A mechanism of chlorotoluron resistance in Lolium rigidum

Many biotypes of Lolium rigidum Gaud, (annual ryegrass) have developed resistance to herbicides; however, few have developed resistance to phenylurea herbicides. Two biotypes with different histories of herbicide selection pressure were six to eight times less sensitive to the phenylurea herbicide, chlorotoluron, than a susceptible biotype. Resistance was not due to differences in the herbicide target site as oxygen evolution by thylakoids isolated from resistant and susceptible biotypes was similarly inhibited by diuron and chlorotoluron. There was no difference in the uptake and distribution of chlorotoluron into resistant and susceptible plants. There was a twofold greater rate of chlorotoluron detoxification in resistant plants with N-demethylation being a major detoxification reaction. Resistant plants treated with a 3-h pulse of 120 M chlorotoluron recovered net carbon fixation after 42 h, half the time taken by susceptible plants. The mixed-function oxidase inhibitor 1-aminobenzotriazole (70 M) intensified the effects of chlorotoluron in resistant plants when applied in combination with the herbicide for 7 d. 1-Aminobenzotriazole also inhibited the metabolism of chlorotoluron in both resistant and susceptible plants. The cytochrome P-₄₅₀ inhibitor, piperonyl butoxide piperonyl butoxide, interacted with chlorotoluron when applied to plants growing in soil. Chlorotoluron applied with reduced plant dry weight to a greater extent than chlorotoluron alone. It appears, therefore, that enhanced detoxification is the major mechanism of resistance to chlorotoluron in the resistant biotypes studied.Abbreviations ABT 1-aminobenzotriazole - VLR1 Victorian L. rigidum biotype 1 — herbicide susceptible - VLR69 Victorian L. rigidum biotype 69 — herbicide resistant - WLR2 Western Australian L. rigidum biotype 2 — herbicide resistant M.W.M.B, was supported by an Australian Postgraduate Research Award and a supplementary scholarship from the Grains Research and Development Corporation. We are very grateful to Dr. E. Ebert, Ciba Geigy, Basal, Switzerland for providing [¹⁴C]chlorotoluron and standards of chlorotoluron metabolites. We express our gratitude to Dr. John Huppatz of the CSIRO Division of Plant Industry for providing ABT. We also thank Ciba Geigy Australia for providing technical-grade chlorotoluron and formulated phenylurea herbicides.     

Light-dependent enhanced metabolism of chlorotoluron in a substituted urea herbicide-resistant biotype ofLolium rigidum Gaud.

A population ofLolium rigidum Gaud. displays resistance to the herbicide chlorotoluron endowed by enhanced metabolism of this herbicide. The level of resistance in intact plants of this population is light dependent. Resistance is about 4-fold at 110 mol photons·m^–2·s^–1, but increases to 11-fold at 600 mol photons·m^–2·s^–1. For seedlings grown in the dark, the rate of chlorotoluron metabolism is identical between biotypes; however, seedlings of the resistant biotype grown in the light display enhanced chlorotoluron metabolism compared to the susceptible biotype. Specifically, light with blue wavelengths induces chlorotoluron metabolism in the resistant biotype. An analysis of the metabolites produced indicates that two routes of chlorotoluron metabolism occur inL. rigidum. These are characterised by initial reactions leading to ringmethyl hydroxylation orN-demethylation of the herbicide. The ring-methyl hydroxylation pathway is increased greatly in light-grown resistant seedlings compared to susceptible seedlings, whereas theN-demethylation pathway is only slightly increased. The differential induction of these two pathways in resistantL. rigidum by light suggests that enhanced activity of two different enzymes may be involved in chlorotoluron resistance.Abbreviations ABT 1-aminobenzotriazole - LD₅₀ dose giving 50% mortality - LSS liquid scintillation spectroscopy     

Genetic control of a cytochrome P450 metabolism-based herbicide resistance mechanism in Lolium rigidum

The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a well-characterized Lolium rigidum biotype. The phenotypic resistance segregation in herbicide resistant and susceptible parents, F1, F2 and backcross (BC) families was analyzed as plant survival following treatment with the chemically unrelated herbicides diclofop-methyl or chlorsulfuron. Dominance and nuclear gene inheritance was observed in F1 families when treated at the recommended field doses of both herbicides. The segregation values of P450 herbicide resistance phenotypic traits observed in F2 and BC families was consistent with resistance endowed by two additive genes in most cases. In obligate out-crossing species such as L. rigidum, herbicide selection can easily result in accumulation of resistance genes within individuals.     

