State of anti-prooxidant balance in soybean plants under the action of synergistic mixture of herbicides pulsar (imazamox) and harmony (thifensulfuron)

Размещено на http://www.allbest.ru/

Размещено на http://www.allbest.ru/

Pavlo Tychyna Uman state pedagogical university

State of anti-prooxidant balance in soybean plants under the action of synergistic mixture of herbicides pulsar (imazamox) and harmony (thifensulfuron)

Svitlana Sorokina,

Ph.D. (biological sciences), associate professor, associate professor of the department of biology and methods of its teaching

Uman

Abstract

soybean herbicide nitrogen fixation

The influence of herbicides inhibitors of acetolactatsynthase (ALS), pulsar (imazamoks) and harmony (tifensulfuron) and their mixtures on the selective phytotoxicity of soybean plants were investigated. It was established that under the conditions of the reduction of herbicide norms in a mixture to 5 and 0.75 l/ha, respectively, the selectivity for culture increased. Proof of this is the increasing contents of photosynthetic pigments and efficiency of soybean-rhizobium symbiosis. It was shown a high level of correlation of data regarding the impact of herbicides on soybean plants and its nitrogen fixation activity and data on changes in status of anti-prooxidant balance. This is perhaps an evidence of the contribution of stress reaction to soybean herbicide action in the negative impact of herbicides on the formation and functioning of the symbiotic nitrogen fixation of soybean.

Keywords: selective phytotoxicity, inhibitors of acetolactatsynthase, photosynthetic pigments, guaiacol peroxidase, hydrogen peroxide, the TBA-active substances, Glycine max L.

Main part

Problem statement. Soybean is a rather sensitive crop to the negative impact of weeds throughout its entire vegetative period [1, 5]. For instance, the average perennial yield losses of soybeans due to weed infestations amount to 9.2 c/ha, which accounts for 54% of the potential yield [4].

The main challenge in protecting soybean crops from weeds lies in the fact that selective and effective herbicides of the ALS inhibitor class, specifically imidazolinone derivatives, which are widely used in soybean cultivation today [12, 13], including PULSAR, are persistent and pose a threat to subsequent crops in the rotation. Selective treatments with different modes of action (bentazone, graminicides) only control a portion of the weed species spectrum, and their interaction when combined is highly undesirable. On one hand, the antagonistic influence of bentazone diminishes the effectiveness of graminicides in eliminating grassy weeds [17]. On the other hand, the synergistic increase of phytotoxicity by graminicides with bentazone leads to soybean damage (unpublished data).

Analysis of recent research and publications. To reduce the application rates of imidazolinones and consequently minimize the likelihood of residue accumulation in the soil, a strategy involves their application in combination with the herbicide Harmony, which shares the same mode of action site. This combination exhibits synergistic interaction. Field trials testing the effectiveness of this mixture confirmed a synergistic increase in phytotoxicity, allowing for effective weed control with reduced component application rates (unpublished data). However, the synergistic interaction of components may lead to reduced selectivity towards soybean since it's known that the selectivity of thifensulfuron towards soybean is low. The absence of visible negative effects of herbicides on soybean plants is insufficient proof of high selectivity, as the suppression of the symbiotic apparatus by herbicides cannot be ruled out [6, 11-12].

A possible mechanism for such negative influence might be that during the formation of the nitrogen-fixing apparatus, plants respond to the entry of nitrogenfixing bacteria in the same manner as they do to pathogen infection [8]. In this context, typical stress reactions are observed, including the stimulation of oxidative processes [2, 14]. Under moderate intensity of this response, the formation of the symbiotic apparatus will proceed normally, whereas excessive intensification of oxidative processes could lead to a pathological nature of macro - and microsymbiont interaction.

Even selective herbicides could affect the state of antioxidant equilibrium and temporarily shift the balance towards oxidative processes [9-10]. Hence, it is plausible that the additional influence of herbicides on the state of antioxidant equilibrium could contribute to a pathological course of processes during the formation of the symbiotic apparatus.

