THE EFFECT OF RHIZOBIUM SEED INOCULATION ON YIELDS AND QUALITY OF FORAGE AND SEED OF BERSEEM CLOVER (TRIFOLIUM ALEXANDRINUM L.) AND ITS IMPACT ON SOIL FERTILITY AND SMALLHOLDER FARMER'S INCOME.
A field experiment was conducted to investigate the effects of Rhizobium seed inoculation on forage and seed yield components and the forage quality of berseem clover (Trifolium alexandrinum). The experiment comprised of two treatments, seed inoculation with Rhizobium trifolii and the non-inoculation (control). The results revealed that the seed inoculation significantly affected (P8), the availability of P can be limited due to its sequestration with Ca in the soils, causing a reduction in crop yields (Jan et al., 2014). Soil salinity can increase rapidly and have a drastic impact on plant growth (Agarwal and Ahmad, 2010).
Salinisation is becoming an acute problem in agriculture in the study region due to the intensive cropping system and excessive use of salty tube well water for crop irrigation (Ghulam et al., 2013). Rhizobia are very sensitive to salt stress and thus the impact of Rhizobium seed inoculation on the growth and yield of berseem clover also varies depending on the salinity of the irrigation water. Agarwal and Ahmad (2010) reported that using low salinity irrigation water (ECa$?8 dS/m) produced the best responses to seed inoculation in berseem clover.
Inoculation not only influences berseem clover production but also contributes to the yields of the crops grown subsequently (Khan et al., 1985). Moreover, inoculation can be performed on different legume crops including pulses and the beneficial effects of inoculation have been found to be persistent more than 2 years post inoculation in low input agricultural systems, making it economically and ecologically sustainable (Pellegrino et al., 2011). Despite the obvious benefits, the inoculation of berseem clover forage crops is currently not being practiced in smallholder production systems in Pakistan due to lack of awareness (ignorance) of farmers. Furthermore, inoculums are often not available in the agricultural markets and when they are, they have not been appropriately stored (Naveed et al., 2015), primarily because of electricity shortage in the country.
This study was designed to investigate the impacts of seed inoculation on forage and seed yields and forage quality of berseem clover. Further, as the N fixation potential can be affected by variety, the opportunity was taken to gather data for the new variety of berseem clover (Agaitti Berseem-2002). Results were extrapolated to investigate the potential income benefits from using seed inoculation.
MATERIALS AND METHODS
The field experiment was conducted at University of Veterinary and Animal Sciences (UVAS) Ravi campus-Pattoki, Pakistan during the 2013-2014 growing season. The experimental site was located at 31o 03' 34.8''N and 73o 52' 42.6''E in the Kasur district of Punjab, Pakistan. The soil analysis results showed that the research site characterised as loamy soil with 0.45% organic matter, having a pH (CaCl2) of 9.1 and EC value of 5.3 dS/m.
Seed inoculation: Agaitti Berseem-2002 variety seed was inoculated with viable Rhizobium trifolii (sourced from the Ayub Agricultural Research Institute (AARI), Faisalabad Punjab, Pakistan) that had been stored at 28 +- 2AdegC. The inoculum was used at the rate 250 g/ha (2.5 bacteria culture bags/ha) for 20 kg of seed, as one culture bag (containing 100 g of culture) was recommended for 1 acre (0.40 ha) (Qureshi et al., 2012). The culture was mixed into a 10% sugar solution to make a slurry for coating (to achieve better adhesiveness) and then sprinkled over the seeds and mixed thoroughly, ensuring that the culture coated all of the seed. Treated seed was left to stand under shade for 2 h before sowing as per the Research Institute recommendations, and as described by Agarwal and Ahmad (2010).
Experimental design: The experiment was laid out in a randomised complete block design at UVAS-Pattoki, district Kasur, Punjab, Pakistan. The block size was 7 m in length x 3 m in width (21 m2) with six replications. The experiment comprised two treatments; use of inoculation and no inoculation (control).
