EFFECTS OF INOCULATION ON THE GROWTH OF SOYBEANS [Glycine max (L.) Merrill] PLANTED IN SOILS FROM DIFFERENT GEOGRAPHICAL LOCATION IN NORTH WESTERN

1AMINU S. M., 2SHAMSUDDEEN U., 3DIANDA M.

1Department of Science Laboratory Technology, Hussaini Adamu Federal Polytechnic Kazaure, Jigawa State 2Department of Microbiology, Bayero University . 3International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria. E-mail: [email protected]

Abstract-A study was conducted to determine the effect of inoculation on the growth of soybeans (Glycine max). Soil samples were collected from seven locations namely; Albasu, , Garko, Gaya (Sudan savanna), Giwa, Soba and Z/kataf (northern guinea savanna). The result showed that across treatments there was no significant difference in the height of soybeans at two, four and eight weeks of planting. The results also showed that; the mean number of nodules was found to be higher in soybeans inoculated with rhizobin inoculants (9.39) followed by the soybeans inoculated with nitrogen (2.96) and control (2.67). However, there was no significant difference between the nitrogen and the control treatments at P < 0.05. The soybeans inoculated with nitrogen have the higher mean root dry weight (2.98g) followed by those inoculated with the rhizobin inoculants (1.39g), and the control has the least mean root dry weight (0.58g). From the result the mean spad value of soybeans across treatments showed that; at fourth week of planting the soybeans inoculated with nitrogen has the highest mean spad value (31.31) followed by the soybeans inoculated with rhizobin inoculants and the control (29.59 and 28.63 respectively). At eighth week the mean spad value of the soybeans inoculated with nitrogen has the highest mean spad value (31.03) followed by those inoculated with rhizobin inoculants (29.73) and the control has the least mean spad value (27.83), and the higher the spad value the higher the nitrogen content of the soybeans. There is need to inoculate the soils since there was significant difference between the treatments.

Keywords: Soil, Soybeans, Inoculation, Inoculants, Nitrogen, Shoot, Root Dry Weight, Spad Values.

I. INTRODUCTION active molecules such as vitamins, minerals and other nutrients (Burris and Roberts, 1993). Legumes are very significant not only ecologically The formation of effective (functional) nodules in but also agriculturally because they are responsible soybean [Glycine max (L.) Merr.] when inoculated for major change of nitrogen from atmospheric N2 to with compatible rhizobia leads to fixation of ammonia (Angelini et al., 2003). The rhizobia widely atmospheric nitrogen (N2) making nitrogenous used in agricultural systems, are represented by fertilization of the soybean unnecessary. Symbiotic approximately 40 species in seven genera including: N2-fixation is a complex physiological process Allorhizobium, Azorhizobium, Bradyrhizobium, influenced by the interaction of genetic elements in Mesorhizobium, Rhizobium, Sinorhizobium (Wei et the higher plant species and rhizobia (Gwata et’ al., al., 2002) and a species in the genus of 2003). Methylobacterium (Giraud et al., 2001). Legumes have been used in agriculture since ancient MATERIALS AND METHODS time and legume seeds or pulses were among the first Collection of Soil Samples: source of human food and their domestication. Soil samples were collected from farm land that Legume plant posses a unique ability to establish legumes were previously planted. Twenty soils were symbiosis with nitrogen fixing bacteria of the family used for the research; the soils were collected from Rhizobiaceae. The bacteria belonging to the genera seven local government areas, four from Rhizobium, Bradyrhizobium, Allorhizobium, and three from Kaduna state. The soils are Garko I, II Sinorhizobium and Mesorhizobium (Martinez and III, Bichi I, II and III, Gaya I, II and III, Albasu I, Romero, 2003; Willems, 2006) which are collectively II and III, Giwa I, II and III, Soba I, II and III, and referred to as rhizobia, are able to form nodules on Z/kataf I and II. The GPS of the sites were also taken their host plants inside of which they fix- and recorded (Somasegaran, and Hoben, 1985). nitrogen.This symbiotic relationship reduces the requirements for nitrogenous fertilizers during the Determination of the effects of inoculation on the growth of leguminous crops and also enrich soil with performance of soybeans. nitrogen. Leguminous plants are also of crucial Sterilization of pots importance as animal feed. Alfaalfa and clovers are Four hundred and fifty pots were washed with water grown over extensive areas as forage crops for and detergent. The pots were air dried and then grazing or as dry hay, and they furnish not only high sterilized by vertical pressure steam sterilizer (LS- quality protein but also a variety of biologically B120L) at 1210C for 15 minutes.

