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Scientia Horticulturae 111 (2007) 371–377

Effect of plant growth promoting rhizobacteria on young apple

tree growth and fruit yield under orchard conditions

Rafet Aslantas a,*, Ramazan Cakmakci b,c, Fikrettin Sahin b,d

a Ataturk University, Faculty of Agriculture, Department of Horticulture, 25240 Erzurum, Turkeyb Ataturk University, Biotechnology Application and Research Center, 25240 Erzurum, Turkey

c Ataturk University, Ispir Technical Vocational School, 25900 Ispir/Erzurum, Turkeyd Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering,

Kayısdagi, 34755 Istanbul, Turkey

Received 18 November 2005; received in revised form 29 October 2006; accepted 5 December 2006

Abstract

The effects of rootstocks (M9 and MM 106), cultivars (Granny Smith and Stark Spur Golden) and growth promoting rhizobacteria (OSU-142,

OSU-7, BA-8 and M-3) on the tree growth and yield at apple (Malus domestica Borkh) trees were studied in a clay loam soil in the eastern Anatolia

region of Turkey in 2002–2004. Plant growth promoting rhizobacteria (PGPR) were capable of producing indole acetic acid (IAA) and cytokinin,

but three of them (OSU-7, BA-8 and M-3) were also able to dissolve phosphate. Maximum shoot number of apple trees was found after inoculation

with BA-8 followed by OSU-7 and M-3. All the inoculated PGPR strains contributed to the increase in fruit yield of apple when compared to

control but it was strongly depended on rootstocks, cultivars and treatments. Plant growth responses were variable and dependent on bacterial

strains, rootstock and cultivar and growth parameters evaluated of young apple trees. Newly planted apple trees inoculated with OSU-142, OSU-7,

BA-8 and M-3 PGPR increased average shoot length by 59.2, 18.3, 7.0 and 14.3% relative to the control and fruit yield by 116.4, 88.2, 137.5 and

73.7%, respectively. Bacterial inoculation increased shoot diameter from 7.0 to 16.3% when compared to control. The production of plant growth

hormones has been suggested as one of the mechanisms by which PGPRs stimulate young apple sapling growth. The growth-promoting effect

appears to be direct, with possible involvement of the plant growth regulators indole-3-acetic acid and cytokinin. In view of environmental

pollution due to excessive use of fertilizers and high costs of the production of fertilizers, PGPR strains tested in our study have potential to be used

for the sustainable and environmentally benign horticultural production.

# 2006 Elsevier B.V. All rights reserved.

Keywords: IAA; Cytokinin; Plant growth promoting rhizobacteria; Apple trees; Tree growth; Fruit yield

1. Introduction

Nitrogen and phosphorus are essential nutrients for plant

growth and development. In irrigated apple orchard systems,

the magnitude and timing of plant demand for nitrogen (N) and

retention of N in the root zone to allow root interception are

important factors for efficient management of N fertilizer

(Neilsen and Neilsen, 2002). While N application can supply

sufficient nutrients to improve plant production, it also leads to

a worldwide concern about environmental contamination

resulting from excessive nitrate leaching (Dong et al., 2005).

Large quantities of chemical fertilizes are used to replenish soil

* Corresponding author. Tel.: +90 442 231 1489; fax: +90 442 236 0958.

E-mail address: aslantas@atauni.edu.tr (R. Aslantas).

0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2006.12.016

N and P, resulting in high costs and severe environmental

contamination. N2-fixing and P-solubilizing bacteria may be

important for plant nutrition by increasing N and P uptake by

the plants, and playing a significant role as plant growth

promoting rhizobacteria (PGPR) in the biofertilization of

crops. Increasing and extending the role of biofertilizers would

reduce the need for chemical fertilizers and decrease adverse

environmental effects.