Land preparation, fertilisers application, sowing and harvesting: The land preparation was undertaken by applying three ploughings with cultivator followed by two plankings to make a fine seedbed. Urea (46% N), di-ammonium phosphate (DAP; 18% N and 46% P2O5) and muriate of potash (MOP; 60% K2O) at 20, 300 and 50 kg/ha respectively were applied to achieve the recommended application of 20, 60 and 30 kg/ha of nitrogen (N), phosphorus (P) and potassium (K) respectively. All the fertilisers were broadcast by hand and then incorporated with ploughing into the soil prior to sowing. A pre sowing irrigation was then applied to the field for wet sowing of berseem seed. The berseem clover seed was cleaned manually and inoculated with Rhizobium trifolii prior to sowing as described by Agarwal and Ahmad (2010). Sowing occurred during the 2nd week of October.
Three forage cuts were taken and forage harvesting was completed during the last week of March and the crop was then left for seed harvesting. The first forage cut was taken 65 days after sowing (DAS), the second was 45 days after the first cut and the third cut was taken 40 days after the second cut. The seed crop was harvested during the third week of May.
Data and sample collection: After laying out the experiment, composite soil samples were collected from different locations at random across the treatments by using a soil auger to a depth of 30 cm before (the application of Rhizobium in oculumand fertilisers) and after sowing and harvesting (Kandil et al., 2005). Soil measurements included organic matter, total N (%) and available P and K (Estefan et al., 2013).
At each harvest three one m2 quadrats were cut from within the plot and individually weighed. Plant height (cm: average of 10 plants per quadrat), and number of stems and heads per m2 were recorded at each harvest. A composite 1.0 kg sample from each plot was oven dried (Hot Air oven/T1-OV-H-250) at 70oC for 72h to determine dry matter (DM) content and calculate DM yield (t/ha).
A second sample was dried at 60oC for 48 h prior to grinding through first a 5 mm and then a 1 mm screen, and a 150 g sub-sample of this material was then dried at 80EC for 24h to determine neutral detergent fibre (NDF), acid detergent fibre (ADF), crude protein (CP), DM digestibility (DMD), digestible organic matter (OM) on a DM basis (DOMD), water soluble carbohydrates (WSC) and metabolisable energy (ME) by near Infrared infrared reflectance spectroscopy (NIRS) using a Bruker multi-purpose analyser (MPA, Bruker Optik GmbH, Ettlingen, Germany) and OPUS software (version 5.1) with calibrations developed by the New South Wales' Department of Primary Industries' Feed Quality Service (FQS).
The calibrations were developed using the following methods: NDF and ADF analysed sequentially (Van Soest et al., 1991) using the filter bag method (AnkomA(r) 200/220 fibre analyser, ANKOM technology, Macedon, NY, USA), CP as N x 6.25 with N determined using the Dumas combustion method (Leco CNS 2000A(r) analyser: Leco, St. Joseph, MI, USA), ash by heating a sample in a muffle furnace at 550oC for 6h (Galyean, 2010, AFIA, 2014), DMD and DOMD by the pepsin cellulase digestibility assay (AFIA, 2014) and ME calculated as DMOD x 0.203 - 3.001 (AFIA, 2014).
At the seed harvest ten seed heads were randomly selected from each plot, and the seeds removed and counted. 1000-seed weight was determined by counting out 1000 seeds and weighing and predicted seed yields (kg/ha) were calculated. In addition, data were also recorded for the number of nodules per plant. Five plants from each treatment plot were uprooted randomly. Roots were washed with clean water to remove all soil particles and then the number of nodules per plant was recorded and the average number of nodules per plant calculated (Hussain et al., 2002).
Statistical analysis: The data were statistically analysed with linear mixed model (ASREML) with the inoculation treatment identified as a fixed effect, and the replications and plots as random effects, by using GenStatA(r) (17thedition) windows software (VSN International, 2014). The least significant differences (LSD) at 5% level of significance were used to compare the treatment means.
Forage production: Inoculation of berseem clover seed with Rhizobium trifolii significantly (P< 0.05) increased plant growth across all plant parameters measured, as shown in Table 1. Inoculation resulted in a 16% increase (P< 0.001) in the number of stems per m2 and the Rhizobium inoculated plants were 22.7% taller (P<0.001) than the non-inoculated plants (Table1; Error! Reference source not found.). In addition, inoculation increased green forage production per ha by 26.3% (P<0.001) and forage DM yield by 38.5% (P <0.05).
Table 1. Effects of Rhizobium trifolii inoculum on forage and seed yield parameters of cv Agaitti Berseem-2002 berseem clover.
