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-endowing ACCase gene mutations in hexaploid wild oat (Avena fatua): insights into resistance evolution in a hexaploid species.

Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids.

Laparoscopic Nissen fundoplication post-oesophageal stenting: an unusual case.

Laparoscopic Nissen fundoplication post-oesophageal stenting is uncommon and yet to be reported. We report the case of a 57-year-old palliative lady who underwent surgery for symptomatic relief of severe gastrooesophageal reflux post-oesophageal stenting. Surgery was carried out successfully with no complications. On the evening post-surgery she was able to lie supine for the first time in months without symptoms of reflux. In conclusion, surgery is still valuable and may play an important role, even in a palliative setting.

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.

Evolution of glyphosate resistance in a Lolium rigidum population by glyphosate selection at sublethal doses.

The majority of the documented cases of field-evolved herbicide-resistant weed biotypes established that single major genes confer glyphosate resistance. However, the contribution of minor genes endowing substantial plant survival at sublethal herbicide doses may be a potential complementary path to herbicide resistance evolution in weed populations under selection. Here, we subjected a number of susceptible individuals of Lolium rigidum to recurrent glyphosate selection to test the potential for sublethal glyphosate doses to additively select for glyphosate resistance. After 3-4 cycles of glyphosate selection in two distinct environments, the progenies of the initially susceptible population were shifted toward glyphosate resistance. The results indicate progressive enrichment of minor gene trait(s) contributing toward plant survival in the glyphosate-selected progenies. After three generations of selection, the estimated LD(50) values were doubled compared with the original population and up to 33% plant survival was obtained in the glyphosate-selected progeny at the recommended glyphosate label rate. This level of resistance probably was the maximum shift achievable with sublethal glyphosate dose selection in this small population. Cross-pollination was a crucial factor enabling the rapid rate of accumulation of minor glyphosate resistance gene trait(s) that are likely to be present at a relatively high frequency in a small susceptible population. The mechanistic basis of the moderate glyphosate resistance level selected by sublethal glyphosate doses remains unknown and warrants future research. Studying the main factors influencing the evolution of resistant weed populations is crucial for understanding, predicting and managing herbicide resistance.

Pollen Expression of Herbicide Target Site Resistance Genes in Annual Ryegrass (Lolium rigidum).

Herbicide resistance can occur either through target-site insensitivity or by nontarget site-based mechanisms. Two herbicide-resistant biotypes of Lolium rigidum Gaud., one resistant to acetolactate synthase (ALS)-inhibiting herbicides (biotype WLR1) and the other resistant to acetyl CoA carboxylase (ACCase)-inhibiting herbicides (biotype WLR96) through target-site insensitivity at the whole plant and enzymic levels, were found to express this resistance in the pollen. Pollen produced by resistant biotypes grew uninhibited when challenged with herbicide, whereas that from a susceptible biotype was inhibited. A third biotype, SLR31, resistant to ACCase-inhibiting and certain ALS-inhibiting herbicides at the whole plant level through nontarget site-based mechanisms, did not exhibit this expression in the pollen. The technique described may form the basis for a rapid screen for certain nuclear-encoded, target site-based herbicide-resistance mechanisms.

Stomatal responses to humidity in Opuntia inermis in relation to control of CO2 and H2O exchange patterns.

At constant cladode temperature the stomatal resistance of O. inermis increased when the cladode-air vapor pressure difference was increased and stomatal resistance decreased when the cladode-air vapor pressure difference was lowered. Net CO2 fixation in the dark was very responsive to these humidity dependent changes in stomatal resistance. Net CO2 fixation and stomatal resistance in the light did not respond to changes in cladode-air vapor pressure differences in the light under the conditions tested. When temperature response functions for dark CO2 fixation were examined at constant ambient humidity, the reduction in dark CO2 fixation at higher temperatures was largely due to stomatal closure in response to the increased vapor pressure difference. The water requirement for net CO2 fixation in the dark at typical nocturnal vapor pressure differences was about 10 times lower than that of net CO2 fixation in the light at vapor pressure differences typical of the late afternoon. The role of the stomatal responses to humidity in determining the patterns and rates of net CO2 exchange in the light or dark, and its possible ecological significance is discussed.

