Growth stimulation of clusterbean (Cyamopsis tetragonoloba) by ...

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Stimulation of root and shoot growth of clusterbean seedlings on water agar plates was observed by ..... J. Chem. Ecol., 25: 2397-2406. Barea, J.M., Navarro, E. and Montoya, E. (1976). .... Rao, A.V., Venkatesarlu, B. and Henry, A. (1984).
Legume Research, 39 (6) 2016 : 1003-1012 Print ISSN:0250-5371 / Online ISSN:0976-0571

AGRICULTURAL RESEARCH COMMUNICATION CENTRE

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Growth stimulation of clusterbean (Cyamopsis tetragonoloba) by coinoculation with rhizosphere bacteria and Rhizobium Sita Ram Chaudhary and Satyavir Singh Sindhu* Department of Microbiology, CCS Haryana Agricultural University, Hisar-1250 04, India. Received: 13-04-2015 Accepted: 12-08-2015

DOI:10.18805/lr.v0iOF.8605

ABSTRACT Clusterbean [Cyamopsis tetragonoloba (L.) Taub.] is an important commercially utilizable crop grown in arid zone of India. Microorganisms present in the rhizosphere of this crop produce various plant growth-promoting substances and enhance the availability of nutrients to the plants. Therefore, fifty five bacterial isolates obtained from the rhizosphere of clusterbean were explored for beneficial characteristics. Twenty rhizobacterial isolates produced indole acetic acid ranging from 3.9 to 24.7 µg/mL. Only six isolates HCS7, HCS19, HFS7, HFS9, HFS10 and HFS12 showed d-aminolevulinic acid production varying from 1.3 to 7.0 µg/mL. Fourteen isolates showed solubilization of potassium on mica containing Aleksandrov medium plates. Stimulation of root and shoot growth of clusterbean seedlings on water agar plates was observed by inoculation of eleven rhizobacterial isolates at 5 and 10 days of growth whereas some isolates showed stunting effect on the growth of shoot and root as compared to uninoculated seedlings. At 60 days of plant growth, inoculation of Bradyrhizobium strain GSA11 and Rhizobium strain GSA110 showed significant nodulation and their inoculation resulted in 141.94 and 151.43% gains in shoot dry weight, respectively under chillum jar conditions. Coinoculation of Bacillus isolate HCS43 with Rhizobium strain GSA110 formed 48 nodules/plant and plant dry weight was enhanced by 190.09% in comparison to uninoculated control plants. Key words: Aminolevulinic acid, Clusterbean, Indole acetic acid, Nodulation, Plant growth, Potassium solubilization, Rhizosphere bacteria. INTRODUCTION Microorganisms and plants are in dynamic equilibrium in the soil and microorganisms of the rhizosphere interact with other microorganisms and plants (Pii et al., 2015). These interactions could be beneficial, neutral or with detrimental effects (Walia et al., 2014). Some microbial populations in the rhizosphere termed as plant growthgrowth promoting rhizobacteria (PGPR) benefit the plant in a variety of ways, including: (i) increased recycling, solubilization and uptake of mineral nutrients such as nitrogen, phosphorus and potassium (Basak and Biswas, 2008; Sindhu et al., 2009; Parmar and Sindhu, 2013), (ii) synthesis of auxins, vitamins, amino acids and gibberlins (Lugtenberg and Kamilova, 2009; Jangu and Sindhu, 2011) and (iii) antagonism with potential plant pathogens by production of antibiotics, siderophores, hydrocyanic acid and/or hydrolytic enzymes (Weller, 2007; Dua and Sindhu, 2012). These different soil microorganisms possessing beneficial characteristics are used as bioinoculants to improve productivity of various crops in sustainable agriculture (Welbaum et al., 2004; Compant et al., 2010). In this context, inoculation of the nitrogen-fixing microorganisms and other PGPR offer an ecofriendly alternative to minimize the use of nitrogenous fertilizers *Corresponding author’s e-mail: [email protected] .

