AGRICULTURAL RESEARCH COMMUNICATION CENTREwww.arccjournals.com/www.legumeresearch.in

*Corresponding author’s e-mail: yadavv39@gmail.com.

Legume Research, 39 (2) 2016 : 256-261Print ISSN:0250-5371 / Online ISSN:0976-0571

Eeffect of biofertilizers and foliar application of organic acids on yield, nutrientuptake and soil microbial activity in soybeanN.N. Lingaraju*, C.S. Hunshal and S.R. SalakinkopUniversity of Agricultural Sciences,Dharwad-580 005, Karnataka, India.Received: 28-09-2014 Accepted: 19-02-2015 DOI:10.18805/lr.v0iOF.6784

ABSTRACTA field experiment was undertaken during kharif 2012 to study effect of biofertilizers and foliar application of organic acidson yield, nutrient uptake and microbial activity of soybean at MARS, Dharwad under rainfed condition. The experiment waslaid out in RCBD factorial having twenty treatment combinations and replicated thrice. The treatments comprised of fourP-Solubilizers (PSB, VAM, PSB + VAM and Control) and five foliar spray of organic acids (Humic acid, Lecithin, Citricacid, Maleic acid and control). The results revealed that higher seed yield of soybean (35.96 q ha-1) was obtained with thetreatment combination of dual inoculation of PSB+VAM with foliar spray of 0.1% humic acid at flower initiation and washigher to an extent 22.5 per cent compared to control (27.90 q ha-1). Similar trend was observed on microbial activity andnutrient uptake (N, P2O5 and K2O) of the soybean crop.

Key words: Nutrient uptake, Organic acids, PSB, Soybean, VAM.

INTRODUCTIONPhosphorus (P) is one of the most important

elements for plant growth and metabolism. It plays a keyrole in many plant processes such as energy metabolism,synthesis of nucleic acids and membranes, photosynthesis,respiration, nitrogen fixation and enzyme regulation.Adequate phosphorus nutrition enhances many aspects ofplant development including flowering, fruiting and rootgrowth. In many soil types, P is the most limiting nutrient forthe production of crops. Maintaining a proper P supplyinglevel at the root zone can maximize the efficiency of plantroots to mobilize and acquire P from the rhizosphere by anintegration of root morphological and physiological adoptivestrategies. Soil type, pH range, fertilizer (rate and type),cropping system etc., influence the fate of P in soil and plant.The use of phosphate solubilizing bacteria and VAM asbiofertilizers and organic acids was suggested as a sustainablesolution to improve plant nutrient and production. Soilmicroorganisms play a key role in soil P dynamics andsubsequent availability of phosphate to plants. Co-inoculationof PSM and arbuscular mycorrhizas (AM) may enhance plantacquisition of P from insoluble P sources.

Phosphate solubilizing bacteria (PSB) have beenused for the crop production since 1903. PSB like Bacillusmegaterium, Bacillus polymyxa, Pseudomonas striata,

Pseudomonas flouresence are important in phosphorusnutrition by enhancing its availability to plants through releasefrom inorganic and organic soil P pools by solubilization andmineralization. Principal mechanism in soil for mineralphosphate solubilization including lowering of soil pH bymicrobial production of organic acids and mineralization oforganic P by acid Phosphatase. Use of phosphorussolubilizing bacteria as inoculants increases P uptake. Greaterefficiency of P solubilizing bacteria has been shown throughco-inoculation with other beneficial bacteria and mycorrhiza.On the other hand use of organic-mineral fertilizers like humicacid has increased with increasing the agricultural productionand the most economical. Humic acid is almost applieddirectly to the soil or as a foliar application to the plants. It isone of the major components of humic substances. Humicmatter is formed through the chemical and biologicalhumification of plant and animal matter and through thebiological activities of microorganisms. The effects of humicsubstances on plant growth depend on the source andconcentration, as well as on the molecular fraction weight ofhumus.

