EFFECT OF RESIDUAL ROCK PHOSPHATE AND BIOFERTILIZERS ( RHIZOBIUM AND BIOPHOSPHATE ) AND THEIR COMBINATIONS ON THE PRODUCTIVITY OF Stylosanthes guianensis CIAT 184

The purpose of this research was to evaluate the agronomic effectiveness of rock phosphate, biological fertilizers (Rhizobium and bio-phosphate) and their combinations on dry matter (DM) production, nutrient content and in vitro dry matter digestibility (IVDMD) of Stylosanthes guianensis CIAT 184. This research was conducted at Forage and Pasture Laboratory, Universitas Gadjah Mada, Yogyakarta, Indonesia from February – October 2010. A strip plot design which consisted of two fertilizer factors with three (3) replicates was used. The first factor was the horizontal factor and consisted of four levels of biological fertilizers (Rhizobium and bio-phosphate) namely: M0 = control, M1 = Rhizobium (0.5 g/plot), M2 = bio-phosphate (0.6 g/plot) and M3 = combinations of Rhizobium and bio-phosphate. The second factor was the vertical factor and consisted of three levels of rock phosphate, namely: P0 = control, P1 = 250 kg/ha (32.5 P kg/ha) and P2 = 500 kg/ha (65 P kg/ha). Defoliation was carried out every three months for 9 months. Results of the study showed that there was no significant difference in DM production (kg/ha) amongst treatments. Although the current research showed no significance difference amongs the treatments, DM production increased in the 2 harvest (12.3%) and 4 harvest (7.1%) between P0 and P1. Combinations treatment P1M2 (21073.63 kg/ha) had the highest average DM production amongs treatments. Statistical analysis followed by Duncan’s new Multiple Range Test (DMRT), showed that rock phosphate can significantly (P<0.05) increase fiber production of Stylosanthes guianensis. It showed that treatment P1 increased crude fiber production by 12.2% from 5864, 47 kg/ha to 6580,19 kg/ha. In addition, tests further revealed that there was a difference between M1 and M2, but had no effect on M0. There was no significant effect on production (kg/ha) of crude protein, phosphorus, nitrogen free extract (NFE) and ash. In conclusion, treatments with rock phosphate and bio-fertilizers tend to increase productivity of Stylosanthes guianensis CIAT 184. (


Introduction
The success of legumes is found in their ability to have a mutualistic symbioses with nitrogen (N)-fixing bacteria to directly capture atmospheric dinitrogen (N 2 ) to support plant growth (Hernandez et al., 2009).Nitrogen is commonly the most limiting plant nutrient in arable farming in the tropics and also the most expensive element as a mineral fertilizer, biological nitrogen fixation (BNF) therefore, holds great promise for smallholder farmers (Tripathi and Psychas, 1992).
However, although leguminous plants are expected to replenish soil nitrogen supplies, they are particularly hard hit by phosphorus (P) deficiency because low supply inhibits effective nodulation and retards the biological nitrogen fixation process (Whiteman, 1980).
The nitrogen fixing systems require more phosphorus (P) than non-N 2 fixing systems, because the process consumes large amounts of energy, and energy-generating metabolism strongly depends upon the availability of P (Schulze et al., 2006)._________________________________ *Korespondensi (corresponding author): Telp.+26 4818 792 289 E-mail: oc_lesedi@yahoo.comPhosphorous is one of important mineral for the plant.Manufactured water soluble phosphorus (WSP) fertilizers such as superphosphates are commonly recommended for correcting P deficiencies.The application of these inorganic fertilizers, results in low efficiency of WSP fertilizers such as triple superphosphate (TSP) or diammonium phosphate (DAP) by crops (Chien et al., 2010).
Another solution can be found in the direct application of rock phosphate which is an agronomic and economically sound alternative to the more expensive superphosphates in the tropics (Zapata and Roy, 2004).Phosphorus however, becomes unavailable to the plants after its application in soils; this is due to the formation of strong bonds between phosphorus with calcium and magnesium in alkaline pH and the same bonds with iron and aluminum in acidic soils (Mehrvars and Chaichi, 2008).
It is therefore, behind this background that this study aimed to investigate the use of biofertilizers (Biophosphate and Rhizobium), to increase the effectiveness of rock phosphate and thus the availability of P.
Stylosanthes guianensis CIAT 184 (stylo) is a legume that has been identified as a potential feed resource that could be used as a supplemental feed during periods of shortages.Another advantage of this legume is that as a N 2 -fixing plant, it has a higher capacity to utilize P from rock phosphate than nitrate-fed plants (Perez et al., 2007), because of their higher cation/anion uptake ratio and corresponding net release of H + .
This research is a continuation of the research conducted by Hutasoit (2010) who aimed at investigating the effect of rock phosphate and biological fertilizers on the production of Stylosanthes guianensis.The purpose of this research is to measure the agronomic effectiveness of rock phosphate, biofertilizers (biophosphate and Rhizobium) and their combinations on: the dry matter yield, nutrient compostion content and in vitro dry matter digestibility of Stylosanthes guianensis.This research would provide valuable information on the agronomic effectiveness of rock phosphate and provide an environmentally and economically attractive means to increase production.

