The cellular components of cucumber seedlings after primed with plant growth promoting rhizobacteria, Bacillus subtilis Bs008

This work aimed at investigating the changes in cellular compositions of cucumber seedlings as enhanced by plant growth promoting rhizobacteria, Bacillus subtilis Bs008 using the Fourier transforminfrared spectroscopy (FT-IR). The objective of this study was to test the hypothesis that Bs008 stimulates production of plant cellular components involved in metabolism and growth development mechanisms. Cucumber vegetative propagation and treatment with Bs008 or with sterile distilled water was done in sterile soil, after incubation for 28 days, Bs008 treated cucumber seedlings had more lateral root, longer roots, shoot length and greater biomass than the control. We also focused on plant cellular composition, and cucumber seedling tissues from the two treatments were harvested for FT-IR analysis. FT-IR analyses revealed that lipid was highly accumulated in response to the strain Bs008. The cucumber seedling treated with the beneficial bacteria B. subtilis strain Bs008 shows the beta sheet secondary structure and apparently appeared with high polysaccharide content but the lipid content was decreased when compared with those of the cucumber seedling treated with distilled water. Our results demonstrate that Bs008 can enhance plant growth under laboratory conditions by direct stimulation of cucumber polysaccharides.


INTRODUCTION
The beneficial bacteria, Bacillus spp.are widely used as commercial bacteria for control of plant pathogens and enhances plant growth promotion.In addition to directly affecting plant growth and development through plant growth regulator, Bacillus spp.can colonize roots and trigger plant biochemical and physiological systems to promote growth enhancement.The plant growth promoting rhizobacterium (PGPRs) such as Bacillus amyloliquefaciens strain KPS46, can enhance growth in several economic crops such as soybean, vegetable soybean, corn, rice, Chinese kale and cauliflower (Prathuangwong and Kasem, 2004;Prathuangwong et al., 2005;Prathuangwong and Buensanteai, 2007), this process is mediated in part by the excretion of phytohormones such as auxin and indole-3-acetic acid (IAA), lipopeptides and extracellular proteins (Buensanteai et al., 2008a).This could account for interacts with plants that the Bacilli group can synthesize phytohormones similar to the plant endogenous growth regulator and enhanced those levels in plant which involved the *Corresponding author.E-mail: natthiya@sut.ac.th.Tel: 66 4 422 4204.Fax: 66 4 422 4281.initial processes of lateral and adventitious root formation and elongation (Buensanteai et al., 2008a, b;Erturk et al., 2010).With regards to the latter mode of action, there is no information as to the proteome expression activated or the growth and development responses triggered in cucumber upon treatment with PGPR as Bacilli group.
Cucumber is a major vegetable crop in Asia, especially Thailand, but there are no studies on the use of PGPR to enhance growth of cucumber seedling (Wan et al., 2005).However, enhanced plant growth promotion studies using the strains of Bacillus spp. on model plants revealed that bacilli can trigger signaling pathways leading to the growth promotion phenotype (Idriss et al., 2002).
Plant response to PGPR requires alterations in the physiology and biochemistry level that directly and indirectly result from the modification of genes and proteins expression (Wan et al., 2005;Buensanteai et al., 2009).Such PGPR-induced modifications may lead to the accumulation of certain metabolites and alterations in the level (increase or decrease) or the presence (appearance or disappearance) of some cellular proteins (Erturk et al., 2010;Wan et al., 2005).The growth and development of plants are the process by which a plant increases in the number, size of leaves, stems, root, and change from one growth stage to another (Buensanteai et al., 2009).The result of plant growth and development is production and the amount of harvested plant yield.
Fourier transform infrared (FTIR) spectroscopy is known to propose the high ability for understanding the total cellular and biochemical components of organism and micro-organism cells (Szeghalmi et al., 2007), because all the bio-organic and cellular composition functional groups absorb specific infrared wavelength.Moreover, there are several publications on the application of this FTIR technique to detect changes in metabolic processes of carbohydrates and lipids under the different stress conditions (Kamnev, 2008;Szeghalmi et al., 2007).
FTIR spectroscopy is also a rapid, versatile and sensitive tool that has been used for elucidating the structure, physical properties and interactions of carbohydrates (Kacurakova and Wilson, 2001).The carbohydrates show high absorbance in the region 1200-950cm -1 that is within the so-called fingerprint region, where the position and intensity of the bands is specific for every polysaccharide (Kacurakova and Wilson, 2001).FTIR spectroscopy may therefore be used to evaluate carbohydrate changes and profiles in plants exposed to biotic and abiotic stresses.For example, FTIR spectro-scopy was used, in association with chemometrics and automatic variable selection, inmetabolic fingerprinting of salt-stressed tomatoes (Johnson et al., 2003) and grapevine (Oliveira et al., 2009).
The aim of this study was to determine whether or not strain Bs008 can interact directly with plants to cause growth promotion.In our experiment, the cucumber was chosen as a economic crop model.The changes in the Buensanteai et al. 1007 cellular composition of the cucumber cell treated with the Bacillus subtilis Bs008 were assessed by using the FT-IR spectroscopy.By selecting the FT-IR procedures related to PGPR-enhance growth promotion, our report display novel perspectives in the use of FT-IR in the change of the cellular composition enhanced by plant growth promoting rhizobacteria.

