**1. Introduction**

In the present intensive agriculture practices leguminous plants play a critical role in natural ecosystem, agriculture and agroforestry because of their ability to fix nitrogen (N2) in symbiotic relationship with *Rhizobium* and *Bradyrhizobium*. In addition to its role as a source of protein in the diet, biologically fixed N2 is essentially free for use in economic terms by the host plant and by associated subsequent crops. This association improves the soil quality vis-à-vis sustainability. Among the legumes, French bean (*Phaseolus vulgaris* L.) is used profusely by the common people as an alternative diet of protein. It is very nutritious and contains 22.9 per cent protein, 1.2 per cent fat, 60.6 per cent carbohydrates and a large number of minerals like Ca (260 mg 100 gm−1 of seed) P (101 mg 100 g−1 of seed) and Fe

(5.8 mg 100 g−1 of seed). French bean is sparsely nodulated throughout the India including North-West Himalayas putting it to disadvantage of biologically fixed-N2 [1, 2] and thus responds to the enhanced levels of nitrogen [3]. In India, it is grown on an area of about 1 lakh hectare (ha) mainly in the states of Maharashtra (60,000 ha), Jammu and Kashmir (10,000 ha), Himachal Pradesh, Uttarakhand, Nilgiri (Tamil Nadu), Palni (Kerala) hills, Chickmagalur (Karnataka) and Darjeeling hills (West Bengal). The sparse nodulation in French bean mainly may be due to lack of threshold level of specific rhizobial cells in soil at the time of sowing. Recently different rhizobial strains have demonstrated various other plant growth promoting activities in addition to biological nitrogen fixation (BNF). This necessitates the isolation and development of the efficient multi-trait rhizobial isolates for French bean for economizing the nitrogen fertilizer, environmental safety and sustainable production.

*Rhizobium* plays a significant role in agricultural ecosystem services due to their ability to form symbiotic association with a wide range of leguminous plants that results in biological nitrogen fixation. Some of the rhizobial strains are reported to enhance the production of phytohormones, mineral uptake and reduce toxic effects of metals, thereby, indirectly promote growth and development of plant in polluted agricultural soils [4]. According to Tsai et al. [5] most commonly used French bean variety exhibit a high dependence on nitrogen fertilizers for growth and yield, and show considerable variation in their ability to nodulate and fix nitrogen, with the nitrogen percentage derived from atmosphere ranging from 68 to 72 per cent for the superior variety. This indicates that French bean needs starter nitrogen fertilization for sufficient nitrogen fixation. Inoculation of French bean with *Rhizobium* increases various plant growths, physiological, quality parameters and grain yield through symbiotic N2-fixation capacity and plant growth promoting abilities. Plant growth promoting rhizobacteria is the beneficial rhizobacteria inoculation of which increases growth and yield of common bean through different direct and indirect mechanisms such as production of IAA, GA, HCN, Ammonia, siderophore and solubilization of phosphorus, potassium and zinc. Co-inoculation of *Rhizobium* with other PGPR; consortium enhanced the growth and grain yield of common bean [6]. By virtue of their rapid colonization of the rhizosphere and stimulation of plant growth, there is currently considerable interest in exploiting such microorganisms for enhanced crop yield. Therefore to harness the benefits of rhizobia and PGPRs in reducing the application of higher doses of inorganic fertilizers; the development of consortium comprising efficient multi-trait rhizobial isolates and efficient PGPRs are need of an hour for sustainable production of French bean. This chapter reviews the research reports relevant on the topic including (i) isolation and characterization of various isolates of French bean rhizobia (ii) authentication and evaluation of the efficacy of rhizobial isolates (iii) evaluation of the efficacy of potential multitrait rhizobial isolates on growth and yield of French bean in relation to N dose (iv) isolation and characterization of PGPRs from the rhizosphere and the impact of various isolates of PGPR on growth of French bean (v) compatibility between selected rhizobial isolates and PGPRs; and development of consortium, and (vi) the interaction effect of efficient multi-trait *Rhizobium* and PGPR on French bean.

