Chlorophyll Fluorescence Assays Research Articles

Triazine resistance in Amaranthus tuberculatus (Moq) Sauer that is not site-of-action mediated.

While surveying Illinois Amaranthus tuberculatus (Moq) Sauer (tall waterhemp) half-sib populations for herbicide response variability, several were observed to segregate for resistance to atrazine. Studies were conducted on greenhouse-grown A tuberculatus plants to compare atrazine responses among populations that were segregating for resistance (SegR), uniformly sensitive (UniS) or uniformly resistant (UniR). In chlorophyll fluorescence assays, leaves of plants from the SegR and UniS populations displayed changes in fluorescence after treatment with atrazine, indicating that atrazine was inhibiting electron transport of photosystem II in chloroplasts. Sequencing of a fragment of psbA, which encodes the D1 protein, revealed that the SegR population did not contain the amino acid substitution that is typically found in triazine-resistant plants. Whole-plant herbicide dose-response experiments revealed that, relative to the UniS population, atrazine resistances in the UniR and SegR populations were > 770-fold and 16-fold, respectively. The SegR population was also resistant to cyanazine (59-fold), but not to metribuzin, linuron or pyridate. Triazine resistance in the SegR population was shown to be a nuclear inherited trait, unlike maternal inheritance of site-of-action mediated triazine resistance found in the UniR population. Taken collectively, these findings confirm the existence of two distinct triazine resistance mechanisms in A tuberculatus.

Comparison of chlorophyll fluorescence and whole‐plant bioassays of isoproturon

Summary The response to aqueous solutions of isoproturon was examined on intact plants of Matricaria perforata, Apera spica‐venti and Sinapis alba in greenhouse tests, comparing visual estimates of shoot phytotoxicity at 30 d after application (DAA) with measurements of fast chlorophyll fluorescence induction at 10 DAA on leaves of the same treated plants. The sensitivity was greatest from pre‐emergence application to intact plants of M. perforata and A. spica‐venti, whereas post‐emergence sprays were relatively more active on S. alba. The use of fluorescence induction was investigated further in Petri‐dish experiments on excised M. perforata leaf segments. The Petri‐dish bioassay enabled detection of isoproturon at concentrations above 9 × 10−7 M within 96 h after herbicide exposure. The potential for using chlorophyll fluorescence assays for the determination of isoproturon content in soil extracts is discussed.

Identification of Two Mechanisms of Atrazine Resistance in Setaria faberi and Setaria viridis Biotypes

Abstract Two resistant (R) biotypes of Setaria faberi and S. viridis have been detected in maize fields continuously treated with triazine herbicides. The R biotypes of S. faberi and S. viridis were 10.0 and 6.5 times, respectively, more resistant to atrazine than their susceptible (S) biotypes. Both R biotype plastids had less affinity for atrazine than the S biotypes based on chlorophyll fluorescence and Hill reaction assays. As expected, they exhibited cross-resistance to a wide variety of photosystem II inhibitors. The hierarchy of resistance level of these two biotypes was chloro- s -triazines ⪢ methoxy- s -triazines > methyltio- s -triazines ≥ as-triazines. There was no difference in the absorption and translocation of [ 14 C]atrazine in R and S biotypes. Fast fluorescence induction curves showed that atrazine inhibited whole-leaf photosynthesis in S biotypes and they later recovered slight fluorescence activity in both populations. All biotypes produced the same pattern of metabolites, but the R biotypes detoxified the atrazine to conjugate-atrazine at a higher rate than the S biotypes (R > 75% > S). The five wild-type Setaria spp. found in Spain showed differences in detoxification of atrazine. The hierarchy of detoxifying level of atrazine of these Setaria spp. was S. verticilata ≃ S. adherens ⪢ S. faberi ≃ S. viridis >⪢ S. glauca .

