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AGRICULTURAL BIOLOGY, ISSN 2412-0324 (ВД* ed. Online)
2015, V. 50, № 5, pp. 673-684
(SEL’SKOKHOZYAISTVENNAYA BIOLOGIYA) ISSN 0i3i-6397 (Russian ed. P™0
v_____________________________________' ISSN 2313-4836 (Russian ed. Online)
UDC 633.31:581.557:631.461.5:57.044 doi: 10.15389/agrobiology.2015.5.673rus
doi: 10.15389/agrobiology.2015.5.673eng
SELECTION OF SALT TOLERANT ALFALFA (Medicago L.) PLANTS FROM DIFFERENT VARIETIES AND THEIR MORFO BIOLOGICAL AND SYMBIOTIC PROPERTIES ANALYSIS
M.L. ROUMIANTSEVA1, G.V. STEPANOVA2, O.N. KURCHAK1,
O.P. ONISHCHUK1, V.S. MUNTYAN1, E.A. DZYUBENKO3,
N.I. DZYUBENKO3, B.V. SIMAROV1
1 All-Russian Research Institute for Agricultural Microbiology, Federal Agency of Scientific Organizations, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail [email protected];
2V.R. Viliams All-Russian Research Institute for Forages, Federal Agency of Scientific Organizations, 1, Na-uchnii Gorodok, Lobnya, Moscow Province, 141055 Russia, e-mail [email protected];
3N.I. Vavilov Institute of Plant Genetic Resources (VIR), Federal Agency of Scientific Organizations, 42-44, ul.
Bol’shaya Morskaya, St. Petersburg, 190000 Russia, e-mail [email protected]
Acknowledgements:
Supported by Russian Foundation for Basic Research 15-04-09295а and 14-04-01441а, State Contract № 16.М04.11.0013 Received August 15, 2015
Abstract
Soils degradation growing in our days is associated with the depletion of their fertility, a result of crop rotation with excessive amounts of mineral fertilizers and chemical plant protection products, as well as it links with widespread worsening climatic conditions and environmental conditions. For this reason, agriculture based on environmentally friendly technologies must be an absolute priority. Legumes can fix atmospheric nitrogen in symbiosis with nodule bacteria and accumulate it in plant biomass. Legumes are unique predecessors for grain crops, as they contribute to the effective restoration of soil fertility by introducing nitrogen into bioavailable form. Pastures based on legumes contribute to the restoration of soils destroyed and excluded from crop rotation, such as desert or saline. In this, the development of pathways to create new productive plant-microbe systems that can grow in adverse conditions, is of great theoretical and practical significance. The objectives of the study was to identify salt-tolerant plants of alfalfa (Medicago L.), to obtain plants of the I1 generation by self-pollination approach and to analyse their morphobiological and symbiotic properties in model experiments. The study was performed on 13 tetraploid and diploid varieties of alfalfa, including commercially valuable varieties Soleustoychivaya and Agniya, both of which were tested without rhizobia inoculation and in symbioses with Sinorhizobium meliloti strains. An analysis of the symbiotic activity of alfalfa varieties showed that they were highly responsible to S. meliloti Rm2011 strain inoculation and formed an effective symbiosis under saline conditions. Geographically different varieties were evaluated for the homogeneity according to dry matter (DM) accumulation at 75 mM NaCl without inoculation, and at 100 mM NaCl with inoculation by S. meliloti. Obtained DM data among the studied cultivars significantly changed only in case of symbiosis that was established with the assistance of the dispersion coefficient (D). Plants of salt tolerant phenotype was obtained for diploid M. caerulea and M. falcata species, as well as for tetraploid M. sativa L. varieties Soleustoychivaya and Agniya in microvegetative experiments done at the All-Russian Research Institute for agriculture microbiology (ARRIAM). Selected salt tolerant plants of both varieties were planted further in greenhouse complex (STC) of V.R. Villiams All-Russian Research Institute for Forages. It was found that salt-tolerant plants of Soleustoychivaya and Agniya are characterized by predominantly purple color of flowers, by a twisted form of bean, by relatively high branching and bushy, by later transition to a period of winter rest according to 3-year vegetation trials in the STC. From seeds obtained from tested plants the I1 plants were grown, which were studied in microvegetative experiments in ARRIAM. Plants I1 of both varieties were analyzed by DM, by growth rate of aboveground and underground parts, by number of nodules formed in typical (salt-free) conditions and under salinity. As a result, it was found that generation I1 plants of both varieties were homogeneous or sufficiently homogeneous according to DM data. Not inoculated generation I1 plants of Soleustoychivaya variety successfully developed in saline conditions (the average increase of DM was 36.92 % in comparison to plants of initial variety). DM of plants I1 of Agniya variety was, on the contrary, lower than that of the plants of the initial variety in the saline conditions, being 16.55 % less. The high level of interaction specificity of both varieties of generation I1 plants with strains CIAM1774 or Rm2011, differing in salt tolerance, was assessed by DM. Thus, under salt stress impact the highest
values of DM was obtained for Soleustoychivaya variety plants in symbiosis with strain Rm2011 characterizing by S. meliloti salt tolerance typical degree. However, the symbiotic system on the basis of salt tolerant genotype of Agniya variety with salt-tolerant strain CIAM1774 may also be promising for cultivation in saline soils. It was found that the length of the root system decreased due to symbiosis, and this parameter depends on the specific plant-microbe interactions. It was concluded that the selection of salt-tolerant genotypes of plants and strains with a certain level of salt tolerance is promising in order to create symbiotic systems with enhanced adaptability.
