Acta agriculturae Slovenica, 119/1, 1–7, Ljubljana 2023 doi:10.14720/aas.2023.119.1.2764 Original research article / izvirni znanstveni članek Mitigation of the negative effect of auxinic herbicide by bacterial suspen- sion of Pseudomonas protegens DA1.2 in wheat plants under drought conditions Sergey СHETVERIKOV 1, Arina FEOKTISTOVA 1, 2, Maksim TIMERGALIN 1, Timur RAMEEV 1, Gaisar HKUDAYGULOV 1, Aliya KENDJIEVA 1, Margarita BAKAEVA 1, Darya СHETVERIKOVA 1, Sergey STARIKOV 1, Danil SHARIPOV 1 Received July 07, 2022; accepted October 21, 2022. Delo je prispelo 7. julija 2022, sprejeto 21. oktobra 2022 1 Ufa Institute of biology – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences, Ufa, Russia 2 Corresponding author, e-mail: feoktistova.arisha@yandex.ru Mitigation of the negative effect of auxinic herbicide by bac- terial suspension of Pseudomonas protegens DA1.2 in wheat plants under drought conditions Abstract: Effect of auxin-producing bacterial strain (Pseudomonas protegens DA1.2) was investigated under condi- tions of drought and herbicide treatment in wheat plants. Posi- tive effect of the bacterial suspension on wheat plants treated with auxinic herbicide under drought conditions was mani- fested in reducing the content of malondialdehyde and proline, preventing inhibition of plant growth and normalizing chloro- phyll content. Under combined stress, changes in concentra- tions and redistribution of phytohormones in plants were de- tected. An imbalance in auxin distribution between shoots and roots could be the reason for the decrease in plant resistance to drought in combination with the herbicide. Treatment of plants with the bacterial suspension restored normal shoot-to- root ratio of auxins in plants. Thus, this bacterial strain showed the properties of synthetic auxin antidotes and can be recom- mended for optimizing the technology of herbicide application under drought conditions. Key words: bacterial strain; water deficiency; auxinic her- bicide; plant hormones; MDA; proline Blaženje negativnih učinkov herbicidov na osnovi auksina s suspenzijo bakterije Pseudomonas protegens DA1.2 pri pšenici v razmerah suše Izvleček: Učinek sevov bakterije, ki tvorijo auksin (Pseu- domonas protegens DA1.2) je bil preučevan pri pšenici v razme- rah suše in obravnavanja s herbicidi. Pozitivni učinki suspenzije bakterij na pšenici, ki je bila tretirana s herbicidi na osnovi au- ksina so se pokazali v zmanjšanju vsebnosti malondialdehida in prolina, kar je preprečilo zaviranje rasti rastlin in normalizi- ralo vsebnost klorofila. V razmerah kombiniranega stresa so se pojavile spremembe v koncentraciji in redistribuciji rastlinskih hormonov v tretiranih rastlinah. Neravnovesje v porazdelitvi auksina med poganjki in koreninami bi lahko bilo vzrok za zmanjšanje odpornosti rastlin na sušo v kombinaciji s herbicidi. Obravnavanje rastlin s suspenzijo bakterij je ohranilo normal- no razmerje v vsebnosti auksinov med poganjki in koreninami. Zaključimo lahko, da je ta bakterijski sev pokazal lastnosti anti- dotov sintetičnih auksinov in bi ga lahko priporočili za optimi- ziranje tehnologije uporabe herbicidov v razmerah suše. Ključne besede: bakterijski sev; pomankanje vode; au- ksinski herbicidi; rastlinski hormoni; MDA; prolin Acta agriculturae Slovenica, 119/1 – 20232 S. СHETVERIKOV et al. 1 INTRODUCTION Drought is a serious stress that reduces the yield of cultivated plants below their genetic potential (Shao et al., 2008; Asseng et al., 2020). Weeds are serious problem in modern agriculture, causing an average decrease in yield by 35 % (Oerke, 2006). The effectiveness of herbi- cides in weed control has allowed them to be widely used to prevent crop losses (Varhney et al., 2015). However, the use of preparations for chemical weeding of plants has a negative effect on cultivated plants, causing “herbi- cidal stress”, which becomes most notable under drought conditions (Varshney et al., 2015). It is known that under herbicidal stresses, photo- synthesis is the most sensitive process contributing to the plant biomass production (Kutilkin et al., 2018). There are also evidences of the effects of herbicides on the anti- oxidant system of plants (Ahemad and Khan, 2010). The role of plant growth regulators, such as hor- mones, in stress tolerance allows plants to choose the necessary strategy to counteract the adverse effects of abiotic stress and to develop stress resistance responses (Han et al., 2015). Hormones are necessary for the nor- mal functioning of growth processes throughout the plant life cycle; as a result they influence the yield and quality of agricultural crops. Recently, bacterial treatments are