CC BY
48
Umarov Bakhtiyor Rakhmatovich, Center of Genomics and bioinformatics AS Republic of Uzbekistan Senior scientists, doctoral science student E-mail: b.r.umarov@mail.ru
STUDY OF WATER STRESS IN SOYBEAN PLANTS WITH JOINT INOCULATION WITH STRAINS BRADYRHIZOBIUM JAPONICUM AND RHIZOBIUM ESPARSETA
Abstract. Among the environmental stress factors the most widely limiting for crop production on a global basis is water. Water stress has been found to decrease productivity of most plant particularly soybean. The aims of the study of nodule bacteria of drought water treatment. The result indicated that nodule bacteria showed as Rhizobium sp. Bradyrhizobium sp. The investigation of drought water stress indicated that, there are significant difference on the treatment of 50 ml water/1000 g soil of drought water stress toward the growth i.e. plant hight, number of leaves and nodule formation on soybean plant.
Keywords: Nodule bacteria, Rhizobium, Bradyrhizobium sp., drought water stress, soybean.
Introduction
Soybean is a major source of vegetable protein for most people. The ability of Rhizobium sp. to survive at a low water potential in soil has been established by many studies in which viability was assessed by determining colony forming ability on agar plates with a high water potential. Very few studies have addressed either the efficiency of Rhizobium growth at low water potential or growth recovery after a rapid water potential increase brought about by a rewetting process [1]. It is unknown what impact a rapid water potential change might have on soil Rhizobium spreading both under free-living conditions and during the period of establishment of the symbiosis. Keeping in view numerous manifestations of a beneficial action of Rhizobium bacteria on plants, present investigation involves isolation and characterization of nodule bacteria Bradyrhizobium japonicum from soybean and Rhizobium Esparseta from roots of wild legume plants of Esparseta. This study was based on the hypothesis that these microbes Rhizobium which are nitrogen fixing and growth promoting exhibit the ability to survive and tolerate moisture stress, should they be implied in imparting tolerance to host plant. To achieve this goal, were conducted isolation of nodule bacteria from root of wild type legume plants Esparseta and characterization to drought water stress.
Material and methods
Research was conducted at the laboratory and greenhouse conditions. The laboratory research included isolation of nodule bacteria from root nodule from soybean and plants Esparseta by using manual of S. Long laboratory (http://cmqm. stanford.edu/biology/long.protocols.htm). Nodule was characterization of greenhouse treated of drought water stress. The nodules soybeans were collected from experimental station were grown where grown of soybean and from Esperseta
planting on dry areas of Kisil Kum, Central Asia. The purification of the nodule bacteria were purified and maintained on yeast extract mannitol, YEM, [2]. Nodule bacteria that have been taken with a needle and entered into sterile (1,5 ml eppendorf tube), and then shaken using a vortex, pipetted 0.1 ml included in petri dish YEMA containing media, and leveled with a spatula, and then incubated at room temperature. Separate colony grows well selected and planted in the media YEMA slant in a test tube (as a pure culture). Characterization of nodule bacteria grown on selective media and studying by electrophoresis with methods T. Eckhardt [3]. Investigation of drought water stress on soybean plant in greenhouse was study uses soybean seed Genetik-1. Soybean seeds were planted in pots (size 20 cm), where each pots filled with 4 soybean seed. Soybean seeds soaked first into sterile distilled water for 2 hours before being planted into pots. After that, the seeds were stocked directly into pots; each pot filled 4 soybean seeds. After one week selected the best seed, while others deprived of pots. Nodule bacteria were identified as Rhizobium isolate, propagated in the medium of YEMA. Applications of Bradyrhizobium japonicum and Rhizobium Esparseta on soybean plant were done at 14 days after planting, by pouring the 2 holes near the rhizosphere. Rhizobium concentration used 105 was cfu/ml by 5 ml each hole, where the inoculation is done only once. The investigation of drought water stress is conducted with five levels of water, namely: WSO = Water Stress 0 ml/1000 g soil; WS1 = Water Stress 25 ml/1000 g soil; WS2 = Water Stress 50 ml/1000 g soil; WS3 = Water Stress 75 ml/1000 g soil; WS4 = Water Stress 100 ml/1000 g soil, where each treatment was the provision of water once every day and performed at 17.00 pm. The investigation was repeated 4 times. The parameter of this investigation is plant height, the number of leaves and the number of nodule. The
Section 1. Biology
observations were made in the afternoon every other day for 4 weeks.
