Improved Plant Growth and Zn Accumulation in Grains of Rice

Article pubs.acs.org/JAFC

Improved Plant Growth and Zn Accumulation in Grains of Rice (Oryza sativa L.) by Inoculation of Endophytic Microbes Isolated from a Zn Hyperaccumulator, Sedum alf redii H. Yuyan Wang,† Xiaoe Yang,† Xincheng Zhang,† Lanxue Dong,† Jie Zhang,† Yanyan Wei,‡ Ying Feng,† and Lingli Lu*,† †

MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China ‡ College of Agriculture, Guangxi University, Nanning, 530005, China ABSTRACT: This study is to investigate the possibility of zinc (Zn) biofortification in the grains of rice (Oryza sativa L.) by inoculation of endophytic strains isolated from a Zn hyperaccumulator, Sedum alf redii Hance. Five endophytic strains, Burkholderia sp. SaZR4, Burkholderia sp. SaMR10, Sphingomonas sp. SaMR12, Variovorax sp. SaNR1, and Enterobacter sp. SaCS20, isolated from S. alfredii, were inoculated in the roots of Japonica rice Nipponbare under hydroponic condition. Fluorescence images showed that endophytic strains successfully colonized rice roots after 72 h. Improved root morphology and plant growth of rice was observed after inoculation with endophytic strains especially SaMR12 and SaCS20. Under hydroponic conditions, endophytic inoculation with SaMR12 and SaCS20 increased Zn concentration by 44.4% and 51.1% in shoots, and by 73.6% and 83.4% in roots, respectively. Under soil conditions, endophytic inoculation with SaMR12 and SaCS20 resulted in an increase of grain yields and elevated Zn concentrations by 20.3% and 21.9% in brown rice and by 13.7% and 11.2% in polished rice, respectively. After inoculation of SaMR12 and SaCS20, rhizosphere soils of rice plants contained higher concentration of DTPAZn by 10.4% and 20.6%, respectively. In situ micro-X-ray fluorescence mapping of Zn confirmed the elevated Zn content in the rhizosphere zone of rice treated with SaMR12 as compared with the control. The above results suggested that endophytic microbes isolated from S. alfredii could successfully colonize rice roots, resulting in improved root morphology and plant growth, increased Zn bioavailability in rhizosphere soils, and elevated grain yields and Zn densities in grains. KEYWORDS: endophyte, Zn hyperaccumulator, rice, Zn biofortification

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INTRODUCTION Zinc (Zn) is an essential micronutrient and fulfills a structural role in over 300 enzymes in eukaryotes.1 Severe deficiency of Zn in the human body causes severe health complications, such as impairments in physical development, immune system, and brain function, especially for children and pregnant women.2 Zinc malnutrition is recognized as a serious threat to both human health and crop production globally3 and is estimated to affect more than 25% of the world’s population.4 Rice (Oryza sativa L.) serves as the dominant staple food for over 21% of the calorie intake needs of the world’s population5 but has the shortcoming, from a nutrition perspective, of being low in Zn and other essential nutrients.6 Therefore, enhancement of micronutrients, especially Zn, in rice grain is viewed to be an important goal as part of global strategies for improving the Zn nutritional quality of human diets.6,7 Genetic biofortification and agronomic biofortification are two important agricultural strategies for Zn biofortification in the edible parts of crops for the benefit of human nutrition. Plant breeding has been suggested as a major strategy to solve micronutrient deficiencies, such as cultivation of Zn efficient crop cultivars on soils with low plant-available Zn and selection of crop genotypes efficient in acquisition and utilization of nutrients, but it is a long-term process requiring a substantial effort and resources.7 The traditional strategy of agronomic biofortification, such as application of Zn fertilizers or Zn© 2014 American Chemical Society

