Quality Herbage Production of Dwarf Napiergrass with Italian Ryegrass Cropping under Digested Effluent Application in Southern Kyushu, Japan

Corresponding Author: Yasuyuki Ishii Faculty of Agriculture, University of Miyazaki, Miyazaki, 889-2192 Japan Tel: +81-985-58-7251, Fax: +81-985-58-2884 E-mail: yishii@cc.miyazaki-u.ac.jp Abstract: Digested effluent produced by a biogas-plant contains essential plant nutrients to solve the treated and disposal problems of livestock manure. Dwarf variety of late-heading type (dwarf) napiergrass as a perennial summer crop and intercropping of over-sown Italian ryegrass as an annual winter crop were applied to 3 levels of digested effluent in southern Kyushu from 2007 to 2009. Digested effluent revealed so effective fertilizer as chemical one to increase dry matter yield, wintering ability and forage quality in crude protein concentration and in vitro dry matter digestibility (digestibility) with increasing application rate. Digestibility in leaf blade of napiergrass and that in whole Italian ryegrass were positively correlated with acid detergent lignin and neutral detergent fiber concentrations, respectively, which might be mediated with reducing tiller size with defoliation proceeded. The present cropping systems have a potential under sufficient application of the effluent to achieve sustainable quality herbage production in southern Kyushu, Japan.


Introduction
Animal manure contains valuable nutrients and organic matter that can be used as a nutritional source for forages to produce high yield with high nutritive value and be suitable to build adequate environments of grassland farming . Livestock wastes can be processed to Digested Effluent of Manure (DEM) by biogas-plant (Thy and Buntha, 2005), which requires arable crops to utilize these nutrients efficiently. Intensive management of grassland systems requires considerable amount of N fertilizer to sustain high production and nutritive value of herbages (Sunusi et al., 1999;Wadi et al., 2003). Defoliation is an important management factor because of the multiple influences on the growth attributes as well as forage qualities, either by cutting operation or by animal grazing (Hodgson, 1979;Thomas, 1980). Napiergrass (Pennisetum purpureum Schumach) is a C 4 tropical grass which produces abundantly nutritious green forage and is considered to be excellent feeds for livestock under both green-chopping and grazing systems in the tropics (Vicente-Chandler et al., 1959;Woodard and Prine, 1993) as well as in temperate Kyushu, Japan (Sunusi et al., 1999;Wadi et al., 2004;Idota et al., 2005;Hasyim et al., 2010), affected by the various conditions such as growth stages, climatic factors and defoliation intensity (Zewdu et al., 2002). Several genotypes of napiergrass were investigated for the potential biofuel production in the tropics (Williams and Hanna, 1995;Rengsirikul et al., 2011) and in temperate Kyushu (Khairani et al., 2013). Napiergrass required heavy fertilization and careful management to maintain a rapid growth with quality forage under the subtropical climate in Australia (Jones, 1985) as well as in southern Kyushu, Japan (Sunusi et al., 1999;Wadi et al., 2003). Dwarf variety of late-heading type (dwarf) napiergrass, bred in Florida, USA (Sollenberger et al., 1988) and introduced from Thailand (Ishii et al., 1998), was evaluated for the adaptability in southern Kyushu areas (Utamy et al., 2011;Ishii et al., 2013) and can be used for grazing of beef cattle . In the temperate Kyushu, lower temperature with shorter optimal growing period requires napiergrass to have a rapid growth with vigorous tillering ability in an optimal growing season and maintain higher wintering ability (Ishii et al., 1995), which might be achievable by the high rate of DEM application.
Since napiergrass, C 4 tropical species, is suffered from low temperature to retard the growth in the wintering season, it is necessary to over-sow temperate species into the interrow space of napiergrass during the winter-spring period, so as to achieve maximum annual herbage yield. Recently, Italian ryegrass (Lolium multiflorum Lam.) has been recognized as the rotation crop for the plowing-down management (Dart and Fulkerson, 2014), since it is a cool-season annual, leafy and palatable grass to livestock and can produce significant amount of herbages under favorable growing conditions. Therefore, this study was examined for the cut-and-carry cropping of dwarf napiergrass with oversown Italian ryegrass under different levels of DEM application compared with chemical fertilization to examine the potential quality herbage production for 2 years after establishment.