being given more attention in reducing plant abiotic stress. In the scientific literature, there are two areas of research aimed on reduc- tion of the damage caused by currently used herbicides. The goal of the first of them is to accelerate the process of herbicide destruction thereby reducing the negative im- pact of herbicides on cultivated plants. Microbiological transformation and detoxification of pesticides is rather fully presented in the literature (Silva et al., 2007; Martins et al., 2011; Kanissery and Sims, 2011). However, the po- tential of bacteria in mitigating pesticide stress in plants (the other area of research) has not been sufficiently stud- ied. There are few publications addressing this question (Ahemad and Khan, 2010; Bourahla et al., 2018; Chen- nappa et al., 2018). And we have not found any work devoted to the use of growth-stimulating bacteria under conditions of combined stress caused by drought and herbicide treatment. The previously isolated strain of Pseudomonas pro- tegens DA1.2 have shown to possess useful properties, namely resistance to herbicides, capacity of auxin synthe- sis, mobilization of inorganic phosphates, antagonism to phytopathogenic fungi, growth in the absence of a nitro- gen source in the medium, growth-stimulating and anti- stress effects on wheat plants (Chetverikov et al., 2021). The aim of the present work was to show the pros- pects of using the bacterial suspension of Pseudomonas protegens DA1.2 as an anti-stress agent when applied to wheat plants against the background of combined “her- bicide + drought” stress. 2 MATERIALS AND METHODS The studies were carried out in laboratory condi- tions on plants of bread spring wheat (Triticum aestivum L.) of a drought-resistant variety, Ekada 109. The bacte- rial strain P. protegens DA1.2 from the collection of mi- croorganisms of the Ufa Institute of Biology, Ufa Federal Research Center, Russian Academy of Sciences and the herbicide (against dicotyledons) based on auxin-like ac- tive substances 2,4-D (2-ethylhexyl ester) and dicamba (sodium salts) – “Chistalan” extra (LLC “AHK-AGRO”, Ufa), were used in the work. Drought conditions were achieved by reducing irrigation to 30 % of the total water capacity of the soil (this was 60 % in the control) and maintaining it throughout the experiment. Vessels with plants were watered daily with distilled water to the re- quired weight every day. Wheat seeds were sterilized and germinated for 3 days in a pallet on wet filter paper. Then they were plant- ed in vessels with a volume of 0.5 l filled with a soil-sand mixture in the ratio of 9 : 1 of 6 plants each, the plants were watered only with distilled water. Seedlings were grown at 14 - h photoperiod, day/night temperature re- gimes of 26 / 22 °C and irradiance of 400 µmol m-2 s-1 from mercury-arc and sodium vapor lamps. Seven days after seed germination, 1 ml of an aqueous solution of “Chistalan” (the working concentration of the herbicide was 0.5 ml l-1) and / or bacterial suspension of P. protegens DA1.2 (108 CFU ml-1) grown in the King B was sprayed on the soil and directly on the plants, using a manual sprayer with a graduated capacity of 20 ml with a divi- sion price of 0.1 ml. Three days after the treatment, shoots and roots of plants were sampled for analysis for hormones (3-indoly- lacetic acid (IAA), abscisic acid (ABA), and cytokinins), proline, malondialdehyde (MDA), and chlorophylls a, b while growth parameters were evaluated 2 weeks later. Determination of hormones content was performed using an enzyme immunoassay method. The plant mate- rial (shoots and roots) was cut and ground in a porcelain mortar to a homogeneous state, then extracted with 80 % ethanol. Alcohol extract was evaporated to an aqueous residue, centrifuged and the aliquots of the supernatant were taken for analysis. Purification and concentration of ABA and IAА was carried out according to a modified scheme with a decrease in volume (Veselov et al., 1992; Vysotskaya et al., 2009). Cytokinins were concentrated Acta agriculturae Slovenica, 119/1 – 2023 3 Mitigation of the negative effect of auxinic herbicide by bacterial suspension of Pseudomonas protegens ... under drought conditions on the C - 18 column and separated using thin-layer chromatography (Kudoyarova et al., 2014). To determine proline content in wheat shoots, the suspension was homogenized in 10 ml of 3 % sulfosali- cylic acid for protein precipitation. Then the homogenate was filtered, 1 ml of filtrate was taken into a clean test tube and 1 ml of acidic ninhydrin reagent (30 ml of gla- cial acetic acid + 20 ml of 6 M phosphoric acid + 1.25 g of ninhydrin) and 1 ml of glacial