for crop plants and resulting in improved crop production (Burris and Roberts, 1993; Franche et al., 2009; Sindhu et al., 2010). The hypothesis of this study is that synergistic interactions of the rhizosphere bacteria with Rhizobium/ Bradyrhizobium may result in growth-promoting effects leading to improved legume productivity. In earlier studies, promotion of plant growth after inoculation with rhizobacteria has been attributed to indole acetic acid (IAA) production in Rhizobium species (Hirsch and Fang, 1994) and Pseudomonas (Xie et al., 1996; Malik and Sindhu, 2011). Similarly, coinoculation of legumes with Rhizobium and IAA-producers such as Azospirillum brasilense, Bacillus and Pseudomonas have been found to increase the number of nodules, nodule fresh weight and nitrogenase activity in comparison to Rhizobium/Bradyrhizobium-inoculated plants (Yahalom et al., 1990). Inoculation with bacteria having the ability to produce ä-aminolevulinic acid (ALA) and potassium solubilization also resulted in growth improvement of different plants (Hotta et al., 1997; Hu et al., 2006; Basak and Biswas, 2010; Liu et al., 2014). Similarly, coinoculation of different PGPR strains with Rhizobium/Bradyrhizobium spp. was found to improve nodulation, root and shoot weight, plant vigor and grain yield in various legumes (Li and

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Alexander, 1988; Dashti et al., 1998; Sindhu et al., 2002; Basak and Biswas, 2010; Malik and Sindhu, 2011). Clusterbean [Cyamopsis tetragonoloba (L.) Taub.], commonly known as guar, is a drought tolerant crop and is grown during the summer season (April-June) in the northern arid zone of India. Clusterbean is a rich source of high quality galactomannan gum which is in great demand in the world market because of it multi-purpose use in textiles, foods, cosmetics, mining, explosives and oil industries. However, its nodulation status is poor with only 5 to 10 nodules per plant (Stafford and Lewis, 1980; Rao et al., 1984). Despite its multipurpose use, little systematic work has been done to improve the nodulation and nitrogen-fixing ability of this crop for increasing its production (Elsheikh and Ibrahim, 1999). Thus, it is desired that efficient rhizosphere bacteria should be characterized and introduced in clusterbean growing areas to improve its nodulation status, seed quality and crop productivity. MATERIALS AND METHODS Isolation of bacteria from rhizosphere soil: Rhizosphere soil samples were collected from seven different field locations of clusterbean grown in CCS Haryana Agricultural University, Hisar farm at 45 and 60 days of plant growth. From each location, samples were collected from six different sites and the samples were pooled to make the composite sample of representative location. The serial dilutions of the composite rhizosphere soil samples (up to 10-5) were plated on King’s B agar medium (Sindhu et al.,1999). Pseudomonas and Bacillus colonies were selected based on morphological and pigment production characteristics after 3 days of incubation at 28±2°C. Fifty five selected bacterial colonies were further purified by streaking on Luria Bertani (LB) medium plates and isolated single colonies were transferred on LB medium slopes. Liquid cultures of all the isolates were preserved in 50% glycerol at -20°C. Screening of bacterial isolates for indole acetic acid (IAA) production: Rhizobacterial isolates were tested for their ability to produce indole acetic acid using Salwoski’s reagent (Gordon and Weber, 1951). Bacterial cultures from LB slopes were transferred into tubes containing 5 ml LB broth supplemented with L- tryptophan (100 µg/mL) and were incubated at 28±2°C for 5 days. The broth was centrifuged for 5 min at 10,000 rpm. Two ml of Salkowski reagent was added to two ml of culture supernatant, mixed and allowed to stand for 30 min for the development of pink colour. Absorbance was estimated at 500 nm wavelength using spectrophotometer (UV-Vis spectrophotometer 117, Systronics, Ahemadabad, India) against a reagent blank. Indole acetic acid (100 µg/mL) was used as standard and results were expressed as µg IAA produced per ml of culture supernatant. Uninoculated broth served as control. Production of d-aminolevulinic acid (ALA): Bacterial isolates were tested for their ability to produce d-

