**9. Authentication and evaluation of** *Rhizobium*

Authentication is the process by which we can ascertain that the isolates are *Rhizobium* or not through its infection capability under gonotobiotic conditions. Authentication of rhizobia to determine their symbiotic efficiency is required to screen out effective native rhizobial isolates [47]. In order to achieve maximum legume productivity, screening of native isolates for their N2-fixation efficiencies [48] is important for the development of effective legume inoculum. Nodulation ability and effectiveness of native rhizobia from the seven districts of Uttarakhand in French bean was determined by Yadav et al. [29] and reported that out of fifty isolates 36 were authenticated as *Rhizobium* based on their ability to nodulate French bean. Morphological assessment and effectiveness of indigenous rhizobial isolates nodulating *Phaseolus vulgaris* in water hyacinth compost testing field in Lake Victoria basin was studied by Muthini et al. [49] and reported that the isolates obtained in his study had the ability to renodulate (Infectiveness) *Phaseolus vulgaris* under bacteriologically controlled conditions. Bala et al. [50], who reported that appropriate rhizobial isolates nodulate and fix N2 on the target host and that each

*Characterization of* Rhizobium *and Plant Growth Promoting Rhizobacteria from French Bean… DOI: http://dx.doi.org/10.5772/intechopen.100592*

isolate was able to form nodules with the host plant was identified as *Rhizobium*. High degree of symbiotic efficiency of the specific indigenous strain S21/6 was recorded indicating different symbiotic potential of indigenous strains and confirmed the importance of rhizobial strain selection [51].

Evaluation or screening of the authenticated isolates can be done on the basis of plant dry matter response (effectiveness), nodule number and nodule dry weight of the inoculated isolates. Isolation, authentication and evaluation of rhizobial isolates from the soils of North-West Himalayas in French Bean (*Phaseolus vulgaris* L.) studied by Yadav et al. [29] and reported that French bean inoculation with the rhizobial isolates significantly increased the dry shoot, root and total biomass; and shoot: root ratio. The inoculation of French bean with rhizobial isolate RA6 produced total dry biomass significantly higher by 154.4 per cent over reference strain i.e. MTCC 10096. This results demonstrated the presence of native rhizobia in soils of N-W Himalayas capable of nodulating French bean that are either superior or at par with reference strain in improving the overall growth and synthesis of higher biomass in French bean. Muthini et al. [49] reported that nodulated plants had higher shoot dry weight, than the non nodulated plants, however, the mean shoot dry weights was not directly related to the nodule number or nodule dry weight.

#### **10.** *Rhizobium* **inoculation, N-levels and interaction impact**

#### **10.1 Rhizobium**

Grain legumes have been recognized worldwide as an alternative means of improving soil fertility through their ability to fix atmospheric nitrogen, increasing soil organic matter and improving soil structure [52]. Several studies have sought to identify efficient and competitive strains of rhizobia to cope the nitrogen requirements of common bean [53]. Deshwal et al. [54] observed that in addition to BNF rhizobia can promote plant growth by different direct or indirect mechanisms such as production of IAA, GA, solubilization of inorganic phosphates and biocontrol of plant diseases. Beneficial effects of rhizobium on French bean have been reported by various workers under different climatic and soil conditions [55, 56]. Ndlovu [57] reported that nodulation was significantly affected by inoculation with *Rhizobium phaseoli*. Nodule dry biomass plant−1 was significantly increased by approximately 51.11 per cent with inoculation compared to uninoculated treatment in 2012–2013. Several workers reported that inoculation of legumes with *Rhizobium* isolates improved nodulation and had a positive effect on a number of plant growth parameters [58]. Das [59] also reported that higher number of nodules plant−1 was observed in inoculated plants than the un-inoculated. This might be due to application or introduction of inoculants that increased number of the *Rhizobium* bacteria which infect the roots to form nodules. The higher number of bacteria resulted in higher number of vigorous nodules plant−1. The number and size of nodules indicated the amount of plant tissue available for nitrogen fixation. Thus, the results of this study also suggested a good symbiotic association between *Rhizobium phaseoli* and the host French bean. The presence of nodules in uninoculated treatments during both seasons might be due to the result of existing indigenous *Rhizobium* present in the soil. The increase in dry biomass of nodules which was formed by the inoculation with *Rhizobium phaseoli* might be results of efficiency of the strain.