Plant Culture and Design of Experiment
The experiment was conducted on andosols at 31 m above sea level in Kibana Field, University of Miyazaki in southern Kyushu, Japan (131.41°E, 31.83°N, 31 m a.s.l.) in the two cropping years from 2007 to 2009. Dwarf napiergrass was grown by transplanting a rooted tiller with 2 plants m −2 on 10 May, 2007 and Italian ryegrass (cv. Ace, Snow Brand Seed Co. Ltd. Sapporo, Japan) was sown into the inter-row space as an intercrop after harvesting dwarf napiergrass in autumn on 30 October, 2007 and27 October, 2008. The plots (13.5 m 2 /plot) were set into a randomized blocked design by 3 replications and were applied into 4 treatments, which have 3 levels of DEM application and chemical compound fertilizer (C) application at the same rate as high DEM level with additional split application.

Fertilizer Application
Application of DEM solution was 3 levels at 5.04, 2.52 and 1.26 g N m −2 time −1 for high (H), medium (M) and low (L) levels, respectively and chemical compound fertilizer was supplied as a check (C: 5.04 g N m −2 time −1 ). Application to napiergrass was conducted 4 times at a monthly interval from June to September in both 2007 and 2008. The same levels of fertilizing treatments were imposed to Italian ryegrass 3 times of application each cropping season on 29 October, 7 November, 2007and 3 May, 2008and on 26 November, 2008, 23 February and 29 April, 2009, for the first and second season, respectively.

Climatic Conditions
Changes in the mean and minimum air temperatures and precipitation were monitored from the data of Miyazaki Meteorological Observatory (JMA, 2013) in the summer (May to November) and winter periods (December to April) for napiergrass and Italian ryegrass, respectively. Climatic conditions of both periods were not significantly different from those in the normal year, obtaining on the 30-year average for 1980-2010(JMA, 2013, except for the variation of summer precipitation, which was extremely higher in July 2007, contrary to the extremely drought condition in July 2008. The minimum monthly temperatures occurred at 1.1°C in February 2008 in the first season, compared with higher wintering temperature at 3.2°C in January 2009 in the second season.

Statistical Analysis
Analysis of variance (ANOVA) was carried out using SPSS software (version 15.0) by one-way analysis procedures for yield and quality attributes of napiergrass and Italian ryegrass in a randomized complete design. Mean separation was tested using the Least Significance Difference (LSD) method at the 5% level.

Wintering Ability of Dwarf Napiergrass in 2008 and 2009
Wintering ability, as assessed by POP and RTN per m −2 in the spring of both 2008 and 2009, is shown in Fig.  1. In the first post-wintered season, POPs were quite stable and almost perfect averaged at 99.2%, while they increased with the increase in DEM application rate from 86.7% (L level) to 93.3% (H level) in the second season. The RTN showed the positive response to the increase in DEM application rate both years, except for the erratic lowest value in M plot in early June 2009. Chemical fertilization caused almost similar effect on POP and RTN with H level of DEM application in mid-May 2008, contrary to the significantly highest POP and RTN across treatments in early June 2009 (Fig. 1).

Yield and Crude Protein of Herbages in Dwarf Napiergrass and Italian Ryegrass
Both of dry matter yield and CP concentration in dwarf napiergrass and Italian ryegrass increased significantly with the increase in DEM application rate across all cuttings both years (Table 1). With cutting, dry matter yield tended to decrease, while CP concentration tended to increase in both dwarf napiergrass and Italian ryegrass both years. Annual total yields increased from the first year to the second year across all treatments and grass species, while CP concentration tended to increase slightly from the first to the second year in dwarf napiergrass, while it decreased from the first to the second year in the DEM-applied Italian ryegrass. Dry matter yields between DEM and chemical fertilization were non-significantly different in any cutting for both species, except for the highest yield of dwarf napiergrass in C plot at the third cutting under the lowest temperature period in 2007.
Annual total yield had a positive and linear regression with annual total N input both for dwarf napiergrass (r = 0.929, p<0.10 and r = 0.965, p<0.05 in the first and second year, respectively) and for Italian ryegrass (r = 0.991, p<0.01 and r = 0.978, p<0.05 in the first and second year, respectively). Correlation coefficient of dry matter yield with CP concentration was significantly negative (r = -0.671, p<0.01) for dwarf napiergrass, while it was non-significantly negative (r = -0.341, p>0.10) for Italian ryegrass (Table 2).