Proceedings of TheIRES 3rd International Conference, Dubai, UAE, 4th July 2015, ISBN: 978-93-85465-46-8 35 Effects Of Inoculation On The Growth Of Soybeans [Glycine max (l.) Merrill] Planted In Soils From Different Geographical Location In North Western Nigeria Preparation of the inoculants RESULT AND DISCUSSION One hundred grams (100g) of the legume fix RESULT (rhizobin) inoculants was dissolved in 400ml of sterile physiological solution and then mixed by using Table 1.0: Mean number of nodules per soils. magnetic stirrer for twenty (20) minutes, until the legume fix (Rhizobin) was dissolved. Preparation of KNO3 solution A 1M solution of KNO3 was prepared in the laboratory by adding 101g of potassium nitrate in 800ml of sterile distilled water; the content was then mixed until the powder was dissolved. Then 200ml of distilled water was added to make it 1Liter. The determination of the effects of inoculation was carried out in the screen house of International Institute of Tropical Agriculture (IITA) Kano station. P < 0.05, means of the same later are not significantly Four hundred (400) sterilized pots were filled with different by Waller separation. raw soils of twenty different sites mentioned above, the pots were grouped in to twenty, and each group Table 2.0: Mean height of soybeans per treatments was filled with one type of soil only. Seedlings of at two, four and eight weeks. soybeans with good root development germinated in the lab were selected and one seedling was transplanted in to each of the pots prepared. The set up was watered daily and after seven days of transplanting fifteen pots out of each twenty pots were selected and grouped in to five pots each. The P < 0.05, means of the same later are not significantly first five pots were labeled as plus Inoculants (+I), the different by Waller separation second five as plus Nitrogen (+N), and the last as Key: LSD; Least Significant Difference. control (C). The first and the second were inoculated with 1ml of legume fix (Rhizobin inoculants) and Table 3.0: Mean shoot dry weights of soybeans per nitrogen from KNO3 stock respectively. soils. Determination of chlorophyll content The chlorophyll content of each plant was measured at 4th and 8th weeks by using SPAD 502 plus chlorophyll meter. The chlorophyll content of the two uppermost leaves of each plant were measured and in each leaf three readings were taken from leaflet one, two and three. This was done by clamping the meter over the leafy tissue of the plant (soybean), and chlorophyll content reading was received in less than 2 seconds. The readings were then recorded in the log book. Measurement of plant height The plant height of each soybean plant at 2nd, 4th and P < 0.05, means of the same later are not significantly 8th weeks was measured and recorded by using a different by Waller separation ruler. The set up was irrigated daily for a period of two months after which nodules were recovered. Table 4.0: Mean root dry weights of soybeans per Nodule Recovery: soils. The plants were transported to the washing ground of IITA Kano station. The soil from the root material was carefully removed without detaching secondary roots from the plant as nodules may be found on the lateral roots as well as the tap root. The roots were then washed using a gentle stream of water from the tap with a sieve of 1mm size underneath; in order to trap nodules that may detach from the roots (Bala, et al, 2010). The nodules number were taken and recorded and then preserved for further research. The shoots and roots of the plants were oven dried; the dry weights of the shoot and roots of all the plants were taken and recorded. P < 0.05, means of the same later are not significantly different by Waller separation

