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RESPONSE OF MAIZE GROWN IN ANALFISOL OF SRI LANKA TO INOCULANTSOF PLANT GROWTH PROMOTINGRHIZOBACTERIAPanachali Katulanda a & Chandanie P. Rajapaksha ba Department of Soil Science , University of Saskatchewan ,Saskatoon , Canadab Deptartment of Soil Science , University of Peradeniya ,Peradeniya , Sri LankaAccepted author version posted online: 03 Aug 2012.Publishedonline: 05 Oct 2012.

To cite this article: Panachali Katulanda & Chandanie P. Rajapaksha (2012) RESPONSE OFMAIZE GROWN IN AN ALFISOL OF SRI LANKA TO INOCULANTS OF PLANT GROWTH PROMOTINGRHIZOBACTERIA, Journal of Plant Nutrition, 35:13, 1984-1996, DOI: 10.1080/01904167.2012.716891

To link to this article: http://dx.doi.org/10.1080/01904167.2012.716891

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Journal of Plant Nutrition, 35:1984–1996, 2012Copyright C© Taylor & Francis Group, LLCISSN: 0190-4167 print / 1532-4087 onlineDOI: 10.1080/01904167.2012.716891

RESPONSE OF MAIZE GROWN IN AN ALFISOL OF SRI LANKA TO

INOCULANTS OF PLANT GROWTH PROMOTING RHIZOBACTERIA

Panachali Katulanda1 and Chandanie P. Rajapaksha2

1Department of Soil Science, University of Saskatchewan, Saskatoon,Canada2Deptartment of Soil Science, University of Peradeniya, Peradeniya, Sri Lanka

� Field experiments were conducted to assess the ability of rhizobacterial inoculants to enhancegrowth and yield of maize. Performances of two phosphorus (P)-solubilizing bacteria in combina-tion with a fertilizer mixture containing rock phosphate and triple super phosphate (PFM), and fivediazotrophs combining either with 150 kg or 100 kg nitrogen (N) ha−1 supplied as urea were com-pared with non-inoculated-fertilized controls. Shoot P and N and soil available P and N contentswere assessed and shoot biomass and ear weights were recorded at harvest. Pseudomonas cepa-cia resulted in significantly higher available P (51 mg P kg−1 soil), P accumulation (3.6 g kg−1

dry matter) and 13% increase in shoot biomass over control. Azospirillum sp. and dual inocu-lant comprising Enterobacter agglomerans + Agrobacterium radiobacter led to significantlyhigher available N (74{94 mg kg−1 soil) and 19 to 26% increase in shoot biomass over the control.However, inoculants did not increase the yield significantly.

Keywords: diazotrophs, maize, phosphorous solubilization, rhizo-bacteria, tropical soil

INTRODUCTION

There is a growing trend in the utilization of biofertilizers in agriculturedue to their low cost and less adverse impacts on environment in comparisonto chemical fertilizers (Brockwell and Bottomley, 1995). Biofertilizers aregenerally formulated with plant growth promoting rhizobacteria (PGPR)that inhabit rhizosphere soil, rhizoplane or endo-rhizosphere (Kloepper,1993). The common beneficial traits exhibited by PGPR are dinitrogen fix-ation (Araragi and Tangcham, 1979; Bashan et al., 2004), solubilizationof sparingly soluble forms of phosphorus (P) (Illmer and Schinner, 1995;Gyaneshwar et al., 2002), and modifyication of root architecture through

Received 10 May 2010; accepted 15 March 2012.Address correspondence to Chandanie P. Rajapaksha, Department of Soil Science, Faculty of Agri-

culture, University of Peradeniya, 20400, Peradeniya, Sri Lanka. E-mail: chandir@pdn.ac.lk

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phyto-hormone production and thereby increasing of nutrient acquisition(El-khawas and Adachi, 1999).

