ВЛИЯНИЕ BACILLUS SUBTILIS НА МИКОРИЗАЦИЮ РАСТЕНИЙ ПШЕНИЦЫ НА ЗАГРЯЗНЕННОЙ ПОЧВЕ КАДМИЕМ

DOI: https://doi.org/10.23670/IRJ.2022.118.4.090

ВЛИЯНИЕ BACILLUS SUBTILIS НА МИКОРИЗАЦИЮ РАСТЕНИЙ ПШЕНИЦЫ НА ЗАГРЯЗНЕННОЙ ПОЧВЕ КАДМИЕМ

Научная статья

Курамшина З.М.^{1, *}, Смирнова Ю.В.², Хайруллина Р.М.³

¹ ORCID: 0000-0001-9506-3458;

^{1, 2} Стерлитамакский филиал Башкирского госудасртвенного университета, Стерлитамак, Россия;

³ Институт биохимии и генетики УФИЦ РАН, Уфа, Россия

^* Корреспондирующий автор (kuramshina_zilya[at]mail.ru)

Аннотация

Целью работы явилось изучение влияния инокуляции семян пшеницы эндофитными штаммами Bacillus subtilis и кадмия на микоризацию корней.

Было показано, что наличие кадмия в почве подавляло образование микоризных грибов в корнях пшеницы в случае, если семена не обрабатывались бактериями. С увеличением концентрации ионов кадмия в почве токсический эффект металла на микоризу усиливался. Показано, что токсический эффект кадмия был менее выражен у растений, инокулированных бактериями B. subtilis, чем у необработанных.

Ростостимулирующий и антистрессовый эффекты исследуемых бактерий, позволяют рассматривать эти бактерии как перспективные средства защиты растений от токсических эффектов кадмия.

Ключевые слова: Bacillus subtilis, кадмий, эндофит, микориза, Triticum aestivum.

INFLUENCE OF BACILLUS SUBTILIS AND CADMIUM ON THE MYCORRHIZATION OF WHEAT PLANTS

Research article

Kuramshina Z.M.^{1, *}, Smirnova Y.V.², Khairullina R.M.³

¹ ORCID: 0000-0001-9506-3458;

^{1, 2}Sterlitamaksky Branch of the Bashkir State University, Sterlitamak, Russia;

³ Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Center of Russian Academy of Sciences, Ufa, Russia

^* Corresponding author (kuramshina_zilya[at]mail.ru)

Abstract

The aim of the work was to study the effect of inoculation of wheat seeds by endophytic strains of Bacillus subtilis on root mycorrhization under the influence of cadmium.

It was shown that cadmium in the soil suppressed the formation of mycorrhizal fungi in wheat roots if the seeds were not treated with bacteria. With an increase in the concentration of cadmium ions in the soil, the toxic effect of the metal on mycorrhiza increased. It was shown that the toxic effect of cadmium was less pronounced in plants inoculated with B. subtilis bacteria than in untreated plants.

The growth-stimulating and anti-stress effects of the studied bacteria allow us to consider these bacteria as promising means of protecting plants from the toxic effects of cadmium.

Keywords: Bacillus subtilis, cadmium, entophyte, mycorrhization, Triticum aestivum.

Introduction

Due to the development of industry and transport networks in the world the pollution of agricultural soils with heavy metals is intensified, which is a serious environmental problem of our time [1, P. 111-121]. Heavy metals, being dangerous toxicants, disrupt interactions in the "soil-plants" system, inhibit the vital activity of macro- and microorganisms, change the properties of the soil. Due to the continuous use of chemical pesticides in crop production the activity of protective physiological processes can decrease and that supports enter in plants toxic elements violating metabolism. [2, P. 84-102]. Therefore, the urgency of developing biological plant protection from pests and immunizers increasing resistance of plant cells to stress factors is increasing. Among these agents, special attention is drawn to the drugs based endophytic microorganisms through their ability to penetrate into the plant tissue and mutualistic coexist with the host plant. Some types of such bacteria, for example, Bacillus subtilis are capable of producing different in structures antibiotics, enzymes and other biologically active substances [3, P. 225-250], which protect plants from phytopathogens, stimulate growth, improve mineral nutrition, increase plant resistance to various stressors including the action of heavy metals [4, P. 648-654]. Despite the positive results of certain drugs on the basis of living cells of endophytic bacterial strains B. subtilis use, their biological properties and ecological role remain poorly understood. For example, in scientific literature there is no information of the effect of endophytic B. subtilis, inhibiting the growth of pathogenic microorganisms, on mycorrhizal fungi. The role of endophytic bacteria in the formation of endomycorrhiza under condition of the toxic effect of various compounds, including heavy metals, is not clear also.