Proceedings of TheIRES 3rd International Conference, Dubai, UAE, 4th July 2015, ISBN: 978-93-85465-46-8 36 Effects Of Inoculation On The Growth Of Soybeans [Glycine max (l.) Merrill] Planted In Soils From Different Geographical Location In North Western Nigeria Table 5.0: The means nodules number, shoot and Soybeans height per treatments root dry weights, and spad values of the soybean Across treatments (Rhizobin, Nitrogen and control) per treatments at four and eight weeks. statistically there was no significant difference in the height of soybeans at two, four and eight weeks (table 2.0). This means that the height of the soybeans was not influenced by rhizobial inoculation. This support the finding of (Bekere et al., 2012); who reported that inoculation did not significantly influence height of soybean when phosphorus was not applied. However, inoculation resulted in significantly (P < 0.05) taller soybeans than the uninoculated check when 60,120 and 180 mg P kg-1 were applied. However Abdul P < 0.05, means of the same later are not significantly jabbar and Saud (2012); reported that the height of different by Waller separation. plant in the inoculated treatment increases Key: Spad4; Spad values at four weeks, Spad8; Spad significantly, compared to non-inoculated treatments value at eight weeks. at (P≤0.01). The increase in phosphorus levels enhanced plant height, and the highest plants were at Table 6.0: Mean organic carbon, Nitrogen content, 120 kg of P/ha, which gave 12.25 cm /plant compared pH, Olsen P, particle sizes and the textural classes with 0, 40, 80 kg of P /ha. of the sampled soils. Shoot and Root dry weights per soils. The soil sampled at Soba produced statistically the highest shoot dry weight followed by soil sampled at Giwa. This was followed by soil sampled at Gaya. There was no significant difference statistically between soils sampled at Bichi and Z/kataf. However soils sampled at Albasu and Garko produced the least shoot dry weights and there was no significant difference statistically between the two soils samples in the rhizobin inoculated soil. (Table3.0). The Key: LOC; Location, OC; organic carbon, N; difference could be due to the result of the MPN of Nitrogen, P; phosphorus. rhizobia and soil analysis which showed that soil sampled at Soba and Giwa has higher MPN/g of DISCUSSION rhizobia and also high organic carbon content, higher Number of nodules per soils. nitrogen content and finer texture than the remaining Even though there was variation in the performance soil samples (Table 6.0). The soil sampled at Gaya of the plants (height, root and shoot dry weights). The has higher olsen P content than all the soils, and the result showed that; statistically there was no soils sampled at Soba and Giwa have higher Nitrogen significant difference in the number of nodules of content than the soils sampled from Sudan savanna. soybeans per soils. This might be because the Soil sampled at Garko and Albasu has the least performance of the plant is not affected by the nodule organic carbon content (Table 6.0). This finding number and sizes as observed in this work. Moreover, opposed the work of (Bekere et ‘al., 2012); who soils with few number and smaller size of nodules reported that Inoculation and phosphorus level may contain active and efficient rhizobia than soils operated independently on shoot dry matter per plant with higher number and bigger nodules (table 1.0). of the soybean. Inoculated soybeans gave However Abdul Jabbar and Saud (2012); reported significantly higher shoot dry matter per plant than that fertilization with phosphorus and bacterial uninoculated ones. According to Abdul Jabbar and inoculation increased the number of nodules and Saud (2012); the difference in increase of plant dry other plant parameters. Hoque and Haq (1994); also weight between inoculated and non-inoculated reported the same results when they treated several treatments was significant (P≤0.01).The interactions legumes with Rhizobium and phosphorus and they between bacterial inoculation and different levels of found an increase in the number of nodules and phosphorus had obvious effect in increasing the maximum growth features with inoculation and weight of the dry matter, whereas interaction between phosphorus. Gwata et’al. (2004); have shown that bacterial inoculation and 120 kg of P /ha showed the promiscuous genotype of soybean produced heavy highest amount 45.75 g /plant followed by 36.54, nodule dry matter than non promiscuous. Other 38.29 and 38.83 g /plant respectively for P workers (Ndakidemi et al. 1998); reported significant fertilization 0, 40 and 80 kg of P /ha. improvement of common bean yields in un- The soil sampled at Giwa statistically produced inoculated treatments following phosphorus soybeans with the highest root dry weight, followed supplementation, and attributed it to the presence of by soil sampled at Z/kataf, Gaya and Garko (Table efficient indigenous rhizobia in the soil. 4.0). This support the finding of (Bekere et ‘al.,