MATERIALS AND METHODSA field experiment was conducted at Main

Agricultural Research Station Dharwad during Kharif 2012.The pH of the soil was neutral (7.3) and low in available

Volume 39 Issue 2 (2016) 257

Tab

le 1

: Num

ber o

f po

ds p

lant

-1, n

umbe

r of

seed

s po

d-1 an

d 10

0 se

ed w

eigh

t (g)

in s

oybe

an a

s in

fluen

ced

by b

iofe

rtiliz

ers

and

folia

r app

licat

ion

of o

rgan

ic ac

ids

Not

e: M

eans

fol

low

ed b

y th

e sa

me

low

er c

ase

lette

rs in

a c

olum

n do

not

diff

er s

igni

fican

tly b

y D

MRT

(P=0

.05)

B1

- PSB

F1- F

olia

r spr

ay o

f H

umic

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

2 - V

AM

F2- F

olia

r sp

ray

of L

ecith

in @

0.1

% a

t flo

wer

initi

atio

n sta

geB

3 - P

SB+V

AM

F3- F

olia

r sp

ray

of c

itric

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

4 - C

ontro

lF4

- Fol

iar

spra

y of

Mal

eic

acid

@ 0

.1%

at f

low

er in

itiat

ion

stage

F5-

Con

trol

nitrogen (238.8 kg N ha-1), medium in available phosphorous(35.22 kg P2O5 ha-1) and high in available potassium (341.3kg K2O ha-1). The experiment was laid out in RCBD factorialhaving twenty treatment combinations and replicated thriceand genotype DSb-21 was used in the study. The treatmentscomprised of four main factors (PSB, VAM, PSB + VAMand Control) and five sub factors of foliar application oforganic acids at flower initiation stage (Humic acid, Lecithin,Citric acid, Maleic acid and control). Recommended fertilizerdose of 40:80:25 kg N, P2O5 and K2O per hectare in the formof Urea, DAP and MOP was applied as a basal at the time ofsowing. PSB (Pseudomonas striata) obtained from theDepartment of Agricultural Microbiology, UAS, Dharwadwas treated at the rate of 6g kg-1 of seeds. Rhizobium(Sb-120) was also applied at the rate of 6 g kg -1 of seeds.VAM @ 20 kg ha-1 was applied to soil at the time of sowing.Five plants from net plot area were randomly selected andobservations on growth and yield parameters were recordedat 30, 60 DAS (days after sowing) and at harvest. At harvest,yield and its components such as number of pods plant-1,number of seeds pod-1, hundred seed weight, seed yield plant-

1, seed yield ha-1 and haulm yield ha-1 were determined atmaturity stage. All the data pertaining to the presentinvestigation were statistically analyzed as per the methoddescribed by Panse and Sukhatme (1967). The level ofsignificance used in ‘F’ and ‘T’ test was P= 0.05. The meandifferences among the treatments were compared by DuncanMultiple Comparison Test (DMRT) at 0.05 level ofprobability.

RESULTS AND DISCUSSIONYield and yield components: Higher seed yield of soybean(35.96 q ha-1) to the extent of 28 per cent was obtained withthe treatment combination of dual inoculation of PSB+VAMwith foliar spray of 0.1% humic acid at flower initiation.Compared to control (27.90 q ha-1) (Table 2). The higherseed yield was mainly due to positive association betweenyield attributing characters viz., Such as higher number ofpods plant-1, number of seeds plant-1 and 100 seed weight.Significantly higher number of pods plant-1 (92.10 g plant-1),number of seeds plant-1 (3.10) and 100 seed weight (13.10 gplant-1) (Table 1) were also recorded by inoculation of PSB+ VAM with foliar spray of 0.1% humic acid at flowerinitiation compared to control (64.9 pod plant-1 2.83 and10.33g plant-1, respectively). This might be due to betternodulation of soybean roots owing to increased availabilityof phosphorus. The improvement in nodulation withinoculationof P-solubilizer might have resulted in highernitrogen fixation and consequent increase in vegetative

258 LEGUME RESEARCH - An International Journal

Treatment Seed yield (q ha-1) Haulm yield (q ha-1)

