Comparison of forage production and nutritive value of 10 Grona spp. accessions in Danzhou, Hainan, China Producción y valor nutritivo de 10 accesiones de Grona spp. en Danzhou, Hainan, China

The demand for high-quality forages is increasing in tropical regions, and could be filled with legume species of the genus Grona, which have good nutritive value. In this study, a comparison of the forage production and nutritive value of 10 accessions of Grona spp. was carried out in the field at Danzhou, Hainan from 2016 to 2018. Yield, plant height, survival rate, leaf:stem ratio and concentrations of crude protein, crude fiber, crude fat (ether extract), nitrogen free extract, crude ash, calcium and phosphorus were measured. Results showed that Grona strigillosa (syn. Desmodium strigillosum) cv. Reyan No. 27 and G. heterocarpa subsp. ovalifolia (syn. Desmodium ovalifolium) cv. Maquenque displayed the best performance, owing to their 261.3% and 235.6% higher dry matter yields, respectively, compared with the Control germplasm, G. heterocarpa subsp. ovalifolia cv. Reyan No. 16 in 2018. Cultivar Maquenque had a higher survival rate than the Control (P<0.05). Regarding nutritive value, cv. Reyan No. 27 exhibited higher crude fat and crude fiber but lower Ca concentrations than the Control (P<0.05). Based on PCA ranking, we concluded that cvv. Maquenque and Reyan No. 27 could be used as suitable candidate materials for livestock production in tropical regions of China. Further studies on their tannin concentrations and their acceptability by animals are needed before practical recommendations can be made.


Introduction
The demand for high-quality animal products in developing countries is increasing year by year with the improvement of living standards and consumption changes (Lee 2018). However, the development of livestock production is usually limited by insufficient high quality forage supply. For example, in the tropical region of Hainan province of China, the main forages are king grass [Cenchrus purpureus × C. americanus (syn. Pennisetum purpureum × P. glaucum)] and Stylosanthes spp., which are not adapted to all edaphic conditions and production systems, resulting in livestock production below its potential (Zi et al. 2018). Therefore, it is crucial to invest in developing and utilizing new forage resources with high production and nutritive value in tropical regions (Kambashi et al. 2014).
Tropical forage legumes have the potential to contribute significantly to sustainable intensification of livestock production (Schultze-Kraft et al. 2018). Based on our prior evaluation of nutritional concentrations (Chen et al. 2010;Liu et al. 2014), an important and well known legume genus is Desmodium, from which the genus Grona was recently separated, based on morphological, palynological and molecular data (Ohashi and Ohashi 2018). Grona comprises currently 21 species and subspecies recognized by GRIN, the taxonomic database of the USDA Genetic Resources Information Network (npgsweb.ars-grin.gov/gringlobal/ taxon/taxonomysearch), among them some species well known by the tropical forages plant research community, such as G. barbata, G. heterocarpa subsp. ovalifolia, G. heterophylla, G. strigillosa and G. triflora.
The Chinese Academy of Tropical Agricultural Sciences (CATAS) introduced more than 800 Desmodium and Grona accessions to China from the International Center for Tropical Agriculture (CIAT ovalifolia is a creeping stoloniferous herb or subshrub, with height ranging from 0.1 to 0.5 (occasionally 1.0) m, which grows well in tropical and subtropical climates, is adapted to infertile acid soils and drought-tolerant (Cook et al. 2020). G. strigillosa has been less researched so far. It is a semi-erect subshrub growing up to about 0.5 m, morphologically similar to G. heterocarpa subsp. heterocarpa (Figure 1). The species has shown promise on acid, low-fertility soils in Colombia and southwestern Nigeria (Thomas and Schultze-Kraft 1990;Larbi et al. 2000). Previous studies have shown that Grona spp. present average tannin concentrations at a relatively moderate level of about 2.09% (Li et al. 2013), but reduction of methanol-extractable condensed tannin concentration in leaves of G. heterocarpa subsp. ovalifolia (syn. D. ovalifolium) by the addition of polyethylene glycol resulted in increased intake and nitrogen retention by sheep (Carulla et al. 2001). Plants of Grona spp. can be used as protein supplements with roughage for ruminants, increasing the value of feed and reducing production costs.
Production and nutritive value of tropical forage legumes can vary widely in different areas with different climatic conditions and soil types. G. heterocarpa subsp. ovalifolia (syn. D. ovalifolium) performed well on a low-phosphorus, acid soil in southern Ethiopia, producing dry matter (DM) yields of 2,977 kg/ha and 2,652 kg/ha in 1989 and 1990, respectively (Larbi et al. 1995). Based on forage production and quality, G. strigillosa (CIAT 13155), along with Centrosema arenarium (CIAT 5236), were considered the most promising species for the development of silvopastoral systems in the west African forest-savanna transition zone and in similar tropical environments (Larbi et al. 2000).
However, little information is available about the relative performance of various Grona spp. accessions in tropical China, both in terms of forage production and nutritive value. To meet the demand for high-quality animal products in China and improve livestock production, it is essential to increase the quantity and quality of forage available, which can be achieved through selecting and breeding the accessions with the highest production and nutritive value. Therefore, the objective of this study was to examine the performance of 9 accessions of G. heterocarpa subsp. ovalifolia (syn. D. ovalifolium) and 1 accession of G. strigillosa in terms of yield and nutritive value, in the tropical region of China, over 3 years.