Keywords: rhizobia, alfalfa (Medicago L.), effective symbiosis, salt tolerance, root and stem length, plant biomass.
Legumes posess the features that can make them promising to restore the degraded soils [1, 2]. They fix atmospheric nitrogen in symbiosis with nodule bacteria and accumulate it in the fertile layer of soil [3]. The area of soils that need restoration, including saline waterlogged and acidified soils is constantly increasing due to worsening the climatic situation both in the world and in Russia [4-6]. The successful development of such areas often depends on the proper selection of crops [7, 8].
Ecotypes of legumes and endemic legumes growing in different ecogeographic regions can be the source of new varieties with higher adaptive capacity [9]. Genus Medicago is represented by species that vary considerably in their adaptability and productivity under different soil and climatic conditions. Alfalfa (Medicago varia) is widespread in central Russia but can grow in low-and moderate-saline soils in the desert zone of the Caspian, and yellow alfalfa M falcate can inhabit slightly acidic soils of the Murmansk and Arkhangelsk regions [10, 11]. Identification of alfalfa plants (phenotypes) differing in their adaptivity (stress resistance) is one of the approaches to the creation of varieties and symbiotic systems with high adaptive capacity.
Inoculation of alfalfa with nodule bacteria strains (Sinorhizobium meliloti) selected genetically has been shown to enhance alfalfa productivity under salinity [12-15]. Estimation of salt tolerance of rhizobia, plants, and their symbioses can be successfully performed in the laboratory models and microvegetative experiments [1, 12, 16].
Herein, we report new data on the specificity of variety-microbe interaction in the systems of high symbiotic efficiency under salt-free conditions and as influenced by salt stress.
So in our study a salt-tolerance of tetraploid and diploid alfalfa species was evaluated under various nitrogen nutrition to identify contrast phenotypes, obtain the offspring from self-pollination, and compare their morphological, biological and symbiotic parameters.
Technique. Alfalfa (Medicago L.) varieties and ecotypes were provided from the VIR collection (N.I. Vavilov All-Russian Research Institute of Plant Genetic Resources) and the V.R. Villiams All-Russian Research Institute for Forages. Sinorhizobium meliloti cultures used for inoculation were Rm 2011 test strain typically resistant to 550 mM NaCl, and CIAM1774 strain (AK23 or A1) with salt tolerance to 700 mM NaCl characteristic of 10 % strains in this species [17]. The strains were cultured in TY medium at 28 °С [16].
Under sterile pot experiments, plants were grown without NaCl (a salt-free standard) or in the presence of 75 or 100 mM NaCl (salinity) with 10-fold replication for 28 or 56 days depending on the aim of the experiment, and КNOз (3 mM) as the source of nitrogen was added to the soil if needed, as described [12, 16]. Vermiculite or 0.6 % agar with Krasil’nikov-Korenyako mineral medium was a substrate. Plant productivity was evaluated by dry matter (DM) accumulation (%) versus control sample. To determine the germination power (GP), the seeds in Petri dishes were exposed to 28 °С, the percentage of germinated seeds to their total number was calculated at day 3. In pot experiments,
vessels were used filled with 6 kg of fertile soil (pH 6.94), at humus content of 4.41 %, total nitrogen of 0.28 %, and mobile phosphorus and potassium of 560.7 and 432.0 mg/kg, respectively. Plants were grown without additional dressing under natural lighting and temperature. Photoperiodicity of plants was analyzed in natural light and controlled temperature conditions.
