and O. ramosa, but not O. cumana. They did not infect any of the vegetable and legume crops tested and thus seem specific to Orobanche. Tomato plant roots dipped into a fungal spore and mycelial suspension and planted in broomrape-infested soil were protected for 6 weeks, as were tomato transplants in pot experiments. About 90% control was also achieved by posttransplant soil drench with fungal suspensions in pot experiments. These pathogens may be effective as seed, transplant, or soil-drench treatments of high-value vegetable and other crops.

Glutathione peroxidase is involved in scavenging free radicals in many biological systems. Its presence in plants has been implicated by identification of genes with nucleotide sequences similar to those of mammals, as well as by indirect, coupled enzyme assays. We directly quantified glutathione peroxidase activity in crude plant extracts using an organic hydroperoxide substrate and measuring GSSG, the direct reaction product, by adapting a sensitive fluorometric method. The constitutive levels of glutathione peroxidase in a paraquat-resistant biotype of Conyza bonariensis (resistant to many oxidative stresses) were almost double those of the sensitive biotype, providing correlative evidence that glutathione peroxidase is part of the system preventing oxidative damage. Glutathione peroxidase activity was separated from both glutathione transferase and glutathione reductase activities by specific antibody immunoprecipitation and by substrate-affinity chromatography. Conyza glutathione peroxidase cross-reacted with citrus polyclonal anti-phospholipid hydroperoxide glutathione peroxidase. The data suggest that this glutathione peroxidase is a component of the systems dealing with oxidative damage protection in plants and is biochemically different from glutathione peroxidases that also have glutathione transferase activity. (C) 2000 Academic Press.

Amphiphilic dithiocarbamate chelators were synthesized to afford the ability to traverse biological membranes. Comparison of copper (II) stability constants for dithiocarbamates was accomplished by a competition reaction between copper oxinates and copper dithiocarbamates using UV/VIS spectrophotometry. The stability constants of mixed copper-hydroxyquinoline-dithiocarbamate complexes were calculated by a continuous variation and subtractive spectrophotometric methods. Using these techniques, the competitive stability constants log K_c were determined as 1.53, 1.95 and 2.03 for amphiphilic dithiocarbamates and 3.52 for dibutyldithiocarbamate.

Root-attaching parasitic flowering broom-rapes (Orobanche spp.) are major constraints to vegetable, legume and sunflower production around the Mediterranean and elsewhere, with banned methylbromide fumigation or land abandonment of these affected crops as the major 'solutions' to the problem. We report the specific generation of transgenic asulam-resistant potatoes as a way to eradicate this pest. The target-site resistance in the crop allows the herbicide to be translocated from treated leaves to the parasite via crop roots. This inhibitor of dihydropteroate synthase lethally then prevents folic acid biosynthesis in the parasite. Additionally, we demonstrate that asulam can be used directly in selecting resistant transformants, without the need for another selectable marker.

Keywords: Agronomy; Plant Sciences

Oxidant stress resistance in Conyza bonariensis and wheat (Triticure 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 μM 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.

Inundatively used mycoherbicides have been uneconomical and ineffectual, mainly because a large inoculum is required to overcome weeds' innate defenses, as well as the need for long dew periods to become established. Weed defenses can be suppressed by specific chemical agents, or overcome biologically (using mixed organisms and/or engineering enhanced pathogenicity), and better formulants replace the long dew periods.

There is a dire, sometimes conflicting need to lower both the economic and the chemical inputs used in weed control. This can be attained by combining rotations, cultural practices, different chemicals, and biologicals, such that an increase cost-effectiveness is achieved; i.e. the combinations lead to $ynergy. $ynergy can be attained by mixing herbicides with each other or with adjuvants that lower inputs, overcome resistance, and/or broaden the spectrum of weeds controlled. Mycoherbicides can be $ynergized by formulants that assist in establishment or in overcoming plant defenses, by engineering genes enhancing pathogenicity and/or by mixing with facilitory organisms.