In light of the aforementioned, the objective of this study was to investigate the selectivity of herbicide mixtures, Harmony and PULSAR, on soybean plants. This was achieved by assessing the content of photosynthetic pigments in soybean leaves and evaluating the indicators of legume-rhizobial symbiosis efficiency. To discern a potential mechanism of herbicide and their mixture's influence on soybean plants, the state of antioxidant equilibrium was investigated.

Material base and methods of research. Vegetation experiments were conducted in the experimental field of the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, with a substrate humidity of 60% and natural illumination. Soybean plants were cultivated in plastic containers, with 6-8 plants per container and 8 kg of soil enriched with a Gelrite mixture containing 0.25 N (1 standard corresponds to 708 mg Ca(NO3) 2x4H2O per 1 kg of soil). Before sowing, the seeds of the Anna variety of soybeans were sterilized with a 70% ethanol solution, washed with running water, and then inoculated with a suspension of the Bradyrhizobium japonicum 634b strain for 1 hour.

The following herbicidal preparations were utilized in the study: PULSAR 40, w.r. (water-based solution) (imazamox, 40 g/l), produced by BASF, and HARMONY 75, w.g. (water-soluble granules) (thifensulfuron-methyl, 750 g/kg), produced by DuPont.

The experiment was established according to the following scheme

To determine the activity of the soluble form of guaiacol peroxidase, the method described by Ridges and Osborne [18] was employed. Plant material was homogenized in 0.06 M Sorensen buffer (pH = 6.2). The activity was measured in the supernatant after centrifugation of the homogenate at 1000g for 15 minutes. In a reaction cuvette, 0.5 ml of 0.007% guaiacol, 1.5 ml of phosphate buffer, 0.5 ml of enzyme extract were mixed, and the reaction was initiated by adding 0.5 ml of 0.15% H2O2. The absorbance was measured at 470 nm, and the obtained data were expressed in pmol/g dry weight-min.

The hydrogen peroxide content was determined using the reaction with titanium sulfate [15]. For the determination of endogenous hydrogen peroxide, 1 ml of the extract was mixed with 3 ml of 0.1% titanium sulfate, and the intensity of color development was measured spectrophotometrically at 410 nm. The hydrogen peroxide content was calculated using a calibration curve constructed based on known concentrations of H2O2.

The content of photosynthetic pigments was determined using the extraction method of weighing plant material in DMSO on a water bath at 67°C for 3 hours [19]. The pigment content was calculated in pg/mg of raw material mass.

The nitrogen fixation activity was determined using the commonly accepted acetylene method, modified in the laboratory of symbiotic nitrogen fixation at the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine [7,16].

Statistical analysis of the results was carried out using the standard computer package Microsoft Excel (mean, standard deviation, correlation analysis, ANOVA). For biochemical analyses, biological replication within each experiment was done three times. Additionally, each experiment was independently replicated 3-4 times.

Results of the study and their discussion. The action of the PULSAR herbicide at the application rate of 0.75 L/ha, despite the high selectivity of imidazolinones towards soybean, led to a certain decrease in the content of chlorophylls a and b in plants (Table 1, variant 2). The reduction in photosynthetic pigment content was most significant in the mixture of HARMONY with PULSAR at their highest application rates (variant 6), and the mixture with the lowest application rates of these herbicides exhibited the highest selectivity in terms of pigment content (variant 3).

