Growth promotion effects of the endophyteAcinetobacter

Growth promotion effects of the endophyte Acinetobacter johnsonii strain 3-1 on sugar beet Yingwu Shi & Kai Lou & Chun Li Received: 24 August 2011 /Accepted: 5 November 2011 /Published online: 18 November 2011 # Springer Science+Business Media B.V. 2011 Abstract An endophytic bacteriumn identified as Acineto- bacter johnsonii strain 3–1 was isolated from surface- sterilized roots of Beta vulgaris. Its effect on sugar beet seedling growth was studied using pot assays and field experiments. This strain promoted beet seedling growth following seed inoculation by seed dipping. Plant height and dry weight of beet increased by 19% and 69%, respectively, compared with controls. Strain 3–1 exhibited the ability to increase absorption of N, P, K, and Mg elements from soil and increase the content of vitamins B and C, and protein within beet. In addition, the strain also produced a phytohormone-auxin, produced nearly twice as much IAA as that produced by strain 2–2, and was able to solubilize phosphates. The concentration of dissolved P in the medium was 180.5 mg L−1 after 4 days of incubation. In field experiments, strain 3–1 significantly increased the content of sucrose, fructose, and the yield of the beet. The growth-promoting properties of Acinetobacter johnsonii strain 3–1 indicates that this promising isolate merits further investigation into its symbiosis with beet plants and its potential application in agriculture. Keywords Sugar beet . Endophytic bacteria . Acinetobacter johnsonii . Growth promotion . Colonization 1 Introduction Sugar beet is the most important sugar crop in the developing world. Fertilizers and pesticides are the most important inputs for beet production but using growth- promoting bacteria can contribute to making beet cultivation sustainable and less dependent on fertilizers. Among such bacteria are rhizobacteria, which colonize plant roots and consume root exudates and lysates (Pieterse et al., 2002; Antoun and Prevost, 2006). Some strains, the growth- promoting rhizobacteria (PGPR), can promote plant growth. They improve the cycling of nutrients and minerals such as nitrogen, phosphate and can be used as biofertilizers (Kennedy et al., 2004). The growth-promoting effects of PGPR are affected by environmental conditions and there- fore are not stable. Endophytes including a variety of bacteria, fungi, and actinomycetes and can be isolated from cultivated crops (Liu and Tang 1996) as well as from wild plants (Brooks et al. 1994) both monocotyledons (Fisher et al. 1992) and dicotyledons (El-Shanshoury et al. 1996). Healthy roots of sugar beet have been shown to harbour endophytic bacteria and healthy leaves to harbour endo- phytic fungi (Jacobs et al. 1985; Larran et al. 2004), little is known about the behaviour of these microorganisms during the growing season. Endophytes occupy microniches within plant tissues and some have been found to be growth- promoting endophytes (PGPE). These endophytes are unaffected by competition from other microorganisms or conditions in the rhizosphere. As a result, a new approach has been developed in recent years to reduce fertilizer use which consists of treating seeds and seedlings with PGPE. Y. Shi :K. Lou (*) Institute of Microbiology, Xinjiang Academy of Agriculture Science, Urumqi, Xinjiang, China e-mail: loukai02@mail.tsinghua.edu.cn Y. Shi Key Laboratory of Green Chemical Processes of Xinjiang Production and Construction Corps, College of Agronomy, Shihezi University, Shihezi, Xinjiang, China C. Li Department of Biotechnology, School of Life Science and Technology, Beijing Institute of Technology, Beijing, China Symbiosis (2011) 54:159–166 DOI 10.1007/s13199-011-0139-x Thus, inoculation of pepper with endophytic Pseudomonas fluorescens significantly increased fresh weight, height, and stem diameter (Lucas-Garca et al. 2003). In an previous study, we isolated 221 endophytic bacterial isolates from tissues sterilized sugar beets from a widely planted beet cultivar (Beta vulgaris L. var. saccharifera) variety KWS2409 in a semi-arid region in east Urumuqi in Xinjiang,China. In the present study, we assess the effect of Acinetobacter johnsonii strain 3–1 on the growth of beet seedlings and on the ability of their root system to absorb mineral nutrients. 