Genotypes with different salt tolerance were selected in pot tests at day 56 in the presence of 75 mM NaCl as described [18]. Selected plants were grown salt-free in 0.5 l containers with sterile vermiculite, and after 2 months they were transplanted into containers with soil and grown in MLR-351H phy-totron («Sanyo Electric Co.», Japan) to obtaining seeds by forced self-pollination. Forced self-pollination and analysis of morphological and biochemical parameters (2012-2014) were performed as described [19] in the breeding greenhouse complex (BGC, V.R. Villiams All-Russian Research Institute for Forages). I1 plants of Soleustoichivaya and Agniya varieties grown from seeds (20 pcs. in each of the 8 variants) were studied for homogeneity, productivity and symbiotic effectiveness in pot tests [16].
Statistical analysis was performed using Statistica v. 6.0 software and Microsoft Excel 2013 software package. Analysis of variance and correlation analysis were performed, coefficient of variation (Cv) and Student 1-test were calculated [20, 21].
Results. Alfalfa varieties and ecotypes were represented by the samples from geographically distant regions with arid conditions and/or salinity, rapid changes in daily and seasonal temperature (Table 1). For the study we selected both cultivated alfalfa varieties (Medicago sativa, M. varia) and populations of wild alfalfa M falcata from the northern and southern regions, one of which, the Aral Sea area, is exposed to extreme salinity (see Table 1). An endemic alfalfa species М trautvetteri grows in the same area. In addition, the populations of М caerulea, an ancient blue-flowering species widespread in the steppe and flooded coastal areas of the Caspian, were analyzed. Salt tolerance was estimated in cultivated alfalfa varieties Soleustoichivaya and Agniya, of which the first one was created based on variety Khivinskaya local by cell technologies under salinity stress [22], and the second one was produced using a combined selection in complex hybrid P211 population obtained from crossing domestic and Canadian varieties (Pasture 88 x North hybrid 69 x Rizoma) [23].
1. Alfaf (Medicago L.) samples of different eco-geographical origin tested for salinity tolerance
Species (ploidy) Alfalfa variety and sample Seed collection area № according to VIR catalogue
М. falcata (2n) subsp. Borealis Grosshm., wild subsp. Romanica prod., wild, Pskov region, Russia k-25557
ecotype Eastern part of Kazakhstan k-49669
М caerulea Less. ex. Ledeb. (2n) Wild, ecotype Dagestan, Russia k-12821
Wild, ecotype Guriev region, Kazakhstan k-28915
Wild, ecotype Azerbaijan k-49904
Wild, ecotype Stavropol’ Territory k-44044
М trautvetteri Sumn. (2n) Wild, ecotype Aktyubinsk region, Kazakhstan k-36579
М sativa L. (4n) Tibetskaya variety Aktyubinsk region, Kazakhstan k-25782
Local cultivated variety Kyrgyzstan k-6376
Uzbekistan k-8913
Libya k-39107
M sativa L. subsp. sativa (4n) M sativa L. nothosubsp. varia Soleustoichivaya variety Moscow region, Russia -
(Martyn) (4n) Agniya variety Moscow region, Russia -
Note. Samples were provided from the VIR collection (N.I. Vavilov All-Russian Research Institute of Plant Genetic Resources, St. Petersburg); dash means that the samples were provided by the V.R. Villiams All-Russian Research
Institute for Forages (Moscow Province).
Fig. 1. Dry matter (DM) dispersion (A) and productivity (B) in various alfalfa (Medicago L.) varieties and ecotypes inoculated with Sinorhizobium meliloti strains under salinity stress: А — 75 mM
NaCl, B — 100 mM NaCl; a, and b, c — control (without inoculation), and inoculation with strains Rm2011 and CIAM1774 (pot tests).