#### **2. French bean and its use**

Gramineae and Leguminosae are two major source of world's food supply, total 15 plant species of which account for more than 90 per cent of the total production of the major seed crops [7]. According to Harlan [8], three major cereal crops such as wheat (*Triticuma estivum* L.), maize (*Zea mays* L.) and rice (*Oryza sativa* L.) account

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

for three quarters of the total food supply. The grains of these cereals provide carbohydrates for human and are complemented by the legumes [9] which vary in their carbohydrate and oil content but have high protein content [7]. In addition to important source of food, feed and fuel legumes are also a renewable source of nitrogen through atmospheric N2-fixation for agriculture [10]. Incorporation of legume crops in field improves soil fertility and yield sustainability. Among the legumes, French bean (*Phaseolus vulgaris* L.), of American origin, is the most edible pulse in the world and is second only to the soybean (*Glycine max* L.) [11]. According to Broughton et al. [12], french bean (*Phaseolus vulgaris* L.) has been reported as an important legume for human nutrition and a major protein and calorie source in the world. It is cultivated in the sub-Himalayan and higher Himalayan altitudes between 1200 and 1800 m. In India, French bean covers an area of 2.3 mha with production of 1.1 million tonnes and productivity of 478 kg ha−1 [13]. French bean is popular among Indian farmers due to its high lucrative features such as short life cycle, good adaptability, high market value and the most important for poor farmers, particularly women, hence it is also known as woman's crop. French bean is a self-pollinating leguminous crop which belongs to the family Fabaceae and considered as an important crop in high population density areas of the world [14]. French bean is used both as a pulse and as a green vegetable [15]. In both the developed and the developing countries, French bean is consumed in different forms [16]. Seeds can be consumed as immature green grain. Dehulled seeds may be boiled, parched, roasted, germinated, fermented or cooked in different ways to suit specific tastes. In some parts of the tropics, the young leaves are used like spinach. Common bean seeds are also cooked with tomato sauce and canned. The residual straw can be used as fodder and forage [16] as well as to incorporate in the soils to improve the soil health.

### **3. Adaptation**

French beans are well adapted to tropics, subtropics, and warm temperate regions, grown from 40°S to 40°N latitude. French bean completes their life cycle within 80 to 110 days which depends on variety and night temperatures during the growing season. Suitable temperature for growth of French bean varies between 20 and 22°C. The maximum temperature during flowering of French bean must be under 28°C. It requires a minimum of 500 to 600 mm of rain during the growing season if the crop is cultivated under rainfed conditions whereas an annual total of 600 to 700 mm is considered ideal. They are planted in warm soils with minimum temperatures preferably above 15°C after all danger of frost has passed. Soil texture such as sandy loam, sandy clay loam or clay loam with good drainage and clay content between 15 and 35 per cent is supposed to be best for cultivation of this crop. Soil pH of 6.0 to 6.5 is considered to be the best for the cultivation of French bean.

### **4. Biological nitrogen fixation**

Biological N2-fixation is a biological phenomenon, which involves some legumes, whether grown as pulses for seed or as pasture in agro-forestry or in natural ecosystems [17]. Biological nitrogen fixation is very efficient in satisfying the high nitrogen requirements of legumes because of the conversion of gaseous nitrogen (N2) to ammonia (NH3) making it available to plant use. Enzyme nitrogenase facilitated the process of BNF. Many N2-fixing prokaryotes are diazotrophic, *i.e.* they can grow using dinitrogen gas as their sole source of N while other organisms can fix N2 only in symbiosis with another eukaryotic organism. The equation for the reaction is.

$$\text{HN} \equiv \text{N} + 8\text{H}^{+} + 8\text{e}^{-} + 16\text{ATP} \rightarrow 2\text{NH}\_{3} + \text{H}\_{2} + 16\text{ADP} + 16\text{Pi} \tag{1}$$

Two protons are reduced by hydrogen for fixation of one molecule of dinitrogen and because of high stability of dinitrogen the reaction needs high energy [18]. Dupont et al. [19] reported that soon after germination of legume seeds, rhizobia present in the soil or added as seed inoculum invade the root hairs and move through an infection thread to the root. The bacteria multiply rapidly in the root, causing the swelling of root cells to form nodules. Nitrogen in the air of soil pores around the nodules is fixed by binding it to other elements and thus changing it into a plant available form. Some of the carbohydrates manufactured by the plant photosynthesis process are transported to the nodules where they are used as a source of energy by the rhizobia. The rhizobia also use some of the carbohydrates as a source of hydrogen in the conversion of atmospheric nitrogen to ammonia. Despite BNF being a naturally occurring process, many soils do not harbor sufficient numbers of appropriate rhizobia for effective symbioses. Inoculation of leguminous crop with appropriate and compatible rhizobia ensures maximum BNF. Inoculation is generally needed when certain new leguminous crops are introduced to new areas.