There were significant differences in shoot, root and total dry biomass of *P. vulgaris* inoculated with variable rhizobial isolates. The significant differences in the shoot dry biomass showed clear differences in the ability of the isolates to

fix nitrogen and are among the preferred methods for determining symbiotic effectiveness of rhizobial isolates [3]. According to Meena et al. [60], application of *Rhizobium* significantly increased the plant height, germination, number of branches plant−1, number of leaves, leaf length and leaf width. The improvement in plant height and dry biomass production at flowering as well as at harvest might be due to the plant growth promoting capabilities, carbohydrates utilization abilities and improved nodulation. The differences in plant growth due to rhizobial inoculation have been attributed to changes in assimilate partitioning [58]. The higher dry shoot biomass of common bean by *Rhizobium* inoculation seems to be due to the supply of N to the crop through symbiotic N2-fixation [61].

Yadegari and Rahmani [62] recorded the positive effect of inoculation with rhizobial strains Rb-133 and Rb-136 on plant growth. Rhizobial strain increased the seed yield, number of pods plant−1, number of seeds pod−1, weight of 100 seeds, seed protein yield, total dry matter over uninoculated control plants. During two years of study they registered the seed yield in inoculated plants ranging from 1221 to 4693 kg ha−1 depending on the strain and cultivars. Inoculation with suitable strains of *Rhizobium* has been recognized as prerequisite for increasing the yield and quality of legumes [63].

Inoculation of seeds by *Rhizobium* sp*.* prior to planting has also been reported to be a key factor in enhancing nodulation, early emergence, crop vigor and high grain yield [64, 65]. Bambara and Ndakidemi [66] also reported high common been seed yield of 1679 kg ha−1 with inoculation compared to 758 kg ha−1 from the uninoculated control. The inoculation of seeds with *Rhizobium* increased nodulation, protein and chlorophyll content, nitrogen uptake, growth and yield parameters of legume crops [67]. In Iran, Namvar et al. [67] reported that *Rhizobium* inoculated plants showed more chlorophyll content and LAI than uninoculated plants. *Rhizobium* inoculation increased chlorophyll content and LAI by 5.43 and 6.99 per cent, respectively as compared to uninoculated plants. In West Bengal, varietal performance of bush type French bean (*Phaseolus vulgaris* L.) for growth, fresh pod yield and quality was studied by Das [59]. Their results revealed that *Rhizobium* inoculation increased the yield and quality parameters *viz*; protein content, vitamin-A content and ascorbic acid content in the fresh pods of the French bean varieties. Under *Rhizobium* inoculation special jhhati beans recorded higher pod yield (23.05 t ha−1) over uninoculated control (20.05 t ha−1). Meena et al. [68] observed and reported, among the six different biofertilizers the best biofertilizer B3 (*Rhizobium*) is recorded significantly improvement in various yield and quality traits. Higher yields obtained with inoculation confirm that the *Rhizobium* technology is efficient in supplying nitrogen to legumes and is a better option for resourcepoor farmers who cannot afford to purchase expensive inputs as well as potential strategy to nullify the adverse impact of chemical fertilizer on environment.

Number of pods, number of grains, grain yield, protein content and protein yield in French bean was influenced significantly due to inoculation with rhizobial isolates over uninoculated control [69]. This positive effect due to inoculation with rhizobial isolates attributed to nodulation and nitrogen fixing capability which enhances the nitrogen supplement to plants, resulted higher vegetative growth and carbohydrate portioning and more protein formation. Phosphorus solubilizing capability of rhizobial isolates helps to solubilize inorganic fixed phosphate and make it available for plant uptake. Higher availability of phosphorus improves N2- fixation, root proliferation which results higher uptake of nutrient from soil and phosphorus also acts synergistically with nitrogen and helps in carbohydrate translocation and protein synthesis. An increase in number of pods plant−1, number of grains pod−1 and pod yield due to *Rhizobium* inoculation might be due to more availability of nitrogen inside the plant bodies [59]. Koskey et al. [30] reported that inoculation of climbing beans in the field

*Characterization of* Rhizobium *and Plant Growth Promoting Rhizobacteria from French Bean… DOI: http://dx.doi.org/10.5772/intechopen.100592*

and green house significantly enhanced nodule and shoot dry biomass, number of pods plant−1, seed yields and nitrogen content in shoot of MAC 13 and MAC 64 climbing beans. Ali (1998) also reported the same results and he concluded that inoculation increased pod yield plant−1. With respect to *Rhizobium* inoculation treatment, higher pod yield was recorded in *Rhizobium* inoculation (20.73 t ha−1) compared to without *Rhizobium* inoculated plants (17.68 t ha−1). Increase in total pod yield is due to more nitrogen availability in the inoculated plants. The higher values of protein content are estimated in *Rhizobium* inoculated plants (2.09%) in comparison to without *Rhizobium* inoculation (1.44%). Increase in protein content due to *Rhizobium* inoculation might be the result of increased nitrogen content inside the plant body which is main element for protein synthesis.