Paraquat resistance in a biotype of Conyza at about 10 weeks of age is genetically and biochemically correlated with constitutively elevated enzyme activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase relative to the sensitive biotype. This high activity of superoxide dismutase and glutathione reductase in total cell extracts was consistent with changes to higher levels of resistance to paraquat and photoinhibition at this stage of plant development, during bolting and flowering, but not at other vegetative stages. We have now measured the constitutive ascorbate peroxidase activity at different ages and compared it with our previous results with superoxide dismutase and glutathione reductase during development (which were also repeated in part). Between the ages of about 9 and 12 weeks, the paraquat-resistant biotype always has higher constitutive ascorbate peroxidase activity than sensitive plants. This parallels and extends our previous findings.

This chapter reviews and summarizes discussions from Symposia No.1, which included presentations on soil and crop responses to alternate tillage practices in Argentina, agroforestry-a mechanistic approach being implemented in Costa Rica, crop sequences for sustaining soil resources in China, and agroecosystem responses to alternate soil and crop management systems in the U.S. Com-Soybean Belt. Dumping at sea is no longer an acceptable practice in Europe, so current debate exists on whether to incinerate the material for its fuel value or to process it as a low nutrient-content soil conditioner. For conditions being evaluated in Costa Rica, agroforestry systems (AFS) ground cover was providing a mulch that effectively maintained the soil resource. Nutrient imbalance, decreasing fertilizer use efficiencies, and declining soil organic matter were identified as serious threats to sustainability. An agroecosystem was defined as a system created by interactions between organisms in the soil, including plants, and their environment.

The severity of fungal infection is usually estimated visually as a rating of injury. A more quantitative method was needed to ascertain the effect of additives used to enhance the virulence of mycoherbicidal preparations. A sensitive and accurate serological method is presented for quantitative measurement of Alternaria cassiae infection of Cassia obtusifolia. An antiserum was prepared against a homogenate of mycelium of A. cassiae. A fast and simple procedure of leaf immunoautoradiography was developed for the visualization of A. cassiae mycelium on inoculated leaves. A radioimmunosorbent assay (RISA) was developed for quantification of the extent of fungal infection. As little as 1. 6 ng/ ml dry weight equivalent of mycelium could be detected by RISA. There was a linear relationship between the logarithm of the RISA values and the logarithm of fungus concentration up to 300 ng/ml. Reduced sensitivity of the assay was pronounced in extracts containing 400 μ g/ml or more fresh weight of leaves. There was 3040 % crossreactivity of the antiserum with two other species of Alternaria, as well as with Monilinia fructicola, and almost no reactivity was found with three other fungi tested.

The attack of weed plants by mycoherbicidal organisms can be inhibited by the antifungal phytoalexins accumulated upon infection. The measurement of phytoalexin levels in inoculated leaves is problematic due to interference by pigments and other plant metabolites. A rapid and simple procedure is presented for measuring flavonoid phytoalexins after TLC in many samples of small amounts of tissue, based on the reaction of flavonoids with AlCl₃, yielding a fluorescent complex. A single flavonoid phytoalexin was isolated from 12- to 14-day-old leaves of Cassia obtusifolia inoculated with the mycoherbicidal pathogen Alternaria cassiae. The phytoalexin reacted with AlCl₃, with a fluorescence emission peak at 472 nm when excited at 340 nm (max), with a linear response between 1 to 200 nmol phytoalexin. The method was qualitatively as sensitive as detection by staining with AlCl₃ on a silica gel thin layer chromatography plate, yet enabled quantification of the phytoalexin from a few milligrams of leaf tissue at varying times after elicitation. An initial small increase in accumulation of the phytoalexin 4 hr after inoculation was detected using this spectrofluorimetric method, but not by measurement of ultraviolet absorbance, which is far less specific and less sensitive.

