www.thaiagj Thai Journal of Agricultural Science 2008, 41(3-4): 101-116
The Biocontrol Bacterium Bacillus amyloliquefaciens
KPS46 Produces Auxin, Surfactin and Extracellular Proteins
for Enhanced Growth of Soybean Plant
个子矮的女生穿衣搭配N. Buensanteai1, G.Y. Yuen2 and S. Prathuangwong1,*
1Department of Plant Pathology, Faculty of Agriculture
Katsart University, Bangkok 10900, Thailand
2Department of Plant Pathology, University of Nebraska-Lincoln
Lincoln, NE 68583-0722, USA
*Corresponding author. Email: agrsdp@ku.ac.th
Abstract
Bacillus amyloliquefaciens strain KPS46, a plant growth promoting rhizobacterium isolated from soybean, was investigated for the cretion of compounds that might be involved in plant growth promotion. Extracts containing indoles, lipopeptides, and proteins were made from the cell-free fluid of KPS46 broth cultures. The extracts, along with a whole culture of KPS46 and the raw fluid and cellular fractions from a KPS46 culture, were applied parately to soybean eds. The eds were planted into plates of a plant growth medium and kept in a growth chamber or into pots of pasteurized planting media maintained in a greenhou. Under both conditions, all of the extracts and fractions, with the exception of a treatment with heat-killed KPS46 cells, incread root and shoot lengths and plant biomass as compared to distilled water control. Thus, indoles, lipopeptides, and proteins creted by KPS46 can directly influence plant growth and development. Analysis of the indole and lipopeptide extracts by HPLC revealed the auxin indole-3-acetic acid and the antibiotic surfactin to be the primary compounds, respectively. To investigate the proteins involved in growth promotion, two-dimensional gel electrophoresis was ud to parate proteins creted by KPS46 and by N19G1, a UV-derived mutant of KPS46 with reduced production of auxins, lipopeptides, extracellular proteins, and lacking growth promotion activity. The identity and putative function of twenty proteins creted by KPS46 but not by N19G1 were determined. The analysis revealed a number of proteins which may be involved in plant growth promotion by acting as plant growth regulators, stimulating metabolism or functioning in defen against stress factors.
Keywords: signal molecules, elicitors, PGPR, proteomic analysis
Introduction
Bacillus amyloliquefaciens KPS46, a strain of plant growth promoting rhizobacteria (PGPR) isolated from a soybean plant in central Thailand (Prathuangwong and Kam, 2004; Prathuangwong et al., 2005), can protect soybean and other crop plants against multiple plant pathogens by induced resistance and cretion of antimicrobial metabolites (Buensanteai et al., 2007a; Prathuangwong and Buensanteai, 2007; Prathuangwong and Kam, 2004; Prathuangwong et al., 2004, 2005). When applied as a ed treatment, it can also promote the growth of soybean (Buensanteai et al., 2007b, 2008). The mechanisms by which KPS46 promotes plant growth are not well understood.
In addition to KPS46, other strains classified in B. amyloliquefaciens and in the cloly related species B. subtilis have been reported to be effective for the biocontrol of phytopathogens and for plant
102 N. Buensanteai et al. Thai Journal of Agricultural Science
growth promotion (Araujo et al., 2005; Kloepper et al., 2004; Idriss et al., 2007). Extrapolating from th
prle accumulative literature on the two related species and on other species of PGPR, numerous mechanisms are possible in KPS46 as to antagonism against pathogens, alteration of nutrient availability, and direct interactions with plants. All of the interactions could potentially lead to plant growth promotion.
As a starting point towards understanding how KPS46 caus enhanced plant growth, we focud this study on factors creted by KPS46 that might directly affect the physiology of soybean plants. In this context, the production of auxins, indolic compounds with phytohormone activity, by Bacillus spp. is well known (Araujo et al., 2005; Kloepper et al., 2004; Idriss et al., 2007). Indole-3-acetic acid (IAA), the main auxin in plants, controls important process including cell enlargement and division and tissue differentiation. Regulation of the process requires a balance between auxins and other phytohormones. Thus, IAA production by strains of PGPR can contribute sufficient auxin to the plant’s auxin pool to have profound effects on the process and potentially allow the microorganisms to redirect a plant’s physiology and biochemistry for their own benefit (Idriss et al., 2007; Leveau and Lindow, 2005; Patten and Glick, 2002).