Treatment###Number of stems (/m2)###Plant height (cm)###Green forage yield (t/ha)###Dry matter yield (t/ha)
Forage quality: The effects of inoculation on the nutritive value of the forage is shown in table 2. The CP content of the forage produced using inoculated seed was significantly higher (P< 0.05) than that produced from non-inoculated seed. Similarly, both the NDF (P< 0.001) and WSC (P0.05) on the ME, and ADF content of the forage.
Table 2. Effects of Rhizobium trifolii inoculum on forage quality parameters of cv. Agaitti Berseem-2002 berseem clover.
Treatment###CP (%)###ME (%)###ADF (%)###NDF (%)###WSC (%)
Seed production: Inoculation of berseem clover seed significantly (P< 0.001) improved seed production and quality across all measured seed parameters (Table 3). The number of seed heads per m2 increased by 43% (P<0.001) in comparison to the non-inoculated control. Additionally, inoculation resulted in a 39.4% increase (P< 0.001) in the number of seeds per head and a 9.8% increase (P< 0.001) in 1000-seed weight. As a consequence of the positive effects of inoculation on these parameters, the predicted seed yield was increased by 118.95% (P< 0.001).
Table 3. Effects of Rhizobium trifolii inoculum on seed yield and yield parameters of cv Agaitti Berseem-2002 of berseem clover.
Treatment###Number of heads###Number of seeds per###1000-seed weight (g)###Predicted seed yield
Nodulation and soil nitrogen content: The nodule count and total available soil N contents for both inoculated and non-inoculated trial plots are presented in Table4. The number of root nodules per plant increased significantly (P< 0.05) over non-inoculated plants, with a 61% increase in the nodule count. Additionally, the nodules present on inoculated roots appeared larger and distributed further along the root system on to secondary and tertiary root axes.
Table 4. Effects of Rhizobium trifolii inoculum on nodule count per plant of cv Agaitti Berseem-2002 of berseem clover, and soil organic matter, nitrogen (N), phosphorus (P) and potassium (K) at UVAS, Pakistan.
Treatment###Number of nodules###Organic matter (%)###Available
###per plant###Total N (%)###P(ppm)###K(ppm)
The chemical analyses of the soil samples showed significant increases (P< 0.05) in the soil OM (from 0.6 to 0.8 %) and nutrients such as N (from 0.02 to 0.04 %), P (from 4.8 to 6.1 ppm) and K (from 140.3 to 199 ppm) with the use of Rhizobium inoculation (Table 4). As shown in Figure 22, inoculation resulted in a significant increase (45.28%) in the available soil N due to an increase in the formation of root nodules (Table 4) which enhanced N fixation resulting in more available N for plant growth in the soil.
Potential impact of inoculation of Berseem clover seed on the income of smallholder farmers: Inoculation of Agaitti Berseem-2002 seed resulted in increased forage and seed production. Using an average market value of 4.04 Rs/kg (PKR) for fresh forage and of 450 Rs/kg for seed (recorded in the study area), inoculation has the potential to significantly increase smallholder farm profitability. Seed inoculation produced 8 tonnes of extra forage and 174 kg seed, and resulted in an additional 111, 913 rupees of net income to farmers. The cost of Rhizobium inoculum was minimal (148 Rs/ha) relative to the financial benefits derived from increased yields and quality of both forage and seed, and added soil N. The increase in N availability of 45% is the equivalent of one 50 kg bag of urea (containing 46% N) which saves the farmers about 2000 Rs.
Forage production: The positive effects of Rhizobium inoculation on overall plant growth and development of Agaitti Berseem-2002 clover plants resulted in higher forage yield. This is consistent with the results of previously reported inoculation trials (Agarwal and Ahmad, 2010, Giambalvo et al., 2011). The differences between inoculated and non-inoculated fresh and DM forage yields occurred as a consequence of the seed inoculation which increased nodulation and N fixation. This enhanced photosynthetic activity by leaves and increased plant growth resulting in higher green forage and DM yields, consistent with the results of Agarwal and Ahmad (2010) and Giambalvo et al. (2011).