Oliveira et al. (2009) found that the diversity of P-solubilizing mi-croorganisms associated with maize cultivars was very high. They suggestedthat the differences in P use efficiency between cultivars may be gov-erned by the interactions between PGPR and plant. The populations ofselected PGPR may increase in the rhizosphere following their inoculationto seeds or planting material resulting in better crop growth and higheryields. The positive impact of PGPR inoculants have been reported onrice (Rodrigues et al., 2008), pulses (Gaind and Gaur, 1991) and lettuce(Chabot et al., 1996). Growth promotion has also been demonstrated bymaize when seeds were inoculated with Rhizobium sp. and Pseudomonas sp.(Chabot et al., 1996), Pseudomonas aurantiaca (Rosas et al., 2009), Enter-obacter agglomerans (Laheurte and Berthelin, 1988) and Azospirillum lipoferum(Zemrany et al., 2007) in vitro and under field conditions. These studiesconcluded that yield and growth enhancement of maize through the in-oculation of PGPR is possible and it is a viable technique that enablesreducing of fertilizer inputs while increasing fertilizer use efficiency bythe crop.

In Sri Lanka, maize is grown in maize-field crop or rice-maize crop rota-tions in fertile to moderately fertile soils using chemical fertilizers as the mainsource of nutrients. Crop residues and manures are rarely incorporated tothese fields although a significant yield increase has been reported due toapplication of poultry manure (de Silva et al., 2005). Although biofertilizershave been identified as a technique of increasing nutrient use efficiencyof maize in different geographical locations (Chabot et al., 1996; Zemranyet al., 2007), the potential of using biofertilizers has not been investigatedin these cropping systems. This study was therefore undertaken to isolate in-digenous PGPR associated with maize and to assess their impact on growthand yield under field conditions.

MATERIALS AND METHODS

Isolation of PGPR from Rhizosphere and Screening

Rhizospheric bacteria were isolated from rhizosphere soils of maizeplants that were at the reproductive stage and grown in several fields inMahailluppallama, Sri Lanka. Soils in this area are classified as Rhodustalfs.The pH (1:2.5 water) of the rhizosphere soils was 7.2 and available P andN contents were 13 mg P kg−1 and 25 mg N kg−1 soil, respectively. Soilsthat were firmly attached to roots were separated and soil dilution series wasprepared using saline water as the diluent. Soil extractions from appropriatedilution levels were inoculated onto agar plates that contained tryptic soybroth (TSB), N-free medium for Azotobacter type free living N fixers (Brown

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et al., 1962), and N-free semi-solid medium for Azospirillum species (Okonet al., 1977).

The most abundant 20 single colonies on agar plates were tested fortheir ability to solubilize P and fix N2. The P solubilizing ability of bacteriawas assessed in solidified and broth media of TSB amended with calciumphosphate [Ca3(PO4)2] (0.4%). Presence of halos around colonies on agarmedium was considered as a positive indication of P solubilizing ability. Thepositive strains were further assessed in Ca3(PO4)2 amended broth mediumby incubating on a rotary shaker for eight days. Aliquots from broths weretaken on 2, 4, 6 and 8 days after inoculation and analyzed for pH using glasselectrodes and for soluble P content by colorimetrically (Murphy and Riley,1962). Atmospheric N fixing ability of the strains isolated from N-free mediawere assessed using the acetylene reduction assay (Rodrigues et al., 2008).Broth medium was incubated under 10% acetylene in the headspace andincubated for 48 hours in the dark. Presence of ethylene was detected by gaschromatograph using flame ionization detector and Poropak column (Shi-madzu 14B, Shimadzu, Kyoto, Japan). In addition, ability to produce indole-3acetic acid (IAA) type compounds was assessed for cell free extracts usingSalkowsky reagent as described by Gordon and Weber (1950). Phospholipidfatty acid (PLFA) of pure cultures were extracted following the proceduredescribed in the MIDI protocol (MIDI, Newark, DE, USA) and the composi-tion of PLFA was analyzed using gas chromatograph (Hewlett-Packard 6890A, Hewlett-Packard Company, Palo Alto, CA, USA) The taxonomic identi-fication of bacteria was carried out based on PLFA profiles using Sherlockmicrobial identification system version 4.5 (MIDI).