Statistical Analysis for the investigation of drought water stress, ANOVA was also performed to determine the effects of plant height, the number of leaves and nodule formation. The percent data were arcsine-transformed before being subjected to ANOVA. When significant differences were detected, means were separated using Tukey's test at 5% probability level.
Result and discussion
Soil bacteria of the genera Rhizobium, Azorhizobium and Bradyrhizobium (collectively referred to as rhizobia) are involved in interaction with legume plants to form N2-fixing nodules. Rhizobia have also been found to be capable of colonizing roots of non-legumes as efficiently as they colonize their legume hosts These organisms are characteristically able to invade the roots hairs of temperate-zone and dry- zone legume plants and incite production of nodules. Rhizobia have great potential to nitrogen fixers. They are rods, cocci, gram negative; colonies are circular, convex, semitranslucent, raised and mucilaginous, usually 2-4mm in diameter within 3-5 days on yeast mannitol-mineral salt agar media, Commonly pleomorphic under adverse conditions [4]. Cells contain plas-mids, including large, naturally-occurring plasmids of 1500, 1000 kb [5]. The nitrogen-fixation (Nif) genes also appear to be plasmid-borne [6]. The present studies found that Rhizobium Esparseta grow less than 2 days. The genus rhizobium can be distinguished by the growth rate which the growth rate of the time needed to establish of a colony, there is growing quite fast, less than 3 days as Rhizobium, Sinorhizobium, and Allorhizobium [7]. There are currently classified as growing, between 4-5 days, such as Mesorhizobium and Azorhizobium, but there is also a relatively slow-growing bacteria, more than 6 days, such as Bradyrhizobium.
Investigation of Drought Water Stress on Soybean Plant in Greenhouse
The means of plant height of soybean at 50% treatment (WS2) showed statiscally significant difference with the treatment of control (WS0) or treatment with 25% (WS1), but did not show significant differences by treatment with 75% (WS3) and 100% (WS4) (Table 1).
Table 1. Means of plant height of soybean (cm)
Treatment Week After Treatment
1 2 3 4
WS0 33 32 6 6
WS1 28 28 28 29
WS2 43 43 43 44
WS3 41 41 42 43
WS4 43 45 46 46
MED - Minimal Essential Difference; P = 0.05
This indicates that simply by giving 50% of water from the minimum requirements with the use of Rhizobium isolate has been able to increase the growth of soybean plants after treatment with Rhizobium isolate. The growth of some strains of Rhizobium isolate greatly influenced by high salinity and low water potential conditions of the cropping land [8]. The developmental stage of a cultivated plant has its influence on multiplication of Rhizobium. Developmental stage of a cultivated plant determines, to a sufficient degree, the number of Rhizobium occurring in a crop [9]. Increased growth rate and metabolism of bacteria is probably related with the development of root system, photosynthetic activity and the amount of exudates associated with this and produced by plants [10].
Effect the number of leaves in the treatment of WS2 (50 ml/1000 soil) is always higher than the other treatments (Figure 1).
2 3 4
Week after treatment
Figure 1. Number of leaves of soybean plant
Maximum leaves in the treatment of WS2 indicated that the optimum growth and development of soybean plant and may be attributed to the symbiotic relationship of rhizobium (bacteria) with the roots of leguminous crops, which fix the atmospheric nitrogen into the roots of soybeans and thus the number of leaves plant was increased.
Effect the number of nodule plant-1 in each treatment and control has no statistically different, while the level is low. The number of nodule plant1 in the treatment ofWS2 (50 ml water/1000 soil) is higher than the other treatments, which 18 nodule plant1 (Table 2).