enriched NPK fertilizers, offers a rapid solution to cope with Zn deficiency and increase grain yield8 and represents a useful complementary approach to ongoing breeding programs.9 However, even Zn added to soil as fertilizer may become rapidly unavailable for plant uptake10 and is not always optimal from an economic perspective.11 Plant growth-promoting rhizobacteria (PGPR), including endophytic and rhizospheric bacteria, are beneficial bacteria that colonize root surface and stimulate the growth of the host.12 The usage of PGPR in agriculture is steadily increasing because it offers an attractive way to reduce the use of chemical fertilizers, pesticides, and related agrochemicals.13 For example, endophytic bacteria were implicated in supplying biologically fixed nitrogen in nonlegumes, thus increasing the nitrogen economy of crop plants and reducing the requirement for N fertilizers.14 Endophytic microbial inoculation significantly increased the crop yield.15 However, the role of endophytes involved in improving the micronutrient nutritional status of plants has been less investigated.16 The possibility of the inclusion of PGPR in micronutrient accumulation in cereals has been proposed to complement the existing biofortification Received: Revised: Accepted: Published: 1783

September 23, 2013 November 27, 2013 January 21, 2014 January 21, 2014 dx.doi.org/10.1021/jf404152u | J. Agric. Food Chem. 2014, 62, 1783−1791

Journal of Agricultural and Food Chemistry

Article

strategies reducing malnutrition in developing countries.16 It has been reported that inoculation of bacteria (Providencia sp. PW5) resulted in a significant increase of Fe, Mn, and Cu in wheat grain.16 Recently, endophytic bacteria colonizing the internal tissues of metal hyperaccumulator or accumulator plants have been gaining increasing attention.17 Endophytic bacteria promoted plant growth, increased heavy metal accumulation, and available heavy metal in the rhizosphere soil. Inoculation of plants with endophytes, which were isolated from metal-resistant or hyperaccumator plant species, is extensively used for phytoremediation of metalliferous soils.18−20 However, there are few reports on whether the bacteria isolated from hyperaccumulator could survive in a crop system and their potential use in biofortification of micronutrients in crop plants. In the present study, five endophytic strains, Burkholderia sp. SaZR4, Burkholderia sp. SaMR10, Sphingomonas sp. SaMR12, Variovorax sp. SaNR1, and Enterobacter sp. SaCS20, were isolated from Sedum alf redii Hance, a Zn hyperaccumulator native to China.21,22 Our previous studies showed that inoculation of the isolated endophytic bacterial strains back to sterilized plants of the hyperaccumulator S. alfredii resulted in elevated biomass and Zn concentrations in both shoots and roots.23 The endophytic microbia were also harmless to crops according to pathogenic detection of tobacco leaf.23 The objective of the study is to investigate the possibility of Zn biofortification in rice grains by inoculation with the endophytic strains isolated from S. alfredii.