### **5.** *Rhizobium*

Rhizobiaceae family is a physiologically heterogeneous and genetically diverse group of soil organisms, which are called rhizobia [20]. Rhizobia include a group of soil bacterial genera viz.; *Rhizobium*, *Bradyrhizobium*, *Sinorhizobium*, *Mesorhizobium*, *Allorhizobium* and *Azorhizobium* which have ability to nodulate symbiotically the members of the plant under Leguminosae family [21, 22]. *Rhizobium*-legume associations are very specific therefore the nodules will be formed in the legume only when infected with a specific *Rhizobium* [23]. According to Broughton et al. [12] specificity involves the recognition of the bacterium by the host and of the host by the bacterium through the exchange of signal compounds which induce differential gene expression in both partners. The bacteria which are able to form root nodules in French bean have been classified into five species of the genus *Rhizobium*, *R. leguminosarum* biovar (bv.) *phaseoli* [24], *R. tropici* [25], *R. etli* bv. *Phaseoli*, *R. gallicum* bvs.*Gallicum* and *phaseoli* and *R. giardinii* bvs.*Giardinii* and *phaseoli.* From various studies, it has been observed that *Phaseolus* rhizobia are very diverse at the species, intra-species and population levels. According to Aguilar et al*.* [26], current evidence refers difficulty to recognize factors which involved in the distribution of the different rhizobial species among sites, although there is increasing evidence in the literature of parallel evolution between bacteria and the French bean.

#### **6. Nitrogen fixation by** *Rhizobium*

French beans have low ability to fix nitrogen symbiotically and surprisingly larger rates of N2-fixation can be obtained under appropriate conditions [27]. In the light of the poor nodulation in French bean, in general in India, it is feasible that under these situations BNF technologies can become extremely important in order to reduce the use of chemical nitrogenous fertilizers, improve the soil health and enhance the yield levels. Hence, inoculation with the effective rhizobial inoculum presents a great potential for increasing food production in N-W Himalayas and other parts of the French bean growing area. The number of nodules in the plant decreases with the

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

higher rates of soil N application at planting. In leguminous plants, Nitrogen fixation is a symbiotic process between nitrogen fixing bacteria and legume roots, and occurs within specialized root nodules. Hungria et al. [28] observed an adverse effect on leguminous root nodule development at low temperature stress.

## **7. Morphological and biochemical characteristics of rhizobia**

Morphological characteristics of rhizobia refers to external appearance of rhizobia *viz*; shape, size and color; and biochemical characteristics refers to different characteristics which are produced by rhizobia through their chemical and microbial activities such as acid or alkali production, CRYEMA test, GPA test, carbohydrate utilization, enzymatic activity and plant growth promoting traits. Yadav et al. [29] studied 50 rhizobial isolates separated from the nodules of French bean (*Phaseolus vulgaris* L.) were tested and exhibited typical characteristics of *Rhizobium* sp. on yeast extract mannitol agar media supplemented with Congo red. In Kenya, genetic characterization and diversity of *Rhizobium* isolated from root nodules of climbing bean (*Phaseolus vulgaris* L.) varieties were studied by Koskey et al. [30] and they found that none isolates absorb Congo red dye when incubated in the dark on CRYEMA medium found Gram +ve rods. All isolates were found to be acid producers and fast growers by turning BTB indicator from deep green to yellow when grown on YEMABTB. Most of the isolates showed a mucoid texture because of the exopolysaccharides production. In the study of biochemical characterization of French bean associated rhizobia, Rai and Sen [31] observed and reported that colonies of *Rhizobium* were circular, convex, semi-translucent, raised, single and mucilaginous in nature. According to Vincent [27] and Holt et al. [32] the colonies were large (2–4 mm in diameter) mucilaginous, circular, convex with smooth edges, glistening translucent or white and precipitated calcium glycerophosphate present in YEM agar. *Rhizobium* test in Congo red showed that the colonies did not absorb the congo red color which differentiates *Rhizobium* from *Agrobacterium* [33]. According to Deka and Azad [34] *Rhizobium* cannot grow in Hoffer's medium, however; in contrast Rai and Sen [31] studied and observed that few of the isolates like S-3, CBR and K-1 showed mild growth. The growth of *Rhizobium* in the Hoffer's medium was also observed by Dubey et al. [1]. Deshwal and Chaubey [33] observed no yellow zone around the colonies of *Rhizobium* and such negative ketolactase activity confirmed the isolates to be free from any contamination of *Agrobacterium*.