Symbiotic efficiency and salt tolerance of different alfalfa species. The variety homogeneity was assessed by the DM in plants grown at 75 mM NaCl which were or were not inoculated with S. meliloti strains. In this, the dispersion coefficient (D) was used according to the guidelines [16]. Without inoculation, the D values ranged from 10.70 to 13.70 in Agniya variety and local M. sativa varieties. Extremely low (2.11) and conversely high values of D (17.50) were observed in varieties Soleustoichivaya (Fig. 1, A) and Tibetskaya, respectively. No dependence between D values and plant geographic origin was found. For example, similar values were obtained in М. caerulea sample pairs from Kazakhstan and Dagestan at an average D of 29.80, and from Azerbaijan and Stavropol’ Territory at an average D of 29.80. Contrasting D values were also noted in M. falcata populations of Kazakhstan and Pskov region (50.30 and 23.20, respectively). The maximum D value was found in the endemic М. trautvetteri species (62.90). Apparently, the low-D varieties are represented by the genotypes with similar tolerance, and in the high-D varieties it varies. The D value was proved to change significantly after inoculation with S. meliloti strains (see Fig. 1, A). In some cases, an inverse relationship between the D values with and without inoculation was found (e.g., in M. caerulea k-1282 or Agniya variety) (see Fig. 1, A). Thus, changes in dispersion reflect the specificity of plant-microbe interactions.
Plant productivity was evaluated in pot tests by DM in studied alfalfa species and varieties in symbiosis with S. meliloti strains at 100 mM NaCl salinity. High gain in DM was noted in a local variety of Uzbekistan (k-8913) and variety Agniya inoculated with Rm2011 or CIAM1774, respectively (an average increase of 57.60 %, see Fig. 1, B). Under saline conditions, these strains formed effective symbioses with M caerulea k-12821 and M. falcata k-25557 samples. The efficiency of symbiotic interaction of M. sativa k-39107 and M. caerulea k-44044 with strain CIAM1774 was found to be twice higher than in the symbiosis of the same host plants with strain Rm2011 (see Fig. 1, B). The local and cultivated alfalfa varieties were concluded to be highly responsive to inoculation and able to form effective symbioses under saline conditions.
Based on the results, it was of interest to identify plant genotypes differing in salt tolerance. The forms contrasting in habitus (i.e., min as low-stem ones, and max as those with a developed high stem) were selected from each of the samples studied. After growing under sterile salt-free conditions, they were moved to phytotron. With forced self-pollination, the seeds from phenotypically different
plants were obtained in the 2nd year in M. caerulea and M. falcata, and also in M. sativa L. varieties Agniya and Soleustoichivaya, but only in Soleustoichivaya plants their number was sufficient for further studying.
Morphological and biochemical parameters in the plants of min- and max-phenotypes. Morphological characteristics are the ones that do not depend on the growth conditions (corolla color, the number of flowers per inflorescence, bean form) but are used to determine alfalfa variety types. In plants of Soleustoichivaya (So) variety of contrasting phenotypes (i.e., So-max and So-min) there was a corolla color from lilac to deep lilac typical of alfalfa (M sativa L.) blue hybrid variety type. At the same time, in the contrasting phenotypes (Ag-max and Ag-min) of the Agniya (Ag) variety the color of flowers varied. It was lilac in max-forms and showed a dominance of purple alfalfa genes, whereas in min-forms a spectrum of shades from yellow-lilac to yellow indicated their genetic relation to northern alfalfa (M borealis L.) which was among the parental forms of the initial variety [23].
2. Morphological and biochemical parameters and productivity in phenotypically contrast alfalfa M. sativa L. varieties Agniya and Soleustoichivaya (pot tests, breeding greenhouse complex, 2012-2014)
Parameter Plant phenotypes tct max/min
max 1 min
Morphological features:
twists per bean, pcs. 2.7 2.s -
flowers per inflorescence, pcs. 2s 18 -
stems, pcs./plant 71 48 1.42
branches, pcs./plant 2s3 124 2.24
stems with flowers, pcs./plant 12 6 1.84
Content per dry matter, %:
raw ash 9.21 8.72 0.94
phosphorus 0.39 0.39 -
potassium 1.41 1.39 0.06
Dry matter, g/plant 1s1 134 0.88
Seeds, g/plant 12.70 11.s0 0.21
N o t е. Max- anf min-phenotype plants are contrasting in habitus (min means low-stem, max means a developed high stem); tact is the actual value of t-test (95 % confidence interval; theoretical value of t-test tos = 2.15). Dashes mean no significant differences between the plants of max and min phenotypes.
In perennial alfalfa species, beans are of the spirally twisted form, the number of twists being species-specific (e.g., 0.5-1.0 in sickle alfalfa, 3.5-4.0 in purple alfalfa). This parameter was 2.1 in the Agniya variety which is one twist less compared to the Soleustoichivaya plants (tact = 2.46 > tos). At the same time, we have not identified significant differences for the above trait between the samples of plants with different phenotypes (Table 2), however, a tendency to the increased bean twistedness was observed in the plants of salinity tolerant (max) phenotype.