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NaCl), then the inoculum density was adjusted to 108 colony-forming units mL−1, and the resulting suspensions were used for inoculation. Roots of rice seedling at five-leaf stage were surface sterilized with 3% chloride for 2 min and four washes with sterile water. A total of 5 mL of bacterial inocula were carefully introduced into 2.5 L of nutrient solution using a pipet avoiding contamination of the above-ground rice tissues. Plants were grown in a growth chamber, and treatments were arranged randomly with four replications. Rice plants were harvested at 30 d after inoculation (DAI). Inoculation with Endophytic Microbia under Soil Conditions. In a separate experiment, the effects of the endophytic strains on rice growth and grain Zn accumulation were further investigated under soil conditions in a greenhouse. Two strains (SaMR12 and SaCS20) selected from the hydroponic experiment were used in the pot experiment. The silty loam soil used in this experiment was collected from the experimental field of Zhejiang University, Zhejiang Province, China. The physical−chemical properties of the experimental soil were pH 6.5, available N, P, and K of 67.7, 37.6, and 62.5 mg kg−1, respectively, DTPA-extractable Zn 4.33 mg kg−1. Soil was air-dried, sieved to 5 mm, and homogenized before filling the pot, each of which contained 8.0 kg of dry soil. Recommended levels of NPK fertilizers were supplied (in mg per kg of dry soil) to each pot at a rate of 160 mg of N (70% as basal and 30% at panicle initiation), 50 mg of P2O5, and 90 mg of K2O before transplanting. Roots of rice seedlings at five-leaf stage were sterilized as described above, and every six seedlings were inoculated by soaking the roots in 500 mL of single bacterial suspension (108 colony-forming units mL−1) for 2 h and then transplanted to each pot. Rice was additionally treated with 20 mL of the bacterial cell suspensions (108 colonyforming units mL−1) by spraying on the plant rhizosphere soil at tillering stage and flowering stage, respectively. The same volume of sterilized deionized water was used as control. Treatments were arranged randomly with four replications. Water content of each pot was maintained at 75% (w/w) with deionized water daily. Plants were grown in greenhouse with day/night temperatures of 30 ± 2 °C/23 ± 2 °C under natural light conditions from June to Oct., 2012, at Zhejiang Province, China. Laser Scanning Confocal Microscopy (LSCM). For hydroponic experiments, rice roots for LSCM were excised from plants at 2, 4, and 6 DAI, and a random sample of root system was assessed for microbial colonization. For pot experiments, rice roots and root with less rhizosphere soil were excised at 3 and 7 DAI for LSCM. LSCM was performed according to Lin et al.25 Root Morphology and Root System Formation. Roots of harvest plants were analyzed with a root automatism scan apparatus (MIN MAC STDI600+, Tokyo, Japan) equipped with the WinRHIZO software (Regent instruments Inc., Quebec, Canada).20 Root system formation of the rice was observed by using a stereomicroscope (Leica MZ9.5) with a DCF300 camera. Individual root was sampled randomly and cut into 10 mm fragments from root tips. Images were captured with a Leica application suite (LAS) software (Leica, Bannockburn, IL). Elemental Determination. At 30 DAI under hydroponic conditions, roots of rice cultured hydroponically were submerged into a 1.0 L bath containing 1.0 mM LaCl3 and 0.05 mM CaCl2 for 10 min to remove apoplastic Zn.27 Plants were separated into roots, stems, and leaves and cleaned with ultrapure water (resistivity ≥ 18.2 MΩ cm−2). Rice plants cultured under soil conditions were harvested at maturity and separated into grains, flag leaves, leaves, and stems. All plant samples were rinsed thoroughly with ultrapure water, oven-dried at 65 °C for 72 h, weighed, and ground with an MM301 (Retsch, Germany) with agate ball and internal wall. Samples were digested in a diacid mixture of HNO3−HClO4 (v/v, 4:1), and the digest was transferred to a 50 mL volumetric flask, made up to volume, and filtered. Samples were digested in triplicate, and Zn concentration was determined using an inductively coupled-plasma mass spectrometer (ICP-MS, Agilent 7500a, CA, USA). DTPA-extractable Zn in the rhizosphere soil was analyzed by ICP-MS (Agilent 7500a, CA), and soil pH was measured in a soil slurry (soil/water ratio = 1:2.5).28

MATERIALS AND METHODS

Bacteria, Plasmids, and Plants. Five single bacterial strains (SaZR4, SaMR10, SaNR1, SaMR12, and SaCS20) used in this study were isolated from S. alfredii as described by Zhang et al.24 The bacterial strains were identified as Burkholderia sp. SaZR4, Burkholderia sp. SaMR10, Sphingomonas sp. SaMR12, Variovorax sp. SaNR1, and Enterobacter sp. SaCS20 based on their 16S rRNA gene sequences and carbon source utilization profiles determined using Biolog GN2 microplates (Biolog Inc., Hayward, CA, USA). SaZR4, SaMR12, SaNR1, and SaCS20 transconjugants constitutively expressing green fluorescent protein (GFP) were obtained by triparental mating with the vector plasmid pHC60 and helper phage pRK2013.23 Bacterial physiology including indole-3-acetic acid (IAA) and siderophore production and phosphate solubilization (except SaNR1) was determined according to Lin et al.25 Japonica rice (Oryza sativa L.) variety Nipponbare obtained from Zhejiang Academy of Agricultural Sciences was used for this study. Surface-sterilized rice seeds were germinated at 30 °C in the dark. After germination, plantlets were sown in a sterile pot that was filled with sterilized quartz sand in a growth chamber. Uniform seedlings at three-leaf stage were transplanted to a 2.5 L black plastic pot with nutrient solution containing (in mM) NH4NO3, 1.43; CaCl2·2H2O, 1.00; MgSO4·7H2O, 1.64; NaH2PO4·2H2O, 0.32; and K2SO4, 1.32; and (in μM) MnCl2·4H2O, 5.0; CuSO4·5H2O, 0.2; (NH4)6Mo7O24· 4H2O, 0.075; H3BO3, 1.90; Fe-EDTA, 20.0; and ZnSO4·7H2O, 2.0.26 The nutrient solutions were replaced every 3 days and adjusted to pH 5.5 ± 0.1 every day. Plants were grown in a growth chamber under a photo flux density of 400 μmol m−2 s−1, a light/dark period of 16/8 h, day/night temperatures of 30/25 °C, and day/night relative humidities of 75%/85%. Inoculation with Endophytic Microbia under Hydroponic Conditions. The five isolated endophytic strains were used in the hydroponic experiment. A single bacterial strain was grown in liquid Luria broth (LB) medium for 48 h at 30 °C on a rotating shaker. Cells were harvested by centrifugation, washed twice with phosphate buffered saline (PBS), and suspended in biological saline (0.85% 1784