The aim of this work is to study the effect of inoculation of wheat by endophytic bacterial strains Bacillus subtilis, capable to serve the basis of commercial Chemical fertilizers, on the mycorrhization of roots under the impact of cadmium.

Research methods and principles

Laboratory experiments were conducted with the plants of Triticum aestivum L. (Omskaya 35). Before sowing, the seeds were washed in water, surface-sterilized with 96% ethanol, rinsed in distilled water, and dried. In the experiments, we used bacteria Bacillus subtilis strain 26D (VNIISKhM, no. 128) and strain 11VM (VNIISKhM, no. 519). The seeds were treated with bacteria in a laminar-flow box; for inoculation, we used 20-h-old culture grown on beef extract agar at 37°С. Bacterial cells were washed with 0.01 М KCl. Cell suspension was adjusted to the required concentration (10⁶ cells/mL) by optical density. Consumption of bacterial suspension was 20 μL per gram of seeds. The seeds were mixed with bacterial suspension in a round-bottomed flask and left for an hour, then used in the experiments. Control seeds were treated with distilled water.Inoculated and control seeds were placed in 8-liter pots containing 2 kg of leached chernozem soil (clay-illuvial chernozems) selected in the forest-steppe zone of the Republic of Bashkortostan (humus content - 9-10%, pH 6, the background content of cadmium is 0.1 mg/kg). The plants were grown at a temperature of 18 to 20 °C under fluorescent lamps (12 KLX) with a 16-hour photoperiod. Each pot accommodated 150 seeds sown at a depth of 1 cm. In the experiments, we used solutions of Сd(NO₃)₂ • 4H₂O at concentrations of 10 and 200 mg/kg soil in terms of metal ions. After 60 days, 50 plants were selected from each variant together with the root system, which was then separated, washed in running water and stained by the Trouvelot method (1986) with trypan blue to visualize the structures of vesicular-arbuscular mycorrhiza. According to the methodology of the same authors, the frequency of occurrence was estimated mycorrhizae, the degree of colonization by mycorrhizae, the prevalence of arbuscules and vesicles. Using the computer program «Mycocalc» the frequency (F%) and intensity (M%) of mycorrhizal colonization in the root system were calculated [5, P. 217–221].

The statistical analysis was performed by standard methods using Microsoft Office Excel. To assess the significance of differences in mean values Student's t-test was used. The tables show mean values and their standard deviations.

Results and discussion

The seed treatment with bacterial spores of both strains decreased the frequency of mycorrhizae in roots of wheat in control values by an average of 32%, the intensity - by 55% (see table 1), which corresponded to the data had been obtained previously [6, P. 172-182]. The presence of heavy metals in the soil suppressed the formation of mycorrhizal fungi in the roots of culture in case it seeds were not treated with any of the bacterial preparations. With increasing concentration of the metal ions such negative effect magnified. The F indicator decreased when exposed to cadmium at a concentration of 10 and 200 mg / kg by 35.5 and 72%, respectively, as compared with the control variant, where  cadmium salt was no added to the soil and seeds were not treated; the M indicator decreased by 58 and 82%, respectively.

Table 1 – The Effect of inoculation by bacteria B. subtilis on the frequency (F%) and intensity of mycorrhization (M%) of plant roots in the conditions of soil contamination with cadmium

Variant	Control	Cadmium mg / kg
		10	200
F%
No treatment	87,3±1,5	56,3±2,6	24,3±1,4
B. subtilis 26D	59,3±2,0	65,7±3,0	46,0±2,1
B. subtilis 11VM	60,0±1,9	53,9±5,2	47,3±2,8
M%
No treatment	1,7±0,1	0,7±0,1	0,3±0,1
B. subtilis 26D	0,7±0,1	1,6±0,2	0,8±0,1
B. subtilis 11VM	0,8±0,1	0,9±0,1	0,8±0,1

The pretreatment of plant seeds with bacterial preparations, in most cases, under the toxic effect of metal ions, especially in their high concentrations, didn’t allow to achieve  mycorrhization indicators equal to those observed in plants pretreated with bacteria and those did not experience the stress of soil contamination.