Stabilization of microbial cytochrome P-450 activity by creation of station-phase conditions in a continuously operated immobilized-cell reactor.

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450-based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in "freezing" of a physiological steady-state analog under stationary phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.     

Growth and Photosynthetic Characteristics of Two Biotypes of the Weed Black-grass (Alopecurus myosuroides Huds.) Resistant and Susceptible to the Herbicide Chlorotoluron

Response of two biotypes of black-grass (Alopecurus myosuroidesHuds.) to the herbicide, chlorotoluron, was characterized inglasshouse and laboratory studies. ED₅₀values, defined as theamount (kg active ingredient ha^-1) of chlorotoluron requiredto reduce fresh mass by 50% under standard conditions, weredetermined for a resistant biotype (39.3 kg a.i. ha^-1) collectedfrom Peldon, Essex, UK and a susceptible biotype (0.93 kg a.i.ha^-1) obtained commercially, giving a resistance factor of 42.The resistance factor was calculated as the ratio of ED₅₀valuesand describes the increase in amount of herbicide needed toreduce fresh mass by 50% in the resistant, compared to the susceptible,biotype. Resistance was further characterized by measurementsof whole plant growth and photosynthesis. Relative growth rate,number of tillers, leaf area and mean fresh mass were the samein untreated plants of both biotypes, and rates of photosynthesisat both high and low photon flux were similar, with no differencein apparent quantum yield. Photosynthesis by whole plants wasstudied over a 24 h period following chlorotoluron treatment.Resistant plants showed no reduction in photosynthesis overthis period, whereas photosynthesis by susceptible plants ceased10 h after treatment and did not recover. Alopecurus myosuroides ; black-grass; herbicide resistance; chlorotoluron     

Enhanced rates of herbicide metabolism in low herbicide‐dose selected resistant Lolium rigidum

Lolium rigidum is an obligately cross‐pollinated, genetically diverse species and an economically important herbicide resistance‐prone weed. Our previous work has demonstrated that recurrent selection of initially susceptible L. rigidum populations with low herbicide rates results in rapid herbicide resistance evolution. Here we report on the mechanisms endowing low‐dose‐selected diclofop‐methyl resistance in L. rigidum. Results showed that resistance was not due to target‐site ACCase mutations or overproduction, or differential herbicide leaf uptake and translocation. The in vivo de‐esterification of diclofop‐methyl into phytotoxic diclofop acid was rapid and similar in resistant versus susceptible populations. However, further metabolism of diclofop acid into non‐toxic metabolites was always faster in resistant plants than susceptible plants, resulting in up to 2.6‐fold lower level of diclofop acid in resistant plants. This corresponded well with up to twofold higher level of diclofop acid metabolites in resistant plants. The major polar metabolites of diclofop acid chromatographically resembled those of wheat, a naturally tolerant species. Clearly, recurrent selection at reduced herbicide rates selected for non‐target‐site‐based enhanced rates of herbicide metabolism, likely involving cytochrome P450 monooxygenases.     

Herbicide resistance of transgenic rice plants expressing human CYP1A1

Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅱ:氧化代谢

氧化代谢的增强是引起家蝇对二氯苯醚菊酯产生抗性的因素之一.抗性家蝇多功能氧化酶的萘羟化活性、对二氯苯醚菊酯的氧化代谢能力和微粒体细胞色素P₄₅₀含量分别是正常家蝇的2、1.48、1.33倍.正常家蝇和抗性家蝇细胞色素P₄₅₀在对增效磷(SV₁)和氧化胡椒基丁醚(Pb)的敏感性上也存在着差异.SV₁与多功能氧化酶专一性抑制剂Pb一样,对该酶系催化的萘羟化活性及二氯苯醚菊酯的氧化代谢有明显的抑制作用,这种抑制作用是SV₁在家蝇体内对二氯苯醚菊酯增效的机理之一.SV₁对氧化代谢的抑制与它和微粒体细胞色素P₄₅₀相互作用形成非活性复合体有关.     