Inoculant Preparation and Field Experiment

Based on the abundance and other beneficial traits, two P solubilizingbacteria and five diazotrophs were chosen to formulate inoculants. Theywere grown in TSB broth medium by incubating on a rotary shaker overnight. The population densities were adjusted to 107–108 mL−1 and dualinoculants were prepared by mixing equal volumes of two auxinic culturesprior to the seed soaking. Then, 70 seeds were soaked in 50 mL of brothfor 2 h period prior to sowing. Those seeds were again mixed with a carrier,which was prepared with finely ground and sterilized maize leaves beforeseeding. About 26 g of carrier was treated with 20 mL of broth and thenmixed with seeds.

The experiments were carried out at the field crop research station,Mahailluppallama, Sri Lanka in a field under field crop rotation in Mahaseason (April–September), 2007 and at the University Sub Campus in Yalaseason (October–March), 2008 in a field on rice-field crop rotation. Meanrainfall and temperature were 227 mm and 28.7◦C, respectively in 2007 and258 mm, and 26.2◦C, respectively in 2008. Soil in the area has a sandy clay

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loam texture and classified as Rhodustalfs. Soil organic matter content andpH of soils used for the first trial were 1.2% and 6.8, respectively, and soil Pand N contents were 16 mg P kg−1 and 22 mg N kg−1 soil, respectively. Soilsof the second trial were characterized by 1.8% organic matter, soil pH of 7.0and 26 mg N kg−1 soil. Both experiments were arranged in a randomizedcomplete block design with three replicates. The plot size was 1.5 m × 2.1 mwith five rows, and spacing for inter-row and intra-row were 60 cm and 30 cm,respectively. Fertilizer recommendation followed was 150:30:45 kg ha−1 asN: P2O5: K2O applied as urea, triple super phosphate (TSP) and muriate ofpotash, respectively. In the field experiment of 2007, P-solubilizing bacterialinoculants were tested in plots added with a P fertilizer mixture (PFM)containing triple super phosphate (TSP) and rock phosphate (RP) at aratio of 1/2 TSP and 1/2 RP, based on P2O5 content. The RP powder wasobtained from a local phosphate deposit located at Eppawela, Sri Lanka,which has about 38% P2O5 and citric acid solubility of less than 12%. Seventreatments were laid down in a randomized complete block design: non-inoculated control combined with recommended fertilizer, non-inoculatedcontrol combined with PFM (PFM-control), two dual inoculants formulatedwith P solubilizing bacteria and combined with PFM, two dual inoculantsand a single inoculant formulated with diazotrophs each combined withrecommended fertilizer. Three seeds (variety ‘Sampath’) were seeded perhill. Weeding was done manually and plots were irrigated twice a week.Seedlings were thinned out at 2–4 leaf stage leaving one per hill. In thesecond experiment, four treatments were laid down; two non-inoculatedcontrol treatments with recommended N fertilizer (150 kg ha−1) or 2/3 ofit and two inoculated treatments with 2/3 of the recommended dose of Nfertilizer. All the plots received the recommended doses of P and K andother agronomic practices imposed were as described for the first field trial.

Sampling and Analysis of Soil and Plant

In the first experiment, soil and plant samples were collected at latevegetative phase with an average of 12 leaves per plant (7th week afteremergence), at the tasseling stage when the last branch of the tassel wasvisible and silk has not been immerged (9th week) and at the initial stage ofmaturity when the grain filling was completed (11th week). In the secondexperiment, available N in soil and shoot N were assessed before applying thetop dressing of urea in the 6th week when plants were having an average of10 leaves and at the tasseling stage, respectively. The ear weight was recordedafter harvesting.