Various mechanisms may be involved, such as the release of

metabolites that directly stimulate growth. The mechanisms by

which PGPR promote plant growth are not fully understood, but

are thought to include: (a) the ability to produce plant

hormones, such as auxins (Jeon et al., 2003; Egamberdiyeva,

2005), cytokinins (Timmusk et al., 1999; Garcıa de Salamone

et al., 2001), gibberellins (Gutierrez-Manero et al., 2001), and

ethylene (Glick et al., 1995); (b) asymbiotic N2 fixation (Sahin

R. Aslantas et al. / Scientia Horticulturae 111 (2007) 371–377372

et al., 2004); (c) solubilization of inorganic phosphate and

mineralization of organic phosphate and/or other nutrients (de

Freitas et al., 1997; Jeon et al., 2003); and (d) antagonism

against phytopathogenic microorganisms by production of

siderophores, the synthesis of antibiotics, enzymes and/or

fungicidal compounds and competition with detrimental

microorganisms (Dobbelaere et al., 2002; Dey et al., 2004;

Lucy et al., 2004). Trials with rhizosphere associated plant

growth-promoting N2-fixing and P-solubilizing bacteria indi-

cated growth and yield increases in apricot (Esitken et al.,

2003), peanut (Dey et al., 2004), cactus (Puente et al., 2004),

blanket flower (Gadagi et al., 2004), holm-oak and pine (Garcıa

et al., 2004) and conifer species (Chanway et al., 2000; Bent

et al., 2002) in long-term condutions.

Economic and environmental benefits can include increased

income from high yields, reduced fertilizer costs and reduced

emission of the greenhouse gas, N2O as well as reduced

leaching of NO3�-N to ground water. Plant growth promoting

bacteria are important in managing plant growth because of

their effects on soil conditions, nutrient availability, tree growth

and yields. However, information is not available on the PGPR

in apple orchard systems under field conditions. This is the first

report yield and growth of young apple trees by phosphate

solubilizer and phytohormones-producing PGPR. The aims of

the present study were to investigate the effectiveness of PGPR

and nutrition on young apple trees under orchard conditions,

and to evaluate the effect of inoculation on growth and fruit

yield of apple cv. Granny Smith and Stark Spur Golden grown

on M9 and MM 106 rootstock.

2. Materials and methods

2.1. Bacterial strains

Four PGPR strains (Bacillus OSU-142, Bacillus M-3,

Burkholderia OSU-7 and Pseudomonas BA-8) originally

selected for their antifungal and antibacterial properties

(Esitken et al., 2003; Sahin and Miller, 1999; Sahin et al.,

2000) were investigated in the study. Nevertheless, Bacillus

OSU-142 was also the most effective N2-fixing bacteria in

previous field experiments with sugar beet and barley

(Cakmakci et al., 2001, 2006; Sahin et al., 2004), tomato

and pepper (Sahin et al., 2000) and apricot (Esitken et al.,

2003).

2.2. Quantification of IAA

All PGPR strains were tested for auxin production (IAA-

like substances), using the method of Bent et al. (2001). The

flasks were incubated for 18 h at 27 8C with 100 rpm rotary

shaking. Following this, 125 ml flasks containing 40 ml half-

strength tryptic soy broth (TSB), supplemented with 0, 0.1

and 25 mg tryptophan/ml were each inoculated with 1 ml of

each strains. After incubation for 48, 72 and 168 h, the

density of each culture was measured spectrophotometrically

at 600 nm, and then the bacterial cells were removed from the

culture medium by centrifugation. The level of indoles

present in the culture fluid was estimated colorimetrically.

The concentration of IAA in the bacterial eluates was

measured by using Salkowski’s reagent (50 ml 35%

HClO4 + 1 ml FeCl3). Each reaction mixture was centrifuged.

Absorbance at 530 nm in a Shimadzu Spectrophotometer UV-

1208 was measured. Bacterial cells were separated from the

supernatant by centrifugation at 10,000 rpm for 30 min. The

concentration of IAA in each culture medium was determined

by comparison with a standard curve. The IAA produced by

each strain was measured in triplicate. Samples were also

taken after 48, 72 and 168 h of growth for determination of

indole acetic acid, thin layer chromatography (TLC) and by

high performance liquid chromatography-mass spectrometry

(HPLC-MS) analysis. Separation of indole in ethyl-acetate

fraction was carried out in chloroform–ethyl acetate–formic

acid.

2.3. Cytokinin extraction and phosphate-solubilizing

capacity

The bacterial strains were also tested for cytokinin

production, using the method of Garcıa de Salamone et al.