Bacterial strains and culture conditions
Bacterial strains used in cucumber experiments as B. subtilis strain Bs008 was isolated from soil around Nakhonratchasima province, Thailand.Cells of the strain Bs008 stored in nutrient glucose broth with 10% glycerol at -80°C were revived by streaking onto nutrient glucose agar (NGA) and cultured at 28 ± 2°C for 48 h.The strain was transferred to 500 ml of nutrient glucose broth (NGB) containing 2% glucose and incubated for 48 h at 28 ± 2°C with constant shaking at 180 rpm.Cells were collected and washed twice by centrifugation at 13,000 rpm for 20 min (Beckman model JS13.1) and the bacterial cell pellet was washed three times in sterile saline (0.85% NaCl).The cells were re-suspended in sterile distilled water, cell concentrations were determined turbidimetrically and adjusted to optical density of 0.2 at 600 nm, corresponding to 1 × 10 8 CFU ml -1 (Buensanteai et al., 2008a, b).

Plant materials and treatment
The cucumber seeds were surface disinfested by treatment with 95% ethanol (v/v) for 2 min, followed by soaking in 20% commercial bleach (v/v) for 20 min.The seeds were then washed with sterile distilled water 5 times in order to remove the bleach.Before planting, 30 g of cucumber seeds were mixed thoroughly with 5 ml of a liquid treatment for 15 min.The cell concentration in the whole culture and cell suspensions were adjusted to 1x10 8 cfu ml -1 on the basis of absorbance.The experiment was conducted under greenhouse conditions, treated cucumber seeds were placed onto sterile soil and place into the plastic pot.There were four replicate pots per treatment with one cucumber seed per pot.The pots were maintained in a greenhouse with a photoperiod of 16 h of light, 8 h of darkness, light intensity of 200 umol m 2 s -1 , and constant temperature of 24°C.At 28 days after germination, cucumber seeds were harvested for measurement of growth parameters (root and shoot lengths; fresh and dry weights; and numbers of lateral roots).The experiment was performed three times.Finally, for cellular components and phytohormone analysis, cucumber seedling from untreated (control) and Bs008 treated plants were used for 28 days old seeds.

Cucumber seedling cellular composition measurement using FTIR
Dried cucumber seedlings were ground in a crystal mortar and pestle.FTIR sample preparation and measurements were performed according to Kamnev et al. (2008).In brief, 1 mg of the resulting dry biomass in a micro sampling cup lightly presses the surface of the powdered sample with a flat glass spatula and mounting the sampling cup into the sample holder of the FTIR spectrometer (Tensor 27).The infrared spectra were collected using the attenuated total reflectance (ATR)-FTIR spectroscopy with single reflection ATR sampling module with and coupled with MCT detector cooled with liquid nitrogen over the measurement range from 4000-600 cm -1 .The measurements were performed with a spectral resolution of 4 cm -1 with 64 scans co-added (Bruker Optics Ltd, Ettlingen, Germany).Spectra from each group were analyzed using Principal Component Analysis (PCA).Individual spectra from each group were analyzed using PCA to distinguish different chemical components of the samples using the Unscrambler 9.7 software.(CAMO, Norway).The spectra were processed using 2nd derivative and vector normalized by the Savitzky-Golay method (3 rd polynomial, 9 smoothing points) and then normalized using Extended Multiplicative Signal Correction in the spectral regions from 1750-850 cm -1 .

