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Leppyanen I.V., Shtark O.Y., Pavlova O.A., Bovin A.D., Ivanova K.A., Serova T.S., Dolgikh E.A. Analysis of the effects of joint inoculation by arbuscular mycorrhizal fungi and rhizobia on the growth and development of pea plants Pisum sativum L. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2021, Vol. 56, № 3, p. 475-486.
(how to cite this article)

doi: 10.15389/agrobiology.2021.3.475eng

UDC: 635.656:579.64:631.811

Acknowledgements:
Supported financially by the Russian Science Foundation (grant No. 17-76-30016)

 

ANALYSIS OF THE EFFECTS OF JOINT INOCULATION BY ARBUSCULAR MYCORRHIZAL FUNGI AND RHIZOBIA ON THE GROWTH AND DEVELOPMENT OF PEA PLANTS Pisum sativum L.

I.V. Leppyanen, O.Y. Shtark, O.A. Pavlova, A.D. Bovin, K.A. Ivanova, T.S. Serova, E.A. Dolgikh ^✉

All-Russian Research Institute for Agricultural Microbiology, Federal Agency for Scientific Organizations, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mailirina_leppyanen@mail.ru, oshtark@yandex.ru, dobbi85@list.ru, andy-piter2007@mail.ru, kivanova@arriam.ru, t_serova@rambler.ru, dol2helen@yahoo.com (corresponding author ✉)

ORCID:
Leppyanen I.V. orcid.org/0000-0002-2158-0855
Ivanova K.A. orcid.org/0000-0003-4915-4126
Shtark O.Y. orcid.org/0000-0002-3656-4559
Serova T.S. orcid.org/0000-0003-4784-1675
Pavlova O.A. orcid.org/0000-0003-0528-5618
Dolgikh E.A. orcid.org/0000-0002-5375-0943
Bovin A.D. orcid.org/0000-0003-4061-435X

Received November 18, 2020

Co-inoculation of plants with arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria of the order Rhizobiales (rhizobia) can have a stimulating effect on plant growth and development. This influence can be considered as the synergistic effect of two microorganisms on a plant in a multicomponent system and as the result of the mutual influence of microorganisms on each other. However, the mechanisms underlying the mutual influence of microorganisms remain insufficiently understood. In the presented work, it was shown for the first time that in the case of joint inoculation of pea plants with fungi of arbuscular mycorrhiza and rhizobia, the method of introduction microorganisms may be important. The results may indicate the presence of competition of microorganisms for a niche in the plant during sequential inoculation. The purpose of our research was to study the possibility of selection of the effective combinations of AM and rhizobia strains for inoculation of such important agricultural crop as Pisum sativum L. as well as estimation of influence on the productivity of plants. In this work, we analyzed the effects of joint inoculation of pea plants Pisum sativum L. with the fungus Rhizophagus irregularis BEG144 and the rhizobial strain R. leguminosarum bv. viciae RCAM 1026. For this purpose, the level of induction of markers development of two types of symbiosis, the degree of root mycorrhization were assessed, as well as the biometric parameters of plants. The research was performed using pea seedlings of the cultivar Frisson grown under sterile conditions. The isolate of the fungus Rhizophagus irregularis BEG144 was used for inoculation. An inoculum was obtained from the mycorrhized roots of Plecthrantus australis. The inoculum was introduced into a moisture substrate before planting the pea seedlings. The scheme of sequential inoculation was used, according to which pea plants were first inoculated with AM fungi R. irregularis BEG144 and 7 days after cultivation, the rhizobial strain Rhizobium leguminosarum bv. viciae RCAM 1026 were introduced into the system. The experimental scheme included the following options: without inoculation (control), R. leguminosarum (Rlv), R. irregularis (AM), R. irregularis + R. leguminosarum (AM + Rlv). 9 and 21 days after planting (2 and 14 days after bacterial inoculation), the material was collected to analyze the expression of genes - markers of the legume-rhizobial symbiosis development. On day 21 after planting, the plants were collected in order to determine their biometric parameters, as well as the markers of the symbiosis development. The lateral roots of the plants were cut off and frozen in liquid nitrogen. After isolation of the total RNA, cDNA was synthesized on the RNA template using reverse transcriptase using oligo(dT) primers. For the analysis, quantitative PCR combined with reverse transcription (RT-PCR) was performed (a CFX96 Real-Time instrument, «Bio-Rad Laboratories», USA). The total weight of plants and the weight of the root system significantly increased in case of monoinoculation with rhizobia, monoinoculation with AM fungi, as well as joint inoculation, compared with the control variant. However, no significant differences in these biometric parameters between the variant with monoinoculation with rhizobia or AM fungi and double inoculation were found. Probably, upon inoculation with several endosymbionts, competition arose between them at the stage of penetration into the plant, which led to a decrease in the intensity of plant infection with rhizobia. This was evidenced by the absence of additional stimulation of the Enod5 and Sym37 gene expression during double inoculation, which are activated in the plant during the development of rhizobial infection. Upon double inoculation, we also did not reveal additional stimulation of the expression of marker genes of symbiosis with AM fungi — PT4, TI, RAM1 and DELLA3. This correlated with the absence of significant differences in all biometric parameters between the variant with monoinoculation with rhizobia or AM fungi and double inoculation, which does not allow us to conclude about the positive effect of double inoculation on the growth and development of pea plants under the conditions of this experiment. The results of data analysis using the experimental scheme used may indicate the presence of competition of microorganisms for a niche in the plant, leading to a decrease in intra-root mycorrhizal colonization and the level of induction of markers that are activated during the development of rhizobial infection.

Keywords: plant-microbe interactions, rhizosphere, symbiosis, arbuscular mycorrhiza, inoculation, rhizobia, Rhizophagus irregularis, gene expression, Pisum sativum.

 

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