Many photosystem II inhibiting herbicides still inhibit this process in triazine-resistant plants; they have no cross resistance with atrazine. Five- to twenty-fold lower concentrations of phenolic type herbicides and 5-fold less of the active ingredient of pyridate and half as much ioxynil are required to inhibit thylakoid PS II in atrazine-resistant biotypes than in sensitive biotypes; i.e., they even show \u201cnegative cross resistance\u201d. Negative cross resistance may be the major reason that atrazine resistance did not evolve where herbicide mixtures were used, when the mixed herbicide (usually a non-PS II inhibiting acetanilide) also controlled triazine-sensitive weeds. Mathematical modeling in principle allows quantification of the very low field levels of herbicides possessing negative cross resistance that could be mixed with atrazine that would stop or delay the evolution of resistant populations without affecting the maize crop. There are few available actual dose response curves of atrazine-resistant vs. susceptible weeds at the whole plant level for herbicides exerting negative cross resistance. Thus, \u201creal situation\u201d modeling cannot be done. Data acquisition is called for so that the model can be extrapolated from the thylakoid to the field.

Keywords: Agronomy; Plant Sciences

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.

The nitrodiphenyl ether herbicide acifluorfen requires light for phytotoxicity even though it alone cannot absorb light. All possible major pigment systems have been implicated as the photoreceptor. We tested whether carotenoids and chlorophyll are essential for phytotoxicity. We used green-photosynthetic (mixotrophic) tomato cell cultures, etiolated cells of the same line (containing carotenoids but no chlorophyll), and carotenoid-free white cells (by continuously culturing etiolated cells on norflurazon). All three cell culture types parallel plants insofar as the first measureable effect is membrane lipoxidation. Acifluorfen at 1 μM had little effect in darkness, but strongly inhibited growth of all cultures in 40 μmol m^-2 sec^-1 white light. Acifluorfen at 0.1 μM did not affect green cells in light, but inhibited the growth of white and etiolated cells. Action spectroscopy showed that 350-nm light was the most effective wavelength inhibiting the growth of white cells with 1 μM acifluorfen, followed by 550-nm, 450-nm, cool-white fluorescent, and 630-nm light, with only a threefold difference between 350-nm and red light. Far-red light was ineffective. These data demonstrate that in this system, chlorophyll, carotenes, cryptochrome, flavins, and phytochrome cannot be the sole photoreceptor for acifluorfen action. Our data, along with all other published findings are consistent with two hypotheses: (a) that acifluorfen interacts with other moieties to produce broad-spectrum chromophore(s) that react(s) with oxygen, forming active-oxygen species in the light; (b) that acifluorfen stimulates the accumulation of chromophoric photodynamic molecules.

Abstract Trichoderma harzianum normally requires light for conidiation. Conidiation of colonies grown in continuous light does not appear to be rhythmic, but sharp banding patterns are formed under light/dark cycles. A single pulse of blue light produces a sharp band of conidia that forms where the growing edge of the mycelia is located at the time when the light is given. A period of about 24 h is required following the light pulse to produce mature conidia. During this time colonies are insensitive to further induction by light. The fluence required to produce 50% saturation varies by a factor of about 3 depending on when the pulse is given. This change of sensitivity is rhythmic with a period length of approximately 27 h when grown on medium containing deoxycholate. The pattern of conidiation in a mutant strain (B119), which is able to form conidia in the dark, is rhythmic and the period length is dependent on the composition of the medium. Addition of deoxycholate to the medium increased the interval between dark bands from 12 to 24 h. The rhythmic banding is suppressed in constant light and a double banding pattern is produced in light/dark cycles. A pulse of blue light induces a band of conidia in this mutant, as in the wild type, but it also delays the reappearance of the dark banding pattern. The extent of this delay depends on when the pulse is given and, although the period length of the dark conidiation rhythm is affected by deoxycholate, the effect of blue light on its phase is not. Of the various rhythmic responses of Trichoderma studied here. the delay in reappearance of the dark banding pattern in B119 is the most promising for further detailed studies, for example of wavelength and temperature dependence.