Other groups of compounds commonly reported to be creted by Bacillus spp. include the lipopeptide families, iturin and surfactin that are well known for their strong antibiotic activity (Araujo
et al., 2005; Bonmatin et al., 2003; Jacques et al., 1999; Ongena et al., 2007). Surfactin, becau of its biosurfactant quality, is directly toxic to diver microorganisms, including fungi, bacteria, and virus (Bonmatin et al., 2003). It was recently to induce resistance in plants (Ongena et al., 2007), and thus, there is evidence that surfactin also has the potential to directly affect the physiology of plants.
Another large group of bacteria-creted compounds is extracellular proteins. Strains in the Bacillus group crete high levels of extracellular proteins as enzymes and condary metabolites (Tjalsma et al., 2004). Some proteins creted by Bacillus spp. are involved in key ecological functions such as biofilm formation (Oosthuizen et al., 2002), but extracellular proteins, as a group, have not yet been investigated in the context of plant growth promotion.
The primary goal of this study was to determine whether or not compounds creted by KPS46 have a direct role in regulating plant growth, i.e., promoted plant growth under optimal nutrient conditions and in the abnce of plant-deleterious organisms. One objective was to determine whether lipopeptides, indoles, and extracellular proteins produced by KPS46 in liquid culture could enhance growth of soybean when applied to soybean eds. Another objective was to analyze the lipopeptide and indole extracts from KPS46 to identify the primary components. Lastly, the proteomic approach
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was ud in analyzing extracellular proteins creted by KPS46 to identify tho that might be important in plant growth promotion. To narrow the spectrum of proteins, creted proteins from wildtype KPS46 were compared with tho from a mutant strain affected in growth promotion traits. Preliminary results have been published (Buensanteai et al., 2007b, 2008).
Materials and Methods
Culture Conditions and Preparation of Extracellular Extracts
Cells of B. amyloliquefaciens strain KPS46 stored in nutrient gluco broth with 10% glycerol at -80o C were revived by streaking onto nutrient gluco agar (NGA) and cultured at 28o C ± 2o C for 48 h. To prepare cultures for extraction of extracellular factors, the strain was transferred to 500 mL volumes of nutrient broth containing 2% gluco (NGB) and incubated for 48 h at 28 ±2o C with constant shaking at 180 rpm. The cultures were centrifuged at 13,000 rpm at 4o C for 20 min and the supernatants were pasd through 0.2 nm nitrocellulo filters and retained for further extraction.
The bacterial cell pellet was washed three times in sterile saline (0.85% NaCl). The cells were re- suspended in sterile distilled water and the density of the suspension was adjusted to 108 cfu mL-1 bad on optical density (OD of 0.2 at 600 nm). A portion of the cell suspension was ud in experim
ents in live form, while another portion was heated in a water bath at 100°C for 30 min to kill the cells prior to u.
你行我也行Extracts containing indoles were made from the cell-free culture supernatant using the method described by Araujo et al.(2005). Cell-free culture
Vol. 41, No. 3-4, 2008KPS46produces auxin, surfactin and extracellular proteins for soybean growth 103
fluid was extracted three times with ethyl acetate after adjusting the pH to 2.8 and then evaporated at 40o C for 20 min. The material was solubilized in sterile distilled water for u in experiments.