Digestibility of Herbages in Dwarf Napiergrass and Italian Ryegrass
Changes in IVDMD of LB, ST and LB + ST in dwarf napiergrass and that of whole plants in Italian ryegrass are shown for each cutting both years in Fig. 2. In dwarf napiergrass, IVDMD of LB maintained above 60 and 65% in 2007 and 2008, respectively and that of ST almost above 70% both years. The IVDMD in Italian ryegrass maintained above 70% both years. The IVDMD in dwarf napiergrass did not respond clearly to the increase in DEM application rate, except for LB at the first cutting in 2007, while positive responses were obtained among treatments at the first cutting both years in Italian ryegrass. Differences in IVDMD between H and C plots were small at all cuttings for both species both years (Fig. 2).
The NDF concentration maintained above 60% for both dwarf napiergrass and Italian ryegrass and ADF concentration maintained below 41 and 40% for dwarf napiergrass and Italian ryegrass, respectively (Table 3). The increase in DEM application rate lead to no consistent effect on NDF or ADF concentration for either species, while ADL concentration tended to decrease with the increase in DEM application rate for both species (Table 3).
The IVDMD was related with fiber concentrations in LB and ST of dwarf napiergrass and in whole plants of Italian ryegrass (Table 4). In LB of dwarf napiergrass, there were negative and positive correlations of IVDMD with NDF and ADL concentration, respectively, contrary to the positive and negative correlations of IVDMD with NDF and ADL concentration, respectively, in whole plants of Italian ryegrass. Among fiber concentrations, positive correlations or tendencies of ADF concentration with NDF and ADL concentrations were obtained in both species (Table 4).