Proceedings of TheIRES 3rd International Conference, Dubai, UAE, 4th July 2015, ISBN: 978-93-85465-46-8 37 Effects Of Inoculation On The Growth Of Soybeans [Glycine max (l.) Merrill] Planted In Soils From Different Geographical Location In North Western Nigeria 2012); who reported that both factors significantly (p to low phosphorus level in the soils used in this < 0.05) influenced root dry matter of soybean. In this research. Low phosphorus level may affect the shoot regard, inoculated soybeans gave significantly higher dry weight of the plant. This finding opposed the root dry matter than uninoculated ones. The soils work of (Bekere et ‘al., 2012); who reported that sampled at Albasu, Bichi, and Soba produced Inoculation and phosphorus level operated soybeans with the least root dry weight and there was independently on shoot dry matter per plant of the no significant difference statistically between the soybean. Inoculated soybeans gave significantly three soils in the control treatment. In the rhizobin higher shoot dry matter per plant than uninoculated and nitrogen inoculated treatments there was no ones. According to Abdul Jabbar and Saud (2012); significant difference statistically between all the soil the difference in increase of plant dry weight between samples in the root dry weights. This could be due to inoculated and non-inoculated treatments was the fact that inoculation did not significantly significant (P≤0.01).The interactions between influence the root dry weight of the plant. This bacterial inoculation and different levels of opposed the work of Abdul jabbar and Saud (2012); phosphorus had obvious effect in increasing the who reported that, the effect of bacterial inoculation weight of the dry matter, whereas interaction between and phosphorus on root dry weight was obvious as bacterial inoculation and 120 kg of P /ha showed the the root dry weight significantly increased under highest amount 45.75 g /plant followed by 36.54, bacterial inoculation (P≤0.01), compared with non 38.29 and 38.83 g /plant respectively for P bacterial inoculation. The root dry weight under fertilization 0, 40 and 80 kg of P /ha. bacterial inoculation was 20.55 g /plant, whereas The soybean inoculated with nitrogen statistically has there was 10.18 g /plant in control. the highest mean root dry weight value, followed by Nodule number per treatments those inoculated with rhizobin inoculants. The control In terms of nodule number across treatments, the has the least mean value (table 5.0). This means that nodules number of the soybeans inoculated with inoculation significantly influence the root dry matter rhizobin inoculants statistically was the highest, of the plant. This support the finding of (Bekere et followed by the soybeans inoculated with nitrogen ‘al., 2012); who reported that Inoculation and and control. However, statistically there was no phosphorus level significantly (p < 0.05) influenced significant difference between the mean nodule root dry matter of soybean. In this regard, inoculated numbers of the soybeans treated with nitrogen and the soybeans gave significantly higher root dry matter control (table 5.0).This means that the nitrogen than uninoculated ones. The three phosphorus levels treatment has no effect on the number of nodules. (60, 120 and 180 mg kg-1) significantly increased root This finding opposed the work of (Bekere et al., dry matter over the control. According to Abdul 2012) that reported; nodules were not generally jabbar and Saud (2012); the effect of bacterial observed in uninoculated treatment. Phosphorus level inoculation and phosphorus on root dry weight was significantly (P < 0.05) affected nodule number per obvious as the root dry weight significantly increased plant. However, (Chowdhury et al., 1983; Peoples et under bacterial inoculation (P≤0.01), compared with al., 1995; Ndakidemi et al., 1998 and 2006; Teymur non bacterial inoculation. The root dry weight under et al., 2012; Gicharu et al., 2013); have reported the bacterial inoculation was 20.55 g /plant, whereas