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.

232Previous chapters have described the technologies available, the results achieved, and the market niches filled by herbicide-resistant crops (HRCs) in U.S. agriculture. This chapter will discuss weed control needs that the author feels could be met, at least in part, by HRC, and further chapters try to assess whether it is economic, ecologically desirable, and safe to use HRCs to fill the needs, although it is hard discussing meeting the needs while ignoring these other issues. This review is an update and extension of longer, more comprehensive reviews by this author on similar subject matter (Gressel 1992a, 1993a, b; Gressel et al. 1994).

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.

It is necessary to control root parasitic weeds before or as they attach to the crop to prevent crop damage. Crop resistance to soil active herbicides or systemic target site herbicide resistances (where crops do not metabolise the herbicide) can be generated by mutagenesis, but using known herbicide resistance transgenes is easier. Herbicides can then be applied as crop seed dressing, lowering amounts and decreasing environmental impact. Crops with two target site resistances will be more resilient to the evolution of herbicide resistance in the parasite, if well managed. Crops could also be directly rendered parasite resistant by genetic engineering using strategies yet to be successfully demonstrated, including engineering the crop not to make germination stimulants, to make allelochemicals toxic to the parasite, have the crop make RNAi's or micro RNAs that are toxic to the parasite, disseminate genes through multi-copy transposons that harm the parasite and/or engineer in genes that confer general resistances.

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.

At very low pesticide rates, a certain low proportion of pests may receive a sublethal dose, are highly stressed by the pesticide and yet survive. Stress is a general enhancer of mutation rates. Thus, the survivors are likely to have more than normal mutations, which might include mutations leading to pesticide resistance, both for multifactorial (polygenic, gene amplification, sequential allelic mutations) and for major gene resistance. Management strategies should consider how to eliminate the subpopulation of pests with the high mutation rates, but the best strategy is probably to avoid too low application rates of pesticides from the outset.

The developing world has many unique constraints to crop production and, lacking inputs, they are best overcome if solutions are seed borne. Classical breeding cannot overcome many of these constraints because the species have attained a 'genetic glass ceiling', the genes are not available within the species. Transgenics can supply the genes, but typically not as 'hand me down genes' from the developed world because of the unique problems: mainly parasitic weeds, and weedy rice, stem borers and post-harvest insects, viral diseases, tropical mycotoxins, anti-feedants, toxic heavy metals and mineral deficiencies. Public sector involvement is imperative for genetically engineering against these constraints, as the private biotechnology sector does not see the developing world as a viable market in most instances. Rice, sorghum, barley, wheat and millets have related weeds, and in certain cases, transgenic gene containment and/or mitigation is necessary to prevent establishment of transgenes in the weedy relatives.

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. Adding virulence factors was considered essential. Exogenous addition of the products of various genes was 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. We have studied these in three Weed/Pathogen systems: Abutilon/Colletotrichum; Senna (Cassia)/Alternaria; and the root parasitic relative of Striga, Orobanche/Fusarium spp. We deal below with the Orobanche, as a model for Striga, a species one does not work with where it might flourish. 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.

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?

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.

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.

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

The other chapters in this book demonstrate the vast array of systems that plants utilize to evolve endoferality and exoferality, and mixtures between the two. This chapter presents short case histories of other crop species that extend this biodiverse array and accentuates other issues and implications that may derive from ferality. Clearly, other cases could be added to this, as many as there are additional other crops, but the idea is not to be exhaustive. Our intent is to supply a feeling of further issues and to provide parameters that must be discerned when analyzing the potential problems, if any, that may arise when a given crop becomes a persistent volunteer or introgresses genes from a wild relative.

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

Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O- neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.

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.

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

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.

High herbicide levels can be localized on or near seed of acetolactate synthase (ALS) resistant maize (Zea mays). The magnesium salt of imazapyr was optimal for seed dressings (drenching, priming, and coating), for preventing field damage from parasitic Striga hermonthica (witchweed) in three seasons. Striga emerged on untreated maize from 6 to 12 weeks after planting. There was almost no Striga emergence for 3 months on imazapyr-dressed homozygous ALS-R 3245IR maize seeds. Occasional Striga that emerged and flowered formed no seed by harvest. Dust or polyvinylpyrrolidone adhesive coatings were safer in maize and as effective in Striga control as priming. Seed dressing coupled with pulling Striga escapes reduces infestation and can be used to deplete the Striga seed bank until genetic crop resistance becomes available.

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.

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

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

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.

Keywords: Agronomy; Plant Sciences

Paraquat-resistant hairy fleabane (Conyza bonariensis L. Cronq.) has been extensively studied, with some contention. A single, dominant gene pleiotropically controls levels of oxidant-detoxifying enzymes and tolerance to many photooxidants, to photoinhibition, and possibly to other stresses. The weed forms a rosette on humid short days and flowers in dry long days and, thus, needs plasticity to photooxidant stresses. In a series of four experiments over 20 months, the resistant and susceptible biotypes were cultured in constant 10-h low-light short days at 25°C. Resistance was measured as recovery from paraquat. The concentration required to achieve 50% inhibition of the resistant biotype was about 30 times that of the susceptible one just after germination, increased to >300 times that of the susceptibles at 10 weeks of growth, and then decreased to 20-fold, remaining constant except for a brief increase while bolting. Resistance increased when plants were induced to flower by long days. The levels of plastid Superoxide dismutase and of glutathione reductase were generally highest in resistant plants compared to those of the susceptibles at the times of highest paraquat resistance, but they were imperceptibly different from the susceptible type at the times of lower paraquat resistance. Photoinhibition tolerance measured as quantum yield of oxygen evolution at ambient temperatures was highest when the relative amounts of enzymes were highest in the resistant biotype. Resistance to photoinhibition was not detected by chlorophyll a fluorescence. Enzyme levels, photoinhibition tolerance, and paraquat resistance all increased during flowering in both biotypes. Imperceptibly small increases in enzyme levels would be needed for 20-fold resistance, based on the moderate enzyme increases correlated with 300-fold resistance. Thus, it is feasible that either these enzymes play a role in the first line of defense against photooxidants, or another, yet unknown mechanism(s) facilitate(s) the lower level of resistance, or the enzymes and unknown mechanisms act together.