Spray of organic acids F1 F2 F3 F4 F5 Mean F1 F2 F3 F4 F5 Mean

B1 31.66bf 29.86ei 31.05cg 30.81fi 29.34be 30.54b 52.39ac 49.41be 50.67bd 49.03be 46.93df 49.68b B2 32.21di 30.07bf 31.44bf 30.67ch 29.58fi 30.79b 53.05ab 49.69be 50.91bd 50.42bd 47.49cf 50.31b B3 35.96a 32.71bc 33.81b 32.94bc 32.47bd 33.58a 55.75a 51.52ad 53.01ab 52.35ac 49.80bd 52.48a B4 30.28di 28.27hi 29.33fi 28.61gi 27.90i 28.88c 46.54df 46.98df 44.82ef 43.07f 43.71f 45.03c Mean 32.53a 30.23c 31.41b 30.76bc 29.82c 51.93a 49.40b 49.85ab 48.72bc 46.98c S.Em+ S.Em+ B 0.331 0.663 F 0.371 0.741 BXF 0.741 1.483

Table 2: Seed yield (q ha-1) and haulm yield (q ha-1) in soybean as influenced by biofertilizers and foliar application of organic acids

Note: Means followed by the same lower case letters in a column do not differ significantly by DMRT (P=0.05)B1 - PSB F1- Foliar spray of Humic acid @ 0.1% at flower initiation stageB2 - VAM F2- Foliar spray of Lecithin @ 0.1% at flower initiation stageB3 - PSB+VAM F3- Foliar spray of Citric acid @ 0.1% at flower initiation stageB4 - Control F4- Foliar spray of Maleic acid @ 0.1% at flower initiation stage

F5- Control

growth and dry matter production and also mediated bybiological process as noticed by increased microbial activity.

Biofertilizers play a key role in enhancing theefficiency of utilization of native as well as applied nutrients.The greater yield response due to dual inoculation of VAMfungus and PSB than with PSB alone can be attributed to theactivity of the VAM fungus in transporting extra phosphorussolubilized by PSB from and beyond the root zone into theplant roots which in the absence of VAM hyphae gets refixedby soil constituents during the course of slower diffusiontowards plant roots. These results are in accordance with thefindings of Chitale et al. (2012) in soybean. They reportedthat higher seed yield of soybean was due to inoculation ofPSB + VAM than inoculation of either PSB or VAM alone.Foliar spray of 0.1% humic acid helps in quick absorption ofnutrients, which enhanced the growth of root and shooteffectively resulting in higher uptake of nutrients. Sprayingof humic acid mainly reduced the flower drop and ultimatelyenhanced the pod setting and resulted in higher seed yield.The results are in line with the findings of Dixit and Elamathi(2007). Combined application of PSB+VAM with foliar sprayof 0.1% humic acid might have helped to enhance thebiological activity in the soil, soil characters improvements,better root development, improved transport of nutritionalelements, enhanced chlorophyll content, protein synthesis andphotosynthesis, solubilization of nutrients resulting in highernutrients uptake by soybean compared to other treatments(Lee and Bartlett, 1976) suggesting existence of synergeticeffect of combined applications of mineral nutrients andhumic acid substances.

Nutrient uptake in soybean: Significantly higher uptake ofnitrogen, phosphorus and potassium by soybean at harvest(223.65, 35.55 and 240 kg ha-1, respectively) (Table 3) bysoybean was recorded with foliar application of 0.1% humicacid at flower initiation as compared to other sprays andcontrol. Higher P uptake might be due to solubilization offixed phosphorus by P-solubilizer due to secretion of organicacids. Similar findings were made by Sharma and Namdeo(1999). Foliar application of humic acid significantlyenhances the presence of various antioxidants in the leavesand stimulates root initiation thus resulting in higher uptakeof nitrogen, phosphorous, potassium, magnesium, calcium,zinc, iron and copper (Schmidt and Zhang, 1998). Combinedeffect from biofertilizers and humic acid might have resultedin the improvement of soil fertility and crop productivity.

Microbial activity: Significantly higher microbial loadof rhizobium population and Rhizosphere P- Solubilizerspopulation (Number X 104 g-1) was influenced by combinedapplication of PSB+VAM. At 30, 60 DAS and at harvest,dual inoculation of PSB+VAM recorded significantlyhigher rhizobium population (18.60, 28.13 and 25.6 X 104

g-1) compared to singular inoculation of VAM (18.60, 22.80and 22.2 X 10 4 g -1) and PSB (16.93, 25.70 and22.9 X 104 g-1) alone over the control (18, 21.01 and 22.1X 104 g-1). Among the foliar sprays, 0.1% humic acidrecorded significantly higher rhizobium population at60DAS (34.23 X 104 g-1) compared to control (27.54 X104 g-1). With respect to interaction, dual inoculation ofPSB+VAM and foliar spray of 0.1% humic acid recorded