At the same time, when the seeds were treated with endophytes, indicators mycorrhization frequency and intensity under the action of metals, generally decreased in comparison with plants which grew in the contaminated soil, but were inoculated with spores of bacteria.

In plants of wheat treated with bacilli, the frequency and intensity of mycorrhization when exposed to cadmium ions at a concentration of 10 and 200 mg / kg were were by 1.8 and 2.6 times, respectively, than those of bacteria uninoculated plants.

As a result of these experiments it was found out that pre-sowing seed inoculation resulted in increase of plant biomass. In wheat the stimulating effect was more pronounced in the root system than in shoots. In treating plants by B. subtilis strain 26D and 11VM the weight of wheat roots was increased by 18%, shoot - by 10-13%, respectively.

Low concentrations of cadmium ions as well as treating by bacteria stimulate plant growth, the effect of metals and bacilli was comparable to each other. It was noted slight synergistic effect of pretreatment endophytic strains seeds and low concentrations of metals. Cadmium at a high concentration (200 mg / kg) inhibited the growth of the plant root system of wheat 14%, the biomass of shoots was almost unchanged. Under exposed to high concentrations of metal, the growth inhibition of plants pretreated with bacilli was not observed.

We have previously suggested that the reduction of mycorrhiza formation in the roots of wheat is a comprehensive indicator of biological activity of endophytic bacilli, such as antagonistic. It is known that B. subtilis can synthesize a large number of various metabolites (different structures antibiotics, enzymes and other biologically active substances), inhibiting the development of pathogenic fungi. B. subtilis strain 26D and 11VM are active endophytes populating the rhizosphere, the root surface (rhizoplane) and penetrating into the root system [6, P. 172-182]. It is likely that there is a primary competition between bacilli and mycorrhizal fungi for colonization of plant tissues.

The growth-stimulating effect of bacteria is probably related to the fact that bacilli can produce phytohormone-like substances, increase the content of nutrients in plant-available form and inhibit the growth of pathogenic micro-organisms [7, P. 507–513], [8], [9], [10, P. 269-277].

Conclusion

Thus, the decrease in mycorrhiza formation occurred in the root system of wheat plants pretreated by bacterial cells, which is a complex indicator of the biological activity of endophytic bacilli. In wheat plants pre-treated with bacilli under cadmium stress, the decrease in mycorrhiza formation was less pronounced than in uninoculated ones. The endophytic strains of B. subtilis 26D and 11BM, as we showed before [10, P. 269-277], reduce the degree of development of oxidative stress in plants under the action of heavy metals. Changes in mycorrhiza of plants treated by bacteria under the action of cadmium prove that bacilli reduce the toxic effect of pollutants and influence complex processes in the rhizosphere, which provides a fine link between the plant, microorganisms and soil.

Конфликт интересов Не указан.

Conflict of Interest None declared.