dx.doi.org/10.1021/jf404152u | J. Agric. Food Chem. 2014, 62, 1783−1791


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Figure 1. GFP-tagged Burkholderia sp. SaMR12 strain colonized on and in the roots of rice. LSCM images present bacterial GFP fluorescence in green and root autofluorescence in red. SaMR12 cells colonized on root cap (A), lateral root (B), and root hairs (C) at 2 DAI. SaMR12 cells colonized at root hairs (D), entered root hair cells (E) and cortical intercellular spaces (F) at 4 DAI. SaMR12 invaded vascular tissues of the primary root (G) at 7 DAI, and arrowhead points to bacterial aggregates. The xy crosshairs present the positions for orthogonal sectioning through the zstrack. The top right orthogonal view presents the yz section of the z-stack; the bottom orthogonal view presents the xz section of the z-strack. Bars: 50 μm in panels A−D and G and 5 μm in panels E and F. DAI, days after inoculation. SRXRF Analysis. Micro-X-ray fluorescence imaging of Zn in rice root and rhizosphere soil was carried out on beamline BL15U at the Shanghai Synchrotron Radiation Facility (SSRF), Shanghai, China. At 30 DAI, root together with its adjacent rhizosphere soil (0.20 cm) was carefully cut with a razor blade. In situ micro-XRF mapping of Zn was analyzed according to Lu et al.29 The electron energy in the storage ring was 3.5 GeV with a current range from 200 to 300(5) mA. The brilliance of the beam in BL15U was 0.5 × 1012 photons s−1 mm−2 mrad−2 (0.1% BW)−1. The microfocused beam of 3.5 μm was provided by a Kirkpatrick−Baez mirror pair (Xradia Inc.) with the sample at 45° to the incident X-ray beam. The fluorescence yield was detected using a seven-element Si (Li) solid state detector, positioned at 90° to the beamline. Dwell time per point was 0.5 s. The step size was set to 10 μm. Scanning areas of the samples were selected and observed using a microscope. BL15U saves full XRF spectrum data at each pixel. Elemental distributions are achieved by windowing on the elements of interest in the XRF spectra. The windows can be applied during data

collection or during data analysis since the full XRF spectrum is saved for each pixel. The beam energy was set to 13 keV during mapping. The fluorescence energy windows for this investigation were Zn, Fe, K, Ca, and Mn. The fluorescence data are presented as color maps, and pixel brightness is displayed in RGB, with the brightest spots corresponding to the highest element fluorescence. Statistical Analysis. All data were statistically analyzed using the SPSS package (version 11.0). Analysis of variance (ANOVA) was performed on the data sets, and the mean and SE of each treatment as well as LSD (P < 0.05 and P < 0.01) for each set of corresponding data were calculated. The figures were made using the software Origin 8.0.

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RESULTS Root Colonization Patterns of GFP-Tagged Endophytic Bacteria. The GFP-tagged endophytic bacteria were inoculated as the treatment of endophytic inoculation for 1785



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Figure 2. Plant growth (A) and biomass (B, shoot; C, root) of rice after 30 days inoculation with different endophytic bacteria isolated from S. alfredii. Different letters indicate statistically significant difference (P < 0.05) among treatments. Error bars indicate standard deviations of three replicate determinations.