Samples of plants of these two varieties significantly differed in the number of flowers per inflorescence and in their tilling capacity (tact > tu), but the max and min samples were not statistically different (tact < tos). However, the salt-tolerant plants tended to increase the average number of flowers per inflorescence and of stems per plant (see Table 2).
The analysis of average number of branches per plant showed that in max phenotype it was greater as twice (tact = 2.24 > tos, see Table 2). Similar were obtained in the analysis of the number of stems with flowers per plant (tact = 1.84; tos = 2.15; see Table 2). Consequently, the plants of salt tolerant phenotype tended to early ripening.
Analysis of photoperiodicity under natural light and controlled temperature showed that the photoperiod reduction (10 hours or less, decade III of October in the Central Region of the Russian Federation) resulted in marcescence
and plant transition to the resting stage. This process is quantified by the DM content. In max-phenotype plants, this value was 11.30 % higher in the marcescence period (data as of October 20, 2014, see Table 2). Upon exiting winter dormancy and with the photoperiod increase to 8 hours, early re-growth was observed in max-forms; when the photoperiod was 1 hour more, re-growth was found in min-phenotypes (tact max = 2.36; tact = 1.97; t)5 = 2.15). The studied samples differed in the raw ash content (see Table 2). Thus, these data support the fact that saline tolerant plants can longer retain activity in the shortened photoperiod.
As the plants of contrasting phenotypes were grown on fertile soil with a neutral pH (pH 6.94), it made it impossible to identify clear differences between the study groups. However, summarizing the findings, we can conclude that the plants of saline tolerant phenotype identified in the varieties studied, are characterized by predominantly lilac colored flowers, more twisted bean form, relatively high branching and tillering, a short-day photoperiodic response and increased DM ash content.
Homogeneity and productivity in the I1 generation. The main features of I1 plants grown from seeds of forced self-pollinated contrasting phenotypes were studied in 8 variants, of 20 pcs. in each.
Evaluation of homogeneity of I1 seeds in both varieties by GP versus the seeds of initial forms (control) revealed no significant differences (average GP value of 95 %). I1 plants without inoculation were estimated by the DM value using a coefficient of variation (Cv). Cv values in both studied alfalfa varieties, Soleustoichivaya (S0-I1) and Agniya (Ag-Ix), were significantly lower compared to that in respective controls (Fig. 2, A). But Cv changed significantly in individual phenotype groups depending on the growing conditions. Particularly, this value was higher with no salinity in Ag-l1-max2 and under salinity in So-l1-min2 (see Fig. 2, A). According to our data, phenotypically non-similar S0-I1 and Ag-I1 plants should be considered as homogeneous (Cv < 17 %) [21], and the above two groups as a fairly homogeneous (Cv in the range of 17-33 %) [21].
Fig. 2. Homogeneity by Cv (A) and productivity (B) by dry matter (DM) in Ii plants of phenotypically contrasting groups in alfalfa (M. sativa L.) varieties Agniya (Ag) and Soleustoichivaya (So) in normal conditions and under saline stress: а — control (salt-free), b — 75 mM NaCl; min and max — low-stem and high-stem forms. Inoculation with Sinorhizobium meliloti and mineral nitrogen were not used (pot tests).
Productivity by DM in S0-I1 and Ag-I1 plants of contrasting phenotypes (no inoculation) in the absence of salinity and under salinity were compared with the parameters found in initial plants of relevant varieties (Ag and So) reported under similar conditions (control, see Fig. 2, B ). The min- and max-Ag-I1 plants varied considerably in their productivity when NaCl was not added. Thus, the genotypes of Ag-l1-min and Ag-l1-max plants were either considerably superior (average of 50.50 %) in DM to control plants, or did not differ signifi-
cantly from them. The plants of only one (Ag-Ii-max1) of the four phenotypic groups were developing better compared to control (DM increase of 22.10 %) under salinity (see Fig. 2, B). Sо-Il plants, on the contrary, were successfully developing in saline conditions (DM increase from 33.85 to 67.69 %), with the exception of the So-I1-min1 in which the DM gain versus control did not exceed 7.69 %. Productivity was an average of 16.55 % lower versus control in all phenotypic S0-I1 groups with no salinity. Adding mineral nitrogen into the substrate promoted an increase in the DM value in all S0-I1 and Ag-Il phenotype groups of an average of 1.90 times regardless of salinity (data not shown).