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.

A single phytoalexin was isolated and purified from 12- to 14-day-old leaves of Cassia obtusifolla L., inoculated with Alternaria cassiae Jurair & Khan. The structure was elucidated by H-1 and C-13-nuclear magnetic resonance and mass spectrometry as 2-(phydroxyphenoxy)-5,7-dihydroxychromone. The compound was shown to be derived in part from phenylalanine. Radial growth of A. cassiae was inhibited 50% by the compound at 0.3 millimolar. This moderate toxicity is compensated for by the relatively high levels (3 millimolar) accumulated. Phenoxychromones have been previously reported only as constitutive secondary metabolites in Artemisla capillaris Thunb, in which their function is unknown.

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.

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil BenAmotz et Avron, a βcaroteneaccumulating halotolerant alga. The large amount of βcarotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phqtoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in βcarotene, with no evident change in other isoprenoid intermediates. Carotenerich Dunaliella bardawil is substantially resistant to highintensity photoinhibition. This resistance is lost in cells grown to contain low & carotene and in the norurazontreated phytoenerich cells. These obseruations are in agreement with the hypothesis that the accumulated βcarotene in Dunaliella bardawil portects the cells against injury by excessive irradiation.

Solar digesters were designed to wet pasteurize straw. Microbial levels were reduced, precluding competition with ligninolytic organisms. The pasteurization treatment alone increased the available cellulose by 20-40%. Ligninolytic organisms produced biomass with 6-8% protein on pasteurized wheat straw.

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil BenAmotz et Avron, a βcaroteneaccumulating halotolerant alga. The large amount of βcarotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phytoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in βcarotene, with no evident change in other isoprenoid intermediates. Carotenerich Dunaliella bardawil is substantially resistant to highintensity photoinhibition. This resistance is lost in cells grown to contain low βcarotene and in the norflurazontreated phytoenerich cells. These observations are in agreement with the hypothesis that the accumulated βcarotene in Dunaliella bardawil protects the cells against injury by excessive irradiation.

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.

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.

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.

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.

Resistances to antibiotics and pesticides except herbicides rapidly developed following their introduction. Despite repeated use of herbicides only a few cases of acquired genetic resistance have been reported. By extrapolation from analogous situations, it is suggested that this is due to a combination of low selection pressure of most herbicides, lower fitness of resistant weed strains in the absence of herbicide, the ability of herbicide thinned strands of susceptible weeds to produce relatively more seeds, as well as to the large soil reservoir of susceptible weed seeds. The few reported cases of resistance are to persistent, high selection pressure herbicides supporting our contentions.

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.

Callus tissue has been isolated from 11 species, of 8 which are 'weeds' from which callus isolation has not been previously described. Continuous suspension cultures of four species have been started. Growth conditions were optimized for high yield. Logarithmic phase and stationary phase cells react differently to some inhibitors. The larger the inoculum size, the greater the concentration of inhibitor that must be used to achieve equal inhibition. Hormonal constitution of the medium affects inhibition by 'auxin' type herbicides. Growth kinetics, delayed response and recovery must be considered in assessing phytotoxicity. Highly uniform and controlled procedures are required for interspecific comparisons of many compounds.

The effects of dikegulac sodium on plastid RNA syntheses were studied, as dikegulac induces the formation of yellow misshapen leaves. It depressed uridine incorporation into both plastid and cytoplasmic ribosomal RNAs of axenically cultured Spirodela (duckweed). With short labeling time (1 hr) dikegulac specifically suppressed the synthesis of a plastid 1.2 × 10⁶ MW RNA species, as well as nonspecifically depressing incorporation. With longer labeling time (24 hr), the incorporation into mature plastid rRNAs was suppressed to a greater extent than that into cytoplasmic rRNAs. The inhibitions of uridine incorporation caused by dikegulac are probably indirect and a reflection of its effect on other growth parameters.

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.

Young and mature fronds of Spirodela oligorrhiza cultured in the light or dark were analyzed for their plastid ribosome distribution by electron microscopy. This distribution was correlated to the loss of integrity (hidden nicking) of the plastid heavy ribosomal RNA. In young fronds there were relatively more plastid ribosomes as well as plastid rRNA in the light than in the dark. Plastid rRNA was synthesized mainly when fronds were young. Chloroplast ribosomes disappeared from the stroma of light grown fronds upon maturation, but the relative amount of chloroplast RNA increased. In the dark the relative etioplast ribosomal density did not change upon maturation. The nicked products of the plastid heavy rRNA were detected by a modified two-dimensional gel electrophoresis. There is already some nicking of the plastid heavy rRNA at a very early stage of frond and plastid development. The rate of hidden nicking of plastid heavy rRNA was greater in the light than in the dark. The rate of nicking in fronds transferred from the dark to the light increased to the level of light grown plants at about the time of the appearance of chlorophyll. The data are discussed in the context of the relationship between plastid ribosomal RNA changes during frond maturation in light and dark grown fronds, as related to protein synthesis and of ribosome binding to thylakoids.

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.