Table 1. Root Morphology of Rice after 30 Days Hydroponic Inoculation of the Endophytesa treatments control SaZR4 SaMR10 SaNR1 SaMR12 SaCS20

tips 7051 7480 8457 7359 7845 8084

± ± ± ± ± ±

468d 473cd 451a 253d 465b 231a

forks 13284 12956 12400 13085 13336 12905

± ± ± ± ± ±

length (cm) 601a 591a 463a 449a 355a 399a

1659 1893 1877 1933 1976 1920

± ± ± ± ± ±

diameter (mm)

136b 167a 189a 158a 195a 382a

0.34 0.43 0.46 0.48 0.46 0.45

± ± ± ± ± ±

0.04b 0.02a 0.02a 0.04a 0.04a 0.03a

surface area (cm2) 253 280 269 267 272 310

± ± ± ± ± ±

34b 43ab 34b 29b 38b 67a

volume (cm3) 2.4 3.1 3.0 3.3 3.3 3.4

± ± ± ± ± ±

0.3b 1.1a 0.4a 0.3a 0.6a 0.6a

Values are presented as mean value ± standard deviation of four replicates. Means followed by different letters in the same column indicate significant difference between the treatments at P < 0.05.

a

fluorescent bacteria observed on or in the uninoculated roots of rice. Plant Growth, Root Morphology, and Zn Uptake of Rice Modulated by Endophytic Bacteria. Modulation of plant growth, root morphology, and Zn uptake of rice plants by endophytic bacteria were investigated hydroponically. Five endophytic strains (SaZR4, SaMR10, SaMR12, SaNR1, and SaCS20) isolated from S. alfredii were inoculated in the rice roots of Nipponbare under hydroponic condition for 30 d. As shown in Figure 2, inoculation with endophytic bacteria, especially SaMR12 and SaCS20, significantly improved the rice growth. Both root and shoot biomass increased markedly with inoculation with each individual endophytic strain, by 29.4− 70.6% in root and 21.1−53.3% in shoot. Among the five

determination of bacterial populations at different days after inoculation (DAI) under hydroponic conditions. Root colonization patterns of other GFP-tagged endophytic bacteria were quite similar to that of SaMR12, which is shown in Figure 1. At 2 DAI, GFP-tagged SaMR12 had successfully colonized the surface of lateral roots (Figure 1A) and primary roots from root caps (Figure 1B) to root hairs (Figure 1C). From 2 to 4 DAI, SaMR12 cells formed biofilm-like structures on the root surface (Figure 1D) and entered into root hair cells and cortical intercellular spaces (Figure 1E,F). By 7 DAI, SaMR12 had invaded vascular tissues of the primary root (Figure 1G). The GFP-tagged SaMR12 heavily colonized the roots of rice after 2 weeks of inoculation (data not shown). There was no 1786



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Figure 3. Root tips of rice after 30 days inoculation without (A) or with different endophytic bacteria, SaMR12 (B) and SaCS20 (C), isolated from S. alfredii.

endophytic strains, the most pronounced effect of inoculation on growth promotion was observed with SaMR12, followed by SaCS20. Significant variation of root morphology was observed for rice plants with endophytic inoculation, especially by SaMR12 and SaCS20 (Table 1). Root length and diameter of inoculated seedlings significantly increased by 11.5−16.4% and 23.9− 35.2%, respectively, compared with the controls, although no differences were recorded among the five strains. After inoculation with individual bacterial strain, root tips significantly increased by 11.3% and 14.7% at the presence of SaMR12 and SaCS20, although no significant difference of root forks was noted. Significantly increased (22.5%) root surface area was also observed with SaCS20 inoculation. Stereomicroscope imaging of single roots showed that endophytic inoculation of SaMR12 and SaCS20 significantly stimulated

root hair production, especially at the 30−40 mm fragment, compared with the controls (Figure 3). Elevated Zn uptake by rice was observed after inoculation with the five individual endophytic strains, among which the effects of SaMR12 and SaCS20 were mostly pronounced (Figure 4). Inoculation with SaMR12 and SaCS20 raised shoot Zn concentration of rice by 44.4% and 51.1%, respectively, compared with the controls (Figure 4A), and also increased Zn concentration in roots by 73.6% and 83.4%, respectively (Figure 4B). Root Zn concentration increased up to 48.1 and 50.8 μg g −1 , respectively, after SaMR12 and SaCS20 inoculation. Calculated from the biomass and Zn concentration, Zn accumulation in shoots and roots of rice increased by approximately 100% and 200%, respectively, in the presence of either SaMR12 or SaCS20. Improved Grain Yield and Zn Accumulation by SaMR12/SaCS20 Inoculation. Since SaMR12 and SaCS20 1787