Thus, the phenotypically non-similar Ag-Il and S0-I1 plant groups characterized as homogeneous were, however, significantly different in their productivity. S0-I1 groups appeared to be more expressed halophytes than the plants of the initial variety. The observed fact is due to the phenotypic segregation in the Soleustoichivaya variety produced from a callus culture using medium with 1.98 M NaCl [5]. Ag-Il plants did not differ in their salinity tolerance from control, however, a possibility of the use of the Agniya variety to search for stress-resistant genotypes has been shown. Therefore, further analysis will be performed in phenotypically different Ag-Il and S0-I1 groups.
Symbiotic systems based on I1 plant s. The effectiveness of Ag-I1 plants in symbiosis with strain CIAM1774 or Rm2011 was similar under salinity and with NaCl-free substrate (average DM gain of 60.05 % and 135.05, respectively) (Fig. 3, A). It also did not differ much in the case of S0-I1 plants’ symbiosis with the same strains in the salt-free variants (an average of 43.80 %), but under salinity the gain with Rm2011 was 23.0 % greater compared to CIAM1774 (see Fig. 3, B). Thus, a triple increase in dry matter in inoculated Ag-Ix plants was observed under normal conditions, and a double increase was found in S0-I1 under salinity stress.
Consequently, symbiosis of alfalfa Soleustoichivaya variety with the strain of typical salinity tolerance (Rm2011) is highly effective under the salinity stress. Nevertheless, the symbiotic system on the basis of salt tolerant genotype of Agniya variety with salt-tolerant strain CIAM1774 may also be promising.
Stems were found to develop better in case of substrates without NaCl in the Ag-I1 group inoculated with strain CIAM1774 (12.04 % length increase versus the variant of strain Rm1021, and 29.62 % compared to non-inoculated control) (see Fig. 3, C, D). Conversely, the stem length in S0-I1 plants was 9.78 % greater with Rm2011, than with CIAM1774. However, under salinity stress, the stem length in Ag-I1 plants with symbiotrophic nutrition was close to or slightly less (by 5.48 %) than in control. At the same time, under salinity stress the stem length increased by 38.85 % in S0-I1 plants in the symbiosis with Rm2011 and by 7.05 % only in the symbiosis with CIAM1774 (see Fig. 3, C, D).
Thus, in the absence of salinity stress, the height of the aerial part of Ag-I1 and S0-I1 plants depend on the inoculating strain, whereas no significant differences in DM were revealed (see Fig. 3, C, D). At that, in symbiosis with Rm2011 or CIAM1774, respectively, the Ag-I1 and S0-I1 plants were low. Similar differences were observed under salinity for S0-I1. The plants inoculated with CIAM1774 were low, in case of Rm2011 they were high, while no significant differences were found in the respective DM values.
Intensive development of the stem and biomass accumulation depends on the process of nitrogen fixation which is due to the nodules formed on the roots [10]. The number of nodules in Ag-I1 and S0-I1 plants inoculated with CIAM1774 under salinity stress-free conditions was 2 times greater compared to Rm2011 versus control plants inoculated with the same strain (29.11 and 10.27 %, respectively). Under salinity, this parameter was 3.02 and 1.34 times
greater in Ag-Ii and S0-I1 plants inoculated with Rm2011 compared to CIAM1774. Our findings suggest that plant-microbe interactions may vary considerably under normal conditions and under salinity.
Fig. 3. Changes in the dry matter (DM) (А, B) content, stem (C, D) and root (E, F) length in Ii alfalfa (M. sativa L.) varieties Agniya (А, C, E) and Soleustoichivaya (B, D, F) at symbiotrophic nutrition in normal conditions (а) and under saline stress (b): Ag and S0 — Agniya and Soleustoyi-chivaya varieties, respectively; CIAM1774 and Rm2011 — Sinorhizobium meliloti strains used for inoculation; A, B, E, F — versus respective non-inoculated or inoculated Ag or So plants, C, D — versus non-inoculated Ag-I1 or S0-I1 plants. Salinity stress with 75 mM NaCl (pot experiments).
The development of the roots which is an important characteristic of successful development of plants was also eveluated [24]. The root length in non-inoculated I1 plants of both varieties was significantly greater versus control plants under normal conditions (see Fig. 3, E, F). For example, this value in Ag-Ix was 2.57 times higher than in S0-I1, whereas under salinity, a 35.48 % root elongation was observed in S0-I1, which is more than 2 times greater compared to the corresponding value in Ag-I1 (see Fig. 3, E, F). Consequently, the root development in alfalfa under salinity stress can serve as a feature of the variety.
