Annotation of the results obtained in 2023
Strains of hydrocarbon-oxidizing bacteria were isolated into pure cultures from various samples of anthropogenically disturbed soils. Of these, 13 strains were selected that have a significant degree of oil biodegradation - 71.8-97.0%. Microorganisms (Acinetobacter calcoaceticus (7 strains), A. seifertii H4.1, A. courvalinii UOM 35, Pseudomonas alcaligenes UOM 10, P. frederiksbergensis UOM 11, P. citronellolis H2, P. schmalbachii H3.1) were identified using MALDI-TOF mass-spectrometry of cellular protein fractions, 16S rRNA gene sequencing (sequences deposited in GenBank, №№ OP686570, OP686572, OP686573, OP692728, OP692729, OR367425-OR367429, OR801644-OR801646) and phylogenetic analysis. Also, bacteria from the collection of microorganisms of the Institute of Biology of the UFRC RAS took part in the experiments, the degree of oil biodestruction of which was above 70%: Pseudomonas putida IB 23, P. chlororaphis IB 93, P. turukhanskensis IB 1.1. The microorganisms were tolerant to herbicides (produced in the Russian Federation) Octapon extra (active ingredient (a.i.) 2,4-dichlorophenoxyacetic acid (2,4-D) (auxin-like)), Spetsnaz (a.i. tribenuron-methyl ( sulfonylurea class)), Tapir (a.i. imazethapyr (imidazolinone class)) in the concentration 1-10 ml (g)/l, but were poorly resistant to the herbicides Chistalan (a.i. 2,4-D and dicamba (auxin-like)) and Phenizan (a.i. dicamba and chlorsulfuron (sulfonylurea class)) (not more than 5 ml/l), probably due to dicamba inhibiting the growth of many bacteria. In the present study, the ability to degrade the active ingredients of herbicides (initial concentration 0.2-0.5 g/l) was found only in bacteria of the genus Pseudomonas (exception of P. frederiksbergensis UOM 11). The maximum degree of destruction of tribenuron-methyl was recorded for P. putida IB 23 (41.5%), for other strains this index was 26.0-30.5%. The degree of destruction of 2,4-D was 3.9-19.4%. The most difficult substrate for biodegradation was imazethapyr - the degree of its biodegradation was no higher than 11.4%, only for P. citronellolis H2 it was 26.9%. The P. chlororaphis IB 93 did not demonstrate the ability to destroy 2,4-D and imazethapyr. Herbicides (1 vol.%) had different effects on the degradation of oil (4 vol.%) by microorganisms. Spetsnaz did not have a significant effect on this process, but Octapon extra and Tapir significantly slowed it down (in some cases, biodestruction decreased by more than 7 times). All microorganisms synthesized the phytohormone indolyl-3-acetic acid (IAA). The lowest level of production was recorded in A. calcoaceticus P21, P25, P32 and C2 (≤ 150 ng/ml of culture liquid); in other strains it was 320-6025 ng/ml. Oil stimulated the production of IAA, especially in P. putida IB 23 and P. frederiksbergensis UOM 11, in which the amount of this phytohormone increased by 6.5 times. Octapone extra increased the formation of IAA in all microorganisms by 1.2-5.1 times, and Tapir suppressed it, with the exception of P. frederiksbergensis UOM 11, whose IAA production increased by 1.8 times. Spetsnaz inhibited the synthesis of phytohormone in some strains by 1.2-2.8 times, in other strains it increased by 1.4-5.7 times. All bacteria grew actively on a medium without a nitrogen source (titer 106-107 CFU/ml), i.e. they were oligonitrophils. In the presence of oil, the nitrogenase activity of the strains remained at the same level or increased, which is indirectly evidenced by an increase in the number of bacteria by 1-2 orders of magnitude. The addition of the herbicides Tapir and Spetsnaz to the medium also increased the number of cells by 1-2 orders of magnitude, however, when Octapon extra was added, the initial titer was retained only in P. chlororaphis IB 93 and P. putida IB 23; in other bacteria it decreased by 3-4 orders of magnitude. The inorganic phosphate solubilization index (SI) in bacteria was 1.3-3.3. A high value (SI>3) was recorded in strains P. chlororaphis IB 93, P. frederiksbergensis UOM 11, A. calcoaceticus C2 and H3.2. In the presence of herbicides or oil, SI in all strains decreased on average by 