aminolevulinic acid by the method as described by Mauzerall and Garnick (1955). Cultures were inoculated in duplicate in 10 ml LB broth supplemented with 15 mM glycine and succinate, and were incubated at 30ºC for 48 hrs under stationary conditions of growth. Culture samples were withdrawn and centrifuged at 10,000 rpm for 15 min (Remi Instruments, Mumbai, India). To 0.5 ml of culture supernatant, 50 µl of acetylacetone and 0.5 ml of 1M sodium acetate buffer were added and then tubes were boiled in a water bath for 15 min. After cooling, 3.5 ml of modified Ehrlich reagent were added. The absorbance of the mixture was measured at 556 nm wavelength after 20 min at room temperature. The concentration of ALA in the culture supernatant was determined by comparison with standard curve. Screening of rhizobacterial isolates for potassium solubilization: Potassium solubilization by rhizobacterial isolates was studied on modified Aleksandrov medium plates by the spot test method (Parmar and Sindhu, 2013). The modified Aleksandrov medium (containing 5.0 g Glucose, 0.5 g MgSO4.7H2O, 0.1 g CaCO3, 0.006 g FeCl 3, 2.0 g Ca 3PO4, 3.0 g insoluble mica powder as potassium source and 20.0 g agar in 1 litre of deionized water) was prepared. A loopful of 48-hour old growth of rhizobacterial isolate was spotted on above prepared plates. Ten bacterial cultures were spotted on each plate and plates were incubated at 28±2°C for 3 days. Detection of potassium solubilization by different rhizobacterial isolates was based upon the ability of solubilization zone formation. Screening of rhizobacterial isolates for root and shoot growth of clusterbean: Clusterbean seeds were surface sterilized with acidic alcohol (concentrated sulphuric acid: ethanol, 7:3) for 3 min and washed thoroughly with several changes of sterilized water. The surface sterilized seeds were inoculated with selected Pseudomonas/Bacillus cultures individually (Khandelwal and Sindhu, 2012). Growth of each bacterial culture was harvested in 3 ml sterilized water and 15 seeds were soaked in bacterial growth suspension for 45 minutes. Inoculated seeds were placed on plain water agar (0.8%) plates in triplicate (five seeds on each plate) and plates were incubated at 28±2°C. Seed germination of clusterbean started on 3rd day on water agar plates. The observations for elongation/retardation of root and shoot growth of seedlings were recorded at 5 and 10 days of growth. Coinoculation of rhizobacterial isolates with Bradyrhizobium/Rhizobium for nodulation and plant growth: Selected three rhizobacterial isolates i.e., Pseudomonas isolate HCS36 and Bacillus isolates HCS5 and HCS43 were used for coinoculation studies with most symbiotically effective Bradyrhizobium strain GSA11 or Rhizobium strain GSA110 (Khandelwal and Sindhu, 2012) in clusterbean variety HG563 under sterilized chillum jar conditions (Dahiya and Khurana, 1981). Chillum jar