A group of 28 dihydropyrimido-benzimidazole analogs and derivatives was tested for herbicidal activities. The 1:1 mixture of 7-fluoro- and 8-fluorodihydropyrimido-benzimidazole had the greatest biological activity. Activity is increased by methylation of nitrogen 2. None of the active compounds was inhibitory to nonphotosynthetic tissue-culture cells in vitro. The I₅₀ of inhibition of growth of green duckweed plants and that of in vitro photosynthesis activity matched the I₅₀ of inhibition of photosystem II activity. Fluorescence analyses of photosystem II activity indicated inhibition at the same step as did many triazine, phenylurea, pyridazinone, and uracil herbicides. As the compounds showed insufficient activity on recently evolved triazine-resistant weed biotypes, their further development was terminated, despite their novel chemistry.

The ligninolytic activities of four cellulolytic organisms were compared using straw. Only Aspergillus japonicus and Polyporous versicolor appreciably degraded lignin with A. japonicus yielding the most protein. In solid culture, most protein was produced by P. versicolor, closely followed by A. japonicus. Pretreatment of the straw by hot water facilitated biodegradation and protein production. The nutritional value of the residual straw was also increased by some fungal cultures. The greatest amount of degradable polysaccharide in the straw was made available by A. japonicus in liquid media and Pleurotus ostreatus in solid media.

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). Complementation in heterokaryons was used to estimate the number of different lesions responsible for the selected phenotypes. As a working hypothesis, each pair that did not complement when tested at a standard light level was assumed to represent a complementation group. When medium depletion, the least stringent stress condition was used, an average of 17 complementation groups was estimated using different mathematical techniques. These groups would represent about 17 gene products; all required for only the light-specific portion of the sporulation pathway. The number of candidates for mutants in the photoreceptor pathway was further reduced by choosing only those strains that responded to a transient stress by a brief incubation in water, which seemed more stringent. The mutants in this subclass mapped into four complementation groups. Two of these groups had both defective absorption and modified action spectra, and are probably defective in the photoreceptor, cryptochrome.

Most lignin research has been on wood-rot fungi and not on other lignolytic organisms. Members of the genus Aspergillus inhabit lignin-rich environments, and we have studied their relative lignin-degrading potential. Aspergillus fumigatus, A. japonicus, A. niger, and A. terreus were tested for their ability to metabolize¹⁴C-labeled aromatic compounds. The species tested decarboxylated, demethoxylated, and cleaved the rings of coumaric, ferulic, vanillic, veratric, and anisic acids. More than 90% of C-ring-labeled ferulic and vanillic acids disappeared from the medium in 96 h of cultivation. More than half of the above was respired, the rest was incorporated in unknown form into the mycelium. Mycelia were homogenized and about 3% of the initial label was found in TCA precipitate of the cell-free supernatant. Protocatechuic acid 3,4-dioxygenase (EC 1.13.11.3) and catechol 1,2-dioxygenase (EC 1.13.11.1) activities were detected in the mycelial extracts of the Aspergillus spp. All the Aspergillus spp. were capable of degrading both aromatic and carbohydrate components of water-soluble lignocarbohydrate complexes (LCC) from wheat straw. The degradation of the aromatic moiety of soluble LCC with apparent molecular mass more than 100,000 daltons was far more active in the Aspergillus spp. than in the whiterot fungi tested; i.e. Polyporus versicolor, Pleurotus ostreatus, and Forties annosus. The aromatics present in the soluble LCC, as well as a variety of lignin-related compounds tested, did not affect the production of hemicellulases by A. japonicus. Aspergillus spp. degraded¹⁴C-dehydrogenative polymerizates, converting carbon from the ring as well as from the -O¹⁴CH₃ groups to¹⁴CCO₂.¹⁴CO₂ release after 21 d did not exceed 10% of the total¹⁴C input. This situation is comparable to some white-rot fungi. Lignosulfonate was poorly degraded by A. japonicus, but clearly modified. Fomes annosus was able to grow much better on lignosulfonate when A. japonicus had previously grown on it. Aspergillus spp. grew efficiently on wheat straw, utilizing lignin and some carbohydrates, and rendering the remaining carbohydrates more available to attack of carbohydrases.