Composite soil sample for each plot was obtained by mixing soils takenfrom the root zone of three plants up to a depth of 10 cm. The availablesoil P was extracted with 0.5 M sodium bicarbonate (NaHCO3) and assayedby colorimetrically (Murphy and Riley, 1962). Soil available N [ammonium

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(NH4)-N and nitrate (NO3)-N] were extracted with 2 M potassium chloride(KCl) and quantified by the Kjeldahl distillation method (Bremer, 1965). Ateach sampling, the 6th leaf from the base was taken for P analysis and ear leafwas taken for N analysis. Leaves were taken from three plants representingeach plot, rinsed with distilled water, dried at 60◦C and ground using amechanical grinder. Ground leaf samples were combusted in a porcelaincrucible at 550◦C for 24 hours. Ash was dissolved in aqua regia and analyzedfor P by colorimetrically (Murphy and Riley, 1962). Ground leaf sampleswere digested in concentrated H2SO4 acid and analyzed for total N by theKjeldahl distillation method. Plant roots were collected at the 7th week fromthree plants of each plot and assessed for mycorrhizae infection by stainingwith trypan blue (Brundrett et al., 1996). Fifteen roots representing eachplot was examined under a compound microscope.

Statistical Analysis

Field experiments were arranged in a randomized complete block designwith three replicates. Analysis of variance was performed using the PROCGLM procedure for each N and P experiments at a given sampling separatelyusing SAS statistical software (SAS Institute, Cary, NC, USA). Means werecompared using Duncan’s New Multiple Range test at a significant level of0.05. Pearson correlation co-efficient were calculated using SPSS (SPSS Inc.,Chicago, IL, USA) for selective variables.

RESULTS AND DISCUSSION

The taxonomy and growth promoting traits of tested rhizobacteria aregiven in Table 1. Pseudomonas cepacia, Pseudomonas caryophylli were the pre-dominant and efficient PSB that inhabit maize rhizosphere whereas Agrobac-terium radiobacter, Enterobacter agglomerans, Flavobacterium jonsoniae, Pseudoal-teromonas haloplanktis and Azospirrllum sp. were the predominant N fixers.Among seven isolates evaluated, only three secrete IAA type compounds

TABLE 1 Taxonomy and growth promoting traits of tested bacterial isolates exhibited in vitro

Taxonomy (Similarity index) P-solubilization¶ N fixation IAA production

Pseudomonas caryophylli (0.75) + − −Pseudomonas cepacia (0.87) ++ − +Enterobacter agglomerans (0.84) + + +Agrobacterium radiobacter (0.65) − + −Flavobacterium jonsoniae (0.72) − + −Pseudoalteromonas haloplanktis (0.50) − + −Azospirillum sp. − + ++

¶− negative reaction; + to ++ high activity.

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FIGURE 1 Changes in pH and soluble P concentration in the broth medium inoculated with P solubi-lizing bacteria. Solid lines and broken lines represent P concentrations and pH, respectively.

(Table 1). Several PGPR isolated in this study have been reported previouslyin the rhizosphere of different maize cultivars. They are Pseudomonas spp.and Enterobacter sp., (Lalande et al., 1988; Chabot et al., 1996), P. putida andAzospirillum (Mehnaz and Lazarovits, 2006), P. cepacia (Oliveira et al., 2009)and E. agglomerans (Laheurte and Berthelin, 1988).

The concentrations of soluble phosphate released from 4 g of Ca3(PO4)2

by P. cepacia and P. caryophylli were 55 and 64 mg L−1, respectively on the 8thday of incubation (Figure 1). These values are higher than 100 mg P L−1 re-leased from 10% Ca3(PO4)2 by Aspergillus niger (Illmer and Schinner, 1994)but lesser than 70.6 mg P L−1 released into a growth medium amended with1.5 g Ca3(PO4)2 by P. cepacia (Oliveira et al., 2009) if the comparisons aremade based on the fraction of P released from the total P in the broth. Theconcentration of soluble phosphate in broth media increased over the timewith a simultaneous reduction in pH from 6.0 to 4.2 in both treatments sug-gesting that acidulation is the main mechanism of P solubilization (Figure 1).This has been demonstrated previously by several authors and accounted forthe release of protons through respiration, NH4

+ assimilation, and secretingof organic acids such as citric (Illmer and Schinner, 1995; Illmer et al. 1995;de Freitas et al., 1997).