Graminicide resistance in a blackgrass (Alopecurus myosuroides) population correlates with insensitivity of acetyl-CoA carboxylase

The appearance of biotypes of the annual grass weed black‐grass (Alopecurus myosuroides L. Huds), which are resistant to certain graminicides, is the most significant example of acquired resistance to herbicides seen so far in European agriculture. An investigation was perfomed into the basis of the specific cross‐resistance to cyclohexanedione (CHD) and aryloxyphenoxypropionoic acid (AOPP) herbicides in the ‘Notts A1’ population of A. myosuroides, which survived treatment of fields with recommended rates of AOPP herbicides. In comparison with the wild‐type ‘Rothamsted’ population, the resistant biotype showed over 100‐fold resistance to these herbicides in a hydroponic growth system. Biosynthesis of fatty acids and activity of crude extracts of acetyl‐CoA carboxylase (ACCase) were commensurately less sensitive to these herbicides in Notts A1 compared with the Rothamsted biotype. These data are consistent with the hypothesis that the highly resistant population has arisen through selection of a mutant ACCase which is much less sensitive to the AOPP and CHD graminicides. Rapidly growing cell suspension cultures established from the Notts A1 population also showed high resistance indices for CHD or AOPP herbicides compared with cultures from the Rothamsted biotype. Fatty acid biosynthesis and ACCase activity in the cell suspensions were similarly sensitive towards the graminicides to those in the foliar tissue counterparts of the resistant and sensitive populations. Moreover, purification of the main (chloroplast) isoform of acetyl‐CoA carboxylase showed that this enzyme from the Notts A1 population was over 200‐fold less sensitive towards the AOPP herbicide, quizalofop, than the equivalent isoform from the Rothamsted population. These data again fully supported the proposal that resistance in the Notts biotype is due to an insensitive acetyl‐CoA carboxylase isoform. Overall, cell suspensions were also demonstrated to be excellent tools for further investigation of the molecular basis of the high level herbicide resistance which is prone to occur in A. myosuroides.     

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F₂ populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.     

Double herbicide-resistant biotypes of wild oat (<Emphasis Type="Italic">Avena fatua</Emphasis>) display characteristic metabolic fingerprints before and after applying ACCase- and ALS-inhibitors

Plant herbicides inhibit specific enzymes of biosynthetic metabolism, such as acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS). Herbicide resistance can be caused by point mutations at the binding domains, catalytic sites and other regions within multimeric enzymes. Direct-injection electrospray mass spectrometry was used for high-throughput metabolic fingerprinting for finding significant differences among biotypes in response to herbicide application. A Mexican biotype of wild oat (Avena fatua) that displays multiple resistances to ACCase- and ALS-inhibiting herbicides was characterized. The dose–response test showed that the double-resistant biotype had a resistance index of 3.58 for pinoxaden and 3.53 for mesosulfuron-methyl. Resistance was accompanied by characteristic mutations at the site of action: an I-1781-L substitution occurred in the ACCase enzyme and an S-653-N mutation was identified within the ALS enzyme. Other mutations were also detected in the genes of the Mexican biotypes. The ionomic fingerprint showed that the multiple-resistant biotype had a markedly different metabolic pattern under control conditions and that this difference was accentuated after herbicide treatment. This demonstrates that single changes of amino acid sequences can produce several holistic modifications in the metabolism of resistant plants compared to susceptible plants. We conclude that in addition to genetic resistance, additional mechanisms of metabolic adaptation and detoxification can occur in multiple-resistant weed plants.     

细胞色素P450酶的结构、功能与应用研究进展

细胞色素P450 (cytochrome P450,CYP)酶是广泛存在于微生物、动植物及人体中与膜结合的血红蛋白类酶,具有氧化、环氧化、羟化、去甲基化等多种生物催化活性。CYP酶在药物、类固醇、脂溶性维生素和许多其他类型化学物质的代谢中具有重要作用,其在异源物质的解毒、药物相互作用和内分泌功能等领域的研究是热点问题。本综述对CYP的结构、功能、临床应用与开发前景进行了概述,并对其最新的研究现状和发展前景进行探讨。     

Inheritance of fenoxaprop-P-ethyl resistance in a blackgrass (Alopecurus myosuroides Huds.) population