Description of the study area
This research was conducted at Pasture and Forage Laboratory, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, Indonesia, for 9 months (February -October 2010).Average rainfall, temperature and humidity during this study was 240.4 mm, 32.1°C (maximum temp.) and 82.4% respectively (Adisutjipto Meteorology Stasion, 2010).

Research method
This research used the Strip Plot Design that consisted of two factors (3 x 4 factors).The first one being the horizontal factor, namely rock phosphate which consisted of three stages, namely: P0 = without rock phosphate P1 = rock phosphate 250 kg/ha (32.5 P kg/ha) P2 = rock phosphate 500 kg/ha (65 P kg/ha) The second factor was the vertical factor namely, biological fertilizers rhizobium and biofosfat (M) which consist of four stages namely: M0 = without biofertilizers M1 = plus rhizobium M2 = plus biophosphate M3 = plus rhizobium dan biophosphate.
The current research did not apply any of the above-mentioned treatments during the course of the 9 month research period.Hence, was only applied once at the time of planting by Hutasoit (2010).

Variables measured
The following were variables observed: dry matter (DM) content, crude protein (CP), crude fiber (CF), content (AOAC, 2005), phosphorus concentration, soil analysis and in vitro dry matter digestibility (IVDMD) (Tilley and Terry, 1963).Data of the proximate analysis and P concentration were used to calculate the production per hectare.Dry matter production was calculated by multiplying the dry matter content by the fresh weight, while the other variables were calculated based on the DM production.

Statistical analysis
Data were analyzed statistically using SPSS for windows version 16 and followed by Duncan's new Multiple Range Test (DMRT) when there was a significant effect.

Soil analysis of research area
The total Ca also reduced from 0.54% to 0.02%, this coincides with the reduced pH level (from 6.9 to 6.7), because as Ca level drops, the more acidic soils become.In addition, as a legume, stylo has high demand for Ca (Zapata and Roy, 2004).The available cation exchange capacity (CEC) recorded was 22.37, this value lies within the normal range 15 -40, and Kettering et al. (2007) reported that at these levels soils are more clay or more organic matter is present with high water holding capacity.Camberato (2001) further reported that pH 6.5 provides a near optimum CEC levels and P availability.
Results in Table 1 show that treatments had no significant effect on the DM production (kg/ha) of stylo.
Although no statistically significance difference, amongs treatments were found production increased in the 2 nd harvest 12.3% and 4 th harvest 7.1% between (P 0 ) and (P 1 ).are high, dry matter can be lost through excessive respiration.Another reason may be due the excess defoliation in the first year (4 times), as according to Tripathi and Psychas (1992) defoliation decreases the photosynthetic ability of legumes and impairs N 2 fixation which can lead to nodule decay.Results also showed that increased levels of rock phosphate (P 2 ) 500 kg/ha (65 P kg/ha) did not increase yield, with treatment (P 2 ) 18470.02 kg/ha having a lower yield as compared to the control.Similarly in their study to test different levels of fertilizer (P) application ranging between 0, 20, 40 -320 kg P/ha, Crespo and Curbelo (1990) found that increased levels more than 23.3 kg P/ha did not increase yields.
Increased production in the 2 nd harvest may be because during the growing period dissolved P could have moved through the soil to the roots (Bushman et al., 2009) and was absorbed by stylo and hence increased growth.In addition, the characteristic of stylo's response to phosphatic fertilization can double its production t' Mannetje (1992).In addition, Mckenzie and Middleton (1997) stated that a good supply of P has been associated with increased root growth, which means the plants, can explore more soil for nutrients and moisture.
There was a trend between rainfall and DM production (kg/ha), this statement is supported by Bushman et al. (2009) who reported that the activity of microorganisms is highly influenced by soil temperature and soil moisture.