In the field experiment conducted with P solubilizing bacteria, avail-able P concentrations at the late vegetative phase (7th week) and tasselingstage were more-or-less comparable but a drastic reduction was observed atthe maturity stage due to translocation of P to pods (Table 2). At the latevegetative phase, available P varied from 36 to 51 mg P kg−1 soil and they

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Inoculants of Rhizobacteria for Maize 1991

were 2-to 3-fold higher than the initial value (16 mg P kg−1 soil). P. cepaciashowed a significantly higher available P (51 mg kg−1 soil) than the PFM-control (37 mg kg−1 soil) and treatment with the recommended fertilizer(44 mg kg−1 soil) demonstrating that it efficiently mobilizes P from RP atthe late vegetative phase. In contrast, available P concentration in soils of P.caryophylli treatment remained comparable to that of the PFM- control (Ta-ble 2) in spite of demonstrating a similar P solubilizing activity to P. cepaciain vitro (Figure 1).

The shoot P contents of the treatment with recommended fertilizer was2.7 mg kg−1 dry matter and remained significantly lower than the inoculatedtreatments (3.6–3.7 mg kg−1 dry matter) and PFM- control (3.4 mg kg−1 drymatter) at the vegetative phase (Table 2). This difference was resulted inirrespective of a higher dry weight observed in ear leaf in inoculated samples(2.3–2.6 g) than the treatment with the recommended fertilizer (1.79 g)(data not shown). Perhaps, plant nutrient acquisition may be facilitated bythe added inoculants, particularly by P. cepacia through releasing of IAA. Itshould also be noted that mycorrhizae infection was negligible in all treat-ments at this growth stage (data not shown). This may imply that the P avail-ability for the crop was high enough to retard infection despite substitutingof TSP partially by RP. Guttay and Dandurand (1989) reported that mycor-rhizae infection in maize was reduced with the increasing soil extractableP but did not affect P uptake by maize. At the maturity stage, inoculatedtreatments showed comparable shoot P contents to that of recommendedfertilizer indicating that both inoculants are competent in mobilizing of Pefficiently at this growth stage. However, in the PFM-control, accumulationof P was comparatively lower (1.8 mg kg−1 dry matter) than the inoculatedtreatments implying that solubilization of RP by the indigenous flora is notefficient in comparison to the tested inoculants.

Differences observed in soil available P and shoot P accumulation led tosignificant differences in plant height and shoot dry weight between treat-ments at harvest (Table 2). Plant height (169 m) and dry matter production(318 g per plant) in P. cepacia treatment were significantly higher than the re-maining treatments. The growth enhancement was about 10% for the heightand 13% for the dry matter over the treatment with the recommended fertil-izer. Nevertheless, ear weight varied from 280 to 290 g per plant and did notdiffer significantly between treatments (Table 2). Interestingly, plant heightmeasured at the late vegetative phase correlated with ear weight (r = 0.69,P = 0.012) implying that crop growth at the late vegetative phase is criticalfor the yield establishment. However, lack of significant relationship betweenplant parameters and P availability or P uptake may suggest that the growthis not limited by the available P in soil. These observations further reveal thatpartial substitution of TSP by RP did not reduce P availability to a level thatwould retard the crop yield. Perhaps the tested variety may be efficient in Pacquisition and may have obtained adequate P from PFM.