Volume 39 Issue 6 (2016) assemblies contained washed river sand in the upper jar and Sloger’s nitrogen-free mineral salt solution (Sloger, 1969) in the lower assembly. Surface sterilized seeds of clusterbean [Cyamopsis tetragonoloba (L.) Taub.] were inoculated with 10 ml of culture (obtained from mixing of 5 ml growth of each bacteria) and having 10 7 - 108 cells/ml of growth suspension. The inoculated seeds were sown in chillum jars (with three replications for each treatment). Uninoculated seeds were sown as control. Quarter strength Sloger’s nitrogen-free mineral salt solution was used for watering as and when required. The observations for nodulation and plant dry weight were recorded at 30 and 60 days of plant growth. Statistical analysis: Completely randomized design (CRD) was used for experimental data analysis. All determinations were carried out in triplicate and data represented are average values of three replications. SEM (standard error of means; ±) values were calculated to determine the significant differences between treatment means. C.D. values represent coefficient of deviation. RESULTS AND DISCUSSION Previous studies have established that some rhizobacterial strains such as Azotobacter, Azospirillum, Arthrobacter, Bacillus, Clostridium, Enterobacter, Pseudomonas, Rhizobium, Bradyrhizobium and Serratia could promote the growth of plants and can be used as biofertilizer. These PGPR improve the plant growth through multitudinous factors viz. production of plant growth promoting substances and hormones (Ahmad et al., 2008; Malik and Sindhu, 2011; Liu et al., 2014), early colonization of root surfaces (Benziri et al., 2001), reducing ethylene level due to synthesis of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Glick, 2014; Chaudhary and Sindhu, 2015), enhancing the availability of nutrients (Sivaramaiah et al., 2007; Sindhu et al., 2010) and by synthesis of antibiotic and other pathogen-depressing substances such as siderophores, cyanide and hydrolytic enzymes (Voisard et al., 1989; Sindhu and Dadarwal, 2001) and stimulation of phytoalexins/flavonoid-like compounds in roots (Goel et al., 2001). The present study was planned with the objective to evaluate the plant growth promotion ability of different rhizobacterial isolates under chillum jar conditions. Fifty five bacterial isolates representing Pseudomonas and Bacillus were selected from rhizosphere soil at 45 and 60 days of clusterbean growth. Based on salient biochemical tests i.e., oxidase test, indole production, catalase utilization, pigment production and Gram negative staining, isolate HCS36 was characterized as Pseudomonas sp. (Palleroni, 1984; Sindhu et al., 1999). Rhizobacterial isolates HCS5 and HCS43 were identified as Bacillus species on the basis of rough, nonmucoid and whitish colonies which showed Gram positive staining and the big rod-shaped cells were found to form spores. Bacterial counts in the rhizosphere soil, collected from clusterbean grown fields,

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ranged from 1.7 to 18.4 x 105 colony forming units (CFU)/g soil at 45 days of plant growth. At 60 days of plant growth, bacterial counts increased and ranged from 19.2 to 76.8 x 105 CFU/g soil. Similarly, Baig et al. (2002) isolated 105 bacteria from rhizosphere and rhizoplane of groundnut. Out of these, 67% isolates were from the rhizosphere and 33% were from the rhizoplane. Pseudomonas was found as the most predominant (42%) followed by Bacillus (28%) and Enterobacter (21%). Screening of 563 bacteria isolated from the roots of pea, lentil and chickpea for plant growth promotion ability and for the suppression of fungal pathogens showed that 76% isolates produced siderophore, 5% isolates showed ACC deaminase activity and 7% isolates were capable of indole production (Hynes et al., 2008). IAA and ALA production by different rhizobacterial isolates: Production of phytohormones has been a dominant mechanism of plant growth promotion by rhizobacteria. Selected rhizobacterial isolates were tested for production of IAA and ALA at 4 days of growth. Twenty rhizobacterial isolates produced IAA ranging from 3.9 to 24.7 µg/mL (Table 1). Two isolates HCS7 and HFS9 produced 24.7 and 23.5 µg/mL of IAA, respectively. ALA production by different rhizobacterial isolates was comparatively less and HCS7, HCS19, HFS7, HFS9, HFS10 and HFS12 showed ALA production varying from 1.3 to 7.0 µg/mL (Table 1). Three rhizobacterial isolates i.e., HCS7, HCS19 and HFS9 produced both IAA as well as ALA. Rhizobacterial isolates Table 1: Production of IAA and ALA by different rhizobacterial isolates Rhizobacterial isolates HCS2 HCS3 HCS7 HCS10 HCS12 HCS14 HCS17 HCS19 HCS22 HCS24 HCS25 HCS26 HCS30 HCS33 HCS34 HCS36 HCS37 HCS39 HCS41 HFS7 HFS9 HFS10 HFS12

IAA production (µg/mL) 9.0 15.9 24.7 22.3 17.7 9.0 9.6 10.2 12.9 22.6 10.8 14.7 3.9 18.3 8.4 19.0 6.9 14.1 19.6 23.5 -

ALA production (µg/mL) 6.5 2.8 1.3 3.8 1.8 7.0

Other 32 rhizobacterial isolates did not show production of IAA and ALA.