Crude protein (CP) content and production
The CP represents a composite analysis of 3 harvests (harvest 2 -4) collected over a 9 month period.Treatment with rock phosphate had no effect on the CP content of stylo recording 17.67% (P 1 ) and 17.84% (P 2 ) as compared to the control 17.96 % (P 0 ).Treatment with bio-fertilizers also had no effect on CP content, with the control (P 0 ) recording the highest CP content at 18.08%.
There was no significant effect amongst the different rock phosphate levels.Bio-fertilizers also had no significant effect on production.The biological fixation of nitrogen may have been affected at earlier stages of plant growth due to the harsh environmental conditions experienced at the initial stage of growth.Zahran (1999) reported that a decrease in the infectivity of cowpea rhizobia was found at 35°C.Serraj and Adu-Gyamti (2004) report that it is well documented that both low and high extremes temperature can prevent nodulation, or if nodulation occurs, can inhibit nitrogen fixation.Nodule functioning in common beans (Phaseolus spp.) is optimal between 25 and 30°C and is hampered by root temperatures between 30 and 33°C (Zahran, 1999).During the research, temperatures recorded an average of 32.1°C; this could have been one of the factors that affected the microbial activity and hampered nodulation.
Sensitivity of symbiotic nitrogen fixation to phosphorus deficiency could have affected the CP production; if bio-phosphate microbes were rendered dormant due to the above mentioned conditions, P-deficiency would drastically cause a reduction in nodulation (Hernandez et al., 2009) and nitrogenase activity.

Crude fiber content and production
The total average CF (%) based on the interaction effects recorded 33.01%and ranged between 31.78 (P 0 M 1 ) -34.11% (P 1 M 1 ).Treatment with P 1 (250 kg/ha) had the highest effect on CF content recorded 33.41%.Keoboualapheth and Mikled (2003) recorded CF of stylo was 30%, which is lower than results in current studies.Keopaseuth et al. (2004) recorded even lower CF at 17.3%.The apparent increasing may be due to the aging of stylo, as sub-samples were collected for a one year period.Maturity through its effects on plant composition is recognized as a major determinant of forage quality (Onyeonagu et al., 2012).Because, as plant matures the cell wall content increases and cell content decreases.
Table 3 shows that the total CF average production (kg/ha) recorded 6186.74 kg/ha and ranged between 5616.40 kg/ha (P 2 M 1 ) -7069.30(P 1 M 0 ).The highest CF was recorded in combination treatment (P 1 M 0 ) 7069.30 kg/ha, similarly Hutasoit (2010) also recorded combination (P 1 M 0 ) as the treatment with the highest CF content.
SPSS statistical analysis followed by DMRT showed that there was a significant effect (P<0.05) of rock phosphate application on CF production, and there was a significant difference between (P 0 ) and (P 1 ).It showed that treatment P 1 increased crude fiber production by 12.2% from 5864.47 kg/ha to 6580.19 kg/ha.The results also showed a significant difference between (M 1 ) and (M 2 ) on CF production (kg/ha).