With I1 plants inoculation, the root length appeared to change significantly. Thus, a similar decrease in this value in Ag-I1 and S0-I1 was observed in their symbiosis with Rm2011 (an average of 20.62 %) both in normal conditions and at salinity. In Ag-I1 plants inoculated with strain CIAM1774, the reduction in root length was not significant if NaCl was not added into the substrate, and amounted to 10.43 % under the salinity stress versus the corresponding control plants. Inoculation of S0-I1 plants with the same strain resulted in the 14.44 and 37.88 % root length reduction (versus control), respectively, in normal conditions and under salinity. Consequently, the salt-tolerant strain of CIAM1774 affected the length of the roots in S0-I1 plants specifically, which resulted in a decrease of this value versus that of Ag-I1 by 2.87
and 3.76 times, respectively, in normal conditions and under the salinity stress. Thus, the effect of strain inoculum on the development of the root system of the host plant was established, but the change in the root length did not correlate with the productivity of plants both normally and under salinity (r = 0.3 and r = 0.5, respectively).
In conclusion, as a result of selection and analysis of plant groups con-trastly differing in salt tolerance, principally new data were obtained about the level of specificity of microbe-variety systems with high symbiotic efficiency under standard conditions and under salinity stress. Symbiotic systems derived from salinity tolerant generation I1 are characterized by increased productivity, active nodule formation, and longer plant stems. A decrease in the length of the root system was first demonstrted under symbiotrophic nutrition which may indicate both an increase the absorptive capacity of the roots (G.V. Stepanov, personal communication), and the activation of the processes of nitrogen transfer from the roots to the aerial parts [25]. This biometric indicator has been found to depend on the specificity of plant-microbe interactions. Interestingly, the strain of salinity tolerant phenotype had a more significant impact on the development of the underground part of the plant in a salinity tolerant phenotype which was especially evident under the salinity stress. This can be explained by the fact that under the interaction of micro- and macrosymbiont with increased salinity tolerance, optimal conditions are formed for the metabolic activity of bacteroides, the internal environment of which (even in standard conditions) is hyperosmotic [26]. The identified combinations of host plants and strain inoculum under the salinity stress suggest that an optimum balance of nitrogen and carbon is maintained in the above systems. It is obvious that the study of metabolic characteristics of symbiosystems formed based on the stress resistant micro- and macrosymbionts is a significant step towards the directed construction of symbiotic systems with a given adaptive potential.
So, our study has demonstrated the prospects of the analysis of stress resistance in both plant and microbial components resulting in the possibility of constructing directed symbiotic systems with a given adaptive potential.
REFERENCES
1. Zahran H.H. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiology Molecular Biology Review, 1999, 63(4): 968-989 (doi: 10.1007/BF00369391).
2. Piskovatskii Yu.M., Lomova M.G., Solozhentseva L.F., Lomov M.V. Selektsiya lyutserny dlya mnogovidovykh kormovykh agrofitotsenozov. V sbornike: Mnogofmk-tsional'noe adaptivnoe kormoproizvodstvo. Vypusk 5(53) [Multifunctional adaptive fodder production. Issue 5(53)]. Moscow, 2015: 159-168.
3. U m a r o v M.M., K u r a k o v A.V., Stepanov A.L. Mikrobiologicheskaya transfor-matsiya azota vpochve [Soil microbial transformation of nitrogen]. Moscow, 2007 (ISBN 5-89118-315-7).
4. Dobrovol'skii G.V. Pochvennye resursy Possii za 150 let. Rossiya v okruzhayushchem mire (analiticheskii ezhegodnik) [Soil resorses of Russia for 150 years. Russia and the world: annual analytics]. Moscow, 2002: 1-16.
5. Reynolds J.F, Smith D.M.S., Lambin E.F., Turner B.L., Mortimore M., Batterbury S.P.J., Walker B. Global desertification: building a science for dryland development. Science, 2007, 316: 847-851 (doi: 10.1126/science.1131634).
6. Rattan L. Restoring soil quality to mitigate soil degradation. Sustainability, 2015, 7: 58755895 (doi: 10.3390/su7055875).
7. Swaraj K., Bishnoi N.R. Effect of salt stress on nodulation and nitrogen fixation in legumes. Indian J. Exp. Biol., 1999, 37(9): 843-848.
