Nitrogen fixation and nodulation of soybean as affected by rhizobial inoculation using different seed adhesives in a sandy clay loam soil

The study evaluated the efficacy of different adhesives added to rhizobial seed inoculum on soybean nodulation and biological nitrogen fixation in a screen house and under field conditions. The experiment was a 6×3 factorial arranged in Completely Randomized Design and Randomized Complete Block Design for the pot and field trials, respectively, with 3 replications. Cassava starch (CSV), corn starch (CS), honey (H), gelatine (G), gum arabic (GA) and water (W) were ® mixed with Rhizo-fix at three ratios: 1: 1, 1:2 and 1:3 (inoculants: adhesive).Viable rhizobial cells -4 on coated seeds were determined using plate count technique from serially diluted 10 aliquot. Nitrogen fixation (NF) was determined using ureide method. Gelatine had most rhizobial cells on seeds (CFU/seed). It had 88, 87 and 84% significant increment when compared with CSV, corn starch CS and GA, respectively. However, CSV and CS considerably had more N fixed than G and was positively correlated with nodule fresh weight. Mixing ratios had no effect on parameters observed. Generally, results obtained by locally sourced adhesives averagely equalled those obtained by the conventional adhesives that had better adhering capability. Thus, locally sourced adhesives could be an alternative to conventional adhesives which are often more expensive and not readily available. Moreover, since mixing ratio had no significant effect on most parameters measured, then the least ratio (1:1) which is probably the most economical should be adopted for rhizobial inoculation.


Introduction
(BNF) in a bid to tackle the insufficiency of N in most Seed rhizobial inoculation is a technology agricultural lands, especially in the tropics can only be that helps provide N to soil biologically and achieved if the appropriate amount of viable rhizobial indirectly reduces the problems associated with the cells are been preserved and kept active before the use of inorganic sources of N and its benefits to both onset of nodulation and nitrogen fixation in order to s o i l a n d t h e e n v i r o n m e n t c a n n o t b e positively affect plant growth. To improve soybean overemphasized. However, many factors such as yield, biological nitrogen fixation, contribution to soil desiccation are militating against the benefits of fertility restoration, inoculation with efficient strains of seed rhizobial inoculation. Ghalamboran and Bradyrhizobia has been tested (Hussain et. al., 2011;Ramsden (2009) reported that death of rhizobial Tairo and Ndakidem, 2014). As the use of efficient cells on seed is majorly attributed to desiccation. strains of inoculants is important in BNF studies (Green This is considered a major problem for African et.al., 1979, Streeter, 2007 so also is the use of farmers owing to the fact that temperature in the adhesive agents (e.g., gum arabic, , methyl cellulose, tropics are very high and further expedite the rate and oil) as it helps in increasing the amount of rhizobial of desiccation and death of rhizobial cells on seeds.
cell that adheres to seed (Rugheim, and Abdelgani, The success of Biological Nitrogen Fixation 2012).
However, most of these adhesive agents inverted in the incubator at 26oC for 5 days and the are not readily available and are expensive for local numbers of rhizobial colonies were counted. The plates farmers. Hence, there is need to assess and validate were counted by diving into a quadrant and multiplying the use of other locally available materials which the numbers of colonies counted by 4. The mean colony can serve as substitute and also enhance (BNF) and forming unit (CFU) was calculated using the convenient most importantly are readily available and relatively formula (equation 1 and 2) for seed and soil B. cheap for the resource-poor small holders famers.
japonicum count. Therefore this study was undertaken to evaluate For seeds: the suitability of locally available material used as adhesives for rhizobium seed inoculation in terms of soybean growth, nodulation and biological nitrogen fixation.