2 Materials and methods 2.1 Sugar beet variety, bacterial strain, and soil The soil for the pot experiments was collected from a fertile field in northern Xinjiang. Chemical and physical proper- ties of soil were analysed (Table 1). Total carbon, C, was determined by muffle furnace heating the soil to 500°C; total nitrogen, N, by the Kjeldahl method; and total phosphorus, P, by molybdenum blue colorimetry (Riehm 1985). Calcium, extractable magnesium, potassium, and sodium were measured using an atomic absorption spec- trophotometer (Schachtschnabel and Heinemann 1974); pH was determined with a pH meter; and total salt content was assessed by measuring the electrical conductivity of soil. Sugar beet (Beta vulgaris L. var. saccharifera) variety KWS2409 and Acinetobacter johnsonii strain 3–1 (GenBank accession number EU594557) were used, with Chryseobac- terium indologenes strain 2–2 (GenBank accession number EU594563) as control for 3–1, in the study assessing the effect of bacteria on plant growth. Sugar beet seeds (Beta vulgaris L. var. saccharifera) variety KWS2409 were obtained from Toutunhe farm in a semi-arid region in east Urumuqi in Xinjiang,China. 2.2 Plant growth and inoculation in pots Both strains 3–1 and 2–2 were grown in a liquid medium (3 g beef extract, 5 g peptone, 5 g NaCl, and 1 g glucose, in 1 L water, pH 7.0–7.2) shaken at 200 rpm at 30°C for 48 h, after which the bacterial cells were precipitated by centrifu- gation at 5,000×g for 5 min. The pellets were suspended in sterile distilled water and the concentration of the suspension adjusted to 108 cfu ml−1 with a spectrophotometer (Mayak et al. 2004). Sugar beet seeds were surface-sterilized with 70% alcohol for 8 min followed by 5% hydrogen peroxide solution for 2 h and then rinsed in sterile water (Caitriona et al. 2004). The disinfected seeds were immersed in the bacterial suspension for 6 h and then sown in pots, 10 seeds per pot. After germination, the stand was thinned to keep six plants in each pot. Ten replicates were maintained, with beet seeds immersed in distilled water instead of the bacterial suspen- sion serving as control (CK). The pots were kept in a growth chamber with the day/night temperatures set at 30°C/25°C and 200 μmol photons m−2 s−1 of light supplied throughout the 12 hday. Soil moisture was maintained at 60% of the soil’s moisture-holding capacity. Germination percentage was recorded 7 days later and data on plant height, fresh weight and dry weight, and the contents of N, P, K, and Mg and of Vitamin B,Vitamin C and protein recorded 4 weeks after germination. 2.3 Re-isolation of endophytic bacteria Endophytic bacteria from the seedling plant tissues were re- isolated 4 weeks after germination. To do this, fresh samples of sugar beet plants were washed thoroughly with tap water to remove adhering soil and debris, rinsed in 95% ethanol, and then flamed. This minimised carry-over of external bacteria when cross-sectioning at various locations using a sterile knife. Samples were aseptically removed from the core, periphery, and secondary root emergence zone (crease) areas. Each sample of root tissue was aseptically weighed, then added to 100 mL of sterile saline (0.85%), and blended for 2 min in a Waring blender. The blended samples were initially diluted to standardise all preparations. This was followed by additional serial decimal dilution in sterile saline. Endophytic bacteria were isolated using an aerobic spread- plate method. Dilution volumes of 0.1 mL were plated in triplicate on nutrient agar (NA) supplemented with 2.0 g/L sucrose. The spread plates were incubated at room tempera- ture for 7 d and colony counts recorded by standard methods (Kodaka et al. 2005). Pure cultures were subsequently isolated and bacterial isolates were stored on agar slants. 