8. Kosolapov V.M., Kostenko S.I., Pilipko S.V. Dostizheniya nauki i tekhniki APK, 2013, 7: 71-73.
9. Dzyubenko N.I., Chapurin V.F., Bukhteeva A.V., Soskov Yu.D. Trudy po prikladnoi botanike, genetike i selektsii (VIR, St. Petersburg), 2007, 164: 153-163.
10. Gasanov G.N., Usmanov R.Z., Musaev M.R., Abasov M.M. Yug Rossii: ekologiya, razvitie, 2007, 1: 79-85.
11. Metodika effektivnogo osvoeniya mnogovariantnykh tekhnologii uluchsheniya senokosov i pastbishch v Severnom prirodno-ekonomicheskom raione [Haw to syplify the effective use of multivariant technologies of hayfield and pasture improvement]. Moscow, 2015.
12. Ibragimova M.V., Rumyantseva M.L., Onishchuk O.P., Belova V.V., Kurchak O.N., Andronov E.E., Dzyubenko N.I., Simarov Mikrobiologiya, 2006, 75(1): 94-100 (doi: 10.1134/S0026261706010140).
13. Tikhonovich I.A., Provorov N.A. Cooperation of plants and microorganisms: Getting closer to the genetic construction of sustainable agro-systems. Biotechnology Journal, 2007, 2(7): 833-848 (doi: 10.1002/biot.200700014).
14. Stepanova G.V. Selektsiya lyutserny na povyshenie effektivnosti simbioticheskikh vzaimodeistvii. Tom 6 [Alfalfa breeding for increased symbiotic effects. V. 6]. Ufa, 2008: 32-38.
15. Gubry - Rangin C., Garcia M., Bena G. Partner choice in Medicago truncatula— Sinorhizobium symbiosis. Proceedings of Biological Science, 2010, 277(1690): 1947-1951 (doi: 10.1098/rspb.2009.2072).
16. Rumyantseva M.L., Simarov B.V., Onishchuk O.P., Andronov E.E., Chizhevskaya E.P., Belova V.S., Kurchak O.N. Biologicheskoe raznoobrazie klubenkovykh bakterii v ekosistemakh i agrotsenozakh: teoreticheskie osnovy i metody [Biodiversity of rhizobia in ecosystems and agrocenoses: theoretical bases and methods]. St. Petersburg—Pushkin, 2011.
17. Rumyantseva M.L., Muntyan V.S. Mikrobiologiya, 2015, 84(3): 263-280 (doi: 10.7868/S0026365615030179).
18. Kurchak O.N., Provorov N.A., Onishchuk O.P., Vorob'ev N.I., Rumyantseva M.L., Simarov B.V. Genetika, 2014, 50(7): 777-786 (doi: 10.7868/S001667581406006X).
19. Metodicheskie ukazaniya po selektsii mnogoletnikh trav [Selection of perennial grasses: guideline]. Moscow, 1985.
20. Lakin G.F. Biometriya [Biolmetry]. Moscow, 1980.
21. Trukhacheva N.V. Matematicheskaya statistika v mediko-biologicheskikh issledovaniyakh s primeneniem paketa Statistica [STATISTICA program for math statistics in medicine and biology]. Moscow, 2012.
22. Katalog raionirovarmykh i perspektivnykh sortov kormovykh kul'tur selektsii Vsemssiiskogo nauchno-issledovatel’skogo institute kormov imeni V.R. Vil’yamsa [Catalog of released and promising varieties of V.R. Villiams All-Russian Research Institute for Forages]. Moscow, 2007.
23. Stepanova G.V., Zolotarev V.N., Muntyan V.S., Rumyantseva M.L. Adaptivnoe kormoproizvodstvo, 2013, 3(15): 43-48 (http://www.adaptagro.ru).
24. Tishchenko E.D., Tishchenko A.V., Nizhegolenko V.M. Kormoproizvodstvo, 2012, Noyabr': 21-22.
25. Onishchuk O.P., Vorob'ev N.I., Provorov N.A., Simarov B.V. Ekolo-gicheskaya genetika, 2009, 7(2): 3-10.
26. Mandon K., Osteras M., Boncompagni E., Trinchant J.C., Spennato G., Poggi M.C., Le Rudulier D. The Sinorhizobium meliloti glycine betaine biosynthetic genes (betlCBA) are induced by choline and highly expressed in bacteroids. Mol. Plant Microbe Interact., 2003, 16(8): 709-719 (doi: 10.1094/MPMI.2003.16.8.709).