Extracellular lipopeptides were extracted from KPS46 culture supernatant by acidifying the fluid to pH 2.0 with concentrated HCl and allowing the formation of a precipitate at 4o C overnight. The precipitate was collected by centrifugation (12,000 rpm, 4o C, 15 min), washed three times with distilled water, and dried by vacuum lyophilization. The dried lipopeptide was extracted three times with 100% methanol for 3 h. The methanol was removed with a rotary evaporator under reduced pressure, yielding a brown-colored crude lipopeptide extract. The extract was solubilized in sterile distilled water prior to u in experiments.现状分析
To prepare the extracellular protein extract, 20 mL 50% trichloroacetic acid (Sigma T6399) was added to 500 mL of culture supernatant, mixed well and placed on ice for 30 min.The aggregated proteins were precipitated by centrifugation at 12,000 rpm at 4o C for 15 min,washed three times in cold 70% ethanol (-20°C), dried and dissolved in IEF (isoelectric focusing) sample buffer consisting of 8 M urea (Sigma U6504),2 M thiourea (Sigma T7875), 2% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Sigma C9426), 2% Triton X-100 (Sigma T8532), 50 mM DTT (dithiothreitol, Sigma D9163), and 0.5% ampholytes (Bio-Rad 163-1152) (Buensanteai et al., 2008), the protein extract was solubilized in sterile distilled water. Total protein content of the sample was quantified by the Bradford assay (Bradford, 1976) prior to u in experiments.
Plant Bioassays for Growth Promotion
平安夜快乐英文Two ts of experiments were conducted to evaluate KPS46 culture fluid extracts for effects on soybean growth. Seeds of soybean cv. Spencer were surface disinfested by treatment with 95% ethanol for 2 min, followed by soaking in 20% (v/v) solution of commercial bleach for 20 min. The eds were then washed with sterile distilled water 5 times in order to remove the bleach. Before planting, 30 g of soybean eds were mixed thoroughly with 5 mL of a liquid treatment. The treatments included a whole culture of KPS46 in NGB, cell-free fluid from a KPS46 culture in NGB, a
suspension of live cells collected from a KPS46 culture, and a suspension of heat-killed cells. Sterile distilled water was ud as the control. Cell concentrations in the whole culture and cell suspensions were adjusted with sterile distilled water to 1 × 108 cfu mL-1, bad on absorbance, while culture fluid was diluted by the same dilution factor as the whole culture. Other treatments included extracts of extracellular proteins (250 µg mL-1), lipopeptides (50 µg mL-1) and indoles (50 µg mL-1) extracted from KPS46. Distilled water was ud to dilute the raw extracts to the concentrations, which roughly correspond to tho found in NGB cultures with 1 × 108 cfu KPS46 mL-1.
In one t of experiments conducted under gnotobiotic conditions, treated eds were placed onto 0.5X MS medium (Murashige and Skoog basal salt mixture, Sigma M5524) in square plates. There were four replicate plates per treatment with three eds per plate. The plates were incubated on edge at a 65◦ angle to allow root growth along the agar surface and unimpeded growth of the tops of the edlings after the ed had germinated. The plates were maintained in a growth chamber with a photoperiod of 16 h of light, 8 h of darkness, light intensity of 200 µmol m2s-1, and constant temperature of 24o C. At 7 days after germination, edlings 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.
In another t of experiments, soybean eds treated with KPS46 culture extracts were planted in pots (30 cm diameter) containing a steam-pasteurized potting medium of Sharpsburg silt clay loam, vermiculite, and sand mixed in equal volumes. There were 10 replicate pots per treatment with two eds per pot. The pots were watered daily with a nutrient solution (20-10-20 Peat-Lite Special, Scotts-Sierra Horticultural Co., Marysville, OH) and kept in a greenhou with a 12-h photoperiod (25o C and 60 to 75% relative humidity during the light period, 15o C and >93% relative humidity during the dark period). At 14 days after edling emergence, edlings were harvested for measurements of root and shoot lengths, along with fresh and dry weights. The experiment was conducted three times.
Data from each experiment was subjected to analysis of variance using SAS version 9.1. Separation
104 N. Buensanteai et al. Thai Journal of Agricultural Science
of treatment means was accomplished by Duncan’s Multiple Range Test, and all tests for significance were conducted at P≤ 0.05.