Unsupervised hierarchical cluster analysis (UHCA)
UHCA was performed on second derivative spectra using Ward's algorithm which utilizes a matrix defining inter-spectral distances to identify the most similar IR spectra.Spectral distances were calculated as D-values.Ward's Algorithm tries to find s homogeneous groups as possible.This means that only two groups merged which show the smallest growth in heterogeneity factor H. Instead of determining the spectral distance, the Ward's Algorithm determines the growth of heterogeneity H. n(i) is the number of spectra that merged in the i cluster.
H(r,i) is calculated according to the following equation: n(p) is the number of spectra which are merged in the p cluster; n(i) is the number of spectra which merged in the i cluster; and n(q) is the number of spectra which merged in the q cluster.The spectral distance between the new r cluster and the i cluster was calculated as follows:

Effect of Bs008 on growth parameter of cucumber seedling
The strain BS008 was effective in promoting the growth of cucumber seedlings under gnotobiotic conditions, the length, weight and lateral root of the seedlings were measured after three days of planting.These strain, when applied to cucumber seeds, increased root and shoot lengths, by more than 55 and 38%, respectively (Figure 1) as compared to the distilled water control.Seed treatment with the strain BS008 also increased the number of lateral root more than 20% as compared to the control (data not shown).Similar results were obtained when the experiment was repeated.In order to investigate the effects of BS008 at the early stage of plant growth and development, the cucumber seedlings of untreated and BS008-treated seedlings were excised, the cellular composition accumulation levels were determined, and proteome and biochemical components analysis were conducted.