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Figure 5. GFP-tagged Burkholderia sp. SaMR12 strain colonized in the roots of rice in soil. LSCM images present bacterial GFP fluorescence in green and root autofluorescence in red. SaMR12 cells colonized on root surface (A) and root cells (B, C) at 3 DAI. Bars: 10 μm.

SaCS20 inoculation. Accordingly, the total Zn accumulation in both brown rice and polished rice increased by 50.9−54.4% and 39.4−41.8%, as the result of endophytic inoculation of SaMR12 and SaCS20, respectively. Due to the elevated biomass, Zn accumulation in plant tissues (stems, leaves, flag leaf) was also significantly improved by inoculation with the two endophytic strains, although no variation of Zn concentrations was observed for leaves and stems (Table 3). Alteration of Rhizosphere Zn by SaMR12/SaCS20 Inoculation. The impacts of SaMR12 and SaCS20 on soil pH and rhizosphere Zn were analyzed for rice cultured in pot experiments. Bacterial inoculation did not change soil pH in the rhizosphere zone cultured with rice plants (Figure 6A). However, in the presence of SaMR12 or SaCS20, rhizosphere soils contained a higher concentration of DTPA-Zn by 10.4− 20.6% compared with the controls (Figure 6B). To further investigate the impacts of endophytic bacterial inoculation on rhizosphere Zn of rice, microscanning XRF mapping of elemental distributions was performed on rice roots and rhizosphere soil nearby with or without SaMR12 inoculation. The integrated intensity for Zn was calculated from the spectrum and normalized by the intensity of the Compton scattering peak. Elemental mapping for the measurement area was obtained from the normalized intensity for the element. The elemental distribution maps of Zn in the scanned area of rhizosphere zone are presented in Figure 7, together with photographs taken using an optical microscope. The XRF maps indicate the relative distribution of Zn, and the normalized X-ray fluorescence intensities are scaled from red (maximum, 1000 counts s−1) to purple (minimum, 0 counts s−1). The XRF image of the controls (Figure 7A) revealed that Zn intensities were very low in the rhizosphere zone nearby the rice roots, while higher intensities of Zn were observed in both roots of rice and their adjacent rhizosphere zone with inoculation with SaMR12 at 30 DAI (Figure 7B). On the basis of the intensity of Zn signal (Figure 7B), the concentration of Zn in the rhizosphere zone in presence of

Figure 4. Zn concentrations in shoots (A) and roots (B) of rice after 30 days inoculation with different endophytic bacteria isolated from S. alfredii. Different letters indicate statistically significant difference (P < 0.05) among treatments. Error bars indicate standard deviations of three replicate determinations.

were of most effective for promotion of plant growth, root morphology, and Zn uptake of rice in solution, their impacts on grain yield and Zn accumulation in rice were further investigated under soil conditions. As shown in Figure 5, SaMR12 began to colonize root rhizosphere soil and root surface at 3 DAI and entered into root cortical intercellular spaces at 7 DAI. SaCS20 showed a similar colonization pattern to that of SaMR12 (data not shown). Endophytic inoculation with SaMR12 and SaCS20 significantly affected the values of yield components and improved grain yields (Table 2). Although no variation of thousand-grain weight (TGW) and grain setting rate was observed, among all the treatments, significant elevation of panicles and grain per spike was noted by inoculation with the endophytes. When compared with the controls, grain yields increased by 25.5% and 26.9%, respectively, after endophytic inoculation with SaMR12 and SaCS20. Inoculation with either SaMR12 or SaCS20 also resulted in significant increase of Zn densities in both brown rice and polished rice (Table 3). For instance, Zn concentrations in brown rice reached up to 30 mg kg−1 with SaMR12 and 1788



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Table 2. Plant Growth and Yield Parameters of Rice Cultivar at Harvest with Two Different Endophytic Inoculation Treatmentsa treatments

panicles per pot

grain per spike (grain per−1)

grain setting (%)