Materials and methods
The experiment was carried out at the 12' to 7 20' N and longitude 3 20' to 3 28' E with a Pot Trial bimodal rainfall distribution with peak in July and Experimental layout October. The experiment was first carried out as the The experiment was a 6 × 3 factorial combination laid pot trial and the result obtained was validated on out in Completely Randomized Design with a total of 18 the field. treatment combination replicated 3 times. The treatment were six adhesives (honey, cassava starch, Seed Inoculation corn starch, water, gelatin and gum arabic) and three ® Carrier-based peat inoculant (Rhizo-fix ) mixing ratios of the inoculants and the adhesives (r = 1 was used, and applied at the rate of 10 g 10:10, r =10:20 and r =10:30).
2 3 inoculant/kg seed (Rice et. al., 2001) using various adhesives. All adhesives were prepared at 20 % w/v ® Soil preparation and planting except honey which was already slurry. Rhizo-fix Soil samples collected from the field location and the prepared adhesives were applied on seeds was air-dried and passed through a 2 mm mesh sieve so at 3 different mixing ratios, first ratio (r ) 10 g of 1 as to remove debris and stone that could serve as ® Rhizo-fix to 10 ml of adhesive, second ratio (r ), 10 2 impediments to root growth. The pots were filled with 5 ® g of Rhizo-fix to 20 ml of adhesive and the third kg of soil. A basal application of Gateway organic ® ratio (r ), 10 g of Rhizo-fix to 30 ml of adhesive.
3 fertilizer was added and thoroughly incorporated at the -1 After inoculation the seeds were exposed to air rate of 2 t ha to give the starter N nutrition to the under room condition so as to allow the inoculants soybean plants (Olayinka et. al., 1998). Water was to properly stick on seeds. Inoculated seeds were applied to each pot to saturation and the organic kept in labeled bags and planted immediately after fertilizer was left to mineralize for another two weeks inoculation.
before the commencement of planting. This was done so as to ensure that the soil condition was favourable for Rhizobial Enumeration seed growth. Three inoculated seeds of TGx-1940-2F The viability of inoculum was determined were planted in each pot; wetting of soil was done every using plate count method as described below other day while thinning to 2 plants per pot was done at before the seed were inoculated. Subsamples of 20 2 weeks after planting (WAP). Weeding was carried out intact seeds were randomly picked with a spatula manually at 3, 7 and 12 WAP. into 20 ml sterile deionised water in a sample bottle, shaken vigorously for 1 -2 minutes so as to desorb Field Trial the rhizobial cells from the surface of the seeds into Experimental layout and soil sampling the stock solution. One millilitre from the stock was The plot size was 3 by 2m with a total of 54 serially diluted into sequentially labelled 9 ml tubes plots. Each plot was separated by a 0.5 m alley, while and 1ml aliquot of appropriate dilutions was spread each replicate was separated by a 1.0 m alley. The field onto the surface of sterilized plates. Congo Red size was 45 by 11m. The treatment involved were the Yeast Mannitol Agar was aseptically poured into the same with that of the pot trial but was laid out using plates in air-flow (at 60°C); plates were carefully RCBD experimental design. Representative soil samples sealed so as to prevent contamination and stored were taken at random from the field, at 0-15 cm Table 1. Physico-chemical properties of the depth using a soil auger. Composite samples were experimental soil bulked, air dried, gently crushed and passed through a 2 mm and 5 mm sieve based on the type of routine analysis to be determined. The physical and chemical properties of processed soil after processing soil physico-chemical properties was determined using standard methods as described by Agbenin (1995).

Planting and other cultural practices
Experimental plots were fertilized uniformly -1 at the rate of 2 t ha with Gateway organic fertilizer to give the starter N effect. Inoculation of seeds took the same trend as that of the screen house. After inoculation the seeds were planted by seed drilling at a space of 60 cm inter row spacing, seeds were covered immediately with soil after sowing to avoid death of cells due to radiation from the sun.
Thinning was done to approximately 5 cm between plants in a row at 2 WAP, when the soil was moist and seedlings well established. Weeding was done rd th The different adhesives varied in their manually using hoe, on the 3 and 6 WAP to control adhering ability. The result for the rhizioal load on weeds.
selected seed prior to planting (Figure 1), gelatine had the highest cfu/g of rhizobial cells on seed surface while Data Collection cassava starch adhered the least cfu/g of seed. Five (5) plants were randomly selected and tagged from two middle rows on the field. Data were Nodulation collected for number and weight of nodules -plants Nodules were formed with all the treatments were carefully uprooted from soil within 2 cm of the (Table 2), but nodule formation at 4 WAP in the pot trial root so as to obtain intact root and nodules, was delayed. In the pot trial for nodule count at 8WAP, adhering soil was removed by washing the roots the various adhesives used had no significant effect with intact nodules gently with water over a metal (p<0.05) on the number of nodule formed but across sieve. Petioles were used for determination of the adhesives there was significant difference on fresh biological nitrogen fixation using ureide method weight of nodule. (Herridge, 1982).
On the field, cassava starch and gum arabic treated plots recorded the highest number of nodules Statistical analysis at 8WAP (p<0.05). The two treatments also Statistical analysis was done using SAS significantly increased the fresh weight of nodule so as (Statistical Analysis System Inc., 2000). The data corn starch. The trend changed at 12 WAP, as water collected was subjected to analysis of variance and cornstarch significantly increased number of (ANOVA) to determine treatment effect at 5% level nodules taking the place of cassava starch and gum of significance. Duncan's Multiple Range Test was arabic. However, cassava starch and corn starch used for mean separation. Pearson's Correlation treated plot recorded nodules with bigger fresh weight analysis was also employed to test relationship when compared to other conventional adhesive tested between components.
in this trial. The mixing ratio had no significant effect on all nodulation parameters observed.