Chemostat studies of bacteria that harbour the prokaryotic transposable elements Tn-5 and TnlO and the temperate phages A, Mu, P1 and P2 have shown that these accessory DNA elements confer a selective advantage on their hosts. We propose that similar selective effects provided the initial impetus for the evolution of nascent accessory DNA elements in primitive bacterial populations. In subsequent evolution the elements acquired or perfected the 'selfish' characteristics of over-replication and horizontal transmission. Such selfish traits led to the dissemination of accessory DNAs among commensal strains, species and genera, genetically interconnecting them to create a 'commonwealth' of species that potentially share a common gene pool. The involvement of accessory DNAs in genetic exchange provides selection at the population level for refinement and diversification of the elements and for regulation of their replication, transposition and transfer among cells. The diversity of intracellular environments encountered by the elements imposes constraints on their evolution while at the same time altering the selection pressures operating on conventional chromosomal genes. This process of coevolution of accessory DNAs with the genomes of their diverse hosts has led to a unique population structure and mechanism of genetic exchange among bacteria, which constitutes the most effective adaptive strategy yet devised by selection.

Clarke: I was fascinated by the use of codons in Streptomyces fradiae, whose DNA has an extremely high guanosine and cytosine (GC) content, and leaves no room for synonymy in the genetic code. This reminded me of some early work by Sueoka (1965) on the amino acid compositions of organisms differing greatly in their GC content. In these organisms the change from one extreme of GC content to the other actually seems to have moved the amino acid compositions of the proteins. The correlations between GC content and the proportion of amino acids with high GC codons were very strong but not sufficient for the changes in amino acid composition to explain the changes in GC. Whatever is driving the DNA and pushing the GC content in one direction or another is so strong that it even moves the protein. So what is it? [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]

Gressel: In the laboratory, the triazine-resistant biotypes are more sensitive to another group of photosystem 11-inhibiting herbicides (Arntzen et a1 1982), which isnt 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: Is it possible to make an anti-ecdysone i.e. an anti-moulting hormone? [first paragraph]

Representative regenerated clonal plants from protoplast fusion of Solanum tuberosum L. and an atrazine resistant biotype of S. nigrum L. were studied to ascertain which plastomes each clone contained. DNA was isolated from fractionated chloroplasts, restricted with DNAases XHO-1, BGL-1, PVU-2 and BAM-H1, and the fragments separated by agarose gel electrophoresis for comparison. No difference could be found between resistant and susceptible biotypes of S. nigrum with all four enzymes. XHO-1, BGL-1, BAM-H1 differentiated between S. nigrum and S. tuberosum. All atrazine resistant regenerants, despite plant morphology, had the plastid DNA pattern of S. nigrum while all sensitive ones resembled S. tuberosum, even the subclone 38S having a S. nigrum morphology and chromosome number.

Lignocellulose from plants is the major organic material in the biosphere and it is slowly biodegraded. The inability of cellulases to degrade the cellulose in the natural complex with lignin could be due to cellulose crystallinity or to a steric hindrance or other factors from the lignin. Digestible cellulose was artificially lignified using peroxidase, H₂O₂ and eugenol. It was less degradable by cellulases and by Trichoderma suggesting that it is the complexed lignin that interferes with cellulose degradation.