(2001) and Arkhipova et al. (2005), and phosphate

solubilization capacity as described by Pal (1998) and

Mehta and Nautiyal (2001). One small colony of each strain

was placed in 9 ml of sterile solution to obtain a

homogeneous bacterial suspension. Three inoculated tubes

per strain were placed in a 30 � 1 8C shaker incubator for 7

days. Then, the tubes were centrifuged at 2800 � g for

20 min. The supernatants were filtered through 0.22 mm

Millipore membranes and stored at �20 8C until cytokinin

dihydrozeatin riboside (DHZR) production (average 96 and

168 h pure cultures). Immunoassays were performed as

described by Garcıa de Salamone et al. (2001), to determine

the amounts of the three cytokinins, isopentenyl adenosine

(IPA), trans-zeatin ribose (ZR), and DHZR, present in the

supernatants. Spots in TLC plates for cytokinin separation

were eluted with 9 ml of absolute methanol–water. The

filtered samples were transferred into injector vials. HPLC

was done in isocratic mode with ethanol–water (90:10, v/v)

with 5 nM ammonium acetate. Multiple reaction monitoring

was used for detection of the HPLC effluent (Prinsen et al.,

1995). The equipment used was a Hewlett Packard HPLC

equipped with an in-house 18 C micro column (10 cm �0.32 mm i.d., 5 mm packing).

All the pure cultures of PGPR strains were examined for

their phosphate solubilizing capacities in sucrose-tricalcium

phosphate agar media (Pikovskaya, 1948) by inoculating 1 ml

of 6-day-old culture (density 4 � 109) into 250 ml Erlenmeyer

flasks in triplicate containing 500 mg/ml of P as Rock

Phosphate (RP) at 30 8C � 1. After incubation for 6 days,

water soluble P was determined colorimetrically by the

vanadomolybdophosphoric yellow colour method (Jackson,

1970). The bacterial strains were characterized by morpholo-

gical, biochemical and physiological tests including pigment

production on nutrient agar medium and the Gram reaction

(Forbes et al., 1998).

R. Aslantas et al. / Scientia Horticulturae 111 (2007) 371–377 373

2.4. Orchard site, design and cultural practices

The experiment was carried out during 2002–2004 in the

Experimental Orchards of the Research Field in Coruh valley in

Erzurum in eastern Anatolia (298550N and 418160E at an

altitude of 1200 m) in order to evaluate the yield and growth of

young apple trees (Malus domestica Borkh., cv. Granny Smith

and Star Spur Golden) in relation to rootstock, cultivar and

plant growth promoting rhizobacteria. Apple is a major fruit

crop grown in the area (Guleryuz et al., 1998; Pırlak et al.,

2003). Plant growth was restricted to the period between April

and October with annual average temperature and total rainfall

of 10.5 8C and 447.8 mm in the region. The experimental soil

was a clay loam with organic matter content of 1.6% and with

2.7% free carbonate (pH 7.8). Available P2O5 and K2O contents

were determined as 51 and 1328 kg ha�1 in experimental area,

respectively.

The experiment was conducted using a completely

randomized factorial design (five treatments and two rootstock

and two cultivars). Treatments with three replicates (each

having three young apple trees) were as follows: (1) control

(without bacteria inoculation), (2) OSU-142, (3) OSU-7, (4)

BA-8, (5) M-3. There were five treatments, two rootstock (M.9

and MM 106), two cultivar (Granny Smith and Stark Spur

Golden) and three replicates totalling 60 plots.

In September 2001, old trees were removed and site was

ripped repeatedly with a chisel plow to 40 cm depth. First year,

the whole orchard received farmyard manure at 40 tonnes ha�1

6 months prior to planting in spring. The soil was ploughed

deeply and left until disk and rotary harrowing in spring. One-

year-old certificated (virus-free) apple trees are planted at

2 m � 2 m spacing in the spring of 2002. Trees were planted

within 1 day of inoculation using standard planting technique.

To homogenize growth in one 2nd year (April 2002), new

shoots at 60 cm above the grafted point were removed. Tree

heights were recorded at the time of planting. Weeds were

controlled around the base of the trees was done by repeated

hoing as required. No pesticide was applied.

2.5. Inoculation of PGPR strains

The bacterial strains were maintained by long-term storage

in nutrient broth with 15% glycerol at �80 8C prior to testing.

