Appl Microbiol Biotechnol (2007) 74:1120–1125 DOI 10.1007/s00253-006-0750-6
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Growth promoting effect of a transgenic Bacillus mucilaginosus on tobacco planting Xin Li & Zhiqiang Wu & Weidong Li & Ruixiang Yan & Li Li & Jun Li & Yihang Li & Minggang Li
Received: 26 August 2006 / Revised: 2 November 2006 / Accepted: 3 November 2006 / Published online: 6 December 2006 # Springer-Verlag 2006
Abstract In this study, we have investigated the plant growth promoting effect of Bacillus mucilaginosus strain D4B1, a rhizosphere soil organism, and its transgenic strain NKTS-3 on tobacco planting. The transgenic strain contains a phytase expression cassette that can express high active phytase extracellularly and hydrolyze phytate in the soil to liberate inorganic phosphorus for the growth of tobacco plants. Greenhouse study and field experiments showed that both wild-type B. mucilaginosus and the transgenic strain could promote tobacco plant growth. Moreover, the transgenic strain promoted tobacco plant growth (235% more than control in pot experiments and 125% more than control in field experiments) was higher than the wild-type B. mucilaginosus (183% more than control in pot experiments and 108% more than control in field experiments). In addition, the inoculation with transgenic rhizobacteria could significantly improve root acquisition of phosphorus and increase the phosphorus content of the plant. Keywords Phytase . Bacillus mucilaginosus . Phosphorus absorbing . Plant rhizosphere X. Li : Z. Wu : W. Li : R. Yan : M. Li (*) The Key Laboratory of Bioactive Material, Ministry of Education, China, Life Science College, Nankai University, Tianjin 300071, China e-mail:
[email protected] L. Li : J. Li Institute of Soil and Fertilizer, Chinese Academy of Agricultural Sciences, No. 30 Baishi QialLu, HaiDian District, Beijing 100081, China Y. Li Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA
Introduction For many years, as a result of inappropriate applications of chemical fertilizers in continuous corn cultivation, the agriculture suffers from problems, such as pollution of arable soils, pollution of water resources, and soil salinization. In an attempt to reduce these chemical inputs and raise soil quality and sustainability, new biotechnological practices, such as the application of bacterial fertilizer, have been investigated to improve crop production. Scientists have isolated numerous rhizobacterium to be used as bacterial fertilizers (Dashti et al. 1997; Groppa et al. 1998; Kozdroj et al. 2004). Generally, the plant growth promoting function of rhizobacteria is carried out by three mechanisms: (1) Bacterium of the genera Rhizobium, Bradyrhizobium,Sinorhizobium and Azorhizobium can fix atmospheric nitrogen in a symbiotic relationship with the root of leguminous plants (Galleguillos et al. 2000). (2) Some rhizobial strains may be able to produce plant growth regulators and promote plant growth (Kloepper & Schroth 1978). (3) A large number of microorganisms possess the capacity of dissolving soil mineral and releasing nutrition elements such as potassium (K) and phosphorus (P), which can be utilized by plants (Suh et al. 1995). Silicate bacterium or Bacillus mucilaginosus is a special bacillus species that produces a variety of exopolysaccharides. It was utilized in agriculture extensively as a multifunctional microbial fertilizer, which can make K, P and other beneficial elements available by dissolving insoluble minerals in soil (Lian et al. 2000). In our previous study, a strain of B. mucilaginosus was isolated from the soil sample of Yunnan Province in China, which is designated as D4B1 (Li et al. 2001). Biological characterization analysis showed that it can dissolve 62.6% of K from feldspar and 61.2% of P from calcium phosphate
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(Wu et al. 2004). Furthermore, this strain could depress the effect of Botrytis cinerea, which affects tobacco as grey mould or gray mold and increase the yield of crop growth. However, the poor ability of D4B1 strain on hydrolyzing phytate, which is the major storage form of organic P in soil (Reddy et al. 1982; Dala 1978), limited its application (Wu et al. 2004). To solve this problem, a phytase expression cassette was inserted into the chromosome of the wild-type B. mucilaginosus D4B1 to construct a transgenic strain NKTS-3, which can express and secrete highly active phytase protein (Li et al. 2005). The objective of the present investigation is to assess the contribution of D4B1 and NKTS-3 to plant growth and yield of tobacco and its effect on the component and content of phosphorus in soil.