Results
The soil used for this study is sandy clay -1 Biological Nitrogen fixation loam containing less than 250 g kg of clay and silt Nitrogen fixation was detected in all treatment ( Table 1). The pH signifies that the soil reaction is examined (Table 3). All treatments had no significant near neutral with low P and N. effect on the amount of nitrogen fixed in both pot and field trial. However, cassava starch treated pots fixed an average amount of N that was higher than the amount fixed by other treatment observed. Regardless been Discussion the one that adhered the least rhizobial load prior to The soil used for the experiment is sandy clay planting, pots with cassava starch had % increment loam containing less than 30 % of clay and silt; implying of 20, 29 , 0.6 , 18 and 7 over honey, corn starch, that the soil is well aerated and the water and nutrient water, gelatine and gum arabic while on the field, holding capacity of the soil is low. The available cassava starch had % increment of 6,11,15,18 phosphorus was relatively low and this was not and 21 in the same sequence as mentioned above.
considered as a limiting nutrient since soybean is more efficient at producing good yield at low soil phosphorous levels than other major agronomic crops (Abassi et. al., 2008). The soil pH was near neutral while the soil nitrogen content was very low. These conditions were favourable as high N soil content tends to circumvent the benefits of BNF and B. japonicum also thrives well under near neutral pH conditions (Lapinskas, 2008).
The adhesives studied all differed in their adhering capability which could be as a result of the adhesives tenacity and viscosity at varying concentrations. Among the adhesives tested under this study, gelatine was the best in terms of adherence of peat inoculant to seeds before planting; as this translated to gelatine having lot more of viable rhizobial cells attached to the seed. This could be as a result of gelatine being able to form a film when it is mixed in thin layer over a surface becoming very tenacious in nature as reported by Johnston-Banks Figure 1. (1990).

Population of B. japonicum on coated
In this study, nodulation and % nitrogen fixed seed in respect to different adhesive used.
varied under the different adhesive treatments. One (Materials with similar alphabets are not statistically would assume that pots treated with gelatine; that different (p <0.05) adhered the most cfu/seed of B. japonicum would have Table 2: a more positive effect on the nodulation parameters Effects of adhesives on nodulation and observed but cassava starch and corn starch which was number of pods per plant at 4, 8 and 12 WAP in a among the adhesives that had least cfu/seed of B. pot and field trial.
japonicum increased these parameters throughout the period of observation. Conversely, nodule fresh weight of cassava starch was the highest as compared to that of water that had the highest number of nodules or with that of gelatine that adhered the most inoculants. Nodule fresh weight per plant is considered the most important nodulation parameter examined as observed by many researchers (Khurana et. al., 2001;Fatima et. al., 2012;Saleh et. al., 2013). The findings also corroborates with that of Zoundji et. al. (2015) and Masresha and Kibebew (2017) that observed a significant and positive correlation existed between nodule number, biomass production, nitrogen uptake and grain yield. So, increases of nodule number involve better nitrogen nutrition which provided a good development of shoot and root of the plant and improved grain yield. In this study, cassava and corn starch considerably had more nitrogen fixed when compared with other conventional adhesives. Cassava and corn starch were least effective in terms of adherence of peat based rhizobium inoculants on the seed but also with the highest percentage of N fixed on the long run, this suggests that the few bacteria that adhered were preserved and active till the onset of