TGW (g)

shoot biomass (g pot−1)

grain yield (g pot−1)

control SaMR12 SaCS20

18 ± 3b 22 ± 2a 20 ± 3ab

57.3 ± 4.1b 63.4 ± 3.5a 66.9 ± 5.8a

0.79 ± 0.03a 0.75 ± 0.02a 0.79 ± 0.04a

25.9 ± 0.7a 26.2 ± 0.9a 25.8 ± 0.8a

32.6 ± 5.6b 45.12 ± 6.9a 46.6 ± 5.7a

21.6 ± 2.9b 27.1 ± 3.0a 27.4 ± 6.2a

Values are presented as mean value ± standard deviation of four replicates. TGW, thousand-grain weight. Means followed by different letters in the same column indicate significant difference between the treatments at P < 0.05. a

Table 3. Zn Concentration (μg g−1) and Zn Accumulation (μg pot−1) of Different Parts of Rice Cultivar at Harvest with Two Different Endophytic Inoculation Treatmentsa treatments

stem


40.3 ± 3.8ab 49.3 ± 6.0a 37.6 ± 4.2b


983.2 ± 24.6c 1481.3 ± 35.3a 1166.8 ± 32.4b

flag leaf

leaf 47.6 ± 3.2a 48.4 ± 2.8a 44.6 ± 2.6a 568.2 ± 26.1b 715.0 ± 32.7a 681.2 ± 31.1a

Zn Concentration (μg g−1) 37.6 ± 4.0a 40.3 ± 3.6a 38.2 ± 2.0a Zn Accumulation (μg pot−1) 45.8 ± 2.3b 60.5 ± 3.0a 59.2 ± 4.3a

brown rice

polished rice

25.1 ± 2.9b 30.2 ± 2.4a 30.6 ± 2.9a

20.5 ± 0.4b 23.3 ± 0.5a 22.8 ± 0.4a

433.3 ± 24.3b 653.9 ± 39.0a 668.8 ± 40.5a

311.8 ± 19.5b 442.2 ± 30.4a 434.5 ± 25.9a

Values are presented as mean value ± standard deviation of four replicates. Means followed by different letters in the same column indicate significant difference between the treatments at P < 0.05.

a

still doubtful even if the microbe could colonize the host plants in hydroponic conditions. Here we also clarified by pot experiments that the isolated endophytic strains from S. alfredii could succeed in surviving in paddy soils and colonize rice root cortical intercellular spaces (Figure 5). These results suggested that these endophytic microbial strains isolated from S. alfredii had a significant effect on plant growth promotion either under hydroponic or soil conditions. Recent investigation on PGPR suggests its promotion of growth by a wide variety of mechanisms, including phytohormone production, rhizosphere engineering, phosphate solubilization, etc.13,16 The main characteristic of PGPR is to promote the growth of plant roots, and this is confirmed by the bacteria-improved root morphology and enhanced root hair production in this study (Figure 2; Table 1). Most commonly, endophytic bacteria synthesize IAA to promote root growth and root length.34 Zhang et al.23 suggested that bacterial IAA production was likely involved in the plant-promoting effects on S. alfredii in hydroponic culture. IAA production of the endophytic microbes ranged from 0.51 to 82.99 μg mL−1,24 with higher IAA production observed for SaMR12 and SaCS20. Although not determined in this study, it is possible that IAA production may be responsible for the enhanced plant growth of rice induced by SaMR12 and SaCS20 inoculation. Distiguished from normal PGPR, endophytic microbial strains applied in this study were isolated from a Zn hyperaccumualtor S. alfredii, which has an extraordinary ability to take up Zn from soils and accumulate large amounts of Zn in its aerial parts.21,22 The endophytic microbial strains were able to increase Zn accumulation in plants of their original host S. alf redii.23 It is therefore not surprising that inoculation of the endophytic microbial strains (SaMR12 and SaCS20) to rice plants not only resulted in promotion of plant growth and grain yields (Table 2) but also elevated Zn densities in rice grains (Table 3). Reduced yield and low harvest index resulting from increased mineral concentrations in the edible parts is a general problem for micronutrient biofortification of crops.35 The positive responses of both rice grains yields and Zn densities

Figure 6. Variations of soil pH and DTPA-Zn (mg kg−1) in rice rhizosphere soil with inoculation of SaMR12 and SaCS20. Different letters indicate statistically significant difference (P < 0.05) among treatments. Error bars indicate standard deviations of four replicate determinations.