Volume 39 Issue 2 (2016) 259

Tab

le 4

: Rhi

zobi

um c

olon

y co

unt (

Num

ber

X 1

04 g-1

in s

oybe

an a

t diff

eren

t gro

wth

sta

ges

as in

fluen

ced

by b

iofe

rtiliz

ers

and

folia

r ap

plic

atio

n of

org

anic

aci

ds

Not

e: M

eans

fol

low

ed b

y th

e sa

me

low

er c

ase

lette

rs in

a c

olum

n do

not

diff

er s

igni

fican

tly b

y D

MRT

(P=0

.05)

B1

- PSB

F1- F

olia

r spr

ay o

f H

umic

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

2 - V

AM

F2- F

olia

r sp

ray

of L

ecith

in @

0.1

% a

t flo

wer

initi

atio

n sta

geB

3 - P

SB+V

AM

F3- F

olia

r sp

ray

of C

itric

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

4 - C

ontro

lF4

- Fol

iar

spra

y of

Mal

eic

acid

@ 0

.1%

at f

low

er in

itiat

ion

stage

F5-

Con

trol

Tab

le 3

: Nut

rient

upt

ake

(N, P

2O5 a

nd K

2O k

g ha

-1) o

f soy

bean

at h

arve

st as

influ

ence

d by

bio

ferti

lizer

s an

d fo

liar

appl

icat

ion

of o

rgan

ic a

cids

Not

e: M

eans

fol

low

ed b

y th

e sa

me

low

er c

ase

lette

rs in

a c

olum

n do

not

diff

er s

igni

fican

tly b

y D

MRT

(P=0

.05)

B1

- PSB

F1- F

olia

r spr

ay o

f H

umic

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

2 - V

AM

F2- F

olia

r sp

ray

of L

ecith

in @

0.1

% a

t flo

wer

initi

atio

n sta

geB

3 - P

SB+V

AM

F3- F

olia

r sp

ray

of C

itric

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

4 - C

ontro

lF4

- Fol

iar

spra

y of

Mal

eic

acid

@ 0

.1%

at f

low

er in

itiat

ion

stage

F5-

Con

trol

DA

S –

Day

s af

ter s

owin

g

260 LEGUME RESEARCH - An International Journal

Tab

le 5

: Rhi

zosp

here

P- S

olub

ilize

rs p

opul

atio

n (N

umbe

r X

104

g-1 in

soy

bean

at d

iffer

ent g

row

th s

tage

s as

influ

ence

d by

bio

ferti

lizer

s an

d fo

liar

appl

icat

ion

of o

rgan

ic a

cids

Not

e: M

eans

fol

low

ed b

y th

e sa

me

low

er c

ase

lette

rs in

a c

olum

n do

not

diff

er s

igni

fican

tly b

y D

MRT

(P=0

.05)