2.4 Identification of endophytic bacteria Pure cultures of the bacterial isolates were prepared for identification purposes. The bacterial re-isolates 3–1 and Table 1 Chemical and physical properties of soila in the top (0–25 cm) layer Parameter Ct g/kg Nt P K Mg Na Ca Total salts pH Value 24.1 1.28 1.1 0.15 0.04 0.14 0.12 1.32 7.85 aThe soil was yellow brown 160 Y. Shi et al. 2–2 were identified using the Vitek AutoMicrobic System (Vitek AMS; Vitek Systems, Inc., Hazelwood, MO.). The Vitek test was repeated twice. Isolates were characterized by Vitek AMS, colony morphology, catalase production, oxidase test, and gram stain. 2.5 Phylogenetic analysis of the isolated bacteral strains The 16S rRNA gene of genomic DNA isolated was extracted from pure bacterial colonies following standard protocols. The primers used for PCR were pF (5 -AGA GTT TGATCC TGG CTC AG-3 ) and pR (5 -AAG GAG GTG ATC CAG CCG CA-3).The PCR products with the expected size (about 1,500 bp) were purified using a DNA Gel Extraction Kit and cloned into pMD18-T vector followed by sequencing. Sequence analysis was performed using the BLAST algorithm (http://www.ncbi.nlm.nih. gov). Bacterial identifications were based on 16S rRNA gene sequence similarity. For further characterization of isolates 3–1 and 2–2, a neighbor-joining phylogenetic tree was constructed with the MEGA 4.0 program. 2.6 Measuring the ability of strain 3–1 to solubilize phosphates Strains 3–1 and 2–2 were grown in PKO (Ca3(PO4)2) medium Pikovaskaia’s medium, Yao 2004, per liter: Ca3(PO4)2 3.0~5.0 g, sucrose 10.0 g, (NH4)2SO40.5 g, NaCl 0.2 g, MgSO4·7H2O 0.1 g, KCl 0.2 g, yeast extract 0.5 g, MnSO41 mL (0.004 g/L), FeSO4 (Fe-EDTA) 0.1 mL (0.002 g/L), agar 15 g, pH value 7.0±0.2 () and incubated at 30°C for 12 h with vigorous shaking. The concentration of the bacterial suspension was adjusted to 108 cfu ml−1 and the suspension used as the primary inoculum. Two millilitres of the primary inoculum were added to 200 ml PKO medium whereas 2 ml distilled water added to 200 ml PKO medium served as control. Each treatment was repeated three times. The phosphates-solubilizing capability of Strains 3–1 and 2–2 were measured every 24 h for 7 days. Each time, a sample of the culture medium was centrifuged at 10,000 rpm for 20 min, and pH of the supernatant determined with a pH meter. Dissolved P (H2PO4 −) was determined using molybdenum blue colorimetry (Zhao et al. 2003; Zhang 2008). The pellet was suspended in 0.1 mol·L−1 HCl, rinsed three times in sterilized water, and optical density (OD660nm) of the suspension was measured. 2.7 Measuring the ability of strain 3–1 to secrete IAA The IAA assay was performed using the method described by Patten and Glick (2002) with some modifications. Both the strains, namely strains 3–1 and 2–2, were multiplied overnight in 5 ml of DF salts minimal medium (Dworkin and Foster 1958) which had the following composition (for 1 L): 4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g MgSO4·7H2O, Fig. 1 Effect of Strain 3–1 on growth of sugar beet. From left CK-W, seeds soaked in water; 3-1-N, seeds soaked in a suspension of Strain 3–1 in nutrient solution; CK-N, seeds soaked in nutrient solution 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 D ry W ei gh t/g 0 5 10 15 20 25 30 CK-W CK-N 3-1-W 3-1-N 2-2-W 2-2-N CK-W CK-N 3-1-W 3-1-N 2-2-W 2-2-N In di vid ua l p la nt h ei gh t/c m Fig. 2 Effect of seed inoculation on growth of beet seedlings. CK-W, seeds soaked in water; CK-N, seeds soaked in Hoagland solution; 3-1-W, seeds soaked in a suspension of Strain 3–1 cells in water; 3-1-N, seeds soaked in a suspension of Strain 3–1 cells in Hoagland solution; 2-2-W, seeds soaked in a suspension of Strain 2–2 cells in water; 2-2-N, seeds soaked in a suspension of Strain 2–2 cells in