It is known that one of the significant criteria for evaluating the effectiveness of soybean-rhizobial symbiosis is nitrogen fixation activity, which correlates with the mass of root nodules [3]. Under the influence of PULSAR, we observed a reduction in nitrogen fixation activity and nodule mass by 1.7 and 1.9 times, respectively (Table 2, variant 2). Decreasing the concentration of this herbicide in the mixture with the lowest application rate of HARMONY partially restored these indicators to the control level (variant 3). In the two other herbicide mixtures of PULSAR and HARMONY, where we reduced the application rates of one of the components, the indicators of nitrogen fixation activity and nodule mass were even lower than in the variant with PULSAR alone, and the lowest values were observed in the case of application rates of 5 g/ha + 0.75 L/ha of these herbicides in the mixture. In terms of the indicators of legume-rhizobial symbiosis efficiency, increasing the PULSAR application rate is relatively safer, while as the HARMONY application rate increases, selectivity towards the crop diminishes.

Under the effect of PULSAR (Table 3, variant 2), we observed a twofold increase in the activity of soluble guaiacol peroxidase compared to the control. When combining the PULSAR herbicide with HARMONY at the highest application rate among those studied, the activity of guaiacol peroxidase increased by an additional 30% compared to using PULSAR alone (variant 6). Conversely, reducing the application rate of at least one component of the mixture led to a decrease in guaiacol peroxidase activity. The most significant reduction in the activity of this enzyme was observed with the lowest application rates of the studied herbicides in the mixture (variant 3). The guaiacol peroxidase activity showed a strong negative correlation with the content of photosynthetic pigments: the correlation coefficient between the activity of this enzyme and, in particular, the content of chlorophyll a was -0.93.

The action of ALS inhibitors led to certain changes in hydrogen peroxide formation. Specifically, only under the effect of PULSAR and when combining PULSAR with HARMONY at the application rate of 5 g/ha, the hydrogen peroxide content increased by 20% compared to the control (variants 2 and 6). This relatively modest increase in hydrogen peroxide content, compared to the peroxidase activity, was likely due to the active detoxification of hydrogen peroxide by this enzyme. In other mixture variants, the hydrogen peroxide content remained at the control level or slightly lower, which also positively correlated with the indicators of guaiacol peroxidase activity.

On the 14th day after the application of herbicides and their mixtures, we observed a decrease in the content of TBK-active substances in soybean leaves compared to the control. This result suggests that the action of these selective herbicides is not associated with the acceleration of lipid peroxidation reactions. Therefore, the stress induced by them, as confirmed by changes in guaiacol peroxidase activity in soybean plants, is minor.

It is worth noting that treating pea plants with another herbicide from the imidazolinone class, imazethapyr, also led to an increase in guaiacol peroxidase activity in the leaves, while changes in other oxidative and antioxidant markers were minor.

Conclusions. According to all the criteria we investigated, the mixture of HARMONY with PULSAR at application rates of 3 g/ha + 0.5 L/ha is the most selective towards soybeans. In terms of selectivity, this mixture even slightly surpasses the selectivity of PULSAR at the application rate of 0.75 L/ha. Increasing the application rates of HARMONY and PULSAR to 5 g/ha and 0.75 L/ha, respectively, leads to a reduction in selectivity. In this regard, increasing the application rate of PULSAR is relatively safer, while increasing the application rate of HARMONY results in the most dramatic loss of selectivity. The high level of correlation between the effects of herbicides on soybean plants and their nitrogenfixing activity, as well as the changes in the state of the anti-peroxidant equilibrium of soybean, indicates that the stress response of soybean to herbicides is indeed a possible factor contributing to the negative impact of herbicides on the formation and subsequent functioning of the soybean's symbiotic nitrogen fixation apparatus.

References

1. Bakay I.D. (2005). Soybean crop weediness. Quarantine and Plant Protection, 3, 24 [in Ukrainian].

2. Vasilieva G.T., Mironova N.V., Luzova G.B., et al. (2004). Influence of inoculation with nitrogen-fixing bacteria of various effectiveness and compatibility on hydrogen peroxide content and catalase activity in pea seedlings. Agrochemistry, 6, 68-73 [in Ukrainian].

3. Datsenko V.K., Laguta S.K., Starchenkov E.P., et al. (1997). Effectiveness of legume - rhizobial symbiosis of different soybean varieties and Bradyrhizobium japonicum strains. Physiology and Biochemistry of Cultivated Plants, 29 (4), 299-303 [in Ukrainian].