B1

- PSB

F1- F

olia

r spr

ay o

f H

umic

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

2 - V

AM

F2- F

olia

r sp

ray

of L

ecith

in @

0.1

% a

t flo

wer

initi

atio

n sta

geB

3 - P

SB+V

AM

F3- F

olia

r sp

ray

of C

itric

aci

d @

0.1

% a

t flo

wer

initi

atio

n sta

geB

4 - C

ontro

lF4

- Fol

iar

spra

y of

Mal

eic

acid

@ 0

.1%

at f

low

er in

itiat

ion

stage

F5-

Con

trol

DA

S –

Day

s af

ter s

owin

g

higher rhizobium count at 60DAS and at harvest (35.0 and27.67 X 104 g-1). Significantly lower rhizobium count wasrecorded by uninoculated control (16 and 17.0 X 104 g-1)(Table 4). Microorganisms with phosphate solubilizingpotential increase the availability of soluble phosphate andenhance the plant growth by improving biological nitrogenfixation. Pseudomonas spp. enhanced the number ofnodules, dry weight of nodules, yield components, seedyield, nutrient availability and uptake in soybean crop asreported by Sharma et al. (2011). Adequate supply of Pwith Rhizobium strains (mixture inoculation) play animportant role in physiological and developmentalprocesses in plant life and the favourable effect on nutrientuptake accelerate the growth processes, which ultimatelyresulted in increased seed yield of the crop. Increase in Ncontent in seed, shoot and total N uptake due to Rhizobiuminoculation was mainly due to significant increase innodulation, resulting in higher accumulation of N due toatmospheric N2 fixation. Higher P uptake with Rhizobiuminoculation was due to the ability of applied rhizobium tosolubilize precipitated P components thereby increased Puptake in plants as reported by Fatima et al. (2007). Higheravailable P due to the solubilization and inoculated PSBmight cause enhancement of nutrient uptake and populationof nitrogen fixers.

Similarly, at 30, 60 DAS and at harvest, dualinoculation of PSB+VAM recorded significantly higher P-Solubilizers population (19.86, 29.05 and 21.46 CFU x104 g -1 soil) than inoculation of PSB (16.10, 25.10 and 20.93CFU x 104 g -1 soil) and VAM alone (11.29, 17.19 and 16.23CFU x 104 g-1 soil) compared to the uninoculated control (9.90,16.08 and 14.67 CFU x 104 g-1 soil) (Table 5). The greateryield response due to dual inoculation of VAM fungus andPSB than with PSB alone can be attributed to the activity ofthe VAM fungus in transporting extra phosphorus solubilizedby PSB from and beyond the root zone into the plant rootswhich in the absence of VAM hyphae gets refixed by soilconstituents during the course of slower diffusion towards plantroots. Increase in the PSB population enhances the phosphorousavailability to plants by lowering soil pH and by microbialproduction of organic acids and mineralization of organic Pby acid phosphatases. The phosphate- solubilizing bacteriapromoted root colonization when associated with mycorrhizalfungi. These organisms besides providing P also facilitate thegrowth of plants by improving the uptake of nutrients andstimulating the production of phytohormones. Son et al., (2007)revealed that inoculation with PSB enhanced beneficialbacterial count and their communities in the soils and improvedthe nutrient uptake.

Volume 39 Issue 2 (2016) 261

CONCLUSIONCombined application of biofertilizers and organic

acids (PSB+VAM in combination with foliar spray of 0.1%humic acid) along with recommended fertilizer proved effective

in significantly enhancing the yield, microbial activity andnutrient uptake by soybean. Dual inoculation of PSB+VAMand foliar spray of 0.1% humic acid recorded significantlyhigher rhizobium and P- solubilizer population in soil.

REFERENCESChitale, S., Sarawgi, S. K. and Sandeep, (2012), Effect of phosphorus application along with PSB, Rhizobium and VAM on

P fractionation and productivity of soybean. Indian J. Agron., 57(1): 55 - 60.Dixit, R. S. and Elemathi, (2007), Effect of foliar application of DAP, micronutrients and NAA on growth and yield of green

gram (Vigna radiata. L). Legume Res., 30(40): 305-307.Fatima, Z., Zia, M. and Chaudhary, M. F., (2007), Interactive effect of Rhizobium strains and P on soybean yield, nitrogen

fixation and soil fertility. Pak. J. Bot., 39: 255-264.Lee, Y. S. and Bartlett, R. J., (1976), Stimulation of plant growth by humic substances. Soil Sci. Soc. Am. J., 40: 876-879.Panse, V. G. and Sukhatme, P. V., (1967), Statistical Methods for Agricultural Workers, ICAR., Publication New Delhi, 359.Schmidt, R. E. and Zhang, Z., (1998), How humic substances help turfgrass grow. Golf Course Management. 66(7): 65-67.Sharma, K. N. and Namdeo. K. N., (1999), Effect of biofertilizer and phosphorus on NPK content, uptake and seed quality

of soybean and nutrient status of soil. Crop Res., 17(2): 164-169.Son, Diep, C. N., Truong, T. M. and Tran, T. A. T., (2007), Effect of co-inoculants (bradyrhizobia and phosphate solubilizing

bacteria) liquid on soybean under rice based cropping system in the mekong delta. Omonrice, 15: 135-143.Sharma, U. C., Datta, M. and Sharma, V., (2011), Effect of phosphorus on the yield and nutrient uptake by soybean cultivars

on acidic soil. J. Soil Sci., 1: 45-48.