SaMR12 was likely to be many times higher than that of the controls.

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DISCUSSION Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria that colonize the rhizosphere and contribute to enhanced plant growth and yield of crop plants.30 The most important characteristic of PGPR is the ability to colonize the roots of host plants.31 Different plant hosts have different ability for the stains to be colonized.32 The present study clearly showed successful colonization of endophytic microbial isolates from the Zn hyperaccumulator S. alf redii,23 resulting in significantly plant growth promotion of rice plants (Figures 1 and 2). It has been reported that the survival of PGPR in soil was greatly influenced by external parameters including water content, pH, soil type, composition of root exudates, and other microorganisms;33 their survival and colonization in soils are 1789



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Figure 7. μ-XRF elemental maps for Zn of rice root and rhizosphere soil treated with control (A) and SaMR12 strain inoculation (B) for 30 DAI.

bacteria increased the root surface area (Table 1) and DTPAZn content in the rhizosphere soil (Figure 6), the same as reported by Orhan et al.,38 further facilitating Zn acquisition by the plants (Table 3). The XRF images (Figure7) further confirmed higher intensities of Zn observed in both roots of rice and their adjacent rhizosphere zone after inoculation of SaMR12 for 30 DAI, compared with the controls. The alteration of rhizosphere Zn was not due to the soil acidification since soil pH did not changed with inoculation of the endophytes. In conclusion, the present study suggest that colonization with the endophytic microbial isolates from hyperaccumulators to rice plants could result in improved root morphology and plant growth, increased Zn bioavailability in rhizosphere soils, and elevated grain yields and Zn densities in both brown rice and polished rice, thus providing a potential strategy for Zn biofortification of rice grains.

with SaMR12 and SaCS20 inoculation suggested that application of the endophytes could be an efficient strategy for Zn biofortification in rice grains. Millions of hectares of cropland are affected by Zn deficiency, and approximately onethird of the human population suffers from an inadequate intake of Zn.36 To the best of our knowledge, the present study for the first time reported the potential use of endophytes isolated from hyperaccumulators on Zn biofortification of rice plants. The possible mechanisms involved in the enhanced Zn accumulation in rice by endophytic inoculation, however, are not well-known. The results in the present study suggested modulation of root morphology might be one of the strategies involved in endophyte-enhanced metal uptake and accumulation in rice plants. In nutrient efficiency conditions, roots play the most important role in nutrient acquisition;37 root morphology (length, surface, volume, and diameter) are of key importance for acquisition of nutrients. In hydroponic experiments, in the presence of endophytic bacteria, root length, surface, volume, and diameter of rice roots were significantly increased (Table 1; Figure 3). The enhancement of root morphology with endophytic inoculation might result in higher Zn uptake by rice plants. Another possible mechanism for enhanced Zn accumulation in rice plants is the alteration of rhizosphere Zn under soil conditions. Biofortification of Zn for food crops is largely dependent on the size of plant-available Zn pools in soil.7 DTPA-Zn is regarded as a mixture of both mobile and potentially mobilizable species and represented the pool of bioavailable Zn in soils. The inoculation of endophytic

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AUTHOR INFORMATION

Corresponding Author

*Dr. Lingli Lu. Mailing address: Department of Plant Nutrition, College of Environmental & Resources Science, Nongshenghuan Building B-517 Zhejiang University, Zijingang Campus, Hangzhou, 310058, P.R. China. Tel: +86-13588356620. Fax: +86-0571-88982907. E-mail: [email protected], linglilulu@ gmail.com. Funding

This research work was financially supported by the HarvestPlus-China Program (Grant 8271), the National Natural 1790



Article

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Science Foundation of China (Grant 31300040), and the Department of Science & Technology of Zhejiang Province (Grant 2011C22077). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Liao Haibing is gratefully acknowledged for ICP-MS analysis. REFERENCES

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Improved Plant Growth and Zn Accumulation in Grains of Rice

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