Site characteristics
The experimental site was located east of Nada, Danzhou, PR China in the province of Hainan (19°30′ N, 109°30′ E; 149 masl). It has a tropical monsoon climate characterized by hot and rainy summers (May-October), and cold, dry winters and springs (November-April). Mean annual rainfall (2010-2018) is 2,153 mm. Mean monthly maximum and minimum temperatures range from 22 to 35 °C and from 14 to 25 °C, respectively. The soil type is a latosol formed on granite, with characteristics of the top 40 cm as follows: pH 4.5-5.5; alkaline hydrolyzable nitrogen 85-100 mg/kg; organic matter 10-12 g/kg; available P 8.5-12.5 mg/kg detected by the hydrochloric acid-ammonium fluoride method (LY/T1233-1999/5); and available K 50-65 mg/kg.

Experimental materials
Ten Grona spp. accessions, including cvv. Reyan No. 16, Reyan No. 27 and Maquenque, were used in this experiment, all of which were obtained from CIAT (Table 1).

Experimental design and management
A single factor randomized block design was used with 3 replications, for a total plot number of 30 (10 accessions × 3 replications). Plot size was 26 m 2 (6.5 × 4 m), and plant spacing was 0.5 × 0.5 m. Guard rows were set up around the experimental site. Seedlings were propagated in a greenhouse by planting seeds on 4 January 2016 and were transplanted into the plots on 25 April 2016. No fertilizer was applied during the experiment, and weeds were removed manually every 2 weeks.

Measurements
The first harvest was carried out 3 months after transplanting in July 2016, leaving a stubble height of 20 cm, and forage yields from the whole plots were recorded subsequently every 4 months until 2018. Fresh material was weighed immediately in the field and random samples of about 1,000 g were collected from each plot and oven-dried at 70 °C until constant weight for determining DM yields.
Before each harvest, 10 plants were selected at random from each plot and height was measured, using a rule to measure from the ground to the highest leaves of the plant. After the final harvest in 2018, all surviving plants were counted, and survival rate was calculated as the ratio of the number of plants remaining and the number transplanted into the plots in April 2016 expressed as a percentage.
Leaf:stem ratio was determined during the first harvest each year, i.e. 25 July 2016, 27 February 2017 and 25 February 2018, respectively. Five to 10 plants (total fresh weight about 1,000 g) were collected at random from each plot and separated into leaf and stem before oven-drying at 70 °C for calculation of leaf:stem ratio.
Samples for determining nutrient composition were collected before the first harvest in 2017 and crude protein (CP), crude fat (ether extract, EE), crude fiber (CF), nitrogen free extract (NFE), crude ash (ash), calcium (Ca) and phosphorus (P) concentrations were determined following the protocols of Owens et al. (2010).

