World Applied Sciences Journal 16 (10): 1368-1374, 2012ISSN 1818-4952© IDOSI Publications, 2012

Corresponding Author: P. Thamizhiniyan, Department of Botany, Annamalai University, Annamalai Nagar-608 002,Tamilnadu, India. Tel: +04144 238282, Fax: +04144 238080.

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Response of Biofertilizers on Growth, Yield Attributes and AssociatedProtein Profiling Changes of Blackgram (Vigna mungo L. Hepper)

G. Selvakumar, S. Reetha and P. Thamizhiniyan

Department of Botany, Annamalai University, Annamalai Nagar-608 002, Tamilnadu, India

Abstract: A field trial was conducted during 2009 growing season at the Botanical garden, Department ofBotany, Annamalai University to study the effect of biofertilizers on the growth, yield as well as nitrogencomponents, total protein and protein profiles in black gram. The treatments consisted of biofertilizers aloneor combination treatment viz., control (without any microbial inoculation), Azotobacter, Azospirillum,Phosphobacteria, Rhizobium, Rhizobium + Azotobacter, Rhizobium + Azospirillum and Rhizobium +Phosphobacteria replicated three times in randomized block design. The results revealed that biofertilizationperform significant improvement in plant productivity and quality. The maximum germination percentage, freshand dry weight, no. of pods per plant, seed yield per plant, hundred seed weight and root nodules per plantwere increased with the inoculation of biofertilizers especially Rhizobium with Phosphobacteria treatment whencompared to others. Generally, biofertilizers induced over or low gene expression of most mid and highmolecular weight protein bands. Hither, they also induced new proteins of low molecular weight. These lowmolecular weights proteins may be used as an adaptive mechanism for biofertilizer application in plants to givethe maximum yield. The molecular weight ranged between 14.3 and 97.4 kDa were observed in treated plants aswell as control. But, Rhizobium with Phosphobacteria inoculated plants produced one new protein band withmolecular weight of 77.5 kDa.

Key words: Vigna mungo Azotobacter Rhizobium SDS-PAGE Phosphobacteria

INTRODUCTION processes and nutrient cycling. Soil is considered a

Black gram is the third important pulse crop in India. occupied by living microorganisms is estimated to be lessIt is annual pulse crop and native to central Asia. It is also than 5% of the total space. Therefore, major microbialextensively grown in West Indies, Japan and other activity is confined to the ‘hot-spot’, i.e. aggregates withtropics/subtropical countries. Black gram seeds are highly accumulated organic matter, rhizosphere [2]. Soil micronutritious containing higher amount of protein (24-26 %) organisms play an important role in soil processes thatand are reported to be rich in potassium, phosphorus and determine plant productivity.calcium with good amount of sodium. It is also reported to Biofertilizer or microbial inoculants can be generallybe rich in vitamin A, B1, B3 besides nutritionally rich defined as preparation containing live or latent cells ofproteins, important mineral and vitamins. It has some efficient strains of nitrogen fixing and phosphatemedicinal properties, like curing diabetes, sexual solubilizing microorganism used for treatment of seed ordysfunction, nervous disorder, hair disorders, soil. They are organic products containing living cells ofdigestive system disorders and rheumatic afflictions [1]. different types of microorganisms, which have the abilityBlack gram seeds have shown antiantherogenic to convert nutritionally important elements fromactivity in guinea pigs. unavailable to available form through biological processes

Soil is a dynamic, living matrix that is an essential part [3]. They are composting the area with the objective ofof the terrestrial ecosystem. It is a critical resource not increasing the number of such microorganisms andonly for agricultural production and food security but accelerate microbial process to augment to extent of thealso towards maintenance of most life processes. availability of the nutrient in a form which can easilyThe functions of soil biota are central to decomposition assimilated by plant [4]. The Rhizobium as fertilizer in

storehouse of microbial activity, though the space

10Carotenoid (mg/g fr. wt.) = = (OD 480 +. 2500

OD 114) ´ (OD 663) - (OD 638 ´ OD 645)

D F Vwt× × ×

×

Sample titre blank titre × Hot HClNitrogen (%) = ×14×100Sample weight × 1000

−

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pulses could fix 50-200 kg of N/ha/season and is able to Estimation of Carotenoid [9]: The same chlorophyllmeet 80-90% of the crop requirement for nitrogen.Inoculation in these crops was found to increase the cropyield by about 10-15% under on farm conditions [5].It includes mainly the nitrogen fixing, phosphatesolubilizing and plant growth-promoting microorganisms[6]. Among biofertilizers benefiting the crop productionare Azotobacter, Azospirillum, blue green algae, Azolla,P-solubilizing microorganisms and mycorrhizae [7].Therefore, an experiment was conducted to study theinfluence of biofertilizer on growth, yield and associatedprotein profiling changes in black gram.

