Pseudomonas fluorescens and Sulfur Application Affect Rapeseed Growth and Nutrient Uptake in Calcareous Soil

International Journal of Agriculture and Crop Sciences. Available online at www.ijagcs.com IJACS/2013/5-1/39-43 ISSN 2227-670X ©2013 IJACS Journal Ps...
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International Journal of Agriculture and Crop Sciences. Available online at www.ijagcs.com IJACS/2013/5-1/39-43 ISSN 2227-670X ©2013 IJACS Journal

Pseudomonas fluorescens and Sulfur Application Affect Rapeseed Growth and Nutrient Uptake in Calcareous Soil Lida Eslamyan, Zarrin Taj Alipour, Shahram Rezvan Beidokhty and Adeleh Sobhanipour Department of Agriculture, Damghan Branch, Islamic Azad University, Damghan Iran Corresponding author email: [email protected] [email protected] ABSTRACT: This experiment was conducted in 2010-2011 at the Agricultural Experiment Research Station of Islamic Azad University, Damghan branch, to evaluate the effect of Pseudomonas fluorescens and sulfur application on growth, yield and nutrient uptake of two rapeseed cultivars. The experiment was conducted in factorial in the form of a completely randomized design with four replications and three factors: Pseudomonas fluorescens (with and without inoculation), sulfur (0 and 1000 kg/ha) and rapeseed cultivars (Brassica napus cv. Hayola 308 and Hayola 401). Results indicated that although few traits were significantly affected by cultivar and sulfur; however, the bacterial inoculation significantly affected nearly all the measured traits. Pseudomonas inoculation increased root weight by about 35% and oil content by about 62%. Moreover, all the measured traits except for harvest index, root weight and root length were the highest in three-fold interactions in Hayola 401 × Pseudomonas inoculation × 1000 kg/ha sulfur. Keywords: Brassica napus; chlorophyll; iron; zinc. INTRODUCTION Soil is a complex medium consist of a physical and a biological phase, with the ability of rehabilitating it self. This ability can be seen in natural ecosystems without human interferences which are yet productive, healthy and balanced. However, in agroecosystems, this ability is disturbed by human activity, extensive application of chemical inputs and interference in soil structure. The result of these human activities is gradual reduction of soil quality and fertility. Considering the mentioned issues, it is required to turn back to the nature and employ the natural processes to sustain soil fertility. Pseudomonas bacteria are categorized under plant growth promoting rhizobacteria (PGPR) and produce abundant siderophores under low Fe content in soil. Siderophores are large organic molecules which severely bound Fe+3; making it available to plants and other microorganisms. Shoda (2000) reported that siderophores transport iron into plant roots; increasing plant growth. PGPR term was first used by Klopper and Schroth (1978). At first, this term was only used to refer to the bacteria involved in biological control; now it refers to all bacteria in rhizosphere which improve plant growth (Weller and Cook, 1982). These bacteria benefit plants in various ways (Fromel et al., 1991). Pseudomonas fluorescens is an important PGPR which affect plant growth through various mechanisms. Shaterabadi et al. (2001) tested the effect of different Pseudomonas fluorescens strains on forage sorghum and reported that all strains increased fresh and dry yield compared with the uninoculated control. Mehran et al. (2011) also reported that Pseudomonas inoculation increased sunflower grain yield, auxin, gibberellin and cytokinin content. Sulfur is an important nutrient for plant growth; however, the oxidation of this element is mainly conducted through the biological processes. So, presence of abundant sulfur oxidizing microorganisms in soil is required. Pseudomonas bacteria are the most important type of these bacteria. Considering the effect of Pseudomonas, sulfur and their interactions on plants growth, this experiment was conducted to test their effect on rapeseed growth, yield and nutrient uptake.