The number of stems per m2 and plant growth/height are important factors affecting forage yields. The positive effect of inoculant on both the number of stems per m2 (16%) and plant height (22.67%) supports the findings of Hussain et al. (2002), Agarwal and Ahmad (2010) and Thalooth et al. (2015) who also demonstrated increases in relative growth rate, number of stems and fresh yield. The magnitude of the responses reported by Agarwal and Ahmad (2010) and Hussain et al. (2002) varied from 7 to 26% and may have varied to those in the present study (42.5 t/ha and 36 t/ha of green forage compared to 39.9 t/ha in the present study) due to the use of different cultivars responding differently to seed inoculant or perhaps the inoculants themselves (Graves et al., 1990).
Both fresh (green) and DM forage yields were increased by inoculation, which is similar to previously reported results (Bajpai et al., 1974, Hussain et al., 2002, Agarwal and Ahmad, 2010) who found a 74% increase in green forage yield, 20% greater green forage and 17% DM yields, and 26% increases in green forage and DM yields, respectively. This is likely due to the increased activity of the plant roots and consequently increased N fixation by nodules, resulting in the enhancement of nutrient availability and nutrient use efficiency; leading to a 21% increase in plant biomass (Thalooth et al., 2015).
Another contributing factor would be the assimilation of higher amounts of carbohydrates in the plant because of increased photosynthetic activity (Agarwal and Ahmad, 2010) resulting from the increased number of stems or leaves, 10% increase in plant height and 13% increase in total DM. Jan et al. (2014) found that shoot yield increased significantly with the use of inoculation. Agarwal and Ahmad (2010) also found that the use of Rhizobium inoculum enhanced shoot weights by 9%, resulting in higher fresh forage yields.
The magnitude of the response to Rhizobium inoculation varies widely across the literature depending on the soil conditions (saline or sodic) and the cultivars used. Agarwal and Ahmad (2010) reported a 14.2% increase in DM yield (10.5 t/ha vs. 12 t/ha), whilst Jan et al. (2014) reported a 2.07-fold increase(2.09 t/ha vs. 4.33 t/ha) and Thalooth et al. (2015) a 2.6-fold increase (3.71 t/ha vs. 9.78 t/ha). Hussain et al. (2002) found that Rhizobium inoculation resulted in a 17% increase in DM yield (72.20 g/pot vs. 84.50 g/pot) compared to the 38.5% increase in DM forage yield recorded in the present study. The differences between inoculated and non-inoculated fresh and DM forage yields occurred as a consequence of seed inoculation increasing nodulation and N fixation. The majority of the nodules were found on the tap root and were of larger size in the inoculated plants compared to non-inoculated, which had smaller nodules that were located on the lateral roots.
The nodules were not dissected for colour assessment; however, the larger nodule size and tap root location was assumed to be indicative of greater activity of the nodules (Hussain et al., 2002, Agarwal and Ahmad, 2010).
Forage quality: The inoculation of berseem clover seed with Rhizobium resulted in increased CP content of the forage, although it did not affect ME. This was similar to the findings of Giambalvo et al. (2011) who reported improved nutritive value by increasing protein levels in forage in response to Rhizobium inoculation. Thalooth et al. (2015) and Hussain et al. (2002) also reported increases in forage CP of 10% and 20%, respectively in response to seed inoculation. Forage CP levels have been shown to increase with access to greater supplies of soil N (Giambalvo et al., 2011). The increase in the CP content of the forage in response to seed inoculation may also have been a consequence of an increase in the number of leaves per plant or an increase in the leaf to stem ratio.
For milk production, NDF is important as dairy cows require sufficient NDF in their diets to maintain rumen function and maximise milk production. Increases in forage NDF can significantly enhance DM intake and NDF digestibility in dairy cows, resulting in increased milk yield. It is recommended that dairy rations contain at least 25% NDF (Oba and Allen, 1999). The forage grown from the non-inoculated seeds (24% NDF) did not meet this recommended level; however, seed inoculation resulted in a 9.19 % increase in the NDF content, which would provide sufficient NDF to dairy animals to maximise milk production. However, inoculation resulted in only a small increase in the ADF content (6.92%) of the forage. As ADF is a measure of the cellulose and lignin contents whilst NDF measures total plant cell wall material which includes hemicellulose, cellulose and lignin (Van Soest et al., 1991), it is likely to be of minimal importance for animal nutrition and productivity.