Materials and methods Strains and growth conditions The transgenic strain NKTS-3 was constructed based on the wild-type B. mucilaginosus D4B1, which was isolated from the soil sample of Yunnan Province in China (Li et al. 2001). The phytase secreting expression vector pSP43 with a miniTn5 transposon was transferred into D4B1 strain by using the particle bombardment method, and three transgenic strains with stable copies of phytase expression cassette integrated into the chromosome of the B. mucilaginosus by Tn5 transposition were selected. The engineered strain whose phytase activity increased 46-fold in comparison to D4B1 was named NKTS-3 described previously (Li et al. 2005). The strains D4B1 and NKTS-3 for pot experiments were cultured at 30 °C for 30–36 h in silicate bacterium growth medium [containing sucrose, 10 g l−1; K2HPO4, 2 g l−1; NaCl, 0.2 g l−1; MgSO4·7H2O, 0.5 g l−1; (NH4)2SO4, 1 g l−1; yeast extract, 1 g l−1; FeCl3, 0.005 g l−1; CaCO3, 0.1 g l−1; pH 7.0–7.5] (Sheng & Huang 2001), and kanamycin was used at a concentration of 50 μg ml−1. The strains for field experiment were cultured at 30 °C for 48 h in a fermentation medium [containing amylum, 8 g l−1; yeast extract, 1.0 g l−1; bean cake, 5.0 g l−1; (NH4)2SO4, 0.5 g l−1; Na2HPO4, 1.5 g l−1; NaCl, 0.2 g l−1; MgSO4·7H2O, 0.5 g l−1; FeCl3, 0.005 g l−1; CaCO3, 1.0 g l−1; pH 7.0–7.5] of 50 l fermenter. The bacterium cells were harvested with a centrifuge at 4,000×g and 4 °C for 5 min, transferred into a sterilized peat moss and well mixed. The mixture was used as the microbial inoculum in which the final population size of bacillus was 1.5×109 CFU g−1. The soil of pot and location of field experimentals The soil used for pot experiment was collected from the farm of Xi qin borough, Tianjin, China. The soil was air
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dried and ground to pass through a 2-mm sieve and mixed thoroughly. The basic properties of the soil were as follows: pH 5.46, organic matter content 1.08%, total N 0.062%, total K 7408 mg kg−1, total P 1,090 mg kg−1, available P (NaHCO3-extractable) 125 mg kg−1 and water-soluble K 134.3 mg kg−1(provided by Soil and Fertilizer, Tianjin Province Academy of Agricultural Sciences, China). The field experiment was conducted at a research farm of Institute of Soil and Fertilizer, Shanxi Province Academy of Agricultural Sciences, China during April–October in 2004. The climate of this area is a semiarid temperate zone, the length of sunlight is 2,400–2,600 h per year with an average temperature of 8.0–12.0 °C and rainfall received of 500–550 mm per annum. During the experiment, the average air temperature was 18.4 °C, and the rainfall was 385.4 mm. The basic properties of the soil were as follows: pH 7.5–8.5, organic matter content 0.6–1.0%, total N 0.05–0.10%, available P (NaHCO3-extractable) 3.0– 5.0 mg kg−1 and water-soluble K 100–150 mg kg−1. Pot experimental procedure To study the influence of NKTS-3 and D4B1 on tobacco growth and soil properties, a randomized block was designed with five treatments (Table 1). The treatment details are presented in Table 1. In the treatments of WTPM, TS-PM and PM-LM, 10-g peat moss-based bacterial (D4B1 or NKTS-3) inoculum or peat moss mixed with liquid medium (no bacterium) were mixed separately with 1 kg of soil and inoculated into the pots. In the treatment of TS-FM, seeds of tobacco (provided by Dr. L. Li, sprouts rate=94%) were inoculated by submersion in 10 ml of bacterial suspension in 50-ml flasks. The flasks were incubated in a rotary shaker (70 rpm, 28 °C) for 2 h to allow bacterial cells to adhere on the seeds. After incubation, the treated seeds were placed in the soil followed by introduction of bacterial suspension on the soil surface of the sown seeds. The treatment without biofertilization was set up as a control group.