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HCS16, HCS36 and HCS40 showed significant growth on ACC supplemented plate, indicating that these bacteria possess the enzyme ACC deaminase (data not shown). Five isolates HCS2, HCS12, HCS30, HCS36 and HCS39 which showed antagonistic activity against fungal phytopathogen R. solani (data not shown) also showed production of IAA. Less production of IAA was observed in rhizobacterial isolates HCS2 and HCS36, which showed more stunting effect on sh oot a nd root gr owt h in clust erbea n. Rhizobacterial isolates HCS12, HCS30 and HCS39, which showed antagonistic activity and promoted the growth of shoot and root in clusterbean, showed moderate production of IAA. Earlier studies have demonstrated that different bacterial strains produce IAA in varying amounts (Keyeo et al., 2011). Barea et al. (1976) reported that among 50 bacterial isolates obtained from the rhizosphere of various plants, 86, 58 and 90% isolates produced auxins, gibberellins and kinetin-like substances, respectively. The production of phytohormones has also been reported in other PGPR strains including Azotobacter chroococcum (Muller et al., 1989), Azospirillum spp. (Remans et al., 2008), Pseudomonas fluorescens (Dubeikovsky et al., 1993) and P. putida (Taghavi et al., 2009). Barazani and Friedman (1999) reported that high levels of IAA (76.6 µm) were excreted by four deleterious rhizobacteria (Micrococcus luteus, Streptoverticillium sp., Pseudomonas putida a nd Gluconobacter sp.) and lower amounts of IAA (16.4 µm) were secreted by plant growth promoting rhizobacterial isolates including Agrobacterium sp., Alcaligens piechaudii and Comamonas acidovorans. Similarly, Hotta et al. (1997) reported that low concentration of ALA (0.01-10 mg/L promoted the plant growth whereas, ALA suppressed the plant growth at higher concentration (> 2 mM). Hyun and Song (2007) showed that Rhodopseudomonas strain BL6 produced low amount of ALA whereas another strain KL9 produced 8.57 mg/L after 48 hours of incubation. Screening of rhizobacterial isolates for potassium solubilization: All the fifty five bacterial isolates were screened for their potassium solubilization ability on modified Aleksandrov medium plates using spot test method (Parmar and Sindhu, 2013). Potassium solubilization was scored after 4 days depending on the ability of bacteria to form zone of solubilization. Only 14 isolates formed significant zone of K solubilization on mica containing medium (Table 2; Figure 1). Five cultures i.e., HCS2, HCS7, HCS36, HCS41 and HCS42 showed more than 2.0 mm solubilization zone. Four isolates HCS44, HFS6, HFS11 and HFS12 showed only 1.5 mm zone of solubilisation. Majority of the isolates did not cause K solubilization on mica containing plates. Similar variation in K solubilization ability has been reported by other workers. Badr (2006) reported that K

Table 2: Formation of potassium solubilization zone by different rhizobacterial isolates Rhizobacterial isolates HCS16, HCS21, HFS5, HFS9, HFS10 HCS44, HFS6, HFS11, HFS12 HCS41 HCS36, HCS42 HCS2, HCS7 HCS1, HCS3, HCS4, HCS5, HCS6, HCS9, HCS10, HCS11, HCS12, HCS13, HCS14, HCS15, HCS17, HCS18. HCS19, HCS20, HCS22, HCS23, HCS24, HCS25, HCS26, HCS27, HCS28, HCS29, HCS30, HCS31, HCS32, HCS33, HCS34, HCS35, HCS37, HCS39, HCS40, HCS43, HFS1, HFS2, HFS3, HFS4, HFS8, HFS7, HFS14

Zone of solubilization (mm) 0.5 - 1.0 1.5 mm 2.0 mm 2.5 mm > 3.0 mm -

Rhizobacterial strains were tested for potassiu m solubilization on modified Aleksandrov medium plates (Hu et al., 2006) supplemented with mica powder (2g/L). K solubilization pattern of different strains was scored on the basis of solubilization zone formed on medium plates after 4 days of growth.