acetic acid were added. The mixture was incubated for 1 hour in a water bath at 100 °C, after which it was quickly cooled in ice. Two ml of toluene was added to the mixture and shaken. The colored organic phase was separated and its optical den- sity measured against pure toluene at a wavelength of 520 nm on a spectrophotometer. The proline content was calculated using a calibration curve, using crystal proline for its construction, with further conversion to dry and fresh mass of the sample (Bates et al., 1973). Lipid peroxidation was determined by the content of malondialdehyde. To achieve this, a sample of plant material was homogenized in 3 ml of distilled water and 3 ml of trichloroacetic acid. The homogenate was centri- fuged at 10000 × g for 10 minutes. Two ml of 0.5 % thio- barbituric acid was added to 2 ml of the supernatant and incubated in a water bath for 30 minutes at 95 °C. The tubes were centrifuged and optical density was measured at wavelengths of 532 nm and 600 nm. The concentration of MDA was calculated using an extinction coefficient of 155 mM-1 cm-1 (Costa et al., 2002). To determine the content of chlorophyll in wheat plants, 100 mg of leaves was taken. The samples were cut with scissors, placed in test tubes and filled with 6 ml of 96 % ethanol. The test tubes were tightly closed with lids and placed in a dark place for 24 - 48 hours. After the time elapsed, the tubes were shaken and the optical den- sity was measured at wavelengths of 665 nm and 649 nm. The content of chlorophylls “a”, “b” was calculated using the formulas: Chl a = 13.7 D665 – 5.76 D649; Chl b = 25.8 D649 – 7.6 D665 where Chl a and Chl b are the concentration of chlorophyll “a” and “b”, and D665, D649 are the values of extinction at the corresponding wavelength. Concentra- tion of chlorophyll was recalculated per gram of fresh or dry mass of the sample (Lichtenthaler and Buschmann, 2001). Data were expressed as means ± S.E., which were calculated in all treatments using MS Excel. Significant differences between means were analyzed by one-way analysis of variance ANOVA test to discriminate means. The data were processed using the Statistica version 10 software (Statsoft, Moscow, Russia). 3 RESULTS AND DISCUSSION Simultaneous exposure to drought and the appli- cation of auxinic herbicide resulted in a decrease in the fresh mass of both roots and shoots of wheat plants (by 24 % and 32 %, respectively). However, plants treatment with bacterial suspension under these conditions con- tributed to an increase in the mass of the shoot (by 13 % compared to stressed plants without bacteria), but with- out an increment in the mass of roots. It is known that under water and / or mineral defi- ciency, relative activation of root growth is a typical adap- tive reaction in plants (Davies et al., 2005; Vysotskaya et al., 2009). In our experiments, the root / shoot mass ra- tio of plants grown under herbicide treatment combined with water scarcity, did not change compared to the con- trol (Figure 1 B), since the fresh mass of both roots and shoots was reduced to the same extent (Figure 1 A), while the addition of growth-stimulating bacteria to the her- bicide solution led to a decrease in this ratio due to the relative activation of shoot growth. Accumulation of low-molecular-weight compounds is one of the early adaptive reactions of plants to the ac- tion of stressors of various nature, amino acid proline being one of such stress metabolites. An increase in the Figure 1: The dry mass of shoots and roots (A) and mass root/ shoot ratio (B) of wheat plants 14 days after treatment. Sig- nificantly different means (n = 20) are labelled with different letters (ANOVA, LSD, p ≤ 0.05) Acta agriculturae Slovenica, 119/1 – 20234 S. СHETVERIKOV et al. concentration of proline in wheat leaves was observed in response to contamination with toxic metals, osmotic and temperature stress (Fatima et al., 2006; Yang et al., 2011; Mwadzingeni et al., 2016). Indeed, in our work, concentration of proline in plant shoots sharply in- creased (by more than 100 %) under combined action of water deficit and herbicide treatment, while introduction of the studied bacteria led to a decrease in proline con- tent to the control level (Figure 2 A). MDA is one of the metabolites of lipid peroxidation and its accumulation in plants shows that they are un- der severe oxidative stress (Reddy et al., 2004; Masoumi et al., 2011). A decrease in the MDA level indicates a decrease in the damaging effect of reactive oxygen spe- cies in leaves of wheat plants inoculated with bacterial suspension under conditions of combined stress, which contributed to maintaining their growth