Data analysis
All data were subjected to analysis of variance (SAS 8.1; SAS Institute Inc., Cary, NC). Treatment means were separated using the least significant difference test at P=0.05.

Comprehensive evaluation of production and nutritive value using PCA
Comprehensive evaluation of the 10 accessions was performed on the data collected using Principal Component Analysis (PCA), and ranking values for each accession were calculated with the formula: , where: x is the contribution of PCi; and n is the number of PCs considered for the ranking (in this case the first 4) (Liu et al. 2015).

Survival rate
Survival rates varied markedly from 13 to 93% with cv. Maquenque having a significantly higher survival rate than the Control cv. Reyan No. 16, while CIAT 13083 had a significantly lower survival rate than all other accessions (P<0.05).

Nutrient composition
Significant differences were observed in CF, EE, ash, NFE, Ca and P concentrations among the 10 accessions (Table 4)

Discussion
Forage quality is determined mainly by DM yield and nutrient composition (Kambashi et al. 2014 accessions of Grona is obviously a reflection of their genetic differences. Our study showed that yields and nutritional composition differed significantly between the 10 accessions tested, which suggests that there are genetic differences between the accessions. Plant height had a marked impact on DM yields of the 10 Grona spp. accessions. The DM yield decrease from 2016 to 2018 may be a reflection of declining survival rates as some plants from all accessions died during the study. CIAT 13083 had the lowest DM yield in 2018 (568 kg/ha) and had the lowest survival rate (13%). In contrast cv. Maquenque had an excellent survival rate of 93% and equal highest DM yield in 2018 (8,956 kg/ha). The differences between accessions in terms of survival rates were possibly a function of the regular 4-monthly harvest times and accessions with higher survival rates may have higher tolerance of regular harvesting (Mukangango et al. 2020).
The fact that G. strigillosa cv. Reyan No. 27 was the tallest accession at 63.1 cm reflects the growth form of this species (semi-erect subshrub vs. the prostrate growth habit of G. heterocarpa subsp. ovalifolia). The somewhat shrubby habit of G. strigillosa was also reflected in the lowest leaf:stem ratio, although differences were not significant.
Interestingly CIAT 13108 had a leaf:stem ratio of 0.98:1, but CP% of forage was not different from that of the other accessions. Despite differences in leaf:stem ratio from 0.70:1 to 0.98:1, no significant differences were detected and no differences in CP concentration.
Crude protein, crude fat and crude fiber are usually used as important indicators of the nutritive value of forages (Lauriault and Kirksey 2004). In this study, average CP, EE and CF concentrations were 12.6, 2.47 and 31.2% for cv. Maquenque and 12.9, 2.81 and 36.1% for cv. Reyan No. 27, respectively. When compared with average CP and ADF concentrations (10.5 and 39.7%, respectively) of Stylosanthes guianensis reported by Li et al. (2014), the Grona spp. accessions tested have a relatively good nutritive value, especially with respect to CP concentration. Our results also showed that G. strigillosa cv. Reyan No. 27 had significantly lower Ca concentration than Control G. heterocarpa subsp. ovalifolia cv. Reyan No. 16.

Conclusions
Based on this comprehensive evaluation of 10 Grona spp. accessions, it appears that G. heterocarpa subsp. ovalifolia cv. Maquenque and G. strigillosa cv. Reyan No. 27 were the most promising accessions in this environment, displaying good DM yields, high CP concentration and good survival rates under regular harvesting. They are possible candidate materials for improving livestock production in tropical regions of China, but further evaluation, e.g. feeding experiments, should be conducted to test acceptability to animals and performance of livestock, especially as forage from Grona spp. can contain high levels of tannins.