Fig 1: Solubilization of potassium by rhizobacterial isolate HCS36

solubilization ranged from the 490 mg/L to 758 mg/L at pH 6.5 to 8.0. Hu et al. (2006) isolated two phosphate and potassium solubilizing Bacillus sp. from the soils in the modified medium containing phosphorite and potassium minerals like kaolinite and potassium feldspar. Sugumaran and Janarthanam (2007) showed that K solubilizing activity of the five slime producing bacterial isolates varied from 1.90 mg/L to 2.26 mg/L from acid leached soil. Parmar and Sindhu (2013) reported that out of 137 rhizobacterial isolates/ strains tested, only 27.7% isolates formed large zone of K

Volume 39 Issue 6 (2016) solubilization on mica containing medium plates and the amount of potassium released by different strains varied from 15 to 48 mg/L. Screening of selected rhizobacterial isolates for effect on root and shoot growth of clusterbean: Twenty two rhizobacterial isolates were selected on the basis of variation in IAA, ALA production, ACC utilization and potassium solubilisation. Selected isolates were screened for root and shoot elongation or retardation effect on clusterbean seedlings on water agar plates. Observations were taken after

H C S3 6

HCS30

HCS2 2

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5 and 10 days of growth. Eleven isolates showed stimulation of root and shoot growth at five days as compared to control uninoculated seedlings (Table 3; Figures 2a, b). Stimulation of root and shoot elongation in clusterbean was observed by inoculation of isolates HCS5, HCS7, HCS10, HCS18, HCS20, HCS23, HCS24, HCS26, HCS37, HFS1 and HFS5 at both 5 and 10 days of growth as compared to control uninoculated seedlings. Rhizobacterial isolates HCS2, HCS4, HCS12, HCS13, HCS30, HCS36, HCS39, HCS43, HFS8, HFS10 and HFS12 showed stunting effect on shoot

HCS2

HCS30

HCS36 Control

HC S43

HCS43

HCS5

H C S5

Control

Fig 2a: Effect of rhizobacterial isolates on root and shoot growth of clusterbean at 5 days of growth

Fig 2b: Effect of rhizobacterial isolates on root and shoot growth of clusterbean at 10 days of growth

Table 3: Effect of rhizobacterial isolates on seedling growth of clusterbean on water agar plates

Rhizobacterial isolates Control HCS2 HCS4 HCS5 HCS7 HCS10 HCS12 HCS13 HCS18 HCS20 HCS23 HCS24 HCS26 HCS30 HCS36 HCS37 HCS39 HCS43 HFS1 HFS5 HFS8 HFS10 HFS12

Root and shoot length (cm) 5 days Root 4.80±0.15 1.00±0.25 3.26±0.41 8.63±0.28 6.96±0.21 5.03±0.38 2.66±0.22 3.33±0.28 6.03±0.18 5.70±0.35 5.60±0.36 5.20±0.35 7.93±0.28 1.96±0.41 1.03±0.68 5.70±0.26 2.26±0.71 2.03±0.38 6.70±0.35 5.00±0.41 3.26±0.26 4.66±0.52 3.76±0.47

10 days Shoot 5.46±0.32 2.36±0.29 3.30±0.20 8.03±0.35 6.46±0.39 4.30±0.55 2.70±0.23 3.96±0.17 7.00±0.05 8.10±0.25 5.23±0.22 5.10±0.15 3.90±0.15 3.56±0.39 2.76±0.51 4.90±0.15 2.80±0.45 4.50±0.30 4.06±0.31 6.26±0.03 2.33±0.31 2.00±0.32 4.23±0.29

Values are average of five replications. SEM (standard error of means) values are represented as (±).

Root 9.66±0.37 1.23±0.28 6.13±0.38 10.67±0.18 11.70±0.45 10.93±0.08 7.76±0.41 7.67±0.61 10.63±0.53 10.10±0.35 10.83±0.47 10.80±0.37 11.37±0.35 7.43±0.43 4.87±0.31 9.37±0.42 5.63±0.58 7.70±0.35 10.07±0.32 8.87±0.26 8.43±0.48 6.97±0.47 4.83±0.48

Shoot 8.60±0.40 5.03±0.38 7.10±0.25 11.67±0.13 9.87±0.37 10.73±0.93 8.03±0.24 8.73±0.38 9.60±0.35 13.90±0.45 8.73±0.58 9.03±0.29 8.97±0.10 8.67±0.51 5.13±0.24 10.10±0.17 6.00±0.45 8.20±0.15 11.70±0.05 9.07±0.31 9.67±0.52 8.17±0.35 6.03±0.58