Table 1 Detail design of pot experiment Treatment code
Fertilizer type
Control PM-LM WT-PM
Control (no amendment) Peat moss mixed with liquid medium (no bacterium) Wild-type B. mucilaginosus D4B1 (mix with peat moss powder) Transgenic B. mucilaginosus NKTS-3 (mix with peat moss powder) Transgenic B. mucilaginosus NKTS-3 ferment medium dilution (1:125) flooding fertilization
TS-PM TS-FM
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The tobacco seeds were disinfected for 30 min in 10% peroxide solution, and then ten seeds were sown in a plastic pot containing 1.5-kg soil. All the pots were randomly placed in a greenhouse (20±4 °C) and watered daily with deionized water to maintain at 60% moisture (water holding capacity of the soil). After 20, 40 and 90 days (for platingbased assessments), tobacco plants were carefully removed from the soil. The plants were divided into shoot and root parts. The shoot fresh and dry weights (drying to constant weight at 105 °C) were measured. The roots with adhering rhizosphere soil were placed in sterilized 0.1% sodium pyrophosphate (pH 7.0), shaken at 20 °C for 30 min at 180 rpm and prepared serial tenfold dilutions for plate counts. Six pots from each treatment were used at each sampling time. Field experimental procedure To study the effect of D4B1 and NKTS-3 on tobacco growth in agriculture system, a field experiment was carried out in a research farm. A randomized block was designed followed by five treatments with four replications and every plot size was 16 m2. Peat moss-based bacterial inoculum of 1.5 kg (Table 1) was mixed with 1.0-kg soil and inoculated into the field soil. Propagules of tobacco plants (25–27 cm, provided by the Institute of Soil and Fertilizer, Shanxi Province Academy of Agricultural Sciences, China) were planted into the soil randomly and irrigated with water. In TS-FM treatment, 1.5-l NKTS-3fermentated medium was diluted with water (1:125) and irrigated into the rhizosphere of the plant. The propagule without bacterial treatment was used as the control. Other parameters of tillage and rainfall were identical for all the blocks, except that the bacterial-type fertilizer treatments were the only difference between the blocks. After 5 months, the shoot and root of plants were collected, and the growth parameters, such as height, fresh biomass and dry weight (drying at 70 °C) of plants under different treatments were recorded. Biological and chemical analyses After the plants were collected, the growth parameters (height and dry weight) of plants under different treatments were recorded. The harvested plants were rinsed with deionized water and oven dried at 80 °C for 72 h. The dried leave tissues were ground and then digested using concentrated HNO3 (Page et al. 1982) for the determination of K using an atomic absorption spectrometer (PEZeemam-5100). Total P was extracted by digesting leave tissue with 3-ml concentrated H2SO4 and 1-ml H2O2 at 360 °C and determined by the molybdenum blue method (Page et al. 1982).
Appl Microbiol Biotechnol (2007) 74:1120–1125
A composite rhizosphere soil sample from experimental pots was collected to determine the nutrient concentrations (P, K) using methods mentioned above (Page et al. 1982). The amount of NaHCO3-extractable P (available inorganic P) from the soil was determined by extracting samples with 0.5 M NaHCO3 (pH 8.5) at a solution/solid ratio of 20:1 for 30 min (Olsen & Sommers 1982). To investigate the content and distribution of D4B1 and NKTS-3 in rhizosphere soil at different periods, soil samples were taken on the 1st, 10th, 20th, 30th, 60th and 90th day after planting, and the amount of introduced bacterium from the fresh soil sample were conducted using suspension dilution techniques on agar plates (Wollum 1982). Replicate aliquots from rhizosphere dilutions was spread plated onto silicate bacterium selection medium agar (containing [g l − 1 ] sucrose 5.0; Na 2 HPO 4 2.0; MgSO4·7H2O 0.5; FeCl3 0.005; CaCO3 0.1; feldspar powder 1.0; pH 7.0–7.5) with either 50 μg ml−1 kanamycin for enumeration of NKTS-3 or without the antibiotic for enumeration of D4B1. All colonies growing on silicate bacterium selection medium agar plate with characterization of silicate bacterium were counted. Data analysis Analysis of variance was performed on all experimental data using the SAS program (SAS Institute, 1996). Correlation analysis was also performed to test for relationships between variables. The significance level was p