A blackgrass population has developed resistance to fenoxaprop-P-ethyl following field selection with the herbicide for 6 consecutive years. Within this population, 95% of the individuals are also resistant to flupyrsulfuron. Both the inheritance(s) and the mechanism(s) of resistances were investigated by making crosses between the resistant and a susceptible biotype. The inheritance was followed through the F₁ and F₂ generations either by spraying the herbicide on seedlings at the three-leaf stage or using a seedling bioassay, based on coleoptile length. No maternal effects were evident in the fenoxaprop-P-ethyl responses of the F₁ plants, suggesting that the inheritance was nuclear. Some F₁ families treated with fenoxaprop-P-ethyl segregated in a 3:1 (resistant:susceptible) ratio, indicating that the resistance was conferred by two dominant and independent nuclear genes. This was confirmed by the 15:1 (R:S) ratio observed in the F₂ generation treated with fenoxaprop- P-ethyl. The use of selective inhibitors of herbicide de-toxifying enzymes (aminobenzotriazole, pyperonylbutoxide, malathion and tridiphane) with the F₂ plants suggested that each of the two genes may govern two different mechanisms of fenoxaprop-P-ethyl resistance: the ACCase mutation previously postulated and an enhanced herbicide metabolism, mediated by cytochrome P 450 mono-oxygenases (P 450) susceptible to malathion. The P 450 activity may also confer resistance to flupyrsulfuron. This study clearly indicates that two distinct mechanisms of resistance may co-exist in the same plant. Received: 18 August 2000 / Accepted: 6 December 2000     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : II. Chlorsulfuron Resistance Involves a Wheat-Like Detoxification System

Lolium rigidum Gaud. biotype SLR31 is resistant to the herbicide diclofop-methyl and cross-resistant to several sulfonylurea herbicides. Wheat and the cross-resistant ryegrass exhibit similar patterns of resistance to sulfonylurea herbicides, suggesting that the mechanism of resistance may be similar. Cross-resistant ryegrass is also resistant to the wheat-selective imidazolinone herbicide imazamethabenz. The cross-resistant biotype SLR31 metabolized [phenyl-U-¹⁴C]chlorsulfuron at a faster rate than a biotype which is susceptible to both diclofop-methyl and chlorsulfuron. A third biotype which is resistant to diclofop-methyl but not to chlorsulfuron metabolized chlorsulfuron at the same rate as the susceptible biotype. The increased metabolism of chlorsulfuron observed in the cross-resistant biotype is, therefore, correlated with the patterns of resistance observed in these L. rigidum biotypes. During high performance liquid chromatography analysis the major metabolite of chlorsulfuron in both susceptible and cross-resistant ryegrass coeluted with the major metabolite produced in wheat. The major product is clearly different from the major product in the tolerant dicot species, flax (Linium usitatissimum). The elution pattern of metabolites of chlorsulfuron was the same for both the susceptible and cross-resistant ryegrass but the cross-resistant ryegrass metabolized chlorsulfuron more rapidly. The investigation of the dose response to sulfonylurea herbicides at the whole plant level and the study of the metabolism of chlorsulfuron provide two independent sets of data which both suggest that the resistance to chlorsulfuron in cross-resistant ryegrass biotype SLR31 involves a wheat-like detoxification system.     

一个致倦库蚊杀虫剂敏感品系的筛选

化学防治是控制蚊虫传播疾病的主要方法, 抗性监测表明我国蚊虫已对有机磷、 有机氯、 氨基甲酸酯和拟除虫菊酯类杀虫剂产生了不同程度的抗性。蚊虫抗药性的分子机制主要包括靶标抗性和三大解毒酶家族带来的代谢抗性。筛选对杀虫剂敏感的品系是抗性监测和抗性机理研究必不可少的材料。本研究通过从一个致倦库蚊Culex pipiens quinquefasciatus野生种群筛选无乙酰胆碱酯酶G119S突变且具有低活性羧酸酯酶、 P450单加氧酶和谷胱甘肽-S-转移酶的单雌系, 建立了一个对杀虫剂敏感的致倦库蚊品系。该品系的羧酸酯酶活性是敏感品系S-lab的2.5倍, P450单加氧酶和谷胱甘肽-S-转移酶的活性与S-lab相当。生物测定表明, 与S-lab相比, 该品系对有机磷杀虫剂有低于2倍的抗性, 对氨基甲酸酯和拟除虫菊酯类杀虫剂没有抗性, 可以作为相对敏感品系用于抗性监测。     

Movement of paraquat in resistant and susceptible biotypes of Erigeron philadelphicus and E. canadensis

