br Conclusions br Conflicts of interest br

Conclusions

Conflicts of interest

Acknowledgements
This work was partially funded by [email protected] (PON a3_00016) – PON Ricerca e competitività 2007–2013. G. Benelli is supported by PROAPI (PRAF 2015) and University of Pisa, Department of Agriculture, Food and Environment (Grant ID: COFIN2015_22). Funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction
Alfalfa (Medicago sativa L.) is a Fabaceae perennial herb and is an important legume used for forage worldwide. Additionally, it is an important source of nectar and pollen for honeybees in several locations around the world, including Al-Ahsa, Saudi Arabia (Taha, 2015a,b). It is a cross-pollinated plant, and pollination occurs with the help of insects, wind, and other external elements. The honeybees Apis mellifera L. and A. florea F. were found to be the most active pollinators in alfalfa abiraterone acetate (Taha et al., 2016). A number of wild pollinators also visit the flowers and participate in pollination (Cane and Schiffhauer, 2003; Hayter and Cresswell, 2006; Cecen et al., 2008; Taha et al., 2016). Different insect pollinators have been shown to vary in how effectively they deposit and remove pollen from individual flowers (Thomson and Goodell, 2001). For example, the tripping rate varies between bee species visiting alfalfa racemes (Cane, 2002), and distinct species deposit different quantities of pollen on cranberry flowers during a single visit to a flower (Cane and Schiffhauer, 2003). Such differences in tripping rates and pollen deposition can be influenced by whether a pollinator forages for pollen or for nectar (Breazeale et al., 2008), and these differences have been shown to influence fruit and seed set (Thomson and Goodell, 2001; Pinheiro et al., 2014).
An insufficient number of suitable pollinators causes a reduction in fruit and seed production (Taha and Bayoumi, 2009; Maiti and Maiti, 2011). Of the total pollination activities, insects represent more than 80% and honeybees represent nearly 80% of the total insect pollinators (Robinson and Morse, 1989; Taha and Bayoumi, 2009; Taha et al., 2016). Alfalfa’s floral structure facilitates cross-pollination. The rate of cross-pollination depends on insect activity, environmental conditions and the availability of other vegetation (Kumar and Lenin, 2000; Breazeale et al., 2008).
Improvements in artificial pollination increase the number of fruits in each branch. In cases where this plant cannot be pollinated, seed production is decreased substantially (Bolanos et al., 2000; Taha and Bayoumi, 2009; Bomfim et al., 2015). In Saudi Arabia, seed production can be achieved using the honeybee (A. mellifera L.) as managed pollinators (Taha et al., 2016). The flowers require bee visits for pollination, and when a bee opens the keel petals, the enclosed stamen and pistil snap forward, forcefully striking the bee (Cecen et al., 2008).
Alfalfa removes large quantities of nutrients from the soil. Phosphorus has been the nutrient needed in the largest quantities for alfalfa production. Low phosphorous (P) availability reduces the yield and persistence of this perennial plant (Berg et al., 2005). Phosphate nutrition interacts with the carbohydrate supply and influences carbohydrate partitioning in plants including alfalfa (Li et al., 1998). The availability of P also influences protein accumulation and utilization in leaves (Rufty et al., 1993) and roots (Li et al., 1998). Phosphorus also enhances the symbiotic nitrogen (N) fixation process in legume crops (Cihacek, 1993). Phosphorus is an essential ingredient for Rhizobium bacteria to convert atmospheric N (N2) into an ammonium (NH4) form useable by plants. Rhizobium are able to synthesize the enzyme nitrogenase that catalyzes the conversion of N2 to two molecules of ammonia (NH3) (James et al., 1995). Phosphorus becomes involved as an energy source when 16 molecules of adenosine tri-phosphate (ATP) are converted to adenosine diphosphate (ADP) as each molecule of N2 is reduced to NH3 (Carroll, 2001; Erdal et al., 2008). The ATP is generated during the process of photosynthesis, when light energy is transformed and stored in the form of ATP for later use by the plant. Phosphorus influences nodule development through its basic functions in plants as an energy source. Inadequate P restricts root growth, the process of photosynthesis, translocation of sugars, and other such functions that directly or indirectly influence N fixation by legume plants (Berg et al., 2005).