For this experiment, pure cultures were grown in 50% strength

tryptic soy broth (TSB) on a rotary shaker (120 rpm; 25 8C) for

3 days. Bacteria were then harvested by centrifugation (ca.

3000 � g for 10 in), washed and re-suspended in 10 mM sterile

phosphate buffer, pH 7 (SPB) to a density of 109 cfu ml�1 for

the bacterial strains. Trees were surface-sterilized prior to

inoculation by soaking in 25% commercial-grade bleach

(sodium hypochlorite) for 5 min, followed by thorough

washing under running tap water and air-drying aseptically

overnight at room temperature. In the field trial, 1-year-old,

average trunk diameter by 0.85 cm, uniform height and virus-

free young apple trees were inoculated with each of the PGPR

strains 1 day prior to planting. The bacterial inoculation

involved dipping the root system of the tree into a suspension of

each PGPR strains for 60 min, prior to planting. For the control

treatment without bacteria, trees were dipped into water

(Heinonsalo et al., 2004). PGPR applications were also made in

the fields by syringe inoculation, in which inoculum was

prepared using TSB cultures of each strain grown to stationary

phase. Cultures were harvested by centrifugation, washed in

20 mM SPB, and resuspended in an equal volume of fresh SPB.

Washed cultures were placed on ice and transported by air for

inoculation. Inoculation involved injection of 5 ml of bacterial

suspension (diluted with water approximately 109 cfu ml�1 for

strains) into the middle of the root plug using a sterile syringe

and needle. Control plants received 5 ml of diluted SPB with no

bacteria (Chanway et al., 2000).

2.6. Data collection and statistics

Shoot number, average and total shoot length, shoot and

trunk diameter and plant height were collected for all apple

trees in the fall of 2002. Yield of apple trees was measured only

at the end of the 2004 growing season since fruit was damaged

by hail in mid July in 2003. Leaf area was measured with a CI

202 portable digital area-meter in 2002. The length of shoots/

tree was measured in late October of each season (Aslantas,

1999). The data were subjected to analysis of variance to test for

rootstock, cultivar and treatment effects and their interactions

by using STATISTICA 5.1. Means were separated according to

Duncan Multiple Range Test.

3. Results

PGPR strains were capable of producing IAA, but the

amounts of IAA varied with bacterial species and tryptophan

concentration ranging from 3.4 to 26.8 mg IAA/ml culture. In

the absence of tryptophan supplements, the four PGPR

produced very low levels of IAA (Table 1). However, when

the four strains grew in the presence of 25 mg tryptophan/ml for

approximately 48–168 h, PGPR strains responded by produ-

cing higher levels of IAA (Table 1). Measurement of IAA by

HPLC confirmed that in the presence of a high concentration of

tryptophan (25 mg/ml), higher levels of IAA were produced by

BA-8 strain (26.8 � 2.7 mg/ml/OD600 unit), and lower level of

IAA produced by OSU-7 (14.3 � 1.7 mg/ml/OD600 unit).

All isolates were oxidase, nitrate reduction and catalase

positive and were also able to grow in N-free basal medium.

Differences in the proportion of each cytokinin produced

relative to the total (IPA + ZR + DHZR) were observed among

the strains data for 96–168 h is presented (Table 1). PGPR strain

Bacillus OSU-142, Burkholderia OSU-7, Pseudomonas BA-8

and Bacillus M-3 produced cytokinin by 8.6, 10.3, 19.8 and

9.7 pmol/ml�1, respectively. OSU-7, BA-8 and M-3 isolates

were capable of dissolving insoluble P, respectively, 13.7, 26.4

and 38.3 mg P solubilized/ml culture/day.