Using chlorophyll a fluorescence to detect the onset of anthracene photoinduced toxicity in Lemna gibba, and the mitigating effects of a commercial humic acid

The influence of a commercial humic acid (Aldrich; AHA) on the development of anthracene photoinduced toxicity to the duckweed Lemna gibba was examined using both room‐ and low‐temperature (77°K) chlorophyll fluorescence assays. Plants were exposed to 2 mg liter‐1 anthracene both with and without 6.2 mg liter‐1 of AHA and grown under either visible light or simulated solar radiation that mimics the natural abundance of UV radiation. Exposure periods ranged from 1 to 48 h to examine temporal changes in chlorophyll degradation and chlorophyll a fluorescence induction in response to these light and HA treatments. The onset of anthracene photoinduced toxicity followed a definite sequence; chlorophyll a fluorescence induction parameters (Fv/Fm , and t½) responded earliest to anthracene exposure, with observable chlorophyll degradation requiring up to 24 to 48 h. Of these, t½ was the most sensitive, with significant inhibition apparent within 1 h of exposure. Throughout the entire 48‐h exposure, 6.2 mg liter‐1 AHA ameliorated the photoinduced toxicity of anthracene, in terms of both chlorophyll degradation and Fv/Fm inhibition. In contrast, AHA delayed the complete inhibition of t½ by only 1 to 24 h rather than permanently protecting the plants from anthracene damage to PS2. This suggests that AHA may slow, but not prevent, the entrance of either intact anthracene or its photooxidized byproducts under these exposure conditions.

A kochia (Kochia scoparia) biotype resistant to triazine and ALS-inhibiting herbicides

A kochia biotype from McDonough County, Illinois, was suspected to be resistant to both triazine and acetolactate synthase (ALS)-inhibiting herbicides. We performed greenhouse and laboratory experiments to confirm, quantify, and determine the molecular basis of multiple herbicide resistance in this biotype. Whole-plant phytotoxicity assays confirmed that the biotype was resistant to triazine (atrazine), imidazolinone (imazethapyr), and sulfonylurea (thifensulfuron and chlorsulfuron) herbicides. Relative to a susceptible kochia biotype, resistance to these herbicides ranged from 500- to > 28,000-fold. The kochia biotype from McDonough County also displayed high levels of resistance (2,000- to 9,000-fold) to ALS-inhibiting herbicides in in vivo ALS enzyme assays, indicating that resistance to these herbicides was site-of-action mediated. Results from chlorophyll fluorescence assays indicated that triazine resistance was also site-of-action mediated. Foliar applications of atrazine had little or no effect on photosynthesis in the resistant biotype, even when atrazine concentrations were 108-fold higher than needed to inhibit photosynthesis in the susceptible biotype. A region of the gene encoding the D1 protein of photosystem II and all of the open reading frame of the gene encoding ALS were sequenced and compared between the resistant and susceptible biotypes. Resistance to triazine and ALS-inhibiting herbicides in the kochia biotype from McDonough County was conferred by, respectively, a glycine for serine substitution at residue 264 of the D1 protein and a leucine for tryptophan substitution at residue 570 of ALS.

A biotype of common waterhemp (Amaranthus rudis) resistant to triazine and ALS herbicides

A common waterhemp biotype that was not controlled by triazine or acetolactate synthase (ALS)-inhibiting herbicides was isolated from a field in Bond County, IL, in the fall of 1996. Greenhouse and laboratory experiments determined resistance to atrazine and three ALS-inhibiting herbicides in this biotype. Based on whole-plant response, the Bond County common waterhemp biotype required over 1,000 times more imazethapyr relative to a susceptible biotype to reduce growth 50%. Cross-resistance to thifensulfuron, a sulfonylurea, and flumetsulam, a triazolopyrimidine sulfonanilide, was also detected. Based on in vivo enzyme assays, ALS in the Bond County common waterhemp biotype was 20-, > 8-, and 68-fold less sensitive than ALS in the susceptible biotype to imazethapyr, thifensulfuron, and flumetsulam, respectively. Whole-plant efficacy trials also indicated that the Bond County common waterhemp biotype required more than 20 kg ha−1of atrazine to inhibit growth 50%. Chlorophyll fluorescence assays revealed that 100 nM atrazine inhibited photosynthesis in the susceptible biotype, whereas 10 M did not affect photosynthesis in the resistant biotype. Regions of the genes encoding ALS and D1 proteins were sequenced to determine the molecular basis for the resistances. Triazine resistance was conferred by a glycine for serine substitution at residue 264 of the D1 protein, while ALS resistance was conferred by a leucine for tryptophan substitution at residue 569 of ALS.