1.7 and 1.3 times, respectively. The sensitivity of plants of the families Fabaceae (alfalfa, white clover, white lupine, meadow clover, chickpeas, field peas) and Poaceae (sudan grass, mogar, spring barley, awnless brome, meadow fescue, oats) to the presence of oil in the soil (20 g/kg) and herbicides Tapir (15 µg/kg), Phenizan (5 µg/kg) and Chistalan (20 µg/kg) was studied. The amount of herbicides was calculated based on the regulations and doubled. It has been established that oil accelerates the germination of seeds of some plants (within 18%), and herbicides, in general, have a relatively neutral effect on the germination of cereal crops and have a differential effect on this indicator in legumes. Phenizan and Chistalan showed greater toxicity compared to Tapir (possibly due to the fact that they contain dicamba), which negatively affected the growth characteristics of plants, especially legumes. Clover had the highest sensitivity to the presence of oil and herbicides. The most promising crops for phytoremediation of oil-contaminated agricultural soils among Fabaceae were lupine, and among Poaceae - oats. They had a consistently high (80% or more) germination rate, which did not decrease when pollutants were added to the soil. They were capable of increasing phytomass in the presence of oil and were resistant to the herbicide Tapir, which manifested itself in the elongation of their shoots and roots. Based on the data obtained, microbial-plant associations were formed, the effectiveness of the relationships in which was assessed by the ability of bacteria to reduce the phytotoxic effect of pollutants on seed germination and seedling growth. For this purpose, we used microorganisms isolated in this project and producing IAA more than 500 ng/ml of culture liquid, as well as clover seeds (the most sensitive to the presence of oil and the herbicide Tapir) and barley, on which oil had a positive effect, and Tapir had a neutral or inhibitory effect. This herbicide is poorly biodegradable and can accumulate in the soil, so the growth-stimulating activity of the strains was assessed in its presence. The germination rate of barley seeds in oil-contaminated soil and in soil with the herbicide Tapir was equally high - 100%. Bacterization reduced it by 4-8%. Against the background of oil, inoculation of barley did not have a significant effect on its morphometric characteristics, and only strains P. schmalbachii H3.1 and P. citronellolis H2 contributed to an increase in the above-ground (by 12.3 and 8.1%, respectively) and underground parts of seedlings (by 13. 7 and 15.2% respectively). In the presence of Tapir, only when treated with bacteria P. putida IB 23, P. turukhanskensis IB 1.1, P. schmalbachii H3.1 and P. citronellolis H2, the length of the shoots increased by 7.7-14.9%, and the roots by 8.5-22.0%. In inoculated clover seeds, germination in soil with oil decreased by no more than 8% (with the exception of P. schmalbachii H3.1), and in soil with herbicide it increased compared to untreated plants. The bacteria P. putida IB 23, P. turukhanskensis IB 1.1, P. schmalbachii H3.1 and P. citronellolis H2 contributed to increased shoot growth (by 20.8-56.9%), and the strains P. putida IB 23, P. turukhanskensis IB 1.1 contributed to root elongation (by 30.9 and 36.4%, respectively). In the soil with the herbicide, an increase in the length of clover shoots (by 17.4 and 18.3%) and roots (by 18.0 and 25.9%) occurred only under the influence of P. schmalbachii H3.1 and P. citronellolis H2. Thus, the use of bacteria P. citronellolis H2 and P. schmalbachii H3.1 had a positive effect on the morphometric parameters of barley and clover plants in oil-contaminated soil, as well as in soil with the herbicide Tapir. This indicates a decrease in the phytotoxic effect of pollutants under their influence. These strains have been deposited in the All-Russian Collection of Microorganisms (VKM). The P. schmalbachii strain H3.1 (VKM B-3755) was placed in public storage, and the P. citronellolis strain H2 (VKM B-3756D) was placed for further patent procedures.

 

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