The rapid effects of the herbicide EPTC (S-ethyl dipropylthiocarbamate) and the protectant DDCA (N,N-diallyl-2,2-dichloroacetamide) on [2-¹⁴C]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 mM and 0.1 mM, increased the incorporation of [2-¹⁴C]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 mM) inhibited overall uptake of [2-¹⁴C]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 [2-¹⁴C]acetate incorporation into different lipid species. EPTC inhibited incorporation of [2-¹⁴C]acetate into both neutral and polar membrane lipids but altered significantly only its distribution into neutral lipids. DDCA (0.1 mM) given together with EPTC (0.2 mM) 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.

The rapid interactions between the herbicide S-ethyl dipropyl thiocarbamate (EPTC) and the structurally similar herbicide protectant N,N-diallyl 2,2-dichloroacetamide (DDCA) at the level of herbicide uptake were examined in maize cell cultures. When the two compounds were given simultaneously, DDCA inhibited uptake of [¹⁴C]EPTC into maize cells measured for 30 min. A Lineweaver-Burk plot indicated this inhibition to be competitive. N,N-Diallyl 2-chloroacetamide (CDAA), a compound similar in structure to DDCA, inhibited uptake to a lesser extent. Other protectants having no similarity in structure to either DDCA or EPTC had no inhibitory effect on the uptake of EPTC. The data suggest that competition between DDCA and EPTC for a site of uptake may be related to their similarity in chemical structure. Experiments with metabolic inhibitors suggested that uptake of EPTC is not via an active transport mechanism. We suggest that competition for uptake between EPTC and DDCA may represent the first step in a complex series of interactions between the herbicide and its protectant that contributes to the protection of maize from herbicide injury.

Triazine herbicides inhibit photosynthesis by binding to thylakoids. However, in certain resistant plant biotypes the thylakoids are somewhat modified. It is possible that experiments with photoaffinity binding of radiolabelled azido-atrazine have led to an erroneous conclusion; i.e. that the specific thylakoid binding site is a 32 000 M_r protein. Alternatively, this 32 000 M_r protein could be adjacent to the binding site and the azido-atrazine acts as a bifunctional reagent. A high-turnover 32 000 M_rm thylakoid protein from Spirodela can be depleted specifically by incubation with low levels of chloramphenicol. Plants depleted of this 32 000 M_r thylakoid protein were not more tolerant to atrazine, as might be expected if this protein bore the electrostatic herbicide binding site.

The extent that green and white calli reflect the phytotoxicities of various compounds on seedlings was measured. Green calli, which were found to photosynthetically fix ¹⁴CO₂ but which were not autotrophic, better mirrored the effects of the same compounds tested on seedlings for two reasons. (a) Inhibitors of photosynthesis inhibited green calli more strongly than white calli; (b) compounds which were inactive in foliar sprays on seedlings but are known to be active through the soil more strongly inhibited white calli than green calli. Thus it is suggested that green cell cultures have potential use in studies of mode of action and for preliminary phytotoxicity screening.

We have previously shown that a wave of enhanced uridine incorporation into RNA occurs in the more vegetative parts of the plumule at the end of the single dark period that evokes flowering in Pharbitis nil. We demonstrate here that a light break that suppresses flowering suppresses this wave as well. It does not shift the kinetics of the wave of uridine incorporation to a different time. The enhanced incorporation is into all RNA fractions. It had been concluded from excision experiments that the floral stimulus reaches the apex much after photoinduction. There is a metabolic shock caused by such excision of the cotyledons or surgical removal of the plumule that can suppress flowering if it is performed near the end of the inductive dark period. The terminal bud is more affected by this shock than lateral buds. Excision of the cotyledons enhances the rate of incorporation of exogenous uridine into the plumule. We propose that the \u201cfloral stimulus\u201d stimulating incorporation into RNA reaches the plumule immediately after the end of the critical dark period.

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.