Chlorophyll fluorescence for rapid detection of propanil-resistant barnyardgrass (Echinochloa crus-galli)

Repeated use of propanil to control barnyardgrass (BYG) and other weeds in rice has led to the development of propanil-resistant barnyardgrass (R-BYG). R-BYG possesses elevated aryl acylamidase activity levels, which cause rapid metabolism of propanil analogous to propanil degradation in rice. The current screening method for determining propanil resistance in BYG requires about 10 mo. The present study examined the use of chlorophyll fluorescence as a more rapid method to identify propanil resistance in BYG soon after it is suspected. Chlorophyll fluorescence data from excised BYG leaf tissue (R-BYG and susceptible-BYG [S-BYG]; 13- to 41-d-old) exposed to 100 μM propanil for 2 h indicated a 95 to 100% inhibition of electron transport (photosynthesis inhibition) in both R- and S-BYG. However, when incubated in water in the dark for 22 h after the initial 2-h treatment, metabolism in R-BYG was sufficient to reduce levels of absorbed propanil and facilitate recovery. Lack of metabolism of propanil prevented recovery in S-BYG, thus allowing the two biotypes to be distinguished easily by the chlorophyll fluorescence assay. Further studies using this 2-h exposure to 100 μM propanil followed by a 22-h recovery period evaluated several assay parameters. A longer recovery time and the effects of various propanil concentrations were also evaluated. A herbicide dose-response curve showed the greatest difference in photosynthesis inhibition for both biotypes at about 100 μM propanil, but both biotypes were inhibited > 95% when treated with 400 μM propanil. Inhibition of photosynthesis in both biotypes was greatest when the recovery incubation temperature was 35 C compared to 20, 25, and 30 C. Fluorescence data from harvested tissue stored moist in plastic bags at 23 C (to simulate shipment) showed that biotypes could be differentiated even when received as late as 4 d after harvest. Thus, samples can be harvested from the field soon after propanil failure and resistance or susceptibility to propanil determined after only a few days. This technique can greatly reduce the time, space, and labor currently required to determine propanil resistance in BYG.

Chlorophyll fluorescence assay for kanamycin resistance screening in transgenic plants.

The applicability of a chlorophyll fluorescence assay for kanamycin (Km) resistance screening in transgenic tobacco (Nicotiana tabacum) and Arabidopsis thaliana plants was investigated. In wild-type leaves incubated in the presence of 200 mg/l Km, a decrease in maximum variable fluorescence ((Fv)m) and a significant increase in constant fluorescence (Fo) were observed. Using (Fv)m/Fo as a screening parameter, we were able to distinguish Km-treated samples from untreated samples within 4 days. This parameter was applied to Km resistance screening using tobacco plants transformed with the nptII gene via Agrobacterium. Among 74 shoots selected on medium containing 200 mg/l Km, 37 plants were scored as Km sensitive by the chlorophyll fluorescence assay. These 74 scorings proved to be accurate, as reconfirmed by (1) polymerase chain reaction amplification of the transgene, (2) enzymatic assay of neomycin phosphotransferase and (3) leaf disc assay. Using the chlorophyll fluorescence assay, we could also screen 3-week old Arabidopsis plants carrying the nptII gene. These results clearly demonstrate the reliability and efficiency of this nondestructive assay for Km resistance screening of transgenic plants.

A high-sensitivity chlorophyll fluorescence assay for monitoring herbicide inhibition of photosystem II in the chlorophyte Selenastrum capricornutum: Comparison with effect on cell growth

A pulse amplitude modulated fluorometer (PAM) was used to measure the chlorophyll fluorescence induction kinetics (OIDP). The increase of I level, recorded at two different excitation light intensities, was used to monitor atrazine, simazine, diuron and isoproturon. The methods used were applied to a sensitive chlorophyte, Selenastrum capricornutum. Concentrations of about 0.01 μM of diuron, 0.04 μM of isoproturon and atrazine and 0.08 μM of simazine can be detected significantly. 150-values obtained by the most sensitive method, corresponding to an excitation light-intensity of 130 μmol photon m -2 . sec -1 , are 0.3 μM for atrazine, 0.5 μM for simazine, 0.05 μM for diuron and 0.2 μM for isoproturon. The inhibition of S. capricornutum growth over a 4 days period was also studied and the I 50 obtained were about 0.4 μM for triazines, 0.03 μM for diuron and 0.2 μM for isoproturon.