#### **10.2 Nitrogen levels**

Nitrogen is an essential nutrient for plant growth and development. Nitrogen deficiency is frequently a major limiting factor for crop production all over the world [70, 71]. Therefore, adequate supply of nitrogen is necessary to achieve high yield potential in plants which usually depend upon combined or fixed form of N such as NH4 + and NO3 − because it is unavailable in its most prevalent form as atmospheric N. The sparse nodulation in French bean needs more amount of nitrogen for growth and development in comparison to other legumes.

In French bean all the plant growth parameters except nodule number and nodule biomass were significantly improved with higher level of nitrogen application (from 0 to 120 kg N ha−1), reported in several studies (**Table 1**). This improvement is attributed to the high vegetative growth and higher formation of photosynthates. Nitrogen plays an important role in the formation of protein and nucleic acids structure, the most important building material for every cell. In addition to it nitrogen is also a component of chlorophyll that enables the plant to capture energy from sunlight thorough photosynthesis. Thus, nitrogen supply to a plant increased the concentration of protein, amino acids, protoplasm and chlorophyll which influenced cell size, leaf area and photosynthetic activity [91, 92]. Increased level of nitrogen application in French bean resulted in increased plant height [81]. The negative effect of N fertilizer on French bean nodulation is well documented [23]. However, farmers have gradually adopted the use of N fertilizers with French bean crops, to maximize yields, particularly when irrigation is used. According to Yadav [69] number of trifoliate leaves, leaf area and chlorophyll content of French bean was significantly increased with higher levels of nitrogen (from 0 to 120 kg N ha−1), the reason behind that N is chief constituent of amino acids which is the building unit of protein, protoplasm leading to improved vegetative growth. Nitrogen being constituent of chlorophyll higher dose of nitrogen increases chlorophyll concentration resulted in more photosynthesis and enhanced number of trifoliate leaves and leaf area which was reported by various workers (**Table 1**). N fertilization upto 120 kg N ha−1 in French bean increased number of pods plant−1 [77–79, 85]. Nitrogen supply affects a wide range of physiological processes in higher plants [87].

#### **10.3** *Rhizobium* **x N-level interaction**

An inoculation of rhizobial isolates in combinations with different levels of nitrogen significantly improved the various plant growth parameters in French bean as compared to uninoculated control [61, 69, 90, 93, 94]. Growth, symbiotic and yield response of N-fertilized and *Rhizobium* inoculated common bean (*Phaseolus vulgaris* L.) was conducted by Yoseph and Shanko [94] at Hawassa University, Ethiopia and reported that N fertilization and *Rhizobium* inoculation had significant effect on


#### **Table 1.**

*List of plant growth, physiological and quality parameters of French bean reported by various workers.*

plant height, dry shoot weight, nodule number plant−1, nodule dry weight plant−1, number of pods plant−1, number of seeds pod−1 and grain yield. Omoregie and Okpefa [95] observed that when initial levels of available soil nitrogen were low, a period of nitrogen hunger can reduce nodulation. Kucuk [96] studied the effect of *Rhizobium* inoculation either alone or in combination with nitrogen applications on French bean (*Phaseolus vulgaris* L.) and reported that plant heights were significantly affected by the control, nitrogen, inoculation and different treatment x variety interactions. Interaction effect between nitrogen and rhizobial isolates significantly improved plant height, number of trifoliate leaves, dry biomass production, and leaf area, chlorophyll content, nitrogen status, grain yield, protein content and protein yield. This could be attributed to additional external supply of nitrogen for vegetative growths of plants and to be used by microbes to fulfill its requirement as starter in initiation of nitrogen fixation. Combined application of rhizobia and nitrogen may be beneficial for improvement of root proliferation and plant growth due to plant growth promoting ability of rhizobial isolates which need more nitrogen for their metabolic activity, might be fulfilled through external supply. Generally, inoculation with *Rhizobium* at all levels of nitrogen application increased biomass production over uninoculated plants [61, 67, 97]. Sajid et al. [98] concluded that the *Rhizobium* inoculation produced higher grain yield than without inoculation. It might also be due to more number of pods and seeds due to *Rhizobium* inoculation and applied N.