Analysis of Indoles
Supernatants from two types of cultures of KPS46 were extracted and assayed for indoles. In one, KPS46 was grown in NGB for 24, 48, 72, 96 and 120 h. In the other, KPS46 was cultured at 28±2o C for 48 h on a shaker incubator in DF salt minimal medium amended with 0, 125, 250, 500, 2,500 and 5,000 mM tryptophan obtained from a filter-sterilized 2 mg mL-1 stock of L-tryptophan prepared in warm water. The indole extract was extracted from cell-free culture supernatants as described above. Indole concentrations were determined bad on the method described by Patten and Glick (2002) with slight modifications. For the colorimetric Salkowski assay for indoles, 1 mL of culture fluid was mixed with 4 mL of Salkowski’s reagent and incubated at room tem- perature for 20 min. The absorbance was measured at 535 nm using a spectrophotometer (Spectronic 20D, Rochester, NY, USA) (Crozier et al., 1988). The quantity of indoles was determined by comparison with a standard curve using purified IAA in the concentration range of 0-50 µg mL-1. Concentration of IAA in the extracts also was determined by rever pha-HPLC by injecting 60 µL aliquots of extracts into an Alltech, type Econosphere C185U column (250 by 4.6 mm) equipped with a differential UV detector absorbing at 280 nm. The isocratic solvent ud for rever-pha chromato- graphy was acetonitrile-glacial acetic acid (1%) in water (1:9). The flow rate was adjusted to 1 mL min-1. Peak retention times were compared with tho of chemically-synthesized IAA standards and quantified by comparison of peak areas.
right是什么意思Analysis of Lipopeptides
A lipopeptide-enriched extract was obtained from 20 mL of the crude lipopeptide extract from cultures of KPS46 in NG
B by purification on an ISOLUTE C-18 CE type cartridge (International Sorbent Technology Ltd., Hengoed, UK) following a modification of the method described by Jacques et al. (1999) and Araujo et al. (2005). The enriched extract was dissolved in butanol and loaded onto a column of a rever-pha HPL
C system. The system was operated at a flow rate of 2.0 mL min-1 with 90% methanol as the mobile pha. HPLC spectra were detected by a UV monitor at 210 nm. The prence of surfactin-type lipopeptides was determined on the basis of retention times compared with tho of purified surfactin standards (Sigma S3523).
Mutagenesis of KPS46 and Selection
of Mutant Strains
Cells were obtained from a culture of strain KPS46 in NGB at stationary pha, 4 h after the optical d
ensity (λ = 600 nm) of cultures stopped increasing. The cells were washed and suspended in 0.05 M potassium phosphate buffer, pH 6.8, to 1 × 108 cfu mL-1. Volumes (200 µL) of this suspension were expod to UV irradiation for various durations in glass petri dishes at a constant distance from the UV source (λ = 254 nm, 10 erg mm-2; BII Illuminator). The samples were frequently agitated during exposure. Aliquots (100 µL) were removed from the irradiated samples at different time intervals and spreaded onto nutrient gluco agar plates. Colonies arising from surviving cells were lected following 48 h of incubation in the dark condition (Whistler et al., 2000; Saxena et al., 2002).
More than 2,490 irradiated strains were screened for loss of or decrea in indole production. Fluid from cultures of the strains in NGB was tested for indole concentration via the Salkowski assay. Tho strains in which indole production was decread by more than 10% compared with wildtype KPS46 were considered to be indole-reduced mutants and were retained for further study. Selected indole-reduced mutants were compared further to wildtype KPS46 with respect to excretion of IAA, using rever-pha HLPC as described above, and production of lipopeptides. Relative lipopeptide production was accesd on the basis of swarming motility across a solid surface, this assay predicated by lipopeptides being required for swarming motility (Kearns and Losick, 2003). This trait was evaluated by eding cell suspensions of stationary-pha cultures (50 µL;
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1 × 108 cfu mL-1) onto the centers of motility test plates (NGB with 1.5% agar; and LB with 0.3, 0.7, or 1.5% agar). Plates were incubated at 37°C,and the diameters of halos due to bacterial migration were measured 24 to 48 h postinoculation, and
Vol. 41, No. 3-4, 2008KPS46produces auxin, surfactin and extracellular proteins for soybean growth 105
swarm cell differentiation was analyzed (Senesi et al., 2002; Kearns and Losick, 2003).