Список литературы / References

1. Wang X. Application and Mechanisms of Bacillus subtilis in Biological Control of Plant Disease Role of Rhizospheric Microbes in Soil / X. Wang, D.-L. Zhao, L.L. Shen et al. – Singapore: Springer, 2018. – 400 p. – [Electronic resource]. URL: https://www.researchgate.net/publication/325132556_Application_and_Mechanisms_of_Bacillus_subtilis_in_Biological_Control_of_Plant_Disease. DOI: 10.1007/978-981-10-8402-7_9 (accessed: 12.03.2022)
2. Trouvelot A. Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthodes d’estimation ayant une significationfication fonctionnelle Physiological and Genetical Aspects of Mycorrhizae / A. Trouvelot, N. Kough, V. Gianinazzi-Pearson – Paris: INRA Press, 1986. – 832 p.
3. Мелентьев А.И. Аэробные спорообразующие бактерии Bacillus Cohn в агроэкосистемах / А.И. Мелентьев – М.: Наука, 2007. – 148 c.
4. Franco-Franklin V. Are endophytic bacteria an option for increasing heavy metal tolerance of plants? A meta-analysis of the effect size. / V. Franco-Franklin, S. Moreno-Riascos, T. Ghneim-Herrera // Frontiers in Environmental Science. – 2021. – 8. – [Electronic resource]. URL: https://www.frontiersin.org/articles/10.3389/fenvs.2020.603668/full. DOI: 10.3389/fenvs.2020.603668 (accessed: 12.03.2022)
5. Chebotar’ V.K. Biodiversity of endophytic bacteria as a promising biotechnological resource. / V.K. Chebotar’, A.V. Shcherbakov, S.N. Maslennikova et al. // Agricultural Biology. – 2015. – 50(5). – p. 648-654. – [Electronic resource]. URL: https://www.researchgate.net/publication/283805342_Biodiversity_of_endophytic_bacteria_as_a_promising_biotechnological_resource. DOI: DOI: 10.15389/agrobiology.2015.5.648eng
6. Kuramshina Z.M. Cadmium and nickel toxicity for Sinapis alba plants inoculated with endophytic strains of Bacillus subtilis. / Z.M. Kuramshina, Yu.V. Smirnova, R.M. Khairullin // Russian Journal of Plant Physiology. – 2018. – 65(2). – p. 269-277. – [Electronic resource]. URL: https://link.springer.com/article/10.1134%2FS1021443718010077. DOI: 10.1134/S1021443718010077 (accessed: 12.03.2022)
7. Mahar A. Challenges and opportunities in the phytoremediation of heavy metals contaminated soils: A review. / A. Mahar, P. Wang, A. Ali et al. // Ecotoxicology Environmental Safety. – 2016. – 126. – p. 111-121. – [Electronic resource]. URL: https://www.sciencedirect.com/science/article/abs/pii/S0147651315302116?via%3Dihub. (accessed: 12.03.2022)DOI: 10.1016/j.ecoenv.2015.12.023
8. Arkhipova T.N. Comparison of the effects of bacterial strains that differ in their ability to synthesize cytokinins on growth and cytokinin content in wheat plants. / T.N. Arkhipova, S.Y. Veselov, A.I. Melentev et al. // Russian Journal of Plant Physiology. – 2006. – 53(4). – p. 507-513. – [Electronic resource]. URL: https://link.springer.com/article/10.1134%2FS1021443706040121. (accessed: 12.03.2022) DOI: 10.1134/S1021443706040121
9. Madhu P.M. Effect of Heavy Metals on Growth and Development of Cultivated Plants with Reference to Cadmium, Chromium and Lead – A Review. / P.M. Madhu, R.S. Sadagopan // Journal of Stress Physiology & Biochemistry. – 2020. – 3. – p. 84-102. – [Electronic resource]. URL: https://cyberleninka.ru/article/n/effect-of-heavy-metals-on-growth-and-development-of-cultivated-plants-with-reference-to-cadmium-chromium-and-lead-a-review. (accessed: 12.03.2022)
10. Egorshina A.A. Phosphate-mobilizing activity of endophytic Bacillus subtilis strains and their effect on the degree of mycorrhization of wheat roots. / A.A. Egorshina, R.M. Khairullin, M.A. Lukyantsev et al. // Journal of Siberian Federal University. A series of "Biology". – 2011. – 4(2). – p. 172-182. – [Electronic resource]. URL: http://journal.sfu-kras.ru/en/article/2555. (accessed: 12.03.2022)DOI: 10.17516/1997-1389-0179