4. Zherebko Y.V. (1998). Soybean weediness. Plant Protection, 8, 12-13 [in Ukrainian].

5. Klischenko S.V., Chernega T.O. (2003). Weed control in soybean crops. Plant Protection, 5, 13 [in Ukrainian].

6. Kovalzhiu A.I., Apostolov S.D., Sabelnikova V.I. (1990). The effectiveness of alfalfa symbiosis with nodulating bacteria under herbicide application. Reports of the Academy of Sciences of the MSSR. Series Biological and Chemical Sciences, 5, 13-18 [in Ukrainian].

7. Krikunets V.M. (1993). Acetylene reduction method in the study of legume-rhizobial symbiosis physiology. Physiology and Biochemistry of Cultivated Plants, 2, 419-430 [in Ukrainian].

8. Kruhova E.D. (2009). Specific strategies of nodulating and phytopathogenic bacteria during plant infection. Physiology and Biochemistry of Cultivated Plants, 41 (1), 3-15 [in Ukrainian].

9. Morderer E. Yu., Palanitsya M.P., Trach V.V. (2009). Synergistic enhancement of phytotoxic action of the graminicide fenoxaprop-P-ethyl in mixtures with metribuzin. Plant Physiology: Problems and Prospects of Development. Proceedings of the Congress of the Plant Physiologists Society, Kyiv, 2, 46-50 [in Ukrainian].

10. Palanitsya M.P., Morderer E. Yu., Rodzevich O.P., et al. (2010). Enhancement of the selective phytotoxicity of the graminicide fenoxaprop-P-ethyl. Weeds and Effective Crop Protection Systems. Proceedings of the VII Scientific-Theoretical Conference of the Ukrainian Herbologists Society, Kyiv, 158-164 [in Ukrainian].

11. Paromenskaya L.N., Kozhemyakov A.P., Chebotar N.I., et al. (1987). Effectiveness of symbiotic nitrogen fixation under herbicide application. Agricultural Biology, 2, 40-42 [in Ukrainian].

12. Patika V.P., Kots S. Ya., Volkogon V.V., et al. (2003). Biological Nitrogen. Kyiv: Svit, 424 p. [in Ukrainian].

13. Ponomarev G.V., Ginevskiy N.K., Altukhova T.V. (2003). Herbicides in soybean crops. Plant Protection and Quarantine, 11, 31 [in Ukrainian].

14. Buffard D., Esnault R., Kondorosi A. (1996). Role of plant defense in alfalfa during symbiosis. World Journal of Microbiology and Biotechnology, 12 (2), 175-188 [in English].

15. Chen L-M. (1999). Effect of excess copper on rice leaves: evidence for involvement of lipid peroxidation. Botanical Bulletin of Academia Sinica, 40, 283-287 [in English].

16. Hardy R.W.F., Holsten R.D., Jackson E.K., et al. (1968). The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiology, 43 (8), 1185-1207 [in English].

17. Jordan D.L. (1995). Influence of adjuvants on the antagonism of graminicides by broadleaf herbicides. Weed Technology, 9 (4), 741-747 [in English].

18. Ridge I., Osborne D. (1970). Hydroxyproline and peroxidases in cell walls of Pisum sativum: regulation by ethylene. Journal of Experimental Botany, 21 (69), 843-856 [in English].

19. Welburn A.R. (1994). The spectral determination of chlorophylls a and b as well as total carotenoids using various solvents with spectrophotometry of different resolution. Journal of Plant Physiology, 144 (3), 248-254 [in English].

20. Zabalza A., Gaston S., Sandalio L., et al. (2007). Oxidative stress is not related to the mode of action of herbicides that inhibit acetolactate synthase. Environmental and Experimental Botany, 59 (2), 150-159 [in English].

Размещено на Allbest.ru