The effects of a broad range of compounds were assessed using both seedlings and callus cultures of five species. An experimentally justified ranking procedure was used to facilitate the comparison of the inhibitory characteristics at multiple concentrations. When the effects on seedlings of Lycopersicon esculentum, Solanum nigrum, Chrysanthemum segetum and Cirsium arvense were compared to those on white calli of the same species, some positive correlations were obtained; photosynthesis inhibitors affected seedlings much more than calli and some compounds affected calli much more than seedlings. Better correlations were obtained between the effects on Rumex obtusifolius seedlings and on green Rumex calli; the only exceptions being those compounds affecting mainly the calli. Thus callus cultures, especially green ones, have a potential for use to assess possible phytotoxicity as well as to detect potential toxicity of compounds not penetrating into or being translocated in whole plants.

The effects of metabolic shifts on nucleic acid syntheses have been widely studied in prokaryotes, but not in plants because of a paucity of suitable systems. Spirodela (Duckweed) was thus used to ascertain the response of the nucleocytoplasmic (nc) and plastid ribosomal RNA metabolisms to partial and total carbon deprivation. The 0.56 × 10⁶ M_m plastid rRNA is the one species of RNA most affected by metabolic shifts; unlike other species, its appearance is delayed by deprivation and it appears more rapidly than other species on transfer from dark to light. The data suggest a discoordination between the transcription and processing of plastid ribosomal precursors. Incorporation into all nc and plastid rRNAs was severely reduced and all rRNA precursors accumulated in green plants that were completely deprived of carbon by transferring to the dark, without sucrose. The amounts of nc and plastid precursors transcribed readjusted to the reduced amounts processed to mature RNA only after long periods in the dark with sucrose. This delay involved the formation of new colorless plants. Less plastid RNAs, compared to nc RNAs are found in the dark steady state.

The electrophoretic mobilities of DNA, ribosomal RNAs, and pulse-labeled RNAs were compared on polyacrylamide gels polymerized at temperatures from 4 to 35°C and subjected to electrophoresis at a fixed temperature. DNA migrated the same distance irrespective of polymerization temperature, the ribosomal RNAs, and the major pulse-labeled species (a putative rRNA precursor) migrated more rapidly in gels polymerized at higher temperatures. The linearity of the migration versus the log of the molecular weight remained for the five rRNA species used, but the extrapolated molecular weight of the putative precursor ranged from 1.8 × 10⁶ to 2.5 × 10⁶ depending on polymerization temperatures. By varying polymerization temperatures, the optimal resolution of various groups of RNA species can be obtained. The results are explained in terms of polymerization temperature effects on gel structure as well as nucleic acid conformation.

The absence of oxygen had no effect on the photoinduced conidiation of Trichoderma, a system (unlike previously used systems) where the transient photoinduction and the prolonged respiratory oxygen requirements are separable.The nature of the presumably ubiquitous blue-light absorbing pigment is not restricted to those requiring oxygen.

A product of the processing of precursor rRNA ("excess" RNA) has been indirectly found to be unmethylated in mammalian systems, but direct measurement was precluded because of its instability. The "excess" RNA of duckweeds is relatively stable allowing direct estimation of its methylation by polyacrylamide gel electrophoresis. The "excess" RNA with an apparent molecular weight 0.5×10⁶ was unmethylated. A pulse labeled RNA with an apparent molecular weight of 1.2×10⁶ (presumably from chloroplasts) was also unmethylated. Under similar conditions the presumed cytoplasmic rRNA precursors, and the mature cytoplasmic and chloroplast rRNAs were methylated.

The rate of ³H-methyl thymidine incorporation increased in two waves following floral induction in Pharbitis. Adenosine only enhanced thymidine incorporation at non-peak times. This enhancement was studied by autoradiography and found to be both nuclear and cytoplasmic. Our interpretation of the results is that there was increased thymidine labeling of nuclei already undergoing DNA synthesis and not an increase of cells in S phase. Various vagaries and possible artifacts in the use of thymidine to measure DNA synthesis quantitatively are pointed out.