Intl J Agri Crop Sci. Vol., 5 (1), 39-43, 2013

MATERIALS AND METHODS This experiment was conducted in 2010-2011 at the Agricultural Experiment Research Station of Islamic Azad University, Damghan branch, Iran. The experiment was conducted in factorial in the form of a completely randomized design with four replications. Treatments of the experiment were Pseudomonas fluorescens (with and without inoculation), sulfur (0 and 1000 kg/ha) and rapeseed cultivar (Hayola 308 and Hayola 401). In order to culture Pseudomonas bacteria, first, five antagonistic strains were located on NA culture medium and kept in 26-28oC for 48 h. Then, the surface of all petri dishes was eluted by 10 ml sterile distilled water to isolate bacteria cells and all suspensions were mixed. The optical absorption of strains' mixture suspension was measured using spectrophotometer, and then 0.5% of Arabic gum was added to it. In seed treatment method, seeds were sterilized by 0.5% sodium hypochlorite (v: v) for two minutes, eluted, and were soaked in the strains suspension for 1 h. After that, seeds were dried under a sterile hood and finally, 5-6 seeds 2 were planted in 0.7 m pots (Weller and Cook, 1982). In soil inoculation method of the greenhouse experiment, 108 cfu ml-1of the mixture of antagonistic strains suspension was added to soil and after 1-7 days, rapeseed was planted in the pots. To determine bacteria CFU, 10 seeds were randomly selected and each was located inside a test tube containing 10 ml sterile distilled water. The test tubes were shook on a shaker and after preparing the concentration series, 10-3, 10-4 and 10-5 concentrations were selected and 1 ml of each was poured on a KB culture medium. For siderophore production assay, bacterial strains were cultured on KB medium containing 0, 25, 50, 100 and 1000 µmol of three valence iron chloride and were kept in 25oC for 48 h. Then, spore suspension of Geotrichum candidum fungus which was produced from a 48 h culture on PDA medium with sterile distilled water, was sprayed on the bacteria inside the petri dishes. Absence of fungal growth around the bacteria is the indicator of siderophore production. In this experiment, three replications were considered for each concentration. In fall 2010, 15 kg of a calcareous soil (20%) was used to fill 32 pots and 1000 kg sulfur/ha was added to soil. Soil and irrigation water analyses are listed below: Table 1. Results of soil sample analysis Texture Loamy silt

EC (ds/m) 1.7

pH 8.1

OC (%) 0.07

TNV (%) 20.7

Gypsum (%) 5

P (mg/kg) 7

Fe (mg/kg) 2.1

Zn (mg/kg) 1.8

Table 2. Results of irrigation water analysis -2

EC (ds/m)

pH

TDS (mg/l

Ca (Meq/l)

Mg (Meq/l)

Co3 (Meq/l)

0.54

7.9

0.6

3

1.5

0

-1

HCo3 (Meq/l) 1.4

-1

Cl (Meq/l) 0.9

At the end of growing season, in May 4th, 2011, leaf chlorophyll content was measured by SPAD chlorophyll meter; then, plants were harvested along with their roots and the required traits were measured. Data were analyzed using SAS and means were compared according to the Duncan's multiple range test. RESULTS AND DISCUSSION Results of this experiment indicated the significant effect of bacterial inoculation on all the measured traits except for the harvest index (Table 3). Among the measured traits, only 1000 kernels weight, oil and Zn contents were significantly different between the cultivars. Results also showed that sulfur application had a significant effect only on chlorophyll, oil, Fe and Zn contents. The three-fold interaction of Pseudomonas × cultivar × sulfur had no significant effect on any of the measured traits. Table 3 (Part 1). Analysis of variance of the effect of treatments on the measured traits SOV

df

Bacteria (B) Cultivar (C) Sulfur (S) BC BS CS BCS Error CV (%)

1 1 1 1 1 1 1 3 -

Mean Squares (MS) 1000 kernels Biologic yield weight ** ** * ns ns ns ns ns ns ns ns ns ns ns 0.08 64.86 8.02 11.55

Number of lateral branches ** ns ns ns ns ns ns 12.03 1.87

Harvest index

Shoot weight

Root weight

Chlorophyll content

ns ns ns ns ns ns ns 15.68 12.68

** ns ns ns ns ns ns 44.06 11.04

* ns ns ns ns ns ns 8.52 29.99

** ns * ns ns ns ns 4.64 3.29

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Intl J Agri Crop Sci. Vol., 5 (1), 39-43, 2013

ns, non significant; *, significant at P 0.05; **, significant at P 0.01. Table 3 (Part 2). Analysis of variance of the effect of treatments on the measured traits SOV

df

Bacteria (B) Cultivar (C) Sulfur (S) BC BS CS BCS Error CV (%)

1 1 1 1 1 1 1 3 -

Mean Squares (MS) Stem diameter Root length * ** ns ns ns ns ns ns ns ns ns ns ns ns 0.01 6.53 12.23 10.41

Plant height ** ns ns ns ns ns ns 28.87 3.71

Oil content ** * * ns * ns ns 0.86 3.01

Fe content ** ns ** ns * ns ns 1.86 1.42

Zn content ** * ** * * * ns 3.03 4.38

ns, non significant; *, significant at P 0.05; **, significant at P 0.01.