Rootstock had no effect on shoot number and diameter, plant

height and leaf area of young trees but there were interactions

between rootstocks and cultivars. These effects depend on

cultivars. The average shoot length and fruit yield of apples

were decreased by stock MM 106. In experiment, stock M9

Table 1

Phosphate solubilization, and cytokinin and IAA production of PGPR strains in the presence of various concentrations of tryptophan in culture media

Tryptophan (mg/ml) Bacillus OSU-142 Burkholderia OSU-7 Pseudomonas BA-8 Bacillus M-3

IAA production (mg/ml/OD600 unit)a

0 6.3 � 0.8 3.4 � 0.3 5.7 � 0.4 4.2 � 0.1

0.1 9.6 � 0.9 4.9 � 0.5 9.7 � 0.8 5.4 � 0.3

25 22.4 � 2.1 14.3 � 1.7 26.8 � 2.7 21.7 � 1.3

Cytokinin production pmol/ml (IPA + ZR + DHZR)b

8.6 � 0.9 10.3 � 1.6 19.8 � 2.7 9.7 � 0.8

P solubilization (mg P/ml culture/day)

ND 13.7 � 1.2 26.4 � 1.9 38.3 � 0.8

ND: not determined.a Data were the means of with three replicates IAA production in average 48, 72 and 168 h.b Cytokinin production in average 96 and 168 h pure cultures. IPA: isopentenyl adenosine, ZR: trans-zeatin ribose, DHZR: dihydrozeatin riboside.

R. Aslantas et al. / Scientia Horticulturae 111 (2007) 371–377374

decreased shoot number by 22% and increased shoot length and

fruit yield compared to MM 106 at 2 years old. Two years after

planting, there were no effects of cultivars on yield, but there

were interaction among R � C � T. These effects consisted of

rootstocks and treatments. However, PGPR still had positive

effects on the tested parameters of apple trees, but Granny

Smith had decreased shoot number, total shoot length, trunk

diameter and leaf area (Table 2).

Shoot number was greatest with BA-8 (e.g. 30.9% of

control) whereas maximal shoot length was with OSU-142

(Table 2). Bacterial strains increased fruit yield of young apple

Table 2

The effect of PGPR, cultivar and rootstock on the measured parameters of yield and v

of young apple trees were collected on 2-year-old apple trees in the fall of 2002,

Shoot

number

Average shoot

length (cm)

Total shoot

length (cm)

Shoot

diameter (

Rootstock

MM 106 5.69 30.3 b 172.2 4.76

M9 5.31 35.7 a 187.0 4.99

Cultivar

GS 4.42 b 33.8 150.1 b 4.98

SSG 6.58 a 32.3 209.1 a 4.77

Treatments

Control 4.79 c 27.63 b 136.8 c 4.48 b

OSU-142 5.22 bc 43.99 a 228.7 a 5.21 a

OSU-7 5.86 ab 32.68 b 190.3 ab 4.94 ab

BA-8 6.27 a 29.57 b 177.8 abc 4.79 ab

M-3 5.37 abc 31.58 b 164.4 bc 4.95 ab

Source d.f. ANOVA

Rootstock (R) 1 NS * NS

Cultivar (C) 1 ** NS **

R � C 1 ** NS **

Treatments (T) 4 ** ** **

R � T 4 NS NS NS

C � T 4 NS NS NS

R � C � T 4 NS NS NS

Error 38

Total 59

Means followed with the same letters (a–d) within each column are not significan* Significant at 0.05 probability level.

** Significant at 0.01 probability level.

trees significantly, especially BA-8 (e.g. 137.5% of control)

followed by OSU-142 (Table 2). Bacterial strain OSU-142,

OSU-7, M-3 and BA-8 inoculations increased average of shoot

length, respectively, by 59.2, 18.3, 14.3 and 7.0% compared

with control young apple trees. However, except from OSU-

142, rootstock inoculation with other bacteria did not differ

from control in term of shoot length and diameter (Table 2).

All bacterial strains significantly increased the fruit yield of

apple. The highest PGPR contribution was obtained with

Pseudomonas BA-8 and Bacillus OSU-142. Yield and shoot

length (r = 0.27*), total shoot length and trunk diameter

egetative growth components of apple trees (vegetative growth parameters data

yield of apple trees was collected for 2004 growing season)

mm)

Trunk

diameter (cm)

Plant

height (cm)

Leaf

area (cm2)

Fruit yield

(kg/cm2)

1.50 121.6 32.7 0.255 b

1.47 127.2 32.6 0.301 a

1.43 b 124.4 32.1 b 0.281

1.54 a 124.4 33.2 a 0.275

1.43 115.7 31.9 0.152 d

1.52 127.5 33.4 0.329 b

1.53 131.0 33.0 0.286 c

1.51 122.3 32.3 0.361 a

1.44 125.6 32.5 0.264 c

NS NS NS NS **

NS ** NS * NS

NS NS NS NS NS* NS NS NS ***

NS NS NS NS **

NS NS NS NS **

NS NS NS NS **

t different. NS: not significant.