Table 2. Means of number of nodule plant
Treatment Number of nodule plant
WS0 1 Low
WS1 7 Low
WS2 18 Low
WS3 8 Low
WS4 10 Low
The low number of nodule plant1showed that the root formation is not well developed. Rhizobium isolates require foods for photosynthesis of soybean plants as an energy
source for the formation of root nodules. The water-stress effect have studied at different stages of N2 fixation and verified the adaptive physiological response to stress. Was found a positive interaction between water stress and development stages of N2 fixation. Water stress applied at the stage of 15-30 days gave the most negative interference, indicating it is possibly a critical period of water stress for cowpea. In our study, the water stress was applied from day 10 to day 30 of growth. The treatment of WS2 (50 ml water/1000 g soil), indicated of the role of Rhizobium isolate as a stimulus to grow of the soybean plant though not much. This is evidenced by the formation of the structure of pods on the plants treated by WS2 at 3 weeks of observation or five (5) weeks after planting. Basically the use of Rhizobium isolate is to increase crop yields, but in achieving optimum outcomes is influenced by several factors such as the number of microorganisms in the soil, pH, soil structure, content of certain chemical elements and temperature [11].
Conclusions
This study shows that there was a treatment of WS2 (50 ml water/1000 g soil) treated with Rhizobium isolate, the optrimum level for the growth of soybean plant i.e. plant hight, number of leaves and nodule formation on soybean plant.
References:
1.
Provorov N. A., Borisov A. Y., Tikhonovich I. A. Developmental genetics and evolution of symbiotic structures in nitrogen-fixing nodules and arbuscular mycorrhiza. J. Theor. Biol. 2002.- V. 214.- No. 2.- P. 215-232. Long S. laboratory Manual URL: http://cmqm.stanford.edu/biology/long.protocols.htm
Eckhardt T. A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid. 1978 - V. 1 -P. 584-588.
Selvakumar G., Mohan M., Kundu S., Gupta A. D., Joshi P., Nazim S., Gupta H. S. Cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC8708) isolated from flowers of summer squash (Cucurbitapepo). Lett. Appl. Microbiol. 2008.- Vol. 46 - P. 171-175.
Holt J. G., Kreig N. R., Sneath P. H.A. Staley J. T. and Williams S. T. 1994. Bergey's Manual of Determinative Bacteriology. 9th ed., Williams and Wilkins, (Eds). Baltimore, USA,- P. 40-169.
Young J. P.W., and Wexler M. Sym plasmid and chromosomal genotypes are correlated in field population of Rhizobium leguminosarium. J. Gen. Microb. 1988.134: 2731-2739.
Young J. P.W. Molecular population genetics and evolution of rhizobia. The Nitrogen Fixation and its Research in China / Hong G. F. (ed.) Springer-Verlag, Berlin, Heidelberg, 1992.- P. 366-381.
Abolhasani M., Lakzian A., Tajabadipour A., and Haghnia G. The Study Salt and Drought Tolerance of Sinorhizobium Bacteria to the Adaptation to Alkaline Condition. Australian Journal of Basic and Applied Sciences, 2010. 4(5): 882-886. Swedrzynska D., and Sawicka A., 2001. Effect of Inoculation on Population Numbers of Azospirillum Bacteria under Winter Wheat, Oat and Maize. Polish J. Environ. Stud., 10: 21-25.
10. Franson R. L., Brown M. S. and Bethlenfalvay G. J. The Glycine-Glomus-Bradyrhizobium symbiosis. XI. Nodule gas exchange and efficiency as a function of soil and root water status in mycorrhizal soybean. Physiol. Plant, 83: 1991.-P. 476-482.
11. Shakirov Z. S., Khakimov S. A., Shomurodov K. F. Effect of salinity and drought on symbiotical and biochemical properties of Onobrychis and alfalfa. Agricultural Sciences, 2012.- Vol. 3.- No. 3.- P. 444-454.
5.
6.
7.
9.