Changes in cucumber seedling cellular components in response to Bs008 treatment using FTIR analysis
In this current study, the FT-IR spectroscopy was performed in order to explore the cellular and biochemical changes of cucumber plant after sensitization with the beneficial bacteria B. subtilis strain Bs008.The IR spectra of cucumber plant reflect the cellular components of the cell wall and membrane such as polysaccharides, proteins secondary structure and lipid content.The conformational change of protein amide noted between 1700-1600 cm -1 can give information on protein seconddary structure such as alpha-helix (centered at 1653 cm - 1 ), beta-sheet (centered at 1635 cm -1 ) and beta-turn (centered at 1685 cm -1 ).The conversion of the original spectra to their second derivatives was used in order to find the exact peak locations and reveal spectral shifting and intensity variations among spectra.Indeed, the second derivative transformation of FT-IR spectra made the differences in two spectral regions more distinctive when cucumber vegetative propagation plants treated with Bs008 were used.Our results indicate that the average FT-IR spectra of cucumber plants (Figure 3) treated with distilled water and the beneficial bacteria B. subtilis strain Bs008 were different in biochemical components upon plant growth promoting bacteria sensitizaton.The cucumber seedling treated with the beneficial bacteria B. subtilis strain Bs008 shows higher content of the polyshaccharides associated with cell membrane structure when compared with those of the cucumber seedling treated with distilled water.The spectra showed in Figure 3 indicated that there are variations in polysacharide component.Clearly, the higher content of polysaccharide in the spectral region of C-O-C stretching (1150-900 cm -1 ) was seen under distilled water negative control when compared with cucumber seedling treated with the beneficial bacteria, B. subtilis strain Bs008 (Figure 3).
Moreover, the multivariate statistical analysis techniques based on PCA was used to statistical analyze the significant spectral data of cucumber seedling (Figure 2).Our results show clearly separate with distinct sample clusters which were observed among the cucumber seedling in two spectral regions.Discrete grouping of samples originating from the use of the beneficial bacteria B. subtilis strain Bs008 and distilled water in these two spectral regions were readily evident within the PC1 and PC2 which appeared as the highest variance, accounting for 92 and 4% of the variability respectively.
Our findings clearly support a specific effect of the beneficial bacteria B. subtilis strain Bs008 on the polysac- Buensanteai et al. 1009 charide that all involve the cellular composition of cucumber seedling, while also affecting another cellular composition in cucumber seedling.Enhanced plant growth promotion effects using PGPR strains in different economic crops were clearly investigated and studied.This current study confirms the earlier experiments which revealed that under the laboratory conditions, cucumber treatment with PGPR as Bacilli group strain Bs008 improved seed germination, seedling vigor and seedling emergence over the control.Similar improvement of seed germination parameters by rhizobacteria has been reported in other field crop such as soybean (Buensanteai et al., 2008(Buensanteai et al., , 2009)).The improvement in seed germination by PGPR was also found in other works, and it was shown that some PGPR induced increases in seed emergence, in some cases achieving increases up to 100%, greater than controls.These findings may be due to the increased synthesis of hormones like IAA and the high lipid band could assume that it is related to the increase of the carbonyl bond around 1743 cm -1 , which would have triggered the activity of specific enzymes that promoted early germination, such as amylase, which have brought an increase in availability of starch assimilation.Beside, significant increase in seedling vigor would have occurred by better synthesis of auxins.These results are also similar to the findings of Buensanteai et al. (2008) who assessed the inoculation effect of PGPR B. amyloliquefaciens strain KPS46 on growth of vegetable soybean.They observed that inoculated plants resulted in better germination, early development and flowering and also increase dry weight of both the root system and the upper plant parts.Similarly, promotion in growth parameters and yields of various crop in response to inoculation with PGPR were reported by other workers.Inoculation of cucumber seeds with Azospirillum strains when compared with Pseudomonas strains under experiment conditions resulted in a more visible increase in shoot development, especially during the establishment of the plant (Wan et al., 2005;Pizzirani-Kleiner and Azevedo, 2004).
In the current study, we also described the changes in the cellular components of cucumber seedlings after sensitization with plant growth promoting rhizobacteria, B. subtilis Bs008 using FTIR spectroscopy and phytohormone analysis.We found that Bs008 can influence the growth and development of cucumber seedlings.The results are consistent with the hypothesis that strain of PGPR promotes the growth of plant seedlings by secretion of several types of compounds interact with directly and indirectly with plant growth enhancement mechanism.Enhanced growth and development in plants inoculated with PGPR have been published.And results of cucumber seedling in present study are consistent with previous documents.The effects of PGPR inoculation on plant growth and development varied depending on the microbial genus and species, B. subtilis, B. amyloliquefaciens, Bacillus polymyxa, Bacillus cereus,  Bacillus megaterium, Pseudomonas fluorescens.The increasing in maize seedling biomass when B. amyloliquefaciens FZB42 treated seeds has been reported previously.And various effects of PGPRs on root morphology have been reported.Enhanced formation of lateral roots leads to increased root surface area and nutrient uptake potential.
This important study examines the interaction of cucumber with a PGPR at the cellular level.In our analysis of cucumber cellular components produced in response to Bs008 treatment, the number of lipid was highly accumulated than untreated treatment.The cellular components we investigated represent only proportion of sample up-regulated cellular components and we also did not consider cellular components that were downregulated by Bs008 treatment.Nevertheless, our results concluded that Bs008 sensitizes and enhances growth of cucumber plants.This most likely reflects the strong positive direct effect that Bs008 has on cucumber growth and development.In conclusion, the results of this experiment suggest that simultaneous finding of beneficial bacteria for growth promotion under pot experiment is a valuable tool to select effective PGPR strain for our microbial biofertilizer in the near future.

Figure 1 .
Figure 1.Effects of B. subtilis BS008 on the growth and development of cucumber seedling under gnotobiotic conditions, as measured at 3 days after inoculation.(a) Shoot and root length treated with water, (b) shoot and root length treated with Bs008.The data are the average of four replications (three plants per replication) for each treatment.Error bars represent the standard deviation.For each growth parameter, different letters indicate significant differenced (P ≤ 0.05) among treatments.

Figure 2 .
Figure 2. PCA analysis of cucumber seedling treated with the beneficial bacteria B. subtillis strain Bs008 and distilled water (a) score plot and (b) loading plot of independent spectra from different conditions.The chemical compositions of two groups were classified with PC1 versus PC2 score plot.PC1 and PC2 explained 92 and 4% of the total variance, respectively.Spectra were derived using second derivative processing with the entire biochemical cell fingerprint region.

Figure 3 .
Figure 3. Average second derivative FTIR spectra of treated sample with the beneficial bacteria B. subtillis strain Bs008 and distilled water in the region of (a) 1800-850 cm -1 and (b) 3000-2800 cm -1 .Spectra were measured with 64 scans co added for each individual spectra.Spectra were preprocessed by taken second derivative spectra after 9 points of smoothing and normalized with EMSC over the range.