**2. Importance of phosphorus for symbiotic nitrogen fixation in legumes**

Phosphorus is the most limiting nutrient for the production of crops especially the nitrogen fixing leguminous crops for adequate growth and nodulation. There are many reports about its direct and positive effect on nodulation of red clover, peas, white clover, *Lupinus* L., soybeans and many more [11–15]. Indirect effect on plant growth has also been reported, thereby increasing the nodulation and stimulating the nitrogenase activity [32]. Reduced root growth and photosynthetic carbohydrate supply to the nodules occurs under P deficient conditions [6, 16] which results in reduced nodule growth and function and ultimately reduced symbiotic N2 fixation [17]. It has been found that N and P control the nodule growth and modulate the symbiotic processes of the legume and Rhizobium [11, 33]. Various morpholog‐ ical and physiological changes occur under P deficient conditions including increase in the root/shoot ratio, changes in root architecture [4, 34], development of root hairs [35], induction of the high-affinity phosphate transporter [36], and synthesis and exudation of the organic acids, phosphatases and ribonucleases (RNases) [37–39]. It has been reported that crop yield is seriously affected under P limited conditions especially during early stage of growth [40].

Phosphorus (P), being the second most important macronutrient after nitrogen, has a critical role in biological nitrogen fixation (BNF). It has been a renowned fact that legumes require more phosphorus (P) compared to non-leguminous crops as they perform the process of BNF through nodulation which is a characteristic property of legumes. Nodulation occurs in almost all the legumes. However, physiology and efficiency of nodules to conduct the process of nitrogen fixation is species specific. So for the process of BNF, P serves as an ultimate source of energy in the form of ATP [5, 41, 42]. It is also needed for signal transduction, membrane biosynthesis, and nodule development and function [8]. Moreover, nodules under P-sufficient conditions have a higher P concentration (up to 1.5% of the total plant P) as compared to shoots and roots [6].

There are many reports about the influence of P on nodulation; ultimately the amount of N fixed by the plants e.g. nitrogen contents of the legume, *Crotalaria micans*, were increased about 4 folds due to increased nitrogen fixation with the application of P at 90 kg ha−1 [43]. Similarly Israel [15], studied the effect of P on accumulation of nitrogen in soybean (*Glycine max* L.). It was found that the concentration of nitrogen was increased with increasing the supply of P that was suggested to be due to increase in the symbiotic nitrogen fixation as P serves as an energy source. Plant dry matter, nodule number, and mass was also increased with increasing P supply. Enzyme activity of nitrogenase was also enhanced with increasing P. Earlier legumes are well renowned as P exhaustive crop plants due to the formation of nodules for symbiotic nitrogen fixation. In another experiment on different legumes (soybean, clover), similar effect of P on growth and nodulation parameters was recorded [44].

Another soil and sand culture experiment to study the interaction of N and P and their effect on growth, nodulation, nitrogen fixation, activity of nitrate reductase, and on the accumulation of nitrogenous compounds (ureides, amino acids, nitrate) in the xylem sap of common bean was conducted [3]. Both the soil and sand culture experiment showed that with increasing levels of N, nodulation parameters such as nodule number and mass, nitrogenase activity, and xylem ureides were decreased, while the concentration of asparagine in the xylem sap increases. Symbiotic nitrogen fixation was only increased at low N concentration with increasing P application. Similarly it was also found that the effect of N on the inhibition of nodulation including nodule number and biomass was systemic, while high dose of P had a systemic stimulatory effect on nodulation parameters as mentioned above. The systemic effect was confirmed by its direct effect on nodulation and not on the plant growth overall. There is still lack of information on whether there is effect of N and P on both the nodulation and nitrogen fixation or not and needs to be explored in future studies [32].

Similarly, another Leonard jar experiment was conducted to study the impact of P (0–2 mM P) on growth, symbiotic nitrogen fixation, N and C metabolism, as well as on the concentration of ATP, N and P contents of common bean (*Phaseolus vulgaris*). With the application of medium to high-P, not only the nodulation (nodule biomass: 4-fold) and growth parameters but also the P contents of the harvested plants were increased at the onset of the flowering. In the case of total soluble sugar and amino acid contents of leaf, root, and nodules of the plant, these were decreased with increasing the level of P application. Moreover, an increase of 20-fold in nodule-ARA and 70-fold in ARA per plant was observed with the application of 1.5 mM P [45]. Another split root experiment found that P has a specific effect on the nodulation not generally on the overall growth of the plant. More P was required in the earlier stage of nodule initiation and growth of the plant. It was also found that P could suppress the effect of N on the inhibition of symbiotic nitrogen fixation [32]. Many other researchers have reported the effect of P on nodulation, growth, and nitrogen fixation in many crops like clover, soybean, red clover, etc. [11, 15, 44, 46].