MATERIALS AND METHODS

Seeds of black gram (Vigna mungo (L.) cv. ADT3)obtained from Rice Research Institute, Aduthurai,Tamilnadu, India. The different types of biofertilizers(Azotobacter, Azospirillum, Phosphobacteria andRhizobium were obtained from Ramvijay Biofertilizers,Puducherry. Seeds were sown in the plotted field withspacing of 10×30 cm between plants and rows.Biofertilizers were used alone as well as in combinationto inoculate the different combination of the field suchas T0-control (without any microbial inoculation),T1-Azotobacter, T2-Azospirillum, T3-Phosphobacteria,T4-Rhizobium, T5-Rhizobium + Azotobacter;T6-Rhizobium + Azospirillum and T7-Rhizobium+ Phosphobacteria. The seeds were grown in arandomized block design in three replications.From each entry, 10 plants were randomly selectedfor recording observations on important yieldattributing characters, plant height, fresh and dryweight, total chlorophyll, carotenoid, no. of pods perplant, seed yield per plant, hundred seed weight, proteincontent and no. of nodules per plant during the plantgrowth period.

Estimation of Chlorophyll [8]: 0.5 mg of fresh leaf wasground in a mortar and pestle with 20 ml of 80 per centacetone. The homogenate was centrifuged at 3000 rpm for15 min. The supernatant was saved. The pellet wasrestricted with 5 ml of 80 per cent acetone each time, untilit become colorless. All the supernatants were pooled andutilized for chlorophyll determination. Absorbance wasmeasured at 645 and 663nm in spectrophotometer.The chlorophyll content was determined by using thefollowing formulae.

Chlorophyll ‘a’ (mg/g fr. wt.) = (0.0127) × (OD663) - (0.00269) × (OD645)Chlorophyll ‘b’ (mg/g fr. wt.) = (0.229) × (OD645) - (0.00488) × (OD663)Total chlorophyll(mg/g fr. wt.)=(0.0202) × (OD645) - (0.00802) × (OD663)

extract was measured at 480 nm, in spectronic-20 toestimate the carotenoid content.

Estimation of Protein [10]: 0.5 mg of plant materials wasmacerated with a pestle and mortar with 10 ml of 20 percent trichloroacetic acid. The homogenate wascentrifuged for 15 min at 600 rpm. The supernatant wasdiscarded. To the pellet, 5 ml of 0.1 N NaOH was addedand centrifuged for 5 min. The supernatant was saved andmade up to 10 ml of 0.1 N NaOH. This extract was used forthe estimation of protein. From this extract, 1 ml of samplewas taken in a 10 ml test tube and 5 ml of reagent wasadded. The solution was mixed well and kept in dark for 10min. Later 0.5 ml folinphenol was added and the mixturewas kept in dark for 30 min. The sample was read at 660nm in the spectronic-20. Blank prepared without proteinsample was used for zero setting. The absorbance valuewas referred to the standard graph proteins prepared byusing 5 fraction of Bovine’s serum albumen.th

Seed Nitrogen Content [11 and 12] and Seed CrudeProtein [13 and 14]: Two hundred mg of dried seedsample was taken in a 100 ml kjeldahal flask. About 5 mgof salt mixture (potassium sulphate, cupric sulphate andselenium powder mixed in the ratio of 50:10:1) was addedwith 3 ml of concentrated sulphuric acid. After digestion,10 ml of distilled water was added. The distillate wascollected in a conical flask containing 10 ml of 4 percent boric acid and 3 drops of mixed indicator(0.3 g bromocresol green and 0.2 g methyl red in 400 ml of90% ethanol). This solution was titrated against 0.05 NHCl. Nitrogen content was estimated using the flowingformula.

Crude protein was calculated by multiplying the totalnitrogen content by 6.25.

Sodium Dodecyl Sulphate-polyacrylamide GelElectrophoresis (SDS-PAGE) for Protein Analysis:At seed maturity harvest stage, seeds from differenttreatments were taken for protein analysis. Proteins wereextracted from the seed samples in liquid nitrogen.The tris buffer system used was that originally devised byLaemmli [15], as described by Dunn [16], with 2% (w/v)

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SDS and 5% (v/v) 2-mercaptoethanol to cleave the P- solubilizing activity of phosphobacteria associateddisulphide bonds. The slurry was centrifuged for 20 minat 12000 rpm. The samples were heated in a boiling waterbath for 15 minutes before loading to ensure dissociation.Gel preparation was carried out according to Hames [17],where 15% resolving gel was used. Bromophenol blue(0.001%) tracking dye was used for marking the bufferfront during electrophoresis. The gel was electophoresedat 25 - 30mA constant current, then at 200 V (for about8 - 9 h). Staining was done using coomassie brilliant blueand a solution of 10% acetic acid and 45% methanol wasused for destaining. In the quantitative analysis of theprotein bands, laser densitometry was used to image thestained profiles. Data were analyzed and identified by geldocumentation system (GDS) which comparingpolypeptide maps, which include the use of bandintensity, molecular weight and the rate of mobility ofeach polypeptide with standard markers.