and indicated a favorable anti-stress effect of DA1.2 bacteria. Thus, the treatment of plants with herbicide during drought led to accumulation of MDA (15 % more than in the control), while introduction of bacterial strain P. protegens DA1.2 leveled the stress by reducing the growth of MDA con- centration (Figure 2 B). Another important indicator of the plant status un- der stress conditions is the content of chlorophylls a, b (Nayyar, Gupta, 2006; Ashraf, Harris, 2013). The content of chlorophylls in wheat leaves was shown to decrease by 8 % (Figure 3) under combined “herbicide + drought” stress. The positive effect of bacteria was manifested in mitigating adverse action of the herbicide on photosyn- thetic apparatus. This was reflected in the quantitative content of pigments in plants: inoculation with bacte- ria led to an increase in the total content of chlorophylls under the combined stress by 17 %. High concentration of chlorophylls is a characteristic of healthy plants, as it is associated with greater efficiency of photosynthesis. Thus, the interaction of wheat with growth-stimulating bacteria of the strain DA1.2 improved plant status com- pared to plants untreated with bacteria under stress con- ditions. Taking into account that under combined stress root mass of plant of both variants (treated and untreated with bacteria) remained equally low compared to the control, we evaluated possible involvement of hormonal regulation in the detected growth responses. The herbi- cide treatment (herbicide from the class of synthetic aux- ins) led to the accumulation of IAA in shoots by more than 2.5 times, while bacterial treatment significantly increased root IAA content (Figure 4 A). We suggested that this could be due to the ability of plants to absorb exogenous auxins. Otherwise, the herbicide could influ- ence the content of endogenous IAA (Kudoyarova et al., 2017). This assumption does not contradict the existing reports (Sung, 1979). The observed imbalance in the auxin distribution between the shoot and root caused by the herbicide may be the reason of the decrease in re- sistance of plants to drought. And accumulation of aux- Figure 2: The content of proline (A) and MDA (B) in the shoots of wheat plants 3 days after treatment. Significantly different means (n = 9) are labelled with different letters (ANOVA, LSD, p ≤ 0.05) Figure 3: The content of chlorophylls a (Chl a) and b (Chl b) in the shoots of wheat plants 3 days after treatment. Signifi- cantly different means (n = 9) are labelled with different letters (ANOVA, LSD, p ≤ 0.05) Acta agriculturae Slovenica, 119/1 – 2023 5 Mitigation of the negative effect of auxinic herbicide by bacterial suspension of Pseudomonas protegens ... under drought conditions ins under the influence of bacteria in the roots serves as one of the mechanisms responsible for improving plant growth, plant status when treated with an herbicide un- der drought conditions. Accumulation of ABA causes stomatal closure un- der conditions of water scarcity (Zhang et al., 2006; Le- ung et al., 2012). The decrease in cell turgor during dehy- dration dramatically activates synthesis of ABA, which accumulates mainly in the chloroplasts of leaf cells (Zhou et al., 2006; Venzhik et al., 2016). Accumulation of ABA in plants under stress was revealed. It was most strongly manifested in the roots, which could be the result of in- hibition of their growth (Figure 4 B). As for cytokinins, stress led to an increase in their content (Figure 4 C), and with bacterial introduction, we observed the great- est accumulation of cytokinins in the shoot. Cytokinins are known to be essential for shoot growth (Martin et al., 2000), inhibit root growth (Werner et al., 2010), and are ABA antagonists (Dodd, 2005). Accumulation of cyto- kinins under the studied stress could compensate for the growth-inhibiting effects of the increased level of ABA, which were most pronounced in plants treated with bac- teria. 4 CONCLUSIONS It was shown that the herbicide-induced imbal- ance in the auxin distribution between shoot and root in plants may be the cause of a decrease in drought resist- ance. The bacterial suspension had a positive effect on the wheat plants treated with the auxinic herbicide under drought conditions, it was expressed in a decrease in the content of biochemical stress markers MDA and proline, preventing plant growth inhibition and normalizing the content of chlorophylls. These positive effects of growth- stimulating bacteria on wheat plants under combined stress caused by drought and herbicide treatment have been described for the first time. 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