significant increase in grain yield, number of nodules, there was 10.18 g /plant in control and nodule fresh weight in legumes following Chlorophyll content (Spad values) of the soybeans inoculation with rhizobium. However, in the current per treatments study nodules were observed in the control treatments The mean spad values of soybeans across treatments which signify that there were native rhizobia in the showed that at fourth week the soybeans inoculated soils which nodulated the soybeans. This so signifies with nitrogen statistically has the highest mean spad that the soybeans were promiscuous variety which value followed by the soybeans inoculated with can be nodulated by any indigenous rhizobia in the rhizobin inoculants and the control. However, soil. statistically there was no significant difference According to Abdul Jabbar and Saud (2012) between those inoculated with rhizobin inoculants fertilization with phosphorus and bacterial and the control at fourth week (Table 5.0). This could inoculation increased the number of nodules and be due to the fact that; inoculant at four weeks does other plant parameters. Hoque and Haq (1994); also not produce nodules that may contain active rhizobia reported the same results when they treated several to contribute in biological nitrogen fixation . legumes with Rhizobium and phosphorus and they Therefore soybeans performance at fourth week may found an increase in the number of nodules and not be enhanced. According to Bagyaraj et ‘al maximum growth features with inoculation and (1979); There was no nodulation at all in the phosphorus. uninoculated control plants and in the plants Shoot and root dry weights per treatments. inoculated only with the mycorrhiza and the plants Across treatments statistically there was no treated with Rhizobium nodulated well. The number, significant difference between the mean shoot dry size, dry weight and nitrogen content of nodules in weights of the soybeans (table 5.0). This could be due plants inoculated with mycorrhiza plus Rhizobium

Proceedings of TheIRES 3rd International Conference, Dubai, UAE, 4th July 2015, ISBN: 978-93-85465-46-8 38 Effects Of Inoculation On The Growth Of Soybeans [Glycine max (l.) Merrill] Planted In Soils From Different Geographical Location In North Western Nigeria were significantly greater than those of plants REFERENCES inoculated with Rhizobium only, 60 days after inoculation but not at 45 days after inoculation. At [1] Abdul Jabbar, B. K., and Saud H. M. (2012). Effect of phosphorus on biological nitrogen fixation in soybean under eight weeks the spad value of the soybeans inoculated irrigation using saline water. Global journal of science frontier with nitrogen statistically has the highest mean spad research. Agriculture and Biology.vol. 12. Pp64-72 value followed by those inoculated with rhizobin [2] Angelini, J., Castro, S., Fabra, A. (2003): Alterations in root colonization and nodC gene induction and in peanut-rhizobia inoculants and the control has the least mean spad interaction under acidic conditions. Plant Physiology and value (table 5.0). This could be due to the fact that at Biochemistry., 41, 289-294. eight week nodules were formed and rhizobia start [3] Bala, A., Abaidoo, R., Woomer, P. (2010): Rhizobia Strain Isolation and Characterisation Protocol, www.N2Africa.org, 16 fixing atmospheric nitrogen to enhance biological pp. nitrogen fixation. Therefore at eighth week there was [4] Bagyaraj, D. J., Mansnath, A. and Patil, R. B. (1978). Interaction high number of nodules and high chlorophyll content between A vesicular arbuscular Mycorriza and Rhizobium and their effects on Soybean in the field. New Phytol. 82, 141-145. of the plant than at fourth week. According to [5] Bekere, W., Wolde-meskel, E. and Kebede T. (2012). Growth Bagyaraj et ‘al (1979); There was no nodulation at all and nodulation response of Soybean (Glycine max L.) to in the uninoculated control plants and in the plants Bradyrhizobium inoculation and phosphorus levels under controlled condition in south western ethopia. African Journal of inoculated only with the mycorrhiza and the plants Agricultural research vol 7 (30) Pp 4266-4270. treated with Rhizobium nodulated well. [6] Burris, R. H. and Roberts, G. P. (1993): Biological nitrogen fixation. Annual Review Nutr. 13:317-335. [7] Chowdhury, M. S., Msumali, G. P., Malekela, G. P. (1983). Need CONCLUSION for seasonal inoculation of soybean with rhizobia at Morogoro, Tanzania. Biol. Agric. and Hortic. 