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length and root growth at 5 and 10 days of growth as compared to control. HCS5 isolate showed maximum growth of root and shoot at 5 days. Pseudomonas isolates HCS2 and HCS36 possessing IAA production and K solubilization showed more retardation effect on root as well as shoot length of clusterbean seedlings at 5 and 10 days as compared to control. Concentration dependent effect of IAA on stimulation or inhibition of root/shoot growth has been reported in earlier studies (Arshad and Frankberger, 1991). The inhibitory effect of some deleterious rhizobacteria (DRB) was also related to their high amount of IAA excretion in Enterobacter taylorae (Sarwar and Kremer, 1995). Loper and Schroth (1986) observed a significant linear relationship between IAA accumulation of the rhizobacterial strains and decreased root elongation of sugar beet seedlings. The initial stunting effect on seedlings could be due to contact of bacterial cell with legume seeds or due to synthesis or secretion of excessive amount of IAA or by production of some inhibitory agent/toxin by the bacterium when grown in synthetic medium or in root exudates of legumes (Bolton and Elliott, 1989; Gealy et al., 1996). Kennedy and Stubbs (2007) reported that inoculation with bacterial isolates caused 23-90% root inhibition of joint goatgrass. It is also possible that phytoalexins produced by seedlings as a host defense response after inoculation (infection) of rhizobacteria could be inhibitory for seedling growth initially (Goel et al., 2001). Moreover, production of toxic metabolites by nonfluorescent Pseudomonas strains has been reported to show an inhibitory effect on wheat root growth (Fredrickson et al., 1987).

number and nodule weight. Coinoculation of Pseudomonas HCS36 isolate and GSA110 showed stimulatory effect on nodule formation (34 nodules/plant) and nodule weight (176.4 mg/plant), and caused 88.47% increase in shoot dry weight as compared to uninoculated plants at 30 days of plant growth. At 60 days of plant growth, Rhizobium isolate GSA110 formed 45 nodules and 151.43% increase in shoot dry weight was observed in comparison to uninoculated control (Table 4). Coinoculation of Rhizobium isolate GSA110 and Bacillus isolate HCS43 increased nodulation (48 nodules/plant) and nodule weight (285.3 mg/plant), leading to 190.09% shoot dry weight gains as compared to uninoculated control (Figure 3). Coinoculation of Bradyrhizobium GSA11 with Bacillus isolate HCS43 also showed increase in nodulation (45 nodules/plant), nodule weight (246.7 mg/plant) leading to 132.59% increase in shoot dry weight in comparison to uninoculated control. Baig et al. (2002) reported that the inoculation effect of different bacterial isolates on plant growth varied and plants showed stunted growth, root and shoot elongation, or a neutral response. Three bacterial isolates increased the root length while 14 isolates increased shoot length over the uninoculated control. An increase in fresh and dry matter was recorded by 16 bacterial strains. Dey et al. (2004) showed that inoculation of peanut with fluorescent pseudomonad isolates viz. PGPR1, PGPR2 and PGPR4 containing ACC deaminase activity significantly enhanced the pod yi eld (23-26, 24-28 a nd 18-24%,

Coinoculation of selected rhizobacterial isolates with Bradyrhizobium/Rhizobium for nodulation and plant growth under chillum jar conditions: Three rhizobacterial isolates i.e., Pseudomonas isolate HCS36 and Bacillus isolates HCS5 and HCS43, having variation in production of IAA, ALA, ACC utilization, antagonistic activity and potassium solubilisation, were evaluated for symbiotic effectiveness on clusterbean variety HG563 in sterilized chillum jar conditions. The observations for nodulation, nodule weight, plant dry weight (for root and shoot) were recorded at 30 and 60 days of plant growth. Rhizobium isolate GSA110 formed 36 nodules at 30 days of plant growth and caused 68.67% increase in shoot dry weight (SDW) as compared to uninoculated control (Table 4). Bradyrhizobium isolate GSA11 formed only 20 nodules and 45.61% increase in SDW was observed in comparison to uninoculated control. Coinoculation of Rhizobium isolate GSA110 with Bacillus isolate HCS43 showed a significant increase in nodulation, nodule weight and shoot dry weight (178.27%) as compared to single inoculation. Coinoculation of Bradyrhizobium isolate GSA11 and Bacillus isolate HCS43 also increased nodule