R. Aslantas et al. / Scientia Horticulturae 111 (2007) 371–377 375

(r = 0.42**), plant height (r = 0.33**), and yield (r = 0.26*) and

trunk diameter and shoot number (r = 0.37**) were significantly

correlated.

Selected PGPR isolates significantly increased shoot

number (up to 30.8%), average shoot length (up to 59.2%),

total shoot length (up to 67.1%), shoot diameter (up to 16.3),

fruit yield (up to 137.5%, respectively) in all the tested cultivars

of young apple trees with different bacterial strains. There was

no significant difference in the shoot and trunk diameter, plant

height and leaf area in young apple trees among the different

treatments.

4. Discussion

The four PGPR strains were able to produce cytokinin and

auxin in pure culture. Inoculation with PGPR strains promoted

significant tree growth in the field, but growth responses were

strain-specific. For example, OSU-142 significantly increased

both shoot length and diameter. In contrast, OSU-7, BA-8 and

M-3 were ineffective for the same parameters. In addition,

saplings inoculated with two of the four strains had significantly

less fruit yield than others. Our results indicate that when plant

growth promotion is induced in the field, yield can be increased

especially OSU-142 and BA-8 treatments by more than 100%

during the harvest season in the field.

The PGPR strains may increase the level of root hormone by

exogenous production of IAA, cytokinin and/or other plant

hormones in the rhizosphere, which are then absorbed by the

root. Our results demonstrated that BA-8 produced the highest

amounts of total cytokinin and IAA. This strain significantly

increased the number of shoots and fruit yield in the field. Thus,

the amount of IAA and cytokinin appeared to be directly

correlated with plant growth and yield. The differential

distribution of cytokinin in the shoot can affect involved in

the pattern of budburst and thus growth habit (Cook et al.,

2001). Many plant-associated bacteria have the ability to

produce the plant growth regulator indole-3-acetic acid and

IAA may play the most important role in plant growth

promotion (Patten and Glick, 2002; Khalid et al., 2004). In

previous studies, it was shown that auxin-producing rhizo-

bacteria influenced root development and had a strong growth-

promoting activity (Probanza et al., 1996; Bent et al., 2001).

Proposed roles for bacterial IAA synthesis include the

determination of rooting capacity (Fogaca and Fett-Neto,

2005), the stimulation of the release of plant metabolites

(Lambrecht et al., 2000), and/or the promotion of root

elongation and shoot growth of inoculated plants (Gadagi

et al., 2004). Application of IAA to P-deficient plants increased

the root surface, carbohydrate release and acid-phosphatase

activity (Wittenmayer and Merbach, 2005). Also, IAA secreted

by a bacterium may promote root growth directly by

stimulating plant cell elongation or cell division or indirectly

by influencing bacterial 1-aminocyclopropane-1-carboxylate

(ACC) deaminase activity (Patten and Glick, 2002), which is

the immediate precursor of the phytohormone ethylene, and

thereby prevents the production of plant growth-inhibiting

levels of ethylene (Penrose et al., 2001). Also, IAA production

contributes to the colonization efficiency and to the growth and

survival of bacteria on its host plants (Vandeputte et al., 2005).

The presence of high number of bacteria in the rhizosphere is

undoubtedly also important, since they may convert organic

and inorganic substances into available plant nutrients.

Bacterial inoculation also affected the pattern of budburst

and growth habit of young apple seedlings. These results

suggest that the production of cytokinins in the PGPRs may be

related to the growth characteristics of apple trees. Similarly,

Watanabe et al. (2004) found a clear connection between

concentration of cytokinins and the growth characteristics of

columnar type apple trees. One of the proposed mechanisms by

which PGPR enhances young apple trees’ growth is through the

production of plant growth regulators. Cytokinins play essential

roles in the regulation of plant growth and development (Zhang

et al., 2003; Jeon et al., 2003). The release of buds from apical

dominance, stimulation of leaf expansion and of reproductive

development, and retardation of senescence (Mok, 1994), play

a central role in budburst in spring and late dormancy in apple

(Faust et al., 1997), and enhance the percentage of plantlets

with lateral and terminal flowers (Galoch et al., 1996).