Hoagland solution. The results are representative of 48 plants. Error bars show the standard deviation Growth promotion effects of the endophyte 161 2.0 g glucose, 2.0 g gluconic acid, and 2.0 g citric acid; trace elements: 1 mg FeSO4·7H2O, 10 mg H3BO3, 11.1 mg MnSO4·H2O, 124.6 mg ZnSO4·7H2O, 78.22 mg CuSO4·5H2O, and 10 mg MoO3; pH 7.2; and 2.0 g (NH4)2SO4 as the nitrogen source. The next day, 20 μL of the primary inoculum was transferred into 5 ml of the DF medium supplemented with 100 μg ml−1 L-tryptophan (Sigma Chemicals) taken from a filter-sterilized 2 mg/ml stock prepared in warm water. After incubation for 48, 72, 96, and 120 h, the density of each culture was measured spectrophotometrically at 600 nm. The bacterial cells were then removed from the culture medium by centrifuging at 6,000×g for 15 min; a 1 ml aliquot of the supernatant was mixed vigorously with 4 ml of Salkowski’s reagent (Gordon and Weber 1951) consisting of 150 ml of concentrated H2SO4, 250 ml of distilled H2O, and 7.5 ml of 0.5 M FeCl3·6H2O, and allowed to stand at room temperature for 20 min before the absorbance at 535 nm was measured. The concentration of IAA in each culture medium was determined by comparing with a standard curve. 2.8 Estimation of internal root colonization Pot trials were designed to assess the internal colonization of sugar beet roots after seedling inoculation through the seed soaking method described above. Rifampicin-resistant mutants of isolates 3–1 and 2–2 were prepared as described by He et al. (2004). The obtained mutants were compared with their wild types in relation to their ability to produce auxins. None of these mutants differed morphologically from their parental strains, and all mutants had identical growth rates and auxin-production capability with their parental strains. Sugar beet seedlings were prepared as described above and inoculated with isolates 3–1 and 2–2. Every week after planting (1–8), roots were sampled from the soil, washed thoroughly in tap water, surface disinfested as described above and the population densities of isolates 3–1 and 2–2 (log10 CFU g−1 fresh root weight) were determined using NA amended with rifampicin (300 μgml−1 , Sigma). Each treatment was replicated five times with two plants in each replicate for each sampling. 2.9 Field experiments to evaluate performance of strain 3–1 Field experiments were conducted at the Toutunhe Farm in Urumuqi, Xinjiang province, China, from 2009 to 2010. Strain 3–1 inoculation was made the same way as pot experiment, inoculated by using the root drench method. Beets were harvested shortly after the 7th week of testing, at which time the plants were about 15 cm at the crown. Table 2 Effect of inoculation with Strain 3–1 on the content (mg/kg fresh weight) of N, P, K, and Mg and of Vb2,Vc, and protein in beet Treatment Nt P K Mg VB2(10 −3) VC Protein CK-W 1789.23±13.56 14.44±0.59 3629.99±61.41 659.61±17.59 1.62±0.05 0.82±0.01 964.01±0.88 CK-N 1828.06±35.09 15.39±0.51 3657.90±9.06 669.64±8.80 1.79±0.07 0.86±0.01 1002.43±1.90 3-1-W 2005.11±5.72* 19.63±0.44* 3823.65±88.53* 857.05±3.30* 1.94±0.07* 0.93±0.02* 1497.25±1.46* 3-1-N 2118.56±14.35* 21.78±0.63* 3949.72±50.98* 877.41±8.65* 2.06±0.05* 0.96±0.01* 1546.67±3.49* 2-2-W 1824.20±28.58 16.39±0.61 3653.14±10.16 664.85±7.75 1.86±0.07 0.85±0.02 1354.39±3.70 2-2-N 1852.63±18.27 17.63±0.62 3701.36±29.83 687.13±10.03 1.95±0.05 0.9±0.02 1405.92±2.19 Average ± standard error from three separate experiments. Values with the symbol * indicate significant differences at P<0.05 from corresponding control A. johnsonii 3P09AC EU977715.1 A. johnsonii 2P08MC EU977619.1 A. johnsonii 2P01AC EU977659.1 A. johnsonii CCA0908 GU320671.1 A. johnsonii CAI-6 DQ257426.1 A. johnsonii CAI-27 DQ257430.1 A. johnsonii UFV-E05 EF114343.1 A. johnsonii 3P02MC EU977623.1 A. johnsonii 3-1 EU594557.1 A. johnsonii 3P08MA EU977634.14 51 30 1 9 76 1 Fig. 3 Phylogenetic analysis of partial 16S rDNA sequence of isolates Acinetobacter johnsonii strain 3–1 obtained from sugar beet. Numbers in parentheses represent the sequencesaccession number in GenBank. The number at each branch points is the percentage supported by bootstrap 162 Y. Shi et al. Beets were fertilized, irrigated, and weeds controlled following typical production practices in northwest, China. The sucrose and fructose assay was performed using a spectrophotometric method (Taylor, 1995). Yield compar- isons among treatments were made at harvest date, and sugar content was determined by refractive method. 