Bacterial inoculation increased root weight by about 35% and oil content by about 62% (Table 4). Pseudomonas fluorescens is an important PGPR which affects plant growth directly and indirectly. Among the indirect mechanisms, is the biological control of plant pathogens as Weller and Cook (1982) reported the inhibition of wheat pythium root rot by P. fluorescens strain Q72-a80. Klopper and Schroth (1978) also found that P. fluorescens strain b10 inhibited barley wilt. Moreover, production of plant growth regulators such as auxin and cytokinin, enhancement of available nutrients to plant roots, improvement of plant root system development, stimulation of other soil microorganisms' activity and siderophores production can be counted as the direct mechanisms by which Pseudomonas bacteria improve plant growth (Molla et al., 2001; Akao et al., 2001). Ansary et al. (2012) conducted an experiment to evaluate the effect of P. fluorescens inoculation on maize phytohormones under normal and drought stress conditions and concluded that the inoculation significantly increased abscisic acid, cytokinin, gibberellin, auxin and praline content, increasing plant resistance to drought stress. Mehran et al. (2011) reported that inoculating sunflower with Pseudomonas increased grain yield, auxin, gibberellin and cytokinin content. Shaterabadi et al. (2001) represented that various P. fluorescens strains increased forage sorghum yield compared with the uninoculated control. Gen and Jordan (1993) also tested the effects of P. fluorescens on spring tomato and reported that the inoculation increased the amount of top quality fruits from 5.6% to 9.6% of the total yield. Moreover, the inoculation increased fruits size by 11.1% and decreased unmarketable fruits from 23% to 12%. Table 4 (Part 1). Effect of the bacterial inoculation on the measured traits Bacterial inoculation With Without

Chlorophyll content

Fe (mg/kg)

Zn (mg/kg)

Oil (%)

86.44a 44.52b

125.94a 65.89b

56.59a 22.98b

38.11a 23.53b

Plant height (Cm) 154.68a 134.56b

1000 kernels weight (g) 4.03a 3.26b

Means in a column followed by the same letter are not significantly different at P 0.05. Table 4 (Part 1). Effect of the bacterial inoculation on the measured traits Bacterial inoculation

Biologic yield (g/pot)

Harvest index (%)

Shoot weight (g/pot)

Root weight (g/pot)

Root length (cm)

With Without

79.47a 59.98b

32.57a 29.86a

68.30a 51.93b

11.16a 8.29a

28.25a 20.81b

Stem diameter (cm) 1.25a 1.00b

Number of lateral branches 12.81a 9.93b

Means in a column followed by the same letter are not significantly different at P 0.05.

Results indicated that sulfur application increased chlorophyll (by 5.94%) and oil content (by 6.08%). Fe and Zn content was also increased when sulfur was applied (Table 5). Sulfur is an important nutrient for plant growth which plays structural roles in several amino acids (e.g., cysteine and methionine) and compounds involved in electron transfers in photosynthesis and respiration. It is also involved in specialized enzymes and related molecules. Sulfur is an acidifier; reducing pH of calcareous and alkaline soils; facilitating nutrients absorption (Epstein and Bloom, 2005; Wiedenhoeft, 2006; Ray and Fritschi, 2009). Kaplan and Erman (1998) found that sulfur application increased sorghum yield and Fe, Zn, P and Mn uptake. Alipour and Sobhanipour (2012) also conducted an experiment to study the effect of microorganisms and sulfur on maize and reported that sulfur application increased plant chlorophyll, Fe and Zn content. However, the oxidation of sulfur mainly occurs through the biological processes. So, presence of abundant sulfur oxidizing microorganisms in soil is required. Pseudomonas bacteria are the most important type of these bacteria.