High survival frequencies at low herbicide use rates in populations of Lolium rigidum result in rapid evolution of herbicide resistance.

The frequency of phenotypic resistance to herbicides in previously untreated weed populations and the herbicide dose applied to these populations are key determinants of the dynamics of selection for resistance. In total, 31 Lolium rigidum populations were collected from sites with no previous history of exposure to herbicides and where there was little probability of gene flow from adjacent resistant populations. The mean survival frequency across all 31 populations following two applications of commercial rates (375 g ha(-1)) of the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicide, diclofop-methyl was 0.43%. Survivors from five of these populations were grown to maturity and seed was collected. Dose-response experiments compared population level resistance to diclofop-methyl in these selected lines with their original parent populations. A single cycle of herbicide selection significantly increased resistance in all populations (LD(50) R:S ratios ranged from 2.8 to 23.2), confirming the inheritance and genetic basis of phenotypic resistance. In vitro assays of ACCase inhibition by diclofop acid indicated that resistance was due to a non-target-site mechanism. Following selection with diclofop-methyl, the five L. rigidum populations exhibited diverse patterns of cross-resistance to ACCase and ALS-inhibiting herbicides, suggesting that different genes or gene combinations were responsible for resistance. The relevance of these results to the management of herbicide resistance are discussed.

Resistance cost of a cytochrome P450 herbicide metabolism mechanism but not an ACCase target site mutation in a multiple resistant Lolium rigidum population.

Costs of resistance are predicted to reduce plant productivity in herbicide-resistant weeds. Lolium rigidum herbicide-susceptible individuals (S), individuals possessing cytochrome P450-based herbicide metabolism (P450) and multiple resistant individuals possessing a resistant ACCase and enhanced cytochrome P450 metabolism (ACCase/P450) were grown in the absence of mutual plant interaction to estimate plant growth traits. Both P450 and ACCase/P450 resistant phenotypes produced less above-ground biomass than the S phenotype during the vegetative stage. Reduced biomass production in the resistant phenotypes corresponded to a reduced relative growth rate and a lower net assimilation rate and rate of carbon fixation. There were no significant differences between the two resistant phenotypes, suggesting that costs of resistance are associated with P450 metabolism-based resistance. There were no differences in reproductive output among the three phenotypes, indicating that the cost of P450 resistance during vegetative growth is compensated during the production of reproductive structures. The P450-based herbicide metabolism is shown to be associated with physiological resistance costs, which may be manipulated by agronomic management to reduce the evolution of herbicide resistance.

Photoinhibition of intact attached leaves of c(3) plants illuminated in the absence of both carbon dioxide and of photorespiration.

When leaflets of bean and leaves of other species of C(3) plants are illuminated in the absence of CO(2) and at low O(2) partial pressure, the capacity for CO(2) assimilation at saturating light and its efficiency at low light intensities are inhibited. This photoinhibition is dependent on leaflet age and period of illumination. In young leaflets and following short exposure to these photoinhibitory conditions, some recovery of CO(2) assimilation capacity is observed immediately after treatment. Following substantial (70 to 80%) photoinhibition of CO(2) assimilation, recovery in fully expanded leaflets is observed only after 48 hours in normal air. The photoinhibition is largely prevented by providing CO(2) at partial pressures equivalent to the CO(2) compensation point, or by >210 millibars O(2) which permits internal CO(2) production by photorespiration. If leaflets are illuminated in 60 microbars CO(2) and 210 millibars O(2) (the CO(2) compensation point in air), no photoinhibition is observed. Electron transport processes and fluorescence emission associated with photosystem II are inhibited in chloroplast thylakoids isolated from leaflets after illumination in zero CO(2) and 10 millibars O(2). These studies support the hypothesis that CO(2) recycling through photorespiration is one means of effectively dissipating excess photochemical energy when CO(2) supply to illuminated leaves is limited.

Evolution of herbicide resistance in weeds: initial frequency of target site-based resistance to acetolactate synthase-inhibiting herbicides in Lolium rigidum.