Discussion
Napiergrass exhibits the best performance in deep and fertile soils with moderate to fairly heavy texture based on its origin (James, 1983) and is tolerant to the wide climatic range of annual precipitation in 2,000-4,000 mm and annual temperature in 13.6-23.7°C (Duke, 1978), which are just fit to the climatic conditions in southern Kyushu, Japan. Cultivation of napiergrass at the border between subtropical and temperate zones such as southern Kyushu, is not so popular among the world, while the grass is available for its cultivation only limited to slightly frosted areas because aboveground herbage is easily suffered to be dead from light frost and underground stem parts remain alive if the soil is not frozen (Burton, 1989). The present study revealed that the wintering ability in dwarf napiergrass was almost perfect under the favorably fertilized H and C plots in Miyazaki both years, as well as in our previous research . The increase in DEM application rate was beneficial for improving wintering ability in dwarf napiergrass, probably due to the increase in the density of stubble tillers, which had underground tiller buds, being potential for spring regrowth (Ishii et al., 1995). Thus, dwarf napiergrass can be grown as perennial cutand-carry herbages under 3 or 4 cuttings per year fertilized at around 20 g N m −2 year −1 .
Napiergrass can be intercropped with herbaceous and tree leguminous crops to increase total yield and nutritive value (Mureithi et al., 1995). In the present study, Italian ryegrass as temperate winter crop has an opposite growing season to tropical species of napiergrass. The combination of dwarf napiergrass with Italian ryegrass cropping proved to be satisfactory in the region with succeeding cropping years that annual total yield increased from 486.2 (L level) -936.9 (H level) g DM m −2 in the 2007-8 season to 795.6 (L level) -1612.2 (H level) g DM m −2 in the 2008-9 season by 64-72% as shown in Table 1. The increase in annual total yields from the year established to the following growing season in this cropping system was brought about by the increasing yielding ability due to the faster spring regrowth of dwarf napiergrass and more stable establishment of Italian ryegrass in the following year. It is a common feature that annual total yields of napiergrass increased from the year of establishment to the following year when the spring regrowth started from the overwintered stubbles (Ishii et al., 1995;Sunusi et al., 1999;Wadi et al., 2004) as well as other perennial tropical grasses, if pasture management was suitable for the grass species. As for the establishment of Italian ryegrass, the way of DEM application was changed from the first to the second year; in the first year, DEM was applied twice just at pre-and post-sowing of Italian ryegrass, which may hinder the emergence of over-sown Italian ryegrass to produce poorer establishment, resulting in lower dry matter yield. However, in the second year, the first application of DEM was carried out one month after the sowing of Italian ryegrass to improve the establishment of the species without disturbing the early growth by DEM solution.
In accordance with the increase in dry matter yield from the first to the second year in dwarf napiergrass, CP concentration increased in a range of 9.3-10.2% in the first year to 10.7-12.2% in the second year as shown in Table 1. The IVDMD in both dwarf napiergrass and Italian ryegrass increased from the first to the second growing season especially in the first-cut plants (Fig. 2), which were composed of principal yielding harvest in the annual yields for both grasses (Table 1). Even though negative correlations were obtained of dry matter yield with CP concentration in both grasses and that with IVDMD in dwarf napiergrass across whole cuttings and treatments (Table 2), dry matter yield was positively correlated with both CP concentration and IVDMD across DEM-applied plots at the same cutting date. Thy and Buntha (2005) examined in Chinese cabbage that both fresh biomass yield and CP concentration increased with the increase in application rate of bio-digester effluent up to 15 g N m −2 . Both crop nutrient requirements and soil properties are crucial factors to judge the proper agronomic practice of DEM as a fertilizer. Application of DEM to soil surface was proved to improve soil chemical properties without major contamination to the environments of arable lands (Hasyim et al., 2014). Several studies in Thailand (Rengsirikul et al., 2011) and Florida, USA (Woodard and Prine, 1993) noted that the recommended application rate at 20-30 g N m −2 are crucial to sustain high yield and quality in napiergrass.
Chemical compositions, particularly IVDMD and CP concentration, become good indices for feeding herbage to dairy and beef cattle. In the present study, IVDMD maintained above 60 and 70% in dwarf napiergrass and Italian ryegrass, respectively and stem inclusive of leaf sheath had always higher IVDMD than the leaf blade in dwarf napiergrass. As the minimum CP requirement for livestock production is 6-8% for maintenance (Minson, 1990), annual average of CP concentration in dwarf napiergrass, ranging in 9.3-10.2 and 10.7-12.2% across DEM-supplied plots in 2007 and 2008, respectively, maintained satisfactory level for feeding to cattle under the examined DEM levels. The situation was the similar to CP concentration in DEM-supplied Italian ryegrass ranging in 9.2-9.7 and 6. 8-8.8% in 2008 and 2009, respectively. Thus, based on IVDMD and CP concentration, sustainable herbage-cropping system of dwarf napiergrass with Italian ryegrass can produce satisfactorily quality herbage to feed cattle.