Seed production: The positive relationship of inoculation with plant nodule formation and forage production helps to explain the significant improvement in all of the seed yield parameters (Table 3), culminating in a 119% increase in seed yield. These results were in line with those of Bajpai et al. (1974) and Agarwal and Ahmad (2010) who also reported increased seed yields in response to inoculation using Rhizobium strains. The increase in seed weights was likely due to increased availability of carbohydrates for seed formation during the reproductive growth stage (Bajpai et al., 1974), resulting from the increased activity of the roots for efficient nutrient (N and P) uptake (Hussain et al., 2002, Jan et al., 2014).
Nodulation, soil fertility and nitrogen content: The N content of the soil increased with the growing of the berseem clover as a result of N fixation and was further enhanced (by 45% over non-inoculated treatment) as a consequence of inoculation of the seed. These results echo those of Jan et al. (2014), Giambalvo et al. (2011) and Hussain et al. (2002). For the non-inoculated plants, nodule counts per plant (63.8/plant) were similar (67.7/plant) to those reported by Hussain et al. (2002); but for the nodule counts for the inoculated plants, results were higher (102.5/per plant) than reported by Hussain et al. (83.3/plant). This may have been associated with varietal differences, the inoculant, and the soils used in the two studies. Hussain et al. also reported a "boosting effect" of using Rhizobium inoculation on root growth, particularly on root dry weights (19% increase) in both non-saline and saline treatments in Pakistan.
The OM, P and K contents of the soils used in the present study increased by 42%, 29% and 42%, respectively (Error! Reference source not found.) as a result of inoculation. The magnitude of these responses is dependent on the level and type of the Rhizobium present in the soil. Similar increases in OM and P contents in soil in response to seed inoculation were reported by Jan et al. (2014). The increased OM levels in the soil were likely associated with increased shoot and root dry weights, as reported by Jan et al. (2014).
The increase in dry weights of shoot and root (see Error! Reference source not found.) with the increased activity of Rhizobium in the root zone resulting in greater availability of soil nutrients to the plant (Hussain et al., 2002, Jan et al., 2014). Moreover, Jan et al. (2014) also reported that nutrient (P and K) uptake of berseem clover plants was increased by seed inoculation under calcareous soil conditions (pH[greater than or equal to]8), and Rhizobium inoculation further increased available soil N by 41% through N-fixation (Hussain et al., 2002).Repeated harvesting of the crop increases utilisation of plant reserves as well as maintaining the plants in the vegetative growth stage for longer (Giambalvo et al., 2011). This increased nutrient (particularly N) demand in turn increases symbiotic N-fixation by Rhizobium present in the root nodules (Graham and Vance, 2000, Bruning and Rozema, 2013).
Alternately, delaying forage cuts results in lower N-fixation due to a decline in the symbiotic N-fixation as the plant progresses to the reproductive growth stage (Giambalvo et al., 2011). Frequent cutting of forage at the optimum times of 65, 110 and 150 DAS is likely to have increased both yields of better quality forage and seed and the N content in the soil.
Conclusion: Both forage and seed yield components increased significantly by inoculation of cv. Agaitti Berseem-2002 berseem clover seed with Rhizobium trifolii. The nutritive value of the forage also increased (4% CP) due to more leaves per plant. The increased forage and seed production could potentially generate an additional net income of PKR 111,913 Rs/ha (US$ 1145/ha) and increase the profitability of the crops grown subsequently in the same field. Therefore, the practice of inoculating seed prior to sowing berseem clover crops in Pakistan offers a cheap and easy method of increasing the productive potential of the forage crop and therefore the profitability of the low input farming systems in which berseem clover dominates winter forage production.
Further studies into the use of seed inoculation, removal of basal N applications and its effects on growth and farm economics as well as barriers to adoption by small-holder farmers now need to be considered to determine why this readily available technology has not been utilised to date.
Acknowledgements: We are thankful to the Australian Centre for International Agricultural Research (ACIAR) for providing the financial support to undertake this research. We are grateful to the School of Animal and Veterinary Sciences, Charles Sturt University for awarding the Faculty of Science Scholarship to the senior author. We also thank the University of Veterinary and Animal Sciences, Lahore for supporting and providing the facilities to undertake the research work.
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|Date:||Oct 31, 2018|
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