Paraquat (1,1'-dimethyl-4,4'-bipyridinium) resistant biotypes of Erigeron philadelphicus and E. canadensis , from fields where paraquat had been used for weed control, showed more than 100 times higher resistance than the susceptible biotype of both plants. Excised leaves of the susceptible biotypes wilted when supplied with more than 5 μ M paraquatat at the cut ends, but those from the resistant biotypes did not wilt even at 500 μ M. Autoradiographs indicated that (¹⁴CH₃)-paraquat taken up through the cut ends was rapidly distributed through the vascular system in leaves of the susceptible biotype, but was barely translocated in leaves of the resistant biotype. The amount of paraquat taken up during 48 h in the resistant biotype was 0.5% of that in the susceptible biotype in light. This difference in paraquat movement may be correlated with paraquat resistance in Erigeron.     

Amylase induction during seed germination in isoproturon susceptible and resistant biotypes of Phalaris minor Retz

Isoproturon resistant biotype of P. minor germinates early, shows higher germination percentage and faster rate of growth as compared to the susceptible biotype. Higher amylase activity is observed in the initial hours of imbibition in the resistant biotype. In the susceptible biotype it is activated at a much later stage.     

Germination and emergence characteristics of triazine-susceptible and triazine-resistant biotypes of Solanum nigrum

1. Seedling emergence patterns of triazine-susceptible and triazine-resistant Solanum nigrum in the field were studied in Wageningen, the Netherlands. Emergence patterns were similar in the first year, but in the second year resistant seedlings emerged faster and the number of resistant seedlings was higher. To explain emergence patterns, a germination experiment was carried out.
2. Seeds from two populations with triazine-susceptible and -resistant biotypes were buried in late autumn and exhumed monthly during spring. Germination was assessed in incubators at different constant temperatures.
3. The lowest temperatures for germination of seeds from the Achterberg population ranged from 20°C on 1 February to 10°C on 1 May for the susceptible biotype, and from 15°C on 1 February to 10°C on 1 May for the resistant biotype. The lowest temperatures for germination of seeds from the Zelhem population ranged from 25°C on 1 February to 10°C on 1 May for the susceptible biotype, and from 15°C on 1 February to 10°C on 1 May for the resistant biotype. The minimum germination temperature of seeds from the resistant biotype appeared to be lower than that of the susceptible biotype.
4. Emergence patterns in the field could be explained by soil temperature and different minimum germination temperature requirements of seeds from the triazine-susceptible and -resistant biotype. This knowledge can be used to manage triazine-resistant biotypes of S. nigrum by the timing of soil cultivation.     

Biochemical characterization of hydrolytic and oxidative enzymes in insecticide resistant and susceptible strains of the German cockroach (Dictyoptera: Blattellidae).

We have identified resistance mechanisms in the German cockroach, Blattella germanica (L.), for propoxur and chlorpyrifos in strains of cockroaches that display multiresistance to several organophosphate and carbamate insecticides. The resistance mechanisms involve the combined effects of increased oxidative and hydrolytic metabolism and both strains are resistant to chlorpyrifos and propoxur. Experiments designed to test for similarity in metabolic enzymes suggest that, although the mechanisms involve similar processes, the enzymes responsible for insecticide detoxification are different in the two strains. Both resistant strains exhibited enhanced activity toward alpha-naphtholic esters relative to a standard susceptible strain; however, analysis of the progeny from resistant X susceptible crosses suggests that this general esterase activity is inherited differently than propoxur or chlorpyrifos resistance. Hybrids of the propoxur-resistant strain displayed the highest activity of all cockroaches tested, in contrast to hybrids of the chlorpyrifos-resistant strain, which were similar to the susceptible strain. Native gel electrophoresis of cytosolic preparations provided further evidence for differences in the pattern of hydrolytic enzymes and inheritance of resistance in the two strains. Analysis of components of the cytochrome P450-dependent monooxygenase system and activities toward model substrates indicate that the two resistance mechanisms also involve different oxidative processes. The propoxur-resistant strain displayed significantly higher levels of total cytochrome P450, but no other components were correlated with resistance. In contrast with the chlopyrifos-resistant strain, which was similar to the susceptible strain in all parameters measured, activity toward model substrates was higher in the propoxur-resistant strain than in any of the other strains and hybrids tested.(ABSTRACT TRUNCATED AT 250 WORDS)