Weedy Adaptation in Setaria Spp.: III. Variation in Herbicide Resistance in Setaria Spp

Setaria is a grass genus of about 125 species that includes both food crops and a number of important agricultural weeds. Setaria viridis, S. faberii, S. glauca, and S. geniculata are major agricultural weeds worldwide and in North America. There is currently an inadequate knowledge of inter- and intraspecific herbicide-resistance variation in these foxtail species despite the importance of this knowledge to understanding evolution of herbicide resistance and improving weed management. Previous isozyme analyses of these species indicate that significant variation in genetic diversity exists among foxtail populations. It is unknown whether this genetic diversity is correlated with variability in important adaptive traits such as herbicide resistance. Studies were conducted to determine if inter- and intraspecific differences in atrazine and metolachlor resistance exist in foxtail species. Three assays were utilized to make these determinations: whole plant dose response, in vivo leaf chlorophyll fluorescence, and glutathione S-transferase (GST) conjugation assays. Significant variations in atrazine and metolachlor resistance were revealed within and among foxtail species. Green and giant foxtail were more resistant to atrazine than was yellow foxtail. Although green and giant foxtail again had a similar level of resistance, yellow foxtail was the most resistant species to metolachlor. These results indicated that the resistance mechanisms (quantitative or qualitative) to these two herbicides may be different in yellow, green, and giant foxtail. Intraspecific differences in atrazine resistance were found within both green foxtail populations and with yellow foxtail populations. Intraspecific metolachlor resistance differences were detected among green foxtail populations, but not in other foxtail species. No evidence for population shifts to more resistant foxtail variants with prolonged atrazine exposure was found in several detailed studies. When populations from several farms with a long history of atrazine use were compared, no differences in atrazine resistance were detected among populations from treated areas and adjacent untreated areas. Chlorophyll fluorescence assays indicated a similar pattern of atrazine resistant among foxtail populations, although it was less sensitive in detecting differences than the whole plant assay. No differences in GST-mediated atrazine or metolachlor conjugation were detected within or between foxtail species. These results may indicate that GST-mediated glutathione-herbicide conjugation may not be the primary detoxification mechanism for these herbicides in these foxtail species. Alternatively, these results may be a reflection of mitigating factors, such as differences at the target site, or in fitness related to growth rate at critical growth stages, among the populations. Several foxtail species had significant inter- and intraspecific differences in GST-mediated 1-chloro-2,4-dinitrobenzene conjugation activity. In some instances these responses were similar to those observed in the whole plant responses to metolachlor, although the significance of these similarities was not clear.

A Chlorophyll Fluorescence Screening Test to Evaluate Chilling Tolerance in Tomato

The chilling tolerance of commercial Lycopersicon esculentum cultivars (H2653, H722), Solanum lycopersicoides, an F1 hybrid of S. lycopersicoides × Sub-Arctic Maxi, and 25 BC2F2 lines of L. hirsutum × H722 (backcrossed twice to H722) was evaluated using a chlorophyll fluorescence assay. The ratio of the initial to the peak fluorescence (Fo: Fp) measured from fully expanded leaves was chosen as an indicator of plant health. Chilling induced an increase in Fo: Fp that was correlated with the sensitivity of the plant to low-temperature stress. Values of Fo: Fp remained low for cold-treated S. lycopersicoides and the F1 hybrid, which showed few symptoms of chilling-related damage, whereas the commercial cultivars, which were essentially intolerant to low temperatures, had large increases in Fo: Fp. A full range of Fo: Fp values was measured in the 25 BC2F2 lines, indicating that some chilling tolerance from the L. hirsutum parent was expressed by plants in these populations.

Comparative Triazine Effects Upon System II Photochemistry in Chloroplasts of Two Common Lambsquarters(Chenopodium album)Biotypes

Biotypes of several annual broadleaved weeds tolerant to 2-chloro-s-triazines have been recorded, including common lambsquarters(Chenopodium albumL.). The mechanism of resistance in common lambsquarter was based on the differential inhibition of the Hill reaction in chloroplasts by atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine]. Chlorophyll fluorescence and electron transport assays were used with isolated chloroplasts of atrazine-tolerant and susceptible biotypes, to determine the effect of atrazine and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] on photosystem II (PS II) activity, differential atrazine penetration of the chloroplast envelope, and relative tolerance to chloro, methoxy, and methylthio triazines. Atrazine and diuron inhibited electron transport on the reducing side of PS II in susceptible biotype chloroplasts. In tolerant biotype chloroplasts only diuron inhibited electron transport whereas atrazine had only slight effects. There were no differences in the chloroplast membrane permeability to atrazine in the two biotypes. Chloroplasts of the atrazine-tolerant biotype of common lambsquarters were also tolerant to the other classes of triazines tested.