Список литературы на английском языке / References in English

1. Wang X. Application and Mechanisms of Bacillus subtilis in Biological Control of Plant Disease Role of Rhizospheric Microbes in Soil / X. Wang, D.-L. Zhao, L.L. Shen et al. – Singapore: Springer, 2018. – 400 p. – [Electronic resource]. URL: https://www.researchgate.net/publication/325132556_Application_and_Mechanisms_of_Bacillus_subtilis_in_Biological_Control_of_Plant_Disease. DOI: 10.1007/978-981-10-8402-7_9 (accessed: 12.03.2022)
2. Trouvelot A. Mesure du taux de mycorhization VA d’un système radiculaire. Recherche de méthodes d’estimation ayant une significationfication fonctionnelle Physiological and Genetical Aspects of Mycorrhizae / A. Trouvelot, N. Kough, V. Gianinazzi-Pearson – Paris: INRA Press, 1986. – 832 p.
3. Melent'ev A.I. Ae'robny'e sporoobrazuyushhie bakterii Bacillus Cohn v agroe'kosistemax [Aerobic spore-forming bacteria Bacillus Cohn in agroecosystems] / A.I. Melent'ev – M.: Nauka, 2007. – 148 p. [in Russian]
4. Franco-Franklin V. Are endophytic bacteria an option for increasing heavy metal tolerance of plants? A meta-analysis of the effect size. / V. Franco-Franklin, S. Moreno-Riascos, T. Ghneim-Herrera // Frontiers in Environmental Science. – 2021. – 8. – [Electronic resource]. URL: https://www.frontiersin.org/articles/10.3389/fenvs.2020.603668/full. DOI: 10.3389/fenvs.2020.603668 (accessed: 12.03.2022)
5. Chebotar’ V.K. Biodiversity of endophytic bacteria as a promising biotechnological resource. / V.K. Chebotar’, A.V. Shcherbakov, S.N. Maslennikova et al. // Agricultural Biology. – 2015. – 50(5). – p. 648-654. – [Electronic resource]. URL: https://www.researchgate.net/publication/283805342_Biodiversity_of_endophytic_bacteria_as_a_promising_biotechnological_resource. DOI: DOI: 10.15389/agrobiology.2015.5.648eng
6. Kuramshina Z.M. Cadmium and nickel toxicity for Sinapis alba plants inoculated with endophytic strains of Bacillus subtilis. / Z.M. Kuramshina, Yu.V. Smirnova, R.M. Khairullin // Russian Journal of Plant Physiology. – 2018. – 65(2). – p. 269-277. – [Electronic resource]. URL: https://link.springer.com/article/10.1134%2FS1021443718010077. DOI: 10.1134/S1021443718010077 (accessed: 12.03.2022)
7. Mahar A. Challenges and opportunities in the phytoremediation of heavy metals contaminated soils: A review. / A. Mahar, P. Wang, A. Ali et al. // Ecotoxicology Environmental Safety. – 2016. – 126. – p. 111-121. – [Electronic resource]. URL: https://www.sciencedirect.com/science/article/abs/pii/S0147651315302116?via%3Dihub. (accessed: 12.03.2022)DOI: 10.1016/j.ecoenv.2015.12.023
8. Arkhipova T.N. Comparison of the effects of bacterial strains that differ in their ability to synthesize cytokinins on growth and cytokinin content in wheat plants. / T.N. Arkhipova, S.Y. Veselov, A.I. Melentev et al. // Russian Journal of Plant Physiology. – 2006. – 53(4). – p. 507-513. – [Electronic resource]. URL: https://link.springer.com/article/10.1134%2FS1021443706040121. (accessed: 12.03.2022) DOI: 10.1134/S1021443706040121
9. Madhu P.M. Effect of Heavy Metals on Growth and Development of Cultivated Plants with Reference to Cadmium, Chromium and Lead – A Review. / P.M. Madhu, R.S. Sadagopan // Journal of Stress Physiology & Biochemistry. – 2020. – 3. – p. 84-102. – [Electronic resource]. URL: https://cyberleninka.ru/article/n/effect-of-heavy-metals-on-growth-and-development-of-cultivated-plants-with-reference-to-cadmium-chromium-and-lead-a-review. (accessed: 12.03.2022)
10. Egorshina A.A. Phosphate-mobilizing activity of endophytic Bacillus subtilis strains and their effect on the degree of mycorrhization of wheat roots. / A.A. Egorshina, R.M. Khairullin, M.A. Lukyantsev et al. // Journal of Siberian Federal University. A series of "Biology". – 2011. – 4(2). – p. 172-182. – [Electronic resource]. URL: http://journal.sfu-kras.ru/en/article/2555. (accessed: 12.03.2022)DOI: 10.17516/1997-1389-0179