RESULTS AND DISCUSSION

Growth and Yield Characters: The growth and yieldparameters of black gram such as germination percentage,plant height, fresh and dry weight, total chlorophyll,carotenoid, no. of pods per plant, seed yield per plant,hundred seed weight, protein content and no. of rootnodules per plant were significantly increased by plantgrowth promoting rhizobacteria (PGPR) application in allconcentrations when compared to control. Utilization ofbiological fertilizer increased fresh and dry weight, no. ofpods per plant, seed yield and hundred seed weight thatit could be due to increasing other nutrient absorption,also biological phosphate fertilizer can be used as asolution for increasing phosphate and micronutrientsorption in the alkaline soil. Zahir et al., [18] in maize bothqualitative and quantitative characteristics weresignificantly increased by phosphate-solubilizingmicroorganisms and also increased the growth andresistance of plants in water deficit conditions(Ehteshami et al., 2007). Hoshang Naserirad et al. [19] andAsad Rokhzadi [20] indicated that inoculation withbiofertilizers containing Azotobacter and Azospirillumincreased plant height, leaf number per plant, fruit meanweight and yield in compare to control (withoutbiofertilizer).

Azotobacter and Azospirillum fixed N from the2

atmosphere and released plant available N forms to soil,resulting in increased uptake and plant height. These dataagree with the previously reported results on the effectsAzotobacter and Azospirillum on the plant height ofbhendi [21].

with the release of organic acids and a drop in the pH ofthe medium. Different kinds of organic acids, namely citricacid, gluconic acid, lactic acid, succinic acid,propionic acid and three other unknown organicacids were produced from the cultures of these isolates.The utilization of phosphate-solubilising microorganisms,account for about 45% of the total biofertiliser productionand use. This bacterium helps in increasing cropproductivity by way of helping in solubilization ofinsoluble phosphorus, stimulating growth byproviding hormones, vitamins and other growth factors[22]. The availability of phosphorus to legume crop is akey constraint to its production. The soil micro organismsare responsible for transfer of the immobilized soilphosphorus into available form through whichphosphorus becomes easily available to these legumecrops [23].

The stimulatory effects of biofertilizers used are inaccordance with the results obtained by Chauhan et al.,[24] who found that inoculation of Azospirillum-as abiofertilizer-markedly increased pods number and seedyield of Brassica napus L. plants over the non inoculatedplants. In addition, Buragohain [25] found that sugarcaneyield was significantly higher in the cultivated crops withAzotobacter than uninoculated crops. In addition,Goel et al., [26] reported that, the inoculation with certainplant growth-promoting rhizobacteria (PGPR) mayenhance crop productivity either by making the othernutrients available or protecting plants from pathogenicmicroorganism (allelopathic effects). Zodape [27] alsoconcluded that, the increase in yield productivity withbiofertilizer application is due to micro-element and plantgrowth regulator contained in the fertilizer.

Co-inoculation can benefit plant growth by differentmechanisms [28]. However one of the most commonlyreported plant growth promotion mechanism by bacteriais the changing of morphological and physiologicalchanges in root system [29]. An increase in the number oflateral roots and root hairs cause addition of root surfaceavailable for nutrients and water uptake. Higher water andnutrient uptake by inoculated roots caused an improvedwater status of plant, which in turn could be the mainfactor enhancing plant growth [30, 31]. Increasing nutrientuptake such as NO , NH , Po , K , Fe in the various3 2 + 2+

4 4

inoculated plants have reported [32]. For example, singleand double inoculation with Azotobacter, Azospirillumand Streptomyces increased P, Mg and N content inwheat grains [33]. Co-inoculation of Sorghum withAzospirillum and Glomus significantly increased P, N, Zn,Cu and Fe content. Thus co-inoculation may substitutepartially as P and N fertilizer [34].