The isolates changed to yellow color showed the production of acid which is the characteristic of *Rhizobium* [35]. Similarly, isolates of *Rhizobium leguminosarum* bv *trifolii* associated with clover showed growth and turned the yeast extract mannitol agar media containing BTB to yellow color indicated all were fast growers and acid producers. It was reported that the utilization of glucose as a carbon source is a confirmatory test for *Rhizobium* [35]*.* Utilization of different carbon sources is an efficient tool to characterize the isolates [36]. Only four isolates obtained in the study were able to use dextrin as a carbon source, which is in accordance with other works indicating that dextrin is rarely utilized by *Rhizobium* [24, 31]. The utilization of majority of carbon and sodium organic salt sources by *Rhizobium* has also been reported [37].

In the glucose-peptone agar medium, growth of the *Rhizobium* has been observed by [1]. Hunter et al. [38] observed the negative gelatinase activity which is a feature of *Rhizobium.* Yellow slants and red butt were obtained showing the utilization of glucose and sucrose in the triple sugar iron agar medium [35]. De Oliveira et al. [39] also observed that *Rhizobium* strains obtained from different sources can utilize starch. Rhizobial isolates may not grow on lactose [35]. As the pH becomes

high, color of the media changes from yellow to pink which indicates the production of ammonia because of urease enzyme secretion by the incubated isolates which is a positive reaction for the test [40]. Gauri et al. [37] observed that all isolates of rhizobia showed a positive test for urease.

Biochemical characterization and protein profile by sds-page of French bean (*Phaseolus vulgaris* L.) associated rhizobia conducted by Kumari et al. [41] in Andhra Pradesh. They isolated total of six isolates. All the rhizobial isolates were positive to the indole acetic acid, nitrate reduction, urease, catalase and oxidase. Some of the isolates did not produced H2S and consumed citrate as a sole source of carbon. Positive results were found from the starch hydrolysis assay. On subjecting inoculated plates to iodine test, clear zones from place to place were observed and the colonies changed to yellow color, however blue color appeared on no growth areas. It designates the isolates have the potential to hydrolyze starch present in the medium.

## **8. Morphological and biochemical characteristics of PGPR**

The plant growth promoting rhizobacteria must be defined by some important attributes such as (a) an efficient tool to colonize the root surface (b) to survive, multiply and compete with other microorganisms and (c) to promote plant growth [42]. Anitha and Kumudini [43] reported that the isolates of fluorescent *Pseudomonas* on King's B agar produced creamy, convex colonies having 1–2 mm diameter and at 265 ηm appeared yellowish-green fluorescence. Microscopic studies showed that the isolates were gram negative and rod shaped. These isolates were found to utilize sucrose, mannitol and lactose to varying extent and showed that the bacterial isolates were positive only for catalase, oxidase, organic acids, citrate, amylase, indole and caseinase. According to Battu and Reddy [44] gram negative and rod shaped colonies, produced yellowish green pigment on King's B medium were positive for gelatinase and oxidase which were identified as *Pseudomonas flurescence*. Rodríguez-Cáceres [45] determined morphology and motility for each isolate and also performed biochemical tests such as nitrate reductase and urea hydrolysis [46]. Qualitative analysis showed that all bacterial isolates produced IAA, ammonia, siderophore and hydrogen cyanide.