2.10 Statistical analysis All experiments were arranged in completely randomised block designs. Population data were transformed into log10 CFU g−1 fresh weight. Data were subjected to analysis of variance, and treatment means were compared using Fisher’s Protected LSD Test at P<0.05. 3 Results 3.1 Effect of strain 3–1 on plant growth Results of seed inoculation are shown in Figs. 1 and 2. Germination percentage was significantly higher in seeds inoculated with Acinetobacter johnsonii strain 3–1 (3–1-W) and so were the two other growth parameters, namely dry weight and plant height. Germination rate of inoculated seeds was 92%, 28.6% (in the text just give percentages to the nearest whole percentage i.e. 92% and 29%) greater than that of seeds that had been soaked in water (CK-W). Dry weight of seedlings from the inoculated seeds was about 3-fold greater, and the seedlings were 18.3% taller, than those from seeds that had been soaked only in water before sowing. Plants from seeds inoculated with strain 3–1 grew significantly better than those growing from seeds that had been soaked in water (W) or in Hoagland solution (N). Plants from seeds inoculated with strain 3–1 were 19.4% taller and 69.8% heavier than those from uninoculated seeds. In comparison, the 2–2 strain did increase the dry weight of beets, and it had little effect on plant height, however. 3.2 Effect of strain 3–1 on plant nutrient uptake The content of N, P, K, and Mg and of vitamin B, vitamin C, and protein was determined 4 weeks after germination (Table 2). Inoculation with Strain 3–1 significantly increased the uptake of all the elements that were analysed: total N and P following inoculation with strain 3–1 were greater by 10.8% and 26.4% respectively than their levels in CK-W, and the levels of K and Mg were also higher. It was also found that salt, that is soaking the seeds in a nutrient solution, affected the uptake of nutrients from soil: levels of N and P in CK-W were lower by 2.1% and 6.2% (In the test just give the % to the nearest whole percentage, i.e. 2% and 6%) respectively than the levels in CK-N. Inoculation with strain 3–1, however, counteracted this effect, levels of K+ and Mg2+ in strain 3–1-N being 7.4% and 23.6% higher respectively than those in CK-N and levels of total Vitamin B,Vitamin C, and protein being 13.1%, 10.4%, and 35.2% Fig. 4 Relationship between growth of Strain 3–1 and level of dissolved phosphorus. The results are representative of three experiments, with distilled water added to PKO medium as control. Error bars indicate standard deviation Fig. 5 Relationship between dissolved phosphorus (in the culture medium inoculated with Strain 3–1) and pH of the culture medium. The results are representative of three experiments. Error bars indicate standard deviation Table 3 Production of IAA by Strain 3–1 in a medium supplemented with tryptophan Time (h) IAA production (μg/ml/OD600 unit) Strain 3–1 Strain 2–2 24 21.53+2.17 10.12+1.01 48 42.19+2.87 15.97+1.60 72 95.84+3.00 20.49+2.05 96 126.97+3.23 25.64+2.56 120 119.45+3.40 33.23+3.32 144 51.07+3.86 33.20+3.32 168 49.56+3.36 29.10+2.91 A average + standard error from three separate experiments Growth promotion effects of the endophyte 163 higher respectively in strain 3–1-N than those in CK-N. Strain 2–2 also influenced the ability of sugar beet to accumulate ions, although not to the same extent as strain 3–1: levels of N, P, and K+ in 2–2-W