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Protein Content: Results obtained from variance analysis culture against native rhizobial population. Since roots areshowed that used bacteria treatment and interaction of the sites for microbial infection, well-developed rootthese agents had significant difference. It is observed in system provides more evidence for infection resulting inmean comparison, that double-inoculation of Rhizobium greater number of nodules.with Phosphobacteria yielded more protein contentthan single and non-inoculation (control). Shehata and NP Contents of Soil: Microbial inoculums not onlyEL-Khawas [35] studied bio-fertilizers including growth increased the nutritional assimilation (total N, P and K) ofprompting bacteria improved the sunflower yield and plants, but also improved soil properties. In this study,qualitative parameters in compared with control soil was tested before sowing and after harvest of plants(non-inoculation) treatment and as a result caused to for residual NPK contents. Available N contentsincreasing protein content. Studying interaction of increased significantly in inoculated soil thanbacteria treatment showed that double-inoculation caused uninoculated control. It may be due to reason that as theto nitrogen content and yielded highest protein content microbes fixes nitrogen and this nitrogen fixationusing double-inoculation. Since Rhizobium with suppresses nitrate uptake as demonstrated by Zarin et al.Phosphobacteria is nitrogen fixing bacteria and nitrogen [39] who reported that soil nitrate was higher whenis basic matter to forming protein treatment. hormones were applied to nitrogen fixing chickpea plants.

Number of Nodules per Plant: The increased nodulation, microbes [40]. Moreover, significant increase in N contentN fixation and yield of legume crops following may be due to the fact that legumes contribute to the total2

inoculation with biofertilizers have been reported by many pool of nitrogen in the soil as observed by Ahmad et al.workers [36]. The number of nodules increased [41]. Some nodules get sloughed off from senescingsignificantly in treatments with pure cultures of plants at harvest and results in increased NO -N and otherRhizobium and carrier based inocula treated plants. macronutrients. Higher P content may be due toIt has already been reported by Hunter et al. [37]. inoculation and availability of P nutrients in soil byThe significant increase in dry matter and nodule weight microbes. Soil pH, organic C, total N, P and K, availablewith Rhizobium inoculation could be higher number of N, P and K content were significantly increased by thebacteria present under inoculated conditions. Vijle and application of biofertilizer application. ConcerningJebaraj [38] also reported similar results. The combined micro-nutrients i.e., iron, zinc and manganese, the data ininoculation of Rhizobium + Phosphobacteria resulting in Table 1 show that the biofertilized treatments inducedthe significant increase in nodule weight than Rhizobium lower values in comparison with the mineral ones for ironor PSB alone. It might have resulted due to more and zinc while little differences between treatments werecompetitive ability of microbes in carrier than in pure found regarding manganese element.

It correlates with maximum nodulation influenced by

3

Table 1: Effect of biofertilizers on seed germination and morphological parameters of Vigna mungo (L.) HepperGermination Plant Fresh Dry Total No. of pods Seed yield Hundred Protein No. of root

Treatments (%) height (cm) weight (mg/g) weight (mg/g) chlorophyll Carotenoid per plant per plant (g) seed wt.(g) content nodules per plantTo 83.8±2.51 45.1±1.35 36.7±1.10 21.0±0.63 2.05±0.06 0.532±0.015 28.9±0.86 6.2±0.18 4.67±0.14 4.17±0.13 20.1±0.60T1 87.2±2.62 50.1±1.50 39.5±1.19 22.7±0.68 2.11±0.06 0.542±0.016 31.5±0.95 7.3±0.21 4.43±0.13 4.20±0.13 76.6±2.30T2 85.5±2.57 53.9±1.61 38.2±1.15 21.9±0.66 2.03±0.06 0.558±0.017 29.1±0.46 6.8±0.20 4.96±0.15 4.22±0.12 73.8±2.21T3 87.2±2.62 54.2±1.63 39.0±1.17 22.4±0.67 2.63±0.08 0.520±0.016 30.0±0.91 7.0±0.21 5.10±0.15 4.34±0.13 76.6±2.29T4 87.6±2.63 53.5±1.61 51.2±1.53 27.7±0.83 2.61±0.07 0.572±0.017 30.9±0.93 7.2±0.22 5.01±0.15 4.61±0.14 79.2±2.37T5 87.8±2.63 53.5±1.61 48.2±1.45 23.9±0.92 2.47±0.07 0.533±0.016 26.6±0.80 7.8±0.23 4.91±0.15 4.29±0.13 70.2±2.11T6 86.4±2.59 55.1±1.65 52.0±1.56 28.7±0.86 2.23±0.06 0.561±0.017 29.6±0.89 8.1±0.24 4.92±0.14 4.49±0.13 74.2±2.23T7 88.5±2.65 56.7±1.70 54.0±1.62 29.6±0.88 2.85±0.08 0.572±0.017 31.8±0.95 8.5±0.26 5.14±0.15 4.81±0.14 80.2±2.41