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Table 5. Effect of sulfur application on some of the measured traits Sulfur 1000 kg/ha 0 kg/ha

Chlorophyll content 67.37a 63.59b

Oil (%) 31.73a 29.91b

Fe (mg/kg) 99.62a 92.21b

Zn (mg/kg) 42.33a 37.24b

Means in a column followed by the same letter are not significantly different at P 0.05.

Among the two cultivars, Hayola 401 had higher 1000 kernels weight, chlorophyll, oil and micronutrient contents (Table 6). Table 6. Variation in some of the measured traits between the two cultivars 1000 kernels weight (g) 3.84a 3.45b

Sulfur Hayola 401 Hayola 308

Chlorophyll content 66.56a 64.40a

Oil (%)

Fe (mg/kg)

Zn (mg/kg)

31.52a 30.12b

96.63a 95.21a

41.05a 38.51b

Means in a column followed by the same letter are not significantly different at P 0.05.

Studying the interactions of the treatments indicated that all the measured traits, except for harvest index, root weight and root length, were the highest in the three-fold interaction of Hayola 401 × Pseudomonas inoculation × 1000 kg sulfur/ha; although the variation was not significant in many cases (Table 7). Table 7 (Part 1). Effect of the three-fold interaction of the treatments on the measured traits Bacterial inoculation

Chlorophyll content

Fe (mg/kg)

Zn (mg/kg)

Oil (%)

C2B1S1 C1B1S1 C2B1S0 C1B1S0 C2B0S1 C1B0S1 C2B0S0 C1B0S0

88.82a 86.81a 85.81a 84.33a 49.48ab 44.38b 42.15c 42.07c

130.32a 127.31b 123.81c 122.33c 70.98d 69.88d 61.40e 61.32e

64.46a 56.60b 53.84bc 51.46c 24.58d 23.68de 21.34e 22.32de

39.36a 37.67bc 38.41ab 37.00c 25.83d 24.07e 22.48f 21.76f

Plant height (Cm) 161.25a 152.50ab 153.75ab 151.25b 136.25c 136.25c 132.50c 133.25c

1000 kernels weight (g) 4.65a 3.75b 3.90b 3.82b 3.50bc 3.20cd 3.32cd 3.05d

Means in a column followed by the same letter are not significantly different at P 0.05.

C1, Hayola 308; C2, Hayola 401. B0, without bacterial inoculation; B1, with bacterial inoculation. S0, 0 kg sulfur/ha; S1, 1000 kg sulfur/ha. Table 7 (Part 2). Effect of the three-fold interaction of the treatments on the measured traits Bacterial inoculation

Biologic yield (g/pot)

Harvest index (%)

Shoot weight (g/pot)

Root weight (g/pot)

Root length (cm)

Stem diameter (cm)

C2B1S1 C1B1S1 C2B1S0 C1B1S0 C2B0S1 C1B0S1 C2B0S0 C1B0S0

83.25a 77.70ab 76.74ab 80.20a 54.62d 58.32cd 67.47bc 59.50cd

33.25ab 34.50a 31.60ab 30.95ab 31.77ab 32.02ab 25.62b 30.02ab

72.42a 66.35ab 64.69ab 69.75a 47.10c 52.05c 57.72bc 50.87c

10.82ab 11.35a 12.05a 10.45ab 7.53c 7.25c 9.77abc 8.62bc

28.50a 28.75a 27.75a 28.00a 22.00b 21.50b 19.25b 20.50b

1.30a 1.27a 1.27a 1.17ab 1.07bc 1.07bc 0.92c 0.95c

Number of lateral branches 13.25a 13.25a 12.25ab 12.50ab 11.00abc 10.75bc 9.25c 8.75c

Means in a column followed by the same letter are not significantly different at P 0.05.

C1, Hayola 308; C2, Hayola 401. B0, without bacterial inoculation; B1, with bacterial inoculation. S0, 0 kg sulfur/ha; S1, 1000 kg sulfur/ha. CONCLUSION Results of this experiment briefly indicated that Pseudomonas florescence inoculation significantly affected nearly all the measured traits, increasing oil content by about 62%. Traits were mostly the same among the two cultivars; however, sulfur application had significant effect on the quality of plant. Finally, the most effective treatment of this experiment was the three-fold interaction of Hayola 401 × Pseudomonas inoculation × 1000 kg/ha sulfur.

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Intl J Agri Crop Sci. Vol., 5 (1), 39-43, 2013

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