Fig 3: Effect of inoculation of rhizobactrial isolates on plant growth at 60 days. I. Control (uninoculated) II. HCS36+GSA11 III. HCS5+GSA110 IV. HCS5+GSA11 V. HCS43+GSA11 VI. HCS43+GSA110

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Table 4: Symbiotic effectiveness of bacterial isolates on coinoculation in clusterbean at 30 and 60 days of plant growth under chillum jar conditions

Treatments

Control (uninoculated) HCS36 (Pseudomonas) HCS5 (Bacillus) HCS43 (Bacillus) GSA11 (Bradyrhizobium) GSA110 (Rhizobium) HCS36+GSA11 HCS36+GSA110 HCS5+GSA11 HCS5+GSA110 HCS43+GSA11 HCS43+GSA110 C.D.

Days of plant growth 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60 30 60

No. of nodules /plant 1 1 2 1 20 40 36 45 22 27 34 38 18 31 22 35 38 45 42 48 6.90 9.60

Nodule weight (mg/plant)

Shoot dry Weight (mg/plant)

4.7 5.2 9.8 5.3 96.7 235.3 207.3 258.3 102.7 177.3 185.7 234.3 88.3 218.3 105.7 224.7 205.7 246.7 234.3 285.3 8.07 8.41

98.0 143.3 105.3 280.7 109.7 215.3 103.0 243.3 142.7 346.7 165.3 360.3 161.3 312.7 184.7 340.3 209.7 286.7 220.3 296.7 247.7 333.3 272.7 415.7 10.45 10.83

Data are average values of three plants. C.D. values represent coefficient of deviation.

respectively), haulm yield and nodule dry weight over the control in field trials. Akhtar and Siddiqui (2008) demonstrated that inoculation of Rhizobium caused a greater increase in plant growth, number of pods, chlorophyll content, nitrogen, phosphorus and potassium contents of pathogen-inoculated plants than caused by P. straita or Glomus intraradices. Mishra et al. (2009) reported that plant growth-promoting bacteria (PGPB) strain B. thuringiensis-CMK1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), promoted plant growth of field pea and lentil (Lens culinaris L.) when coinoculated with R. leguminosarum-PR1. Similarly, coinoculation of IAA producing wild type Pseudomonas strains and their mutants on green gram enhanced nodule number, nodule fresh weight and plant dry weight in comparison to inoculation with Bradyrhizobium alone (Malik and Sindhu, 2011). In other studies also, mostly synergistic effects have been observed on nodulation and plan t growth of l egumes by mixed inoculation of B. japonicum and PGPR in soybean (Li and Alexander, 1988; Masciarelli et al., 2014), R. leguminosarum with an antibiotic-producing P. fluorescens strain F113 in pea

(Andrade et al., 1998) and Bradyrhizobium/Mesorhizobium strains with Pseudomonas sp. in green gram and chickpea (Sindhu et al., 1999; 2002). CONCLUSION Plant growth promoting rhizobacteria affect plant growth either directly or indirectly. The direct promotion of plant growth by PGPR may include the production of plant growth regulators or facilitating the uptake of certain nutrients from the root environment. Indirect promotion of plant growth occurs when introduced PGPR prevents deleterious effect of phytopathogenic organisms in the rhizosphere. In this study, rhizobacterial isolates showed IAA and ALA production, and were found to solubilize potassium. Some isolates stimulated the growth of clusterbean seedlings whereas other isolates showed retardation effect on seedlings’ growth. Coinoculation of Bacillus isolate HCS43 with Rhizobium strain GSA110 improved the nodulation and caused 190.09% increase in plant dry weight in comparison to uninoculated control plants. Thus, coinoculation of legumes with rhizobia and PGPR could be an ecofriendly and cost effective technology for improving nodulation and growth of legumes.

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