Table 2: The effect of biofertilizers oh physico-chemical analysis of soil before sowing and after harvestingEC Available Available Available Available Available Available Available Available Available

Treatments pH (dS m ) N (kg/ha) P (kg/ha) K (kg/ha) Ca (ppm) Mg (ppm) Zn (ppm) Cu (ppm) Fe (ppm) Mn (ppm)1

Before sowing 7.67 0.41 112.6 4.5 100.7 2.09 9.15 4.70 0.20 7.72 4.32After harvesting

T0 7.60 0.44 119.2 5.9 114.2 2.18 9.37 5.72 0.26 8.16 5.01T1 7.26 0.74 147.6 6.09 12.05 2.26 9.57 5.82 0.29 8.99 5.24T2 7.52 0.98 162.7 8.12 120.1 2.32 9.61 5.44 0.23 8.26 5.33T3 7.46 0.95 149.1 9.03 122.1 2.06 9.74 5.24 0.24 8.34 5.16T4 7.38 0.94 160.1 8.2 120.6 2.87 9.62 6.07 0.32 9.26 5.21T5 7.42 0.93 162.0 8.01 124.1 2.36 9.59 5.68 0.27 9.01 5.26T6 7.46 0.93 152.0 7.6 121.0 2.44 9.62 5.71 0.31 8.39 5.06T7 7.33 0.98 159.6 9.1 125.1 2.90 9.70 6.04 0.38 9.10 5.47

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Fig. 1: Protein patterns of biofertilizer treated seeds in biofertilizers with single and combined treatments inVigna mungo L. The control (T0) represents addition to increased yield could be a strategy to achievewithout any microbial inoculation. The other sustainable agriculture.groups of seeds are T1-Azotobacter,T2-Azospirillum, T3-Phosphobacteria, REFERENCEST4-Rhizobium, T5-Rhizobium + Azotobacter,T6-Rhizobium + Azospirillum and T7-Rhizobium 1. Anonymous, 2010. http//:wik.onse.conr.+ Phosphobacteria. M=marker 2. Lynch, J.M., 1990. Introduction: some consequences

SDS-Protein Electrophoresis: Little investigations were In The Rhizosphere (ed. Lynch, J.M.), John Wiley &carried out to study the effect of biofertilizer application Sons, Chichester, UK, pp: 1-10.on the protein content and protein profile. In this 3. Vessey, J.K., 2003. Plant growth promotinginvestigation, the total soluble protein content was rhizobacteria as biofertilizers. Plant Soil., 255: 571-586.significantly increased, the increase was more 4. Subba-Rao, N.S., 1986. Phosphate Solubilization bypronounced with biofertilizers (Table 2). Seed protein soil Micro organisms. In: Advances in Agriculturalcontent was increased in response to biofertilizer Microbiology, Subba-Rao, N.S. (Ed.). Oxford andapplication to soybean [40] and these results are in IBH, New Delhi, pp: 295-303.accordance with those obtained by Sharma and Namdeo 5. Khurana, A.L. and S.S. Dudeja, 1997. Biological[41] in mungbean and blackgram. The SDS-electrophoretic Nitrogen Fixation Technology for Pulses Productionpattern showed that one new band having molecular in India. Indian Institute of Pulses Research, Kanpur,weight 77.5 kDa that appeared by the effect of India.biofertilizer (Rhizobium with Phosphobacteria) treated 6. Goel, A.K., R.D. Laura, D.V. Pathak, G. Anuradha andseeds among the treatments under investigation in A. Goel, 1999. Use of biofertilizers: potential,Figure 1. This combination treatment is characteristic by constraints and future strategies review. Inter. J.the appearance of one new inducible protein and this may Trop. Agric., 17: 1-18.be an adaptive mechanism for the biofertilizer absorption 7. Hedge, D.M., B.S. Dwivedi and S.N.S. Babu, 1999.in plants to give higher yield productivity. Biofertilizers for cereal production in India - A review.

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obtained maximum performance. Co-inoculation caused toincreasing yield through synergistic effects by improvinggrowth prompting hormones, controlling pathogenesisand growth reducing agents due to producing fungicideantibiotics and compounds (antagonistic effect) and alsoair molloculal nitrogen fixing and also producing growthprompting hormones such as auxin, cytokinin andgibberellin and solving mineral compound. The higheststimulatory effect of root associative beneficial bacteria,especially strains of Rhizobium + Phosphobacteria havepotential to be used as biofertilizer increase theproductivity of blackgram. Overall utilization of

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