In addition, an experiment was conducted to determine whether or not extracellular proteins produced by UV mutant strain N19G1 derived from KPS46 can enhance plant growth. As in the gnotobiotic and greenhou experiments described above, 30 g of soybean eds was mixed thoroughly for 5 min with 15 mL of protein extract from cultures of wild type strain KPS46 and mutant strain N19G1, and the ed germinated and edlings grown in plates with 0.5X MS medium or planted in pots of planting medium. Biomass, number of lateral root, and root and shoot lengths were determined at 7 days after ed germination. Each experiment was conducted three times.
Proteomic Analysis of Extracellular
Proteins of KPS46
The intent of this experiment was to identify some of the specific components of the extracellular proteome of KPS46 that may have some relationship to IAA biosynthesis, lipopeptide production and plant growth promotion. To reduce the range of proteins to tho more cloly involved in the process, extracellular proteins of KPS46 were compared with tho creted by mutant strain N19G1 which was reduced in growth promotion ability. Phenylmethylsulphonyl fluoride (Sigma) was added to late-exponential pha cultures of KPS46 and N19G1 to a final concentration of 5 mM to prevent proteolytic digestion. Proteins were then extracted from the culture fluids as described above. The extracellular protein samples were ud directly to passively rehydrate isoelectrically focud on an Electro Immobiline Dry Strip pH 3–10 and pH 4-7 (11 cm; Bio-Rad, USA) by applying 185 µL of each sample (equivalent to 250 µg of protein). Extracellular preparations dissolved in the 10X of IEF sample solution was applied to the first dimension. The IPG strips were focud for 15 h at 400V followed by 1 h at 600V using a Multiphor II (Amersham Pharmacia). After placing IPG strips in equilibration buffer A [50 mM Tris/HCl, pH 6.8, containing 8 M urea, 30%glycerol, 2.5% SDS (sodium dodecyl sulfate, Sigma-Aldrich 436143) and 0.25% DTT] for 15 min, they were transferred into buffer B [50 mM Tris/HCl, pH 6.8, 8 M urea, 30% glycerol, 2.5% SDS, 0.25% DTT and 4.5%iodoacetamide (Sigma, 1149)] for 15 min. The isoelectric focusing gels were embedded in gels (0.25 M Tris/HCl, pH 6.8,0.25% SDS, 1% agaro onto 14% SDS polyacrylamide). The proteins wer
blastcodee resolved in the cond dimension by a constant current of 100 volts until the bromphenol blue marker entered the stacking gel, followed by 140 volts until the blue dye reached the bottom of the gel (Antelmann et al., 2003; Duy et al., 2007; Lai et al., 2003). The 2D gels were visualized by staining with colloidal Coomassie blue G-250 [17% ammonium sulphate, 34% methanol, 3.6% orthophosphoric acid, 0.1% Coomassie blue G-250 (Sigma B0770)] (Voigt et al., 2006). The gels were fixed in destaining solution (80% ethanol and 20% acetic acid mixture) and washed with 70% ethanol.The gels were analyzed using ImageMaster software for protein spots visible exclusively or at higher intensity in the gel containing extracellular proteins from wildtype strain KPS46 as compared to the UV mutant strain N19G1. Proteins showing more than 2-fold increa in expression in the wildtype compared with the mutant were lected for identification. Amino acid quences of the protein spots were identified by the Protein and the Mass Spectrometry Core Facility of the Center for Biotechnology, University of Nebraska-Lincoln, using mass spectrometry analysis methods modified from tho described by Voigt et al. (2006) and Domon and Aebersold (2006). The quence data were compared with the NCBI and SwissProt databas using local MASCOT to identify possible protein names and functions.
Results
Effects of KPS46 Extracellular Factors on
Plant Growth
Crude extracellular lipopeptide, proteins, and indole extracts from cultures of strain KPS46 were effective in promoting the growth of soybean edlings under gnotobiotic conditions. The culture extracts, when applied to soybean eds, incread root and shoot lengths, by more than 40 and 20%, respectively (Figure 1A), and incread fresh and dry weights by more than 30% (Figure 1B) compared to the distilled water control. Seed treatment with the extracted factors of KPS46 also incread the number of lateral root more than 20% compared to the control (Figure 2). Treatment with the lipopeptide,