Phosphorus content and production
Treatment P 2 had the highest effect on phosphorus concentration, and M 2 had highest concentration 0.42% amongst the bio-fertilizer treatments, as compared to the control (M 0 ) 0.38%.The total average P based on the interaction effects recorded 0.40% and ranged between 0.35% (P 1 M 3 ) -0.50% (P 2 M 3 ).Compared to Hutasoit (2010) who recorded an average P of 0.13% and ranged between 0.12% (P 2 M 2 ) -0.16% (P 2 M 0 ).
The highest P content 0.50% (P 2 M 3 ) showed that combinations of rock phosphate levels at 500 kg/ha (65 P kg/ha) and bio-fertilizers combinations (bio-phosphate and Rhizobium) had an increasing effect on P content.Crespo and Curbelo (1990) in their research to determine the response of Stylosanthes guianensis CIAT-184 to phosphoric fertilization, recorded critical minimum P concentration in the plantamounted to 0.16%, while P phytotoxicity symptoms were present when 320 kg P/ha was applied and a P content of 0.71% was attained.Furthermore, they reported that increased application of fertilizer increased P concentration in plant.Similarly, Underwood and Suttle (1999) reported that phosphate application increases herbage phosphorus concentrations.
The phosphorus production (kg/ha) of stylo as per treatment with rock phosphate, biological fertilizers and their interaction effect recorded an average P (kg/ha) 75.51 and ranged between 61.43 (P 1 M 3 ) -89.77% (P 2 M 3 ).Statistical analysis showed that interaction treatment with rock phosphate and bio-fertilizers had no significant effect on P production (kg/ha) of stylo.
The low digestibility was a direct effect of the increased fiber content.This increase in fiber content influenced the digestibility of stylo, because a fiber fraction of a feed is closely related to its digestibility (McDonald et al., 1995).Buxton and Redfearn (1997) reported that the major factor lowering digestibilites of forages as they mature is the higher fiber and lower cellsoluble concentrations of mature forages.In addition, lignin is also primarily responsible for limiting digestibility of fiber, as lignin interferes with microbial degradation of fiber polysaccharides by acting as a physical barrier.Legume stems have a ring of thick-walled, lignified cells resistant to digestion.

Conclusion
Results of the study showed that residual rock phosphate and bio-fertilizers application tended to increase re-growth productivity of Stylosanthes guianensis CIAT 184.The most effective interaction between treatments was (P 1 ) 250 kg/ha (32.5 P Kg/ha) and (M 2 ) (bio-phosphate).Harvest 3 showed that (P 1 ) and (P 2 ) had lower yields as compared to P 0 .Rock phosphate significantly increased fiber production of stylo by 12.2%, while bio-fertilizers seemed not to influence the fiber production in statistical analysis.In vitro dry matter digestibility was not significantly affected by interaction treatment of rock phosphate, biophosphate and Rhizobium.Average P concentration increased from 0.13% (harvest 1) -0.40% (in current research).

Recommendation
Further analysis on the chemical composition, mineralogy and its influence on the reactivity of rock phosphate can further be researched.Also, a detailed economic analysis including cost of production can be formulated to compare the cost of rock phosphate to that of watersoluble-phosphate fertilizer.The neutral detergent fiber (NDF) and acid detergent fiber (ADF) using the Van Soest fiber fractionation system can be analyzed, as this estimates the structural carbohydrates and evaluates the usable area of the plant cell wall.

Table 1 .
Combinations treatment rock phosphate 250 kg/ha+ biophosphate (21073.63kg/ha)had the highest average DM production amongst treatments.Temperature probably had one of the greatest effects on the low DM average.If soil temperatures Effect of rock phosphate, bio-fertilizers and their interactions on DM production (kg/ha) of stylo

Table 2 .
reported CP figures ranged from 12.1 to 18.1% for the whole plant, this record Effect of rock phosphate, bio-fertilizers and their interactions on CP content of stylo

Table 3 .
Effect of rock phosphate, bio-fertilizers and their interactions on CF contents of stylo

Table 4 .
Effect of rock phosphate, bio-fertilizers and their interactions on in vitro dry matter digestibility of stylo