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Chabot et al. (1998) reported that P solubilizing Rhizobium strain en-hanced dry matter content of maize in soil with a very high P availability(400 kg ha−1) than in nutrient poor soils. In the view of this finding, soilswhere the field trials were conducted may be designated as a high fertilesoil for the maize crop. Poor infection of roots by mycorrhizae may be con-sidered as a supportive evidence in this regard. There is also evidence thata Rhizobium strain enhanced maize growth only when the recommendedfertilizer was applied whereas Pseudomonas sp. performed well at half therecommended P fertilizer (Chabot et al., 1998). Accordingly, difference inthe plant growth promotion by the two strains tested in this study may bepartly attributed to the difference in their optimum nutrient requirementsnecessary for an efficient P mobilization. Perhaps, the nature of interactionmay also differ between the tested maize variety and two inoculants. Mehnazand Lazarovits (2006) demonstrated that P. putida had enhanced growth ofonly selective varieties of corn. Several studies reported that yield has beenincreased in lettuce and canola by P solubilizing bacterial inoculants in lessfertile soil without increasing the P uptake (de Freitas et al., 1997; Chabotet al., 1998). In this study however, there was no yield increase despite anincrease in P uptake. Perhaps further increase in yield may be limited by theavailability of nutrients other than P, particularly by micronutrients, whichhas been observed previously (de Silva et al., 2005).

In the experiment conducted with the diazotrophs in 2007, compara-tively high N levels (80–108 mg kg−1) were recorded at the late vegetativephase than the maturity phase (43–70 mg kg−1) (Table 3). At the late vege-tative phase, significantly higher available N was reported for the treatmentswith dual inoculant comprising A. radiobacter + E. agglomerans (94 mg kg−1

soil) and single inoculant Azospirillum sp. (74 mg kg−1 soil) in comparison to50 mg kg−1 soil reported for the treatment with the recommended fertilizer.This may imply that N fixation is enhanced as soil depletes with N. However,variability in tissue N contents among treatments was not parallel to thatof available N at all three sampling (Table 3). The tissue N contents weresignificantly higher for the treatment with recommended fertilizer than theinoculated treatments at the vegetative phase despite the fact that the latterreceived the same quantity of N fertilizer and recorded high available Ncontents. One possible explanation for this is the dilution effect resulted bytheir higher shoot biomass contents.

Significant differences were observed in plant height and shoot dryweight in inoculated treatments at harvest implying that added inoculantshave enhanced crop growth. Dual inoculant 1 showed the highest plantheight (159 cm) and was comparable to treatments with dual inoculant2 (147 cm) and recommended fertilizers (149 cm) (Table 3). Increase inshoot dry weight was 19% for the treatment with dual inoculant 1 (336 g perplant) and 26% for Azospirillum sp. (356 g per plant), respectively. Growthenhancement by Azospirillum sp. has been well document for a range of crops

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ulan

t1∗

80±

4.6

94a±

4.9

54±

2.0

14ab

±1.

421

b±

2.1

26±

2.7

159a

±0.

935

6a±

3328

8±

6.3

Dua

lin

ocul

ant2

∗∗10

4±

5.2

57c±

3.1

43±

5.4

15ab

±1.

524

ab±

2.4

24±

2.4

147ab

±5.

126

9b±

2629

6±

13.2

Azo

spir

illum

sp.

108

±4.

274

b±

8.0

70±

3.3

13b±

1.3

24ab

±2.

425

±2.

513

5b±

15.3

336ab

±4

303

±11

.4—

93±

1250

c±

7.2

48±

3.2

18a±

1.8

26a±

2.6

25±

2.6

149ab

±2.

928

2b±

1128

7±

6.8

∗ Eag

glom

eran

s+

A.r

adio

bact

er.

∗∗F

joho

nson

iae

+P.

halo

plan

ktis

.V

alue

ssh

arin

gth

esa

me

lett

erin

aco

lum

ndo

not

diff

ersi

gnifi

can

tatP

<0.