Cultivars significantly affected shoot number, total shoot

length, trunk diameter and leaf area of apple trees, which was

previously reported (Khan et al., 1998; Bianco et al., 2003).

Trees on M9 rootstock had greater average shoot length and

fruit yield. Trees on MM 106 had more shoots and larger stem

diameter. Rootstocks affected response to nitrate nitrogen

fertilization of fruit, surface colour, total soluble solids, and

acidity (Motosugi et al., 1995). Rootstock genotype affected

tree growth and bacterial rhizosphere community composition

in an experimental orchard (Rumberger et al., 2004). In

contrast, Al-Hinai and Roper (2004) could not find an effect

between M9 rootstock and fruit growth. Our results showed that

leaf area was not significantly different for each rootstock,

consistent with previous research (Li et al., 2002).

The inoculation modes with PGPR and rootstock played a

very important role in the effects observed. Thus, plant growth

promoting bacteria may interact synergistically with stock and

cultivar selected. Our results also suggest that a synergistic,

metabolic interaction involving existing rootstock � cultivar

and PGPRs. Also, rootstock � treatment and cultivar � treat-

treatment interactions were observed for fruit yield. Indicating

both apple cultivar and rootstock tested responded differently to

inoculation with different rhizobacterial strains. Bacterial

efficiency in M9 rootstock was higher than MM 106. Late

season fruit growth and final fruit size were correlated

(r = 0.27*), with average shoot length. Growth promotion

effects were seen early in young apple trees development, and

these subsequently translated into higher yield, which was

reported also by Dobbelaere et al., 2002. PGPR inoculation

strongly influenced shoot number, shoot length and diameter

and yield during the early stages of growth. Indicating the tested

PGPR strains tested are capable of promoting plant growth in

the early years after planting in apple.

PGPR inoculation could be increased not only growth but

also yield of young apples trees. The optimum amount of

nitrogen application for young dwarf apple trees was estimated

R. Aslantas et al. / Scientia Horticulturae 111 (2007) 371–377376

to be less than 10 g/year/tree (Neilsen et al., 2001).

Furthermore, apple trees grown with sufficient soil nitrogen

can grow vigorously and produce fruits for long-term without

supplementing nitrogen fertilization. Excessive nitrogen

application increases growth and leaf nitrogen content, but

negatively affects fruit quality (Komamura et al., 2000). In

addition to, based on tree vigour, budgets for N, P, and K and

soil N status, N concentration of medium would be adequate for

maiden apple trees, without over-fertilizing and excessive

growth (Ro and Park, 2000). The amount of shoot growth is

often used as an indicator of plant N requirement in orchards.

The growth of 25–30 cm indicates a good N fertilization

program in young apple trees locally (Guleryuz et al., 1998).

The shoot growth of inoculated trees with PGPR strains was

just in the range, which may indicates the PGPR treatments

match tree N requirement in the first 2 years. These

observations indicated that mechanisms of growth promotion

other than N2 fixation such as phytohormone production,

improved nutrient uptake balance, may be attributable to these

PGPR.

Obtaining maximum benefits on farms from diazotrophic,

plant growth promoting biofertilizers will require a systematic

strategy designed to fully utilize all these beneficial factors,

allowing crop yields to be maintained or even increased while

fertilizer applications are reduced (Kennedy et al., 2004). Our

results indicated that selected PGPR are able to promote tree

growth and fruit yield and reduce the need for chemical

fertilizers for young apple trees. Among the various PGPR

isolates tested, BA-8 was found the most effective in promoting

growth and yield of different cultivars. In contrast, OSU-142

was most effective in promoting average and total shoot length,

shoot diameter and leaf area of different cultivars of apple

compared to control. Overall, the response to inoculation with

various PGPR isolates varied with cultivars. The plant growth

promoting effect of bacterial applications appeared to be

related to phytohormone production and phosphate solubiliza-

tion activities of the PGPR strains tested. Further studies are

necessary to determine the residual effect of PGPR treatments

on the growth, fruit yield and quality of apple trees as well as

soil microbial communities in the orchards for over longer time

periods.

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