The frequency of individuals resistant to two acetolactate synthase (ALS)-inhibiting herbicides in three previously untreated populations of Lolium rigidum was determined. The frequency of individuals resistant to the sulfonylurea herbicide sulfometuron-methyl varied from 2.2 x 10(-5) to 1.2 x 10(-4) and the frequency of individuals resistant to the imidazolinone herbicide imazapyr varied from 1 x 10(-5) to 5.8 x 10(-5) depending on the population. Application of sulfometuron-methyl selected individuals with a herbicide-insensitive ALS, which was also cross-resistant to imazapyr. The high initial frequency of individuals resistant to ALS-inhibiting herbicides in L. rigidumpopulations never previously exposed to these herbicides helps explain the rapid evolution of herbicide resistance in this species once ALS-inhibiting herbicides were used.

Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L.

A biotype of Sonchus oleraceus L. (Compositae) has developed resistance to herbicides inhibiting acetolactate synthase (ALS) following field selection with chlorsulfuron for 8 consecutive years. The aim of this study was to determine the inheritance and mechanism of resistance in this biotype. Determination of ALS activity and inhibition kinetics revealed that Km and Vmax did not vary greatly between the resistant and susceptible biotypes. ALS extracted from the resistant biotype was resistant to five ALS-inhibiting herbicides in an in vitro assay. ALS activity from the resistant biotype was 14 19, 2, 3 and 3 times more resistant to inhibition by chlorsulfuron, sulfometuron, imazethapyr, imazapyr and flumetsulam, respectively, than the susceptible biotype. Hybrids between the resistant and a susceptible biotype were produced, and inheritance was followed through the F1, F2 and F3 generations. F1 hybrids displayed a uniform intermediate level of resistance between resistant and susceptible parents. Three distinct phenotypes, resistant, intermediate and susceptible, were identified in the F2 generation following chlorsulfuron application. A segregation ratio of 1∶2∶1 was observed, indicative of the action of a single, nuclear, incompletely dominant gene. F3 families, derived from intermediate F2 individuals, segregated in a similar manner. Resistance to herbicides inhibiting ALS in this biotype of S. oleraceus is due to the effect of a single gene coding for a resistant form of the target enzyme, ALS.

Effect of Light Intensity during Growth on Photoinhibition of Intact Attached Bean Leaflets.

In the study reported here, two different photoinhibitory phenomena were compared within a single plant species. Bean plants were grown in three different light intensities to simulate sun and shade environments. The effects of photoinhibitory treatments on in vivo CO(2) assimilation rates and in vitro chloroplast electron transport reactions were investigated and the extent to which carbon metabolism served to prevent photoinhibition was characterized. It was shown that the photoinhibition which follows exposure of intact leaflets of low light-grown bean plants to high light intensity in normal air is essentially similar to that which occurs when leaflets of plants grown in full sunlight are illuminated in the absence of CO(2) at low O(2) partial pressures.

Effect of high-light treatments in inducing photoinhibition of photosynthesis in intact leaves of low-light grown Phaseolus vulgaris and Lastreopsis microsora.

Photoinhibition studies, using gas-exchange techniques, were conducted with leaflets of Phaseolus vulgaris L. plants that were grown under low photonfluence rates. Comparative measurements were made on attached, intact leaflets and in subsequently isolated chloroplasts. Photoinhibition studies were also conducted with attached fronds of the deep-shade fern Lastreopsis microsora (Endl.) Tindale. Leaflets of lowlight-grown Phaseolus vulgaris and fronds of the shade fern were found to be subject to similar photoinhibition when exposed to photon-fluence rates in excess of those at which they were grown. Photoinhibition following exposure to a photon fluence-rate approximating full sunlight is manifested as a reduction in the capacity for both light-saturated and light-limited carbon uptake and is reflected at the chloroplast level as substantial inhibition of electron flow through photosystem (PS) II, with little effect on PS I. The extent of photoinhibition is markedly dependent on the length of exposure to a high-light regime and on the actual photon-fluence rate maintained during treatment. A greater degree of photoinhibition is evident if carbon metabolism is prevented by the removal of CO2 than when maximum rates of CO2 uptake prevail throughout the exposure to a high photonfluence rate. Apparently a certain level of CO2 turnover is beneficial in providing a sink for photochemically generated energy. When leaf material is exposed to photon-fluence rates well in excess of the rate present during growth apparently the potentials of the various biophysical and photochemical means of dissipating excitation energy are exceeded and photoinhibition of photosynthesis results.

Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander.

The effect of exposing intact leaves and isolated chloroplast membranes of Nerium oleander L. to excessive light levels under otherwise favorable conditions was followed by measuring photosynthetic CO2 uptake, electron transport and low-temperature (77K=-196°C) fluorescence kinetics. Photoinhibition, as manifested by a reduced rate and photon (quantum) yield of photosynthesis and a reduced electron transport rate, was accompanied by marked changes in fluorescence characteristics of the exposed upper leaf surface while there was little effect on the shaded lower surface. The most prominent effect of photoinhibitory treatment of leaves and chloroplasts was a strong quenching of the variable fluorescence emission at 692 nm (Fv,692) while the instantaneous fluorescence (Fo,692) was slightly increased. The maximum and the variable fluorescence at 734 nm were also reduced but not as much as FM,692 and Fv,692. The results support the view that photoinhibition involves an inactivation of the primary photochemistry of photosystem II by damaging the reaction-center complex. In intact leaves photoinhibition increased with increased light level, increased exposure time, and with decreased temperature. Increased CO2 pressure or decreased O2 pressure provided no protection against photoinhibition. With isolated chloroplasts, inhibition of photosystem II occurred even under essentially anaerobic conditions. Measurements of fluorescence characteristics at 77K provides a simple, rapid, sensitive and reproducible method for assessing photoinhibitory injury to leaves. The method should prove especially useful in studies of the occurrence of photoinhibition in nature and of interactive effects between high light levels and major environmental stress factors.

Inhibition of photosynthetic reactions under water stress: interaction with light level.

When the shrub Nerium oleander L., growing under full natural daylight outdoors, was subjected to water stress, stomatal conductance declined, and so did non-stomatal components of photosynthesis, including the CO2-saturated rate of CO2 uptake by intact leaves and the activity of electron transport by chloroplasts isolated from stressed plants. This inactivation of photosynthetic activity was accompanied by changes in the fluorescence characteristics determined at 77 K (-196°C) for the upper leaf surface and from isolated chloroplasts. The maximum (F M) and the variable (F V) fluorescence yield at 692 nm were strongly quenched but there was little effect on the instantaneous (F O) fluorescence. There was a concomitant quenching of the maximum and variable fluorescence at 734 nm. These results indicate an inactivation of the primary photochemistry associated with photosystem II. The lower, naturally shaded surfaces of the same leaves were much less affected than the upper surfaces and water-stress treatment of plants kept in deep shade had little or no effect on the fluorescence characteristics of either surface, or of chloroplasts isolated from the water-stressed leaves. The effects of subjecting N. oleander plants, growing in full daylight, to water stress are indistinguishable from those resulting when plants, grown under a lower light regime, are exposed to full daylight (photoinhibition). Both kinds of stress evidently cause an inactivation of the primary photochemistry associated with photosystem II. The results indicate that water stress predisposes the leaves to photoinhibition. Recovery from this inhibition, following restoration of favorable water relations, is very slow, indicating that photoinhibition is an important component of the damage to the photosynthetic system that takes place when plants are exposed to water stress in the field. The underlying causes of this water-stress-induced susceptibility to photoinhibition are unknown; stomatal closure or elevated leaf temperature cannot explain the increased susceptibility.

Resistance to the herbicide paraquat and increased tolerance to photoinhibition are not correlated in several weed species.