In general, the level of CP concentration of herbages is affected by direct and indirect factors with the application of fertilizer including DEM. The former direct factor appears rapidly in forages after the supply of inorganic fertilizer (Moss, 2009), as well as liquid DEM which contained mainly inorganic elements, in accordance with the change of leaf color to become dense green. The latter indirect factor is mainly attributable to dilution of CP concentration by the rapid accumulation of cell wall carbohydrates at the latter stages of growth (Van Soest, 1994), which appeared typically in the first-cut Italian ryegrass in 2009 due to the heavy accumulation of biomass ( Table 1). One of the other indirect factors is the change of leaf percentage with advance in growth stage. It is a frequent phenomenon in napiergrass that CP concentration is higher in the early growth stage than in the late and senescent growth stage, as reported in Africa (Seyoum et al., 1998;Tesema et al., 2002). In the temperate southern Kyushu, regrowth rate in dwarf napiergrass was suppressed by the decline in temperature and solar radiation as well as in Australia (Russel and Webb, 1976) after the first and second defoliation in 2007 and 2008, respectively. The optimal temperature for napiergrass growth is above 25°C up to 40°C and its growth ceases when temperature falls below 10°C (Bogdan, 1977). Under the sub-optimal temperature below 25°C, stem elongation of napiergrass is suppressed severely, causing the higher leaf percentage to lead to the increase in CP concentration, which happened in dwarf napiergrass after August both years. The similar situation of sub-optimal temperature and termination of spring vigorous growth in the second-cut Italian ryegrass proved to increase CP concentration to the large extent, compared with the first-cut plants ( Table 1).
The compositions of fiber fraction can be classified into NDF, which is a measure of the amount of structural carbohydrates, including digestible (hemicellulose), less digestible (cellulose) and indigestible (lignin) components and ADF, which is a measure of less digestible and indigestible components. In napiergrass, ADF and NDF concentrations are reported at 36.6 and 63.4%, respectively (NARO, 2001), which are almost corresponded with the current average of ADF concentration at 38 and 40% and NDF concentration at 62 and 63% in 2007 and 2008, respectively (Table 3). Fiber fractions, such as NDF, ADF and lignin concentrations, are negatively correlated with dry matter digestibility of forages in general (Minson, 1990). However, in the present study, positive correlations were obtained of IVDMD with ADF and ADL concentrations in leaf blade of napiergrass and of IVDMD with NDF concentration in whole plants of Italian ryegrass (Table  4). In leaf blade of napiergrass, IVDMD tended to increase seasonally from the first to the last defoliation both years (Fig. 2), which were concurrent with the increase in ADL and ADF concentrations and the decrease in NDF concentration across all treatments both years (Table 3). The seasonal diverse trend of NDF and ADL concentrations in leaf blade of dwarf napiergrass may be derived from the reduction of tiller size with time. The first-cut plant was mainly composed of primary tillers, which was emerged from the underground stem and had large size of leaf, while the third-or fourth-cut plants were composed of large number of daughter tillers with smaller leaf size. The difference between NDF and ADF concentration can be estimated as hemicellulose concentration, which tended to decrease seasonally from the first to the last defoliation, suggesting poor cell wall components accumulated in the third-or fourth-cut plants. In Italian ryegrass, IVDMD increased from the first to the second defoliation across all treatments both years (Fig. 2), which was associated with the slight increase in NDF and ADF concentrations and the great decrease in ADL concentration with time (Table 3). The seasonal diverse trend of NDF and ADL concentration in Italian ryegrass may be caused by the reduction of heading tiller size with time. The first-cut plants were composed of large heading tillers, possessing of high lignin concentration to support stem-hardened heading tiller, while the secondcut plants had a smaller size of heading tiller possessing poor accumulation of non-structural carbohydrates, resulting in the slight increase of NDF and ADF concentrations with time.
Rate of DEM application will be determined by several agronomic and economic factors. In the present study, DEM solution was produced by a biogas-plant, built in Faculty of Agriculture, University of Miyazaki and was transported to the experiment field by the plastic tank. Therefore, authors cannot estimate costs of processing DEM by biogas-plant and transporting DEM to the field, but can do the fertilizing effect on dwarf napiergrass and Italian ryegrass in the present cropping system. This standpoint has some sort of reasonable aspect that biogas-plant is used to build at the region where the density of livestock is relatively higher than the area of fields and suitable manure treatment is highly required without influencing on the environments neighboring to the field. Thus, it is worthwhile to omit the cost of purchasing DEM. From the present study on herbage yield and quality in dwarf napiergrass with Italian ryegrass, combining with the former study in the effect of DEM on soil and soil water environments and plant production (Hasyim et al., 2014), herbage yield and quality of dwarf napiergrass and Italian ryegrass increased in accordance with the increase in plant persistence of dwarf napiergrass with increasing DEM application rate up to 35 g N m −2 yr −1 , without influencing on the major changes in the environments.

Conclusion
The application of DEM to dwarf napiergrass with Italian ryegrass cropping system had a comparable effect with chemical fertilization on herbage yield and quality in both grasses and DEM had a positively linear effect on enhancing dry matter production within the examined level of 20 and 15 g N m −2 for summer and winter crop, respectively. Plant regrowth after defoliation was almost perfect both in the growing and post-wintering seasons for 2 years. Thus, the present cropping system can be maintained for several years only by the over-sowing of Italian ryegrass just after the final defoliation of dwarf napiergrass under the sufficient use of DEM to achieve sustainable quality herbage production system in southern Kyushu, Japan.