1, 219–228. Based on the result obtained, inoculation significantly [8] Gicharu, G. K., Gitonga, N. M., Boga, H., Cheruiyot, R. C, Maingi, J. M. (2013). "Effect of inoculating selected climbing influences the performance of the soybeans since; bean cultivars with different rhizobia strains on nitrogen fixation. there was significant difference between the Int. J. of Micr. Res. 1(2), 25-31. [9] Giraud, E., Jourand, P., Garcia, N., Willems, A., De Lajudie, P. treatments. From the result at two months, the Y., Prin, Y., Neyra, M., Gillis M., Boivin- masson, C., Dreyfus, chlorophyll content (spad values) of the soybeans B. (2001): Methylotrophic Methylobacterium bacteria nodulate inoculated with nitrogen statistically was the highest and fix nitrogen in symbiosis with legumes. The J. Bact., 183, followed by those inoculated with rhizobin inoculants 214-220. [10] Gwata, E. T., Wofford, D. S., Boote, K. J., and Mushoriwa, H. and the control has the least mean spad value. The (2003). Determination of effective nodulation in early juvenile soils inoculated with rhizobin inoculants produced soyabean plants for genetic and biotechnology studies. African soybeans with the higher number of nodules followed jounal of Biotechnology vol 2(11), pp417-420. [11] Gwata, E. T., Wofford D. S., Pfahler, P. L. and Boote, K. J. by those inoculated with nitrogen and the control has (2004). Genetics of promiscuous nodulation in soybean: nodule the least mean nodules number. Also the result dry weight and leaf color score. J. Hered. 95(2):154-157. indicated that the root dry weight and the chlorophyll [12] Hoque, M. and Haq, M. F. (1994). Rhizobial inoculation and fertilization of lentil in Bangladesh. Lens Newsletter. 21(2): 29- contents of the soybeans inoculated with nitrogen and 30. inoculants are higher than the non inoculated [13] Martínez-Romero, E. (2003): Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives. Plant Soil. (control). 252:11-23. [14] Ndakidemi, P. A, Nyaky, A. S, Mkuchu, M., Woomer, P. L. RECOMMENDATIONS (1998). Fertilization and inoculation of Phaseolus vulgaris in Arusha, Tanzania. In ‘Proceedings of the 8th congress of the African Association for Biological Nitrogen Fixation’. (Ed. Increasing and extending the role of biofertilizer such Dakora FD). (University of Cape Town: Cape Town). pp. 166- as rhizobium would decrease the need for chemical 167. fertilizers and reduce adverse environmental effects. [15] Peoples, M. B., Herridge, D. F., and Lahda, J. K. (1995): Biological nitrogen fixation: an efficient source of nitrogen for Based on the result obtained in this research, the sustainable agricultural production. Plant Soil 174:3–28. following recommendations are made: [16] Somasegaran P., Hoben H. J. (1985). Methods in legume-  There is need to be producing inoculants using our Rhizobium technology. Hawaii Institute of Tropical Agriculture and Human Resources. p.550. indigenous rhizobia, in order to avoid producing [17] Teymur K. B, Raouf, S. S., Mohammad, S., Ali, N. (2012). inoculants that is less effective in our farms. Effects of plant density, Rhizobium inoculation and  The farmers should be encouraged to use microelements on nodulation, chlorophyll content and yield of chickpea (Cicer arietinum L.). Annals of Biological Research. 3 inoculants to increase their agricultural production. (2):951-958.  There is need for the Government to enlighten the [18] Wei, G. H., Wang, R. T., Tan, Z.Y., Zhu, M.E., Chen, W.X. public on the benefits of using inoculants and the (2002): Rhizobium indigoferae sp. nov. and Sinorhizobium kummerowiae sp. nov. respectively isolated from Indigofera spp. effects of chemical fertilizers on our lands. and Kummerowia stipulacea. Int. J. Syst. Evol. Micr., 52, 2231-  More researches should be conducted to compare 2239. [19] Willems, A. (2006). The taxonomy of rhizobia: an overview. the benefits of using inoculants over the use of Plant Soil 287: 3–14. chemical fertilizers.



Proceedings of TheIRES 3rd International Conference, Dubai, UAE, 4th July 2015, ISBN: 978-93-85465-46-8 39