Photoinhibition was examined in paraquat-resistant and paraquat-susceptible biotypes of Hordeum glaucum Steud., Hordeum leporinum Link., Arctotheca calendula (L.) Levyns., and Conyza bonariensis (L.) Cronq. Plants were photoinhibited at low temperature, and the extent of photoinhibition determined by O(2) evolution and 77 K fluorescence. No difference in the degree of photoinhibition was detected between paraquat-resistant and paraquat-susceptible biotypes for any of the species examined. C. bonariensis plants were also photoinhibited by treatment without CO(2) at either 21% (volume/volume) O(2) or 4% (volume/volume) O(2), and again no difference was observed between the paraquat-resistant and paraquat-susceptible biotypes in reduction of the ratio of variable fluorescence to maximal fluorescence. This is in contrast to a recent report (MAK Jansen, Y Shaaltiel, D Kazzes, O Canaani, S Malkin, J Gressel, [1989] Plant Physiol 91: 1174-1178 in which it was claimed that a paraquat-resistant biotype of C. bonariensis was more tolerant of photoinhibition than a paraquat-susceptible biotype. We conclude that paraquat-resistant biotypes of these plant species are not more tolerant of photoinhibition when compared with the paraquat-susceptible biotypes.

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): IV. Correlation between Membrane Effects and Resistance to Graminicides.

The herbicidally active aryloxyphenoxypropionates diclofop acid, haloxyfop acid, and fluazifop acid and the cyclohexanedione sethoxydim depolarized membranes in coleoptiles of eight biotypes of herbicide-susceptible and herbicide-resistant annual ryegrass (Lolium rigidum). Membrane polarity was reduced from -100 millivolts to -30 to -50 millivolts. Membranes repolarized after removal of the compounds only in biotypes with resistance to the compound added. Repolarization was not observed in herbicide-susceptible L. rigidum, nor was it observed in biotypes resistant to triazine, triazole, triazinone, phenylurea, or sulfonylurea herbicides but not resistant to aryloxyphenoxypropionates and cyclohexanediones. Chlorsulfuron, a sulfonylurea herbicide, at a saturating concentration of 1 micromolar, reduced membrane polarity in all biotypes studied by only 15 millivolts. The recovery of membrane potential following the removal of chlorsulfuron was restricted to chlorsulfuron-susceptible and -resistant biotypes that did not exhibit diclofop resistance. These differences in membrane responses are correlated with resistance to dicloflop rather than with resistance to chlorsulfuron. It is suggested that the differences may reflect altered membrane properties of diclofop-resistant biotypes. Further circumstantial evidence for dissimilarity of properties of membranes from diclofop-resistant and diclofop-susceptible ryegrass is provided by observations that K(+)/Na(+) ratios were significantly higher in coleoptiles from diclofop-resistant biotypes than in coleoptiles from susceptible plants. Intact and excised roots from susceptible biotypes were capable of acidifying the external medium, whereas roots from resistant biotypes were unable to do so. The ineluctable conclusion is that in L. rigidum the phenomena of membrane repolarization and resistance to aryloxyphenoxypropionate and cyclohexanedione herbicides are correlated.

Inheritance of bipyridyl herbicide resistance in Arctotheca calendula and Hordeum leporinum.

The mode of inheritance of resistance to bipyridyl herbicides in bipyridyl-resistant biotypes of Arctotheca calendula and of Hordeum leporinum was investigated. F1 plants from reciprocal crosses between diquat-resistant and -susceptible plants of A. calendula showed an intermediate response to diquat application that was nuclearly inherited. Treatment of F2 plants with 100 g ai ha(-1) of diquat or 800 g ai ha(-1) of paraquat killed all homozygous-susceptible plants, caused severe injury to heterozygous plants but only slight or no injury to homozygous-resistant plants. Back crosses of F1 to susceptible plants exhibited intermediate and susceptible phenotypes. The observed segregation ratios in F2 and test-cross populations fitted predicted segregation ratios, 1:2:1 (R:I:S) and 1:1 (I:S) respectively, showing that bipyridyl resistance is conferred by a single incompletely-dominant gene. Biotypes of paraquat-resistant and -susceptible H. leporinum were crossed reciprocally. F1 plants from reciprocal crosses showed an intermediate response to paraquat application. The F2 progeny showed segregation ratios that fitted the predicted segregation ratio of 1:2:1 (R:I:S) forinheritance of resistance being governed by a single partially-dominant gene.