05

1993

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4

1994 P. Katulanda and C. P. Rajapaksha

including maize. Mehnaz and Lazarovits (2006) demonstrated that growthof selective varieties of corn was enhanced by Azospirlillum lipoferum. Zemranyet al. (2007) also reported that an increase in root biomass, length and sur-face of maize plants inoculated with Azospirlillum lipoferum CRT1. In anotherstudy, a significant impact of PGPR was evident in the early growth stages ofbeetroot and later resulted in higher yield as well (Cakmakci et al., 2005). Inthis study however, a significant growth enhancement was not accompaniedwith an increase in the ear yield, which varied from 287 to 303 g per plant(Table 3). Although yield data illustrate that N was not limiting for the yieldestablishment, soil available N content in the 9th week at tasseling correlatedwith the plant height (r = 0.74, P = 0.009) and dry matter production (r =0.71, P = 0.009) at harvest.

In the second field experiment, dual inoculant 1 and Azospirillum sp.which promoted plant growth were re-tested for their ability to provideadequate N to the crop when soil was applied with 1/3 of recommendedN fertilizer (Table 4). Soil samples were taken before applying urea as thetop dressing. Consequently, positive impacts of Azospirillum treatment wereevident for available N concentration, which remained significantly higherthan the non-inoculated treatment added with the 2/3 of recommendedN dose (Table 4). At the tasseling stage, Azospirillum treatment resulted insignificantly higher N accumulation than the treatment with recommendedfertilizer suggesting that they improve N acquisition by the maize crop. Bothatmospheric N fixation and enhanced N uptake through a modified rootsystem may be responsible for the observed increase in shoot N but theirrelative contribution is not known. Similar to the previous season, there wasno significant difference in the ear weight between treatments, which variedfrom 201 to 227 g per plant. Yield data thus suggest that reduction in Nfertilizer dose by 1/3 did not affect yield significantly in the experimentalsoils because the studied soil was able to provide required N to support thecrop.

Results in general provide evidence that yield retardation due to substi-tuting 50% of the recommended P2O5 by RP or reducing recommended N

TABLE 4 Mean values (n = 3) of available N in soil and ear weight of maize in treatments inoculatedwith diazotrophs, in Yala 2008

N content

N-fertilizer Inoculant Soil Shoot Ear weight

(mg kg−1) (g per plant)2/3 N E agglomerans+ A. radiobacter 65ab ± 4.0 24b ± 0.22 203 ± 9.12/3 N Azospirillum sp. 67a ± 1.3 27a ± 0.41 227 ± 6.32/3 N — 60b ± 1.0 21b ± 0.13 191 ± 17.1Recommended — 70a ± 1.3 28a ± 0.33 216 ± 9.4

Means with different letters in a given column are significantly different at P <0.05.

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Inoculants of Rhizobacteria for Maize 1995

fertilizer by 1/3 was not significant in the experimental soils which is partlybe attributed to inherently high fertility of experimental soils resulted bythe building up of nutrients supplied through the chemical fertilizer. Poorroot infection by mycorrhizae also support the explanation that plant growthwas not limited by the P availability. Repeating the experiment in the samelocation for several seasons in order to exclude the residual effect of P andN fertilizers may be necessary to assess the true impact of inoculants. Inaddition, the competence of inoculants to promote plant growth needed tobe evaluated in the absence of fertilizer.

CONCLUSIONS

Neither reduction in N application from 150 kg ha−1 to 100 kg ha−1 norsubstitution of 50% of the recommended TSP (30 kg) by RP affect yieldof maize significantly in experimental soils. P. cepacia, Azospirillum and dualinoculant E. agglomerans + A. radiobacter enhanced shoot biomass produc-tion by 9 to 26% over the fertilized control but not the ear yield. Furtherstudies are necessary to assess the potential of growth promotion under fieldconditions with variable application rates of chemical fertilizers.

ACKNOWLEDGEMENTS

We acknowledge C.J. Perera, K.J. Jayasinghe and R.T. Ekanayake, FieldCrop Research Station, Mahailluppallama, Sri Lanka and Farm manager,University Sub-campus Mahailluppallama, for their corporation in conduct-ing field trials.

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